Histology. A Text and Atlas [8 ed.] 9781496383426

Table of contents :
Cover
Copyright
Preface
Acknowledgments
Contents
1 Methods
OVERVIEW OF METHODS USED IN HISTOLOGY
TISSUE PREPARATION
HISTOCHEMISTRY AND CYTOCHEMISTRY
MICROSCOPY
2 Cell Cytoplasm
OVERVIEW OF THE CELL AND CYTOPLASM
MEMBRANOUS ORGANELLES
NONMEMBRANOUS ORGANELLES
INCLUSIONS
CYTOPLASMIC MATRIX
3 The Cell Nucleus
OVERVIEW OF THE NUCLEUS
NUCLEAR COMPONENTS
CELL RENEWAL
CELL CYCLE
CELL DEATH
4 Tissues: Concept and Classification
OVERVIEW OF TISSUES
EPITHELIUM
CONNECTIVE TISSUE
MUSCLE TISSUE
NERVE TISSUE
HISTOGENESIS OF TISSUES
IDENTIFYING TISSUES
5 Epithelial Tissue
OVERVIEW OF EPITHELIAL STRUCTURE AND FUNCTION
CLASSIFICATION OF EPITHELIUM
CELL POLARITY
THE APICAL DOMAIN AND ITS MODIFICATIONS
THE LATERAL DOMAIN AND ITS SPECIALIZATIONS IN CELL-TO-CELL ADHESION
THE BASAL DOMAIN AND ITS SPECIALIZATIONS IN CELL-TO-EXTRACELLULAR MATRIX ADHESION
GLANDS
EPITHELIAL CELL RENEWAL
6 Connective Tissue
OVERVIEW OF CONNECTIVE TISSUE
EMBRYONIC CONNECTIVE TISSUE
CONNECTIVE TISSUE PROPER
CONNECTIVE TISSUE FIBERS
EXTRACELLULAR MATRIX
CONNECTIVE TISSUE CELLS
7 Cartilage
OVERVIEW OF CARTILAGE
HYALINE CARTILAGE
ELASTIC CARTILAGE
FIBROCARTILAGE
CHONDROGENESIS AND CARTILAGE GROWTH
REPAIR OF HYALINE CARTILAGE
8 Bone
OVERVIEW OF BONE
GENERAL STRUCTURE OF BONES
TYPES OF BONE TISSUE
CELLS OF BONE TISSUE
BONE FORMATION
BIOLOGIC MINERALIZATION AN DMATRIX VESICLES
BONE AS A TARGET OF ENDOCRINE HORMONES AND AS AN ENDOCRINE ORGAN
BIOLOGY OF BONE REPAIR
9 Adipose Tissue
OVERVIEW OF ADIPOSE TISSUE
WHITE ADIPOSE TISSUE
BROWN ADIPOSE TISSUE
TRANSDIFFERENTIATION OF ADIPOSE TISSUE
10 Blood
OVERVIEW OF BLOOD
PLASMA
ERYTHROCYTES
LEUKOCYTES
THROMBOCYTES
COMPLETE BLOOD COUNT
FORMATION OF BLOOD CELLS (HEMOPOIESIS)
BONE MARROW
11 Muscle Tissue
OVERVIEW AND CLASSIFICATION OF MUSCLE
SKELETAL MUSCLE
CARDIAC MUSCLE
SMOOTH MUSCLE
12 Nerve Tissue
OVERVIEW OF THE NERVOUS SYSTEM
COMPOSITION OF NERVE TISSUE
THE NEURON
SUPPORTING CELLS OFTHE NERVOUS SYSTEM:THE NEUROGLIA
ORIGIN OF NERVE TISSUE CELLS
ORGANIZATION OF THE PERIPHERAL NERVOUS SYSTEM
ORGANIZATION OF THE AUTONOMIC NERVOUS SYSTEM
ORGANIZATION OF THE CENTRAL NERVOUS SYSTEM
RESPONSE OF NEURONS TO INJURY
13 Cardiovascular System
OVERVIEW OF THE CARDIOVASCULAR SYSTEM
HEART
GENERAL FEATURES OF ARTERIES AND VEINS
ARTERIES
CAPILLARIES
ARTERIOVENOUS SHUNTS
VEINS
ATYPICAL BLOOD VESSELS
LYMPHATIC VESSELS
14 Immune System and Lymphatic Tissues and Organs
OVERVIEW OF THE IMMUNE AND LYMPHATIC SYSTEMS
CELLS OFTHE IMMUNE SYSTEM
LYMPHATICTISSUESAND ORGANS
15 Integumentary System
OVERVIEW OF THE INTEGUMENTARY SYSTEM
LAVERS OFTHE SKIN
CELLS OFTHE EPIDERMIS
STRUCTURES OF SKIN
16 DIGESTIVE SYSTEM I: Oral Cavity and Associated Structures
OVERVIEW OF THE DIGESTIVE SYSTEM
ORAL CAVITY
TONGUE
TEETH AND SUPPORTING TISSUES
SALIVARY GLANDS
17 DIGESTIVE SYSTEM II: Esophagus and Gastrointestinal Tract
OVERVIEW OF THE ESOPHAGUS AND GASTROINTESTINAL TRACT
ESOPHAGUS
STOMACH
SMALL INTESTINE
LARGE INTESTINE
18 DIGESTIVE SYSTEM III: Liver, Gallbladder, and Pancreas
LIVER
GALLBLADDER
PANCREAS
19 Respiratory System
OVERVIEW OF THE RESPIRATORY SYSTEM
NASAL CAVITIES
PHARYNX
LARYNX
TRACHEA
BRONCHI
BRONCHIOLES
ALVEOLI
BLOOD SUPPLY
LYMPHATICVESSELS
NERVES
20 Urinary System
OVERVIEW OF THE URINARY SYSTEM
GENERAL STRUCTURE OF THE KIDNEY
KIDNEY TUBULE FUNCTION
INTERSTITIAL CELLS
HISTOPHYSIOLOGY OF THE KIDNEY
BLOOD SUPPLY
LYMPHATICVESSELS
NERVE SUPPLY
URETER, URINARY BLADDER, AND URETHRA
21 Endocrine Organs
OVERVIEW OF THE ENDOCRINE SYSTEM
PITUITARY GLAND (HYPOPHYSIS)
HYPOTHALAMUS
PINEAL GLAND
THYROID GLAND
PARATHYROID GLANDS
ADRENAL GLANDS
22 Male Reproductive System
OVERVIEW OF THE MALE REPRODUCTIVE SYSTEM
TESTIS
SPERMATOGENESIS
SEMINIFEROUS TUBULES
INTRATESTICULAR DUCTS
EXCURRENT DUCT SYSTEM
ACCESSORY SEX GLANDS
PROSTATE GLAND
SEMEN
PENIS
23 Female Reproductive System
OVERVIEW OF THE FEMALE REPRODUCTIVE SYSTEM
OVARY
UTERINE TUBES
UTERUS
PLACENTA
VAGINA
EXTERNAL GENITALIA
MAMMARY GLANDS
24 Eye
OVERVIEW OF THE EYE
GENERAL STRUCTURE OF THE EYE
MICROSCOPIC STRUCTURE OF THE EYE
ACCESSORY STRUCTURES OFT HE EYE
25 Ear
OVERVIEW OF THE EAR
EXTERNAL EAR
MIDDLE EAR
INTERNAL EAR
Index

Citation preview

EIGHTH EDITION

HISTOLOGY A TEXT AND ATLAS With Correlated Cell and Molecular Biology

WOJCIECH PAWLINA

t:. ®Wolters Kluwer

EIGHTH EDITION

HISTOLOGY A TEXT AND ATLAS With Correlated Cell and Molecular Biology

Wojciech Pawlina Driving in his classic red 1972 Buick Skylark convertible, contemplating red histologic structures: red blood cells (RBC), red pulp (RP) of the spleen, red bone marrow (RBM), red muscle fibers (RMF), red margin (RM) of the lip ..• and a red squirrel (RSQ) running between the trees (out of the plane of vision).

EIGHTH EDITION

HISTOLOGY A TEXT AND ATLAS With Correlated Cell and Molecular Biology

WOJCIECH PAWLINA, MD, FAAA Professor ofAnatomy and Medical Education Fellow of the American Association ofAnatomists Chair, Department ofAnatomy Department of Obstetrics and Gynecology Director of Procedural Skills Laboratory nr::.r~·"Nr.,_,.5. ~~~ Mayo Clinic College of Medicine and _,.,..,,....,....:; Rochester. Minnesota

r. .Wolters Kluwer Philadelphia • Baltimore • New York • London Buenos Aire.s • Hong Kong • Sydney • Tokyo

Acquisitions Editor: Crystal Taylor Dtvtlopmmt Editor: Andrea Vosburgh melance Dtvtlopmmt Editor: Kathleen H. Scogna Editorial CoordiTUltor: Alexis Pozonsky Production Project Manager: Marian Bellus Design Coordinator: Steve Druding 1/lurtration CoordiTUltor: Jennifer Clements Manufacturing CoordiTUltor: Margie Orzech-Zarenlro Prepress Vendor: Absolute Service, Inc. 8th edition Copyright © 2020 Wolters Kluwer Copyright © 20 16 Wolters Kluwer Health. Copyright © 20 11, 2006, 2003 Lippincott Williams & Wilkins. Copyright© 1995, 1989 Williams & Wilkins. Copyright© 1985 Harper & Row, Publisher, J. B. Lippincott Company. All rights reserved. This book is protected by copyright. No pan of this book may be reproduced or transmitted in any form or by any means, including as photocopies or scanned-in or other electronic copies, or utilized by any information storage and retrieval system without written permission from the: copyright owner, c:xcept for brief quotations embodied in critical aniclc:s and reviews. Materials appearing in this book prepared by individuals as pan of their official duties as U.S. government employees are not covered by the above-mentioned copyright. To request permission, please contact Wolters Kluwer at Two Commerce Square, 2001 Market Street, Philadelphia, PA 19103, via email at [email protected], or via our website at shop.lww.com (products and services).

9 8 7 6 5 4 3 2 Printed in China

Library of Congress Cataloging-in-Publication Data Names: Ross, Michael H., author. I Pawlina, Wojciech, author. Title: Histology : a text and atlas : with correlated cell and molecular biology I Wojcic:ch Pawlina, Michael H. Ross. Description: Eighth edition. I Philadelphia : Wolters Kluwer Health, [2020] I Includes index. I Michael H. Ross' name appears first in the previous edition. Identifiers: LCCN 20180411541 ISBN 9781496383426 Subjects: I MESH: Histology ITissues I Intracellular Space IAclasc:s Classification: LCC QM551I NLM QS 5171 DDC 611/.018---dc23 LC record available at https://lccn.loc.gov/2018041154 This work is provided "as is," and the publisher disclaims any and all warranties, express or implied, including any warranties as to accuracy, comprehensiveness, or currency of the content of this work. This work is no substitute for individual patient assessment based upon healthcare professionals' examination of each patient and consideration of, among other things, age, weight, gender, current or prior medical conditions, medication history, laboratory data, and other factors unique: to the patient. The publisher does not provide medical advice or guidance, and this work is mc:rely a reference tool. Healthcare professionals, and not the publisher, are solely responsible for the use of this work including all medical judgments and for any resulting diagnosis and treatments. Given continuous, rapid advances in medical science and health information, independent professional verification of medical diagnoses, indications, appropriate pharmaceutical selections and dosages, and treatment options should be made and healthcare professionals should consult a variety of sources. When prescribing medication, healthcare professionals are advised to consult the product information sheet (the manufucturer's package insett) accompanying each drug to verify, among other things, conditions of use, warnings, and side e1fects and identify any changes in dosage schedule or contraindications, patticularly if the: medication to be administered is new, infrequendy used, or has a narrow therapeutic range. To the maximum extent permitted under applicable law, no responsibility is assumed by rhe publisher for any injury and/or damage to persons or propetty, as a matter of products liability, negligence law or otherwise, or from any reference to or use by any person of this work. shop.lww.com

1his edition is dedicated to Teresa Pawlina, my wife, colleague, and best friend, whose love, patience, and endurance created a safe haven for working on this textbook and to my children Conrad Pawlina and Stephanie Pawlina Fixell and her husband Ryan Fixell whose stimulation and excitement are always contagious.

PREFACE This eighth edition of Histology: A Tea and Atlas With Corwlated Cell and MDkcular BiDiogy continues its tradition of introducing health science students to the world of histology. In addition, to better undei'SWld the nature of cells and tissues, the presented histology knowledge is immersed in basic anatomy, embryology. and physiology and is accompanied by relevant clinical commentaries. .AJ; in previous editions, this book is a combination "text-atlas" in that the standard textbook descriptions ofhistologic concepts are supplemented by an array of schematics, tissue and cell images, and clinical photographs. The separate atlas sections conclude each chapter to provide large-fonnat, labeled atlas plates with detailed legends that highlight and summarize the elements of microscopic anatomy. HisttJ!bgy: A Tat and Atlas is, therefore, "'two books in one." This edition of Histology: A Text and klas With CDmlattd Cell and MDkcular BiDiogy is intended to serve as a reliable resource for those who seek to understand histology both from basic science as well as clinical perspective. Its inclusion of current and up--to-date infonnation provides a solid framework on which to build further scientific exploration and clinical application. As a student resource, it should not be approached with the goal of memorizing detailed facts but rather as a guide and explanation to key concepts that will serve future academic pursuits. The following improvements have been made to this edition: "H#Hiog:y lOP'~ INtw !JHtt. Mli#tlll1UI twktlpMI. These sections contain clear and concise summaries for a quick review of the material listed in a "'sticky notes" format posted on the notebook pages at the end of each chapter. The bullet-point format is designed specifically for students needing a quick review and is especially useful for quiz and examination preparation. These reader-friendly sections allow fast information retrieval with concepts and facts grouped on separate sticky notes. The sticky notes design has ample free space that gives Students room to write their own notes to complement the bulleted points. All.Jigw;tw n. tllil book hiiH b.,. emwfolly rmmlll1UI ~ Several new figures have been added to show the latest interpretation of important concepts based on recent discoveries in molecular and cellular research. All drawings maintain a uniform style throughout the chapters with a palette of eye-pleasing colors. Several conceptual drawings have been aligned side by side with photomicro~hs, a feature carried over from previous editions that has received wide acclaim from reviewers, students, and faculty members.

vi

(AlluLr,r MUl ~ ~ t:OIU'InJt btu lwn ... JMtJ. Text material introduced in the seventh edition has been updated to include the latest advancements in cellular and molecular biology, stem cell biology, cellular markers, and cell signaling. The eighth edition focuses on target concepts to help students with overall comprehension of the subject matter. To accommodate reviewers' suggestions, the eighth edition integrates new information in cell biology with clinical correlates, which readers will see as new clinical infonnation items highlighted in blue rextand in clinical boxes (called "Folders"). For example, within the discussion on bone formation, the reader might also discover a new cell biology explanation related to matrix vesicles secreted in the process of bone mineralization. A new discwsion on vesicular transport in the cell, cytoskeleton in nerve cells, and tricellular junctions in epithelial cells are among many topics that were supplemented and updated. Also added is a basic discussion on new developments in tissue preparation and super-resolution microscopy methods that bring optical microscopy into the subcellular level. l~Juukr...frhtully ~tu biiH Htm htt.pltmutt.Ntl. Similar to the previous editions of this book, the aim is to provide ready access to important concepts and essential infonnation. Changes introduced in previous editions, such as bolded key terms, clinical information in blue text, pages with color-coded edges, and a fresh design for clinical correlation folders, were all enthusiastically approved by the new generation of textbook users and have been maintained in this edition. Essential terms within each specific section are introduced within the text in eye-catching, oversized, bold, red font. Text containing clinical information and the latest research findings is presented in blue, with terminology pertaining to diseases, conditions, symptoms, or causative mechanisms in oversized bold blue font. Each clinical folder contains updated clinical text with even more illustrations and drawings and is easily found within each chapter. A bright, energetic: new text design that sets off the new illustrations and photos makes navigation of the text even easier than be· fore. All of these visually appealing elements will keep readers turning page after page. As in previous editions, all changes have been made with students in mind. The author-editor team strived for clarity and concision to aid student comprehension of the subject matter, familiarity with the latest information, and applica· tion of newfound knowle~.

Wojciech Pawlina

ACKNOWLEDGMENTS I remain grateful to the creator of this book, Dr. Mic:hael H. Rou, my mentor, colleague, and dear friend, for the ability to carry on his vision for teaching histology. There are many changes in histology education that have occurred in the last decade or so. However, Dr. Ross's vision to provide the best quality histology text with superior imaging integrated with the most recent advances in molecular and cell biology and supporting clinical f.r.cts remains unchanged. In today's medical curricula, as histology courses continue to lose their identity as they become integrated into larger didactic blocks, there is a need for a comprehensive textbook &om which swdents can pick small chunks of knowledge for their specific learning assignments. While editing and writing new sections for this eighth edition, I always wondered how Dr. Ross would explain this particular concept or idea. He will forever be present in my heart and thoughts. Changes to the eighth edition arise largely &om comments and suggestions by stUdents &om all around the world who have taken the time and effort to send me e-mails of what they like about the book and, more important, how the book might he improved to help them better learn histology. I have also received thoughtful comments from my first-year histology swdents who often direct me to explore new discoveries and achievements in the many fields related to histology. I am grateful to them for the keen sense by which they sharpen this work. Also, many of my colleagues who teach histology, cell biology, immunology, and physiology courses all over the world have been helpful in improving this new edition. Many have suggested a stronger emphasis on clinical relevance and new discoveries, especially in the areas of cell and molecular biology, which I strive to continually engage as new research makes itself known. Others have provided new photomicrographs, electron micrographs, access to their virtual slide collections, or new tables or have pointed out where existing diagrams and figures need to be redrawn. Specifically, I owe my thanks to the following reviewers, who have spent time to provide me with constructive feedback in planning this current edition. Stefanic Auardi, PhD Oakland University William Beaumont School of Medicine Rochester, Michigan

Pike See Cheah, PhD Universiti Putra Malaysia Serdang. Selangor, Malaysia

Btaris Baylml, MD

:K:mn. N. C~, MD Winona Health Winona, Minnesota

Gii.lhane Military Medical Academy Ankara, Turkey

Paul B. Bell, Jr., PhD University of Oklahoma Norman, Oklahoma

John Clancy, Jr., PhD

Jraltduddin Bin Mohamed, MBBS, PhD National Defence University of Malaysia Kuala Lumpur, Malaysia

Rita Col~ PhD University of Louisville School of Medicine Louisville, Kentucky

David E. Dirk, PhD University of South Florida, College of Medicine Tampa, Florida

lria M. Cook, PhD State University of New York Westchester Community College Valhalla, New York

Christy Bridges, PhD Mercer University School of Medicine Macon, Georgia

Dongmei Cui, MD, PhD University of Mississippi Medical Center Jackson, Mississippi

Craig A. Canby, PhD Des Moines University Des Moines, Iowa

Andrea Deyrup, MD, PhD University of South Carolina School of Medicine Greenville Greenville, South Carolina

Smpben. W. Carmichael, PhD Mayo Clinic College of Medicine and Science Rochester, Minnesota

Jc.onifu Ea.ttwoocl, PhD

Loyola University Medical Center Maywood, Ulinois

Burrell College of Osteopathic Medicine Las Cruces, New Mexico

vii

viii

Rodrigo Enrique Elizondo--Om.a6ra, MD, PhD Autonomous University of Nuevo Le6n, School of Medicine Monterrey, Nuevo Le6n, Mexico

Christopher Hont Lillig, PhD University of Greifswald Greifswald, Gennany

Michael Hortsch, PhD Tamira Elul, PhD Touro University College of Osteopathic Medicine Vallejo, California

University of Michigan Medical School Ann Arbor, Michigan

Jim Hunon, PhD Francia A. Fakoyra, MBChB, MSc, PhD St. George's University School of Medicine True Blue, Grenada, West Indies Bruce E. Felgenhauer, PhD University of Louisiana at Lafayette Lafayette, Louisiana

G. ltan. Gallic:ano, PhD Georgetown University School of Medicine Washington, DC Joaquin J. Gucia, MD Mayo Clinic College of Medicine and Science Rochester, Minnesota Mathangi Gilbs, MBBS, MSc St. George's University School of Medicine True Blue, Grenada, West Indies

Ferdinand Gomez, MS Florida International University, Herbert Wertheim College of Medicine Miami, Florida Amoa Gonra, PhD University of Medicine & Dentistry of New Jersey New Brunswick, New Jersey

Texas Tech University Lubbock, Texas John-Olov jtuWOn, MD, PhD University of Gothenburg Gothenburg, Sweden CynthiaJ. M. Kane, PhD University of Arkansas for Medical Sciences Little Rock, Arkansas G. M. Kibria, MD National Defence University of Malaysia Kuala Lumpur, Malaysia

ThomaS. King, PhD University ofTexas Health Science Center at San Antonio San Antonio, Texas Penprapa S. Klinkbachom, PhD West Virginia University Morgantown, West Virginia Bruce M. Koeppen, MD, PhD Quinnipiac University Frank H. Netter MD School of Medicine North Haven, Connecticut

Andrew Koob, PhD .Errin M. Gore, PhD Middle Tennessee State University Murfreesboro, Tennessee

Joseph P. Grande, MD, PhD Mayo Clinic College of Medicine and Science Rochester, Minnesota Joseph A. Gruso, PhD University of Connecticut Health Center Farmington, Connecticut Brian H. Halla, PhD New York Institute ofTechnology Old Westbury, New York Arthur R. Hand, DDS University of Connecticut School of Dental Medicine Farmington, Connecticut

University ofWisa>nsin~River Falls River Falls, Wisa>n.sin Beftrley Kramer, PhD University of the Witwatersrand Johannesburg, South Africa

Craig Kuehn, PhD Western University of Health Sciences Pomona, California Nlruaba Lachman, PhD Mayo Clinic College of Medicine and Science Rochester, Minnesota

Gavin R. :t.aw.on, PhD Western University of Health Sciences Pomona, California

Susan LeDoux, PhD Clwlene Hoegler, PhD Pace University- Pleasantville Campus Pleasantville, New York

University of South Alabama Mobile, Alabama

Karen Leong, MD Drexel University College of Medicine Philadelphia, Pennsylvania

Siobhan Moya, PhD University of Plymouth Plymouth, United Kingdom

Kenneth M. Lerea, PhD New York Medical College Valhalla, New York

Christine E. Niekruht DMD Quinnipiac University Frank H. Netter MD School of Medicine North Haven, Connecticut

Frank Liuzzi, PhD Lake Erie College of Osteopathic Medicine Bradenton, Florida

Donaldj. Lowrie, Jr., PhD University of Cincinnati College of Medicine Cincinnati, Ohio

JCno..Shyrm L~ PhD National Taiwan University College of Medicine Taipei, Taiwan AndrewT. Mariusy, PhD Nova Southeastern University College of Medical Sciences Fort Lauderdale, Florida

John P. Marinelli, MD Mayo Clinic College of Medicine and Science Rochester, Minnesota

Geoffrey W. Mc:Auliffe, PhD Rutgers Robert Wood Johnson Medical School Piscataway, New Jersey Kevin J. Mc:Ctuthy, PhD Louisiana State University Health Sciences Center Shreveport Shreveport, Louisiana

David L. McWhortu, PhD Georgia Campus - Philadelphia College of Osteopathic Medicine Suwanee, Georgia Fabiola Medeiros, MD University of Southern California Keck School of Medicine Los Angeles, California

William D. Meek, PhD Oklahoma State University, College of Osteopathic Medicine Tulsa, Oklahoma Bjiim Master, MD, PhD Karolinska lnrtitutet Stockholm, Sweden Amir A. Mbawi, DVM, PhD

Saba University School of Medicine Saba, Dutch Caribbean

Diego F. Nino, PhD Louisiana State University Health Sciences Center, Delgado Community College New Orleans, Louisiana

Suha N. Noe, DO, PhD Saint Leo University Saint Leo, Florida Mohammad (Reza) Nourbakh.sh, PhD University of North Georgia Dahlonega, Georgia Iwn T. C. Novak, PhD National University of C6rdoba C6rdoba, Argentina Joanne Otth, PhD Temple University School of Medicine Downingtown, Pennsylvania

Fauziah Otbmtm, DVM, PhD Universiti Putra Malaysia Serdang, Selangor, Malaysia

CLuu Oxvig, PhD Aarhus University Aarhus C, Denmark

Scott Pateraon, PhD University of Bristol Bristol, United Kingdom Ndini Patber, PhD

University of New South Wales Sydney, Australia 'Ihom.u E. Phillips, PhD University of Missouri Columbia, Missouri Stephen R.. Planck, PhD Oregon Health & Science University Portland, Oregon Harry H. Plymale, PhD San Diego State University San Diego, California

ix

X

Rebec:c:a L. Pmtt, PhD Oakland University Wllllam Beawnont School of Medicine Rochester, Michigan

.. sorbs ultraviolet light and emits green light. Antibodies con· jugated with fluorescein can be applied to sections of lighdy fixed or frozen tissues on glass slides to localize an antigen in cells and tissues. The reaction of antibody with antigen can then be examined and photographed with a fluorescence microscope or confocal microscope that produces a three· dimensional reconstruction of the aamined tissue (Fig. 1.4).

binds to it, and the reaction is visualized by fluorescence microscopy. Monoclonal antibodies (Folder 1.3) are those pro~ duced by an antibody-producing cell line consisting of a single group (clone) of identical B lymphocytes. The single done that becomes a cell line is obtained from an individual

Two types of antibodies are used in immunocytochemistry: polyclonal antibodiesthatara produced by immunized animals and monoclonal antibodies that are produced by immortalized (continuously replica1ing) antibodyproducing cell lines. In a typical procedure, a specific protein, such as actin, is isolated from a muscle cell of one species, such as a rat, and injected into the circulation of another species, such as a rabbit. In the immunized rabbit, the rat's actin molecules are recognized by the rabbit immune system as a foreign antigen. This recognition triggers a cascade of immunologic reactions involving multiple groups (clones) of immune cells called B lymphocytes. The cloning ofB lymphocytes eventually leads to the production of anti·actin antibodies. Collectively, these polyclonal antibodies represent mix· tures of different antibodies produced by many clones of B lymphocytes that each recognize dUferent regions of the actin molecule. The antibodies are then removed from the blood, purified, and conjugated with a fluorescent dye. They can now be used to locate actin molecules in rat tissues or cells. If actin is present in a cell or tissue, such as a fibroblast in connective tissue, then the fluorescein·labeled antibody

FIGURE 1.4. Confocal mlcn:M~copy Image of • rat cardiac muacle c1ll. This image was obtained with the confocal microscope using the indirect immunofluorescence method. Two primary antibodies were used. The first primary antibody recognizes a specific lactate transporter IMCT1) and is detected with a secondary antibody conjugated with rhodamine (red). The second primary antibody is directed against the transmembrane protein CD147. which is tightly associated with MCT1. This antibody was detected by a secondary antibody labeled with fluorescein (green). The yellow color is visible at the point at which the two labeled secondary antibodies exactly colocalize within the cardiac muscle cell. This three-dimensional image shows that both proteins are distributed on the surface of the muscle cell. whereas the lactate transporter alone is visible deep to the plasma membrane. !Courtesy of Drs. Andrew P. Halestrap and Catherine Heddle.)

9 Monoclonal antibodies are now widely used in immun~ cytochemical techniques and also have many clinical appl~ cations. Monoclonal antibodies conjugated with radioactive compounds are used to detect and diagnose tumor metastasis in pathology, differentiate subtypes of tumors and stages

of their differentiation, and in infectious disease diagnosis to identify microorganisms in blood and tissue fluids. Monoclcr nal antibodies conjugated with immunotoxins, chemotherapy agents, or radioisotopes are now being used to deliver therapeutic agents to specific tumor cells in the body.

n

~.. :II

with multiple myeloma, a rumor derived from a single antibody-producing plasma cell. Individuals with multiple myeloma produce a large population of identical, homogeneous antibodies with an identical specificity against an antigen. To produce monoclonal antibodies against a specific antigen, a mouse or .rat is inununized with that antigen. The activated B lymphocytes are then isolated from the lymphatic tissue (spleen or lymph nodes) of the animal and fused with the myeloma cell line. This fusion produces a hybridoma, an immortalized individual antibody-secreting cell line. To obtain monoclonal antibodies against rat actin molecules, for example, the B lymphocytes from the lymphatic organs of immunized rabbits must be fused with myeloma cells.

Bath direct and indirect immunocytochemical mathods are used to locate a target antigen in cells and tissues. The oldest immunocytochemistry technique used for identifying the distribution of an antigen within cells and tissues is known as direct immunofluorescence. This technique uses a fluorochrome-labeled primary antibody (either polydonal or monoclonal) that reacts with the antigen within the sample (Fig. 1.5a) . .Ar. a one-step procedwe, this method involves only a single labeled antibody. Visualization of strUCtUreS is not idea.l because of the low intensity of the

signal emission. Direct immunofluorescence methods are now being replaced by the indirect method because ofsuboptimal sensitivity. Indirect immunofluorescence provides much greater sensitivity than direct methods and is often referred to as the "sandwich" or "double-layer technique." Instead of conjugating a fluorochrome with a specific (primary) antibody directed against the antigen of interest (e.g., a rat actin molecule), the fluorochrome is conjugated with a secondary antibody directed against a rat primary antibody (i.e., goat anti-rat antibody; Fig. 1.5b). When the fluorescein is conjugated directly with the specific primary antibody, the method is direct; when fluorescein is conjugated with a secondary antibody, the method is indirect. The indirect method considerably enhances the fluorescence signal emission from the tissue. An. additional advantage of the indirect labeling method is that a single secondary antibody can be used to localize the tissu~specific binding ofseveral different primary antibodies (Fig. 1.6). For microscopic studies, the secondary antibody can be conjugated with different fluorescent dyes so that multiple labels can be shown in the same tissue section (see Fig. 1.4). Drawbacks ofindirect immunofluorescence are that it is expensive, lahor~intensive, and not easily adapted to automated procedures.

DIRECT IMMUNOFLUORESCENCE

-~~

c: : :=~:~· 1~~.It:::::==~=====:i ~ c::::::::::=.

a

Antigen

Fluorescent secondary antibody

IND1~;;~U7CENC~ Il-l --

8

()V[RV1~ OF MnHOOO U~ED IN HlmtlLOGY

...J

~

I

•~

•

0 ~ ------------------------------------~ 2 --------------------------------------~

p·---------------------------------------------

~ 1&.1

----~

~ ------------------------------------------------~.

e

Histology (microscopic anatomy) is the

scientific study of microscopic structures of tissues and organs of the body. Ught microscopy (for viewing glass slich:s) and virtual microscoPY (for viewing digitized microscopic specimens on a computer screen or mobile device) are the most commonly taught methods for examining cells, tissues, and organs in histology courses.

Ill(

::c u TI~U( PRDJARATION

• Routindy prepared hematoxylin and eosin (H&.E)-st:ained sections of formalin-fixed tissue are the specimens most commonly examined for histologic studies with the light microscope. • The first step in preparation of a tissue sample is fixation, which preserves structure and prevents enzymatic degradation. • In the second step, the specimen Is dehydrated, cleared. and then embedded In paraffin or epoxy resins to permit sectioning. • In the third step, the specimen Is mounted on a glass slide and stained to permit light microscope examination. • Specific preparations are required for expansion microscopy (ExM) in which specimens are infiltrated with hydrogds that cause ph}'!ical o:pansion of specimens. • Steps in specimen preparation for the transmission electron microscope (TEM) are similar to that for light microscopy except that they require different fixatives (glutaraldehyde and osmium tetroxide), embedded media (plastic and epoxy resins), and staining dyes (heavy metals).

grAINING PROC(DURS! • •

Eosin is an addle dye (pink) and carries a net negative charge. It reacts with positively charged cationicgroups in cdls and tissues, particularly amino groups ofproteins (eosinophilic st:ructures). Hematoxylin acts as a basic dye (blue) and carries a net poslttve charge. It reacts with

negativdy charged ionized phosphate groups in nucleic acids (basophtlic structures).

e The periodic acid-Schift (PAS) reaction stains carbohydrates and carbohydrate-rich molecules a distinctive magenta color. It is used to demonstrate glycogen in cells, mucus in cells and tissues, the basement membrane, and reticular fibers in connective tissue. e Immunocytochemistry is based on the specificity of the reaction between an antigen and an antibody that is conjugated either to a fluorescent dye (for llght microscopy) or gold partides (for electron microscopy). Both direct and indirect Immunocytochemical methods are used to locate a target antigen in cells and tissues. • Histochemical and cytochemical procedures are based on specific binding of a dye with a particular cell component exhibiting inherent enzyma~c activity. e Hybridization is a method of localizing mRNA or DNA by hybridizing the sequence of interest to a complementary strand of a nucleotide probe. e Fluorescence In situ hybridization (FISH) procedure utillzcs fl.uorescent dyes combined with nucleotide probes to visualize multiple probes at the same time. This technique is wed extensively in genetic testing. e Autoradiography makes we of a photographic emulsion placed over a tissue section to localize radioactive material within tissues. '-

27

MIC.ROOCOPY • Correa interpretation ofmicroscopic images is important because ozgans are th.ree-d.im.ensional,

whereas histologic sections an: only two-dimensional. • Resolving power is the ability of a microscope lens or optical system to produce separate •

• •

•

images of closely positioned objects. 1he resolving power of a bright-field microscope (most commonly used by students and researchers) is about 0.2 J.Lrn. In addition to bright-field microscopy, other optical synems include phase contrast microscopy, dark-field microscopy, fluorescence microscopy, confocal scanning microscopy, ulti'IIViolet microscopy, and supel'-resolution microscopy. lhlnsmlsslon electron microscopes (TEMs; theoretical resolving power of 0.05 nm) use the interaction of a beam of electrons with a specimen to produce an image. Scanning electron microscopes (SEMs; resolving power of2.5 om) use decttons reflected or forced out of the specimen surface that are collected by detectors and reprocessed to form an image of a sample surface. Atomic force microscopes (AFMs; resolving power of 50 pm) are nonoptical microscopes that utilize an ultrash.arp, pointed probe (cantilever) that is dragged across the surface of a specimen. 1he up and down movements of the cantilever are recorded and transformed into a graphic image.

n :z:

,..

~ m ::a ~

1:

------- i 0

i

• :I:

~r-

8-< ..... g

CELL CYTOPLASM OVERVIEW OFTHE CELL AND CYTOPLASM I 28 MEMBRANOUS ORGANELLES 131 Plasma Membrane /31 Signaling Processes /35 MembranaTransport and Vesicula rTra nsport /36 Endosomes /43 Lysosomes I 45 Proteasome-Mediated Degradation /50 Rough Endoplasmic Reticulum /51 Smooth Endoplasmic Reticulum /55 Golgi Apparatus /56 Mitochondria /59 Peroxisomes (Microbodies) /61 NONMEMBRANOUS ORGANELLES I 62 Microtubule& /62 Actin Filaments /65

• OVERVIEW OFTHE CELL AND CYTOPLASM Cells are the basic structural and functional units of all multicellular organisms. The processes we normally associate with the daily activities oforganisms-protection, ingestion, digestion, absorption of metabolites, elimination of wastes, movement, reproduction, and even death-are all reflections of similar processes occurring within each of the billions of cells that constitute the human body. To a large extent, cells of different types use similar mechanisms to synthesize protein, transform energy, and move essential substances into the cell. They use the same kinds of molecules to engage in contraction, and they duplicate their genetic material in the same manner. Specific functions are identified with specific structural components and domains within the cell. Some cells devdop one or more of these functions to such a degree ofspecialization that they are identified by the function and the cell structures associated with them. For example, although all cells contain conttacti.lc filamentous proteins, some cells, such as muscle cells, contain large amounts of these proteins in specific arrays. lhis allows them to carry out their speciallzed function of contraction at both the cellular

28

Intermediate Filaments /68 Centrioles and MicrotubuleOrganizing Centers /71 Basal Bodies /75 INCLUSIONS I 11 CYTOPLASMIC MATRIX I 79 Folder 2.1 Clinical Correlation: Lysosomal Storage Diseases /48 Folder 2.2 Clinical Correlation: Abnormalities in Microtubules and Filaments /76 Folder 2.3 Clinical Correlation: Abnormal Duplication of Centrioles and Cancer /79

HISTOIDGY 101/80

~

and tissue levd. The specialized activity or function of a cdl may be reflected not only by the presence of a larger amount of the specific structural component performing the activity but also by the shape of the cell, its organization with respect to other similar cells, and its products (Fig. 2.1).

Cells can ba divided into two major compartments: the cytoplasm and the nucleus. In general, the cytoplasm is the part of the cell located outside the nucleus. The cytoplasm contains organelles ("little organs}, cytoskeleton (made of polymerized proteins that form microrubules, intermediate filaments, and actin filaments), and lnclusrons suspended in an aqueous gel called the cytoplasmic matrix. 1he matrix consists of a variety of solutes, including inorganic ions (Na+, K+, Ca2 +), and organic molecules such as intermediate metabolites, carbohydrates, lipids, proteins, and RNAs. The cell controls the concentration of solutes within the matrix. which infiuences the rate of metabollc activity within the cytoplasmic compartment. The nucleus is the largest organelle within the cell and contains the genome along with the enzymes necessary for DNA replication and RNA transcription. The cytoplasm and nucleus not only play distinct functional roles but also work in concert to maintain the cdl's viability. The structure and function of the nucleus is discussed in Chapter .3, The Cell Nucleus.

29

FIGURE 2.1. Hhrtologlcfeaturaa of dllleNnt cell typel. These three photomicrographs show different types of cells in three different organs of the body. The distinguishing features include size, shape, orientation, and cytoplasmic contents that can be related to each cell's specialized activity or function. •· Epithelial cells in the kidney. Note several shapes of epithelial cells: columnar cells with well-defined borders in the collecting duct (CD), squamous cells in the thin segment ITS) of the nephron. and even more flattened cells lining blood vessels. the vasa rec1a (VR) in the kidney. x380. b. Dorsal root ganglion cells. Nota the large size of these nerve cell bodies and the large, pale {euchromatic! nuclei INl with distinct nucleoli. Each ganglion cell is surrounded by flattened satellite cells IS'l. The size of the ganglion cell and the presence of a euchromatic nucleus. prominent nucleolus. ~nd ~iss! bodies ~

m

:II ~

n m rr-

~

"a

E .:

• 3:

m

s: OJ :::D )>

z

0 0

Ci)

)>

'Myoclonic epilepsy end ragged red fibers syndrome. "Mitochondrial myopathy, encephalopathy, lactic: acidosis, and stroke-like episodes syndrome. ATP, adenosine triphosphate; mRNA. messenger RNA; rER, rough endoplasmic: reticulum; rRNA, ribosomal RNA; sER, smooth endoplasmic reticulum.

• MEMBRANOUS ORGANELLES

n

many physiologic and biochemical activities essential to cell function and survival. When the plasma membrane is properly fixed. sectioned, stained, and viewed in cross section with the transmission dectron microscope (TEM). it appears as two dectron-dense layers separ.tted by an intermediate, electron-lucent (nonstaining) layer (Fig. 2.2). The total thickness of the plasma membrane is about 8 to 10 run.

z

m r rm

(n

making the inner portion of the membrane hydrophobic (i.e., having no affinity for water). The swfaces of the membrane are formed by the polar head groups of the lipid molecules, thereby making the swfaces hydrophilic (i.e., they have an affinity for water). Lipids are distributed asymmetrically between the inner and outer leaflets of the lipid bilayer, and their composition varies considerably among d.Uferent biologic membranes. In most plasma membranes, protein molecules constirute approximately half of the total membrane mass. Most of the proteins are embedded within the lipid bilayer or pass through the lipid bilayer completely. These proteins are called inta· gral membrane proteins. The other types of proteinperipheral membrane proteins-are not embedded within the lipid bilayer. They are associated with the plasma membrane by strong ionic interactions, mainly with integral proteins on both the extracellular and intracellular surf.tces of the membrane (see Fig. 2.3). In addition, on the extracellular surface of the plasma membrane, carbohydrates may be attached to proteins, thereby forming glycoproteins, or to lipids of the bilayer, thereby forming glycolipid&. These surface molecules constirute a layer at the surface of the cell, referred to as the cell coat or glycocalyx (see Fig. 2.2). They help estab~ lish extracellular microenvironments at the membrane surface that have specific functions in metabolism, cell recognition, and cell association and serve as receptor sites for hormones.

32

(f)

w

...J ...J

w

~a:

0

(f)

::> 0

z ~

al

:::2: w ~

•

I

Microdomains of the plasma membrane, known as lipid rafts, control the movement and distribution of proteins within the lipid bilayer. The fluidity of the plasma membrane is not revealed in static electron micrographs. Experimenu reveal that the membrane

FIGURE 2.2. Electron micrograph of microvilli on the apical surface of an ablorptlve cell. This electron micrograph shows the apical portion of absorptive cells with microvilli. Note that at this magnification, the plasma membrane displays its characteristic appearance, showing two electron-dense lines separated by an electron-lucent intermediate layer. The glycoproteins of the glycocalyx can be seen extending from the tips of the microvilli into the lumen. The relationship between the outer plasma membrane leaflet and the glycocalyx is particularly well demonstrated. Glycoproteins of the glycocalyx include terminal digestive enzymes such as dipeptidases and disaccharidases. X100,000. (Courtesy of Dr. Ray C. Henrikson.)

The plasma membrane is composed of an amphipathic lipid layer containing embedded integral membrane proteins with peripheral membrane proteins 8Uached to its surfaces. The current interpretation of the molecular organization of the plasma membrane is referred to as the modified fluidmosaic model (Fig. 2.3). The membrane consists primarily of phospholipid, cholesterol, and protein molecules. The lipid molecules form a lipid b.ilayer with an amphipathic ch.aracter (it is both hydrophobic and hydrophilic). The fatty-acid chains of the lipid molecules face each other,

behaves as though it were a two-dimensional lipid fluid. For many years, it vr.lS thought that integral membrane proteins moved freely within the plane ofthe membrane; this movement vr.lS compan:d to the movement of icebergs floating in the ocean (see Fig. 2.3). However, the distribution and movement of proteins within the lipid bilayer is not as random as once thought. The plasma membrane appears to be patchy with Iocalm:d regions that are distinct in sttucrure and function and vary in thickness and composition. These localW:d regions contain high concentrations of cholesterol and glycosphingolipids and are called lipid rafts. Because ofthe high concentration ofcholesterol and the presence of longer, highly saturated &tty-acid chains, the lipid raft area is thicker and cxh.ibiu less fluidity than the surrounding plasma membrane {Ftg. 2.4). Cholesterol is the dynamic "glue" that holds the raft together; its removal from the raft results in dispersal of raft-associated lipids and proteins. In general, there are two types of lipid rafts:

• Planar lipid rafts contain a fumily of 47 kDa proteins known as ftotillins as wdl as specific lipids and choles· terol. Flotillins are regarded as the molecular markers of lipid rafts and are considered to be scaffolding proteins. They also participate in the recruitment of specific mem· branc proteins into the rafts and work as active partners in various signaling pathways. • Caveolar rafts, or caveolae ("little caves"). represent small (50 to 100 nm in diameter). Bask-shaped invagi· nations of the plasma membrane containing small (18 to 24 kDa) integral membrane proteins called caveolins.

carbohydrates

cholesterol

molecule hydrophobic

fatty-acid chain

hydrophilic polar head

FIGURE 2.3. Diagram of a plasma membrane showing1he modified fluid-mosaic model. The plasma membrane is a lipid bilayer consisting primarily of phospholipid molecules. cholesterol, and protein molecules. The hydrophobic fatty-acid chains of phospholipids face each other to form the inner portion of the membrane, whereas the hydrophilic polar heads of the phospholipids form the extracellular and intracellular surfaces of the membrane. Cholesterol molecules are incorporated within the gaps between phospholipids equally on both sides of the membrane. Note the elevated area of the lipid raft that is characterized by the high concentration of glycosphingolipids and cholesterol. It contains large numbers of integral and peripheral membrane proteins. The raft protrudes above the level of asymmetrically distributed phospholipids in the membrane bilayer !indicated by the ddferent colors of the phospholipid heads). Carbohydrate chains attach to both integral and peripheral membrane proteins to form glycoproteins as well as to polar phospholipid heads to form glycolipids.

These proteins have the capacity to bind cholesterol and a vuiety of proteins that are involved in signal ttansduc· tion. lnvaginations formed by caveolar rafts initiate vesicle formation in mic.ropinocytosis, a process described later in the section on endocytosis (page 37). Lipid rafts contain a variety of integral and peripheral membrane proteins involved in cell signaling. They can be viewed as "signaling platforms" floating in the ocean of lipids. Each individual raft is equipped with all of the necessary elements (receptors, coupling factors, efFector enzymes, and substrates) to receive and convey specific signals. Signal transduction in lipid rafts occurs more rapidly and efficiently because of the close proximity of interacting proteins. In addition, different signaling rafts allow for the separation of specific signaling molecules from each other.

FIGURE 2.4. Image of lipid nrft1 obtained with tapping-mode atomic force microscopy (AFM). This image shows a 5-nm-thick lipid bilayer spread on a mica support. The bilayer is composed of dioleoylphosphatidylcholine ldioleoyi-PCI, sphingomyelin, and cholesterol. Sphingomyelin and cholesterol together form lipid rafts represented on the image by the pink areas; the blue-purple areas are the nonraft background of the bilayer. Because the sphingomyelin molecules are longer than the dioleoyi-PC molecules, the rafts protrude from the nonraft background by about 0.8 nm, and the AFM is sensitive enough to detect this protrusion. The black regions represent the mica support. The image also shows molecules of the Helicobscter pylori toxin VacA (white particles), which preferentially bind to protein receptors on the raft domains. The area depicted in this image is 800 nm 2• (Courtesy of Drs. Nicholas A. Geisse,limothy L. Cover, Robert M. Henderson, and J. Michael Edwardson.)

In bacterial and viral infections, the initial contact of the microorganism with the cell occurs at the raft. For example, some bacteria {e.g., Shigella flexneri, Salmonella typhimurium) hijack the rafts with their signaling mechanism and use them to support their own entry into the cell. Many bacteria use rafts to avoid phagocytosis and subsequent destruction in lysosome&. In other cases, invading bacteria use raft-associated receptors to generate vacuoles made of raft components. These vacuoles are then used to transport bacteria into the cell without the risk of being detected by phagocytic compartments.

Integral membrane proteins can be visualized with the freeze-fracture tissue preparation 1echnique. The existence of proteins within the substance of the plasma membrane (i.e., integral proteins) was confirmed by the

33

integral membrane proteins

34

(f)

w

...J ...J

w

~a:

0

(f)

::> 0

z ~

al

:::2: w

Inner leaflet of lipid layer depression left by P-face protein --integral membrane proteins

~

•

I

I

P-face

I

I

cytoplasm

b

c

FIGURE 2.1. Freeze fnacture examination of the pluma membrane. a. ViffW of the plasma membrane seen on edge, with amJw indicating the preferential plane of splitting of the lipid bilayer through the hydrophobic portion of the membrane. When the membrane splits, some proteins are carried with the outer leaflet, although most are retained within the inner leaflet. b. View of the plasma membrane with the leaflets separating along the cleavage plane. The surfaces of the cleaved membrane are coated, forming repliC6s; the repliC6s are separated from the tissue and examined with the transmission electron microscope (TEMI. Proteins appear as bumps. The replica of the inner leaflet is called the P.face; it is backed by cytoplasm (protoplasm). A view of the outer leaflet is called the E-face; it is backed by the extracellular space. c. Electron micrograph of a freeze fracture replica shows the E-face of the membrane of one epithelial cell and the P.face of the membrane of the adjoining cell. The cleavage plane has jumped from the membrane of one cell to the membrane of the other cell, as indicated by the clear space {intercellular space) across the middle of the figure. Note the paucity of particles in the E-face compared with the P.face, from which the majority of the integral membrane proteins project. !Courtesy of Dr. Giuseppina d'Eiia Raviola.)

preparation technique known as freeze fracture. When tissue is prepared for electron microscopy by the freeze fracture process (Fig. 2.5a), membranes typically split or cleave along the hydrophobic plane (i.e., between the two lipid layers) to expose two interior faces of the membrane, an E-face and a P-Eace (Fig. 2.5b). For details on tissue preparation using freeze fracture technique, see Chapter 1, Methods, page 22. The E-face is backed by the auace.llular space, whereas the P..face is backed by the cytoplasm (protoplasm). The numerous particles seen on theE- and P-faces with the TEM represent the integral proteins of the membrane. Usually. the P-face displays more particles, thus more protein, than the E-face (Fig. 2.5c).

Integral membrane proteins have important functions in cell metabolism, regulation, integration, and cell signaling. Six broad categories of membrane proteins have been defined in terms oftheir function: pumps, channels, receptors, Unkers, enzymes, and sttuctu.I31 proteins (Fig. 2.6). These catego· ries are not mutually exclusive (e.g., a sttuctutal membrane

protein may simultaneously serve as a receptor, an enzyme, a pump, or any combination of these functions):

• Pumps transport certain ions, such as Na+, actively across membranes. Pwnps also transport metabolic precursors of macromolecules, such as amino acids and sugars, across membranes, either by themselves or linked to the Na+ pump. • Channels allow rhe passage of small ions, molecules, and water across the plasma membrane in either direction (i.e., passive diffusion). Gap junctions fonned by aligned channels in rhe membranes ofadjacent cells permit passage ofions and small molecules involved in signaling padr.w.ys &om the cytoplasm of one cell to the cytoplasm ofadjacent cells. • Receptor proteins allow recognition and localized binding ofligands (molecules that bind to the extracellular surface of the plasma membrane) in processes such as hormonal stimulation, coated-vesicle endocytosis, and antibody reactions. Receptors that bind to signaling molecules transmit the signal through a sequence of molecular

cell membrane

FIGURE 2.6. Different function• of Integral membrane proteins. The six major categories of integral membrane proteins are shown in this diagram: pumps, channels, receptors, linkers, enzymes, and structural proteins. These categories are not mutually exclusive. A structural membrane protein involved in cell-to- 0

z ~ ro

~

w

early

•

endosome

~

I

The fate of the internalized ligand-recaptor complex depends on the sorting and recycling ability of the early endosome.

portant because it allows surface receptors to be recycled. Most ligand-receptor complexes dissociate in the acidic pH of the early endosome. The receptor, most likely an integral membrane protein (see page 34), is recycled to the surface via vesicles that bud off the ends of narrowdiameter tubules of the early endosome. ligands are usually sequestered in the spherical vacuolar part of the endosome that will later form MVBs, which will uansport the ligand to late endosomes for further degradation in the lysosome (Fig. 2.18a). This pathway is utilized by the

low-density lipoprotein (LDL)-receptor complex. late endosome

lysosome FIGURE 2.18. hthways for delivery of newly syntheslud lysosomal enzym-. Lysosomal enzymes (such as lysosomal hydrolases) are synthesized and glycosylated within the rough endoplasmic reticulum (rER). The enzymes then fold in a specific way so that a signal patch is formed to which M-6-P is added. This additional modification allows the enzyme to be targeted to specific proteins that possess M-6-P receptor activity. M-6-P receptors are present in the trsns-Golgi network. (TGM of the Golgi apparatus, where the lysosomal enzymes are sorted and packaged into vesicles later transported to the early or late endosomes.

lysosomes, they ate also called prelysosomes. Late lyso· somes may fuse with each other or with manu:e lposomes. VJ.dcomicroscopy allows researchers to observe the complex behavior of these organelles.

late endosome

The major function of early endosomes is 1o sort and recycle proteins internalized by andocytatic pathways. Early endosomes sort proteins that have been internal· W:d by endocytotic processes. The morphologic shape and gl:!Ometry of the tubules and vesicles emerging from the early endosome create an environment in which localized changes in pH constitute the basis of the sorting mechanism. This mechanism includes dissociation of ligands from their receptor protein; thus, in the past, early endosomes were referred to as compartments of uncoupling receptors and ligands (CURLs). In addition, the narrow diameter of the tubules and vesicles may also aid in the sorting of large: molecules, which can be mechanically prevented from entering specific sorting compartments. After sorting, most of the protein is rapidly recycled, and the excess membrane is returned to the plasma membrane.

lysosome

FIGURE 2.17. Schematic dlagNm of endosomal compartment. of the call. This diagram shows the fate of protein Ired circles) endocytosed from the cell surface and destined for lysosomal destruction. Proteins are first found in endocytotic (coated! vesicles that deliver them to early endosomes, which are located in the peripheral part of the cytoplasm. Because of the sorting capability of early endosomes, receptors are usually recycled to the plasma membrane, and endocytosed proteins are transported via multivesicular bodies {MVBI to late endosomes positioned near the Golgi apparatus and the nucleus. The proteins transported to late endosomes eventually will be degraded in lysosomes. Note the acidification scale (/efll that illustrates changes in pH from early endosomes to lysosomes. The acidification is accomplished by the active transport of protons into endosomal compartments.

coated vesicle LDL receptor ~

• • • '\. •

transfen1n \ Fe

....... LDL r-

~

\._r~-{ early

c'YJ -

-< )

-= 1'

r '

.l..

;:-'\ ""~ ') ~

\_:~ •,:;

• ... ,

endosome

I ++~

\ "" v ... ---*

• • ' ''1- •

ru

EGF receptor • , \ transferrin receptor • •

•I•-...._ ' ~

~~~

MVB , •

• •• .J

metabolism

I!.

_..,.... EGF •

' ' ;.. "

'-.. • )

~c

...4 • /

,

secretory component oflgA ~ ._. • I

I~

r

T\ ---~ )

(~ ·')

~

~

•

late endosome )

lysosome

a

b

FIGURE 2.18. Fate of receptor and ligand In receptoJ'omedlated endocytosis. This diagram shows four major pathways along which the fate of internalized ligand-re 0

Lysosomes have a unique membrane that is resistant to the hydrolytic digestion occuft'ing in their lumen.

ro

Lysosomes contain a collection of hydrolytic enzymes and are surrounded by a unique membrane that resists hydrolysis by their own enzymes (Fig. 2.19). The lysosomal membrane has an unusual phospholipid structure that contains cholesterol and a unique lipid called lysobisphosphatidic acid. Most of the structural lysosomal membrane proteins are classified into lysosome-associated membrane pro-

z ~

:::2: w :::E

•

I

teins (LAMPs}, lysosomal membrane glycoprotelns (LGPs), and lysosomal Integral membrane proteins (LIMPs}. LAMPs, LGPs, and LIMPs represent more than 50% of the total membrane proreins in lysosomes and are highly glycosylated on the lwninal surface. Sugar molecules cover almost the entire lwninal swface of these proteins, thus protecting them from digestion by hydrolytic enzymes. Lysobisphosphatidic acids within the lysosomal membrane may play an important role in restricting the activity of hydrolytic enzymes directed against the membrane. The same f.unily of membrane proteins is also detected in late endosomes.

polysaccharides

lipids nucleic acids

Certain drugs can affect lysosomal function. For example, chloroquine, an agent used in the treatment and prevention of malaria, is a lysosomotropic agent that accumulates in thelysosomes. It raises the pH of the lysosomal content thereby inactivating many lysosomal enzymes. The action of chloroquine on lysosomes accounts for its antimalarial activity; the drug concentrates in the acidic food vacuole of the malaria parasite (Plasmodium falciparum) and interferes with its digestive processes, eventually killing the parasite.

Lysosomal membrane proteins are synthesized in the rER and have a specific lysosomal targeting signal. All mentioned previously. the intracellular uafficking leading to the delivery of many soluble lysosomal enzymes to late endosomes and lysosomes involves the M-6-P signal and its receptor. All membrane proteins destined for lysosomes (and late endosomes} are synthesized in the rER and transported to and sorted in the Golgi apparatus. However, because they do not contain M-6-P signals, they must be targeted to lysosomes by a different mechanism. The targeting signal for integral membrane proteins is represented by a short cytoplasmic Cterminus domain, which is recognized by adaptin protein complexes and packaged into clathrin-coated vesicles. These proteins reach their destination by one of two pathways: In the constitutive secretory pathway, UMPs exit the Golgi apparatus in coated vesicles and are delivered to the cell surface. From there, they are endocytosed and, via the early and late endosomal compartments, finally reach lysosomes (Fig. 2.20). • In the Golgi-derived coated vesicle secretory pathway, LIMPs, after sorting and packaging, exit the Golgi apparatus in clathrin-coated vesicles (see Fig. 2.20). These transport vesicles travel and fuse with late endosomes as a result of intetaction between endosome-specific com· ponents of v·SNARE and t·SNARE docking proteins (see page 41).

•

Three dHferent pathways deliver material for intracellular diges1ion in lysosomu.

organio-linked phosphates

membrane impenneable to enzymes; contains lysosomal-specific membrane proteins LAMP, LIMP, and LGP FIGURE 2.19. Schematic diagram of a lysasome. This diagram shows a few selected lysosomal enzymes residing within a lysosome and their respective substrates. The major lysosomal membranespecific proteins, as well as a few other proteins associated with membrane transport, are also shown.

Depending on the nature of the digested material, different pathways deliver material for digestion within the lysosomes (Fig. 2.21). In the digestion process, most of the digested material comes from endocytotic processes; however, the cell also uses lysosomes to digest its own obsolete parts, nonfunc· tiona! organdies, and unnecessary molecules. Three pathways for digestion cnst:

• Extracellular large particles such as bacteria, cell debris, and other foreign materials are engulfed in the process of phagocytosis. A phagosome, formed as the material is internalized within the cytoplasm, subsequendy receives hydrolytic enzymes to become a late endosome, which matures into a lysosome.

lysosomalspecific membrane proteins

~ Golgi

_/:.@ { /

.._//

~

apparatus

CONSTITUTIVE SECRETORY PATHWAY

phagocytized bacteria and fragments of damaged cells are often recognized in macrophages. Hydrolytic breakdown of the contents of lysosomes often produces a debris-filled vacuole called a residual body that may remain for the entire life of the cell. For example, in neurons, residual bodies are called age pigment or lipofuscin granules. Residual bodies are a normal feature of cell aging. The absence of certain lysosomal enzymes can cause the pathologic accwnulation of undigested substrate in residual bodies. This can lead to several disorders collectively termed lysosomal storage diseases (see Folder 2.1).

Autophagy

late endosome

FIGURE 2.20. Lytotome blogenMit. This

diagram shows r~ ulated and constitutive pathways for delivery of lysosomal-specific membrane proteins into early and late endosomes. The lysosomal membrane possesses highly glycosylated specific membrane proteins that protect the membrane from digestion by lysosomal enzymes. These lysosome-specific proteins are synthesized in the rough endoplasmic reticulum, transported to the Golgi apparatus. and reach their destination by two pathways. Blue arrows indicate the constitutive secretory pathway in which certain lysosomal membrane proteins exit the Golgi apparatus and are delivered to the cell surface. From there. they are endocytosed and, via the early and late endosomal compartments. finally reach lysosomes. Green arrows indicate the endosomal Golgi.. 0

z ~ ro

:::2: w :::2:

•

I

apparatus, rER, or nuclear envelope (these structures are discussed in the next sections) or integral components of the plasma membrane.

Coatomers mediate bidirectional traffic between the rER and Golgi apparatus. Two classes of coated vesicles are involved in the transport of protein from and to the rER. A protein coat similar to dath· rin surtounds vesicles transporting proteins between the rER

and the Golgi apparatus (page 40). However, unlike clathrins, which mediate bidiJ:ectional transport from and to the plasma membrane, one class of proteins is involved only in anterograde transport from the rER to the cis-Golgi network (CGN), the Golgi cisternae closest to the rER. Another class of proteins mediates retrograde transport from the CGN back to the rER (Fig. 2.28). These two classes of proteins are called coatomers or COPs.

ANTEROGRADE TRANSPORT

cis-Golgi

.• •

rEA

...• •"••~

·~.

J•

~

•~

l •

• •

•

• I

•

)..

.

• • • ••

•~

•

COP-I

COP-II

r"

>--

I '"'(

~

J._y

recycle\... ,_,

•

-< -
0

matrix---~~

z ~

crista

ro

----H"""'-..

:::2: w :::E

•

I b FIGURE 2.37. Structure of the mitochondrion. •· This electron micrograph shows a mitodlondrion in a pancreatic acinar cell. Note that the inner mitochondrial membrane forms the cristae (C) through a series of infoldings, as is evident in the region of the arrow. The outer mitochondrial membrane is a smooth continuous envelope that is separate and distinct from the inner membrane. X200,000. b. Schematic diagram showing the components of a mitochondrion. Note the location of the elementary particles (inset}. the shape of which reflects the three-dimensional structure of adenosine triphosphate (ATP) synthase.

in the cytoplasm. Mitochondria can acxumulate c:atioru against a concentration gradient. Thus, in addition to ATP production, mitochondria also regulate the concentration of certain ions of the cytoplasmic maaix, a role they share with the sER. The matrix also contains mitochondrial DNA,. ribosomes, and tRNA$.

Mitochondria contain the enzyme system that generates AlP by means of the citric acid cycle and oxidative phos-

phorylation. Mitochondria generate ATP in a variety of metabolic path· ways, including oxidative phosphorylation, the ciaic acid cycle, and ~-oxidation of fatty acids. The energy generated from these reactions, which take place in the mitochondrial matrix, is represented by hydrogen ions (H+) derived from reduced NADH. These ions drive a series of proton pumps located within the inner mitochondrial membrane that transfer H+ from the maaix to the intermembrane space (Fig. 2.38). These pumps constitute the electron-transport chain of respiratory enzymes (see Fig. 2.37). The transfer of H+ across the inner mitochondrial membrane establishes an electrochemical proton gradient. This gradient creates a large proton motive force that causes the movement ofH+ to occur down its decttochemical gradient through a large, membrane-bound enzyme called ATP synthase. ATP

synthase provides a pathway across the inner mitochondrial membrane in which H+ ions are used to drive the energetically unfavorable reactions leading to synthesis of ATP. This movement of protons back to the mitochondrial matrix is .r:c:fened to as chemiosmotic coupling. The newly produced ATP is transported from the mat.tix. to the intermembranc space by the voltage gradient-driven ATP/ADP exchange protein located in the inner mitochondrial membrane. From here, ATP leaves the mitochondria via voltage-dependent anion channels (WAC) in the outer membrane to enter the cytoplasm. At the same time, ADP produced in the~ plasm rapidly enters the mitochondria for rechaJ:ging. Several mitochondrial defects are related to defects in enzymes that produce ATP. Metabolically active tissues that use large amounts of ATP, such as muscle cells and neurons, are the most commonly affected. For example,

myoclonic epilepsy with ragged red fibers (MERRF) is characterized by muscle weakness, ataxia, seizures, and cardiac and respiratory failure. Microscopic examination of muscle tissue from affected patients shows aggregates of abnormal mitochondria that provide a ragged appearance of red muscle fibers. MERRF is caused by mutation of the mitochondrial DNA gene encoding tRNA for lysine. This defect produces two abnormal complexes in the electron-transport chain of respiratory enzymes affecting ATP production.

61 ADP

- .. - ... - .. - .. - .. .. .. -

~

:!1 m

::a l'f

n

intermembrane space

m

ATP/ADP

exchange

+ ATP ATP ADP

I

protein

+ + + +

ADP

F

~

E 31: •s: m

s: OJ

::D )>

z

0

c en 0

FIGURE 2.38. Schematic diagram Illustrating how mitochondria generate energy. The diagram indicates the adenosine triphosphate (ATP) synthase complex and the electron-transport chain of proteins located in the inner mitochondrial membrane. The electron-transport chain

generates a proton gradient between the matrix and intermembrane space that is used to produce ATP. Numbers represent sequential proteins involved in the electron-transport chain and ATP production: 7, NADH dehydrogenase complex; 2, ubiquinone; 3, cytochrome b-e, complex; 4. cytod'lrome c; 5, cytochrome oxidase complex; and 6, ATP synthase complex. ADP. adenosine diphosphate.

Mitochondria dacida whathar1ha call Iivas or diu. Experimental studies indicate that mitochondria sense cellu~ Jar stress and are capable of deciding whether the cell lives or dies by initiating apoptosis (programmed cell death). The major cell death evmt generated by the mitochondria is the release of cytochrome c from the mitochondrial intermembranous space into the cell cytoplasm. Changes in the voltage-dependent anion channels (VDAC) at the outer mitochondrial membrane are responsible for this release. This event, rcgulated by the proapoptotic Bcl-2 protein family (see page 100), initiates the cascade of proteolytic enzymatic reactions that leads to apoptosis. The Bcl-2 family of proteins thus controls cell death primarily by regulating permeability of the outer mitochondrial membrane, which lead to the ~ersible release of cytochrome c, subsequent c:aspase activation, and apoptosis. However, in certain conditions (e.g., translational modifications), the: Bcl-2 proteins may act as antiapoptotic: agenrs. Mitochondria undergo morphologic changes related to their functional state. TEM studies show mitochondria in two distinct configurations. In the orthodox configuration, the cristae are prominent, and the matrix compartment occupies a large part of the: total mitochondrial volume. This energized mitochondrion connguration is observed in healthy cells. In this configuration, most of the cytochrome cis sequestered within

the cristae and is resistant to rdeasc: by agenrs that disrupt the mitochondrial outer membrane. Matrix remodeling to the condensed configuration resulrs in depolarization of mitochondrial membranes. This configuration is characterized by unfolded cristae that are not easily recognized in the TEM. The .matrix is ttduc:cd in volume and appears more concentrated, whereas the intermembrane space increases to as much as 50% of the total volume of the organelle. These: c.hanges expose cytochrome c to the intermembrane space, facilitating irs release from the mitochondria during apoptosis.

Peroxisomes (Microbodies) Peroxisomas are single-membrana-bounded organelles containing oxidative enzymes. Peroxisomes (microbodies) are small (0.5 mm in diameter), membrane~Umited spherical organelles that contain oxidative enzymes, particularly catalase and other peroxi.dases. Vtrruall.y, all oxidative enzymes produce hydrogen peroxide (H2~) as a product of the oxidation reaction. Hydrogen peroxide is a toxic substance. The catalase univt:rsally present in peroxisomes carefully regulates the cellular hydrogen peroxide content by breaking down hydrogen peroxide, thus protecting the cell. In addition, peroxisomes contain D-amino acid oxidases, 13-oxidation enzymes, and numerous other enzymes. Oxidative enzymes are particularly important in liver cells (hepatocytes), where they perform a variety of detoxification

~ zm r

r m

en

62

(I)

w

:::1 w

~a:

0

(I)

::> 0

~

ro

:::2: w :::E

z z

0

•

::E

~

~:I ~ N

a:

i u

processes. Peroxisomes in hepatocytes are responsible for detoxification of ingested alcohol by converting it to acetaldehyde. The Ji·oxidation of fatty acids is also a major function of peroxisome&. In some cells, peroxisomal fatty-acid oxidation may equal that of mitochondria. The proteins contained in the peroxisome lwnen and membrane are synthesized on cytoplasmic ribosomes and imported into the peroxisome. A protein destined for peroxisomes must have a peroxisomal targeting signal attached to its carboxy-terminus. Although abundant in liver and kidney cells, peroxisomes are also foWld in most other cells. The nwnher ofperoxisomes present in a cell increases in response to diet, drugs, and hormonal stimulation. In most animals, but not hwnans, peroxisomes also contain urate oxidase (uricase), which often appears as a characteristic crystalloid inclusion (nucleoid). Various human metabolic disorders are caused by the inability to import peroxisomal proteins into the organelle because of a faulty peroxisomal targeting sig· nal or receptor. Several severe disorders are associated with nonfunctional peroxisomes. In the most common inherited disease related to nonfunctional peroxisome&, Zallwager syndrome, which leads to early death, peroxisomes lose their ability to function because of a lack of necessary enzymes. The disorder is caused by a mutation in the gene encoding the recaptor for the peroxisome targeting signal that does not recognize the signal SerLys-Leu at the carboxy-terminus of enzymes that act on peroxisome&. Therapies for peroxisomal disorders have bean unsatisfactory to date.

• NONMEMBRANOUS ORGANELLES Microtubules Microtubules are nonbranching and rigid hollow tubes of polymerized protein that c:an rapidly assemble and equally rapidly disassemble. In general, .mi.c:rotubules are found in the cytoplasm, where they originate from the microtubule organizing center (MTOC). They grow from the MTOC located near the nucleus and extend toward the cell periphery. Mic:rotubules are also present in cilia and flagella, where they form the axoneme and its anchoring basal body; in centrioles and the mitotic: spindle; and in elongating processes of the cell, such as those in growing axons. Mic:rotubules are involved in numerous essential cellular functions: • Intracellular vesicular transport (i.e., movement of secretory vesicles, endosome&, and l~somes). Microtubules create a system of connections within the cell, frequendy compared. with railroad tracks originating from a central station, along which vesicular movement occurs. • Movement of cma and flagella • Attachment of chromosomes to the mitotic spin· die and their movement during mitosis and meiosis • Maintenance of call shape, particularly its asymmetry • Regulatory effect on of cell elongation and move· mant (migration) Although microtubules may exert a regulatory efkct on cell elongation and movement, they are not essential for

these functions, which are mediated by actin polymerization (see page 67). Microtubules play an indirect role by regulating actin polymerization, organizing transport of vesicles to the leading edge of migrating cells, and &cilitating disassembly of focal adhesions (see page 155). In addition, microtubule& may restrain cell locomotion by slowing the retraction of the trailing edge (tail) of the migrating cell, thus influencing the direction of cell migration.

Microtubules are elongated polymeric structures composed of equal parts of u-tubulin and f:J-tubulin. Microtubulea measure 20 to 25 nm in diameter. The wall of the microtubule is approximately 5 nm thick and consists of 13 circularly arrayed globular dim eric tubulin molecules. The tubulin dimer has a molecular weight of 110 kDa and is formed from an a~tubulin and a (3~tubulin molecule, each with a molecular weight of 55 kDa (Fig. 2.39). The dimers polymerize in an end~to-end fashion, head to tail, with the a molecule of one dimer bound to the (3 molecule of the next dimer in a repeating pattern. Longitudinal contacts between dimers link them into a linear structure called a protofilament. Axial periodicity seen along the 5~nm~diameter dimers corresponds to the length of the pro~ tein molecules. A small, l~f.Lm segment of microtubule con~ tains approximately 16,000 tubulin dime.rs.

Microtubules grow from y-tubulin rings within the MTOC that serve as nucleation sitas for each microtubule. Microtubule formation can be traced to hundreds of1·tubulin rings that furm an integral part of the MTOC and function as templates for the correct assembly of mic:rotubules. Their nucleation pattern initiated in the MTOC can be studied in vitro (Fig. 2.40). The a- and (3·tubulin dime.rs are added to a 'Y·tubulin ring in an end-to-end fashion. The most simplistic modd used in the past described microtubule assembly as a process of adding tubuUn di.mers one by one onto the growing end of a fully formed .microtubule. However, a nwnber of experimental studies using cryoelectron microscopy have shown that the initial assembly occurs from a curved sheet made of tubulin dimers, which in tum doses into a tube at the growing end of the microtubule (see Fig. 2.39). Polymerization of tubulin dimers requires the presence of guanosine triphosphate (GTP) and Mg2 +. Eac:h tubulin molecule binds GTP before it is incorporated into the funning .microtubule. The tubulin dime.rs containing GTP have a conformation that favors stronger lateral interactions between dimers resulting in polymerization. At some point, GTP is hydrolp.ed to guanosine diphosphate (GDP). As a result ofthis polymerization pattern, microtubules are polar structures because all of the dimers in each protofilament have the same orientation. Each microtubule possesses a nongrowing (-) end that corresponds to a·tubulin; in the cdl, it is usually embedded in the MTOC and often stabilized by actin-capping proteins (see Fig. 2.39). The growing(+) end ofmicrotubules corresponds to P·tubulin and extends the cell periphery. Tubulin dime.rs dissociate from microtubules in the steady state, which creates a pool of free tubulin dime.rs in the cytoplasm. This pool is in equilibriwn with the polymerized tubulin in the microtubules; therefure, polymerization and depolymerization are in equilibrium. The equi· librium c:an be shifted in the direction of depolymerization

63

' I I ~-tubulin ' I ~ u II u-tubulin

n

~

:II ~

Q F=

FIGURE 2.40. Nudelltlon activity of mlcrotubules observed in vitro using immunocytochemic:al mllthods. The behavior of mi-

(+end)

Tubulin dimer

crotubules obtained from human breast cancer cells can be studied in vitro by measuring their nucleation activity. Microtubules were labeled with a mixture of anti-«-tubulin and anti-P-tubulin monoclonal antibodies (primary antibodies) and visualized by secondary antibodies conjugated with fluorescein dye (fluorescein isothiocyanate-goat anti-mouse immunoglobulin G). Polymerization of tubulin dimers is responsible for the formation of more than 120 microtubules visible on this image. They originate from the microtubule-organizing center IMTOC) and extend outward approximately 20 to 25 jiiTI in a uniform radial array. x 1,400. (Photomicrograph courtesy of Drs. Wilma L. Lingle and Vivian A. Negron.)

boundtoGTP

~

i.:

•z 0

z

s: m s: OJ

~

0

c

Tubulln dlmer boundtoGDP

{-end) proteins

FIGURE 2.39. Polymerization of mlcrotubules. On the left, the diagram depicts the process of polymerization of tubulin dimers during microtubule assembly. Each tubulin dimer consists af an a.-tubulin and jHubulin subunit. The plus(+) end of the microtubule is the growing end to which tubulin dimers bound to guanosine triphosphate IGTPI molecules are incorporated into a curved sheet, which in turn closes into a tube. Incorporated tubulin dimers hydrolyze GTP. which releases the phosphate groups to form polymers with guanosine diphosphate (GDPI-tubulin molecules. The minus 1-1 end of the microtubule contains a ring of 'Y"tubulin, which is necessary for microtubule nucleation. This end is usually embedded within the microtubule-organizing center IMTOC) and possesses numerous capping proteins. On the right is a diagram showing that each microtubule contains 13 tubulin dimers within its cross section.

by exposing the cell or isolated .microtubules to low tem· perarurcs or high pressure. Repeated exposure to alternating low and high tempcrarures is the basis of the purification technique for rubulin and .mictotubules. The speed of po· lymerization or depolymcrization can also be modified by in· teraction with specific microtubule-associated proteins (MAPs). These proteins, such as MAP·l, MAP·2, MAP·3, and MAP4; MAP4; and TOG·p, regulate microtubule assembly and anchor the mictotubulcs to specific organelles. MAPs arc also responsible for the existence of stable popula· tions of nondepolymcr.izing microtubules in the cell, such as those foWld in cilia and flagella. The length of microtubules constantly changes as dimers are added or removed in a process of dynamic instability. Microtubules observed in culrurcd cells with real·time video microscopy appear to be constantly growing toward the cell periphery by addition (polymerb:ation) of rubulin di.mers and then suddenly shrinking in the d.irc:.;tion of the MTOC by removal (depolymerh:ation) of tubuli.n dimers (Fig. 2.41). This constant remodeling ptocess, known as dynamic instability, is linked to a pattern ofGTP hydtoly· sis during the microtubule assembly and disassembly ptocess. The tubulin dimers boWld to GTP at the growing (+) end of the microrubule protect it from disassembly. In contrast, rubulin di.mers boWld to GOP ate prone to depolymerization that leads to rapid microtubule disassembly and shrinking. During disassembly, the tubulin dimers boWld to GOP lose lateral interaction with each other and protofilaments of the rubulin dimers curl away from the end of the microrubule, producing "split ends" (see Fig. 2.41). The process of switch· ing from a growing to a shrinking microrubule is often called a microtubule catastrophe.

(/)

0

:JJ

~

m

F

m

(/)

64

Vl

w

...J ...J

w

z ...J

u

z

•

I

in nondividing cells such as neurons and skeletal and cardiac muscle cells. Lipofuscin at:CUmulates over time in most euk:aryotic cells as a result ofcellular senescence (aging); thus, it is often called the •weafoand-tear"' pigment. Lipofuscin is a conglomerate of oxidized lipids, phospholipids, mecds, and organic molecules that accumulate within the cells as a result of oxidative degradation of mitochondria and lysosomal digestion. Phagocytotic cells such as macrophages may also contain lipofuscin, which accumulates &om the digestion of bacteria. foreign particles, dead cells, and their own organelles. Experimental Stlldies indicate that lipofuscin accumulation may be an accurate indicator ofcellular stress. • Hemosiderin is an iron-storage complex foWld within the cytoplasm of many cells. It is most likely formed by the indigestible residues ofhemoglobin, and its presence is related to phagocytosis of red blood cells. Hemosiderin is most easily demonstrated in the spleen, where aged erythrocytes are phagocytosed, but it can also be found in alveolar macrophages in the lung tissue, especially following pulmonary infection accompanied by a small hemorrhage into the alveoli. It is visible in light microscopy as a deep brown granule, more or less indistinguishable &om lipofuscin. Hemosiderin granules can be differentially stained using histochemical methods for iron detection. • Glycogen is a highly branched polymer used as a storage material for glucose. It is not stained in routine H&E preparations. However, it may be seen in the light microscope with special fixation and staining procedures (such as toluidine blue or the periodic acid-Sch.ifF [PAS] method).

Liver and striated muscle cells, which usually contain large amounts of glycogen, may display unstained regions where glycogen is located. Glycogen appe:m in the EM as granules 25 to 30 nm in diameter or as dusters ofgranules that often occupy significant portions of the cytoplasm (Fig. 2.60). • Upid inclusions (fat droplets) are usually nutritive inclusions that provide energy for cellular metabolism. The lipid droplets may appear in a cell for a brieftime (e.g., in intestinal absorptive cells) or may reside for a long period (e.g., in adipocytes). In adipocytes,lipid inclusions often constitute most of the cytoplasmic volume, compressing the other formed organelles into a thin rim at the margin ofthe cdL Lipid droplets are usually cruacted by the organic solvents used to prepare tissues for both light and elecaon microscopy. What is seen as a fat droplet in light microscopy is a.aually a hole in the cytoplasm that represents the site from which the lipid was eruacted. In individuals with genetic defects of enzymes involved in lipid metabolism, lipid droplets may accumulate in abnormal locations or in abnormal amounts. Such diseases are classified as lipid storage diseases. • Crystalline inclusions contained in certain cells are recognized in the light microscope. In humans, such inclusions are found in the Senoll (sustentacular) and Leydig (interstitial) cells of the testis. With the TEM, crystalline inclusions have been found in many cell types and in virtually all parts of the cell, including the nucleus and most cytoplasmic organelles. Although some of these inclusions contain viral proteins, storage material, or cellular metabolites, the significance of others is not clear.

,..~

...

,.,.~,.

>'w

~

FIGURE 2.60. Electron micrographs of a liver cell with glycogen inclulions. a. Low-magnification electron micrograph showing a portion of a hepatocyte with part of the nucleus (N, upper leffl. Glycogen (G) appears as irregular electron-dense masses. Profiles of rough endoplasmic reticulum lrER'J and mitochondria {IW) are also evident. X10,000. b. This higher magnification electron microscope (EM) reveals glycogen {G) as aggregates of small particles. Even the smallest aggregates (arrows) appear to be composed of several smaller glycogen particles. The density of the glycogen is considerably greater than that of the ribosomes (lower leffl. X52,000.

FOLDER 2.3

CLINICAL CORRELATION: ABNORMAL DUPLICATION OF CENTRIOLES AND CANCER One of the critical components of normal cell division is the precise redistribution of chromosomes and other cell organelles during mitosis. Following replication of dlromosomal DNA in the S phase of the cell cycle, centrioles undergo a single round of duplication that is closely coordinated with cell-cycle progression. During mitosis, centrioles are responsible for forming the bipolar mitotic spindle, which is essential for equal segregation of chromosomes between daughter cells. Alterations of mechanisms regulating centriole duplication may lead to multiplication and abnormalities of centrioles and surrounding centrosomes (MTOCs). Cells with multiple centrosomes that overcome tumor-suppressing protein (p53J-mediated cell cycle arrest and spindleassembly checkpoint inhibition can enter cell divisions with distortions of the mitotic spindle (i.e., the presence of multipolar or misoriented spindles) (Fig. F2.3.1 J. leading to abnor ::D ('")

0

:s:: ""C

eu00~~7 metaphase chromosome

a

0

T 1

1400 nm

b FIGURE 3.3. Packaging of chromatin Into th• chromosomal stnlcture. •· Sequential steps in the packaging of nuclear chromatin are shown in this diagram, beginning with the DNA double helix and ending with the highly condensed form found in chromosomes. b. Structure of human metaphase chromosome 2 as visible in atomic force microscopic image. x20,000. (Courtesy of Dr. Tatsuo Ushiki.)

As a result of meiosis,~ and sperm have only 23 chromosomes, the haploid (1n) number, as wdl as the haploid {1d) amount of DNA. The somatic chromosome number (Zn) and the diploid (2d) amount of DNA are restored at fertilization by the fusion of the sperm nucleus with the egg

nucleus.

In a karyotype, chromosome pairs are sorted according to their size, shapa. and aminad fluorescant calor. A preparation of chromosomes derived from mechanically ruptured, dividing cells that an: then fixed, plated on

z m z -4

en

86

~

zUJ z

0 a.. ~

0

u

~

....J

u::> z

•

en

:»

~

u

:» z

~

u

w

~

cij

i

a microscope slide, and stained is called a metaphase spread. In the past, chromosomes were routinely stained with Giemsa stain; however, with the recent development of in situ hybridization techniques, the fluorescent in situ hybridization (FISH) procedure is now more often used to visual.ize a chromosomal spread. These spreads are observed with fluorescence microscopes, and computer-controlled cameras are then used to capture images of the chromosome pairs. Image-processing software is used to sort the chromosome pairs according to their morphology to form a karyotype (see Fig. F3.1.la). A variety of molecular probes that are now commercially available are used in cytogenetic testing to diagnose disorders caused by chromosomal abnormalities such as nondisjunctions, transpositions (see Fig. F3.1.1 a), deletions (see Fig. F3.1.1 b}, and duplications of specific gene sites. Karyotypes are also used for prenatal determination of sex in fetuses and for prenatal diagnosis for certain genetic diseases {see Fig. 1.7).

The Barr body represen1S a region of facultative heterochromatin that can be used 10 identify the sex of a fetus. Some chromosomes are repressed in the interphase nucleus and exist only in the tightly packed heterochromatic form. One X chromosome of the female is an example of such a chromosome and can be used to identify the sex of a fetus. This chromosome was discovered in 1949 by Barr and Bartram in nerve cells of female cats, where it appears as a weltstained roood body; now called the Barr body, adjacent to the nucleolus. In females, the Barr body represents a region of facultative heterochromatin that is condensed and not involved in the transcription process. During embryonic development, one randomly chosen X chromosome in the female zygote undergoes chromosome-wide chromatin condensation, and this state is maintained throughout the lifetime of the organism. Although the Barr body was originally found in sectioned tissue, it was subsequently shown that any relatively large number ofcells prepared as a smear (e.g., scrapings of the oral mucous membrane from the inside of the cheeks or neutrophils from a blood smear) can be used to search for the Barr body. In cells of the oral mucous membrane, the Barr body is located adjacent to the nuclear envelope. In neutrophils, the Barr body forms a drumstick-shaped appendage on one of the nuclear lobes (Fig. 3.4). In both sections and smears, many cells must be examined to find those whose orientation is suitable for the display of the Barr body.

RGURE 3.4. Photomicrograph of a neutrophil from a female patient's blood smear. The second X chromosome of the female patient is repressed in the interphase nucleus and can be demonstrated in the neutrophil as a drumstick-appearing appendage (am>~ on a nuclear lobe. X250.

• Fibrillar material {pars fibrosa} contains ribosomal genes that are actively undergoing transcription and large amounts of rRNA. • Granular material (pars granulosa) represents the site of initial ribosomal assembly and contains densely packed preribosomal particles. The network furmed by the granular and the fibrillar ma· terials is called the nucleolonema. rRNA is present in both granular and fibrillar material and is organized, respectively, as both granules and extremely fine filaments packed tighdy together. Genes fur the ribosomal subunits are localized in the interstices of this network and are transcribed by RNA poly· merase I. After further processing and modification of rRNA

Nucleolus The nucleolus is1he site of ribosomal RNA (rRNA) synthe· sis and initial ribosomal assembly. The nucleolus is a norune.rnbranous region of the nucleus that surrounds transcriptionally active rRNA genes. It is the primacy site of ribosomal production and assembly. The nu· cleolus varies in size but is particularly wdl devdoped in cells active in protein synthesis. Some cells contain more than one nucleolus (Fig. 3.5). The nucleolus has three morphologically distinct regions:

• Fibrillar centers contain DNA loops of five diiferent chromosomes(l3, 14, 15,2l,and22)thatcontainrRNA genes, RNA polymerase I, and transcription factors.

RGURE 3.1. Electron micrograph of the nucleolus. This nucleolus from a nerve cell shows fibrillar centers {FC) surrounded by the fibrillar (F) and granular IGl materials. Such a network of both materials is referred to as the nucleolonema. The rRNA, DNA-containing genes for the rRNA, and specific proteins are localized in the interstioes of the nucleolonema. X15.000.

87 inset in Figure F3.1.1 a shows a translocation between chromosome 8 and 14 (t8;14l. It is clearly visible on this color image that a part of the original chromosome 8 (aqua blue region) is now attached to chromosome 14, and a small portion of chromosome 141red region) is now part of chromosome 8. Such chromosomal translocations are present in lymphomas (cancers of blood cells), such as acute myeloid leukemia (AMU. non-Hodgkin lymphoma INHLJ, and Burkitt lymphoma. In Figure F3.1.1b, a metaphase spread obtained from cultured lymphocytes of a patient with suspected Prader-Willi/Angalman syndrome (PWS/AS) has been hybridized with several DNA probes reacting with chromosome 15 (an enlarged chromosomal pair from chromosome 15 is shown in the yellow box inset). The green probe (D15Z1) indicates the centromere of chromosome 15. The adjacent orange probe (D15S10l reacts with the PWS/AS region of chromosome 15. Deletion of this region is associated with PWS/AS. Note that one homologue of chromosome 15 has lost that region (no orange signal is visible). The third red probe (PMU recognizes the distal long arm of chromosome 15 and is visible in both chromosomes. Severe intellectual disability, muscular hypotonia, short stature, hypogonadism, and insulin-resistant diabetes are characteristics of PWS/AS. When the deletion is inherited from the mother, patients develop Angelman syndrome; when inherited from the father, patients develop Prader-Willi syndrome. This preparation is counterstained with DAPI that reacts with double-stranded DNA and exhibits blue fluorescence.

Cytogenetic testing is an important component in the diagnosis and evaluation of genetic disorders and refers to the analysis of chromosomes. Chromosome abnormalities occur in approximately 0.5% of all live births and are detected in approximately 50% of first-trimester miscarriages (spontaneous abortions) and 95% of various tumor cells. Chromosome analysis can be performed on peripheral blood, bone marrow, tissues (such as skin or chorionic villi obtained from biopsies), and cells obtained from amniotic fluid during amniocentesis. Studies of chromosomes begin with the extraction of whole chromosomes from the nuclei of dividing cells. These chromosomes are then placed on glass slides, hybridized with special fluorescence probes (FISH technique), and examined under a microscope. A single fluorescent DNA probe produces a bright microscopic signal when the probe is hybridized to a specific part of a particular chromosome. To obtain an image of all of the chromosomes, a mixture of different probes is used to produce different colors in each chromosome. Karyotypes labeled by this method allow cytogeneticists to perform a comprehensive analysis of changes in the number of chromosomes and chromosomal abnormalities such as additions or deletions. The paired chromosomes are numbered in the karyotype, and the male sex is indicated by the presence of chromosomes X andY (Fig. F3.1.1 a). The white box inset in Figure F3.1.1 a shows the XX chromosome pair as it appears in the female. Sometimes, part of a chromosome will break off and attach to another chromosome. When this happens. it is referred to as a translocation. Note that the red box

'' \' f

'

.

.... ..

1

6

. .. 13

~

'

• ' >I ••• • II Jl •• 2

3

~~

..

7

8

•• '' :: 14

11

15

•• 21

20

.J ~ ~

14

•)

10

t

• ,!

8

•p • I'

16

.

11

I' 1

4

5

r

't

1

11

••. " 17

,,

.. •

•

12

li

.._,

18

.:..-

~

22

...

...

X y

·01

' '.....

~'" 4t

'~

~or,

D • G:J 0

~

FIGURE F3. 1.1. Karyotypes obtained with the fluorucent in litu hybridization IFISHI technique. a. Karyotype of a normal male. The white box inset shows the XX chromosome pair of a normal female. The red box inset reveals an abnormality in chromosomes 14 and 8. (Courtesy of the Applied Imaging lntemational Ltd., Newcastle upon Tyne, United Kingdom.) b. A metaphase spread from a patient with Prader-Willi/Angelman syndrome. The yellow box inset shows the enlarged pair of chromosome 15. (Courtesy of Dr. Robert B. Jenkins.)

n :z:

,.. ~

m

::a ~

:i!m

n m I"" I""

z

c

n

!;;

c (I)

•z

c

n r

~

::D

n 0

s:

"0

0

z

m

z

-I

{/)

88

~

zLU z

0

Cl...

~

0

u

c:

z

•

en

:»

~

u

:» z

~

u

w

~

cij

i FIGURE 3.7.

EJ~n micrograph oUhe nuclenenvelope.

Note

the visible nuclear pore complexes (BITOINS} and the two membranes that constitute the nuclear envelope. At the periphery of each pore. the outer and inner membranes of the nuclear envelope appear continuous. X30,000.

exhibits additional structural detail (see Fig. 3.8). Eight multidomain protein subunits arranged in an octagonal central framework at the periphery of each pore form a cylinder-like structure known as the nuclear pore complex (NPC). The NPC, which has an estimated total mass of 125 X 106 Da, is composed of approximately 50 different nuclear pore complex. proteins collectively referred to as nucleoporins (Nup proteins). This central framework. is inserted between the cytoplasmic ring and the nuclear ring (Fig. 3.9). From the cytoplasmic ring, eight short protein fibrils protrude into the cytoplasm and point toward the center of the structu.te. The nucleoplasmic ring complex anchors a nuclear basket (or nuclear "cage, that resem· bles a fish trap) assembled from eight thin 50-nm-long fila· ments joined distally by an adjustable tenninal ring 30 to 50 nm in diameter (see Fig. 3.9). The cylindeNhapc:d central framework. encircles the central pore of the: NPC, which

• Large molecules (such as large proteins and macromolecular complex:es) depend on the presence of an attached signal sequence called the nuclear localization signal {NLS) for passage through the pores. Labeled NLS proteins destined for the nucleus then bind to a soluble cytosolic receptor called a nuclear import receptor (importin) that directs them from the cytoplasm to an appropriate NPC. They are then actively transported through the pore by a GTP energy-dependent mechanism. Export of proteins and RNA from the nucleus is similar to the import mechanism into the nucleus. Proteins that possess a nuclear export sequence (NES) bind in the nucleus to exportin (a protein that moves molecules from the nucleus into cytoplasm) and to a GTP molecule. Protein-exportin-GTP complexes pass through the NPC into the cytoplasm where GTP is hydrolyzed and the NES protein is released. The NPC transports proteins and all forms of RNA as well as ribosomal subunits in their fully folded configurations. • Ions and smaller wateJ~osoluble molecules (less than 9 Da) may cross the wata~~ofilled channels of the NPC by simple diffusion. This process is nonspecific and does not require nuclear signal proteins. The effective size of the pore is approximately 9 nm for substances that cross by diffusion rather than the 70- to 80-nm measurement of the pore boundary. However, even smaller nuclear proteins that are capable of diffusion are selectively transported, presumably because the rate is f.tster than with simple diffusion. During cell division, the nuclear envelope is disa11embled to allow chromosome separation and is later rea11embled as the daughter cells form. In late prophase of cell division, enzymes (kinases) are activated that cause phosphorylation of the nuclear lamias and other lamina-associated proteins of the nuclear envelope. After phosphorylation, the proteins become soluble, and the nuclear envelope disassembles. The lipid component of the nuclear membranes then disassociates from the proteins and is retained in small cytoplasmic vesicles. The replicated c.h.romosomes then attach to the microtubules of the mitotic spindle and undergo active movement.

91

n

~

:II ~

;! m

~

z

c

FIGURE 3.8. Clyoelactron tomography of the nudear pore complex {NPC). These surface renderings of electron tomograms obtained from the frozen-hydrated Dictyostelium nuclei show detailed structure of the NPC. X320,000. a. Cytoplasmic face of the NPC shows eight protein fibrils arranged around the central channel. They protrude from the cytoplasmic ring subunits and point toward the center of the structure. Note a presence of the central plug or transporter within the central pore. which represents either ribosomes or other protein transporters captured during their passage through the NPC. b. Nuclear face of the NPC shows the nucleoplasmic ring subunits connected by nuclear filaments with the basket indicated in brown color. !Adapted from Beck M. FOrster F. Ecke M, et al. Nuclear pore complex structure and dynamics revealed by cryoelectron tomography. Science 2004;306:1387-1390.1

Reassembly of the nuclear envelope begins in late anaplwe, when phosphawes are activated to remove the phosphate residues from the nuclear lamins. During telophase. the nuclear lamins begin to .repolymerize and fonn the nuclear lamina material around each set of daughter chromosomes. At the same

time, vesicles containing the lipid componenu of the nuclear membranes and strucrural membrane protein components fuse, and an envelope is formed on the sur&ce of the alreadyreassembled nuclear lamina. By the end oftelophase. formation ofa nuclear envelope in each daughter cell is complete.

FIGURE 3.9. Sagittal "etion of the nuclear pore complex. Cryoelectron tomographic view of a sagittal section of the nuclear pore complex-shown in Figure 3.8-is compared with a schematic drawing of the complex. Note that the central plug/transporter has been removed from the central pore. x320,000. Each pore contains eight protein subunits arranged in an octagonal central framework at the periphery of the pore. These subunits form a nuclear pore complex that is inserted between two cytoplasmic and nucleoplasmic rings. Eight short protein fibrils protrude from the cytoplasmic rings into the cytoplasm. The nuclear ring anchors a basket assembled from eight thin filaments joined distally into the terminal ring. The diameter of the ring can be adjusted to meet nuclear pore transport requirements. The cylindrical central framework encircles the central pore, which acts as a close-1itting diaphragm. !Adapted 1rom Beck M, Forster F. Ecke M, et al. Nuclear pore complex structure and dynamics revealed by cryoelectron tomography. Science 2004;306:1387-1390.1

e c

tn

•z c

nr ~ :lJ n 0

~

(3 z

m

~

92

....w

~

....w ...J

u

•

0

~:» z

:I

~

1&1

~

"

I

Nucleoplasm Nucleoplasm is the material enclosed by the nuclear envelope exclusive of the chromatin and the nucleolus. Although crystalline, viral, and other inclusions are sometimes found in the nucleoplasm, until recently, morphologic tech· niques showed it to be amorphous. It must be assumed, how· ever, that many proteins and other metabolites reside in or pass through the nucleus ia .n:lation to the synthetic and metabolic activity of the chromatin and nucleolus. Structures that have been identified within the nucleoplasm include iattanuclear lamin·based anays, the protein filaments emanating inward from the nuclear pore complexes, and the active gene-tethered RNA transcription and processing machinery itsel£

• CELL RENEWAL Somatic cells in the adult organism may be classified ac· cording to their mitotic activity. The lc:vel ofmitotic activity in a cell can he assessed by the number ofmitotic metaphases visible in a single higlHnagnification light microscopic field or by autoradiographic studies of the incorporation oftritiated thymidine into the newly synthesized DNA before mitosis. Using these methods, cell populations may be classified as static, stable, or renewing:

vessels, and fibroblasts of the connective tissue may be in· eluded in this category. • Renewing cell populations may be slowly or rapidly renewing but display regular mitotic activity. Division of such cells usually results in two daughter cells that differ· entiate both morphologically and functionally or two cells that remain as stem cells. Daughter cells may divide one or more times before their mature state is reached. The differentiated cell may ultimately be lost from the body. • Slowly renewing populations include smooth muscle cells of most hollow organs, fibroblasts of the uterine wall, and epithellal cells of the lens of the eye. Slowly renewing populations may acrually slowly increase in size during life, as do the smooth muscle cells of the gastrointestinal tr.r.ct and the epithelial cells of the lens. • Rapidly renewing populations include blood cells, epithelial cells and dennal fibroblasts of the skin, and the epithelial cells and subepithelial fibroblasts of the mucosal lining of the alimentary tract.

•cELL CYCLE

Phases and Checkpoints Within the Cell Cycle

• Static cell populations consist of cells that no longer

The cell cycle represents a aeH-regulated sequence of events that controls cell growth and cell division.

divide (postmitotic cells), such as cells of the central nervous system and skeletal or catdiac muscle cells. Under certain cin;umstances, some ofthese cells (i.e., cardiac myoc:ytes) may eater mitotic division. • Stable cell populations consist of cells that divide ep· isodically and slowly to maintain normal tissue or organ structure. 'These cells may be stimulated by injury to be· come more mitotically active. Periosteal and perichon· drial cells, smooth muscle cells, endothelial cells of blood

For renewing cell populations and growing cell populations, including embryonic cells, and cells in tissue culture, the goal of the cell cycle is to produce two daughter cells, each con· tain.ing chromosomes identical to those of the parent cell. The cell cycle incorporates two principal phases: interphase, representing continuous growth of the cell, and M phase (mitosis), characterized by the partition ofthe genome. Three other phases, G, (gap 1) phaae, S (synthesis) phase, and G2 (gap 2) phase, further subdivide interphase (Fig. 3.10).

spindle-assembly

chromosome-segregation

checkpoint

checkpoint

G2 DNA-damage checkpoint

FIGURE 3.10. Cell cycle and checkpoints. This diagram illustrates the cell cycle of rapidly dividing cells in relation to DNA synthesis. After mitosis, the cell is in interphase. G1 represents the period during which a gap oocurs in DNA synthesis. S represents the period during which DNA synthesis occurs. G2 represents a second gap in DNA synthesis. Go represents the path of a cell that has stopped dividing; however, such a cell may reenter the cell cycle after an appropriate stimulus. The cell residing in Go may undergo terminal differentiation (G 70) and produce a population of permanent nondividing cells (e.g., marure fat cells). The average timing of each phase of the cell cycle is indicated on the diagram. Each phase contains several checkpoints that ensure that the system only proceeds to the next stage when the previous stage has been completed and no damage to the DNA is detected.

Rapidly renewing populations of human cells progress through the full cell cycle in about 24 hours. Throughout the cycle, several internal quality control mechanisms or checkpoints represented by biochemical pathways control transition between celt cycle stages. The cell cycle stops at several checkpoints and am only proceed if certain conditions are met-for example, if the cell has reached a certain size. Checkpoints monitor and modulate the progression of cells through the cell cycle in response to intracellular or environmental signals. The G1 phase is usually the longest and the most variable phase of the cell cycle, and it begins at the end of M phase.

During the G1 phase, the cell gathers nutrients and synthesizes RNA and proteins necessary for DNA synthesis and chromosome replication. The cell's progress through this phase is monitored by two checkpoints: (1) the restriction checkpoint, which is sensitive to the size of the cell, the state of the celt's physiologic processes, and its interactions with extracellular matrix; and (2) the G1 DNA-damage checkpoint, which monitors the integrity of newly replicated DNA. For instance, if the DNA has irreparable damage, the G 1 DNA-damage checkpoint detects high levels of tumo,..suppressor protein p53 and does not allow the cell to enter the S phase. The celt will then most likely undergo programmed celt death (apoptosis). The restriction checkpoint (or •point of no retum•) is the most important checkpoint in the cell cycle. At this checkpoint, the cell sdf-ev:aluates its own replicative potential before deciding to either enter the S phase and the nat round of cell division or to retire and leave the cell cycle. A cell that leaves the cycle in the G 1 phase usually begins temalnal dlf· ferentlatlon (Gro) by entering the Go phase ("0" stands for "outside" the cycle). Thus, the G 1 phase may last for only a few hours (average 9 to 12 hours) in a rapidly dividing cell, or it may last a lifetime in a nondividing cell. This checkpoint is mediated by inte.ractions between the retinoblastoma susceptibility protein (pRb) and a family of essential transcription factors (E2F) with target promoters. In normal cells, proper interaction between pRb and E2F turns off many genes and blocks cell cycle progression. In the S phase, DNA is replicated.

Initiation of DNA synthesis marks the begiMing of the

S phase. which is about 7.5 to 10 hours in duration. The DNA of the cell is doubled during the S phase, and new chromatids are formed that will become obvious at prophase or metaphase of the mitotic division. Chromosome replication is initiated at many different sites called replicons along the chromosomal DNA. Each replicon has a specifically assigned time frame for replication during S phase. Presence of the S DNA-damage checkpoint in this phase monitors the quality of replicating DNA.

are arrested in G 2 for extended periods, such as primary oocytes. Two checkpoints monitor DNA quality: the G 2 DNA-damage checkpoint and the unreplicated-DNA checkpoint. The latter checkpoint prevents the progression of the cell into theM phase before DNA synthesis is complete. Mitosis occurs in the M phase.

Mitosis nearly always includes both karyokinesis (division of the nucleus) and cytokinesis (division of the cell) and lasts about 1 hour. Mitosis takes place in several stages described in more detail bdow. Separation of two identical daughter cells concludes the M phase. The M phase possesses two checkpoints: the spindle-assembly check· point, which prevents premature entry into anaphase, and the chromosome-segregation checkpoint, which prevents the process of cytokinesis until all of the chromosomes have been correctly sepuated. A mitotic ca1astrophe caused by malfunction of cell cycle checkpoints may lead to cell death and 1umor cell development.

Malfunction of any of the three DNA-damage checkpoints at the G., S, and G 2 phases of the cell cycle and the spindleassembly checkpoint at M phase may lead to a mitotic catastrophe. Mitotic catastrophe is defined as the failure to arrest the cell cycle before or at mitosis, resulting in aberrant chromosome segregation. Under normal conditions, death in these cells will occur by activation of the apoptotic cycle. Cells that fail to execute the apoptotic cycle in response to DNA or mitotic spindle damage are likely to divide asymmetrically in the next round of cell division. This leads to the generation of aneuploid cells (cells containing abnormal chromosome numbers). Thus, a mitotic catastrophe may be regarded as one of the mechanisms contributing to oncogenesis (tumor cell development). Malfunction of the restriction checkpoint at the G 1 phase may also result in malignant transformation of cells. Malignant cells lose contact inhibition, a normal process in which c:ells inhibit their division when they contact other cells. Malignant cells in culture continue to divide and may grow on top of one another rather than discontinuing growth when the plate is fully covered in a monolayer of cells. The malfunction of the resuiction checkpoint may be facilitated by viral proteins of several cancer-causing viruses, such as the T-antigen of simian virus (SV40) that binds to pRh. This binding alters the configuration of the pRb-T-antigen complex and renders the resuiction checkpoint inoperable, thus facilitating the c:ell's progression from the G 1 to S phase of the c:ell cycle. This mechanism of carcinogenesis occurs in mesothelioma {cancer of the lining epithelium of the pleural cavities in the thorax), osteosarcoma (a type of bone cancer), and ependymoma (a type of childhood brain tumor}.

In the G2 phase, 1he cell prepares for cell division.

The reserve stem cell popula1ion may become activated and r11nter the call cycle.

During this phase, the cell examines its replicated DNA in preparation for cell division. It is also a period of cell growth and reorganization of cytoplasmic: organelles. The Gz phase may be as short as 1 hour in rapidly dividing cells or of nearly indefinite duration in some polyploid c:ells and in cells that

Cells identified as reserve stem cells are essentially Go cells that may be induced to reenter the c:ell cycle in response to injury. Activation of these cells may occur in normal wound healing and in repopulation of the seminiferous epithelium after intense acute exposure of the testis to

93

n

~

:II ~

;! m

~

z

c

e c

tn

•n m rr-

n~

rm

94

X-irradiation or during regeneration of an organ, such as the liver, after removal of a major portion. If damage is too

Cyclin A-Cdk1

extensive, even the reserve stem cells die, and there is no potential for regeneration.

Regulation of the Cell Cycle UJ .....J

~

u

.....J .....J LLJ

u

•~ w

.... u :;) z .... .... w u

w

...cii :::r::::

f5

t:

c

iS

Pa1111ga through the cell cycle is driven by proteins that ara cyclically syn1hasizad and dagraded during each cycle. A number of cytoplasmic protein complexes regulate and control the cell cycle. Some of these proteins function as biochemical oscillators, whose synthesis and degradation are coordinated with specific phases of the cycle. Cellular and molecular events induced during the increase and decrease of diH"erent protein levels are the basis of the cell cycle "engine." Other proteins actively monitor the quality of the molecular processes at the different checkpoints distributed throughout the cycle (described above). The protein complexes at the checkpoints may drive the cell into and out of the cell cycle, stimulating growth and division when conditions are favorable and, conversely, stopping or reducing the rate of cell division when conditions are not favorable.

A two-protein complex consisting of cyclin and a cyclin· dependent kinase (Cdk) helps power the cells through the checkpoints of cell cycle division. The first milestone in understanding the regulation of the cell cycle was the discovery in the early 1970s of a protein called maturation promoting factor (MPF). MPF appeared to control the initiation of mitosis. When injected into the nuclei of immature frog oocytes, which are normally arrested in G21 the cells immediately proceeded through mltosis. MPF was eventually found to consist of two proteins:

• Cdc2 (also known as Cdk-1), a 32 kDa member of the Cdk family of proteins

TABLE3.1

D-Cdk4/8 Cyclin E-Cdk2 ~le by ~lirt-Cdk complexes. This diagram shows the changing pattern of cyclin-Cdk activities during different phases of the cell cycle .

FIGURE 3.11. Regulation Df the aell

• Cyclin B, a 45 kDa member of the cydin family, which are key regulators of the cell cycle. Cydins are synthesized as constitutive proteins; however, their levels during the cell cycle are controlled by ubiquitin·mediated degradation. It is now known that the cyclin-Cdk complex acts at different phases of the cdl cycle and targets different proteins to control cell cycle-dependent functions. Table 3.1 shows the combination of the different types of cyclins with different types of Cdks and how interactions between these two proteins affect cells progressing through the cell cycle. Passage through the cell cycle requires an increase in cyclinCdk activity in some phases followed by decline of activity in other phases (Fig. 3.11). The increased activity of cydinCdk is achieved by the stimulatory action of cyclins and is

Functional Summary of Cyclin-Cyclin-Dependent Kinase Complexes Used in Regulating the Human Cell Cycle

CyclinType

Associated Cyclin-Depandent Protein Kinase

Targeted Phase of Cell Cycle

Cyclin D

Cdk4/6

G, phase progression

Tumor-suppressor protein p53, retinoblastoma susceptibility protein {pRb)

Cyclln E

Cdk2

S phase entry

ATM or ATR protein kinases, tumor-suppressor protein p53

CycllnA

Cdk2

S phase progression

Replication protein A, DNA polymerase, minichromosome maintenance (MCMJ protein

CyclinA

Cdk1

S phase through G2

Cdc25 phosphatase, cyclin B

Targeted Effector Proteins

phase and M phase entry Cyclin E

Cdk1

M phase progression

Chromatin-associated proteins, histone H1, nuclear lamins, myosin regulatory proteins, centrosomal proteins, transcription factors c-fosnun. c-myb, oct-1, SWI5; p60src protein kinases, casein kinase II, c-mos protein kinases

ATM. ataxia-telangiectasia mutated protein kinase; ATR, ATM- and Rad3·related kinase; Cdk, cyclin-depandent kinase.

counterbalanced by the inhibitory action of proteins such as Inks (inhibitors of kinase), Cips (Cdk inhibitory proteins), and Kips (kinase inhibirory proteins).

Mitosis Cell division is a crucial process that increases the number of cells, pennits renewal of cell populations, and allows wound repair.

Mitosis is a process of chromosome segregation and nuclear division followed by cell division that producu two daughter cells with the same chromosome number and DNA content as the parent cell. The tenn mitosis is used to describe the equal partitioning of replicated chromosomes and their genes into two id.enti.cal groups. The process of cell division includes division of both the nucleus (karyokinesis) and the cytoplasm (cytokinesis). The process of cytokinesis results in distribution of nonnuclear organelles into two daughter cdls. Before entering mitosis, cells duplicate their DNA. This phase of the cell cycle is called the S or synthesis phase. At the beginning of this phase, the chromosome number is {2n), and the DNA content is also (2d}; at the end, the chromosome number remains the same (2n}, and the DNA content doubles to (4d).

Mitosis follows the S phase of tile cell cycle and is divided into four phases. Mitosis consists of four phases (Fig. 3.12):

• Prophase begins as the replicated chromosomes condense and become visible. As the chromosomes continue to condense, each of the four chromosomes derived from each homologous pair consists of two chromatids. The sister chromatids are held together by a ring of proteins called cohesins and the centromere. In late prophase or prometaphase (sometimes identified as a separate phase of mitosis), the nuclear envelope begins to disintegrate into small transport vesicles and resembles the sER. The nucleolus, which may stUI be present in some cells, also completely disappears in prometaphase. In addition, a highly specialized protein complex called a kinetochore appears on each chromatid opposite to the centromere (Fig. 3.13). The protein complexes that form kinetochores in the centromere region of the chromatids are attached to specific repetitive DNA sequences known as satellite DNA, which are similar in each chromosome. Microtubules of the developing mitotic spindle attach to the kinetochores and thus to the chromosomes. • Metaphase (Fig. 3.14) begins as the mitotic spindle~ comes organized around the microtubule-organizing centers (MTOCs) located at opposite poles of the cell. The mitotic spindle consists of three types of microtubules: • Astral microtubules that are nucleated from 'Y·tubulin rings in a star-like fashion around each MTOC (see Fig. 2.55). They are responsible for positioning the spindle within the cell. • Polar microtubulea, also originating from the MTOC. They constitute all rnicrotubules that lie between spindle poles which are not connected to

kinetochores. Polar microtubules from opposite poles interact with each other via motor proteins in an antiparallel fashion pushing the spindle poles apart to ensure its bipolarity. • Kinetochore microtubule& that emanate from the MTOC to probe the cytoplasm in search of kinetochores. When a kinetochore is finally captured by a kinetochore microtubule, it is pulled toward the MTOC, where additional microtubules will attach. The kinetochore is capable of binding between 30 and 40 microtubules to each chromatid. In some species, kinetochore microtubules are formed by MTOCindependent mechanisms that involve kinetochore&. Kinetochore microtubules and their associated motor proteins direct the movement of the chromosomes to a plane in the middle of the cell, the equatorial or

metaphase plate. • Anaphase (Fig. 3.15) begins at the initial separation of sister chromatids. This separation occurs when the cohesins that have been holding the chromatids together break down. The chromatids then begin to separate and are pulled to opposite poles of the cell by the molecular motors (dyneins) sliding along the kinetochore microtubules toward the MTOC. • Telophase (Fig. 3.16) is marked by reconstitution of the nuclear envelope around the chromosomes at each pole. The chromosomes uncoil and become indistinct except at regions that will remain condensed in the interphase nucleus. The nucleoli reappear, and the cytoplasm divides (cytokinesis) to form two daughter cells. Cytokinesis begins with the furrowing of the plasma membrane midway between the poles of the mitotic spindle. Separation at the cleavage furrow is achieved by a contractile ring consisting of a very thin array ofactin filaments positioned around the perimeter of the cell. Within the ring, myosin II molecules are assembled into small filaments that interact with the actin filaments, causing the ring to contract. As the ring tightens, the cell is pinched into two daughter cells. Because the chromosomes in the daughter cells contain identical copies of the duplicated DNA, the daughter cells are genetically identical and contain the same kind and number of chromosomes. The daughter cells are (2d) in DNA content and {2n) in chromosome number.

Meiosis Meiosis involves two sequential nuclear divisions followed by call divisions tllat produce gametes containing haH tile number of chromosomea and half the DNA found in somatic calla. The zygote (the cell resulting from the fusion ofan ovum and a sperm) and all the somatic cells derived from it are diploid (2n) in chromosome number; thus, their cdls have two cop·

ies of every chromosome and every gene encoded on this chromosome. These c.hromosomes are called homologous chromosomes because they are simUar but not identical; one set of chromosomes is of maternal origin, the other is from the male parent. The gametes, having only one mem· her of each chromosome pair, are described as haploid (1 n).

95

n

~

:II ~

;! m

~

z

c

e c

tn

•n m rr

nr~

m

96

w

...J

~

PREMITOTICIMEIOTIC EVENTS

hh

two pairs of homologous chromosomes

0

...J ...J

w

0

•

0

:)

1&1 ...J

CJ

:)

......z

1&1

CJ

1&1

:

~

" IL a:

1&1

c

:

CJ

MEIOSIS

Mn'OSIS

@ leptotene

prophase

zygotene

pachytene

diplotene

diakinesis ~

~

continuation of

continuation of

metaphase

0

anaphase

"2" 0 'in

;::E _-c

!iii -c:: CDO

e:g

"0

~

telophase

daughter cells

spermatozoa

•note: prophase II, anaphase II, and telophase II are not shown

ovum

FIGURE 3.12. Comparllon of mltota and melo111 In an kiHIIzad cell with two paiN of chromo10m11 (2n). The dlromosomes of maternal and paternal origin are depicted in red and blue, respectively. The mitotic division produces daughter cells that are genetically identical to the parental cell (2n). The meiotic division, which has two components, a reductional division and an equatorial division, produces a cell that has only two chromosomes 11 n). In addition. during the dlromosome pairing in prophase I of meiosis. dlromosome segments are exchanged, leading to further genetic diversity. It should be noted that in humans, the first polar body does not divide. Division of the first polar body does occur in some species.

97

n

~

:II ~

;! m

~

z

c

FIGURE 3.13. Atomic fon:e microscopic Image of the centro· meric l"'gion of a human metaphase chromosome. The facing surfaces of two sister chromatids visible on this image form the cen· tromere, a point of junction of both chromatids. On the opposite side from the centromere, each chromatid possesses a specialized protein complex, the kinetochore. which serves as an attachment point for kinetochore microtubules of the mitotic spindle. Note that the surface of the chromosome has several protruding loop domains formed by chromatin fibrils anchored into the chromosome scaffold. X40,000. (Courtesy of Dr. Tatsuo Ushiki.l

FIGURE 3.14. Mitotic spindle In metaphase. Using indirect immunofluorescence techniQues. the mitotic spindle in a Xenopus Xl-177 cell was labeled with an antibody against a.·tubulin conju· gated wi1h fluorescein !green). DNA was stained blue with fluorescent DAPI stain. In metaphase, the nuclear membrane disassembles, DNA is condensed into chromosomes. and microtubules form a mi· totic spindle. The action of microtubule-associated motor proteins on the microtubules of the mitotic spindle creates the metaphase plate along which the chromosomes align in the center of the cell. X 1,400. !Courtesy of Dr. Thomas U. Mayer.)

e c

tn

•n m rr-

nr~

m

During gametogenesis, reduction in chromosome number to the haploid state (23 chromosomes in hwnans) occurs through meiosis, a process that involves two successive divisions, the second of which is not preceded by an S phase. 1h.is reduction is necessary to maintain a constant nwnber ofchromosomes in a given species. Reduction in chromosome nwnber to (1n) in the first meiotic division is followed by reduction in DNA content to the haploid (1d} amoWlt in the second meiotic division. During meiosis, the chromosome pair may exchange chromosome segments, thus altering the genetic composition of the chromosomes. This genetic exchange, called crossing-over, and the random assortment of ea£:b. member of the chromosome pairs into haploid gametes give rise to infinite genetic diversity.

FIGURE 3.11. Mitotic eplndleln anaphaM. This immunofluorescent image comes from the same cell type and identical preparation as in Figure 3.13. Connections that hold the sister chromatids together break at this stage. The chromatids are then moved to opposite poles of the cell by microtubule-associated molecular motors ldyneins and kinesins) that slide along the kinetochore microtubules toward the centriole and are also pushed by the polar microtubules (visible between the separated chromosomes) sway from each other, thus moving opposite poles of the mitotic spindle into the separate cells. X 1,400. {Courtesy of Dr. Thomas U. Mayer.)

The cytoplasmic events auociated with meiosis dHfar in 1he male and female. The nuclear events of meiosis are the same in males and females, but the cytoplasmic events are markedly different. Figure 3.12 illustrates the key nuclear and cytoplasmic evenu of meiosis as they orxur in spermatogenesis and oogenesis. The events of meiosis through metaphase I ate the same in both sexes. Therefore, the figure illustrates the differences in the process as they diverge after metaphase I. In males, the two meiotic divisions of a primary spel'matocyte yield four strUCturally identical, although genetically unique, haploid spermatids. Each spermatid has the capacity to differentiate into a spermatozoon. In contrast,

FIGURE 3.16. Mitotic tplndle In telophtee. In 1his phase, DNA is segregated, and a nuclear envelope is reconstituted around the chromosomes at each pole of the mitotic spindle. The cell divides into two during cytokinesis. In the middle of the cell. actin, septins. myosins, microtubules, and other proteins gather as the cell establishes a ring of proteins that will constrict, forming a bridge between the two sides of what was once one cell. The chromosomes uncoil and become in· distinct except at regions that remain condensed in interphase. The cell types and preparation are the same as those in Figures 3.13 and 3.14. X1,400. (Courtesy of Dr. Thomas U. Mayer.)

98

w

...J

~

...J ...J

w u

•

0

~:» z

:I

~

1&1

~

"

I

in females, the two meiotic divisions of a primary oocyte yield one haploid ovum and three haploid polar bodies. The ovum receives most of the cytoplasm and becomes the functional gamete. The polar bodies receive very little cytoplasm and degenerate.

The nuclear evems of meiosis are similar in males and females. Meiosis consists of two successive mitotic divisions without the additional S phase between the two divisions. Owing the S phase that precedes meiosis, DNA is replicated forming sister chromatids (two parallel strands of DNA) joined together by the centromere. The DNA content becomes (4d), but the chromosome number remains the same (2n). The cells then undergo a reductional division (meiosis I) and an equatorial division {meiosis II). During meiosis I, as the name reductional division implies, the chromosome nwnber is reduced from diploid (2n) to haploid (1n), and the amount of DNA is reduced from the {4d) to (2d). During prophase I, double-stranded chromosomes condense, and homologous chromosomes (normally; one inherited from the mother and one from the father) are paired at centromeres. At this point, recombination of genetic material between the maternal and paternal chromosome pair:s may occur. In metaphase I, the homologous chromosomes with their centromeres line up along the equator of the mitotic spindle and in anaphase I are separated and distributed to each daughter cell. This results in reduction of both the chromosome number (1n) and the DNA to the (2d) amount. No DNA replication precedes meiosis II. The division during meiosis II is always equatorial because the number of chromosomes does not change. It remains at (1n), although the amount of DNA represented by the number of chromatids is reduced to (1d). During metaphase II, each chromosome aligns along the equator of the mitotic spindle, and at anaphase II, sister chromatids are separated from each other. Thus, each chromosome splits into two single-stranded chromosomes that are then distributed to each haploid daughter cell.

Phases in tile process of meiosis are similar to the phases of mitosis. Prophase I The prophase of meiosis I is an extended phase in which pairing of homologous chromosomes, synapsis (close association of homologous chromosomes), and recombination of genetic matedal on homologous chromosomes is observed. Prophase I is subdivided into the following five stages (see Fig. 3.12).

• Leptotene. Th.is stage is characterized by the condensation of chromatin and appearance ofchromosomes. Sister chromatids also condense and become connected with each other by meiosis-specific cohesion complexes (Rac8p). At this phase, pairing of homologous chromo~ somes of maternal and paternal origin is initiated. Ho~ mologous pairing can be described as a process in which chromosomes actively search for each other. After finding their mates, they align themselves side by side with a slight space separating them.

• Zygotene. Synapsis, the dose association of homologous chromosomes, begins at this stage and continues throughout pachytene. This process involves the formation of a synaptonamal complex, a tripartite structure that binds the chromosomes together. The synaptonemal complex is often compared to railroad tracks with an additional third rail positioned in the middle between two others. The cross ties in this track are represented by the transverse filaments that bind the scaffold material of both homologous chromosomes together. • Pachytene. At this stage, synapsis is complete. Crossing-over occurs early in this phase and involves transposition of DNA strands between two different chromosomes. • Diplotene. Early in this stage, the synaptonemal complex dissolves, and the chromosomes condense further. Homologous chromosomes begin to separate from each other and appear to be connected by newly formed junctions between chromosomes called chiasmata (sing., chiamut.). Sister chromatids still remain closely associated with each other. Chiasmata indicate that crossing-over may have occurred. • Diakinesis. The homologous chromosomes condense and shonen to reach their maximum thickness, the nu~ cleolus disappears, and the nuclear envelope disintegrates.

Metaphase I Metaphase I is similar to the metaphase of mitosis except that the paired chromosomes are aligned at the equatorial plata with one member on either side. The homologous chromosomes are still held together by chiasmata. At late metaphase, chiasmata are cleaved and the chromosomes separate. Once the nuclear envelope has broken down, the spindle microtubules begin to interact with the chromosomes through the multilayered protein structure, the kinetochore, which is usually positioned near the centromere (see Fig. 3.13). The chromosomes undergo movement to ultimately align their centromeres along the equator of the spindle.

Anaphase I and Telophase I Anaphase I and telophase I are similar to the same phases in mitosis except that the centromeres do not split. The sister chromatids, held together by cohesin complexes and by the centromere, remain together. A maternal or paternal member of each homologous pair, now containing exchanged segments, moves to each pole. Segregation or random assortment occurs because the maternal and paternal chromosomes of each pair are randomly aligned on one side or the other of the metaphase plate, thus contributing to genetic diversity. At the completion of meiosis I, the cytoplasm divides. Each resulting daughter cell (a sac· ondary spannatocyte or oocyte) is haploid in chromosome number (1n) and contains one member of each homologous chromosome pair. The cell is still diploid in DNA content (2d).

Meiosis II After meiosis I, the cells quickly enter meiosis II without pass~ ing through an S phase. Meiosis II is an equatorial division

and resembles mitosis. During this phase, the proteinase enzyme separase cleaves the cohesion complexes between the sister chromatids. Cleavage of the cohesin complexes in the region of the centromere releases the bond between both cent.tomeres. 1his cleavage allows the sister chromatids to separate at anaphase II and move to opposite poles of the cell. During meiosis II, the cells pass through prophase II, metaphase II, anaphase II, and tdophase II. These stages are essentially the same as those in mitosis except that they involve a haploid set of chromosomes (1 n) and produce daughter cells that have only haploid DNA content (1d). Unlike the cells produced by mitosis, which are genetically identical to the parent cell, the cdls produced by meiosis are genetically unique.

Overview of Characteristic:

TABLE3.2

Features Distinguishing Necrosis from Apoptosis

Features of Dying Cells Cell swelling

Necrosis Apoptosis

+

Cell shrinkage Damage to the plasma membrane

+ +

Plasma membrane blabbing Aggregation of chromatin Fragmentation of the nucleus

In humans, as in all other multicellular organisms, the rates of cell proliferation and cell death determine the net cell production. An abnormality in any of these rates can cause disorders of cell accumulation (e.g., hyperplasia, cancer, autoimmune diseases) or disorders of cell loss (atrophy, degenerative diseases, AIDS, ischemic injury). Therefore, the balance (homeostasis) between cell production and cell death must be carefully maintained (Fig. 3.17).

Cell death may occur as a resuh at acute cell injury ar an intamally encoded suicide program. Cell death may result from accidental cell injury or mechanisms that cause cells to self-destruct. The major two different mechanisms of cell death are necrosis and apoptosis:

• Necrosis, or accidental cell death, is a pathologic process. It occurs when cells are exposed to an unfavorable physical or chemical environment (e.g., hypothermia, hypoxia,

CELL DIVISION

CELL DEATH

f·w~.~

~ HOMEOSTASIS

~r l 8 1t.:'l~~~·..···~t~ 1 CELL ACCUMULATION DISORDERS: • cancer • lupus erythematosus • glomerulonephrttls • viral infections

CELL LOSS DISORDERS: • AIDS • Alzheimer disease • Parkinson disease • aplastic anemia • myocardial infarction

FIGURE 3.17. Schematic diagram showing the relationship b.twe1n cell death and cell division. Under normal physiologic conditions (homeostasis), the rates of cell division and cell death are similar. If the rate of cell death is higher than that of cell division, then a net loss of cell number will occur. Such conditions are categorized as cell loss disorders. When the siruation is reversed and the rate of cell division is higher than the rate of cell death, then the net gain in cell number will be prominent leading to a variety of disorders of cell accumulation.

Random DNA degradation Caspase cascade activation

~

:!1 m

:a

+ + + +

Oligonucleosomal DNA fragmentation

• CELL DEATH

99

+I-

~

-t

:I:

m

Q r-

r-

z c: n r-

m

+

c:

en

• n

m r

r

radiation, low pH, cell trauma) that causes acute cellular injury and damage to the plasma membrane. Under physiologic conditions, damage to the plasma membrane may also be initiated by viruses, or proteins called pnforim. Rapid cell swelling and lysis are two characteristic features of this process. • Apoptosis [Gr., foiling off. as pnais from jlowen} was referred to in the past as programmed cell death. Today, the term programmed ceO death is applied more broadly to any kind of cdl death mediated by an inuacellular death program, irrespective of the uigger mechanism. Apoptosis represents a physiologic process. During apoptosis, cells that are no longer needed are diminated from the organism. This process may occur during normal embryologic development or other normal physiologic processes, such as follicular atresia in the ovaries. Cells can initiate their own death through activation of an internally encoded suicide program. Apoptosis is characterized by controlled autodiges· tion, which maintains cell membrane integrity; thus, the cell "dies with dignity" without spilling its contents and damaging its neighbors. In addition, certain cells or their secretions found in the immune system are toxic to other cells (e.g., cytotoxic T lymphocytes, natural killer [NK] cells); they initiate processes that destroy designated cells (e.g., cancertransformed or virus-infected cells). In contrast to necrosis and apoptosis, cytotoxic death does not involve one specific mechanism. For example, cell death mediated by cytotoxicT lymphocytes combines some aspects of both necrosis and apoptosis. For an overview of the apoptosis and necrosis, see Table 3.2.

Necrosis begins with impairment of the call's ability to maintain homeostasis. As a result of cell injury, damage to the cell membrane leads to an influx of water and cxtracellular ions. Intracellular

0

~

::I:

w

organelles, such as the mitochondria. rER, and nucleus, undergo irreversible changes that are caused by cell swelling and cell membrane rupture (cell lysis). & a result of the breakdown of the plasma membrane, the cytoplasmic contents, including lysosomal enzymes, are released into the extracellular space. Therefore, necrotic cell death is often associated with extensive surrounding tissue damage and an intense inflammatory response (Fig. 3.18).

::1

Apoptosis

u •

Apoptosis is a mode of cell death that occurs under normal physiologic conditions.

100

:::c

~

NECROSIS

APOPTOSIS

0

w

In apoptoais, the cell is an active participant in its own demise ("cellular suicide"). This process is activated by a variety of extrinsic and intrinsic signals. Cells undergoing apoptosis show the following characteristic morphologic and biochemical features (see Fig. 3.18):

• DNA fragmentation occurs in the nucleus and is an irre-versible event that commits the cell to die. DNA fragmentation is a result of Ca2+-dependent and Mi+ -dependent activation of nuclear endonucleases. These enzymes selectively cleave DNA. generating small oligonuc:leosomal fragments. Nuclear chromatin then aggregates, and the nuc:leus may divide into several discrete fragments bounded by the nuc:lear envelope. • Decrease in cell volume is achieved by shrinking of the cytoplasm. The cytoskeletal dements become reorganized in bundles parallel to the cell sur&c:e. Ribosomes become clumped within the cytoplasm, the rER fonns a series of concentric whorls, and most of the endocytotic vesic:les fuse with the plasma membrane. • Loss of mitochondrial function is caused by changes in the permeability of the mitochondrial membrane channels. The integrity of the mitochondrion is breached, the mitochondrial transmembrane potential drops, and the electron-transport chain is disrupted. Proteins from the mitochondrial intermembrane space, such as cytochrome c and SMAC/DIABLO (second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with low isoelectric point [pi]), are released into the cytoplasm to activate a cascade of proteolytic enzymes called caspases that are responsible for dismantling the cell. The regulated release of cytochrome c and SMAC/DIABLO suggests that mitochondria, under the influence of Bcl-2 proteins (see page 101), are the decision makers for initiating apoptosis. Thus, many researchers view mitochondria either as the "headquarters for the leader of a crack suicide squad" or as a "high-security prison for the leaders of a military coup." • Membrane blabbing results from celt membrane aJ.. terations. One alteration is related to translocation of certain molecules (e.g., phosphatidylserine) from the cytoplasmic surface to the outer surface of the plasma membrane. These changes cause the plasma membrane to change its physical and chemical properties and lead to blebbing without loss of membrane integrity (see Fig. 3.18). • Fonnation of apoptotic bodies, the final step of apoptosis, results in cell breakage (Fig. 3.19a-c). These

injury at cell

membrane

swelling

DNA

fragmentation

~

dac:reasa of cell volume

1 /

membrane membrane breakdown

disintegration

blabbing

formation of apoptotic bodies

and lnftammatfon FIGURE 3. 18. Schem.& diagram of dlanges occurring in n• crolis and apoptalis. This diagram shows the major steps in necrosis and apoptosis. In necrosis (/eft side), breakdown of the cell membrane results in an influx of water and extracellular ions, causing the organelles to undergo irreversible dlanges. Lysosomal enzymes are released into the extracellular space, causing damage to neighboring tissue and an intense inflammatory response. In apoptosis {right sidel, the cell undergoes characteristic morphologic and biochemical dlanges such as DNA fragmentation, decrease in cell volume, membrane blebbing without loss of membrane integrity, and formation of apoptotic bodies, causing cell breakage. Apoptotic bodies are later removed by phagocytotic cells without causing an inflammatory reaction.

101

n

~

:II ~

;! m

~

z

c

e c

tn

•n

a

m rr0 m

~

::I:

FIGURE 3.19. Electron mlcrogNph• of epoptotlc celll. e. This electron micrograph shows an early stage of apoptosis in a lymph~ cyte. The nucleus is already fragmented, and the irreversible process of DNA fragmentation is turned en. Nota the regions containing condensed heterochromatin adjacent to 1he nuclear envelope. x5,200. b. Further fragmentation of DNA. The heterochromatin in one of the nuclear fragments (left) begins to bud outward through the envelope, initiating a new round of nuclear fragmentation. Nota the reorganization of 1he cytoplasm and budding of the cytoplasm to produce apoptotic bodies. X5,200. c. Apoptotic bodies containing fragments of the nucleus, organelles. and cytoplasm. These bodies will eventually be phagocytosed by cells from the mononuclear phagocytotic system. x5,200. !Courtesy of Dr. Scott H. Kaufmann.) d. This photomicrograph taken with light microscopy of intestinal epithelium from the human colon shows apoptotic bodies (AB) within a single layer of absorptive calls. BM. basement membrane. X750.

membrane-bounded vesicles or&gmate from the cyto· plasmic bleb containing organelles and nuclear material. They are rapidly removed without a trac:e by phagocytotic cells. The removal of apoptotic bodies is so efficient that no inflammatory response is elicited. Apoptosis occurs more than 20 times faster than mitosis; therefore, it is clJallenging to find apoptotic cells in a routine H&E preparation (Fig. 3.19d).

Apoptosis is regulated by external and internal stimuli. Apoptotic processes can be activated by a vatiety of ex· ternal and internal stimuli. Some factors, such as tumor necrosis factor (TNF), acting on cell membrane re· ceptors, trigger apoptosis by recruiting and activating the caspasc cascade. Conscquendy, the TNF receptor is known as the "death receptor." Other external activa· tors of apoptosis include transforming growth factor 13 (TGF·J3), certain neurouan.smitters, free radicals, oxidants,

and ultraviolet (UV) and ionizing radiation. Internal activators of apoptosis include oncogenes (e.g., myc and rei), tumor suppressors suclJ as p53, and nutrientdeprivation antimetabolites (Fig. 3.20). Apoptotic pathways arc also activated by the events leading to mi· totic catasuophe-namely, malfunction of specific DNAdamage checkpoints in the cell cycle (see page 93). Mitotic catastrophe is accompanied by chromatin condensation, mitoclJondrial release of cytoclJrome c, activation of the caspase cascade, and DNA fragmentation. Apoptosis can also be inhibited by signals from other cells and the surrounding environment via sO-

(!)

0

...J

~ ~

OVERVIEW OF THE NUCLBJ~

_________________

Cl)

•

a ::l

:;,

e

Z

~

U

!I!

The nudeus is a membrane~limited compart~ ment that contains the genome (genetic in~ formation) in eukaryotic cells. 1he nucleus of a nondividing cell consim of chromatin (contains DNA) and the nudeolus (site of rRNA synthesis), which are suspended in the nucleoplasm and surrounded by the nucleer envelope.

~ -------------------------------------------~~==~--------~~~--------------­

a:

~

NUC!LQR CHROMATIN

c(

:z: u

Chromatin, a complex of DNA and associated proteins, is responsible for basophilic staining of the nucleus in H&E preparation. • Two forms ofchromatin are found in the nucleus: a dispersed form called euchromatin and a condensed form called heterochromatin. • Nucleosomes are the smallest units of chromatin structure. They represent the initial folding of the DNA molecule. • In dividing cells, chromatin is condensed and organized into discrete bodies called •

chromosomes.

• B

NUCl[OW~

The nucleolus is the site of rRNA synthesis and initial ribosomal assembly, and it is involved in regulation of the cell cycle. • The nucleolus has three distinct regions: fibrillar centers (include DNA loops of chromo~ ------------------~ somes containing rRNA genes), fibrillar material (includes actively transcribed ribosomal -------------------"1 genes), and granular material (site of initial ribosomal assembly).

NUCLEAR ENVELOPE e

- - - --ill •

- - - - - - - --'ill •

The nuclear envelope, formed by two membranes with a perinuclear cisternal space b~ tween them, separates the nucleoplasm from the cytoplasm. The outer nuclear membrane binds ribosomes and is continuous with the rER membrane. The inner nuclear membrane is supported by the nuclear (fibrous) lamina. The nuclear lamina is composed of nuclear lamina, a specialized type of intermediate filaments, and lamln-assoclated proteins. Lamins disassemble during mitosis andreas~ semble when mitosis ends. The nuclear envelope has an array of openings called nuclear pores. Nuclear pores contain a cylinder-like: structure known as the nuclear pore complex (NPC), which mediates bidirectional nucleocytoplasmic transport.

105 cruevcu~: • The call cycle represents a sdf·regulated sequence of events that controls cell growth and - - - - - - - -cell division. Progress through the cell cycle is monitored at different checkpoints. • The G, phase is usually the longest and the most variable phase of the cell cycle; it begins - - - - - - - ----n :::1: at the end of mitosis (M phase). During the G1 phase, the cell gathers nutrients and syn· > thesizes RNA and proteins necessary for DNA synthesis and chromosome replication. This ~ m phase also contains the most important checkpoint in the cell cycle, the restriction point, :a at which the cell evaluates its own replicative potential. w :..:. • In the S phase, DNA is replicated, and the quality of DNA synthesis is monitored at the :::1: m S DNA-damage checkpoint. n • In the G2 phase, the cell prepares for division during mitosis (M phase) and continues to m rassess the quality of the newly synthesized DNA (at the G1 DNA-damage checkpoint and rthe unreplicated-DNA checkpoint). --------- ~ • Mitosis occurs in the M phase and is controlled by thespindle-assembly and chromosomen rsegregation checkpoints. m c • Passage through the cell cycle is driven by a two·protein complex consisting of cyclln and en cyclin-dependent kinase (Cdk). These proteins are synthesized and degraded at regular intervals during each cycle.

• I

~

MITOru~

• MIIDels is a process ofchromosome segregarlon. nuclear division, and evmwal cell division that produces two daughttT cdls with the same c:hrornosornt: number and DNA rocn:nt as the parent cdL • Mitosis follows ttl a S phase of the cell cycle and contains four phases: prophase, during which chromosomes condense and become visible, the nuclear envelope disassembles, and the mitotic spindle d.evdops from microtubules; metaphase, which involves the alignment of chromosomes in the equatorial plate; anaphase, during which the sister chromatids begin to separate and are pulled to opposite poles ofthe cdl; and telophase, which involves the: reconstruction of the nuclear envelope and the division of cytoplasm. _ _ _ _ _ _ _ _......,. • Mitosis ends with formation of two daughter cdls that are genetically identical (containing the: same number of chromosomes and amount of DNA).

M~IO~I~

• MeiosiS involves two sequential nuclear divisions followed by cell divisions that produce gametes containing half the amount of chromosomes and DNA found in somatic cells. • During the prophase of meiosis I (reductional division), homologous chromosomes are paired and the: recombination of genetic material occurs between maternal and paternal pairs. These pairs (with exchanged segments) form two daughter cells that contain a haploid number of chromosomes and a diploid amount of DNA. • MeiosiS II occurs quickly without passing through the S phase. The second meiotic division separates the sister chromatids into two final cdls, each containing a haploid number of chromosomes and a haploid amount of DNA. C~LLDQTH

• Cell death may occur as a result of acute cdl injury (necrosis) or programmed cell death (apoptosis). • Apoptoals occurs under normal physiologic conditions to diminate defective or senescent -------------! cells without an inflammatory response: by the tissue. _ _ _ _ _ _ _ _ ____,. • Molecular rc:gulation ofapoptosis involves a cascade ofevents controlled by the proapoptotic Bcl-2 family of proteins, which increase the: permeability of the mitochondrial membrane by releasing cytochrome c and SMAC/DIABLO. - - - - - - - - ---ill • Cytochrome c and &MAC/DIABLO activate: the cascade: of cytoplasmic protc:ases called caspases. They dismantle the cell by digesting cytoplasmic proteins. - - - - - - - - --111 • Anoikis is a form ofapoptosis that is induced by a lack ofcdl-ro--c:xtracdlular matrix inter.u:tions. ~

0 r 0

G)

-< ....... S!

TISSUES:

CONCEPT AND CLASSIFICATION OVERVIEW OFTISSUES /108 EPITHELIUM /107 CONNECTIVE TISSUE /108 MUSCLE TISSUE I 108 NERVE TISSUE /109 HISTOGENESIS OFTISSUES /110 Ectodermal Derivatives /110 Mesodermal Derivatives /110 Endodermal Derivatives /111

• OVERVIEW OF TISSUES Tissues are aggregates or groups of calls organized to parfonn one or mora specific functions. At the light microscope level, the cells and extracellular components of the various o.tgans of the body exhibit a recognizable and often distinctive pattern oforganization. This organized arrangement reB.ecm the cooperative effort of cells performing a particular function. An organized aggregation of cells that function in a collective manner is called a tissue [Fr., tissu, wovm; L, texo, to Ulellvt]. Although it is frequendy said that the cell is the basic functional unit of the body, it is really the tissues, through the collaborative efforts of their individual cells, that are responsible for maintaining body functions. Cells within tissues are connected to each other by specialized anchoring junctions (cell-to-cell attachments, page 107). Cells also sense their surrounding extracellular environment and communicate with each other by special.ized intercellular junctions (gap junctions, page 107); facilitating this collaborative effort allows the cdls to operate as a functional unit. Other mechanisms that permit the cells ofa given tissue to function in a unified manner include specific membrane receptors that generate responses to various stimuli (i.e., hormonal, neural, or mechanical).

Despite their disparate structure and physiologic properties. all organs are made up of only four basic tissue types. The tissue concept provides a basis for understanding and recognizing the many cell types within the body and how

106

IDENTIFYING TISSUES /113 Folder 4.1 Clinical Correlation: Ovarian Teratomas /112

HISTOIDGY 101/ 114

~

they intc:rrd.ate. Despite the variations in general appearance, structural organization, and physiologic properties of the var~ ious body organs, the tissues that compose them are classified into four basic types:

• Epithelium (epithelial tissue) covers body sw:faces, lines body cavities, and forms glands.

• Connective tissue underlles or supports the other three basic tissues, both structurally and functionally. • Muscle tissue is made up of contractile cells and is responsible for movement.

• Nerve tissue receives, transmits, and integrates information from outside and inside the body to control the ~tiesofthebod~

Each basic tissue is defined by a set of general morphologic characteristics or functional properties. Each type may be further subdivided according to specific characteristics of its various cell populations and any special extracellular sub~ stances that may be present. In classifying the basic tissues, two different definitional parameters are used. The basis for definition of epithelial and connective tissue is primarily morphologic; for muscle and nerve tissue, it is primarily functional. Moreover, the same parameters exist in designating the tissue subclasses. For example, whereas muscle tissue itself is defined by its function, it is subclassified into smooth and striated catcgo· ries: a purely morphologic distinction, not a functional one. Another kind of contractile tissue, myoepitheliwn, functions as muscle tissue but is typically designated epithellum because of its location.

For these reasons, tissue classification cannot be reduced to a simple formula. Rather, stUdents are advised to learn the features or characteristics of the different cell aggregations that define the four basic tissues and their subclasses.

• EPITHELIUM Epithelium is characterized by close cell apposition and presence at a frte surface. Epithelial cells, whether arranged in a single layer or in multiple layers, are always contiguous with one another. In addition, they are usually joined by specialized cell-to-cell junctions that create a selective barrier between the external environment and the underlying connective tissue. The Intercellular space between epithelial cells is minimal and devoid of any structure except where junctional attachments are present. Free surfaces are characteristic of the exterior of the body, the outer surface of many internal organs, and the lining of the body cavities and rubes that ultimately communicate with the exterior of the body. The enclosed body cavities and tubes include the pleural, pericardia!, and peritoneal cavities as well as the cardiovascular system. All of these structures are

lined by epithelium. The epithelium also forms glands and their ducts that help secrete their products onto a free swface or into the lumen of a rube. Classifications of epithelium are usually based on the shape of the celJs and the number of cellla:yets rather than on function. Cell shapes include squamous (flattened), cuboidal, and columnar. La:yer:s are described as simple (single layer) or stratified (multiple layers). Figure 4.1 shows epithelia &om three sites. Two of them (see Fig. 4.la and b) are simple epithelia (i.e., one cell layer) that line a free surface that is exposed to the lumen of the strw::tu.re. The major distinction between these two simple epithelia is the shape of the cells: cuboidal (see Fig. 4.la) versus columnar (see Fig. 4.1b). The third example (see Fig. 4.lc) is a stratified squamous epithelium that contains multiple layers of cells. Only the top layer of squamous cells is in contact with the lumen; the other celJs are connected with each other by specialized cell-to-cell anchoring junctions or to the underlying connective tissue (lower darkstained bottom layer) by specialized cell-to-extracellular matrix anchoring junctions. The free surface of the epithelium exhibits special structural surface modifications that perform specific functions. Simple epithelia may possess microvilli, stereocilia, or cilia. Stratified epithelia may be keratinized on the exterior

RGURE 4.1. Simpl• 41Pith•li•. a. An H&E-stained section showing a pancreatic duct lined by a single layer of contiguous cuboidal epithelial cells. The free surface of the cells faces the lumen; the basal surface is in apposition to the connective tissue. x 540. b. An H&E-stained section showing a single layer of tall columnar epithelial cells lining the gallbladder. Note that the cells are much taller than the lining cells of the pancreatic duct. The free surface of the epithelial cells is exposed to the lumen of the gallbladder, and the basal surface is in apposition to the adjacent connective tissue. X540. c. An H&E-stained section showing the wall of the esophagus lined by stratified squamous epithelium. Only the top layer of the squamous cells is in contact with the lumen. Note that not all of the cells in this epithelium are squamous. In the lower portion of the epithelium. cells are more rounded. and at the boundary between the epithelium and connective tissue. the basal cell layer appears as a dark band due to smaller cell size and high nucleusto-cytoplasmic ratio. X240.

107

108

of the body or nonkeratinized within the lumen of internal organs. All epitheUa rest on the basal lamina, the structural attachment site for overlying epithelial cells and Wlderlying connective tissue.

• CONNECTIVE TISSUE Connective tissue is characterized on the basis of its extracellular matrix. Unlike epithelial cells, connective tissue cells are conspicu· ously separated from one another. The interverung spaces are occupied by material produced by the cells. This cruacellular material is called the extracellular matrix. The nature of the cells and matrix varies according to the function of the tissue. Thus, classification of connective tissue takes into account not only the cells but also the composition and organi· zation of the cruacellular matrix. Embryonic connective tissue derives from the meso· derm, the middle embryonic germ layer, and is present in the embryo and within the umbilical fold. It gives rise to various connective tissues in the body. A type ofconnective tissue found in close association with most epithelia is loose connective tissue (Fig. 4.2a). In fact, most epithelia rest on connective tissue. The extracel· lular matrix of loose connective tissue contains loosely ar· ranged collagen fibers and numerous cells. Some of these cells, the fibroblasts, form and maintain the extracellular matrix. However, most of the cells are migrants from the vascular system and are associated with the immWle system. In contrast, where only strength is required, collagen fibers are more numerous and densely packed. Also, the cells are relatively sparse and limited to the fiber·forming cell, the fibroblast (see Fig. 4.2b). This type of connective tissue is described as dense connective tissue.

Examples of specialized connective tissues include bone, canilage, and blood. These connective tissues are characterized by the specialized nature of their exrracellular matrix. For instance, bona has a matrix that is mineralized by calciwn and phosphate molecules that are associated with collagen fibers. Cartilage possesses a matrix that contains a large amoWlt of water bound to hyaluronan aggregates. Blood consists ofcells and an extracellular matrix in the form of a protein-rich fluid called plasmtz that circulates throughout the body. Again, in all of these tissues, it is the extracellular material that characterizes the tissue, not the cells.

• MUSCLE TISSUE Muscle tissue is categorized on the basis of a functional property, the ability of its cells to contract. Muscle cells are characterized by large amounts of the con· uaccile proteins actin and myosin in their cytoplasm and by their particular cellular arrangement in the tissue. To func· tion efficiently to effect movement, most muscle cells are ag· gregated into distinct bundles that ate easily distinguished from the surroWlding tissue. Muscle cells are typically elon· gated and oriented with their long axes in the same direction (Fig. 4.3}. The arrangement of nuclei is also consistent with the parallel orientation of muscle cells. Although the shape and arrangement of cells in specific muscle types (e.g., smooth muscle, skeletal muscle, and cardiac muscle) are quite different, all muscle types share a common characteristic. The bulk. of the cytoplasm consists of the contractile proteins actin and myosin, which form thin and thick myofilaments, respectively. Skeletal muscle (see Fig. 4.3a) and cardiac muscle (see Fig. 4.3b) cells ahibit crosNtriations that are produced. largely by the specific urangement of myofilament&. Smooth muscle

FIGURE 4.2. Loose and dense connective titsue. a. Mallory-Azan-stained specimen of a section through the epiglottis. showing the lower part of its stratified epithelium (Epl. subjacent loose connective tissue {LCn. and dense connective tissue IDCn below. Loose connective tissue typically contains many cells of several types. Their nuclei vary in size and shape. The elongated nuclei most likely belong to fibroblasts. Because dense connective tissue contains thick collagen bundles, it stains more intensely with the blue dye. Also, note the relatively fewer nuclei. X 540. b. A Mallory-stained specimen of dense connective tissue, showing a region composed of numerous, densely packed collagen fibers. The few nuclei (M that are present belong to fibroblasts. The combination of densely packed fibers and the paucity of cells characterize dense connective tissue. Relatively few small blood vessels (8Vl are shown on this section. x 540.

109

n

~

:II ~

ic

Rl

in RGURE 4.3. Muscle tiuue. a. An

H&E-stained specimen showing a portion of three longitudinally sectioned skeletal muscle fibers !cells). Two striking features of these large, long cells are their charaoteristic croSS-$triations and the many nuclei located along the periphery of the cell. X420. b. A MallofY-$1ained specimen showing cardiac muscle fibers that also exhibit striations. These fibers are composed of individual cells that are much smaller than those of skeletal muscle and are arranged end to end to form long fibers. Most of the fibers are seen in longitudinal array. The organized aggregation-that is, the parallel array of the fibers in the case of muscle tissue. allows for collective effort in performing their function. Intercalated discs la"ows) mark the junction of adjoining cells. X420. c. An H&E-stained specimen showing a longitudinal layer of smooth muscle cells from the wall of 1he intestine. More intensely stained tissue at the top and bottom of this photomicrograph represents connective tissue. Note that all nuclei of smooth muscle cells !middle) are elongated, and their cytoplasm does not exhibit crosS-$triations. X512.

~

Ic

i l!

~

0

z

•z m

~

m

:::! (/) (/)

cells (see Fig. 4.3c) do not exhibit ctoss-suiations because the myofilaments do not achieve the same degree oforder in their arrangement. The contractile proteins actin and myosin are ubiquitous in all cells. But only in muscle cells are they present in such large amounts and organized in such highly ordered arrays that their contractile activity c:an produce movement in an organ or organism.

• NERVETISSUE Narve1issua consists of nerve cells (neurons) and associated suppor1ing cells of several1ypes. Although all c:ells exhibit electrical properties, nerve cells or neurons are highly specialized to uansmit electrical impulses from one site in the body to another; they are also specialized to integrate those impulses. Nerve c:ells receive and process information from the external and internal environment and may have specific sensory receptors and sensory organs to accomplish this function. Neurons are chatac:tcrized by two different types of pro· c:esses through which they interact with other nerve cells and with cells of the epithelia and muscle. A single, long axon (sometimes longer than a meter) carries impulses away

from the cell body, which contains the neuron's nucleus. Multiple dendrites receive impulses and carry them toward the cell body. (In histologic: sections, it is usually impossi· ble to differentiate axons and dendrites because they have the same structural appearance.) The axon terminates at a neuronal junction called a synapse at which electrical im· pulses are transferred from one cell to the next by secretion ofneuromediators. These chemical substances are released at synapses to generate ele r

~

5 z {/) z ()

m r

lj""

9 ()

m

I

r

)>

0

:I:

m

{/)

0 z

134

z

0

(/)

w

:::c

~ ::1 w

y

~w

0

z z

(/)

0

however, the dot-like component is seen as a dense bar or line between the apposing cells (Fig. 5.13). The bars, in fact, form a polygonal structUre (or band) that encircles each cell to bind them together. Arrangement of this band can be compared to the plastic rings that hold together a six-pack of canned beverages. Because of its location in the tenninal or apical portion of the cell and its bar-like configuration, the stainable material visible in light microscopy was called the terminal bar. It is now evident that intercellular cement as such does not exist. The terminal bar, however, does represent a significant strUCtUral complex. Electron microscopy has shown that it includes a specialized site that joins epithelial cells (Fig. 5.14a). It is also the site of a considerable barrier to the passage (diffusion) of substances between adjacent epithelial cells. The specific structUral components that make up the barrier and the attachment device are readily identified with the EM and are collectively referred to as a junctional complex (see Table 5.5, page 146). These complexes are responsible

RGURE 5.13. Tennlnal ban. In paeudoltnrtlfted epithelium. Photomicrograph of a hematoxylin and eosin (H&E)-stained specimen showing the terminal bars in a pseudostratified epithelium. The bar appears as a dot {arrowheads) when seen on its cut edge. When the bar courses parallel to the cut surface and lies within the thickness of the section, it is seen as a linear or bar-like profile (arrows). X550.

~ ~

0

w

a..

(/)

~

APICAL DOMAIN

0

~

z

~

0

0

~

w

~

w

:::c ~

•

1&1

I...

a ~

1ft

i

maculae adherantes (desmosomes)

FIGURE 5.14. Junctional complex. a. Electron micrograph of the apioal portion of two adjoining epithelial cells of the gastric mucosa. showing the junctional complex. It consists of the zonula occludens (ZO), zonula adherens (ZA), and macula adherens {MA). x30,000. b. Diagram showing the distribution of cell junctions in the three cellular domains of columnar epithelial cells. The apical domain with microvilli has been lifted to better illustrate the spatial arrangements of junctional complexes within the cell.

for joining individual cells together. There are three types of junctional complexes (Fig. 5.14b):

• Communicating junctions allow direct communication between adjacent cells by d.iffiJsion of small (< 1.2 kDa) molecules (e.g., ions, amino acids, sugars, nucleotides, second messengers, metabolites). This type of intercellular communication permits the coordinated cellular activity that is important for maintaining organ homeostasis.

• Occluding junctions, also called tight junctions, are essential for establishing a barrier between different com· partments ofthe: body and allow epithelial cells to function as a barrier. Occluding junctions form the: primary paracellular diffusion barrier between adjacent cdls. By limiting the movement of ions, water, and other macromolecules through the intercellular space, they main· tain physicochemical separation of tissue compartments. Because they are located at the most apical point between adjoining epithelial cells, occluding junctions act as fences to prevent the migration of lipids and specialized mem· brane proteins between the apical and lateral surfuces, thus maintaining cell polarity and integrity of these two domains. In addition, occluding junctions recruit various signaling molecules to the cell surfuce and link them to the actin filaments of the cell cytoskeleton. • Anchoring junctions provide mechanical stability to ep-ithelial cells by linking the cytoskeleton of one cell to the cytoskeleton of an adjacent cell. These junctions are im· portant in cn:ating and maintaining the strucrural unity of the epithelium. Anchoring junctions interact with both actin and intennediate filaments and can be found not only on the lateral cell surface but also on the basal domain of the: epithelial cell. Through their signal transduction ca· pabillty. anchoring junctions also play important roles in cell-to-cell recognition, morphogenesis, and diffi:rentiation.

Occluding Junctions The zonula occludens (pl., unultu occhulmtes) represents the most apical component in the junctional complex between epithelial cells.

The zonula occludens is created by localized sealing of the plasma membrane between two adjacent cells. Examination of the zonula occludens or tight )unction with the transmission electron microscope (TEM) reveals a narrow region in which the plasma membranes of two adjoining cells come in close contact to seal off the intercellular space (Fig. 5.15a). At high resolution, the zonula ocdudens appears not only as a continuous seal but also as a series of focal fusions between the cells. These focal fusions are created by transmembrane proteins of adjoining cells that join in the intercellular space (Fig. 5.15b). The arrangement of these proteins in forming the seal is best visualized by the freeze fracture technique (Fig. 5.15c). When the plasma membrane is fractured at the site of the zonula occludens, the junctional proteins are observed on the P-face of the membrane, where they appear as ridge-like structures. The opposing surface of the fractured membrane, the E.-face, reveals complementary

135

n

~

:II

!-!'

.,m ;! m

~

:::!

c = m

•~ m

~

m

~

r

8 ~

z )> z 0

adjacent cell membranes

~ U> "tJ

m

apical surtaca

_ /.................

0

)>

_

r

/

~

0

z z

zo---

U>

ZA --f+~O:.l"'l

0 m r

"d0 m r r

)>

b

0 ::I:

m

~

0

z

FIGURE 5.15. Junctional complex. a. This diagram shows the location of anchoring cell·tcx:ell junctions in the epithelial cell. The junctional complex near the apical (luminal) surface comprises zonula occludens (ZO). zonula adherens IZA). and macula adherens (MAJ. also called desmosome. Below the MA. note the communicating junctions. Also, cell-to-extracellular matrix junctions (hemidesmosomes and focal adhesions! are visible on the basal cell membrane. b. Diagram showing the organization and pattern of distribution of the transmembrane proteins within the oocludin junction. Compare the linear pattern of grooves with the ridges detected in the freeze fracture preparation on the right side. c. Freeze-fracture preparation of zonula occludens shown here reveals an anastomosing network of ridges {arrows} located on the fractured membrane surface near the apical part of the cell !note presence of microvilli at the cell surface). This is the P-face of the membrane. {TheE-face of the fractured membrane would show a complementary pattern of grooves.! The ridges represent linear arrays of transmembrane proteins (most likely oocludins) involved in the formation of the zonula occludens. The membrane of the opposing cell contains a similar network of proteins. which is in register with the first cell. The actual sites of protein interaction between the calls form the anastomosing network. x100,000. (Reprinted with permission from Hull BE, Staehelin LA. Functional significance of the variations in the geometrical organization of tight junction networks. J Cell Biol1976;68:~704.)

136

z

0

(/)

w

:::c

~ ::1 w

y

~w

0

z z

(/)

0

~ ~

u

w a..

(/)

~ 0

~

z

~

0

c

~

grooves resulting from detachment of the protein particles from the opposing swface. The ridges and grooves are arranged as a honeycomb-like network of anastomosing in· tramambranous particle strands, creating a belt of junctions that surrounds each cell and seals the whole intercellular space witllln the epithelial sheets. The number of strands as well as the degree of anastomosis vary in different cells. The zonula occludens undergoes modification in areas where the comers of three epithelial cells meet together. The zonula occludens has been classically described as a bicellular contact structure because it only seals the space between two adjacent cdls. However, because most apical domains of epithelial ceUs are polygonal in shape, only the sides of these cells form a zonula ocdudens with this classically bicellular contact with. neighboring ceUs. Their vertices form a modified tricellular zonula occludens junction at a tri· cellular contact, where the corners of three epithelial ceUs meet together (Fig. 5.16). k the zonula occludens approaches a tricellular contact region, apical extensions of the intramembranous particle strands that have run horizontally tum vertically to run along the lateral domain at the comer of each epithelial cell, thus forming a pair of vertical strands. These two vertical strands are rererred as central sealing elements (see Fig 5.16) and contain unique proteins, different than those found in bilateral contacts. These dements are more effective in maintaining the epithelial barrier in the specific areas of tricdlular contacts. The comers of the plasma membranes from all three ceUs never completely seal the extracellular space at their meeting points. Their comers containing central sealing dements border a long and narrow space called the central tube, which is an integral part of the e:xtta.cellular space. Because the

central tube represents a weak point for the epithelial barrier, unique proteins (i.e., tricellulin) are required to seal this area and maintain the epithelial permeability barrier.

Several proteins are involved in the formation of zonula occludens strands. Zonula occludans strands correspond to the location of the rows of transmembrane proteins. Four major groups of transmembrane proteins are found in the zonula occludens (Fig. 5.17; Table 5.4):

• Claudins constitute a family of proteins (20-27 kDa) that form the backbone of tight junctions and are integral components ofzonula ocdudens strands. In addition, claudins (especiallyclaudin-2 and claudin-16) are able to form extracellular aqueous channels for the paracellular passage ofions and other small molecules. About 24 different members of the claudin fumily have been characterized to date. Mutations in the gene encoding claudin-14 have been linked to human hereditary deafness. A mutated form of claudin-14 causes an increased permeability of zonula occludens in the organ of Corti (receptor of hearing), affecting generation of action potentials. • Occludln, a 60 kDa protein, participates in forming and maintaining the barrier between adjacent cells and acts as a fence to resuict movement of lipids and proteins between the apical and lateral domains. Occludin is present in most occluding junctions. However, several types ofepithelial ceUs do not have occludin within their strands but still possess well-developed and fully functional zonulae occlud.entes. Multiple viruses exploit tight junctions to invade cells and tissues by binding to zonula occludens

w

~

e ocdudin e claudin e tricellulin

w :::c ~

•

1&1

I... a ~

1ft

I

d bicellular junction

b

horizontal strands of zonula occludens (bicellular contact)

FIGURE &.18. lHcellular zonula ocdudena )unction•. a. This image of the apical surface of mesothelial cells impregnated with silver salts clearly shows the polygonal shape of epithelial cells. Their boundaries are delineated by black: lines marked by the precipitated silver. Note that in addition to regions where the oells are in close apposition to one another, there are also regions where three cells come together to form tricellular contacts. X700. b. This schematic drawing shows the polygonal shape of epithelial cells and areas of tricellular and bicellular contacts. c. The horizontal strands of zonula occludens (formed by occludins and claudinsl seal only the space between two adjacent cells. In corners where three epithelial cells meet, the zonula occludens is modified to form a tricellular junction. The vertical strands of the zonula occludens approaching this tricellular contact tum vertically to run along the corner of each epithelial cell. A pair of these vertical strands is composed of unique proteins that include tricellulin. d. In this cross section of a tricellular junction, note the central sealing elements formed by vertical strands of tricellulins that border a narrow space between all three cells. This space, called the central tube, represents a potential place for intercellular (paracellular) passage of water and solutes.

137

n

~

:II

!-!'

.,m ;! m

~

:::!

c = m

•~ m

~

m

~

TABLE5.4

Major Proteins Localized within the Zonula Occludens Junction

r

8 ~

Zonula Occludens Protein

Associated Protein Partners

Function

ClaudIn

Claudin, Z0-1, JAM

Forms backbone of ZO strands; forms and regulates aqueous channels used for parecelluler diffusion

Occludln

Occludin, Z0-1, Z0-2, Z0-3, Vep33, actin

Is present in most occluding junctions; maintains barrier between apical and lateral cell surface

JAM

JAM, Z0-1, claudin

Present in occluding junctions in endothelial cells; mediates interactions between endothelial cells and monocyte adhesions

1HceUulln

Tricallulin, angulins. claudin. occludin

Present in specific areas of ZO at tricellular contacts

z )> z 0

vJ U> \1

m 0

)>

r

~

0

z z

Z0-1

Z0-2. Z0-3, oocludin. claudin. JAM, cingulin. actin. ZONAB, ASIP, AF-6. afadin. a-

Z0-2

Z0-1. occludin. cingulin, 4.1 R

Required in the epidermal growth factor-receptor signaling mechanism

0 m

Z0·3

Z0-1. occludin. actin

Interacts with Z0-1, occludin, and actin filaments of cell cytoskeleton

AF-6

RAS. Z0-1

Small protein involved in molecular transport system and signal transduction

d

Clngulln

Z0-1. Z0-2, Z0-3, cingulin, myosin II

Acidic, heat-stable protein that cross-links actin filaments into sedimentabla complexes

m r r

Symplekin

CPS~100

Dual-location protein: localized in ZO and in the interchromatin particles of the karyoplasm

0 ::I:

Controls relocation of asymmetrically distributed proteins

0

ASIP/Par3

PKC-t

Rab3b

GTPase

Rab13

8-PDE

Rab8

G/C kinase. Sec4

Sec4

RabB

GTPasa required for polarized delivery of cargo vesicles to plasma membrana

SeeS

SeeS

Participates in fusion of Golgi vesicle with the plasma membrane

Sec8

Sees

Inhibits basolateral translocation of LDLP receptors after formation of ZO

Members of the RAS oncogene family of proteins; control the assembly of protein complexes for docking of transport vesicles

AF. anti secretory factor; ASIP. agouti signaling protein; CPSF, cleavage and polyadenylation specificity factor; G/C. genninal center; GTPase, guanosine triphosphatase; JAM, junctional adh&Sion molecule; LDLP, low-density lipoprotein; POE. phosphodi&Sterases; PKC, protein kinase C; RAS. rat sarcoma; ZO. zonula occluclens; ZONAB. zonula occluden&-1-essoc:iatecl nucleic acicl binding.

r li"'

0

)>

m

~

z

138

z

0

(/)

w

:::c

~ ::1 w

y

~w

0

z z

(/)

0

~ ~

0

w a..

(/)

~ 0

~

z

~

0

proteins (e.g., hepatitis C virus, adenovirus). For example, a viral envelope protein of hepatitis C virus binds to occludin to disrupt the integrity of the zonula occludens, allowing the virus to invade the cell. • Junctional adhesion molecule (JAM) is a 40 kDa protein that belongs to the immunoglobulin superfamily (IgSF). JAM does not iuelfform a zonula occludens strand but is instead associated with claudins. It is responsible for increasing the dectrical resistance of the cell membrane, thereby reducing paracellular permeability. JAM is involved in the formation ofoccluding junctions in endothelial cells as wdl as between endothelial cells and monocytes migrating fiom the vascular space to the connective tissue. • Tricellulin, a 64 kDa protein, is localized in specific areas of the zonula occludens at tricellular conta.ca. Tricdlulin is a component ofthe junction and is recruited to this junction by the angulin family of proteins (angulin-1, angulin-2, and angulin-3), whose members are also expressed. in the comers wbm: three epithdial cdls meet. Tricellulin plays a critical role in maintaining the epitbdial barrkr and organizing actin filam.ents in aicdlular contacts, forming aucial points that support the tensile fon:es generated by the actin cytoskeleton.

claudins, and tricellulins interact with the actin cytoskeleton through Z0-1 and Z0-3. The Z0-1 protein binds the zonula a.dherens junction proteins af.uiin and a.-catenin, thus providing an important llnk between the zonula occludens and zonula a.dherens junctions. Regulatory functions during the formation of the zonula occludens have been suggested for all ZO proteins. In addition, Z0-1 is a twnor suppressor, and Z0-2 is required in the epidermal growth factorreceptor signaling mechanism. The Z0-3 protein interacts with Z0-1 and the cytoplasmic domain of occludins. The proteins localized in the region of the zonula occludens are summarized in Table 5.4. Many pathogenic agents, such as cytomegalovirus, dengue virus, and cholera toxins, act on Z0-1 and Z0-2, causing the junction to become permeable.

The extracellular portions ofthese transmembrane proteins function as a zipper and seal the intercellular space between adjacent cells, thus creating a battier against paracellular diffusion. The cytoplasmic portions of all four proteins contain a unique amino acid sequence that attracts regulatory adaptor and signaling proteins called PDZ-domain proteins. These proteins include the zonula occludens proteins Z0-1 , Z0-2, and Z0-3 (see Fig. 5.17). Occludin,

• The transcellular pathway occurs across the plasma membrane ofthe epithdial ceU. In most of these pathways, transport is active and requires specialized energy-dependent membrane transport proteins and channels. These proteins and c.hannels move selected substances across the apical plasma membrane into the cytoplasm and then across the lm:ral membrane below the levd of the occluding junction into the intercellular compartment.

The zonula occludens separates the luminal space from the intercellular space and connective tissue compartment It is now evident that the zonula occludens plays an essential role in the selective passage ofsubstances from one side of an epithdiwn to the other. The ability of epithelia to create a diBUsion barrier is controlled by two disrinct pathways for transport ofsubstances across the epithelia (Fig. 5.18a):

0

~

w

~

w :::c ~

•

zonula oocludens

paracellular pathway

1&1

I... a

PDZdomains of associated

Intracellular

attachment proteins

~

1ft

i

r----l-zonula oocludens

strands lateral domain basal domain

a

b

paracellular pathway FIGURE 5.18. Two traRSCellular and paracellular pathways for 1ransport of substances acron the epithelia. a. The transcallular path· way occurs across the plasma membrane of the epithelial cell and represents an active transport system that requires specialized energydependent membrane transport proteins and channels. The paracellular pathway occurs across the zonula occludens between two epithelial cells. The amount of water. electrolytes. and other small molecules transported through this pathway is contingent on the tightness of the zonula ocx:ludens. b. Structure of the extracellular and cytoplasmic portions of tight junction strands. Two zonula occludens strands from neighboring cells fuse in a zipper-like fashion and create a barrier to movement between the cells. Aqueous pores allow water to move between the cells. The permeability of the barrier depends on the mixture of claudins and occludins in the zipper seal. The cytoplasmic portion of the strand attracts PDZ-domain proteins that function in cell signaling.

• The paracellular pathway occws across the zonula occludens between two epithelial cells. The amount of water, electrolytes, and other small molecules transponed through this pathway is contingent on the tightness of the zonula occludens and tricellular zonula occludens junctions. The permeability of an occluding junction depends on the molecular composition of the zonula occludens strands and thus the number of active aqueous channels in the seal (see the following section). Under physiologic conditions, substances transponed through this pathway may be regulated or coupled to transcellular transpon.

Penneabi lity of the zonula occludans depends not only on the complexity and number of strands but also on characteristics of proteins involved in their formation. Microscopic observations ofdifferent kinds of epithelia reveal that the complexity and number ofstrands forming the zonulae ocdudentes varies. In epithelia in which anastomosing strands or fusion sites are sparse, such as certain kidney tubules, the intercellular pathway is panially permeable to water and solutes. In contrast, in epithelia in which the strands are numerous and extensively intertwined, such as intestinal and urinary bladder epithelia, the intercellular region is highly impermeable. However, in some epithelial cells, the number of strands does not din:c:tly correlate with the tightness of the seal. Differences in tightness between different zonulae oc:cludentes could be explained by the presence ofaqueous pores within individual zonula occludens strands (Fig. 5.18b). Claudine not only form the backbone of the individual zonula occludens strand but also are responsible for the formation ofextracellular aqueous channels. For instance, claudin-16 func-tions as an aqueous Mg'+ channel between specific kidney epithelial ceUs. Similarly, claudin-2 is responsible for the pres-ence of high-conductance aqueous pores in outer kidney epithelia. Therefore, the combinations and ratios of claudins to occludins and other proteins found within individual paired zonula occludcns strands determine tightness and selectivity of the seal between ceUs. Tricellular zonula occludens junctions are uniquely placed permeability barriers formed at the corners of epithelial cells. & discussed earlier, the central tube at these junctions represents a weak point for the epithelial barrier and a potential conduit for parac:eUular passage of water and solutes. Tightness of these junctions is regulated by tricellulin and other structural proteins found in central sealing elements (see page 136). Recent experimental studies involving epithelia that do not express tricellulin molecules (tricellulin-knock.down cells) found decreased transepithelial electrical resistance and increased penneabillty of water, solutes, and other macromolecules in these triceUulin-free cells.

The zonula occludans establishes functional domains in the plasma membrana. & a junction, the zonula occludens controls not only the passage of substances across the epithelial layer but also restricts the movement of lipid rafts containing specific proteins within the plasma membrane itself. The cell sep gates certain internal membrane proteins on the apical (free)

surfuce and restricts others to the lateral or basal surfaces. In the intestine, for instance, the enzymes for terminal digestion of peptides and saccharides (dipeptidase& and disaccharidases) are localized in the membrane of the microvilli of the apical surface. The Na+fK+ -ATPase that drives salt and transcellular water transpon, as well as amino acid and sugar transpon, is restricted to the lateral plasma membrane below the zonula occludens.

139

n

~

Anchoring Junctions

:II

Anchoring junctions provide: lateral adhesions between epithelial cells using proteins that link into the cytoskeleton ofadjacent ceUs. Two types ofanchoring cell-to-cell junctions can be identified on the lateral cell surface:

.,m

• Zonula adherens (pl., Ztmuku adhmntes), which inter•

acts with the network of actin filaments inside the cell Macula adherene (pl., macul4e adhmntes) or desmosome, which interacts with intermediate filaments In addition, two other types of anchoring junctions

can be found where epithelial cells rest on the connective

focal adhesions (focal contacts) and hemide1mosomes are discussed in the section on the basal

tissue matrix. These

!-!'

;! m

~

:::!

c = m

•~ m

~

domain (see pages 147-157).

m

Cell adhesion molecules play important roles in cell-tocall and cell-to-extracellular matrix adhesions. Transmembrane proteins known as cell adhesion molecules (CAMs) form an essential pan of every anchor-

r

ing junction on both lateral and basal cell surfaces. The extracellular domains of CAMs interact with similar domains belonging to CAMs of neighboring cells. If the binding occurs between difFerent types of CAMs, it is described as hetervtypic binding; homotypic binding occurs between CAMs of the same type (Fig. 5.19). CAMs have a selective adhesiveness of relatively low strength, which allows cells to easily join and dissociate. The cytoplasmic domains are linked through a variety of intracellular proteins to components of the cell cytoskeleton. Through the cytoskeleton connection, CAMs are able to control and regulate diverse intracellular processes associated with cell adhesion, cell proliferation, and cell migration. In addition, CAMs are implicated in many other cellular functions, such as intercellular and intracellular communications, cell recognition, regulation of intercellular diffusion barrier, generation of immune responses, and apoptosis. From early embryonic development, every type of tissue at every stage of differentiation is defined by the expression of specific CAMs. Changes in the expression pattern of one or several CAMs may lead to pathologic changes during tissue differentiation or maturation. To date, about 60 CAMs have been identified, and they are classified on the bases of their molecular structure into five major families: cadherins, nec:tins, integrins, selectins, and the immunoglobulin superfamily (see Fig. 5.19).

• Cadherins are represented by the family of transmem~ brane Ca2+-dependent CAMs localized mainly within the zonula adherens. At these sites, cadherins maintain homotypic interactions with similar proteins from the neighboring cell. They are associated with a group

~

8 ~

z )> z 0

~ U> \1

m 0

)>

r

~

0

z z

U>

0 m r

II

d 0

m r r )>

0 ::I:

m

~

0

z

140

z

0

HOMOPHIUC INTERACTIONS

(/)

w

:::c

~ ::1 w y

~w

0

z z

(/)

0

~ ~

0

w

......-.-++-- lg-superfamily (lgSF)

selectin

receptors (mucinlike CAMs)

HETEROPHIUC INTERACTIONS

selectlns

j

integrins

fibronectin

a..

(/)

~ 0

~

z

~

0

0

~

w

~

w

:::c ~

•

1&1

I... a ~

1ft

i

Extracellular space FIGURE 5.19. Cell adh..ion molec:uln (CAMs). Cadherin and immunoglobulin superfamily {lgSF) CAMs exhibit homotypic binding in which two identical molecules from the neighboring cells interact. Binding that occurs between different types of CAMs {e.g., selectins and integrins) is considered heterotypic binding (no identical pair of molecules reacts with each other). Note that integrins bind to the extracellular matrix proteins (e.g., fibronectin). For simplicity of this drawing, the associated intracellular attachment proteins are not shown.

of intracellular proteins (catenins) that link cadhedn molecules to actin filaments of the cell cytoskeleton. Through this interaction, cadherins convey signals that regulate mechanisms of growth and cell differentiation. Cadherins control cell-to-cell interactions and partici· pate in cell recognition and embryonic cell migration. Epithelial, or E·cadharin, the most studied member of this family, maintains the zonula adherens junction between epithelial cells. It also acts as an important suppressor of epithelial tumor cells.

• Nactins are represented by a f.unily of transmembrane Ca1+·independent immunoglobulin-like CAMs. In contrast with cadherins, necrins are able to establish both hemophilic interactions with the same family member and heterophilic interactions with related nectin f.unily members. They are also unable to establish strong adhering junctions. These types ofweaker junctions might he favorable to the dynamic regulation of cell-to-cell adhesion during embryologic development, rapid tissue remodeling, and regeneration. • lntagrins are represented by two transmembrane glycoprotein subunits consisting of 15 a and 9 p chains. This composition allows for the formation of different combinations of integrin molecules that are able to interact with various proteins (heterotypic inter~ actions). lntegrins interact with extracellular matrix molecules (such as collagens, laminin, and fibronectin)

and with actin and intennadiate filaments of the cell cytoskeleton. Through these interactions, integri.ns regulate cell adhesion, control celt movement and shape, and participate in cell growth and differentiation. • Salactins are expressed on white blood cells (leukocytes) and endothelial cells and mediate neutrophilendothelial call recognition. This heterotypic binding initiates neutrophil migration through the endothelium of blood vessels into the extracellular matrix. Sdectins are also involved in directing lymphocytes into accumulations of lymphatic tissue (a process called lymphocyte homing). • Immunoglobulin superfamily {lgSF). Many molecules involved in immune reactions share a common precursor element in their structure. However, several other molecules with no known immunologic function also share this same repeat element. Together, the genes encoding these related molecules have been defined as the immunoglobulin gena superfamily. It is one of the largest gene families in the human genome, and its glycoproteins perfonn a wide variety of important biologic functions. IgSF members mediate homotypic cell-to-cell adhesions and are represented by the intercellular cell adhesion molecule (ICAM), cell-cell adhesion molecule (C~CAM), vascular cell adhesion molecule (VCAM), Down syndrome cell adhesion molecule (DSCAM), platelet endothelial cell adhesion molecules (PECAM), junctional adhesion molecules QAM), and many others. These proteins play key roles in cell adhesion and differentiation, cancer and tumor metastasis, angiogenesis (new vessel formation), inflammation, immune responses, and microbial attachment as well as many other functions.

The zonula adherans provides lateral adhesion between epithelial calls. The integrity of epithelial surfaces depends in large part on the lateral adhesion of the cells with one another and their ability to resist separation. Although the zonula ocxludens involves close contact of adjoining cell membranes, their rep sistance to mechanical stress is limited. Reinforcement of this region depends on a strong bonding site below the zonula ocdudens. Like the zonula occludens, this lateral adhesion device occurs in a continuous bandp or beltplike configuration around the cell; thus, the adhering junction is referred to as a zonula adherans.

Zonula adherens is composed of two families of transmembrane proteins: the cadherinsand the nectins. The zonula adherens in most epithelial cells is composed of the transmembrane family of cell adhesion molecules, the cadharins. On the cytoplasmic side, the tail ofE-cadherin is bound to catanin proteins (a· and p-catanin) (Fig. 5.20a). The resulting E·cadharin-catenin complex binds to vinculin and a·actinin and is required for the interaction ofcadherins with the actin filaments of the cytoskeleton. The extracellular components of the E-cadherin molecules from adjacent cells are linked by Ca2 + ions or an additional extracellular link protein. E-cadherin-catenin complexes form strong adhering junctions; however, their morphologic and functional integrity is calcium..

0 ::I:

m

~

0

z

142

FOLDER5.3

CLINICAL CORRELATION: JUNCTIONAL COMPLEXES AS A TARGET OF PATHOGENIC AGENTS

z

0

Cl.l

UJ

:I:

0

0 ::I:

m

~

0

z

144

membranes of adjacent cells

z

0

(/)

w

:::c

~ ::1 w

y

~w

0

z z

(/)

0

~ ~

u w

a..

(/)

~

desmocollin and dasmoglain

b

FIGURE 5.22. Molecular structure of the macula adherens (Desmosome). e. Electron micrograph of a macula adherens. showing the intermediate filaments {a"ows} attaching to a dense. intracellular attachment plaque located on the cytoplasmic side of the plasma membrane. The intercellular space is also occupied by electron-dense material {arrowheads} containing desmocollins and desmogleins. The intercellular space above and below the macula adherens is not well defined because of extraction of the plasma membrane to show components of this structure. X40,000. {Courtesy of Dr. Emst kallenbach.J b. Schematic diagram showing the structure of a macula adherens. Note the intracellular attachment plaque with anchored intermediate filaments. The extracellular portions of desmocollins and desmogleins from opposing cells interact with each other in the localized area of the desmosome, forming the cadherin zipper.N N

0

~

z

~ 0 0

~

w

~

w

:::c ~

•

1&1

I... a ~

1ft

i

Communicating Junctions Communicating junctions, also called gap junctions or nexuses, are the only known cellular strucrures that pennit the direct passage of signaling molecules fi:om one cell to another. They are present in a wide variety of tissues, including epithelia, smooth and cardiac muscle, and nerves. Gap junctions are important in tissues in which activity ofad· jacent cells must be coordinated, such as epithelia engaged in fluid and electrolyte ttansport, vascular and intestinal smooth muscle, and heart muscle. A gap junction consists of an accumulation of transmembrane channels or pores in a tightly packed array. Cells cxcb.ange ions, regulatory molccul.cs, and small metabolites through these pores. The number of pores in a gap junction can vary widely. as can the number of gap junctions between adjacent cells. A variety of methods are used to study structure and function of gap junctions. Various procedures have been used to study gap junctions, including the injection of dyes and fluorescent or radiolabeled compounds and the measurement of electric current flow between cells. In dye studies, a fluorescent dye is injected with a micropipette into one cell. After a short period, the dye can be readily visualized in immediately adjacent cells. Electrical conduc:tance studies show that neighboring cells joined by gap junctions exhibit a low electrical resistance between them when curn:nt flow is high; then:fore, gap junctions are also called low-resistance junctions. Cum:nt molecular biology techniques allow for isolation of eDNA c:lones encoding a &mily of gap junction proteins (connexins) and expressing them in tissue culture c:ells. Connexins expressed in transfected cells produce gap junctions, which can be isolated and studied by molecular and

biochemical methods as well as by the improved imaging techniques ofelectron crystallography and atomic force microscopy. Gap junctions are fonnad by 12 subunits of the connexin protein family. When viewed with the TEM, the gap junction appears as an area of contact between the plasma membranes of adjacent cells (Fig. 5.23a). High-resolution imaging techniques such as cryoelcctron microscopy have been used to examine the struaun: of gap junctions. These stUdies reveal groups of tightly packed channels, each formed by two half-channels called connexons embedded in the facing membranes. These channels are represented by pairs of connexons that bridge the extracellular space between adjacent cells. As the name implies, the connexon in one cell membrane is precisely aligned to dock with a corresponding connexon on the membrane of an adjacent cell, allowing communication between the cells. Each connexon contains six symmetrical subunits of an integral membrane protein called connexin (Cx) that is paired with a similar structure from the adjacent membrane; thus, each channel consists of 12 subunits. The subunits are configured in a circular arrangement to surround a 10-nmlong cylindrical transmembrane channel with a diameter of 2.8 nm (Fig. 5.23b). Cunently, 21 members of the connexin family of proteins have been identified. All traverse the lipid bilayer four times (i.e., they have four transmembrane domains). Most connexons pair with identical connexons (homotypic interaction) on the adjacent plasma membrane. These channels allow moleades to pass evenly in both directions; however, heterotypic channels can be asymmetrical in function, passing certain moleades &stcr in one direction than in another.

145

n

~

cell membrane I

:II

!-!'

.,m ;! m

oonnexons

~

:::!

c = m

I

extracellular space

•~ m

b

c

RGURE 5.23. Structu,. of a g1p junction. a. Electron mic:rogn~ph showing the plasma membranes of two adjoining cells forming a gap junction. The unit membranes (a/TOWS) approach one another, narrowing the intercellular space to produce a 2-nm-wide gap. x76,000. b. Drawing of a gap junction showing the membranes of adjoining cells and the structural components of the membrane that form channels or passageways between the two cells. Each passageway is formed by a circular array of six subunits, dumbbelkhaped transmembrane proteins that span the plasma membrane of each cell. These complexes, called connexons, have a central opening of about 2 nm in diameter. The channels formed bv 1he registration of the adjacent complementary pairs of connexons permit the flow of small molecules through the channel but not into 1he intercellular space. Conversely, substances in the intercellular space can permeate the area of a gap junction by flowing around 1he connexon complexes, but they cannot enter the channels. c. The diameter of the channel in an individual connexon is regulated bv reversible changes in the conformation of the individual connexins.

~

m

~

r

8 ~

z )> z 0

Atomic force microscopy reveals confonnational changas in cannexins1hat causa gap iunction channels 1o opan or close. Earlier electron microscopy studies of isolated gap junctions suggested that the gap junction channels open and dose by twisting of the connexin subunits (Fig. 5.23c). Recent atomic force microscopy (AFM) studies provide a dynamic view of the conformational changes that take place in connexons. Channels in gap junctions fluctuate rapidly between an open

and a closed state through reversible changes in the confor~ mation of individual connexins. The conformational change in connexin molecules that triggers closure of gap junction channels at their extracellular surface appears to be induced by Cal+ ions (Fig. 5.24). However. other calcium-independent gating mechanisms responsible for dosing and opening of the cytoplasmic:: domains of gap junction channels have also been identified.

vJ U> \1

m 0

)>

r

~

0

z z

U>

0 m r

li"'

d 0

m r r )>

0 ::I:

m

~

0

z

FIGURE &.24. Atomic fon:e microscopic [AFM) image of a gap Junction. These images show the extracellular surface of a plasma membrane preparation from the HeLa cell line. Multiple copies of the connexin-26 gene were incorporated into the HeLa cell genome to achieve overexpression of the connexin protein. Connexin-26 proteins self-essemble into functional gap junctions, and they were observed with AFM in two different buffer solutions. a. Gap junction containing individual connexons in a calcium·free buffer solution. X500,000. Inset shows a single connexon at higher magnification. Note the clear profiles of individual connexin molecules assembled into the connexon. The open profile of the channel is also visible. X2,000,000. b. The same preparation of connexons in a buffer containing Ca2 +. X500,000.1naet. Note that the conformational change of the connexin molecules has caused 1he channel to close and has reduced the height of 1he connexon. X2,000,000. {Courtesy of Dr. Gina E. Sosinsk:y.)

146

TABLE5.5

Summary of Junctional Features

z

Classification

en UJ

Zonula occludana (tight Junction)

0

:I:

~

...J ...J UJ

u

Major Link Proteins

AHociated Intracellular Extracellular Cytoskeleton Attachment Functions Ligands Components Proteins Z0-1, Z0-2, Z0-3,

Occludins. claudins. JAMs. tricellulin (in tricellular junctions)

Occludins. claudins. JAMs. tricellulin in adjacent cell

Actin filaments

E- ~

m

:II

!-!' Simple coiled tubular

Skin: Eccrine sweat gland

Coiled tubular structure is composed of the secretory portion located deep in the dermis

..,m =I

:z: m

i,..

:::!

fl

'a

c (3

•

Simple branched tubular

Stomach: Mucus-secreting glands of the pylorus Uterus: Endometrial glands

tt

'ii.

E

Branched tubular glands with wide secretory portion are formed by the secretory cells and produce a viscous mucous secretion

(i

.; c ij

Simple acinar

Urethra: Paraurethral and periurethral glands

Simple acinar glands develop as an outpouching of the transitional epithelium and are formed by a single layer of secretory cells

Branched acinar

Stomach: Mucus-secreting glands of cardia Skin: Sebaceous glands

Branched acinar glands with secretory portions are formed by mucus-secreting cells; the short, single-duct portion opens directly into the lumen

Compound tubular

Duodenum: Submucosal glands of Brunner

Compound tubular glands with coiled secretory portions are located deep in the submucosa of the duodenum

Compound acinar

Pancreas: Exocrine portion

Compound acinar glands with alveolar-shaped secretory units are formed by pyrami~shaped seroussecreting cells

Compound tubuloaclnar

Neck region and oral cavity: Submandibular salivary gland

Compound tubuloacinar glands can have both mucous branched tubular and serous branched acinar secretory units; they have serous en~aps (demilunes)

~.·

•

'a

c

::II

8.

E

8

c = m

• C')

z~ a (I)

160

....J

~

w

zw

CI: ....J ....J

w

0

....J

~

S'~AMIIIIAS' mRNA

rER

· ~naJ) ~ \Y sequence

---

1-

w

>

t>w z z

8

extracellular matrix

•

1&1

I 1&1

~z z

8

•

I Intracellular events 1. Formation of mRNA In the nucleus 2. Initiation of synthesis of prCHX chains with signal sequences by ribosomes 3. Synthetlis of pro-a chains on the rEA 4. Hydroxylallon of proline and lysine residues (vitamin C requirtld) and cleavage of signal sequence from pro-a chain 5. Glycosylation of apecific hydroxylysyl residues In the rEA 6. Formation of procollagen triple helix molecules from a C terminus toward the N tenninus in a zipper-like manner

extracellular events 7. S1ablllzatlon of Ule triple helix by formation of intra- and interc:hain hydrogen and disulfide bounds and chaperone proteins (e.g., hep-47) 8. Transport of procollagen molec:Qies to Golgi apparab.ts 9. Packaging of procollagen molecules by Golgllnto aecrell:lry veslclee 10. Mcwarnent of vesicles to plasma membrane, &881sted by molecular molor pro1eins associated with miCI'Ot:ubules

11. Exocytoals of procollagen molecules 12. Cleavag& o1 trirtlllfic globular C- and helleal N1Jrocollagen domains by procoii&IJ8fl N- and C1Jroteinases 13. Polymerization (self-essembly) of collagen molecules into ccllagen fibrils (In cove of fibroblast) with development of covalent C1'088-llnklng 14. Incorporation of other ccllagens (e.g., type V. FACI'Tll, e1c.) Into ccllagen fibrils

FIGURE 8.8. Collagtn biasynth-.is. Schematic representation of the biosynthetic events and organelles participating in collagen synthesis. Bold numbers correspond to the numbered events in collagen biosynthesis listed at the bottom.

Collagen molecule biosynthesis involves a number of intracellular aventJ. The steps in biosynthesis of almost all fibrillar collagens are simUar, but type I collagen has been studied in the most detail. In genetal, the synthetic pathway for collagen molecules is simUar to other constitutive secretory pathways used by the cell. The unique features of collagen biosynthesis are expressed in multiple posttranslational processing steps that are required to prepare the molecule for the extracellular assembly procc:ss. Intracellular procc:ssing events are as follows:

• Collagen a chains are synthesized in the rough endoplasmic reticulum (rER) as long precursors containing

Luge

globular amino- and carboxy-terminus propeptides called pro-a chains (preprocollagen molecules). The newly synthesized polypeptides are simultaneously discharged into the cistttn.ae ofthe rER. where intracdlular processing begins. • Within the cisternae of the rER, a number of posttranslational modifications of the preprocollagen molecules occur, including the following: • The amino-terminus signal sequence is cleaved. • Proline and lysine residues are hydroxylated while the polypeptides are still in the nonhelical conformation. Ascorbic acid (vitamin C) is a required cofactor for the addition of hydroxyl groups to proline and lysine residues in pro-a: chains by the enzymes prolylhydroxylase and lysylhydroxylase; without hydroxylation of proline and lysine residues, the hydrogen bonds essential to the final structure of the collagen molecule cannot form. This explains why wounds fail to heal and bone formation is impaired in scurvy (vitamin C deficiency). • ()..linked sugar groups are added to some hydroxy-

lysine residues (glyrosylation), and N-linkcd sugars are added to the two terminal positions. • The globular structure is formed at the carboxyterminus, which is stabilized by disulfide bonds. Formation of this structure ensures the correct alignment

of the three a chains during the formation of the triple helix. • A triple helix (beginning from the carboxy-terminus) is formed by three a chains, except at the terminals where the polypeptide chains remain uncoiled. • Inuachain and interchain hydrogen and disulfide bonds form that influence the shape of the molecule. • The triple--helix molecule is stabilized by the binding ofthe chaperone heat shock protein 47 {hsp47), which also prevents the prematun: aggregation of the t:rime1's within the celL The resultant molecule is procollagen. • The folded procollagen molecules pass to the Golgi apparatus and begin to associate into small bundles. 1his bundling is achieved by the lateral associations between uncoiled terminals of the procollagen molecules. Free and small aggregates of procollagen molecules are packaged into secretory vesicles and transporred to the cell surface. Fonnatian of collagen fibrils (fibrillagenasis) involves the following extracellular events. • & procollagc:n is~ from the cell, it is converted into a matutt collagen molecule by procollagen peptidascs associated with the cell membrane, which cleave the uncoiled ends of the procollagen (Fig. 6.9). Serum levels of pro· collagen type I N-terminal propeptide (PINP) can be measured and used as indicators for collagen type I metabolism. An elevated level of PINP is indicative of increased production of collagen type I, which is associated with bone metastases in breast and prostate cancer.

• The aggregated collagen molecules then align to form the final collagen fibrils in a process known as fibrillogenesis. The cell controls the orderly array of the newly formed fibrils by directing the secn:tory vesicles to a localized sutfacc: site for disch.arge. The cell simultaneously creates specialized collagen assembly sites called coves. These inwginarions of the cell su.r&ce allow molecules to accumulate and assemble (see F'lg. 6.8). Within the cove, the collagen molecules

procollagen molecule

Gic ; Gal

' Gal

I

-ss

'

s

l~~~·J~ • (Man)n

1 ~GicNac FIGURE 1.8. Cleavage of the prooollagen molecule. Illustration showing the procollagen molecule with Nand C termini. Scissors in the top illustration show where C and N terminals are cleaved by carboxy- and aminopeptidase, respectively, from the procollagen molecule to form the collagen molecule. On the C terminus of 1he molecule, the sugar subunit is GlcNac (N-acetylglucosamine) attached to mannose (Man ln. Globular N-terminal propeptide is smaller and has short triple-helical and nontriple-helical domains, whereas C-terminal propeptide is larger with a single nontriple.flelical domain.

procollagen N-terminal

propeptlde (15X2nm)

collagen molecule (300 X 1.5 nm)

procollagen C-tannlnal propeptide (10 X 10 nm)

179

~

:!1 m

:a ~

n 0

z zm

!:l ~

-t

~

m

• n 0

z

zm

~

< m -t en Cl)

c

m

Tl

OJ

m

::D

en

180

~ w ro

LL.

w

::>

~

1-

w

>

t>w z z

8

•

1&1

I 1&1

~z z

8

•

I

align in rows and self..assemble longitudinally in a head~ to~Wl fashion. They also aggregate laterally in a quarter~ staggered pattern (see Fig. 6.7). The collagen molecules are then cross-linked by covalent bonds that are formed be~ tween the lysine and hydroxylysine aldehyde groups. Collagen biogenesis results in the formation of highly organized polymers called fibrils. The fibrils further associate with each other to form larger collagen fibers, which on a per weight basis have the tensile strength comparable to that of steel. For example, collagen type I fiber of 1 mm in diameter can withstand a load of 10 to 40 kg before it breaks. Collagen fibrils often consist of more than one type of collagen. Usually, different types of fibrillar collagens assemble into fi~ brils composed of more than one type of collagen molecule. For example, type I collagen fibrils often contain small amounts of types II, III, V. and XI. Current studies indicate that assembly oftype I collagen fibrils is preceded by formation of a fibrillar core containing type V and type XI molecules. Subsequently, type I collagen molecules are deposited and polymerized on the surface ofthe fibrillar core (Fig. 6.10). In ad· dition, small amounts oftype II and III collagen molecules are incorporated into type I collagen fibrils. Collagen types V and XI are important regulators offibrillogenesis. 'They control the thickness oftype I fibrils by limiting the deposition ofcollagen molecules after the fibril has reached the desired diameter. Fully mature collagen fibers are usually associated with the FACIT f.unily of collagen molecules that reside on their surfaces. For c::xample, type I fibrils are associated with type XII and type XIV collagens. 'These collagens contribute to the threedimensional organization of fibers within the ECM. Type II collagen fibrils, which are abundant within the cartilage, are usually smaller in diameter than type I fibrils. However, these fibrils are also associated with type IX collagen (another member of the FACIT subgroup). Collagen type IX resides on the sur&c.c of the type II fibril and anchors it to proteoglycans and other components ofthe cartilaginous ECM (Fig. 6.11). Collagen molecules are synthesized by various types of connective1issue and epithelial cells. Collagen molecules are largely synthesized by connective tissue cells. 'These cells include fibroblasts in a variety of tissues

~Vcollagen

type I collagen fibril

N terminus of type V collagen type Ill collagen FIGURE 8.10. Type I collag•n fibril. The type I collagen fibril contains small amounts of other collagen types such as types II, Ill, V. and XI. Note that the core of the fibril contains collagen types V end XI, which help initiate the assembly of the type I fibril.

type IX collagen

RGURE 8.11. Type II collagen fibril. This diagram illustrates the interaction between type II collagen fibrils and type IX collagen molecules in the cartilaginous matrix. Collagen type IX provides the link between the collagen fibrils and GAG molecules, which stabilizes the network of cartilage fibers.

(e.g., chondrocytes in cartilage, osteoblasts in bone, and pericytes in blood vessels). In addition, the collagen molecules of the basement membrane (see page 151) are produced by epithelial cells. The synthesis ofcollagen is regulated by com~ plex interactions among growth factors, hormones, and cyto~ ltines. For example, transforming growth factor J3 (TGF-~) and platelet~derived growth factor (PDGF) stimulate col~ lagen synthesis by fibroblasts, whereas steroid hormones (glucocorricoids) inhibit its synthesis. Collagen fibers art degraded either by proteolytic or phagocytic pathways. All proteins in the body are being continually degraded andre· synthesized. 'These processes allow tissues to grow and remodel. Collagen fibers also undergo constant but slow turnover. The half.Jife ofcollagen molecules varies from days to several years (e.g., in skin and cartilage). Initial fragmentation of insoluble collagen molecules occurs through mechanical wear, the action of free radi.cals, or proteinase cleavage. Further degradation is continued by specific enzymes called protelnases. The resulting collagen fragments are then phagocytosed by cells and degraded by their lysosomal enzymes. Excessive collagen degradation is observed in several diseases (e.g., deg· radation of cartilage collagen in rheumatoid arthritis or bone collagen in osteoporosis). Secreted collagen molecules are degraded mainly by two different pathways: • Proteolytic degradation occurs outside the cells through the activity of enzymes called matrix metalloproteinases (MMPs). 'These enzymes are synthesized and secreted into the ECM by a variety ofcorutective tissue cells (fibroblasts, chondrocytcs, monocytes, neutrophils, and macrophages), some epithelial cells (keratinocytes in the epidermis), and cancer cells. The MMPs include collageneses (which degrade type I, II, In, and X collagens), gelatinases (which degrade most types ofdenatured collagens, laminin, fibronectin, and elastin), stromelysins (which degrade proteoglycans, fibronectin, and denatured collagens), matrilysins (which degrade type IV collagen and proteoglycans), membrane-type MMPs (which are produced by cancer cells and have a potent pericellular

181 The important role of collagens in the body can be illustrated by collagenopathles (collagen diseases), which are caused by a deficit or abnormality in the production of specific collagens. Most collagenopathies are attributed to mutations in genes encoding the u chains in the var=ious collagens. Mutation of collagens produces a wide variety of genetic disorders that range from mild to lethal,

depending on the mutation of the collagen gene and its subsequent effect on the molecular structure of the collagen and its function in the body. In the future, gene therapy could potentially be used either to control deposition of faulty collagen or to reverse the disease process caused by the mutated genes. The following table lists the most common collagenopathies that occur in humans.

n :z:

•~ m

::a ~

The Most Common Collagenopathies in Humans Type of Collagen Disease

z

m

Symptoms

Osteogenesis imperfects

Repeated fractures after minor trauma, brittle bones, abnormal teeth, thin skin, weak tendons, blue sclerae, and progressive hearing loss

II

Kniest dysplasia; achondrogenesis, type 2

Short stature, restricted joint mobility, ocular ctlanges leading to blindness, wide metaphyses, and joint abnormality seen in radiographs

Ill

Hypermobility Ehlers-Danlos syndrome, type 3 (has additional mutation of tenascin X gene); vascular Ehlers-Danlos syndrome, type 4

Type 3: Hypermobility of all joints, dislocations, deformity of finger joints. and early onset of osteoarthritis Type 4: Pale, translucent, thin skin; severe bruising, and early morbidity and mortality (resulting from rupture of vessels and internal organs)

Alpert's syndrome

Hematuria resulting from structural changes in the glomerular basement membrane of the kidney, progressive hearing loss. and ocular lesions

IV

8 z

v

Classical Ehlers-Danlos syndrome, types 1 and 2 (includes additional mutations of type I collagen gene)

Same symptoms as type 3 but with additional skin involvement (fragility, hyperelasticity, delayed wound healing); type 1 manifests with more severe skin abnormalities than type 2.

VII

Kindler's syndrome

Severe blistering and scarring of the skin after minor trauma, resulting from absence of anchoring fibrils

IX

Multiple epiphyseal dysplasia (MED)

Skeletal deformations resulting from impaired endoctlondral ossification and dysplasia (MED), premature degenerative joint disease

X

Sctlmid metaphyseal chondrodysplasia

Skeletal deformations characterized by modifications of the vertebral bodies and chondrodysplasia metaphyses of the long bone

XI

Weissenbacher-Zweymuller syndrome, Stickler's syndrome (includes additional mutations of type II collagen gene)

Similar clinical features to type II collagenopathies in addition to craniofacial and skeletal deformations, severe myopia, retinal detachment, and progressive hearing loss

XVII

Generalized atrophic benign epidermolysis bullose IGABEBI

Blistering skin disease with mechanically induced dermalepidermal separation. epidermolysis bullose resulting from faulty hemidesmosomes, skin atrophy, nail dystrophy, and alopecia

fibrinolytic activity), and macrophage metalloelastases (which degrade elastin, type IV collagen, and laminin). In general, triplerhelical undenatured. forms of collagen molecules are resistant to degradation by MMPs. In contrast, damaged or denatured collagen (gelatin) is degraded by many MMPs, with gelatinases playing the prominent role. MMP activity can be specifically inhibited by tissua Inhibitors of matalloprotalnasas (TIMPs). Because MMPs are secreted by invasive (migrating) cancer cells, researchers are investigating synthetic therapeutic agents that inhibit the activity of MMPs to control the spread of cancer cells.

• Phagocytic degradation occurs intracellularly and involves macrophagcs to remove components of the ECM. Fibroblasts are also capable of phagocytosing and degrading collagen fibrils within the lysosomes of the cell.

Reticular Fibers R1licular fibers provide a supporting framework far the cellular constiblents of various tissues and organs. Reticular fibers and collagen type I fibers share a prominent feature in that they both consist of collagen fibrils. Unlike collagen fibers, however, reticular fibers are composed of type Ill collagen. The individual fibrils that constitute

!l ~

•• en cm

n 0

z zm

~

< m :::!

(/') (/')

c

m :!! [JJ m ::0

(/')

w

type I collagen fibers. Reticular fibers also function as a supporting stroma in hemopoietic and lymphatic tissues (but not in the thymus). In these tissues, a special cell type, the reticular cell, produces the collagen of the reticular fiber. This cell maintains a unique relationship to the fiber. It surrounds the fiber with its cytoplasm, thus isolating the fiber from other tissue components. In most other locations, reticular fibers are produced by fibroblasts. Important exceptions to this general rule include the endoneurium of peripheral nerves, where Schwann cells secrete reticular fibers; tunica media of blood vessels; and muscularis of the alimentary canal, where smooth muscle cells secrete reticular and other collagen fibers.

t>w

Elastic Fibers

8

Elastic fibers allow tissues to respond 1o stre1ch and distension. Elastic fibers are typically thinner than collagen fi-

182

~ w

co

LL.

w

=> ~ ~

> z z

•

1&1

I 1&1

~z z

8

•

I

FIGURE 8.1 2. Reticular fibers in a lymph node. Photomicrograph of a lymph node silver preparation showing the connective tissue capsule at the top and a trabecula extending from it at the left. The reticular fibers (arrows) form an irregular anastomosing network. X650.

a rcticul.aJ: fiber ahibit a 68-nm banding pattern (the same as the fibrils of type I collagen). The fibrils have a narrow diameter (about 20 nm), ahibit a branching pattern, and typically do not bundle to form thick fibers. In routinely stained H&:E preparations, reticular fibers cannot be identified positivdy. When visualized in the light microscope with special techniques, reticular fibers have a thread-like appearance. Because they contain a much greater relative concentration of sugar groups than do collagen type I fibers, reticular fibers are readily displayed by means of the periodic acid-Sehiff (PAS) reaction. They are also revealed with special silver-staining procedures such as the Gomori and Wilder methods. .After silver tteatmcnt, the fibers appear black; thus, they arc said to be argyrophilic (Fig. 6.12). The thicker collagen fibers in such preparations are colored brown.

Reticular fibers are named for their arrangement in a meshlika pattern or network. In loose connective tissue, networks of reticular fibers arc found at the boundary of connective tissue and epithelium as well as sunounding adipocytes, small blood vessels, nerves, and muscle cells. They are also found in embryonic tissues. The prevalence of reticular fibers is an indicator of tissue maturity. They are prominent in the initial stages of wound healing and scar tissue formation, where they pro· vide early mechanical strength to the newly synthesized ECM. As embryonic development or wound healing pro· gresses, reticular fibers are gradually replaced by the stronger

bers and are arranged in a branching pattern to form a three-dimensional network. Because elastin is roughly 1,000 times more flexible than collagen, elastic fibers are interwoven with collagen fibers to limit the distensibility of the tissue and prevent tearing from excessive suetching (Plate 6, page 208). Elastic fibers are produced by many of the same cells that produce collagen and reticular fibers, particularly fibroblasts, smooth muscle cells, endothelial cells, and chondrocytes. Elastic fibers stain with eosin but not well, so they cannot always be distinguished from collagen fibers in routine H&:E preparations. Because elastic fibers become somewhat refractile with certain fixatives, they may be distinguished from collagen fibers in specimens stained with H&:E when they display this characteristic. Elastic fibers can also be selectively stained with special dyes, such as orcein or resorcin-fuchsin, as shown in Figure 6.13.

Elastic material is a major extracellular substance in ver· tebralligamems, larynx.. and elastic arteries. In elastic ligaments, the elastic material consists of th.ick fibers interspersed with collagen fibers. Examples of this material are found in the ligamenta ftava of the vertebral column and the ligamentum nuchae of the neck. Finer fibers are present in elastic Ugaments of the vocal folds of the larynx. In elastic arteries, the elastic material is present as fenestrated lamellae, which are sheets of elastin with gaps or openings. The lamellae are arranged in concentric layers between layers of smooth muscle cells. Like the collagen fibers in the tunica media of blood vessel walls, the elastic material of arteries is produced by smooth muscle cells, not by fibroblasts. In contrast to elastic fibers, microfibrils are not found in the lamellae. Only the amorphous elastin component is seen in electron micrographs.

The elastic property of the elastin molecule is related to its unusual polypeptide backbone. which causes random coiling. Elastin (tropoelastin, 72 kDa) is one of the most hydrophobic proteins in the body. It is charactt':rized by the presence of hydrophobic regions (which comprise more than 80% of entire protein structure) alternating with hydrophilic regions.

Because the introns of the elastin gene contain large amounts of repetitive sequences, the likelihood of replication errors is increased. These errors may lead to diseases such as supravalvular aortic stenosis and cutis taxa syndrome. In supravalvular aortic stenosis (SVAS), mutated elastin forms thinner elastic fibers and disorga· nized elastic lamellae in the wall of ascending aorta. This triggers a compensation reaction in which increased pro· duction and deposition of smooth muscle in aortic wall thickens the artery wall and progressively narrows the lumen. Cutis laxa is an inherited or acquired disease characterized by wrinkled, redundant, sagging, and inelas· tic skin caused by defective dermal elastic fibers synthesis. Most inherited forms of cutis taxa cause associated multi· pie organ systems abnormalities due to the prevalence of mutated elastin in the body.

Elastic fibers are composed of cross-linked elastin molecules and a network of fibrillin microfibrils with associated proteins. Fibrillin-1 (350 leDa) is a glycoprotein that polymerizes in the

extracellular space in a head·to-tail arrangement to form fine

fibrillin microfibrils measuring 10 to 12 nm in diameter.

183

n

~

:II

fP

iz

~ e c m

•n 0

z z

m

Q single elastin molecule

(I) (I)

a mesentery spread stained with resorcin-fuchsin. The mesentery is very thin, and the microscope can be focused through the entire thickness of the tissue. The delicate thread-like branching strands are the elastic fibers {E). Collagen fibers (C} are also evident. They are much thicker; although they cross one another. they do not branch. X200.

Like co~n, elastin is rich in proline and glycine; however, unlike collagen, it is poor in hydroxyproline and completely lac:ks hydro:xylysine. The distribution of nonpolar amino acids such as glycine, valine, proline, and leucine, often arranged in repetitive motifs, makes the elastin molecule hydrophobic and allows for random coiling of its fibers. 'This permits elas· tic fibers to "slide" over one another or to be stretched and then recoil to their original state. 'The hydrophilic domains of elastin are rich in lysine and alanine, and they participate in cross-linking. Elastin also contains desmosine and isodesmosine, two large amino acids unique to elastin, which are also re-sponsible for the covalent bonding of elastin molecules to one another. 'These covalent bonds link four sites on elastin molecules into either desmosine or isodesmosine cross-links (Fig. 6.14). Elastin forms fibers of variable thicknesses, or lamellar layers (as in elastic arteries). Elastin is encoded by one ofthe largest genes in the human genome. 'The elastin (ELN) gene consists of approximately 48 kilobases of genomic DNA on chromosome 7. Analysis of the human ELN gene has revealed that functionally distinct hydrophobic and hydrophilic domains of the elastin are en· coded in separate exons that alternate in the genes. Because the ELN gene has 34 c::xons with the cxon/intron ratio ap· proximately 1:20; therefore, less than 10% of the kllobases cany the sequence that encodes elastin.

~

-1

FIGURE 8.13. Collagen and elastic fibers. Photomicrograph of

c

d&Smosine

.,m m m

:JJ

(I)

a

b FIGURE

6.14. Diagram of elastin moleculu and their

Interaction. a. Elastin molecules are shown joined by covalent bonding between desmosine and isodesmosine (purple} to form a crosslinked network. Inset shows enlargement of the elastin molecule in its individual and random-coiled conformation with the covalent bond formed by desmosine. b. The effect of stretching is shown. When the force is withdrawn. the network reverts to the relaxed state as in panel a. (Modified with permission from Alberts B. et al. Essential Cell Biology. p. 153. Copyright 1997. Routledge, Inc., part of The Taylor & Francis Group.)

184

its periphery. The presence of microfibrils within the fiber is associated with the: growth process; thus, as the fiber is formed and thickens, the microfibrils become: entrapped within the newly deposited elastin. In addition to fibrillin micro6brils, several associated proteins are involved in the regulation and assembly of elastin fibers. These include the following:

head region (N tennlnus)

(/)

a:

UJ al

folded back tail region

u:::

(C tenninus)

UJ

::;, (/) (/)

i= UJ

> i=

u

UJ

z z

b

0

u

•

dastogenesis.

w :I en en j:

I

c

z z

8

• a: w

t: ~

u

EMILIN-1 (elastin microfibril interface-located protein, 106 kDa) is a glycoprotein found at the elastin-fibrillin microfibril interface that most likely regulates the: production of dastin aggregates during the formation of fibers. • MAGP.1 (microfibril-assoc.iared glyooprotein, 20 to 30 kDa), another glycoprotein, is a component of almost all elastinassociated fibrillin microfibrils. It binds to elastin molecules, fibrillin-1, and various cnraa:J..lular ma.trix proteins. Both EMll.IN-1 and MAGP·l play a major role: in regulating

•

d fibrill in micr'ofibril FIGURE B.15. Diagram Df elaatagen..is. a. Elastic fibers are formed in the extracellular matrix of connective tissue. This model shows the molecular structure of fibrillar microfibrils. The fibrillin-1 molecule polymerizes in a head-to-tail arrangement to form fibrillin microfibrils. The regular densities (beads) are formed by the interactions between the folded-back: C terminus tail domain of one fibrillin-1 molecule with the head domain (N terminus) of another molecule. b. Formation of the elastin fiber is initiated by MAGP..1 molecules, which are associated with microfibrils at the bead region. The presence of MAGP..l allows the tropoelastin molecule to be deposited on the microfibril, cross-linlced to the fibrillin microfibril and MAGP.1. c. Tropoelastin molecules are laid down in small clumps. They are cross-linlced by lysyl oxidase. d. Mature elastin fibers show gradual fusion of tropoelastin molecules to form amorphous elastin fibers incorporating fibrillin microfibrils in its structure.

In dcctron microscopy, a fibrillin microfibril exhibits reg· ular densities (beads) in 56-nm intervals, which are most likely attributed to the three-

~z

also unique among the GAGs in that it does not contain sulfate. Each hyaluronan molecule is always present in the form of a free carbohydrate chain; in other words, it is not cova~ lently bound to protein, so it does not fonn proteoglycans. By means of special link proteins, however, proteoglycans indirectly bind to hyaluronan, forming giant macromolecules called proteoglycan aggregates (Fig. 6.17). These molecules are abundant in the ground substance of cartilage. The pressure, or turgor, that occurs in these giant hydrophilic proteoglycan aggregates accounts for the ability of cartilage to resist compression without inhibiting flexibility, making them excellent shock absorbers. Another important function of hyaluronan is to immobilize certain molecules in the desired location of the ECM. For instance, ECM contains binding sites for several growth factors, such as TGF-f!. The binding of growth factors to proteoglycans may cause either their local aggregation or dispersion, which in turn either inhibits or enhances the movement of migrating macromolecules, microorganisms, or metastatic cancer cells in the extracellular environment. In addition, hyaluronan molecules act as efficient insulators because other macromolecules have difficulty diffusing through

the dense hyaluronan network. With this property, hyaluronan (and other polysaccharides) regulates the distribution and transport of plasma proteins within the connective tissue.

Proteoglycans are composed of GAGs covalendy attached to core proteins. The majority of GAGs in the connective tissue are linked to core proteins, forming proteoglycans. The GAGs extend perpendicularly from the core in a brush-like structure. The linkage of GAGs to the protein core involves a specific trisaccharide composed of two galactose residues and a xylulose residue. The trisaccharide linker is coupled through an 0-glycosidic bond to the protein core that is rich in serine and threonine residues, allowing multiple GAG attachments. Proteoglycans are remarkable for their diversity (Fig. 6.18). The number of GAGs attached to the protein core varies from 1 (i.e., decorin) to more than 200 (i.e., aggrecan). A core protein may have identical GAGs attached to it (as in the case of fibroglycan or versican) or different GAG molecules (as in the case of aggrecan or syndecan). Proteoglycans are found in the ground substance of all connective tissues and also as membrane-bound molecules

z

8

•

I type I

collagen fibril

FIGURE 6.17. Proteoglycan stru;ture. This schematic drawing shows. on the right. a proteoglycan monomer and its relationship to the hyaluronan molecule as represented in the ground substance of cartilage. The proteoglycan monomer is composed of a core protein to which GAGs ere covalently bound. The proteoglycan monomer consists of different numbers of GAGs joined to the core protein. The end of the core protein of the proteoglycan monomer interacts with a link protein, whidl attaches the monomer into the hyaluronan forming 1he proteoglycan aggregate. On the left. hyaluronan molecules forming linear aggregates, each with many proteoglycan monomers, are interwoven with a network of collagen fibrils.

Aggrecan is another important extracellular proteoglycan. Its molecules are noncovalently bound to the long molecule of hyaluronan (like bristles to the backbone in a bottle brush); this binding is f.r.cilltated by linking proteins. To each aggrecan core protein, multiple chains of chondroitin sulfate and keratan sulfate are covalently attached through the trisaccharide linker. The most common proteoglycans are summarized in Table 6.4. Multiadhesive glycoproteins play an important role in stabilizing the ECM and linking it to cell surfaces.

eggrecen

N-linked oligosaccharide

heparan sulfate chain

vereican

syndecan decorln

FIGURE 1.18. Common proteoglycan monomers of the con· nective titsue matrix. Not& the diversity of prot&oglycan molecules; the number of GAGs attached to the protein core varies from one in deoorin to more than 200 in aggrecan. Note also that versican has identical GAG molecules (chondroitin sulfate) attached to the core molecule, whereas aggrecan has a mixture of chondroitin sulfate and keratan sulfate attached to the core protein. Syndecan is a transmembrane proteoglycan that attaches the cell membrane to the extracellular matrix.

on the surface of many cell types. Transmembrane proteoglycans, such as syndecan, link cells to ECM molecules (see Fig. 6.18). As an example of this function, synd.ecan is expressed on the surface of B lymphocytes at two different phases of their development. Syndecan molecules are first expressed during early development when lymphocytes are attached to the matrix protein of the bone marrow as they Wldergo differentiation. The loss of expression of this proteoglycan coincides with the release of the B lymphocyte into the circulation. The second time B lymphocytes express syndecan is during its differentiation into a plasma cell within the connective tissue. Synd.ecan anchors the plasma cell to the ECM proteins of the connective tissue.

Multiadhesive glycoproteins represent a small but important group of proteins residing in the ECM. They are multidomain and multifunctional molecules that play an important role in stabilizing the ECM and linking it to the cell surface. They possess binding sites for a variety of ECM proteins such as collagens, proteoglycans, and GAGs; they also interact with cell-surface receptors such as integrin and laminin receptors (Fig. 6.19). Multiadhesive glycoproteins regulate and modulate functions of the ECM related to cell movement and cell migration as well as stimulate cell proliferation and differentiation. Among the best characterized multiadhesive glycoproteins are the following:

• Fibronectin (250 to 280 kDa) is the most abundant glycoprotein in connective tissue. Fibronectins are dimer molecules formed from two similar peptides linked by disulfide bonds at a c:arboxy-tenninus to form 50-nm-long arms (see Fig. 6.19). Each molecule contains several binding domains that intetact with d.Uferent ECM molecules (e.g., heparan sulfate; collagen types I, II, and lll; fibrin; hyaluronan; and fibronectin) and integrin, a cdl-su.tfacc receptor. Binding to a c:ell-surfuc:e receptor activates fi. bronectin, which then assembles into fibrils. Fibroneain plays an important role in cell attachment to the ECM. At least 20 different fibronectin molecules have been identified to date. • Laminin (140 to 400 kDa) is present in basal and external laminae. It possesses binding sites for collagen type IV molecules, heparan sulfate, heparin, entactin, laminin, and the laminin n::ceptor on the cell surface. The process of basal lamina assembly and the role of the laminin in this process are described in Chapter 5, Epithelial Tissue (see p • 151). • Tenascin (280 kDalmonomer) appears during embryo· genesis, but its synthesis is switched off in mature tissues. It n:appears during woUild healing and is also found within musculotendinous junctions and malignant tu· mors. Tenascin is a disulfide-linked dimer molecule that consists of six chains joined at their amino-terminus (see Fig. 6.19). It has binding sites for fibrinogen, hepatin, and EGF-like growth factors; thus, it participates in cell attachment to the ECM. • Osteopontin (44 kDa) is present in the ECM of bone. It binds to osteodasts and attaches them to the underlying bone surface. Osteopontin plays an important role in sequestering calcium and promoting calcification of theECM. Important multiadhesive glycoproteins foWld in the ECM of connective tissue are summarized in Table 6.5.

189

190

TABLE6.4

Proteoglycans

Name

Molecular Weight (kDaJ

Aggrecan

Molecular Composition

Localization

Function

250

Linear molecule; binds via a link protein to hyaluronan; contains 100-150 molecules of keratan suifate and chondroitin sulfate chains

Cartilage, chondrocytes

Responsible for hydration of extracellular matrix of cartilage

Decorin

38

Small protein that contains only one chondroitin sulfate or dermatan sulfate chain

Connective tissue, fibroblasts, cartilage, and bone

Functions in collagen fibrillogenesis; by attaching to neighboring collagan molecules, helps to orient fibers; regulates thickness of the fibril and interacts with transforming growth factor~ CTGF-~)

Versican

260

Associated with a link protein; contains main and 12-15 chains of chondroitin sulfate attached to core protein

Fibroblasts, skin, smooth muscle, brain, and mesangial cells of the kidney

Possesses EGF-Iike domains on the core protein; participates in cellto-cell and cell-to-extracellular matrix interactions; binds to fibulin-1

Syndecan

33

Family of at least four different types of transmembrane proteoglycans, containing varying amounts of both heparan sulfate and chondroitin sulfate molecules

Embryonic epithelia, mesenchymal cells, developing lymphatic tissue cells. lymphocytes, and plasma cells

The extracellular domain binds collagens. heparin, tenascin, and fibronectin; intracellular domain binds to cytoskeleton via actin

~ _J L1J

u

L1J

:J

(I) (I)

i= L1J

>

Ei L1J

z z

0

u

•::»w

Cl) Cl)

I=

~

tiw z

EGF. epithelial growth factor; kDs, ldlodaltons.

z

0

u fij

• CONNECTIVE TISSUE CELLS

a: w

~

Cannactivatissua calls can ba residant ar wandaring. The cells that make up the resident cell population are relatively stable; they typically exhibit little movement and can be regarded as permanent residena of the tissue. These resident cells include

• fibroblasts and a closely related cell type, the myofibroblast; • macrophagas; • adipocytes; • mast cells; and • adult stem calls.

collagen VII collagen I fibronectin

I ~

The wandering cell population or trantdent cell population consists primarily of cells that have migrated into the tissue from the blood in response to specific stimuli.

v

These include

/

tenascin

FIGURE 1.19. Common mullllldhallve glycoprotalns. These proteins reside in the extracellular matrix and are important in stabilizing the matrix and linking it to the cell surface. They are multifunctional molecules of different shapes and possess multiple binding sites for a variety of extracellular matrix proteins such as collagens, proteoglycans, and GAGs. Note that multiadhesive proteins interact with basal membrane receptors such as integrin and laminin receptors.

• lymphocytes, • plasma cells,

• neutrophlls, • eosinophils, • basophils, and • monocytas.

Fibroblasts and Myofibroblasts Tha fibroblast istha principal call of cannactivatissua. Fibroblasts arc responsible for the synthesis of collagen, elastic, and reticular fibers and the complex carbohydrates of the ground substance. Research suggesa that a single 6.bro~ blast is capable of producing all of the ECM components.

TABLE8.5

191

Multiadhesive Glycoproteins

Name

Molecular Molecular Weight (kDa) Composition

Fibronectin

25~280

Laminin

14o-400

Localization

Function

Dimer molecule formed from two similar peptides linked by a disulfide bond

Present in the ECM of many tissues

Responsible for cell adhesion and mediates migration; possesses binding sites for integrins, type IV collagen, heparin, and fibrin

Cross-shaped molecule formed from three polypeptides (a. chain and two ~ chains)

Present in basal laminae of all epithelial cells and extemallaminae of muscle cells, adipocytes. and Schwann cells

Anchors cell surfaces to the basal lamina; possesses binding sites for collagen type IV. heparan sulfate, heparin, entactin. laminin, and integrin receptors on the cell surface.

Tenascin

1,680

Giant protein formed from six chains connected by disulfide bonds

Embryonic mesendlyme, perichondrium, periosteum, musculotendinous junctions, wounds, tumors

Modulates cell attachments to the ECM; possesses binding sites for fibronectin, heparin, EGRike growth factors, integrins. and CAMs

Osteopontin

44

Single"(;hain glycosylated polypeptide

Bone

Binds to osteoclasts; possesses binding sites for calcium, hydroxyapatite, and. integrin receptors on the osteoclast membrane

Single"(;hain rod-like suifated glycoprotein

Basal lamina-specific protein

Entactin/ nidogen

150

Links laminin and type IV collagen; has binding sites for perle can and fibronectin

n

~

:II ~

iz

~

ic •n m 0

z z

m

CAM, cell adhesion molecule; ECM, extracellular matrix; EGF. epithelial growth factor; kDs, kilodaltons.

Q ~

Fibroblasts reside in close proximity to collagen fibers. In routine H&E preparations, however, often only the nucleus is visible. It appears as an elongated or disc-litre structure, sometimes with a nucleolus evident. The thin, pale·staining, flattened processes that form the bulk of the cytoplasm are usually not visible, largely because they blend with the colla· gen fibers. In some specially prepared specimens, it is possible to distinguish the cytoplasm of the cell from the fibrous components (Fig. 6.20a). When ECM material is produced dwing active growth or in wound repair (in activated fl· broblasts), the cytoplasm of the fibroblast is more extensive and may display basophilia as a result of increased amounts of rER associated with protein synthesis (Fig. 6.20b). When examined with the TEM, the fibroblast cytoplasm exhibits profiles of rER and a prominent Golgi. apparatus (Fig. 6.21).

The myofibroblalt displays properties of both fibroblasts and smooth muscle cells. The myofibroblast is an elongated. spindly connective tissue cell not readily identifiable in routine H&E preparations. It is characterized by the presence of b1mdles of actin filaments with associated actin motor proteins such as nonmuscle my· osin (page 66). Expression of a.-smooth muscle actin (a.-SMA; actin isoform found in vascular smooth muscles) in myofibroblasts is regulated by TGF-Pl. The actin bundles aansverse the cell cytoplasm, originating and terminating on the opposite sides of the plasma membrane. The site where actin fibers attach to the plasma membrane also serves as a cellto-ECM anchoringj1mction and is called a flbronexus; it resembles the focal adhesion fo1md in epithelial cells (page 155). The arrangement of actin bundles and their atta.chment sites

FIGURE 8.20. Fibroblllts i" connective tissue. •· Photomicrograph of a connective tissue specimen in a routine H&E-stained, paraffinembedded preparation shows nuclei of fibroblasts IF). x600. b. During wound repair, the activated fibroblasts {F) exhibit more basophilic cytoplasm, which is readily observed with the light microscope. X500.

-1 (I) (I)

c n

m m r-

(i;

192

~ ...J w 0 w

::>

en en

i= w

~w z z

0 0

•

Ill

::::»

~

~ z z

8

......_;;~ __ FIGURE 8.21. Electron micrograph of fibroblasts. The processes of several fibroblasts are shown. The nucleus of one fibroblast is in the upper right of the micrograph. The cytoplasm contains conspicuous profiles of rER. The cisternae of the reticulum are distended, indicating active synthesis. The membranes of the Golgi apparatus (G) are seen in proximity to the rER. Surrounding the cells are collagen fibrils (CF), almost all of which have been cut in cross section and thus appear as small dots at this magnification. x 11,000.

fonn a mechanotransduction system. in which forces generated by the contraction of intracellular actin bundles are transmitted to the ECM. With the TEM. the myofibroblast displays characteristics typical of a fibroblast along with characteristics of smooth muscle cells. In addition to rER and Golgi profiles. the myofi· broblast contains bundles oflongitudiaally disposed actin fil. aments and dense bodies similar to those observed in smooth muscle cells (Fig. 6.22). .& in the smooth muscle cell. the nu· cleus often shows an undulating surface profile. a phenome· non associated with cell contncti.on. The myofibroblast differs

RGURE 8.22. Electron micrograph of a myofibroblaat. The cell exhibits some features of a fibroblast. such as areas with a moderate amount of rER. Compare with Figure 6.21. Other areas, however, contain aggregates of thin filaments and cytoplasmic densities (arrows), features that are characteristic of smooth muscle cells. The arrowheads indicate longitudinal profiles of collagen fibrils. x 11,000.

from the smooth muscle cell in that it lacks a surrounding basal lamina (smooth muscle cells are surrounded by a basal or cxte.mallamina). Also. it usually exists as an isolated cell. although its processes may contact the processes of other my· ofibroblasts. Such points of contact exhibit gap junctions. in· dicating intercellular communication.

Macrophages Macrophagas ara phagocytic calls darivad fram monocvtes that contain an abundant number af lysosomes.

Connective tissue macrophages. also known as tissue his· tiocytes. are derived from blood cells called monocytes.

Monocytes migrate from the bloodstream into the connective tissue, where they differentiate into macrophages. In the light microscope and with conventional stains, tissue maaophages are difficult to identify unless they display obvious evidence of phagocytic activity-for example, visible ingested material within their cytoplasm. Another feature that assists in identifying maaophages is an indented or kidney-shaped nucleus (Fig. 6.23a). Lysosomes areabWldantin the cytoplasm and can be revealed by sW.ning for acid phosphatase activity (both in the light microscope and with the TEM); a positive reaction is a fwther aid in identification of the macrophage. With the TEM, the surface of the macrophage exhibits numerous folds and finger-like projections (Fig. 6.23b). The swf.tce folds engulf the substances to be phagocytosed. The lysosomes of the macrophage, along with the swface cytoplasmic projections, are the structures most indicative of the specialized phagocytic capability of the cell. The macrophage may also contain endocytotic vesicles, phagolysosomes, and other evidence of phagocytosis (e.g., residual bodies).

The rER, sER, and Golgi apparatus support the synthesis of proteins involved in the cell's phagocytic and digestive functions, as well as its secretory functions. Secretory products leave the cell by both the constitutive and regulated ex:ocytotic pathways. Regulated secretion can be activated by phagocytosis, immune complexes, complement, and signals from lymphocytes (including the release of lymphokines, biologically active molecules that influence the activity of other cells). The secretory products released by the macrophage include a wide variety of substances related to the immune response, anaphylaxis, and inflammation. The release of neutral protease& and GAGases (enzymes that break down GAGs) facilitates the migration of the macrophages through the connective tissue.

Macrophages are antigen-prtsenting cells and play an important role in immune response reactions. Although the main function of the macrophage is phagocytosis, either as a defense activity (e.g., phagocytosis of bacteria)

193

n

~

:II ~

iz

~

ic •n m 0

z z

m

Q ~

-1 (I) (I)

c n

m m r-

(i;

FIGURE 8.23. Photomicrograph and el.aron micrograph af a macrophage. a. This photomicrograph shows several macrophages IMl in the connective tissue from an area of wound healing. They can be distinguished from other cells by an presence of an indented or kidney-shaped nucleus (similar to that of monoc:ytes in the blood vessels). Note several mature neutrophils (M with segmented nuclei located in the connective tissue that surround blood vessel {8Vl filled with red and white blood cells in the center of 1he image. X480. b. The most distinctive EM features of the macrophage are its population of endoc:ytotic vesicles, early and late endosomes, lysosomes. and phagolysosomes. The surface of the cell reveals a number of finger-like projections, some of which may be sections of surface folds. X 10,000.

194

~ ...J w 0 w

::>

en en

i= w

~w z z

0 0

•

Ill

::::»

~

~ z z

8

or as a cleanup operation (e.g., phagocytosis of cell debris), it also plays an important role in immune response reactions. Macrophages have specific proteins on their surface known as major histocompatibility complex II (MHC II) molecules that allow them to interact with halparcD4+T lymphocytes. When maaophages engulfa foreign cell, antigens-short polypeptides (7 to 10 amino acids long) from the foreign~ displayed on the surfu.ce of the MHC II molecules. If a CD4+ T lymphocyte recognizes the displayed antigen, it becomes activated, triggering an immWle response (see Chapter 14, Immune System and Lymphatic TISSues and Organs). Because macrophage& "present" antigens to helper CD4+ T lymphocytes, they are known as antigen-presenting cells (APCs). Macrophages arrive after neutrophils to the site of tissue injury and und11go dHf11entiation. At the site of tissue injury, the first cells to reach the injured area are neutrophils. They are the first cells to recognize foreign organisms or infectious agents and initiate destruction either by reactive oxygen intermediates or oocygenindependent killing mechanisms (see pages 302-304). During this destruction process, large amounts of secretory products and cellular debris are generated at the site of injury. In addition, microorganisms that survived the action of neutrophils may also be present. After 24 hours, monocytes from blood vessels enter the site of injury and differentiate into macrophages, where they remain until inflammation resolves. Initially, the macrophage's objective is to kill microorganisms that have survived the attack of neutrophils. Simultaneously, macrophages are activated by the interaction with several molecules produced by neutrophils and invading microorganisms. During this process, macrophages undergo a series of functional, morphologic, and biochemical modifications triggered by various gene activations. Classically activated macrophages (M1 macrophages) promote inflammation. the dNtruction of ECM. and apoptosis. Activation by interferon 'Y (IFN--y), tumor necrosis fuctor a. (TNF-a), or by bacterial lipopolysaccharide (LPS) creates the classically activated macrophage or M1 macrophage. These macrophages have the capacity. through the production of nitric oxide (NO) and other intermediates, to destroy microorganisms at the site ofinflammation. They also secrete interleukin (IL)-12, which stimulates helper CD4+ T lymphocytes. In turn, helper T cells secrete IL-2, which stimulates cytotoxic cos+ T lymphocytes to arrive at the site of inflammation. In summary. M1 macrophages eUcit chronic inflammation and. tissue injury. When macrophages encounter large foreign bodies, they may fuse to form a large cell with as many as 100 nuclei that engulfs the foreign body. These multinucleated cells are called foreign body giant cells (Langhans cells). AHernatiVBiy. activated macrophages (M2 macrophages) assist in the resolution of inflammation and promote rebuilding of ECM, cell proliferation. and angiogenesis. When the inflammatory stimulus is removed from the site of tissue injury, the body switches into a repair mode that includes the removal of cell debris, synthesis of components of new ECM, and revascularization ofthe injured tissue. Owing this period, macrophages are activated by cytok:ines IL-4,

IL-5, IL-10, or IL-13. These types of cells are called alteflo natively activated macro phages or M2 macrophages. M2 macrophage& are anti-inflammatory in that they assist in resolution of inflammation. They secrete IL-4 to promore differentiation of B lymphocytes into plasma cells and vascular endothelial growth factor (VEGF) to stimulate angiogenesis. M2 macrophages also secrete ECM components (e.g., fibronectin and other multiadhesive glycoproteins). They promote wound repair due to their anti-inflammatory, proliferative, and angiogenic activities. M2 macrophages are also efficient at combating parasitic infections (i.e., schistosomiasis). In addition to their beneficial activities, M2 macrophages are involved in pathogenesis of allergy and asthma.

Mast Cells Mast cells develop in bone marrow and differentiate in connective tissue. Mast cells are large, ovoid, corutective tissue cells (20 to 30 Jl.D1 in diameter) with a spherical nucleus and cytoplasm filled with large, intensely basophilic granules. They are not easily identified in hwnan tissue sections unless special fixatives ate used to preserve the granules. After glutaraldehyde fixation, mast cell gr.mules can be displayed with basic dyes such as toluidine blue. It stains the granules intensely and metaclJtomatically because they contain heparin, a highly sulfated proteoglycan (Fig. 6.24a). The cytoplasm displays small amounts of rER. mitochondria, and a Golgi apparatus. The cell surface contains numerous microvilli and folds. The mast cell is related but not identical to the basophil, a white blood cell that contains similar granules (Table 6.6). They both arise from a pluripotential hemopoietic stem cell (HSC) in the bone marrow. Mast cells progenitors (MCPs) initially circulate in the peripheral blood as agranular cells of monocytic appearance. After migrating into the connective tissue, these immature cells differentiate and produce their characteristic granules (Fig. 6.24b). In contrast, basophils progenitors (BaPs) differentiate and remain within the circulatory system. The surface of mature mast cells expresses a large number of high-affinity Fe receptors (FceRI) to which immunoglobulin E (IgE) antibodies arc: attached. Binding ofa spccific antigen to exposed IgE antibody molecules on the mast cell swface leads to an aggregation of F, receptors. This triggers mast cell activation, which results in granule exocytosis (degranulation) and the release of granule content into the ECM. Mast cells can also be activated by an 1gB-independent mechanism during complement protein activation. Two types of human mast cells have been identified based on morphologic and biochemical properties. Most mast cells in the connective tissue of the skin, intestinal submucosa. and mammary glands and axillary lymph nodes contain cytoplasmic granules with a lattice-like internal structure. These cells contain granule-associated tryptase and chymase and are referred to as MCrc mast cells or connective tissue mast cells. In conttast, mast cells in the lungs and intestinal mucosa have granules with a scroll-like internal structw:e. These cells produce only ttypt:ase and are tenned MCr mast cells or mucosal mast cells. Nearly equivalent concentrations of each type are found in nasal mucosa.

'\ ' '

'

195

n :z:

•~ m

::a ~

8 z z

m

a

!l ~

•• en cm

n 0

FIGURE 8.24. Mast •II. a. Photomicrograph of a mast cell stained with toluidine blue. The granules stain intensely and, because of their numbers, tend to appear as a solid mass in some areas. The nucleus of the cell is represented by the pale-staining area. x 1,250. b. This electron micrograph shows the cytoplasm of a mast cell that is virtually filled with granules. Note a small lymphocyte present in the upper left of the figure. X6,000.

z zm

~

< m :::!

(/') (/')

c Malt calls are especially numerous in the connective tis- within the brain and spinal cord is devoid of mast cells. sues of skin and mucous membranes but are not present in The absence of mast cells protects the brain and spinal cord from the potentially disruptive effects of the edema the brain and spinal cord. caused by allergic reactions. Mast cells are also numerous in ConnectiV\': tissue mast cells (MCrc mast cells) are distribthe thymus and, to a lesser degree, in other lymphatic organs, uted chiefly in the connective tissue of skin in the vicinity of but they are not present in the spleen. small blood vessels, hair fulllcles, sebaceous glands, and sweat glands. Mast cells are also present in the capsules of organs Most mast call secretory products (mediators of inflammaand the connective tissue that surrounds the blood vessels of tion) are starad in granolas and ara ralaasad atthatima of internal organs. A notable exception is the central nervous mast cell activation. system. Although the meninges (sheets of connective tis- Mast cells contain intensely basophillc granules that store sue that surround the brain and spinal cord) contain mast chemical substances known as mediators of inflammation. cells, the connective tissue around the small blood vessels

TABLE &.6

Mediators produced by mast cells arc divided into two

Comparison of Features Characteristic of Mast Cells and Basophils Mast Cells

Basophils

Origin

Hemopoietic stem cell

Hemopoietic stem cell

Site of differentiation

Connective tissue

Bona marrow

Cell divisions

Yes (occasionally)

No

Cells in circulation

No

Yes

Characteristic Features

Life span

Weeks to months

Days

Size

2D-30 ).l.m

7-10 ,...m

Shape of nucleus

Round

Segmented (usually bilobar)

Granules

Many, large, metachromatic

Few, small, basophilic

High-affinity surface receptors for lgE antibodies (Fe&Ril

Present

Present

Marker of cellular activity

Tryptasa

Not yet established

lgE, immunoglobulin E; FcsRI, Fe receptors.

m

n m r

~

196

~

...J UJ

u

UJ

::::l C/l C/l

i= UJ

~

u UJ

z z

0

u

• w

::::) U) U)

I=

~

~

z

An important role of myofibroblasts occurs during the process of wound healing. A clean surgical skin incision begins the healing process when a blood clot containing fibrin and blood cells fills the narrow space between the edges of the incision. The inflammatory process. which begins as early as 24 hours after initial injury, contains the damage to a small area, aids in the removal of injured and dead tissues, and initiates deposition of new ECM proteins. During the initial phases of inflammation, neutrophils and monocytes infiltrate the injury (maximum infiltration by neutrophils occurs in the first 1 to 2 days after injury). Monocytes transform into macrophages (they usually replace neutrophils by day 3 after injury; page 192). At the same time, in response to local growth factors. fibroblasts and vascular endothelial cells begin to proliferate and migrate into the delicate fibrin matrix of the blood clot. forming the granulation tissue. a specialized type of tissue characteristic of the repair process. Usually by day 5 after injury, the fully developed granulation tissue bridges the incision gap. It is composed mainly of large numbers of small vessels, fibroblasts, and myofibroblasts, and variable numbers of other inflammatory cells. Migrating fibroblasts exert

z

8 Vi

a:

~ ~

u

FIGURE F8.3. 1. Fibrabl1sts 1nd myofibrablasts in cuHura. This immunofluorescence image shows wild-type 3T3 fibroblasts cultured on a collagen lattice. Under the stimulation of certain growth factors such as TGF~1. some fibroblasts differentiate to myofibroblasts, expressing a-SMA. the marker of myofibroblast differentiation. Cells were stained with fluorescein-labeled phalloidin to visualize Filctin filaments (green), and a:-SMA were labeled with primary antibodies against a-SMA and visualized with secondary goat anti-mouse antibodies conjugated with FITC (red). Co-localization of a-SMA with Filctin is indicated by the yellow color. Note that some cells have completed their differentiation, and others are in the early stages. X1,000. (Courtesy of Dr. Boris Hinz.)

tractional forces on the ECM, reorganizing it along lines of stress. Under the influence of growth factors such as TGF-P1 and mechanical forces, fibroblasts undergo differentiation into myofibroblasts. This process can be visualized by monitoring the synthesis of cx-SMA. This type of actin is not present in the cytoplasm of fibroblasts (Fig. F6.3.1). The myofi broblasts generate and maintain a steady contractile force (similar to that of smooth muscle cells) that causes shortening of the connective tissue fibers and wound closure. At the same time, myofibroblasts synthesize and lay down collagen fibers and other ECM components that are responsible for tissue remodeling. During the second week of wound healing, the number of cells in tissue undergoing repair decreases; most of the myofibroblasts undergo apoptosis and disappear, resulting in a connective tissue scar that has very few cellular elements. In some pathologic conditions, myofibroblasts persist and continue the process of remodeling. This continued remodeling causes hypertrophic scar formation, resulting in excessive connective tissue contracture. Extensive numbers of myofibroblasts are found in most contractive diseases of connective tissue (fibromatoses). For example, palmar fibromatosis (Dupuytren's disease) is characterized by the thickening of palmar aponeurosis, which leads to progressive flexion contracture of the fourth and frfth digits of the hand (Fig. F6.3.2J. If scar tissue grows beyond boundaries of the original wound and does not regress, it is called a keloid. Its formation is more common among African Americans than other ethnic groups.

FIGURE F8.3.2. Hand of 1 patient with Dupuytren's disease. Dupuytren's disease is an example of a contractive disease of connective tissue of the palm. The most commonly affected areas-near the crease of the hand close to the base of the ring and small fingers-form contracted fibrous cords, which are infiltrated by an extensive number of myofibroblasts. Most patients report problems when they try to place the affected hand on a flat surface. In severe cases, the fingers are permanently flexed and interfere with everyday activities, such as washing hands or placing the hand into a pocket. (Courtesy of Dr. Richard A. Berger.)

197 The cells that are included in the mononuclear phagocyte system (MPS) primarily are derived from monocytes and denote a population of antigen-presenting cells involved in the processing of foreign substances. These cells are able to phagocytose avidly vital dyes such as trypan blue and India ink. which malc:es them visible and easy to identify in the light microscope. The common origin of MPS cells from monocytes serves as the major distinguishing feature of the system, although there are some exceptions (see below). In addition, with the exception of osteoclasts, cells of the M PS display receptors for complement and Fe fragments of immunoglobulins. The various cells of the MPS are listed in the following table. Most cells of the MPS become fixed in specific tissues and may adopt a variety of morphologic appearances as they differentiate. The main functions of MPS cells are phagocytosis, secretion (lymphokines), antigen

processing, and antigen presentation to other cells of the immune system. Some functionally important phagocytic cells are not derived directly from monocytes. For example, microglia are small, stellate cells located primarily along capillaries of the central nervous system that function as phagocytic cells. They arise from hemopoietic progenitor cells that are recruited from the blood vessels to differentiate in the central nervous system during the embryonic and perinatal stages of development; nevertheless, they are included in the MPS. Similarly, ostaoclasts derived from the fusion of granulocyte/macrophage progenitor cells (GMPJ that give rise to granulocyte and monocyte cell lineages are also included in the MPS. Also, fibroblasts of the subepithelial sheath of the lamina propria of the intestine and uterine endometrium have been shown to differentiate into cells with morphologic, enzymatic, and functional characteristics of connective tissue macrophages.

n :z:

•~ m

::a ~

8 z z

m

!l ~

•• en

cm

n 0

Calls of the Mononuclear Phagocyte System Nama of Cell

Location

Monocyte and its precursors in bone marrow: monoblast and promonocyte

Blood and bone marrow

Macrophage

Connective tissue, spleen, lymph nodes, bone marrow, and thymus

Parisinusoidal macrophage (Kupffer cell)

Liver

Alveolar macrophage

Lungs

Fetal placental antigen-presenting ceii(Hofbauer cell)

Placenta

Pleural and peritoneal macrophage

Serous cavities

Osteoclast (originates from hemopoietic progenitor cells)

Bone

Microglia (originate from hemopoietic progenitor cells)

Central nervous system

Langerhens cell

Epidermis of skin, oral mucosa, foreskin, female genital epithelium

Fibroblast-derived macrophage (originates from mesenchymal cells)

Lamina propria of intestine, endometrium of uterus

Dendritic call

Lymph nodes. spleen

Multinucleated giant cells (e.g., foreign body giant calls, Langhans giant cells; originate from fusion of several macrophagesl

Pathological granulomas: suture granuloma. tuberculosis

z zm

~

< m :::!

categories: prafonned mediators that are ston:d in secretory granules and released upon cell activation and newly synthesized mediators (mostly lipids and cytokines) that are often absent in the: resting cells, although they are produced and secreted by activated mast cells. Preformed mediators found inside mast cell granules are the following: • Histamine is a biogenic amine that increases the permeabUity ofsmall blood vc:ssels, causing edema in the surrounding tissue and a skin reaction demonstrated by an itching sensation. In addition, it increases mucus production in the

(/') (/')

c

m

n m r

~

bronchial tree and prompts contraction of smooth muscle in the pulmonary airways. Histamine's effects can be blocked by antlhlstamlnlc agents. These competitive inhibitors have a similar chemical structure and bind to histamine receptors without initiating histamine's effects.

•

Heparin is a sulfated GAG that is an anticoagulant. Its expression is limited essentially to the granules of mast cells

and basophUs. When heparin unites with antithrombin III and platelet factor IY. it can block numerous coagulation factors. On the basis of its anticoagulant properties, heparin is useful for treatment of thrombosi8. It also

198

~

...J

w

u

w

::J (f.l (f.l

i= w

~

u w

z z

0

u

• w

:I

!~

M z

interacts with fibroblast growth factor (FGF) and its receptor to induce signal transduction in the cells.

including eosinophil migration and the increase ofvascular permeability. Similar to histamine, leukotrienes trigger

• Serine proteases (tryptase and chymase). Tryptase is sdectivdy concentrated in the secretory granules of human mast cells (but not ba.sophils). It is released by mast cells together with histamine and serves as a marker of mast cell activation. Chymase plays an important role in generating angiotensin II in response to vascular tissue injury. Mast cell cb.ymase also activates MMPs and induces apoptosis of vascular smooth muscle cells, particularly in the area of atherosclerotic lesions. • Eosinophil chemotactic factor (ECF) and neutrophil chemotactic factor (NCF), which attract eosinophils and neutrophils, respectively. to the site of inflammation. The secretions of eosinophils counteract the effects of the hista-

prolonged constriction of smooth muscle in the pulmonary airways, causing bronchospasm. The bronchoconstrictive effects of leukotrienes develop more slowly and last much longer than the effects of histamine. Bronchospasm caused by leukotrienes can be prevented by leukotriene receptor antagonists (blockers) but not by antihistaminic agents. The leukotriene receptor antagonists are among the most prescribed drugs for the management of asthma; they are used for both treatment and prevention of acute asthma attacks.

mine and leukotrienc:s. Newly synthesized mediators include the following: • Leukotriene C (LTC..) is released from the mast cell and

then cleaved in the ECM, yielding two active leu1rotrienes-D (LTD,J and E (LTE.). They represent a fam-

ily of modified lipids conjugated to glutathione (LTCJ or cysteine (L1D4 and Ln). LeuJmtriencs are released from mast cells dwing anaphylaxis (see Folder 6.5 for a description of anaphylaxis) and promote inflammation,

• Tumor necrosis factor a (TNF-a) is a major cytokine

produced by mast cells. It increases expression of adhesion molecules in endothelial cdls and has antitumor effects. • Several lntarteuklns (ll.r4, -3 ·5, ·&, ·8 and -16), growth factors (GM-CSF), and prostaglandin 0 2 (PGD2J are also released during mast cell activation. These mediators are not stored in granulc:s but are synthesized by the cell and released immediately into the ECM. Mediators released during mast cell activation as a result of interactions with allergens are responsible for a variety of symptoms and signs that are characteristic of

allergic reactions.

z

8 U:i

a:

~ ~

u

FOLDER6.5

CLINICAL CORRELATION:THE ROLE OF MAST CELLS AND BASOPHILS IN ALLERGIC REACTIONS Exposure to a specific antigen (allergen) that reacts with lgE antibodies bound to the surface of mast cells or basophils via their high-effinity receptors (FceRIJ initiates mast cell activation. This type of lgE-dependent activation triggers a cascade of events, resulting in an allergic reaction. These reactions can occur as immediate hypersensitivity reactions (usually within seconds to minutes after exposure to an allergen), late-phase reactions, or chronic allergic inflammations. The immediate hypanensitivity reaction involves lgE-mediated release of histamine and other mediators from mast cells and also from basophils. The clinical symptoms caused by these mediators vary, depending on which organ system is affected. The release of mediators in the superficial layers of the skin can manifest as erythema (redness), swelling and itching, or pain sensations. Respiratory symptoms include sneezing, rhinorrhea (runny nose), increased production of mucus, coughing, bronchospasm (constriction of bronchi), and pulmonary edema. Individuals with these symptoms often complain of tightness in the chest. shortness of breath. and wheezing. The gastrointestinal tract can also be affected with symptoms of nausea, vomiting, diarrhea, and abdominal cramping. In highly sensitive individuals, the antigen injected by an insect can trigger a massive discharge of mast cells and basophil granules that affect more than one system. This condition is known as anaphylaxis. Dilation and increased permeability of systemic blood vessels can cause anaphylactic shock. This often-explosive, life-threatening reaction is characterized by significant

hypotension (decreased blood pressure), decreased circulating blood volume (leaky vessels), and smooth muscle cell constriction in the bronchial tree. The individual has difficulty breathing and may exhibit a rash as well as have nausea and vomiting. Symptoms of anaphylactic shock usually develop within 1 to 3 minutes, and immediate treatment with vasoconstrictors such as epinephrine is required. Clinical assessment of the activation of basophils in systemic anaphylactic reactions is not possible because an assay for a specific cellular marker released by basophils (and not by other cells such as mast cells) has not yet been developed. Following resolution of the signs or symptoms of an immediate hypersensitivity reaction, an affected individual may develop a late-phase allergic reaction 6 to 24 hours later. The symptoms of these reactions may include redness, persistent swelling of the skin, nasal discharge, sneezing, and coughing, usually accompanied by an elevated white blood cell count. These symptoms usually last a few hours and then disappear within 1 to 2 days of the initial allergen exposure. In the respiratory system, the late-phase reaction is believed to be responsible for the development of persistent asthma. If the exposure to an allergen is persistent (for instance, by a dog-owning patient who is allergic to dogs), it can result in chronic allergic inflammation. Tissues in such individuals accumulate a variety of immune cells such as eosinophils andT lymphocytes that cause more tissue damage and prolong inflammation. This can lead to permanent structural and functional changes in the affected tissue.

Basophils Basophils that develop and differentiate in bone marrow share many features with mas1 cells. Basophils are granulocytes that circulate in the bloodstream and comprise less than 1% of peripheral white blood cells (leukocytes). Developmentally, they represent a separate lineage from mast cells, despite shuing a common precursor cell in the bone marrow. Basophils develop and matute in the bone marrow and are released into the circulation as matutc: cells. They also have many other common fi:atures with mast cells, such as basophilic secretory granules, an ability to secrete similar mediators, and an abundance of high-affinity Fe receptors for lgE antibodies on their cell membranes. They participate in allergic reactions (see Folder 6.5) and together with mast cells release histamine, heparin, heparan sulfate, ECF, NCF, and other mediators of inflammation. In contrast to mast cells, basophils do not produce prostaglandin 0 2 (PGD~ and interleukin-5 (IL-5). Basophils and their fi:atures are discussed in more detail in Chapter 10, Blood.

Adipocytes The adipocyte is a connective tissue cell specialized to store neutral fat and produce a variety of hormones. Adipocytes differentiate from mesenchymal stem cells and gradually accumulate fat in their cytoplasm. They are located

connective tissue of adults. These cells give rise ro differentiated cells that function in the repair and formation of new tissue, such as in wound healing and in the development of new blood vessels (neovascularization).

The vascular pericytes that partially wrap around capillaries and venule• are mesenchymal stem cells. Pericytes, also called adventitial cells or per"IY8scular cells, are typically wrapped, at least partially, around capUlaries and venules (Fig. 6.25). Their nuclei are shaped simUarly ro that of endothelial cells (i.e., Battened but curved to conform to the tubular shape of the vessel). Several observations support the interpretation that vascular pericytes are indeed mesenchymal stem cells. Experimental studies show that in response to external stimuli, pericytes express a cohort of proteins similar to those of stem cells in the bone marrow. Pericytes are surrounded by basal lamina material that is continuous with the basal lamina of the capillary endothellum; thus, they are not truly located in the connective tissue comparnnent. The role of pericytes as mesenchymal stem cells has been confirmed experimentally in studies that demonstrate the ability of cultured pericytes from retinal capillaries to differentiate into a variety of cells, including osteoblasts, adipocytes, chondrocyte&, and fibroblasts.

n

~

:II

fP

iz

~ e c m

•n 0

z z

m

Q ~

-1 (I) (I)

throughout loose connective tissue as individual cells and groups ofcells. When they accumulate in large numbers, they are called adipose tissue. Adipocytes are also involved in the synthesis of a variety of hormones, inflammatory mediators, and growth factors. This specialized connective tissue is discussed in Chapter 9, Adipose TISSUe.

c n

m m r-

li)

Adult Stem Cells and Pericytes Niches of adult stem cells are located in various tissues and organs. Many tissues in mature individuals contain reservoirs of stem cells called adult stem cells. Compared with embryonic stem cells, adult stem cells cannot differentiate into multiple lineages. They usually are capable of differentiating only into lineage-speci1ic cells. Adult stem cells are found in many tissues and organs, residing in specific sites referred to as niches. Cells residing within niches in various tissues and organs (excluding bone marrow) are called tissue stem cells. They have been identified in the gas· trointestinal tract-for instance, in the swmach (isthmus of the gastric gland), small and large intestines (base of the intestinal gland), and many other areas. Bone marrow represents a unique reservoir of stem cells. In addition to containing HSCs (see Chapter 10, Blood), bone marrow also contains at least two other populations of stem cells: a het· erogenous population of multipotent adult progenitor cells (MAPCs) that appear to have broad developmental capabilities and bone marrow stromal cells (BMSCs) that can generate chondrocytes, osteoblasts, adipocytes, muscle cells, and endothelial cells. MAPCs are adult counterparts of embryonic stem cells. Niches of adult stem cells called mesenchymal stem cells are found in the loose

199

FIGURE 8.2&. Electron mlcrogl'lph of a small blood veeNI. The nucleus at the upper teftbelongs to the endothelial cell that forms the wall of the vessel. At the right is another cell. a pericyte. which is in intimate relation to the endothelium. Note that the basal lamina IBLI covering the endothelial cell divides lsrro!M to surround the pericyte. X11,000.

200

~ ...J w 0 w

=>

VJ VJ

i= w

TEM stUdies demonstrate that pericytes surrounding the smallest venules have cytoplasmic characteristics almost identical to those of the endothelial cells of the same vessel. Pericytes associated with larger venules have characteristics of smooth muscle cells of the tunica media of small veins. In fortUitous sections cut parallel to the long axis of venules, the distal portion and proximal portion of the same pericyte exhibit characteristics of endothelial cells and smooth mus~ de cells, respectively. These studies suggest that during the development of new vessels, cells with characteristics of paricytes may differentiate into smooth muscle found in the vessel wall.

5 z

The fibrobla11s end blood vessels within healing wounds develop from mesenchymal stem cells associated with 1he tunica adventitia of venules.

0

Antoradiographic studies of wound healing using parabiotic (crossed-circulation) pairs of animals have established that mesenchymal stem cells located in the tunica adventitia of venules and small veins are the primary source of new cells in healing wounds. In addition, fibroblasts, pericytes, and endothelial cells in portions of the connective tissue adjacent to the wound divide and give rise to additional cells that form new connective tissue and blood vessels.

w

z

0

•:»

w

~

~ z z

8

FIGURE 8.28. Plasma C~ell. •· This photomicrograph shows the typical features of a plasma cell as seen in a routine H&E preparation. Note clumps of peripheral heterochromatin alternating with clear areas of euchromatin in the nucleus. Also note the negative Golgi (a"owsl and basophilic cytoplasm. X5,000. b. Electron micrograph shows that an extensive rER occupies most of the cytoplasm of the plasma cell. The Golgi apparatus (G) is also relatively large, a further reflection of the cell's secretory activity. X15,000.

Lymphocytes, Plasma Cells, and Other Cells of the Immune System Lymphocytes are principally involved in immune responses. Connective tissue lymphocytes are the smallest of the wan· dering cells in the connective tissue (see Fig. 6.24b). They have a thin rim of cytoplasm surrounding a deeply staining. heterochromatic nucleus. Often, the cytoplasm of connec· tive tissue lymphocytes may not be visible. Normally, small numbers of lymphocytes are found in the connective tis· sue throughout the body. The number increases dramatically, however, at sites of tissue inflammation caused by pathogenic agents. Lymphocytes are most numerous in the lamina propria of the respiratory and gastrointestinal tracts, where they are involved in immunosurveillance against pathogens and foreign substances that enter the body by crossing the epithelial lining of these systems.

Lymphocytes are a heterogeneous population of at least three maior func1ional cell 1ypes: T cells, B cells, end natural killer (NK) cells. At the molecular levd, lymphocytes are characterized by the expression of specific molecules on the plasma membrane known as cluster of differentiation {CD} proteins.

CD proteins recognize specific ligands on target cells. Because some CD proteins are present only on specific types of lymphocytes, they are considered specific marker proteins. On the basis of these specific markers, lymphocytes can be classified into three functional cell types.

The plasma cell is a relatively large, ovoid cell (20 ..,m) with a considerable amount of cytoplasm. The cytoplasm displays sttong basophilia because of an c::nensive rER (Fig. 6.26a). The Golgi apparatus is usually prominent because of its relatively large size and lack of staining. It appears • T lymphocytes are character.iz.ed by the presence of the in light microscope preparations as a clear area in contrast to CD2, CD3, CD5, and CD7 marker proteins and T-cell the basophilic cytoplasm. The nucleus is spherical and typically offset or ecreceptors (TCRs). These cells have a long life span and centrically positioned. It is small-not much larger than are effectors in cell-mediated immunity. the nucleus of the lymphocyte. It exhibits large clumps • B lymphocytes are characterized by the presence of of peripheral heterochromatin alternating with clear areas CD9, CD19, and CD20 proteins and attached immuof euchromatin. This urangement has traditionally been noglobulins lgM and lgD. These cells recognize antigens, described as resembling a cartwheel or analog clock have a variable life span, and are effectors in antibodyheterochromatin resembling the spokes face. with the mediated (humoral) immunity. of the wheel or the numbers on a clock (Fig. 6.26b). The • NK lymphocytes are non-T, non-B lymphocytes that express CD16a, CD56, and CD94 proteins not found heterochromatic nucleus of the plasma cell is somewhat on other lymphocytes. These cells neither produce immu- surprising given the cell's function in synthesizing large noglobulins nor express TCR on their surface. Thus, NK amounts of protein. However, because the cells produce lymphocytes are not antigen specific. Similar in action to large amounts of only one type of protein-a specific T lymphocytes, however, they destroy virus-infected cells antibody-only a small segment ofthe genome is aposed for transcription. and some tumor cells by a cytotoxic mechanism. Eosinophils, monocvtas, and nautraphils are also observed In response to the pn:scnce ofantigens, lymphocytes become in connective tissue. activated and may divide several times, producing clones ofthemselves. In addition, clones ofB lymphocytes mature into plasma Certain cells rapidly migrate from the blood to enter the cdls. A description ofB and T lymphocytes and their functions connective tissue, particularly neutrophils and monocytes during immune response reactions is pn:scnted in Chapter 14, in response to tissue injury. Their presence generally indi· cates an ac:ute inflammatory reaction. In these reactions, Immune System and Lymphatic TISSUes and Organs. neutrophils migrate into the connective tissue in substan· Plasma calls are antibody-producing cells derived from tial numbers, followed by large numbers of monocytes. As B lymphocytes. noted, the monocytes then differentiate into macrophages. Plasma cells are a prominent constituent of loose connec- A description of these cells and their roles is found in tive tissue at sites where antigens tend to enter the body (e.g., Chapter 10. Blood. The eosinophil, which functions in the gasttointestinal and respiratory tracts). They are also a allergic reactions and parasitic infections, is also denormal component of salivary glands, lymph nodes, and he- scribed. Eosinophils may be observed in normal connecmatopoietic tissue. Once derived from its B lymphocyte tive tissue, particularly the lamina propria of the intestine, precursor, a plasma cell has only limited migratory ability and as a result of chronic immunologic responses that occur a somewhat short life span of 10 to 30 days. in these tissues.

201

n

~

:II

fP

iz

~ e c m

•n 0

z z

m

Q ~

-1 (I) (I)

c n

m m r-

li)

202

...

6

>-

- ---'l't

8 ...J

~

:r:

•w - - - - - - - - - - - - - - - :;)

rn

f!

OV£RV1£W OF CONNECTM TI~UE .

of various epithelia and by the exter

LLI

>

aranent throughout the body

• Connective tissue forms a conttn~ous co~pb ded by th basal laminae It IS oun e that connects and supports other tiSSUe. na11am· f uscle cells and nerve~

e

mae o m

suCopporrlJ_tg ~· mpriscs a diverse amup of cells within a tissue-specific nnccove tJSSue co "". fib d nd extracellular matrix (ECM}. ECM contains protem ers an grou

~ LLI z z - - - - - - - - - - - - ---1 • ~=~:a.C:~n of connective tissue is primarily based. on the composition and

8

o

·zation ofits extracellular components and on its ~ctions: embryonic,

co~ective tissue proper, and specialized connective tissue.

cG

a: LLI

b: ~

u

EMBRYONIC CONNECTIVE TI~U~ • Mesenchyme is derived from embryonic mesoderm and gives rise to various connective tissues of the body. It contains a loose network ofspindle-shaped cells that are suspended in a viscous ground substance containing fine

collagen and reticular fibers.

I

• Mucous connective tissue is present in the wnbilical cord. It contains widely separated. spindle-shaped cells em~ bedded in a gelatin~like, hyaluronan~rich ECM: its ground substance is called Wharton's jelly.

CONNECTM TI~UE PROPER • Connective tissue proper is divided into loose and dense connective tissue. Dense connective tissue is further subdivided into dense lnagular and dense regular connective tissue. • Loose connective tissue is characterized by large numbers of cells of various types embedded in an abundant

gel-like ground substance with loosely arranged fibers. It typically surrounds glands, various tubular organs, and - - - - · blood vcssds and is found beneath the epithelia that cover intemal and external body surfaces. e Dense irregular connective tissue contains few cells (primary nbroblasts), randomly distributed bundles of collagen fibers, and relatively little ground substance. It provides significant strength and allows organs to resist acessive stretching and distension. • Dense regular connective tissue is characterized by densely packed, parallel arrays of collagen fibers with cells (tcndinocytes) aligned between the fiber bundles. It is the main functiocal component of tendons, ligaments, and aponeuroses.

CONNreTM TI~U( FIBDU:

203

• There are three principal types of connective tissue fibers: collagen, reticular, and elastic fibers. • Collagen fibers are the most abundant structural components of the connective tissue. They are flexible, have a - - high tensile strength, and are formed from collagen fibrils that exhibit a characteristic 68-nm banding pattx:rn. n • Collagen fiber fonnatlon involves events that occur both within the fibroblasts (production of procollagen mole:z: cules) and outside the fibroblasts in the ECM (polymerization of collagen molecules into fibril, which are assembled into larger collagen fibers). ~ m • Reticular fibers are composed of type Ill collagen and provide a supporting framework for cells in various tissues ::a ~ and organs (abundant in lymphatic tissues). • In the lymphatic and hemopoietic tissues, reticular fibers are produced by spc:cial.izc:d reticular cells. In most other tissues, reticular fibers are produced by fibroblasts. z z _ _ _.._,. • Elastic fibers are produced by fibroblasts, chondrocytes, endothelial cells, and smooth muscle cells. They allow m tissues to respond to stretch and distension. ~ - - - -• • Elastic fibers are composed of cross-linked elastin molecules associated with a network of flbrlllln mlcroflbrlls, ~ which are made of fibrillin and fibrillin-associated proteins (EMILINs and MAGPs).

,..

8

•• en

cm

OO"RAC(LUJlAR MATRIX • The ECM provides mechanical and structural suppon for connective tissue, influences extracellular communication, and provides pathways for cell migration. In addition to protein fibers, the ECM contains ground substance that is rich in proteoglycans, hydrated glycou.minoglycans (GAGs), and multiadhesive glycoproteins. • GAGs are the most abundant heteropolysaccharide components of ground substance. These molecules are composed of long-chain unbranched polysaccharides containing many sul&te and carboxyl groups. They covalently bind to core proteins to form prateoglycans that are responsible for the physical properties of ground substance. • The largest and longest GAG molecule is hyaluronan. By means ofspecial link proteins, proteoglycans indirectly bind to hyaluronan, forming giant macromolecules called proteoglycan aggregates. • The binding of water and other molecules (e.g., growth factors) to proteoglycan aggregates regulates movement and migration of macromolecules, microorganisms, and metastatic cancer cells in the ECM. • Multiadhesive glycoproteins (e.g., fibronectin,laminin, and tenascin) are multifunctional molecules that possess binding sites for a variety ofECM proteins (e.g., collagens, proteoglycans, and GAGs). They also interact with cell-surface receptors such as integrin and laminin receptors.

CONN(C.TM TI~U£ CEll~ • Connective tissue cells are classified as part of the resident call population (relativdy stable, nonmigratory) or the wandering (or transient) cell population (primarily cells that have migrated from blood vessels). • Resident calls include fibroblasts (and myofibroblasts), macrophages, adipocytes, mast cells, and adult stem cells. Wandering (transient) cells include lymphocytes, plasma cells, neutrophils, eosinophils, basophils, and monocytes (described in Chapter 10, Blood). • Fibroblasts are the principal cells ofconnective tissue. They are responsible for the synthesis ofcollagen and other - components of the ECM. • Fibroblasts that express actin filaments and associated actin motor proteins such as nonmuscle myosin are called myofibroblasts. • Macrophages are phagocytic cells derived from monocytes that contain an abundant number oflysosomes and

play an important role in immune response reactions. are specialized connective tissue cells that store neutral fat and produce a variety of hormones (see Chapter 9, Adipose Tissue). • Mast cells develop in bone marrow and differentiate in connective tissue. They contain basophilic granules that store mediators of in:Bammation. Upon activation, mast cells synthesize leukotrienes, intcrleukins, and other in:Bammarion-promoting cytokines. • Adult stem cells reside in specific locations (called niches) in various tissues and organs. They are difficult to distinguish from other cells of connective tissue. •

I

Adipocytes

I

~r

0

G')

-< .....

...

0

204

PLATE 4

LOOSE AND DENSE IRREGULAR CONNECTIVETISSUE

Loose and dense irregular connective tissue represents two of the several types of connective tissue. The others ere namely cartilage, bona, blood, adipose tissue, end reticular tissue. Loose connective tissue is characterized by a relatively high proportion of cells within a matrix of thin and sparse collagen fibers. In contrast, dense irregular connective tissue contains few cells, almost all of which are fibroblasts that are responsible for the

Loose and dense inegular connective tissue, mammary gland, human, H&E X175; insets X350_ This micrograph shows at low magnification both loose connective tissue (LCT) and dense irregular coaoective tissue (DICT) for comparative purposes. The loose connective tl• sua surrounds the glandular epithelium (GE). The danae Irregular connective tissue consists mainly of thick bundles of collagen fibers

Loose connective tissue, colon, monkey, Mallory trichrome, X250. This micrograph reveala an ~ely cellular loose connective tissue (LC71, also called lamina propria, whkh is located between the intestinal glands of the colon. The aimple, columnar, mu.cus-cecreting epithelial cells seen here represent the glandular tissue. The Mallory stain colors cell maclci reel and

Loose connective tissue, colon, monkey, Mallory trichrome, X700. Shawn at higher magnification is the bozed area in the adjacent figure. The base of the epithelial cells (Ep) is seen on each side of the micrograph. 1he collagen flbers (CF) CF, collagen fibers

DICT, dense irregular connective tissue Ep, epithelial cells

formation and maintenance of the abundant collagen fibers that form the matrix of this tiaaue. The cells that are typically associated with loose connective tissue are fibroblasts, the collagen-forming cells, and those cells that function in the immune system end those of the body's general defense system. Thus, in loose connective tissue, there ere, to varying degrees, lymphocytes, macrophages, eosinophils, plasma cells, and mast cells. with few cells present, whereas the loose connccti~ tissue has a rclati~ paucity of fibers and a considerable number of cells. The upper iNet is a higher magnification of the dense connective tissue. Note that only a few cell nuclei are present relative to the larger expanse of collagen fibers. The IDwer insa, revealing the glandular epithelium and surrounding loose connective tissue, shows very few fibers but large numbers of cells. Typically, the cellular component of loose connective tissue contains a relatively small proportion of fibroblasa h11t large numbers of lymphoc:ytes, plasma cells, and other connective dssue cell types.

collagen blue. Non: how the cells are surrounded by a framework of the blue-mined collagen fibers. Also shown in this micrograph is a band of smooth muscle, the muscularis mUCOIJa (MM) of the colon and below that. seen in pan, is dense irregular connective tissue (DICT) that forms the submucosa of the colon. Typically, the collagen fibers (C) that lie juat below the epithelial cells lf1>) at the luminalsur&ce are more concentrated and thus appear prominently in the micrograph.

appear as thin threads that form a stroma surrounding the cells. The mixture of cells that are present here consists of lymphocytes (L), plasma calla (P), fibroblasts, smooth mwcle cells, macrophagcs (M), and occasional mast cells.

GE, glandular epithelium L, lymphocyte LCT, loose connective tissue

M, macrophage MM, muscularis mucosa P, plasma cells

205

206

PLATE 5 •

DENSE REGULAR CONNECTIVE TISSUE, TENDONS,

AND LIGAMENTS Dense regular connective tissue is distinctive in that its fibers are very densely packed and are organized in parallel array into fascicles. The collagen fibrils that make up the fibers are also arranged in an ordered parallel array. Tendons, which attach muscle to bone, and ligaments, which attach bone to bone, are examples of this type of tissue. Ligaments are similar to tendons in most respects, but their fibers and the organization of the fascicles tend to be lass ordered. In tendons as well as ligaments, the fascicles are separated from one another by dense irregular connective tissue, the endotendineum, through which travel veaaels and nerves. Also, a fascicle may be partially divided by connective tissue septa that extend from the endotendineum and contain the smallest vessels and nerves. Some of the fascicles may be grouped into larger functional units by a thicker, surrounding connective tissue, the peritendineum.

Dense regular connective tissue, tendon, longitudinal section, human, H&E X100.

@

This specimen includes the surrounding dense im:gular connectm: tissue ofthe tendon, the epitendineum (Ept). Th.e tendon fascicles (1F) that make up the tendon arc swrounckd by a less dense connective tissue than that associated with the epitendinmm. In longitudinal sections such as this, the connective tissue that surrounds the individual fascicles, the endotandlneum

6a

Dense regular connective tissue, tendon, longitudinal section, human, H&E X400. This higher magnllit.ation mkrograph shows the ordered single-file array of the tendinocyta nuclei (TC) along with the intervening collap. The latter hal a homogeneow

Dense regular connective tissue, tendon, cross section, human, H&E X400.

@

This specimen is well preserved, and the densely pacla:d collagenous fibers appear as a homogeneous 6.eld, even though the fibers arc viewed on their cut ends. The nuclei appear

BV, blood vessel Ent. endotendineum Ept. epitendineum

Finally, the fascicles and groups of fascicles are surrounded by dense irregular connective tissue, the epitendineum. The fibroblasts, also called tendinocytes in tendons, are elongated cells that possess exceedingly thin, sheet-like cytoplasmic processes that reside between and embrace adjacent fibers. The margins of the cytoplasmic processes contact those of neighboring tendon cells, thus forming a syncytiumlike cytoplasmic network. The most regular dense connective tissue is that of the stroma of the cornea of the eye (see Chapter 24, Eye). In this tissue, the collagen fibrils are anranged in parallel in lamellae that are separated by large, flattened fibroblasts. Adjacent lamellae are arranged at approximately right angles to one another, thus forming an orthogonal arqy.The extreme regularity of fibril size and fibril spacing in each lamella, in conju nc:tion with the orthogonal array of the Ia mellae, is believed to be the basis of corneal transparency.

(EIIt), seems to disappear at cc.rta.in points, with the result that one fascicle appears to merge with a neighboring fascicle. This is due to an obliqueness in the plane of section rather than an actual merging of fascicles. Th.e collagen that mahs up the bulk of the tendon fascicle has a homogcneaw appearance as a rcsult of the orderly packing of the individual collagen fibrils. The nuclei of the tendinocytes appear as elongate profiles arranged in linear rows. The cytopla.sm of these cells blends in with the collagen, leaving only the nuclei as the representative fi:atun: of the cdl.

appearance. The cytopla.sm of the c:dU II indistinguishable &om the collagen, as is typic:al in H&E paraffin specimens. The variation in nuclear appearance is due to the plane ofsection and the position of the nuclei within the thickness of the section. A small blood vessel (BV) coursing within the endotendineum is also present in the specimen.

irregularly scattered. as opposed to their more uniform pattern in the longitudinal plane. 1h.is is explained by examining the tlmhed lint in the lower left figure. which is meant to represent an arbitrary cmaa.sectional cut of the tendon. Note the irregular spacing of the nuclei that arc in the plane of the cut. Lastly. ~ small blood vessels (BV) are present within the endotandlneum (E111) within a fascicle.

TC, tendinocyte nuclei TF. tendon fascicle

d81had line, arbitrary cross-sectional cut of tendon

207

' - Epf .. -

/ · BV •

·

.

,• ·.·r

.~

••

'

--

... .. ~

.

.

·. ..... ,

.. . , ' I

I

' '

,

..

••

•

. ,. .

I

•

. .. ,

....

""';.

..

..

-.- . ... .

r

.;_&

•

•

• j

. .

-- ,...

• "

I

I

I

.

\

.

.

•

•• \

•

;

.

•

•

.. .

•

•

·. ,. ,... .

-• ..

208

PLATE 6

ELASTIC FIBERS AND ELASTIC LAMELLAE

Elastic fibers are present in loose and dense connective tissue throughout the body but in lesser amounts than collagenous fibers. Elastic fibers are not conspicuous in routine H&E sections but are visualized readily with special staining methods. (The following selectively color elastic material: Weigert's elastic tissue stain, purple-violet; Gomori's aldehyde fuchsin stain, blue-black; Verhoaff's hematoxylin elastic tissue stain, black; and modified Taenzei'-Unne orcein stain, red-brown.) By using a combination of the special elastic steins and counterstains, such as H&E, not only the elastic fibers but also the other tissue components may be revealed, thus allowing study of the relationships between the alastic material and other connective tissua components. Elastic material occurs in both fibrous and lamellar forms. In loose and dense connective tissue and in elastic

Elastic fibers, dermis, monkey, Weigert's X160. 1his shows the connective tissue of the skin, referred to as the dennis, stained to show the nature and distribution of 1...-...J...---l the elastic fibers (E), which appear purple. The collagen fibers (C) been stained by eosin, and the two fiber types

rum:

m easily differentiated. The conneAIR OF HYAUNE: CARTILAGE:

Due to its avascular natwe, cartilage has limited ability for repair. Repair mosdy involves - - - - - --:1 the production of dense connective tissue. _ _ _ _ _ _..,.._• In the aging process, hyaline cartilage is prone to calcification and is replaced by bone.

~

0

r

0

G)

-
osteoid deposition occurs, the osteoblast is eventually surrounded by osteoid matrix and thereby becomes an osteocyte. Not every osteoblast is designated to become an osteocyte. Only 10% to 20% of osteoblasts differentiate into osteocytes. Most osteoblasts undergo apoptosis. Others transform

i

into inactive cells and become either periosteal or endosteal bone-lining cells (see Fig. 8.8).

Osteoblast processes communicate with other ostaoblasts and with osteocytn by gap junctions. At the electron microscope levd, osteoblasts exhibit thin cytoplasmic processes that penetrate the adjacent osteoid produced by the cell and are joined to similar processes of adjacent osteocytes by gap junctions. 1h.is early establishment of junctions between an osteoblast and adjacent osteocytes (as well as between adjacent osteoblasrs) allows neighboring cells within the bone tissue to communicate. The osteoblast cytoplasm is characterized by abundant rER and free ribosomes (Fig. 8.10). These features are consistent with its basophilia observed in the light microscope as well as with its role in the production of collagen and proteoglycans for the extracellular matrix. The Golgi apparatus and surrounding regions of the cytoplasm contain numerous vesicles

. .. .. .... . . .... , ··.,

'

' •"•

~.

'

.. ·'"-~.

.

..... ...

FIGURE 8.1 0. Electron micrograph showing active bone formation. This electron micrograph is similar to the growing surface of the bone spicule in the preceding light micrograph !see Fig. 8.9). The marrow cavity (Ml with its developing blood cells is seen in the tower right corner. Osteoprogenitor cells {Opci are evident between the marrow and the osteoblasts {Ob). They exhibit elongated or ovoid nuclei. The osteoblasts are aligned along the growing portion of the bone. which is covered by a layer of osteoid (Os). In this same region. one of the cells (upper right corner) embedded within the osteoid exhibits a small process (err~. This cell. because of its location within the osteoid, can now be called an osteocyte (Ocl. The remainder of the micrograph (upper lefO is composed of calcifl&d bone matrix ICBl. Within the matrix are canaliculi !Cl containing osteocyte processes. The boundary between two adjacent lamellae (L) of previously formed bone is evident as an irregular dark line. X9,000.

with a flocculent content that is presumed to consist of ma~ trix precursors. These vesicles are the PAS~staining granules seen in light microscopy. The matrix vesicles, also produced by the osteoblast, appear to arise by an ectosomal pathway, originating a.s sphe~li.ke outgrowths that pinch off from the apical plasma membrane or microvilli to become free in the matrix. Other cell organelles include numerous rod~shaped mitochondria and occasional dense bodies and lysosomes.

ground sections, the canaliculi are readily evident (Plate 11, page 264). Osteocytes are typically smaller than their precursor cells because of their reduced perinuclear cytoplasm. Ofren, in routinely prepared microscopic specimens, the cell is highly distoned by shrinkage and other artifacts that result from decalcification of the matrix prior to sectioning the bone. In such instances, the nucleus may be the only prominent feature. In well-preserved specimens, osteocytes exhibit less cytoplasmic basophilia than osteoblasts, but little additional Osteocytes cytoplasmic detail can be seen (Plate 12, page 266). The osteocyte is the mature bone cell enclosed by bone Osteocyte• are metabolically actin and multifunctional matrix that was previously secreted as an osteoblast. cells 1hat respond to mechanical forces applied 1o the When completely surrounded by osteoid or bone matrix, the bone. osteoblast is refu.rred. to as an osteocyte (see Fig. 8.9). The In the past, osteocytes were considered passive cells responsible process of transformation from osteoblast to osteocyte takes ap- only for maintaining the bone matrix. Recent discoveries show proximately 3 days. Dwing this time, the osteoblast produces a that osteocytes are metabolically active and multifunctional large amount of cnracellular matrix (nearly 3 times its own cel- cells. They are involved in the process of mec:hanotransduclular volume), reduces its cell volume by roughly 70% in com- tion in which they respond to mechanical forces applied to the parison to the volume of the original osteoblast. decreases size bone. Decreased mechanical stimuli (e.g., immobilizarlon, musand number oforganelles, and develops long cell processes that cle wealcn.ess, weightl.C$Sness in space) causes bone loss, whereas radiare from its cell body. Each osteocyte develops on average increased mechanical stimuli promotes bone formation. about 50 cell processes. Following bone matrix miner.Wzation, Due to the slight flexibility of bone, mechanical forces apeach osteocyte occupies a space, or lacuna, that conforms to the plied to the bone (e.g., to the kmur or tibia during walking) shape of the cell. O.steocytcs' cytoplasmic processes are enclosed cause interstitial fluid to flow out of the canaliculi and lacunae by canaliculi within the matrix. (Fig. 8.11 ). They communicate on the compressed side of the bone. Movement of interstitial with processes ofneighboringosteocytes and bone-lining cells by fluid through the canalicular system generates a transient elecmeans ofgap Junctions formed by a fumily of bone-expressed trical potential (streaming potential) at the moment when connexins. Osteocytes also communicate indirectly with distant the stress is applied. The streaming potential opens voltage--gated osteoblasts, endothelial cells of bone marrow v:asculatu.re, peri- calcium channels in the membranes ofthe oste

~

:lJ

Ci)

~

• BONE AS A TARGET OF ENDOCRINE HORMONES AND AS AN ENDOCRINE ORGAN

,

0

m

z

0

0 0

FIGURE 8.21. Microradiograph of the cross section of a bone. This 200-p.m-thick cross section of bone from a healthy 19-year-old male shows various degrees of mineralization in different osteons. Mature compact bone is actively replacing immature bone, which is seen on the periosteal (upp91'! surface. The degree of mineralization is reflected by the shade of light and dark in the microradiograph. Thus. very light areas represent the highly mineralized tissue that deflects the X-ravs and prevents them from striking the photographic film. Conversely, dark areas contain less mineral and, thus, are less effective in deflecting the X-rays. Note that the interstitial lamellae {the older bone) are very light. whereas some of the osteons are very dark (these are the most newly formed). The Haversian canals appear blsck. as they represent only soft tissue. X 157. (Courtesy of Dr. Jenifer Jowsey.l

bind to type I collagen, which anchors the vesicle to the extracellular matrix. Anne:rin A5 is a channel protein for Ca2+ entry into the matrix vesicles. Influx of Ca2+ into the matrix vesicle is accompanied by simultaneous transport ofP04 ions via the Na+·phosphate cotranaporter 3 (NPT3}. The initial matrix veaicl&-mediated mineralization phase takes place within the matrix vesicles (Fig. 8.26). In this phase, the following events occur: • The matrix vesicles accumulate Ca2+ and P04 ions that cause the local isoelectric point to increase, which results in formation ofsmall (10 nm), nonc.rymlllne spheroidal particles of amorphous calcium phosphate [Ca,(HP04) (P04MOH)], also called calcium-ddicient hydroxyapatite. • Amorphous calciwn phosphate undergoes funher crystallization to octacalcium phosphate [CaaH2(POJ6 • SH20]. • In the presence of a high concentration of Ca2+ and P04 ions, the octacalcium phosphate crysl31 grows within the matrix vesicle into insoluble, needle-like hydroxyapatite crystals [Ca10(P04)t;;(OH),J. Hydroxyapatite crystals accumulate within the matrix vesicle.

Bone serves as a reservoir for body calcium. The maintenance of normal blood calcium levels is critical to health and life. Because bone serves as a reservoir for body calciwn, its release and retrieval from the blood is closely monitored by endocrine hormones such as parathyroid hormone and calcitonin. Calciwn may be delivered &om the bone matrix to the blood. if the circulating blood levels of calcium full below a criri.cal point (ph)'!iologic calcium concentration ranges from 8.9 to 10.1 mgldl). Conversely; excess blood calcium may be removed from the blood and stored in bone. These processes are regulated by parathyroid honnone (Plli}, secreted by the principal (chief) cells of the parathyroid glands, and calcitonin. secreted by the parafollicular cells of the thyroid gland (Folder 8.4).

• Plll acts on the bone to in~fl!~ lbw blood calcium kvtls to normal. PTH release results in the rapid mobilization of eat. from bone. • Calcitonin acts on the bone to tk~as~ ~kvattd blood calcium kv~ls to normal.

PTH regulates the distribution of total body Ca2+. It stimulates both osteocytes and osteoclasts (indirectly via RANKRANKL signaling pathways because osteoclasB do not have PTH receptors) to resorb bone, thereby releasing calciwn into the blood. .Ar. described previously (see page 241-242), resorption of bone by osteocytes oa:urs during osteocytic remodeling. PTH also reduces c:xc.retion of calciwn by the kidney and stimulates absorption of calcium by the small intestine. PTH further acts to maintain homeostasis by stimulating the kidney to excrete the c:xcess phosphate produced by bone resorption. Calcitonin inhibits bone resorption, specifically inhibiting the effects of PTH on osteodasts. It is highly active in young individuals but decreases in activity as individuals age.

:JJ

z m

::I:

0

:JJ

3:: 0

z

m

(/)

~

0

~

~

m

z

0

0

n

:lJ

z

m

0

:JJ

~

Aagen

256 organic and Inorganic substrates

z < (!I

fibrils

P034

a:

matrix

0

vaslcl•medlaled mineralization

w z a: u

phase

0 0

\tHzO

zw z

-< 0

Tl

CD

0

zm

::D

m

~ :ti

d

c

hard callua aoftcallua FIGURE 8.28. Bone fracture and ateges of bone heeling proceu. a. View of healthy bone before fracture. b. The initial response to the injury produces a fracture hematoma that surrounds the ends of the fractured bone. The ends of bone fragments undergo necrosis. An acute inflammatory reaction develops and is manifested by infiltration of neutrophils and macrophages, activation of fibroblasts, and proliferation of capillaries. The fracture hematoma is gradually replaced by granulation tissue. c. Fibrocartilage matrix is deposited. Newly formed fibrocartilage fills the gap at the fracture site producing a soft callus. This stabilizes and binds together the fractured ends of the bone. d. The osteoprogenitor cells from the periosteum differentiate into osteoblasts and begin to deposit new bone on the outer surface of the callus (intramembranous process) until new bone forms a bony sheath over the fibrocartilaginous soft callus. The cartilage in the soft callus calcifies and is gradually replaced by bone as in endochondral ossification. Newly deposited woven bone forms a bony hard callus. e. Bone remodeling of the hard callus transforms woven bone into the lamellar mature structure with a central bone marrow cavity. The hard callus is gradually replaced by compact bone through the action of osteoclasts and osteoblasts, which restores bone to its original shape.

Hormones other than PTH and calcitonin have major effects on bone growth. One such hormone is pituitary growth honnona (GH, somatotropin). This hormone stimulates growth in general and. especially, growth of epiphyseal cartilage and bone. It acts directly on osteoprogenitor cells, stimulating them to divide and differentiate. Chondrocytes in epiphyseal growth plates are regulated by insulin-like growth factor I (IGF-1), which is primarily produced by the liver in response to GH. In addition to IGF-1, insulin and thyroid hormones also stimulate chondrocyte activity. Oversecretion in childhood, caused by a defect in the mechanism regulating GH secretion or a GH-secreting tumor in the pituitary gland, leads to gigantism, an

abnormal increase in the length of bones. Absence or hyposecretion of GH in childhood leads to failure of growth of the long bones. resulting in pituitary dwarfism. Absence or severe hyposecretion of thyroid hormone during development and infancy leads to failure of bone growth and dwarfism, a condition known as congenital hypothyroidism. When oversecretion of GH occurs in an adult, bones do not grow in length as a result of epiphyseal closure. Instead, abnormal thickening and selective overgrowth of hands, feet, mandible, nose, and intramembranous bones of the skull occurs. This condition, known as acromegaly, is caused by increased activity of osteoblasts on bone surfaces.

260

a:

~

w w

a:

z

0 co LL.

0

>-

C)

g 0

co

1&1

z

i

•

a:

5 ::c u

FIGURE 8.28. Photomicrograph of fractured long bone undergoing rep1lr. •· This low-magnification photomicrograph of a 3-week-old bone fracture, stained with H&E. shows parts of the bone separated from each other by the fibrocartilage of the soft callus. At this stage, the cartilage undergoes endochondral ossification. In addition. the osteoblasts of the periosteum are involved in secretion of new bony matrix on the outer surface of the callus. On the right of the microphotograph, the soft callus is covered by periosteum, which also serves as the attachment site for the skeletal muscle. X35. b. Higher magnification of the callus from the area indicated by the upper rectangle in panel • shows osteoblasts lining bone trabeculae. Most of the original fibrous and cartilaginous matrix at this site has been replaced by bone. The early bone is deposited as an immature bone, which is later replaced by mature compact bone. x300. c. Higher magnification of the callus from the area indicated bV the lower rectangle in panel •· A fragment of old bone pulled away from the fracture site bV the periosteum is now adjacent to the cartilage. It will be removed by osteoclast activity. The cartilage will calcify and be replaced by new bone spicules as seen in panel b. X300.

Bane fracture initiates an acuta inflammatory responsa that is necessary for bone healing. The initial response to bone fracture is similar to the response to any injury that produces tissue destruction and hemotthage. Initially, a fracture hematoma (a collection of blood that surrounds the fracture ends of the bones) is fo.rmcd (Fig. 8.28b), and bone necrosis is seen at the ends of the frac.. tured bone fragments. Injury to nearby soft tissues and degranulation of platelets from the blood dot secrete cytokines (e.g., TNF-a, IL-l, IL-6, n,..11, IL-18) and initiate an acute inflammatory mponse. This process is reflected by the infiltta· tion of neutrophlls followed by the migration of macrophages. Fibroblasts and capillaries subsequently proliferate and grow into the site of the injury. Also, specific mesenchymal stem cells arrive at the site of injury from the suttounding soft tissues and bone marrow. The fracture hematoma, which initially contained entrapped erythrocytes within a network of fibrin, is gradually replaced by granulation tissue, a type of newly fonned loose coMective tissue containing collagen type III

and type II fibers. Both fibroblasts and periosteal cells participate in this phase ofhcallng. Granulation tissue 1ransforms into a fibrocartilaginous soft callus, which gives 1he fracture site e stable, semirigid structure. As the granulation tissue becomes denser, chondroblasts differentiate from the periosteal lining, and the newly pro· duced canilage matrix invades the periphery of granulation tissue. The dense connective tissue and newly formed carti· lage grows and covers the bone at the fracture site, producing a soft callus (Fig. 8.28c). This callus will form even if the fractured parts are not in immediate apposition to each other, and it hdps stabilize and bind together the fracnu:ed bone (Fig. 8.29). A bony callus replaces fibrocartilage at the fracture site and allows for weight bearing. While the callus is forming, osteoprogenitor cells of the peri· osteum divide and diffe.rentiate into ostcoblasts. The newly

formed osteoblasu begin to deposit osteoid on the outer c:allus must occur in order to transform the newly deposited surface of the c:allus (intramembranous process) at a di$.- woven bone into a lamellar mature bone. Subsequently. the tance from the fractu~. This new bone formation progresses bone marrow cavity must be restored. While compact bone is toward the fracture site until new bone forms a bony sheath being formed, remnants of the hard callus are removed by the over the fibrocartilaginous callus. Osteogenic buds from the action ofosteoclasts, and gradual bona remodeling restores new bone invade the callus and begin to deposit bone within the bone to its original shape (Fig. 8.28e). In healthy individuals, bona healing usually takes 6 to the c:allus, gradually ~placing the original fibrous and cartilaginous c:allus with a hard callus (Fig. 8.28d). In addition, 12 weeks, depending on the severity of the break and the endosteal proliferation and diffe~tiation occur in the m.al'- specific bone that is fractured. The inflammatory process row cavity; and bone grows from both ends of the fracture lasts approximately 1 week. It is typically accompanied toward the center. When this bone unites, the bony union of by pain and swelling, and it leads to granulation tissue the fractured bone, produced by the ostcoblasts and derived formation. The soft callus is formed approximately 2 to from both the periosteum and endosteum, consists of spongy 3 weeks after fracture. The hard callus in which the fracbone. .& in normal endochondral bone formation, the spongy tured fragments are firmly united by new bone requires 3 bone is gradually replaced by compact bone. The hard callus to 4 months to develop. The process of bone remodeling may last from a few months to several years until the becomes more solid and mechanically rigid.

The remodeling proceu restores the original shape of the bona. Although the hard c:allus is a rigid sttucn:u:e providing mechanical stability to the fracture site, it does not fully restore the properties of normal bone. Bone remodeling of the hard

bone has completely returned to its original shape. Setting the bone (i.e., reapproximating the normal anatomic configuration) and holding the parts in place by internal fixation (by pins, screws, or plates) or by external fixation (by casts or by pins and screws) expedites the healing process.

261

m

0

5 Q 0, m 0

z

m ::D

m

~

::D

262

6 ----~--=~~!!!!!!~· g ---~

~ ~

OVERVIEW OF BON~

------"'

I w_

•

Bone is a specialized type of connective tissue characterized by a mineralized

•

extracellular matrix that stores calcium and phosphate. Bone contributes to the skeleton, which supports the body,

'des the mechanical basis for body movement,

Z

structures, proVI

0

·tat

pandrote~~

rs

m~--------------------------~--1~~ bo~n~e~m ~ u~ro~w~·------------------------------------------·-------

m

-

11:11;

w t: ---~--

c

:::r:::: _ __

G~N(RAL ~TRUC.TUR~ OF BONS!

u

Bones are classified according to shape into long, shon, flat or irregulu bones. Long bones ate tubular in shape and consist of two ends (proximal and distal epiphyses) and a long shaft (diaphysis). Metaphysis is the junction between the diaphysis and the epiphpis. • Bone is covered by periosteum, a connective tissue membrane that attaches to the outer surface by Sharpey • •

•

J • - - -00011

fibers. Periosteum contains a layer of osteoprogenitor (periosteal) cells that can differentiate into osteoblasts. Bone cavities are lined by endosteum, a single layer of cells that contains osteoprogenitor (endosteal) cells, osteoblasts, and osteoclasts. Bones articulate with neighboring bones by synovial joints, a movable connection. The articular surfaces that form contact areas b~ two bones are covered by hyaline (articular) cartilage.

Bone tissue formed during development is called immature (woven) bone. It differs from mature (lamellar) bone in its collagen fiber arrangement. • Bone tissue is classified as either compact (dense) or spongy (cancdlow). Compact bone lies outside and be•

neath the periosteum, whereas an internal, sponge-like meshwork of trabeculae forms spongy bone. •

j • ---"'"~~

Mature (lamellar) bone is mostly composed of ostaons (Haversian systems). These concentric lamellar structures are organized uound an osteonal (Haversian) canal that contains the wscu1ar and nerve supply of the osteon. Perforating (Volkmann) canals are perpendicululy arranged and connect osteonal canals to one another. The lacunae between concentric lamellae contain osteocytes that are interconnected with other osteocytes and the osteonal canal via canaliculi.

C.~ AND ~LLULAR MATRIX • • •

• _ __

1

•

cells in the bone marrow. They differentiate under the influence of the CBFAl transcription factor (RUNX2) into osteoblasts. Osteoblasts differentiate from osteoprogenitor cells and secrete osteoid, an unmineralized bone matrix that undergoes mineralization triggered by matrix vesicles. Osteocytas are mature bone cells enclosed within lacunae of bone matrix. They communicate with other osteocytes by a network of long cell processes occupying canaliculi, and they respond to mechanical forces applied to the bone. Osteoclasis differentiate from hemopoietic progenitor cells; they ttsorb bone matrix during bone formation and remodeling. They differentiate and mature under the control of the RANK-RANKL signaling mechanism. Bone matrix contains mainly type I collagen along with other noncollagenow proteins and regulatory proteins. Osteoprogenitor cells derive from mesenchymal stem

263 BON~ FORMATION • The development of bone is classified as endochondral (a cartilage model serves as the precursor of the bone) or intramembranous ostification (without involvement of a carttlage precursor). • Flat bones of the skull, mandible, and clavicle develop by intramembranous Ostification; all other bones develop by endochondral ossification. • In endochondral ossification, the hyaline cartilage model is formed. Next, osteoprogenitor cells surround.ing this model differentiate into bone~forming cells that initially deposit bone on the cartilage surface (periosteal bony collar) and later penetrate the diaphysis to form rhe prlmary ossification center. • Secondary ossHication centers develop later within the epiphyses. • Primary and secondary ossification centers are separated by the epiphyseal growth plate, seen in children and adolescents, that provides a source for new cartilage involved in bone

-------------------n

:::1:

>

~

m

:a ~ ----------------~m

-----------------z0m

growth.

en

d r 0

G")

• The epiphyseal growth plate has several zones (reserve cartilage, proliferation, hypertrophy, calcified cartilage, and resorption). Resorbed calcified cartilage is replaced by bone.

BONE GROWTH. REIVIODEUNG. AND REPAIR

-------------------'!1

:I:

• Elongation of endochondral bone depends on the interstitial growth of cartilage on rhe epiphyseal growth plate. • Bone increases in widrh (diameter) by appositional growth of new bone that occurs between the compact bone and the periosteum. • Bone is constantly being remodeled throughout life by bon~remodeling units composed of osreoclasts and osteoblasts. This process allows bone to change shape in response to mechanical load. • Bone can repair itself after injury either by a direct (primary) or Indirect (secondary) bone healing process. .After injury, periosteal cells become activated to produce a soft (fibrocartilage) callus, which is subsequently replaced by a hard (bony) callus.

----------------11 •

PIM:IOLOGie ~PEeT~ OF BONE • Bone serves as a reservoir for Ca2 + in the body. Cal+ may be removed from bone if rhe circulating kvd of Ca2 + in the blood &J.ls below the critical value. Likewise, a.cess c~+ may be removed from the blood and stored in bone. e Maintenance of blood Ca2 + levels is regulated by parathyroid hormone (PTH), secreted by the parathyroid glands, and calcitonin, secreted by the thyroid gland. • PTH stimulates both osteocytes and osteodasts (indirectly via RANK-RANKL signaling pathways because osteoclasts do not have PTH receptors) to resorb bone, thereby increasing Cal+ levels in rhc blood. • Calcitonin inhibits bone resorption by inhibiting the effects of PTH on osteoclasts, thereby l~g blood Ca2 + levels.

-


398

w w

z

:::c

1-

:::2:

~

(f)

:::>

g

cytoplasm of the Schwann cell, as noted, is extruded from between the opposing layers of the plasma membranes. Electron micrographs, however, typically show small amounts of cytoplasm in severa.l locations (Figs. 12.14 and 12.15): the inner collar of Schwann cell cytoplasm, between the axon and the myelin; the Schmidt·Lantennan clefts, small islands within successive lamellae of the myelin; perinodal cytoplasm, at the node of Ranvier; and the outer collar of perinuclear cytoplasm, around the myelin (Fig. 12.16). These areas of cytoplasm are what light microscopists identified as the Schwann sheath. However, if one conceptually unrolls the Schwann cell process, as shown in Fig. 12.17, its full extent can be appreciated, and the inner collar of Schwann cell cytoplasm can be seen to he continuous with the body of the Schwann cell through the Schmidt-Lanterman clefts and through the perinodal cytoplasm. Cytoplasm of the clefts contains lysosomes and occasional mitochondria and microtuhules, as

a: w w

z

well as cytoplasmic inclusions, or dense bodies. The number of Schmidt-Lanterman clefts correlates with the diameter of the axon; larger axons have more clefts.

Unmyelinated axons in the peripheral nervous system are enveloped by Schwenn cells and their external lamina. The nerves in the PNS that are described as unmyelinated are nevertheless enveloped by Schwann cell cytoplasm as shown in Fig. 12.18. The Schwann cells are elongated in parallel to the long axis of the axons, and t:b.e axons fit into grooves in the surfuce of t:b.e celL The lips of the groove may be open, exposing a portion of the axolemma of t:b.e axon to the adjacent external lamina of the Schwann cell, or the lips may be closed, forming a mesaxon. A single axon or a group ofamns may be enclosed in a single invagination of the Schwann cell swface. Large Schwann cells in the PNS may have 20 or more grooves, each containing one or more axons. In the ANS, it is common for bundles of wunyellnated axons to occupy a single groove.

Satellite Cells

:::c

The newonal cell bodies ofganglia are surrounded by a layer of small cuboidal cells called satellite cells. Although they form a complete layer around the cell body, only their nuclei are typically visible in routine H&E preparations (Fig. 12.19a and b). In paravertebral and peripheral ganglia, neural cell processes must penetrate between the satellite cells to establish a synapse (there are no synapses in sensory ganglia). They help to establish and maintain a controlled microenvironment around the neuronal body in the ganglion, providing electrical insulation as well as a pathway for metabolic exchanges. Thus, the functional role of the satellite cell is analogous to that of the Schwann cell except that it does not make myelin.

ILL

0

~ ...J w u (!)

z

li: 0

D... D...

:::>

(f)

•

1&1

::::)

Enteric Neuroglial Cells

!

Neurons and their processes located within ganglia of the enteric division of the ANS are associated with enteric neuroglial cells. These cells are morphologically and functionally similar to eatrocytes in the CNS (see below). Enteric neuroglial cells share common functions with astrocytes, such as structural, metabolic, and protective support of neurons. However, recent studies indicate that enteric glial cells may also participate in enteric neurotransmission and help coordinate activities of the nervous and immune systems of the gut.

1&1

~ z

1&1

.. N

Central Neuroglia There are four types of central neuroglia:

• Aatrocytes are morphologically heterogeneous cells that provide physical and metabolic support for neurons of the

CNS. FIGURE 12.14. El-.:tron micmgn~ph of an axon in th• pracns of myelination. At this stage of development, the myelin (M) shea1h consists of about six membrane layers. The inner mesaxon (/Ml and outer mesaxon (OM) of the Schwann cell (SCI represent parts of the mesaxon membrane. Another axon (see upper left A) is present that has not yet been embedded within a SC me saxon. Other notable features include the SC basal (extemal) lamina (8LI and the considerable amount of Schwann cell cytoplasm associated with the myelination process. X50,000. (Courtesy of Dr. Stephen G. Waxman.)

• Oligodendrocytes are small cells that are active in the formation and maintenance of myelin in the CNS. • Microglia are inconspicuous cells with small, dark, elongated nuclei that possess phagocytotic properties. • Ependymal cells are columnar cells that line the ventricles of the brain and the central canal of the spinal cord. Only the nuclei of glial cells are seen in routine histologic preparations of the CNS. Heavy metal staining or

399

~

:!I m

:a ..a

!lo1

zm ~

m -t

~

m

•c (/)

""C

(g ~ zCi) (")

m FIGURE 12.1&. Electron micrograph of a mature myelinated axon. The myelin sheath (M) shown here consists of 19 paired layers of Schwenn cell membrane. The pairing of membranes in each layer is caused by the extrusion of the Schwann cell cytoplasm. The axon displays an abundance of neurofilaments, most of whid1 have been cross-sectioned, giving the axon a stippled appearance. Also evident in the axon are microtubules (Mn and several mitochondria (Mil). The outer collar of Schwann cell cytoplasm (OCS) is relatively abundant compared with the inner collar of Schwann cell cytoplasm (ICSl. The collagen fibrils (C) constiMe the fibrillar component of the endoneurium. BL, basal (external! lamina. X70,000. lns.t. Higher magnification of the myelin. The arrow points to cytoplasm within the myelin that would contribute to the appearance of the Schmidt-Lanterman cleft as seen in the light microscope. It appears as an isolated region here because of the thinness of the section. The intercellular space between the axon and Schwann cell is indicated by the arrowhead. A coated vesicle (C\.1 in an early stage of formation appears in the outer collar of the Schwenn cell cytoplasm. X130,000. (Courtesy of Dr. George D. Pappas.)

r ~ 0

"TI

-I I

m

z

m

::c

cs

c

(/)

~

m

~

-I I

FIGURE 12.18. Dillgnm af the node of Ranvier and auociated Schwann cells. This diagram shows a longitudinal section of the axon and its relationships to the myelin, cytoplasm of the Schwann cell, and node of Ranvier. Schwann cell cytoplasm is present at four locations: the inner and the outer cytoplasmic collar of the Schwann cell, the nodes of Ranvier, and the Schmidt-Lanterman clefts. Note that the cytoplasm throughout the Schwann cell is continuous (see Fig. 12.171; it is not a series of cytoplasmic islands as it appears on the longitudinal section of the myelin sheath. The node of Ranvier is the site at which successive Schwenn cells meet. The adjacent plasma membranes of the Schwann cells are not tightly apposed at the node, and extracellular fluid has free access to the neuronal plasma membrane. The node of Ranvier is also the site of depolarization of the neuronal plasma membrane during nerve impulse transmission and contains clusters of higiHiensity, voltage-gated Na+ channels.

m

zm

c

::c 0

Ci)

r

>

voltage-gated Na+ channels perinodal cytoplasm

of Schwann cell

inner collar of Schwarm cell cytoplasm

400

interdepandence of neuroglial cells and neurons. The

~~ perinodal - cytoplasm of Schwann cell

w w

SchmiatLanterman clefts

z

:::c

1-

~

~

::::>

g a:

z

1-

nucleus of Schwarm cell

~ ...J w u

FIGURE 12.17. Three-dimeMional diagrams conceptualizing the relatlonahlp of myelin end cytoplaam of a Schwann cell. This diagram shows a hypothetically uncoiled Schwann cell. Note how the inner collar of the Schwann cell cytoplasm is continuous with the outer collar of Sc:hwann cell cytoplasm via Schmidt-Lanterman clefts.

:::c u.. 0

(!)

z

li: 0

D... D...

::::>

(f)

•

Astrocytes are closely associated with neurons to support and modulate their activities. Aatrocytea are the largest ofthe neuroglial cells. They form a network ofcells within the CNS and communicate with neu-

(f)

w w

most obvious example of physical support occurs during development. The brain and spinal cord develop &om the embryonic neural tuba. In the head region, the neural tube undergoes remarkable thickening and folding, leading ultimately to the final structure, the brain. During the early stages of the process, embryonic glial cells extend through the entire thickness of the neural tube in a radial manner. These radial glial celis serve as the physical scaffolding that directs the migration of neurons to their appropriate position in the brain.

immunocytochemical methods are necessary to demonstrate the shape of the entire glial cell. Although glial cella have long been described as supporting cells of nerve tissue in the purely physical sense, current concepts emphasize the functional

rons to support and modulate many of their activities. Some astrocytes span the entire thickness of the brain, providing a scaffold for migrating neurons during brain development. Other astrocytes stretch their processes &om blood vessels to neurons. The ends of the processes expand, forming end feet that cover large areas of the outer surface of the vessel or axolemma. Astrocytes do not form myelin. Two kinds of astrocytes are identified:

• Protoplasmic astrocytea are more prevalent in the outermost covering of brain called gray matter. These astrocytes have numerous, short, branching cytoplasmic processes (Fig. 12.20). • Fibrous astrocytea are more common in the inner core of the brain called white matter. These astrocytes have fewer processes, and they are relatively straight (Fig. 12.21).

1&1

::::)

! 1&1

~ z

1&1

.. N

FIGURE 12.18. Electron micrograph of unmyelinated nerve flbera. The individual fibers or axons !A) are engulfed by 1he cytoplasm of a Schwann cell. The arrows indicate the site of mesaxons. In effect, each A is enclosed by the Schwann cell cytoplasm, except for the intercellular space of the mesaxon. Other features evident in the Schwenn cell are its nucleus (NJ. the Golgi apparatus {G), and the surrounding basal (external} lamina {BLJ. In 1he upper part of the micrograph, myelin {M) of two myelinated nerves is also evident. x27.000. Inset. Schematic diagram showing the relationship of A engulfed by 1he Sc:hwann cell.

401

n

~.. :II ~

z m

~

:::1

c = m

• (I)

c

"'t]

~ ~

z C) 0 m r

(;; 0

FIGURE 12.19. Photomlcroai'8Ph of a nerve ganglion. a. Photomicrograph showing a ganglion stained by the Mallory-Azan method. Note the large nerve cell bodies !arrows) and nerve fibers (NF) in the ganglion. Satellite cells are represented by the very small nuclei at the periphery

,

of the neuronal cell bodies. The ganglion is surrounded by a dense irregular connective tissue capsule (C7) that is comparable to, and continuous with, the epineurium of the nerve. x200. b. Higher magnification of the ganglion, showing individual axons and a few neuronal cell bodies with their satellite cells (a/TOWS). The nuclei in the region of the axons are mostly Schwann cell nuclei. X640.

m

~ z m

~ 0 c(I)

~

m

~

~ z m m

c

8 r

)>

blood vessel FIGURE 12.20. Protoplasmic mro~ In the gray m.U.r of the brain. a. This schematic drawing shows the foot processes of a protoplasmic astrocyte terminating on a blood vessel and the axonal process of a nerve cell. The foot processes terminating on the blood vessel contribute to the blood-brain barrier. The bare regions of the vessel as shown in the drawing would be covered by processes of neighboring astrocytes, thus forming the overall barrier. b. This laser-scanning confocal image of a protoplasmic astrocyte in the gray matter of the dentate gyrus was visualized by intracellular labeling method. In lightly fixed tissue slices, selected astrocytes were impaled and iontophoretically injected with fluorescent dye (Alexa Fluor 568) using pulses of negative current. Note the density and spatial distribution of cell processes. X480. (Reprinted with permission from Bushong EA. Martone ME, Ellisman MH. Examination of the relationship between astrocyte morphology and laminar boundaries in the molecular layer of adult dentate gyrus. J Comp Neurol 2003;462:241-251.)

402

w w

z

:::c

1-

~

~

(f)

::::>

g a: w w

z

:::c

ILL

FIGURE 12.2 t. Fibrous astrocytes In the white matter of the brain. a. Schematic drawing of a fibrous astrocyte in the white mater of the brain. b. Photomicrograph of the white matter of the brain, showing the extensive radiating cytoplasmic processes for which astrocytes are named. They are best visualized, as shown here, with immunostaining methods that use antibodies against glial fibrillary acidic protein (GFAP). x220. {Reprinted with permission from Fuller GN, Burger PC. Central nervous system. In: Sternberg SS, ed. Histology for Pathologists. Philadelphia: Lippincott-Raven, 1997.1

0

~ ...J w u (!)

z

li: 0

D...

D...

::::>

(f)

•

Ill

:::::»

! Ill

~ z

Ill

.. N

Both types of asttocytes contain prominent bundles of in the brain's extracellular space by asuocytes is called potasintermediate filaments composed of glial fibrillary acidic sium spatial buffering. protein (GFAP). The filaments are much more numerous Oligodendracvtes produce and maintain the myelin sheath in the fibrous asttocytes, however, hence the name. Antibod· in the CNS. ies to GFAP are used as specific stains to identify asttocytes in sections and tissue cultures (see Fig. 12.21b). Tumors The oligodendrocyte is the cell responsible for producarising from fibrous astrocytes, fibrous astrocytomas, ing CNS myelin. The myelin sheath in the CNS is formed account for about 80% of adult primary brain tumors• by concentric layers of oligodendrocyte plasma membrane. They can be identified microscopically and by their GFAP The formation of the sheath in the CNS is more complex, however, than the simple: wrapping of Schwaan cell mesaxon specificity. Astrocytes play impon:an.t roles in the movement of me· membranes that occurs in the PNS (pages 193-194). Oligodendrocytes appear in specially stained light micro· tabolites and wastes to and from neurons. They hdp maintain the tight junctions of the capillades that form the blood- scopic preparations as small cells with relatively few processes brain barrier (see page 415). In addition, asuocytes provide compared with asttocytes. They are often aligned in rows be· a covering for the "bare areas" of myelinated axons-for tween axons. Each oligodendrocyte gives off several tongu~ example, at the nodes of Ranvier and at synapses. They may like processes that make contact with nearby axons. Each confine new:ouansmitters to the synaptic clefi: and remove process wraps itself around a portion of an axon, forming an excess neurotransmitters by pinocytosis. Protoplasmic internodal segment of myelin. The multiple processes of astrocytes on the brain and spinal cord surfaces extend a single oligodendrocyte may myelinate one axon or several their processes (subpial feet) to the basal lamina of the pia nearby axons (Fig. 12.23). The nucleus--containing region of mater to form the glia limitans, a relatively impermeable the oligodendrocyte may be at some distance from the axons it mydinates. barrier surrounding the CNS (Fig. 12.22). Because a single oligodendrocyte may myelinate sev· Astrocvtes modulate neuronal activities by buffering the eral nearby axons simultaneously. the celt cannot embed tc+ concentration in the extracellular space of the brain. multiple axons in its cytoplasm and allow the mesaxon It is now generaiiy accepted that astrocytes regulate K+ membrane to spiral around each axon. Instead, each concentrations in the brain's extracellular compartment, tongue·lik.e process appears to spiral around the axon, al· thus maintaining the microenvironment and modulating ways staying in proximity to it, untU the myelin sheath is activities of the neurons. The astrocyte plasma membrane formed. contains an abundance of K+ pumps and K+ channels that mediate the transfer of K+ ions from areas of high to low The myelin sheath in the CNS differs from that in the PNS. concenttation. Accumulation of large amounts of intracellu· There are several other important differences between the mylar K+ in asttocytes decreases local extracellular K+ gradients. elin sheaths in the CNS and those in the PNS. OligodendroThe: astrocyte membrane becomes depolarized, and the charge cytes in the CNS express different myelin-specific proteins is dissipated over a large area by the extensive network of~ during myelination than those expressed by Schwaan cells in trocyte processes. The maintenance of the K+ concentration the PNS. Instead of PO and PMP22, which are expressed only

basement membrane subpial foot process

gila llmltans

403

pia mater

I I

n

~.. :II !~!:'

z m

~

:::1

c = m

• (I)

c

"'tt

~

neuron oligomicroglial cell myelin dendrocyte

astrocyte FIGURE 12.22. Distribution of glial cells in the brain. This diagram shows the four types of glial cells--estrocytes, oligodendrocytes, microglial cells, and ependymal cells-interacting with several structures and cells found in the brain tissue. Note that the astrocytes and 1heir processes interact with 1he blood vessels as well as with axons and dendrites. Astrocytes also send 1heir processes toward the brain surface, where 1hey contact the basement membrane of the pia mater, forming the glia limitans. In addition, processes of astrocytes extend toward the fluid-filled spaces in the central nervous system (CNSI. where they contact the ependymal lining cells. Oligodendrocytes are involved in myelination of the nerve fibers in the CNS. Microglia exhibit phagocytotic functions.

~

z G) 0 m r

In

,0

~ z m m

in myelin of the PNS, other proteins, including proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), and oligodendrocyte myelin glycoprotein (OMgp), perform similar functions in CNS myelin. Defi· ciencies in the expression of these proteins appear to be

nerve~

fibers

~

~~

~myelin

node of Ranvier (showing axon in contact with extracellular space)

FIGURE 12.23. Three-dimensional 'VIew of an oligodendrocyte as it na.te. to "veral axons. Cytoplasmic processes from the oligodendrocyte cell body form flattened cytoplasmic sheaths 1hat wrap around each of the axons. The relationship of cytoplasm and myelin is essentially the same as that of Schwann cells.

important in the pathogenesis of several autoimmune

demyelinating diseases of the CNS. On the microscopic level, myelin in the CNS exhibits fewer Schmidt-Lantennan clefts because the asuocytes provide metabolic support for CNS neurons. Unlike Schwann cells of the PNS, oligodendrocytes do not have an exte.rna1 lamina. Furthermore, because of the manner in which oligodendrocytes fonn CNS myelin, Uttle or no cytoplasm may be present in the outennost layer of the myelin sheath, and with the absence ofexternal lamina. the myelin ofadjacent axons may come into contact. Thus, where myelin sheaths of adjacent axons touch, they may share an inuaperiod line. Finally. the nodes ofR.mvier in the CNS are luger than. those in the PNS. The larger areas of exposed axolemma thus make saltatory conduction (see below) even more efficient in the CNS. Another difference between the CNS and the PNS in regard to the relationships between supporting cells and neurons is that unmyelinated newons in the CNS are often found to be bare-that is, they are not embedded in glial cell processes. The Ia.ck of supporting cells around unmyelinated axons as well as the absence of basal Iantina material and connective tissue within the substance of the CNS helps to distinguish the CNS from the PNS in histologic sections and in TEM specimens.

Microglia possess phagocytotic properties. Microglia are phagocytotic cells. They nonnaiiy account for about 5% of all glial cells in the adult CNS but proliferate and become actively phagocytotic (reactive microglial cells) in regions of injury and disease. Microglial cells are

~ 0 c(I)

~

m

s: ~ z m m

c

8 r

)>

404

w w

z

:::c

1-

~

~

(f)

::::>

g a: w w

FIGURE 12.24. Microglial call in the tray matter of the brain. a. This diagram shows the shape and characteristics of a microglial cell. Note the elongated nucleus and relatively few processes emanating from the body. b. Photomicrograph of microglial cells (arrows) showing 1heir characteristic elongated nuclei. The specimen was obtained from an individual with diffuse microgliosis. In this condition, the microglial cells are present in large numbers and are readily visible in a routine hematoxylin and eosin IH&E) preparation. X420. (Reprinted with permission from Fuller GN. Burger PC. Central nervous system. In: Sternberg SS. ed. Histology for Pathologists. Philadelphia: Lippincott-Raven. 1997.)

z

:::c

1u.

0

~ ...J w u (!)

z

li: 0

D...

D...

::::>

(f)

•

Ill

:::::»

! Ill

considered part of the mononuclear phagocytotic system (see Folder 6.4, page 197) and originate from granulocyte/ monocyte progenitor (GMP) cells. Microglia precursor cells enter the CNS parenchyma from the vascular system. Recent evidence suggests that microglia play a critical role in defense against invading microorganisms and neoplastic cells. They remove bacteria, injured cells, and the debris of cells that undergo apoptosis. They also mediate neuroimmune reactions, such as those occurring in chronic pain conditions.

Microglia are the smallest of the neuroglial cells and have relatively small, elongated nuclei (Fig. 12.24). When stained with heavy metals, microglia exhibit short, twisted processes. Both the processes and the cell body are covered with numerous spikes. The spikes may be the equivalent of the ruffled border seen on other phagocytotic cells. The TEM reveals numerous

lysosomes, inclusions, and vesicles. However, microglia contain little rER and few microtubules or actin filaments.

Ependymal cells fonn the epithelial-like lining of the ventricles of the brain and spinal canal• Ependymal cells form the epithelium-like Uning of the fluid-filled cavities of the CNS. They form a single layer of cuboidal-to-columnar cells that have the morphologic and physiologic characteristics of fluid-transporting cells (Fig. 12.25). They are tightly bound by jWlctional complexes located at the apical surfaces. Unlike a typical epithelium, ependymal cells lack an external lamina. At the TEM Ievd, the basal cell surf.u:e exhibits numerous infoldings that interdigitate with adjacent astrocyte processes. The apical surface of the cell possesses cilia and microvilli. The latter are involved in absorbing cerebrospinal fluid.

~ z

Ill

.. N

FIGURE 12.2&. Ependymal lining of the aplnal canal. a. Photomicrograph of the central region of the spinal cord stained with toluidine

blue. The an-ow points to the central canal. X20. b. At higher magnification, ependymal cells, which line the central canal, can be seen to consist of a single layer of columnar cells. X340. (Courtesy of Dr. George D. Pappas.) c. Transmission electron micrograph showing a portion of the apical region of two columnar ependymal cells. They are joined by a junctional complex {JC) 1hat separates the lumen of the canal from the lateral intercellular space. The apical surface of the ependymal cells has both cilia (C) and microvilli {M). Basal bodies {88) and a Golgi apparatus (G) within the apical cytoplasm are also visible. X20,000. (Courtesy of Dr. Paul Reier.)

Tanycytes are specialized types of ependymal cells. They are most numerous in the floor of the third ventricle. The free surface of tanycyte.s is in direct contaCt with cerebrospinal 8uid, but in contraSt to the ependymal cell$, they do not possess cilia. The cell body of tanyqtes gives rise to a long process that projecu into the brain parenchyma. Their role remains unclear; however, they are involved in the transport ofsubstances from the cerebrospinal8uid to the blood within the portal circulation of the hypothalamus. Tanycytes are sensitive to glucose concentration; therefOre, they may be involved in detecting and responding to changes in energy balance as well as in monitoring other circulating metabolites in the cerebrospinal fluid. Within the system of brain ventricles, the epitheliumlike lining is further modified to produce the cerebrospinal 8uid by transport and secretion of materials derived from adjacent capillary loops. The modified ependymal cells and associated capillaries are called the choroid plexus.

Impulse Conduction An action potential is an electrochemical process triggered by impulses carried to the axon hillock after other impulses are received on the dendritu or the cell bodyibeH.

A nerve impulse is conducted along an axon much as a flame ttave4 along the fuse ofa fi.recra.cker. This electrochemical process involves the generation of an action potential, a wave of membrane depolarization that is initiated at the initial segment of the axon hillock. Its membrane contains a large number of voltage-gated Na+ and K+ channels. In response to a stimulus, voltage-gated Na+ channels in the initial segment of the axon membrane open, causing an in8ux of Na+ into the axoplasm. This influx of Na+ briefly reverses (depolarizes) the negative membrane potential of the resting membrane (-70 mV) to positive (+30 mV). After depolarization, the voltage-gated Na+ channels dose and voltage-gated K+ channels open. K+ rapidly exits the axon, returning the membrane to its resting potential. Depolarization of one part of the membrane sends electrical current to neighboring portions of unstimulated membrane, which is still positively chazged. This local current stimulates adjacent portions of the axon's membrane and repeats depolarization along the membrane. The entire process takes less than 1,OOOth of a second. After a very bdef (refractory) period, the neuron can repeat the process of generating an action potential once again. Rapid conduction af the action potential is attributable to the nodes of Ranvier. Myelinated axons conduct impulses more rapidly than unmyelinated axons. Physiologists describe the nerve impulse as '"jumping'" from node to node along the myelinated axon. This process is called saltatory {L. taltus, to jump] or discontinuous conduction. In myelinated nerves, the myc:Un sheath around the nerve does not conduct an electric current and forms an insulating layer around the axon. However, the voltage reversal can only occur at the nodes of Ranvier, where the axolemma lacks a myelin sheath. Here, the axolemma is exposed to cxt:J:acc:llular fluids and possesses a high concentration of voltage-gated Na+ and K+ channels (see Figs. 12.16

and 12.23). Thus, the voltage reversal (and, thus, the impulse) jumps as current flows from one node of Ranvier to the next. The speed of saltatory conduction is related not only to the thickness of the myelin but also to the diameter of the axon. Conduction is more rapid along amns of greater diameter. In unmyelinated axons, Na+ and K+ channels are distributed uniformly along the length of the fiber. The nerve impulse is conducted more slowly and moves as a continuous wave ofvoltage reversal along the axon.

405

n

~.. :II

• ORIGIN OF NERVETISSUE CELLS CNS neurons and central glia. except microglial cells, are derived from neuroectodermal cells af the neural tube. Neurons, oligodendrocytes, asttocytes, and ependymal cells are derived from cells of the neural tube. After developing neurons have migrated to their predestined locations in the neural tube and have differentiated into mature neurons, they no longer divide. However, in the adult mammalian brain, a very small number of neural stem cells retain the ability to divide. These cells migrate into sites of injury and differentiate into fully functional nerve cells. Oligodendrocyte precursors are highly migratory cells. They appear to share: a developmental lineage with motor neurons migrating from their site of origin to developing axonal projections (tracts) in the white matter of the brain or spinal cord. The: precursors then proliferate in response to the local expression of mitogenic signals. The matching of oligodendrocytes to axons is accomplished through a combination of local regulation of cell proliferation, differentiation, and apoptosis. Astrocytes are also derived from cells of the neural tube. During the embryonic and early postnatal stages, immature astrocytes migrate into the cortex, where they differentiate and become mature asttocytes. Ependymal cells are derived from the proliferation of neuroepithelial cells that immediately surround the canal of the developing neural tube. In contrast to other central neuroglia, microglia cells are derived from mesodertnal macrophage precursors, specifically from granulocyte/monocyte progenitor {GMP) cells in bone marrow. They infiltrate the neural tube in the early stages of its development and under the influence of growth factors such as colony stimulating factor-1 (CSF-1) produced by developing neural cells as they undergo proliferation and differentiation into motUe ameboid cells. These motile cells are commonly observed in the developing brain. As the only glial cells of mesenchymal origin, microglia possess the vimentin class of intermediate filaments, which can be useful in identifying these cells with immunocytochemical methods. PNS ganglion calls and peripheral glia are derived from the neural crest. The development of the ganglion cells of the PNS involves the proliferation and migration of ganglion precursor cells from the neural crest to their future ganglionic sites, where they undergo further proliferation. Here, the cells develop processes that reach the cells' target tissues (e.g., glandular tissue or smooth muscle cells) and sensory territories. Initially, more cells ate produced than are needed. Those that do not m~ functional contact with a target tissue undergo apoptosis.

!~!:'

z m

~

:::1

c = m

• 0

:JJ G')

z

., 0

z

m

~

m

-1

~

c

m 0

m r

In

406

~

~

~

(f.)

::::::1

52 a: w

z

_J

~ w

::I: a_

a: w

a_

w

::I:

ILL

0

z

0

~ z

....,

axon degeneration

z

~ z

0

oligodendrocyte ·shedding myelin debris

resident macrophage

c::

::::> w

z

apolis

Schwann cell •Shedding myelin debris

1.1..

0

w

(/)

z

a.. (/)

reactive microglia

w

c::

•

w

J

myelin

= i=

~

~

- /rapid clearance

w

/

w

z

15

n

persistance of myelin debris

r-'

~~ myelin debris

!

...

N

It: c

myelin

axon injury

::;)

t

C

microglia

- -.... • ·dedifferentiation ·divisions

0

~

!

(/)

••

axon regeneration

failure or axon regeneration

bloodnerve barrier

bloodbrain barrier

FIGURE 12.37. Schematic diagram of .-.pun.. to neuronal injury within peripheral and ~ntral nervous sys•ms. Injuries of nerve processes (axons and dendrites) both in the peripheral nervous system (PNSl and central nervous system (CNS) induce axonal degeneration and neural regeneration. These processes involve not only neurons but also supportive cells such as Schwann cells and oligodandrocytes as wall as phagocytic cells such as macrophages and microglia. Injuries to axons in PNS lead to their degeneration, which accompanies divisions and dedifferentiation of Schwann cells and disruption of the blood-nerve barrier along the entire length of the injured axon. This allows massive infiltration of monocyte-derived macrophages, which are responsible for the process of myelin removal. Rapid clearance of myelin debris allows for axon regeneration and subsequent restoration of the blood-nerve barrier. In the CNS, limited disruption of the blood-brain barrier restricts infiltration of monocytEH:Ierived macrophages and dramatically slows the process of myelin removal. In addition. apoptosis of oligodendrocytes. inefficient phagocytic activity of microglia, and the formation of an astrocytEH:Ierived scar lead to failure of nerve regeneration in the CNS.

perinuclear cytoplasm and organelles statts within a few days. The cell body of the injured nerve swells, and its nucleus moves peripherally. Initially, Nissl bodies disappear from the center of the neuron and move to the periphery of the neuron in a process called chromatolysis. Chromatolysis is first observed within 1 to 2 days after injury and .reaches a peak at about 2 weeks (see Fig. 12.36b). The changes in the cell body are proportional to the amount of axoplasm destroyed by the injury; extensive loss of axoplasm can lead to death of the cell. Before the development of modern dyes and radioisotope tracer techniques, Wallerian degeneration and chromatolysis were used as research tools. These tools allowed researchers to trace the pathways and destination of axons

and the localization of the cell bodies of experimentally injured nerves.

Regeneration In tba PNS. Scbwann calls divide and develop cellular

bands that bridge 1 newly farmed scar and direct growth of new nerve proceaes. & mentioned above, division of dedifferentiated Schwann cells is the first step in the regeneration of a severed or crushed peripheral nerve. Initially, these cells arrange themselves in a series of cylinders called endoneuria! tubes. Removal of myelin and axonal debris from inside the tubes causes them to eventually collapse. Proliferating Schwann

FOLDER 12.3

419

CLINICAL CORRELATION: REACTIVE GLIOSIS: SCAR FORMATION IN THE CENTRAL NERVOUS SYSTEM When a region of the CNS is injured, astrocytes near the lesion become activated. They divide and undergo marked hypertrophy with a visible increase in the number of their cytoplasmic processes. In time, the processes become densely packed with GFAP intennediate filaments. Eventually, scar tissue is formed. This process is referred to as reactive gliosis, whereas the resulting permanent scar is most often called a plaque. Reactive gliosis varies widely in duration. degree of hyperplasia. and time course of expression of GFAP immunostaining. Several biological mechanisms for induction and maintenance of reactive gliosis have been proposed. The type of glial cell that responds during reactive gli-

osis depends on the brain structure that is damaged. In addition, activation of the microglial cell population occurs almost immediately after any kind of injury to the CNS. These reactive microglial cells migrate toward the site of injury and exhibit marked phagocytic activity. However, their phagocytic activity and ability to remove myelin debris is much less than that of monocyte-derived macrophages. Gliosis is a prominent feature of many diseases of the CNS. including stroke, neurotoxic damage, genetic diseases, inflammatory demyelination, and neurodegenerative disorders such as multiple sclerosis. Much of the research in CNS regeneration is focused on preventing or inhibiting glial scar formation.

n :z:

,.

~ m ::a ~

"' 2

~

m -1

c = m

• ::0

m

Cl)

cells organize themsdves into cellular bands resembling longitudinal columns called bands of BU.ngner. Cellulae bands guide the growth of new nerve processes (neurites or sprouts) of regenerating axons. Once the bands ace in place, large numbers of sprouts begin to grow &om the proximal srump (see Fig. 12.36c). A growth cone develops in the distal portion of each sprout that consists of filopodia rich in actin filaments. The tips of the filopodia establish a direction for the advancement of the growth cone. 1hey preferentially interact with proteins of the extracellular matrix such as fibronectin and laminin found within the external lamina of the Schwann cell. Thus, if a sprout associates itsdf with a band of Bii.ngner, it regenerates between the layers of external lamina of the Schwann cell. This sprout will grow along the band at a rate of about 3 mm per day. Although many new sprouts do not make contact with cellulae bands and degenerate, their large: number increases the probability of reestabl..is1llng sensory and motor connections. After crossing the site of injury, sprouts enter the surviving cellular bands in the distal stump. These bands then guide the neurites to their destination as well as

provide a suitable microenvironment for continued growth (Fig. 12.36d). Axonal regeneration leads to Schwann cell redifferentiation, which occurs in a proximal-to-distal direction. Redifferentiated Schwann cells upregulate genes for myelin-specific proteins and downregulate c-jun.

H physical contact is raastalllishad batwaan a motor neuron and its muscle, function is usually reestablished. Microsurgical techniques that rapidly reestablish intimate apposition of severed nerve and vessel ends have made reattachment of severed limbs and digits, with subsequent reestablishment of function, a relatively common procedure. If the axonal sprouts do not reestablish contact with the appropriate Schwann cells, then the sprouts grow in a disorganized manner, resulting in a mass of tangled axonal processes known as a traumatic neuroma or amputation neuroma. Clinically, a traumatic neuroma usually appears as a freely movable nodule at the site of nerve injury and is characterized by pain, particularly on palpation. Formation of a traumatic neuroma of the injured motor nerve prevents reinnervation of the affected muscle.

"1J

0

z

(/)

m

0

"T1

z c

m ::0

0

z

Cl)

d t:

c ~

420

....

0

>-

(!)

g 0

~

OV£RV1~ OF TH( N[RVOU~ m'~M

:I:

. ------------------------

• The nervous system enables the body to respond to changes in its external

w ---------------------------

i

e

I!11.1 ~

11.1

..ffi

z

__________________________, e

N

e

li: ~

u

environ~~~t

:f::~:t 7~~tha:'!.~:~ervous

andth controls functi:: system Anatomlowy, e nervous sys (PNS· peripheral (CNS; brain and spinal cord) and the peripheral nervous system ' and cranial nerves and ganglia). . ..is divided into the somatiC nervous • .,.._m Functionally, the n~ous system l) d the autonomic nervous system (SNS; under consc1ous voluntary centro an (ANS· under involuntary control). . d nta · ' • ~--Lbdi 'ded into sympathetic, parasympathetiC, an e nc The ANS lS runw;r su Vl • canal and lates the function ---• regull ll gl dular divisions The enteric division serves the alimentary . . . oth and cardiac mu.5ae cc s as we as an ofinternal organs by mnervatmg smo epithelium.

~UPPORTING cau! OF TH( NERVOU~ m1!HM:

-

N(UROGLIA

• Peripheral neuroglia includes Schwann cells and satellite cells. • In myelinated nerves, Schwann cells produ.a: the myelin sheath from compacted layers of their own cell membranes that are wrapped concentrically around the nerve cell process. • The junction between two adjacent Schwann cells, the node of Ranvier, is the site when: the electrical impulse is regenerated for high-speed propagation along the axon. • In unmyelinated nerves, netvt: processes are enveloped in the cytoplasm of Schwann cdls. • Satellite cells maintain a controlled microenvironment around the nerve cell bodies in ganglia of the PNS. • There are four types ofcentral neuroglia: astrocytas (provide physical and metabolic support fur newons of the CNS), oligodendrocytes (produce and maintain the myelin sheath in the CNS), microglia (possess phagocytotic properties and mediate neuroimmWle reactions), and ependymal calls (form the epithelial-like lining of the ventricles of the brain and spinal canal).

NEURON~ • Nerve tluue consists of two principal types of cells: neurons (specialized cells that conduct impulses) and supporting cells (nonconducting cells in close proximity to nerve cells and their processes). • The newon is the structural and functional unit of the nervous system. • Neurons do not divide; however, in certain regions of the brain, neural stem calls may divide and differentiate into new neurons. • Neurons are grouped into three categories: sensory neurons (catty impulses from receptors to the CNS), motor neurons (carry impulses from the CNS or ganglia to effector cells), and intemeurons (communicate berween sensory and motor neurons). • Each neuron consists of a call body or perikaryon (contains the nucleus, Nissl bodies, and other organelles), an axon (usually the longest process of the cell body; transmit! impulses away from the cell body), and several dendrites (shorter processes that transmit impulses toward the cell body). • Neurons communicate with other neurons and with dfector cells by specialized junctions called synapses. • Chemical synapses are the most common type of synapse. Each has a presynaptic element containing vesicles filled with neurotransmitter, a synaptic cleft into which neurotransmitter is released from the presynaptic vesicles, and a postsynaptic membrana containing receptors to which the neurotransmitter binds. • Electrical synapses are less common and are represented by gap junctions. • lhe chemical structure of a neurotransmitter determines either an excitatory (e.g., acetylcholine, glutamine) or Inhibitory (e.g., GABA, glycine) response from the postsynaptic membrane.

421 ORIGIN OF N(RV( TI~U( C(W! • CNS neurons and central gl.ia (except microglial cells) are derived from neuroectodermal cells of the neural tube. _ _ _ ____,. • PNS ganglion cells and peripheral gl.ia an: derived from the neural crest.

n :z:

,..

~ m ::a ~

ORGANIZATION OF TH~ P~IPH~L N~U~ ~n;M • The PNS consists of peripheral nerves with specialized nerve endin~ (synapses) and ganglia containing nerve cell bodies. • Motor neuron cell bodies of the PNS lie in the CNS and sensory neuron cell bodies are located in the dorsal root ganglia. - - - --1111 • Individual nerve fibers are hdd together by connective tissue organized into endoneurium (surrounds each individual nerve fiber and associated Schwann cell), perineurium (surrounds each nerve fascicle), and epineurium (surrounds a peripheral nerve and :fills the spaces betwc:c:n nerve fascicles). • Perineurial cells are connected by tight junctions and contribute to the formation of the blood-nerve barrier.

----~&---------------~~------------------------------

"' 2

~

m

....

c = m

• :I:

en

d;0

G)

ORGANIZATION OF TH( C(NTRAL N~RVOU~ W~TJ;M

- - - -"1

• The CNS consists of the brain and spinal cord. It is protected by the skull and vertebrae and is surrounded by three connective tissue membranes called meninges (dura matter, arachnoid, and pia matter). • The cerebrospinal fluid (CSFI produced by the choroid plexus in the brain ventricles occupies the subarachnoid space located between arachnoid and pia matter. CSF surrounds and protects the CNS within the cranial cavity and the venebral column. • In the brain, the gray matter forms an outer layer of the cerebral cortex, whereas the white matter forms the inner core that is composed of axons, associated glial cells, and blood vessels. • In the spinal cord, gray matter exhibiu a butterfly-shaped inner substance, whereas the white matter occupies the periphery. • The cerebral cortex contains nerve cell bodies, axons, dendrites, and central glial cells. • The blood-brain barrier protects the CNS from B.ucruating levels of dectrolytes, hormones, and tissue metabolites circulating in the blood.

ORGANIZATION OF TH£ AUTONOMIC NEMJU~ ~

- - - --ill

• The ANS controls and regulates the body's internal environment. Its neural pathways are organized in a chain of two neurons (presynaptic and postsynaptic neurons) that convey impulses from the CNS to the visceral effectors. • The ANS is subdivided into sympathetic, parasympathetic, and enteric divisions. • Presynaptic neurons ofthe sympathetic division are located in the thoracolumbar portion ofthe spinal coni., whereas the presynaptic neurons of the parasympathetic division are located in the brainstem and sa.cJ:al spinal cord. • The enteric division of the ANS consists of ganglia and their processes that innervate the alimentary canal.

RS:PO~E OF NEURON~ TO

INJURY

• Injured axons in the PNS usually regenerate, whereas axons severed in the CNS do not regenerate. 1his difference is related to the inability of oligodendrocytcs and microglia cells to efficiently phagocytose myelin debris. • In the PNS, neuronal injury initially induces complete degeneration of an axon distal to the site of injury _ _ _ _..,. (Wallerian degeneration). _ _ _ _.....,. • Traumatic degeneration occurs in the proximal part of the injured nerve, followed by neural regeneration, in which Schwann cells divide and develop cellular bands that guide the growing axonal sprout& to the effector site.

-< ..... g

422

PLATE 27

SYMPATHETIC AND DORSAL ROOT GANGLIA

Ganglia are clusters of neuronal call bodies located outside the central nervous systBm (CNS); nerve fibers lead to and from them. Sensory ganglia lie just outside the CNS and contain the cell bodies of sensory nerves that carry impulses into the CNS. Autonomic ganglia are peripheral motor ganglia of the autonomic nervous system (ANSI and contain the cell bodies of postsynaptic neurons that conduct nerve impulses to smooth muscle, cardiac muscle, and glands. Synapses between presynaptic neurons (all of which have their cell bodies in the CNS) and postsynaptic neurons occur in autonomic ganglia. Sympathetic ganglia constitute a major subclass of autonomic

tE

Sympathetic ganglion, human, silver and H&E stains, X160.

tE tE

Sympathetic ganglion, human, silver and H&E stains, X500.

This micrograph shows a sympathetic ganglion stain!Cd with silver and countcmain!Cd with H&E is illuruated here. Shown to adwn.tage arc several disctetc bundles of ru:rvc fibers (NF) and numerow large citcular suuctures, namely, the cell bodies (CBJ of the postsynaptic neurons. Random parterDS of nerve

The cell bodies of the sympathetic ganglion are typically I:uge, and the one labeled here shows several processes (P). In addition, the cell body contains a large, pale-staining sphcrlw nucleus (N); this, in turn, contains a sphcrlw, inn:nsdy

Dorsal root ganglion, cat, H&E, X 160.

Dorsal root ganglia differ from autonomic ganglia in a number of ways. Whereas the latter contain multipolar neworu and have synaptic connecti0118, dorsal root gaDglia contain pseudounipolar sc:nsory neurons and have no synaptic connections in the ganglion.

Dorsal root ganglion, cat, H&E, X350.

EB

At higher magnification of the same ganglion, the cowtituents of the nerve fiber show their characteristic structure. namely, a centrally locat!Cd axon (A) surrounded by an empty space after myclin 'NliS washed out during slide preparation (not labeled), which, in turn, is bounded on ia outer border by the thin cytoplasmic stta.nd of the neurilemma

(arrowheds).

A. axon BY, blood vessels CB, cell body of neuron CT, connective tissue

ganglia; parasympathetic ganglia and enteric ganglia constitute the other subclasses. Sympathetic ganglia are located in the sympathetic chain (paravertebral ganglial and on the anterior surface of the aorta (prevertebral ganglia). They send long postsynaptic axons to the viscera. Parasympathetic ganglia (tenninal ganglia) are located in, or close to, the organs innervated by their postsynaptic neurons. The enteric ganglia are located in the submucosal plexus and the myenteric plexus of the alimentary canal. They receive parasympathetic presynaptic input as wall as intrinsic input from other enteric ganglia and innervate smooth muscle of the gut wall.

fibers arc also seen. Moreover. cardUI aarnination of the cdJ bodies shows that some display several proccsses joinca to them. Thw, these are multipolar neurons (one contained within the m14ng/e is shown at higher magnification). Generally, the connective tissue is not conspicu· ow in a silver preparation, although it can be idcotifi!Cd by virtue of its loc:a.tion around the larger blood vessels (BV), particularly in the upptr pllrtof this figure.

staining nudco!Q& (NL). These: features, namdy, a large pale·.naining nucleus (indicating much extended chromatin) and a large nucleolus, rdlca a cell active in protein synthesis. Aha shown in the cell body arc ~uladons of lipofwcln (L), a yellow pigment that is darkenca by the silver. Because of the large s1ze of the cell body, the nucleus is not always included in the section; in that case, the cell body appears as a roundca cytoplasmic mass.

Part ofa dorsal root ganglion sta.inca with H&E is shown in this fig· ure. The specimen includes the edge of the ganglion, where it is covered with connective tissue (CT). The dorsal root ganglion coatairu large cdJ bodies (CB) that arc typically arranged as closely pada:d clwtcrs. Also, b~ and around the cdl clwtcrs, there arc bundles of nerve fibers (NF). Most of the fiber bundles ind.ic:a.t!Cd by the label have been section!Cd longitudinally. The cell bodies of the sensory neurons display largt:, pale--staining spherical nuclei (N) and inten1cly staining nucleoli (NL). Also seen in this H &:E preparation are the nuclei of satellite cells (Sat C) that completely swround the cdl body and arc contiouow with the Schwann cells that invest the uon. Note how much smaller these cells arc compared with the neurons. Ouaters of cells (amriskr) within the ganglion that have an epithelioid appearance arc en face views of satellite cells where the section tangentially includes the satdlite cdJs but barely grazes the adjacent cell body.

L, lipofuscin N, nucleus of nerve cell NF. nerve fibers NL. nucleolus

P. processes of nerve cell body S.t C, satellite cells arrowheads, neurilemma astarlllb, clusters of satellite cells

423

424

PLATE 28 •

PERIPHERAL NERVE

Peripheral nerves are compoaed of bundles of nerve fibera held together by connective tissue and a specialized layer (or layers) of cells, the perineurium. The connective tissue consists of an outer layer, the epineurium, sur· rounding the whole nerve; the perineurium, surrounding bundles of nerve fibers; and the endoneurium, associated with individual neurons. Each nerve fiber consists of an axon that is surrounded by a cellular investment

tE

Peripheral nerve, cross section, femoral nerve, H&.E, X200 and 640. This aoss section shows several bundles of ~ fibers (BNF). The external cover for the en~ nerve is the eplneurtum (Epn), the layer of dense connective tissue

that one touches when a nerve has been etposed. during a dissection. The epineurium may also serve as part of the outennost cover of indi· vidual bundles. It contains blood veuels (BV) and may contain some fat a:J.Ls. Typially. adipose tissue CAn surrou.nd.s the nerve. The figun: on the ri,h# mom, u hlgher magnification, the perineur· ial septum (muked with llmnliS on the kft image. which is now rotated and vercically diapo~ed). The layer beneath the epineurium that directly IUIIOundJ the bundle of nerve Sben il the perineurium (h). As seen in the crou section throup the nerve, the nuclei of the perineurial ccl1s appeu flat and elongated; they arc actually being viewed on edge and belong to flat cella that arc also being viewed on edge. Again, as noted by the distribution of nuclei, it can be ascertaincd that the perineurium is only a few cells thick. The perineurium is a specialaed laytr of cells and enracellular

tE

Peripheral nerve, longitudinal section, femoral nerve, H&.E, X200 and 640.

lhe ~ of a longitudlnally aectioned nerve bundle ls shown on the kft; a portion of the ume netve bundle ls shown u higher magnification on the rlf)t. The boundary between the epineurium (Ep,.) and perineurium is ill-defined. Within the nerve bundle. the nerve fibers show a characteristic wavy pattern. Included among the wavy nerve fiben are nuclei belonging to Sohwann cells and to cdls within the endoneurium. ffisher magnification allows one to identify certain specific components of the nerve.

A. axon AT. adipose tissue BNF, bundle of nerve fibers BV, blood vessels C, capillary

called the neurtlemma or the sheath of Schwann. The fiber may be myelinated or unmyelinated. The myelin, if present, is immediately around the axon and is formed by the concentric wrapping of the Schwann cell around the axon. This, in turn, is surrounded by the major portion of the cytoplasm of the Schwenn cell, forming the neurilemma. Unmyelinated axons rest in grooves in the Schwenn cell.

material whose arnngement is not evident in H&E sections. The per· ineurium (PrJ) and epineurium (Ep•} are readily distinguished in the triangular area formed by the divctging perineurium ofthe two adjacent nerve bundles. The nerve fibers included in the figure on the ri,h# are mostly myelinued, and because the nerve is cross-sectioned. the nerve fiben arc also seen in this plane. They have a characteristic cro.u-sccrlonal profile. Each nerve fiber shDWII a c:cnually placed axon ~}; this is surrounded by a myelin space (M) in wbkh some radially di.po~ed precipitate may be retained, u in this •pedmen. External to the mydin space is a thin c1lar surfAce is the pia mater (Pw). Cerebellar blood vC88cls (BY') navel in this layer. (Shrinkage artifact has separated the pia mater &om the ccrd>c1lar surface.) The rraanplar aru is shown at higher magnification in the figure on the right.

surrounding the nucleus. In contrast. the granule cell layer presents an overall spotted-blue appearance due to the staining of numcrow small nuclei with hematoxylin. These small neurons, called granule cells, receive incoming impulses &om other parts of the CNS and send axons into the molecular layer, where they branch in the form of a T, 80 that the axons contact the dendrites ofseveral Purkinje cells and basket cells. Incoming (mossy) fibers contract granule cells in the lighdy !taincd areas called tlomn"Uii (11770WS). Careful examination of the granule cell layer where it meets the molccular layer will reveal a group of nuclei (G) that~ larger than the nuclei of granule cells. These belong to Golgi type II cells.

magnification shown here. however, the Purki.nje cells can be recognized in the sUvcr preparation because of their large size, characteristic shape, and location bci:'M:en an outer molecular layer (Mol) and an inner gran· ulc c:dllayer (Gr). The main advantage of this silver preparation is that the white matter {WM) can be recognized as being composed of fibers; they haft: been blackened by the silver--staining procedure. The pia mater (Pill) and cerebellar blood vessels (B\1 ~ also evident in the preparation.

the granule cell layer (Gr), about the Purkinje cell bodies, and in the molecular layer (Mol) disposed in a horizontal direction (relatift: to the cerebellar sur&ce). Ba.sket cells (BCJ ~ the mast common neurons that ~ visible in the molecular layer. The lilmiW indicates a T turn characteristic of the turn made by uons of granule cells. h these axonal branches navel horizontally, they make synaptic contact with numerous Purkinje ccll1. ai'I'OWS, upper right figure, glomeruli; lower right figure, T branching of axon in molecular layer ntertangular ...... areas shown at higher magnification

429

430

PLATE 31

SPINAL CORD

The spinal cord is organized into two discrete parts. The outer part, called the white matter of the cord because of its appearance in unfixed specimens, contains ascending and descending nerve fibers. Soma of the fibers go to and from the brain, whereas others connect different levels of the spinal cord. The inner part of the spinal cord, called the gray matter because of its appearance in unfixed specimens, contains the cell bodies of neurons as well as nerve fibers. The gray matter forms an H- or butterflyshaped pattern surrounding the central canal. The gray matter is described as having dorsal (posterior) homs

Spinal cord, human, silver stain, X16. A cross section through the lower lumbar region of the spinal cord is shown here. The preparation is designed to stain the gray manu that is $urtounded by the a.stending and destending ~ fibers. Although the 6ben that have common origins and destinations in the physiologic: sellSe m ar~ iD tracts, these ttacu cannot be distinguished UD!ess they have been marked by special n:chniques, such as causing injury to the cell bodies from which they arise or by using special dyes or radioisotopes to label the: axons.

Ventral hom. spinal cord, human, silver stain, X640. This pn:paration showa a n:gion of a ventral horn. The nucleua (N} of the ventral hom cell (ventral motor neuron) is the large, spherical, pale-staining structure

Ventral hom, spinal cord, human, toluidine blue, X640.

@

This preparation of the spinal c:ord is from an area comparable to the left image. Three ventral hom cells (ventral motor ncwollS) are visible. Due to plane of the section,

BV, blood vessels DH, dorsal horn DR. dorsal root GC, gray commissure

and ventral (antertor) homs. The ventral horns contain the Ia rge call bodies of ventral motor neurons, whereas the dorsal horns contain neurons that receive, process, and retransmit information from the sensory neurons whose cell bodies are located in the dorsal root ganglia. The size of the gray matter {and, therefore, the size of the spinal cord) is different at differant levels. Where the gray matter contains many large motor nerve cells that control the movement of the upper and lower limbs, the gray matter and the spinal cord are considerably larger than where the gray matter contains only the motor neurons for the muscle of the torso.

The gray matter of the: spinal cord appears roughly in the form of a butterfly. The anterior and posterior prongs are referred to as vmtnzl horns (VH) and dtJrstd horns (DH), respectively. The connecting bar is called the: gnty crJmmisturff (GC). The neuron cell bodies that are within the ventral horns (ventral horn cells) are so large that they c:a.n be seen even at this crtremely low magni6cation (tln'bWS). The pale-staining fibrous material that surrounds the spinal cord i5 the: pia mater (Pill). It follows the s1Uf3cc of the spinal c:ord intimately and dips into the large ventral fissure (VF) and into the ahallower sulci. Blood vessels (BV) m present in the pia mater. Some donal roots (DR) of the: 5pinal nerves m included In the sec:tfon. within the: cell body. The ventral horn cell has many obvious processes. A number of other nuclei bdong to neuroglial cells. The cytoplasm of these cells i5 not evident. The remainder of the 6.dd c:ollSists of nerve fibers and neuroglial cells whose organization is hard to interpret. This is called the neuropil (Np).

only two of th.c:m exhibit large pale-staining nuclei (N) with da.dr.-stain· ing nucleoli in the tenter. The toluidine blue reveals the Nlssl bodies (NBJ that appear as the large. dark-staining bodies iD the cytoplasm. Nissl bodies do not cnend into the amn hillock. The amn leaves the cell body at the amn hillock. The nuclei of neuroglial cells (NN) are also evident here,

N, nucleus of ventral horn cell NB. Nissl bodies NN, nucleus of neuroglial cell Np, neuropil

Pia, pia mater VF. ventraI fissure VH, ventral hom arrows, cell bodies of ventral horn cell

431

CARDIOVASCUtAR SYSTEM OVERVIEW OFTHE CARDIOVASCULAR SYSTEM/432 HEART /433 Wall of the Heart /434 Heart Valves /436 Intrinsic Regulation of Heart Rate /438 Systemic Regulation of Heart Function 1439 GENERAL FEATURES OF ARTERIES AND VEINSI440 Layers ofVascularWall I 440 Vascular Endothelium /442 ARTERIES I 447 Large Arteries (Elastic Arteries) I 447 Medium Arteries (Muscular Arteries) I 450 Small Arteries and Arterioles I 451

CAPILLARIES /452 Classification of Capillaries /453 Functional Aspects of Capillaries 1454 ARTERIOVENOUS SHUNTS I 455 VEINS /455 Venules and Small Veins 1455 Medium Veins 1456 Large Veins I 457 ATYPICAL BLOOD VESSELS I 458 LYMPHATIC VESSELS /459 Folder 13.1 Clinical Correlation: Atherosclerosis 1442 Folder 13.2 Clinical Correlation: Hypertension /448 Folder 13.3 Clinical Correlation: Coronary Heart Disease I 460

HISTOLOGY 101/462

• OVERVIEW OFTHE CARDIOVASCULAR SYSTEM The cardiovascular system is a transpon system that carries blood and lymph to and from the tissues of the body. The constitutive dements of these fluids include cells, nutrients, waste products, hormones, and antibodies.

Tha cardiavascular system includaa tha haart blood VBIIBII, and lymphatic VBIIBII. The cardiovascular system consists of a pump, represented by the heart, and the blood vessels, which provide the route by which blood circulates to and from all parts of the body (Fig. 13.1). The heart pumps the blood through the anerial system under significant pressure; blood is returned to the bean under low pressure with the assistance of negative pressure in the thoracic cavity during inspiration and compression of the veins by skeletal muscle. The blood vessels are arranged so that blood delivered from the heart quickly reaches a network of narrow, thin-walled vessels--the blood capillaries -within or in proximity to the tissues in every part of the body. In the capillaries, a two-directional exchange of fluid occurs between the blood and tissues. The fluid, called blood filtrate, carries oxygen and metabolites and passes

432

8

through the capillary wall. In the tissues, these molecules are exchanged for carbon dioxide and waste products. Most of the fluid reenters the distal or venous end of the blood capillaries. The remaining fluid enters lymphatic capillaries as lymph and is ultimately returned to the bloodstream through a system of lymphatic vessels that join the blood system at the junction of the internal jugular veins with the subclavian veins. Normally, many of the white blood cells conveyed in the blood leave the blood vessels to enter the tissues. This occurs at the level of the postcapillary venules. When pathologic changes occur in the body, such as an inflammatory reaction, large numbers of white blood cells emigrate from thasa venules.

Arteries are the vessels that deliver blood to the capillaries. The smallest arteries, called arterioles, are functionally associated with networb of capillaries into which they ddiver blood. The anerioles regulate the amount of blood that enters these capillary networb. Together, the arterioles, associated capillary network, and postcapillary venules fonn a functional unit called the microcirculatory or microvascular bed of that tissue. Veins, beginning with the postcapillary venule, collect blood from the microvascular bed and carry it away.

left atrium

433

left pulmonary vein

aorta

valve

FIGURE 13.1. Photograph of the human heart. This specimen was sectioned in the oblique plane to visualize all of the chambers of the heart. The posterior part of the heart is on 1he left; the anterior part has been removed and is shown on the right. Note the thickness of the ventricular walls and the intermuscular septum. The interatrial septum. which separates the atria, is also visible.

Two circuits distribute blood in the body: tile sys1emic and tile pulmonary circulations. Two pathways of circulation arc formed by the blood vessels and the heart:

preventing baddlow of blood. An interatrial septum and an interventricular septum separate the right and left sides of the heart. The right side of the heart pumps blood through the pul· monary circulation. The right atrium receives deoxygenated

• Pulmonary circulation conveys blood from the heart to the lungs and from the lungs to the heart (Fig. 13.2). • Systemic circulation conveys blood from the heart to other tissues of the body and from other tissues of the body to the heart.

arch of

aorta

Although the general arnmgement of blood vessels in both circulations is from arteries m capillaries m veins, in some pam of the systemic circulation, it is modified so that a vein or an artel'iole is interposed between two capillary networks; these vessels constitute a portal system. Venous portal systems occur in vessels carrying blood to the .lM:.r, namdy, the hepatic portal system (portal vein), and in vessels leading to the pituitary, the hypothalamic-hypophyseal portal system.

left atrium

•HEART The heart lies obliquely, about two·thirds into the left side of the thoracic cavity, in the middle mediastinum-the space enclosed by the sternum, vertebral column, diaphragm, and lungs. It is surrounded by a tough fibrous sac, the pericafdium, from which the great vessels enter and leave the heart. Through the pericardium, the heart is attached to the dia· pb.ragm and neighboring organs that lie in the thoracic cavity. The heart is a muscular pump that maintains unidirectional flow of blood. The heart contains four chambers-the right and left aaia and right and left ventricles-through which blood is pumped (see Fig. 13.1). Valves guard the exits of the chambers,

right

left ventricle

ventricle FIGURE 13.2. Diagram deplc11ng circulation of blood through tile heart. Blood returns from 1he tissues of the body via the superior vena cava and inferior vena cava. These two major venous vessels carry the blood to the right atrium. Blood then passes into the right ventricle and is pumped into the pulmonary trunk before flowing into the pulmonary arteries. which convey the blood to the lungs. The blood is oxygenated in the lungs and is then returned to the left atrium via the pulmonary veins. Blood then passes to the left ventricle and is pumped into the aorta, which conveys the blood to the tissues of the body. From the heart to the lungs and from the lungs to the heart constitutes the pulmonary circulation; from the heart to 1he tissues and from the tissues to the heart constitutes the systemic circulation.

434

pulmonary

valve

right

ventrlcfe

(high pressure) FIGURE 13.3. Diagram of the blood circulation. This diagram shows the right and left sides of the heart artificially separated. The right side of the heart pumps blood through the low-pressure pulmonary circulation. The right atrium receives deoxygenated blood returning from the body via the inferior and superior venae cavae. The right ventricle receives blood from the right atrium and pumps it to the lungs for oxygenation via the pulmonary arteries. The left side of the heart pumps blood through the high-pressure systemic circulation. The left atrium receives the oxygenated blood rerurning from the lungs via the four pulmonary veins. The left ventricle receives blood from the left atrium and pumps it into the aorta for systemic distribution.

blood returning from the body via the inferior and superior venae cavae, the two largest veins of the body (Fig. 13.3). The right ventricle receives blood from the right atrium and pumps it to the lungs for oxygenation via the pulmonary arteries. The left side of the heart pumps blood through the systemic circulation. The left atrium receives the oxygenated blood returning from the lungs via the four pulmonary veins. The left ventricle receives blood from the left atriwn and pwnps it into the aorta for distribution to the body. The heart contains the following: • A musculature of cardiac muscle for contraction to propel the blood • A fibrous skeleton that consists of four fibrous rings surrounding the valve orifices, two fibrous trigones connecting the rings, and the membranous part of the interventricular and interatrial septa. The fibrous rings are composed of dense irregular connective tissue. They encircle the base of the two arteries, leaving the heart (aorta and pulmonary trunk) and the openings between the atria and the ventricles (right and left atrioventricular [AV] orifices) (Fig. 13.4). These rings provide the attachment site for the leaflets of all four valves of the heart that allow blood B.ow in only one direction through the openings. The membranous part of the interventricular septum is devoid of cardiac muscle; it consists of dense connective tissue that contains a short length of the atrioventricular bundle of the condUcting system of the heart. The fibrous skeleton provides independent attachments for the atrial and ventricular myocardium. It also acts as an electrical insulator by preventing the free B.ow ofelectrical impulses between atria and ventricles. • A conducting system for initiation and propagation of rhythmic depolarizations, which results in rhythmic cardiac muscle contractions (Fig. 13.5). This system is

formed by modif11d cardiac muscle cells (Pultcin)e fibers}, which generate and conduct electrical impulses rapidly through the heart. In cardiac atTeSt, the sudden cessation of normal heart rhythm leading to abrupt cessation of blood circulation, the conducting system of the heart fails to produce or conduct electrical impulses that cause the heart to contract and supply blood to the body. Sudden cardiac arrest is a medical emergency; first-aid treatment such as cardiopulmonary resusci· tation {CPR) and defibrillation (delivering a therapeutic dose of electrical energy to the heart) can improve the chances of survival. If not treated, cardiac arrest leads to sudden cardiac death. Heart rhythm pathologies associated with cardiac arrest include tachycardia (accelerated heart rhythm), fibrillation {rapid, irregular, and ineffective contractions), bradycardia (decelerated heart rhythm), and asystole (total absence of heart rhythm}. • A coronary vasculature that consists of two coronary arteries and cardiac veins. The right and left coronary arteries provide the arterial blood supply to the heart. They originate from the initial part of the ascending aorta near the aortic valves and circle the base of the heart, with branches converging toward the apex of the heart. Venous drainage of the heart occurs via several cardiac veins, most ofwhich drain into the coronary sinus located on the posterior surface of the heart. The coronary sinus drains into the right atrium.

Wall of the Heart The wall of the heart is composed of three layers: epicardium, myocardium, and endocardium. The structural organization of the wall of the heart is continuous within the atria and ventricles. The wall of the heart is

fibrous ring of

fibrous ring of pulmonary

conus

trunk

ligament

membranous part of interventricular

septum

opening for atrioventricular

bundle (of His) FIGURE 13.4. F1brou• 1keleton of the heart u teen with the two at1111 removed. This fibrous network {indicated in light blue) serves for the attachment of cardiac muscle; it also serves for the attachment of the cuspid valves between the atria and ventricles and for the semilunar valves of the aorta and the pulmonary artery. The atrioventricular bundle passes from the right atrium to the ventricular septum via the membranous septum of the fibrous skeleton.

tissue. The blood vessels and nerves that supply the heart lie in the epicardium and are surrounded by adipose tissue that cushions the heart in the pericardia! cavity. The epicardium is reflected back at the great vessels entering and leaving the heart as the parietal layer of serous pericardium, which lines the inner surface of the pericardium that surrounds the heart and roots of great vessels. Thus, there is a potential space containing a minimal amount (15 to 50 ml) of serous (pericardia!) fluid between the visceral and parietal layers of the serous pericardium. This space is known as the pericardial cavity; its lining consists of mesothelial cells (see Fig. 13.6). • Cardiac tamponade is a condition in which excess

right atrium

FIGURE 13.&. Chambers of the heart and the impuiMconductlngaystem. The heart has been cut open in the coronal plane to expose its interior and the main parts of its impulse-conducting system (indicated in yellow!. Impulses are generated in the sinoatrial (SA) node. transmitted through the atrial wall to the atrioventricular (A\I) node, and then sent along the AV bundle to the Purkinje fibers.

composed of three layers. From the outside to the inside, they are as follows: • The epicardium, also known as the visceral layer of serous pericardium, adheres to the outer surface of the heart (Fig. 13.6). It consists of a single layer of mesothelial cells and underlying connective and adipose

fluid (blood or pericardia! effusion) rapidly accumulates in the pericardia! cavity. It is commonly caused by both blunt and penetrating chest injuries and by myocardial rupture or pericarditis (inflammation of pericardium). This condition is potentially life-threatening because the accumulating fluid may compress the heart and prevent the heart's chambers from filling properly with blood. Relieving the pressure is usually accomplished with pericardiocentesis (a procedure to drain the fluid from the pericardia! cavity). • The myocardium, consisting of cardiac muscle, is the

principal component of the heart. The detailed histologic strUCture and function of cardiac muscle is discussed in Chapter 11, Muscle Tissue. The myocardium of the atria is substantially thinner than that of the ventricles. The atria receive blood from the large veins and deliver it to adjacent ventricles, a process that requires relatively low pressure. The myocardium of the ventricles is substantially myocardium

epicardium

--~adipose tissue ~--+-visceral

layer of serous pericardium

1!-,---~ parietal

layer of

serous pericardium I- - - + - fibrous pericardium

right ventricle

RGURE 13.8. Layers of the heart aftd pericardium. This schematic diagram shO'Ns the anatomic relationship between the layers of the heart. In the middle mediastinum, the heart and roots of the great vessels are surrounded by the pericardium, which is often covered by highJv variable amounts of adipose tissue. The pericardium has two layers: a tough external fibrous layer called the fibrous pericardium and a parietal layer of serous pericardium that lines its inner surface. The parietal layer of the serous pericardium is reflected back at the great vessels entering and leaving the heart as the visceral layer of the serous pericardium or epicardium. The epicardium lines the outer surface of the heart. The pericardia! cavity is a space between the visceral and parietal layers of the serous pericardium, and it is lined by the mesothelial cells. Deep to the epicardium is the myocardium, which consists of cardiac muscle. Note the small amount of adipose tissue in the epicardium, which contains the coronary arteries and cardiac veins. The inner layer of the myocardium is called the endocardium. which is lined by the mesothelium with an underlying thin layer of connective tissue.

435

layer of connective tissue and smooth muscle cells, and a deeper layer of connective tissue, which is also called the subendocardial layer. The latter is continuous with the connective tissue of the myocudium. The conducting system of the heart is located in the subendocardial layer of the endocardium (see the following section called "Intrinsic R£gulation of Heart Rate").

436

The interventricular septum is the wall between the right and left ventricles. It contains cardiac muscle in all but the membranous portion. Endocardium lines each surface of the interventricular septum. The interatrial septum is much thinner than the interventricular septum. Except in certain localized areas that contain fibrous tissue, it has a cen· ter layer of cardiac muscle and a lining ofendocardium fucing each chamber.

Heart Valves ROURE 13.7. tloltzontal section through the ventrtdes of the heart This photograph shows a cross section of the human heart at the level of the wntricles. Cusps of bath the tricuspid valve in the right ventricle and the mitral valve in the left ventricle are visible with their attadlments to the dlordae tendineae. Cross sections of the papillary muscles in both ventricles are visible. Note the differences in the thiacness between the wall of the right and left ventricles. Adipose tissue of the epicardium contains .brandles of the coronary arteries and tributaries of the cardiac veins. RV. right ventricle; LV. left ventricle. !Courtesy of Dr. William D. Edwards.)

thicker because of the higher pressure required to pwnp the blood through the puhnonary and systemic circulations (Fig. 13.7). • The endocardrum consists of an inner layer of endothelium and subendothelial connective tissue, a middle

Heart valves are composed of three distinct layers of connective tissue with overlying endocardium. The heart valves attach to the complex framework of dense irregular connective tissue that forms the fibrous rings of the heart and surrounds the orifices containing the valves (Fig. 13.8). Each valve is composed of three distinct layers: the fibrosa, spongiosa, and either ventricularis (on the ventricular su.dace of the aottic and pulmonary semllunar valves) or the atrialie (on the atrial sur&c:e of the mitral and tricuspid atrioventricular valves): • The fibrosa is situated on the ventricular surface of atrioventricular valves and the arterial surface (facing aorta or pulmonary trunk) of semllunar valves. This

.,.4-+.~E.;w.. circumflex

branch of left coronary artery

FIGURE t3.8. Photomlaorraph of the left 81JIIIand left ventricular walls. •· This photomicrograph shows a sagittal section of the posterior wall of the left atrium and left ventricle. The line of section crosses the coronary (AV) groove containing the coronary sinus and circumflex branch of the left coronary artery. Note that the section has cut through the fibrous AV ring of the mitral valve. which provides the attachment site for the muscle of the left atrium and the left ventricle and the cusp of the mitral valve. The ventricular wall consists of three layers: (1} endocardium (arrowheads), 12) myocardium. and {31 epicardium. The visible blood vessels lie in the epicardium and are surrounded by adipose tissue. The layers of the mitral valve are shown at higher magnification in Figure 13.9b. X35. b. This high magnification of the area indicated by the rectangle shows the characteristic features of the inner surface of the heart. Note that the endocardium consists of a squamous inner layer of endothelium {Enol. a middle layer of subendothelial dense connective tissue !DCTJ containing smooth muscle cells (SMCl. and a deeper subendoC6rdiallayer containing Purkinje fibers (PF). The myocardium contains cardiac muscle fibers !CMFl and is seen on the left. X120.

437

a

b

FIGURE 13.9. Mimi valve In the human heart. a. This photograph shows a sagittal section of the posterior wall of the left ventricle and the posterior cusp of the mitral valve. The chordae tendineae extend from the papillary muscle to the ventricular side of the mitral valve cusp. Note the thickness of the myocardium in the left ventricle. The glistening inner surface of the heart represents the endocardium; the outer surface of the myocardium is covered by the epicardium. x2. (Courtesy of Dr. William D. Edwards). b. Photomicrograph of a mitral valve. This photomicrograph shows a section through one of the two cusps of the mitral valve. Both sides of the cusp are covered by the endothelium. Note that the valve exhibits a layered architecture. Beginning at the atrial side Itop of the image), the first layer underlying the endothelium is the arterialis composed of densely packed collagen and elastic fibers. The second (middle} layer is the spongiosa. which forms the majority of the core of the valve end contains loosely arranged collagen fibers embedded in ground substance rich in proteoglycans and gtycosaminoglycans. This layer gets thinner toward the attachment of the mitral valve to the annulus fibrosus and becomes more prominent toward the leaflet free edge. The third layer, the fibrosa. is formed by dense connective tissue containing layers of elastic lamellae and collagen fibers. At this magnification, nuclei of valvular interstitial cells that resemble fibroblasts are difficult to identify. x 125.

layer is derived from the dense irregular connective tissue of the skeletal rings of the heart. It is predomi· nantly composed of densely packed type I (74%) and type III (24%) collagen fibers and elastic fibers that are arranged parallel to the leaflet free edge. On the ven· tricular/arterial surface, the fibrosa is covered by a layer of endothelial cells. The fibrosa provides tensile stUf· ness to the leaflet. In atrioventricular valves, the fibrosa continues into the chordae tendineae, which are fi. brous, thread-like cords also covered with endothelium (Fig. 13.9). At the sites of chordae tendinae insertion, the fibrosa changes from a Hat layer to a cylindrical chord that enables the gradual transition of forces he· tween the chordae and leaflet without its deformation. Chordae tendineae extend from the ventricular surfaces of the mitral and tricuspid valves into muscular projec· tions from the wall of the ventricles, which are called

papillary muscles. • The spongiosa comprises the middle layer of the valve leaflet. It is composed of loosely arranged collagen and elastic fibers infiltrated by large amounts of ground substance containing proteoglycans and glycosaminoglycans. The spongiosa acts as a shock absorber to dampen vibrations associated with the closing of the valve. It also confers flexibility and plasticity to the valve cusps. Spongiosa is thin at the base of the leaflet but becomes very prominent toward the leaflet's free edge, where it contributes to the correct apposition of leaflets during valve closure that helps prevent valve leakage (regurgitation).

• The ventricularis/atrialis layer is immediately adjacent to the ventricular or atrial surface of each valve and is covered with endothelium. It represents a dense connective tissue layer with wdl-organized collagen fibers containing a large number of elastic fibers and elastic lamellae. The atrialis/ventricularis layer facilitates valve movement by allowing extension and recoil of the valve leaflet during the cardiac contraction cycle. In atrioven· tricular valves, this layer also contains cardiac muscle cells derM:d from atria (not ventricles) and small bundles of smooth muscle cells that may modulate leaflet stiffi:u:ss and defurmation during valve closure. Although heart valves share a basic structural pattem and common functional requirements, each valve is structurally different, and emerging evidence suggests that molecular variations maintain distinct structural and biomechanical characteristics of individual valves.

Valve cusps are avascular and contain unique valwlar interstitial cells that maintain die valve's intemal structure throughout life. Valve cusps are normally avascular. Small blood and lymphatic vessels, nerves, and smooth muscle can be fuund only at the base of the mitral and tricuspid valves. The surfaces of the valve are c:xposcd to blood, and the cusps arc thin enough to allow nutrients and oxygen to diffuse from the blood. 1heleafletsofvalves are populated byvalvular interstitial cells that have unique features and sustain valve homeostasis throughout life. These cells originate from endocardial endothelial cells, but in microscopic examination, they

438

resemble fibroblasts. They are positive for vimentin and chondromodulin 1, wruch inhibit blood vessel formation. Under normal conditions, they maintain basellne levels of extracellular matrix gene expression necessary for repair and synthesis of connective tissue fibers and exaacellular maaix protcins. However, in activated conditions (e.g., during valve development or heart valve diseases), valvular interstitial cells transition into activated myofibroblast·like cells expressing genes that encode protcins necessary for synthesis of collagens, elastin, smooth muscle a.-actin, proteoglycans, matrix metalloproteinases, and inflammatory cytokines, which rapidly remodel the extracellular matrix of the valve. Several diseases affect the valves of the heart, causing their degeneration and resulting in heart malfunction because of insufficiency or stenosis of valvular orifices. These conditions, known collectively as heart valve diseases, include myxomatous mitral valve disease, rheumatic heart disease, vegetative endocarditis, degenerative calcific aortic valve stenosis, and mitral annular calcification. At the cellular level, heart valve diseases are characterized by activation of valvular interstitial cells as well as by in· creased expression of genes encoding extracellular matrix proteins and remodeling enzymes. Pathologic changes to the valves can be divided into three categories basad on the type of valvular damage. The first category includes degeneration of extracellular matrix by accumulation of pathologic proteoglycans, collagen degradation, and elastic fiber fragmentation. These changes are characteristic of myxomatous mitral valve disease and result in a •floppy,. valve that is prone to prolapse and regurgitation. The second category includes fibrosis, which is characterized by accumulation of collagen, degradation of proteoglycans, and elastic fiber fragmentation. These changes occur in rheumatic heart disease and result in a thick, rigid, and inflexible valva that is prone to restricted movement and stenosis. The fibrosis is initiated by inflammation of the valves (valvulitis} that occurs during the bacterial infection known as rheumatic fever. Inflammation induces angiogenesis in the valva and vascularization in the normally avascular layers of the valva. These changes most commonly affect the mitral valve (65% to 70%) and aortic valve (20% to 25%}. Inflammation can lead to progressive replacement of elastic tissue by irregular masses of collagen fibers, causing the valve to thicken. The third category includes nodular calcification that begins within valvular interstitial cells. Such changes occur in degenerative calcific aortic valve stenosis that is characterized by thickening of the valve leaflets and formation of calcium nodules. Valvular calcification is also a common late finding in chronic kidney disease and in the elderly.

Intrinsic Regulation of Heart Rate

the heart is initiated and propagated by the conducting system of the heart. The rate of depolarization of cardiac muscle varies in different parts of the conducting system; the fastest is in the aaia, the slowest in the ventricles. The contraction cycle of the heart is initiated in the atria, forcing blood into the ventricles. A wave ofconaaction in the venaicles then begins at the apex of the heart, forcing blood from the heart into the aorta and pulmonary trunk. The conducting system of the heart consists of two nodes-the sinoatrial (or sinu-atrial) node and the atrioventricular node-and a series of conduction fibers or bundles (tracts). Electrical impulses are generated at the sinoatrial (SA) node, a group of specialized nodal cardiac muscle cells located near the junction of the superior vena cava and the right atrium (see Fig. 13.5). Because the SA node has the fastest rate of depolarizations, it is referred to as the dominant pacemaker of the heart. The pacemaker rate of the SA node is about 60 to 100 beats per minute. The SA node initiates an impulse that spreads along the cardiac muscle fi. hers of the atria and over internodal tracts composed of modified cardiac muscle fibers. The impulse is then picked up at the atrioventricular (AV) node and carried across the fibrous skeleton to the venaides by the AV bundle (of His). The bundle divides into smaller right and left bundle branches and then into subendothelial branches, commonly called Purkinje fibers. The components of the conducting system convey impulses at a rate approximately four times faster than the cardiac muscle fibers and are the only elements that can convey impulses across the fibrous skeleton. If the SA node fails to function (e.g., because of insufficient blood supply}, then the area with the next fastest intrinsic rate of depolarization will take over. In this situation, the AV node will drive the heart contractions at a rata of about 50 beats per minute. In complete heart block, in which the conduction of electric impulses to the ventricles is interrupted, the ventricles will beat at their own rata of about 30 to 40 beats par minute, driven by depolarization of Purkinje fibers. Purkinja fibers have the slowest rata of intrinsic depolarization of the entire conducting system. The spread of electrical impulses through the myocardium can ba monitored and recorded by an electrocardiogram (ECG). The ECG is obtained by placing electrodes at differant points on the skin at specific distances from the heart. Electrodes record electrical activity of the heart by measuring voltage differences between different points. The coordinated spread of the electrical activity through the heart is responsible for the shape of the ECG waveform, careful analysis of which can provide information about heart rate, cardiac rhythm, conduction times through various parts of the heart, effects of electrolyte concentration, effects of cardiac medication, and location of pathologic (ischemic) damages in the heart.

The nodal cardiac muscle cells in both the SA and AV nodes are modified cardiac muscle fibers that are smaller than the surrounding atrial cardiac muscle cells. They contain Cardiac muscle can conaact in a rhythmic: manner without fewer myofibrils and lack typical intercalated discs. The AV any direct stimulus from the nervous system. For the heart to bundle, the bundle branches, and the Purkinje fibers are also be an effective pump, it is necessary for the atria and ventric:les composed of modified cardiac muscle cells, but they are larger to contract in a coordinated rhythmic manner. The electric:al than the surrounding ventricular muscle cells (Fig. 13.10 and activity (impulses) that results in the rhythmic pulsations of Plate 32, page 464). Electrophysiologic studies of the cells in

Contraction of the heart is synchronized by specialized cardiac conducting cells.

of pacemaker cells is manife.ted by a sinus pause or arrest of up to 3 seconds or more without generating impulses. Failure of transitional cells results in SA block, in which the cells are unable to transmit generated impulses into the atrial musculature. Symptoms of SSS include palpitations (irregular heartbeat) and tissue hypoperfusion leading to fatigue, presyncope (light-headedness, muscular weakness, blurred vision, and feeling faint), and syncope (fainting). Recent genetic studies of patients with SSS have identified several gene mutations associated with familial and congenital forms of SSS. The main treatment of SSS is permanent placement of an electronic pacemaker.

The tenninal ramifications of the conducting system consist of Purkinje fibers. Cardiac conducting cells that make up the bundle of His

FIGURE 13. 10. Photomicrotraph of the ventricul•r wall containing the conducUng 1yatam. This photomicrograph shows a Mallory-Azan-$tained section of the ventricular wall of a human heart. The upper twa-thirds of the micrograph is occupied by the endocardium (E) containing a 1hick layer of Purkinje fibers. The free luminal surface of the ventricle (top) is covered by endo1helium and an underlying layer of subendothelial connective tissue (stained blue). The deep layer of endocardium contains the Purkinje fibers. Note 1he intercalated discs in the fibers {a/TOws). The Purkinje fibers contain large amounts of glycogen, which appear as homogeneous, pale-staining regions that occupy the center portion of the cell surrounded by the myofibrils. The nuclei (N) are round and are larger than the nuclei of the cardiac muscle cells in the myocardium {M). They are frequently surrounded by the lighter stained cytoplasm, which represents the ju:xtanuclear region of the cell. Because of the considerable size of the Purkinje cells. the nuclei are often not included in the section. Among the Purkinje fibers are course nerves INFl that belong to the autonomic nervous system. X320.

the SA node reveal the exUt:ence of two different fun~tional groups ofcells. These inc::lude the pacemaker cells (P-cells) with intrinsic spontaneous pacemaker function that generate impulses and transitional cells (T-cells). which are responsible for propagating impulses into the right atrium. P-cell.s are grouped in elongated dusters in the middle of SA node. Dysfunction of the nodal cardiac muscle cells, known as a sick sinus syndrome (SSS), is primarily a disease of the elderly and is the most common indication for the implantation of an electronic pacemaker worldwide. It results from age-related degeneration of nodal cardiac muscle cells in the SA node that affects its ability to generate and transmit impulses to the atrial musculature. SSS is characterized by abnormal heart rhythm disturbances, which include a slow abnormal heart rate (bradyarrhythmia) alternating with a fast abnormal heart rate (tac:hyarrhythmia). Failure

originate at the AV node, pass through the fibrous skeleton of the heart, course along both sides of the interventricular septum (sec Fig. 13.5). and tetminate as Purkinje fibers in the myocardium of the ventricles. The cells that form the Purkinje fibers are larger than ventricular muscle cells. Their myofibrils are located at the periphery of the cell. The nuclei are round and are larger than the nuclei of the cardia~ muscle cells in the myoc:ardium. Because of the conside.table size of the cells. the nuclei are often not included in the section. Intercalated disks are present in Purkinje fibers, but they are variable in appearance and number depending on their location. They are positive for periodic acid-Schiff (PAS) staining because of the large amount of glycogen they contain. With hematoxylin and eosin (H&E) and most other stains, the glycogen~rim center portion of the cell appears homogeneous and stains pale (sec Fig. 13.10). Because of the stored glycogen. Purkinje fiber cells are more resistant to hypoxia than arc ventricular muscle cells.

Systemic Regulation of Heart Function & mentioned above, the heart beats independently ofany nervous stimulation. This spontaneous rhythm of the heart~ be altered by nerve impulses from both sympathetic and parasympathetic divisions of the autonomic nervous system. The autonomic nerves do not initiate contraction of the cardiac muscle but rather regulate the heart .tate (a chronotropic effect) ac:cording to the body's immediate needs.

Stimulation of the parasympathetic nerves decreases the heart rata. The parasympathetic nerve supply to the heart originates in the vagus nerve (cranial nerve X). Presynaptic parasympathetic fibers synapse with postsynaptic neurons within the heart. Their short postsynaptic fibers tetminate ~hiefly at the SA and AV nodes but also extend into the coronary arteries that supply the heart. The release of the neurotransmitter acetylcholine from the terminals of these fibers slows the heart rate (an effect known as bradycardia), reduces the force of the heartbeat, and constricts the coronary arteries ofthe heart.

Stimulation of the sympathetic nerves increases the

heart rate. The sympathetic presynaptic fibers that supply the heart originate in the late.ral horns at the level of the Tl to T6

439

440

(/)

z >

jjj

0

~

(/)

w

a:

~

LL.

0

(/)

w

a:

::::>

!a: w LL.

~

w

zw

(!)

•m

!

~ ~

u

I I.. c.;

segments of the spinal cord. They conduct electrical signals to the cell bodies of postsynaptic neurons located in the cervical and thoracic paravertebral ganglia of sympathetic trunks (see Fig. 12.28, page 410). The postsynaptic fibers terminate at the SA and AV nodes, extend into the myocardiwn, and also pass through the epicardium to reach. the coronary arteries. The autonomic fibers secrete norepinephrine that regulates the rate of impulses emanating &om the SA node. The sympathetic component causes the rate of contraction to increase (an effect known as tachycardia} and increases the force of muscle contraction. Sympathetic stimulation produces dilation of the coronary arteries by inhibiting their constriction. The heart rate end the force of contraction can be regu· lated by circule1ing hormones and other substances.

Changes in the force and rate of cardiac muscle contractions are regulated by hormones secreted from the adrenal medulla. These hormones include epinephrine and norepinephrine that reach. the heart muscle cells via the coronary cilculation. Activation of adrenergic receptors (mainly p1 type) by epinephrine and less efficiently by norepinephrine increases the force of contraction (a positive Inotropic effect) and the heart rate (a positive chrono· tropic effect). Other substances that have positive inotropic and chronotropic effects on the heart include Ca2 +) thyroid hormones, calfe.ine, theophylline, and the cardiac glycoside digoxin. These substances all increase intracellular Ca2 + levels in cardiac myocytes. Substances that have negative lno· tropic and chronotropic actions on the heart muscle include adrenergic-receptor antagonists such as propranolol or Ca2+ channel blockers. These substances decrease the heart rate and the force of cardiac muscle contraction.

• GENERAL FEATURES OF ARTERIES AND VEINS Layers of Vascular Wall The walls of arteries and veins are composed of three layers called tunics. The three layers of the vascular wall, from the lumen outward (Fig. 13.11 and Plate 33, page 466), are as follows: • The tunica Intima, the innermost layer of the vessel, consists ofthree components: (1) a single layer ofsquamous epithelial cells, the endothelium; (2) the basal lamina of the endothelial cells (a thin enracellular layer composed chiefly of collagen, proteoglycans, and glycoproteins); and (3) the subendothelial layer, consisting of loose connective tissue. Occasional smooth muscle cells are found in the loose connective tissue. The subendothelial layer of the intima in arteries and arterioles contains a sheet-like layer or lamella of fenest:J:ated elastic material called the lntemal elastic membrane. Fenesuations enable substances to diffuse readily through the layer and reach cells deep within the wall of the vessel. VEINS

ARTERIES

large vein

large (elastic) artery

The central nervous system monitors arterial pressure and heart function through specialized receptors located within the cardiovascular system.

The activity of the au:di.ovascular system is monito.ted by specialized centers in the centtal. nervous system (CNS). Specialized sensory nerve receptors that supply afferent information about blood pressure are located in the walls oflarge blood vessels near the heart and within the heart itsd£ The infonnation received from all types of cardiovascular receptors initiates the appropriate phpiologi.c reflexes. The receptors function as follows: • Baroreceptors (high-pressure receptors) sense arterial

blood pressure. These receptors are located in the carotid sinus and aortic arch. • Volume receptors (low-pressure receptors) located within the walls of the atria and ventricles sense central venous pressure and provide the CNS with information about cardiac distention. • Chemoreceptors detect alterations in oxygen, carbon dioxide tension, and pH. These receptors are the carotid and aortic bodies located at the bifurcation of the com· mon carotid arteries and in the aortic arch, respectively. The carotid bodies consist of cords and irregular groups of epithelioid cells. A rich supply of nerve fibers is associated with these cells. The neural elements are both afferent and efferent. The structure of the aortic bodies is essentially sim· Uar to that of the carotid bodies. Both receptors function in neural rdlexes that adjust cardiac output and respiratory rate.

capillaries

-......;.,..;

venule mlcroc:ln::ulatory bed

~ smoo1h~ muscle cell

~ ~

RGURE 13.1 1. Scbematic diagram of the major ttructural fee. ture• of blood veeael1. The layers or tunics of the blood vessel walls are labeled in 1he upper two panels. The arrangement of the microcirculatory bed in certain parts of the body is shown in 1he lowest panel. Note the location of pericytes and their relationship to the basal lamina. Also, an arteriovenous (A\11 anastomosis is shown within the microcirculatory bed.

• The tunica media. or middle layer. consists primarily of circwnferentially arranged layers of vascular smooth muscle cells. In arteries. this layer is relatively thick and extends from the internal elastic membrane to the extemal elastic membrane. The external elastic membrane is a layer of elastin that separates the tunica media from the tunica adventitia. Variable amowu:s ofelastin, reticular fibers, and proteoglycans are inrerposed between the smooth muscle cells of the tunica media. The sheets or lamellae of elastin are fenestrated and arranged in circular concentric layers. All of the extracellular components of the tunica media are produced by the vascular smooth muscle cells. • The tunica adventitia, or outermost connective tissue layer, is composed primarily of longitudinally arranged collagenous tissue and a few elastic fibers. These connective

TABLE 13.1

tissue dements gradually merge with the loose connective tissue surrounding the vessels. The tunica adventitia ranges from relatively thin in most of the arterial system to quite thick in the venules and veins. where it is the major component of the vessel wall. In addition. the tunica adventitia of large arteries and veins contains a system of vessels called the vasa vasorum that supplies blood to the vascular walls themselves as well as a network of autonomic nerves called nervi vasorum {vascularis) that conuols contraction of the smooth muscle in the vessel walls. Histologically, the various types of arteries and veins are distinguished from each other by the thickness of the vascular wall and differences in the composition ofthe layers. Table 13.1 summarizes the features of the various types of blood vessels.

Vessel

Diameter

1\lnlca Intima (Inner Layer}

1\lnlca Media {Middle Layer}

1\lnlcaAdventltla (Outer Layer)

Large artery (elastic artery)

>10mm

Endothelium Connective tissue Smooth muscle

Smooth muscle Elastic lamellae

Thinner than tunica media Connective tissue Elastic fibers

Medium artery (muscular artery)

2-10mm

Endothelium Connective tissue Smooth muscle Prominent internal elastic membrane

Smooth muscle Collagen fibers Relatively little elastic tissue

Thinner than tunica media Connective tissue Some elastic fibers

Small artery

0.1-2 mm

Endothelium Connective tissue Smooth muscle Internal elastic membrane

Smooth muscle (8-10 cell layers) Collagen fibers

Thinner than tunica media Connective tissue Some elastic fibers

Endothelium Connective tissue Smooth muscle

Smooth muscle (one or two cell layers)

Endothelium

None

Capillary

10-100 p.m 4-10 p.m

n

~.. :II ~

~

:II Cl

i 2

E

Characteristics of Blood Vessels

Arteries

Arteriole

441

I• (j)

m z m

~

.,mr ~

c:

:D

rn

., 0

Thin. ill-defined sheath of connective tissue

)>

~

m None

~

m

(I)

Veins

)>

Vessel

Diameter

1\lnlca Intima (Inner Layer}

1\lnlca Media {Middle Layer}

l\ln1ca Adventitia (Outer Layer)

Paatcapillary venule

10-50 p.m

Endothelium Pericytes

None

None

Muscular venule

50-100p.m

Endothelium

Smooth muscle (one or two cell layers)

Thicker than tunica media Connective tissue Some elastic fibers

Small vein

0.1-1 mm

Endothelium Connective tissue Smooth muscle (two or three layers)

Smooth muscle (two or three layers continuous with tunica intima)

Thicker than tunica media Connective tissue Some elastic fibers

Medium vein

1-10mm

Endothelium Connective tissue Smooth muscle Internal elastic membrane in some cases

Smooth muscle Collagen fibers

Thicker than tunica media Connective tissue Some elastic fibers

Large vein

>10mm

Endothelium Connective tissue Smooth muscle

Smooth muscle (2-151ayers) Collagen fibers

Much thicl::er than tunica media Connective tissue Some elastic fibers, longitudinal smooth muscles Cardiac muscle extensions (myocardial sleeves} into great veins near the heart

z < m z (I) 0

442

CIJ

z

LU

>

0

z

Vascular Endothelium In the adult human body, the circulatory system consists of about 60,000 miles of different-sized vessels that are lined by a simple squamous epithelium called endothelium. The endothelium is formed by a continuous laycr of flattened, dongated, and polygonally shaped endothelial cells that are aligned with their long axes in the direction of the blood flow. At the lwninal surface, they express a variety ofsurface adhnion molecules and receptors (i.e., low-density

lipoprotein [LDL], insulin, and histamine receptors). Endothdial cdls play an important role in blood homeostasis. The functional properties of these cells change in response to various stimuli. This process, known as endothelial activation, is also responsible for the pathogenesis of many vascular diseases (e.g., atherosclerosis; Folder 13.1). Inducers of endothelial activation include bacterial and viral antigens, cytotoxins, complement products, lipid products, and hypoxia. Activated endothelial cells exhibit new

surface adhesion molecules and produce different classes

of cvtokines, lymphokines, growth factors, and vasoconstrictor and vasodilator molecules as well as molecules that control blood coagulation.

Endalhalial calls canlributa to lha structural and functional integrity of the vascular wall. Endothelial cells are active participants in a variety of interactions between the blood and underlying connective tissue and arc responsible for many properties of the vessels (Table 13.2). These properties include the following: • Maintenance of a selective penneability barrier allows selective movement of small and large molecules from the blood to the tissues and from the tissues to the blood. The barrier is mediated by endothelial cell-cell adhesion complexes including tight junctions, zonula adherens junctions, and a variety of other adhesion molecules that are connected to the actin cytoskeleton. This movement across the endothelium is related to the size and charge of the molecules. The endothelium is permeable to small hydrophobic (lipid-soluble) molecules

(e.g., oxygen, carbon dioxide) that readily pass through the lipid bUayer of the endothelial cell membrane (a process called simple diffusion). However, water and hydrophilic (water-soluble) molecules (e.g., glucose, amino acids, electrolytes) cannot diffuse across the endothelial cell membrane. 1hese molecules and solutes must be either actively transported across the plasma membrane and released into the extracellular space (transcellular pathways) or transponed across the zonula occludens between two epithelial cells (paracellular pathway; see Chapter 5, Epithelial Tissue). 1he transcdlular pathway uttlizes micropinocytotic and macropinocytotic vesicles (a clathrin-independent fonn of endocytosis) to transpon bulk. material from the blood into the cell. In addition, some specific molecules (e.g., LDL, cholesterol, transferrin) are transported via receptor-mediated endocytosis (a clathrin-dependent process), which uses endothelial-specific sur&ce receptors. In some blood vessels, larger molecules are transported through fenestrations within the endothelial cells visible in transmission electron microscope (TEM) preparations.

z 0 < m z

en

444

(/)

z >

jjj

0

~

(/)

w

a:

~

LL.

0

(/)

w

a:

::::>

!a: w LL.

~

w

zw

(!)

•m

!

~ ~

u

I I.. c.;

• Maintenance of a nonthrombogenic banier between blood platelets and subendothelial tissue results from production of anticoagulants (agents that prevent coagulation such as thrombomodulin and others) and antithrombogenic substances (agents that prevent or interfere with platelet aggregation and release of &ctors that cause formation of clots, or thrombi, such as prostacydin [PGI1) and tissue plasminogen activator). In addition, the endothelial cell surf.r.ce is rich in heparin· like sulphated glycosaminoglycans that bind and activate circulating antithrombogenic substances. Normal endothelium does not support the adherence of platelets or the formation of thrombi on its surface. Damage to endothelial cells causes them to release prothrombogenic agents (agents that promote thrombi formation} such as von Willebrand factor or plasminogen-activator inhibitor.

• Modulation of blood flow and vascular resistance is achieved by the secretion of vasoconstrictors (endothelins, angiotensin-converting enzyme [ACE], prostaglandin H2> thromboxane A,) and vasodilators (nitric oxide [NO], prosta.cydin). This subject is discussed in more depth in the next section.

• Regulation and modulation of immune responses occurs through the interaction of lymphocytes with the endothelial surface, which is mainly achieved through the expression of adhesion molecules and their receptors on the endothelial-free surface as well as by secretion of three classes ofinterleukins (IL-l, IL-6, and IL-8).

• Hormonal synthesis and other metabolic activities occur through the synthesis and secretion of various growth facto~for example, hemopoietic colony-stimulating factors (CSFs) such as granulocytemacrophage CSF (GM-CSF), granulocyte CSF (G-CSF), and macrophage CSF (M-CSF); fibroblast growth factor (FGF); and platelet-derived growth fuctor (PDGF). Endothelial cells also synthesize growth inhibitors such as heparin and transforming growth factor 13 (TGF-~). Endothelial cells function in the conversion of angiotensin I to angiotensin II in the renin-angiotensin system that controls blood pressure as well as in the inactivation or conversion of a several compounds conveyed in the blood (norepinephrine, thrombin, prostaglandins, bradykinin, and serotonin) to inactive forms. • Modification of the lipoproteins occurs by oxidation. Lipoproteins, mainly LD Ls with a high cholesterol content and very low-density lipoproteins (VLDLs), are oxidized by free radicals produced by endothelial cells. Modified LDLs, in turn, are rapidly endocytosed by macrophage& to form foam cells (see Fig. F13.1.1). Foam calls are a characteristic feature in the formation of atheromatous plaques.

Endothelium of blood vesstls controls contraction and relaxation of vascular smooth muscle cells in the tunica media, influencing local blood flow and pressure. Endothelial-derived relaxing factor (EDRF) was historically one of the early compounds discovered in endothelial cells that cause relaxation of blood vessels. For years, researchers had difficulty characterizing EDRF chemically.

It is now known that most of the vascular effects of EDRF can be attributed to nitric oxide (NO} and its related compounds, which are released by endothelial cells in arteries, blood capillaries, and even lymphatic capillaries. As a chemical compound, NO is a gas with a very short physiologic half-life measured in seconds; hence, the difficulty with its discovery.

Shear stress produced during the interaction of blood llow with vascular endothelial cells initiates nitric oxide (NO)·derived relaxation of blood vessels. Vasodilation (the relaxation of vascular smooth mus· de cells) increases the lwninal diameter of the vessels, decreasing vascular resistance and systemic blood pressure. Endothelium-derived nitric oxide (NO) is one of several critical regulators of cardiovascular homeostasis. It regulates the blood vessel diameter, inhibits monocyte adhesion to dysfunctional endothelial cells, and maintains an antiprolifera· tive and antiapoptotic environment in the vessel wall. NO is an endogenous vasodilatory gas continuously syn· thesized in endothelial cells by endothelial nitric oxide synthase (eNOS). This Cal+-dependent enzyme catalyzes oxidation of L-arginine and acts through the G-proteinsignaling cascade. Endothelial cells are constandy subjected to shear stress, the dragging force generated by the blood flow. Shear stress increases synthesis ofa potent eNOS stimulator, vascular endothelial growth factor (VEGF), and triggers a variety ofother molecular and physical changes in endothelial cell structure and function. Once NO is produced by endothelial cells, it dilfuses out through the cell and basement membrane to the underlying tunica media and binds to guanylate cyclase in smooth muscle cytoplasm. This enzyme increases production of cyclic guanosine monophosphate (cGMP), which activates smooth muscle protein kinase G (PKG). Activation of protein kinase G has a negative effect on intracellular concentration of Ca2+, causing smooth muscle relaxation (Fig. 13.12). NO is a signaling molecule in many pathologic and physiologic processes. It acts as an anti-inflammatory agent under normal physiologic conditions, although its overproduction induces inflammation. NO is also involved in immune reactions (it stimulates macrophages to release high concentrations of NO), is a potent neurotransmitter in the nervous system, and contributes to the regulation of apoptosis. The pathogenesis of inflammatory disorders of the joint, gut, and lungs is linked to local overproduction of NO. Researchers are studying the pharmaceutical applications of NO inhibitors to treat a variety of disorders, including inflammatory diseases, migraines, and traumatic brain injury. Metabolic stress in endothelial cells also contributes to smooth muscle relaxation. Endotheliwn-derived relaxing factors include prostacyclln (PGI2 ) , which in addition to relaxing smooth muscles is a potent inhibitor of platelet aggregation. PGh binds to receptors on the smooth muscles; stimulates cAMP-activated protein kinase A (PKA), which in tum phosphorylates myosin light chain kinase (MLCK); and prevents activation of the calcium-calmodulin complex. This type of relaxation occurs without changing the intracellular CaH concentration. Endothelium-derived hyperpolariz· ing factor (EDHF) represents another endotheliwn-derived

b:;i't.

l •~

y

E;dolhelial cells

+

~

EDHF

PGI2

r

"T1

m

rdaxing &ctor that acts on Ca2 +-dependent potassiwn channds causing hyperpolarization of vascular smooth muscle: cdls and their relaxation (sec: Fig. 13.12).

Endalhalins produced by vascular endothelial calls play

an important rala in both physiologic and pathologic mechanisms of the circulatory system. Vasoconstriction (contraction of smooth muscle) in the: tunica media of small arteries and arterioles reduces the lwninal diameter of these vt:SSels and increases vascular resistance. Vasoconstriction increases systemic blood pressure. [n the past, vasoconstriction was thought to be mainly induced by nc:rvc: impulses or circulating hormones. It is now known that endothelium-derived factors play an important role in both physiologic and pathologic mechanisms of the circulatory S}'5tcm. Members of the endothelin family of 21 amino acid peptides produced by vascular c:ndothc:llal cells arc the most potent vasoconstrictors. The family consists of three members: endothelin-1 (ET-1), endothelin-2 (ET-2), and endothelin-3 (ET-3). Endothelin.s act mainly as paracrinc and autocrine agents and bind to their own receptors on the epithelial cells and vascular smooth muscles (Fig. 13.13). ET-1 is the most potent naturally occurring vasoconstticting agent that interacts with its ETA receptor on vascular smooth muscles. High levels of ET-1 gene expression are associated with many diseases that are caused in part by sustained endothelium-induced vasoconstriction. lhasa include systemic hypertension (sea Folder 13.2), pu Imona ry hypertension, atherosclerosis, congestive heart failure, idiopathic cardiomyopathy, and

renal failure. It is interesting to note that snake venom from the Israeli burrowing asp (Atractaspis engaddansis) contains sarafotoxin, a highly toxic protein that exhibits a high degree of sequence homology with ET-1. After it entars the circulation, the toxin binds to ETA receptors and causes life-threatening, intense coronary vasoconstriction. This is remarkable because endothelin is a natural compound of the human vascular system, whereas sarafotoxin is a toxin in snake venom. The other endotheliumdcrivM vasoconstrictors include thromboxane A2 and prostaglandin H2 • Thromboxane A2 is synthesized from prostaglandin H 2 • In addition, decreased rate of NO production or inactivation of NO by the superoxide anion (02-) has a stimulating effect on smooth muscle contraction (see Fig.

13.13).

In swnmary, under normal phpiologic conditions, vascular endothdial cells become activated by environmental &ctots such as mechanical stimuli (pressure and shear stress) and chemical compounds (hormones and locally secreted vasoactive substances). In response to these stimuli, the endothdium. rdeases factors that tcgulatc vasomotor function, inflanunatory processes, cell growth, and hemostasis. However, endothelial dysfunction, a term that comprises multiple potential defects of the endothelial cells, may shift the actions of the endothelium toward reduced vasodilation and various proliferative, prothrombotic, and proinflammatory conditions. Endothelial dysfunction is an important early evant that may lead to many pathologic conditions, such as progressive atherosclerotic disease (see Folder 13.1).

~ c

::lJ

m

tf)

0

"T1 )>

~ m ~

m

C/l )>

z < m

0

z

C/l

446

(/)

z

w

> Cl z !'

j .___ _... j

'15

' .S!

salivon

parotid

submandibular sublingual

FIGURE 16.22. Dlagr'llm comparing the components of the salivon in the thi'M major salivary glands. The four major pans of the salivon-the acinus. intercalated duct. striated duct. and excretory duct-are color-coded. The three columns on the right of the salivon compare the length of the different ducts in the three salivary glands. The reck:olored cells of the acinus represent serous-secreting cells, and the yellow-colored cells represent mucus-secreting cells. The ratio of serous-secreting cells to mucus-secreting cells is depicted in the acini of the various glands.

As noted above, each mixed acinus, such as those found in the sublingual and submandibular glands, contains serous and mucus·producing cells. In routine preparations for both light and electron microscopy, serous cells have traditionally been regarded as the structures that make up the demilune. Recent electron microscopic studies now challenge this classic interpretation of the demilune. Rapid freezing of the tissue in liquid nitrogen, followed by rapid freeze substitution with osmium tetroxide in cold acetone, reveals that both mucous and serous cells are aligned in the same row to surround the lumen of the secretory acinus. No serous demilune is found. Sections prepared from the same specimen by conventional methods show swollen mucous cells with enlarged secretory granules. The serous cells form typical demilunes and are positioned in the peripheral re-gion of the acinus with slender cytoplasmic processes interposed between the mucous cells. These findings indicate that the demilune observed in light or electron microscopy is an artifact of the routine fixation method (Fig. 16.23).

Dental caries is an infectious microbial disease of teeth that results in the destruction of affected calcified tissues, that is, enamel, dentin, and cementum. Carious lesions generally occur under masses of bacterial colonies referred to as "dental plaque." The onset of dental caries is primarily associated with bacterial colonies of Streptococcus mutans, whereas lactobacilli are associated with active progression of the disease. These bacterial colonies metabolize carbohydrates, producing an acidic environment that demineralizes the underlying tooth structure. Frequent sucrose ingestion is strongly associated with the development of these acidogenic bacterial colonies. Trace amounts of fluoride, from sources such as water supplies 10.5 to 1.0 ppm is optimal), toothpaste, and even diet, can improve resistance to the effects of cariogenic bacteria. Fluoride improves the acid resistance of the tooth structure, acts as an antimicrobial agent, and promotes remineralization of small carious lesions. Resistance to acid breakdown of enamel is facilitated by the substitution of fluoride ion for the hydroxyl ion in the hydroxyapatite crystal. This decreases enamel crystal solubility in acid. Treatment of cavitated lesions, or "tooth cavities" (Fig. F16.3.1 ), includes excavation of the infected tooth tissue and replacement with dental materials such as amalgam, composite, and glass ionomer cements. Microbial invasion of tooth structure can reach the "pulp" of the tooth and elicit an inflammatory response. In this

FIGURE F16.3. t. Photamicmgraphaofceriouslesions.a. Photomicrograph of an unstained ground section of a tooth sh r

~

610

• Microvilli are tighdy packed, microscopic projections of the apical surface of intestinal absorptive cells. They further increase the surface available for absorption.

In addition, the glycocalyx consists of glycoproteins that project from the apical plasma membrane of epithelial absorptive cells. It provides additional surface for adsorption and includes enzymes secreted by the absorptive cells that are essential for the final steps of digestion of proteins and sugars. The epithelium selectively absorbs the products of digestion both for its own cells and for transport into the vascular system for distribution to other tissues.

The secretory function of the mucosa provides lubrication and delivers digestive enzymes. honnonas, and antibodies into the Iuman of the alimentary lube. Secretion .is carried out largely by glands distributed throughout the length of the digestive tube. The various secretory products provide mucus for protective lubrication as wdl as buffering of the tract lining and substances that assist in digestion, including enzymes, hydrochloric acid, peptide hormones, and water (see Fig. 17.1). The mucosal epitheUwn also secretes antibodies that it receives from the underlying connective tissue. The glands of the alimentary tract (see Fig. 17.1) develop from invaginations of the luminal epithelium and include the following:

-

• Mucosal glands that extend into the lamina propria • Submucosal glands that either deliver their secretions directly to the lumen of mucosal glands or via ducts that pass through the mucosa to the luminal surface • Extramural glands that lie outside the digestive tract and deliver their secretions via ducts that pass through the wall of the intestine to enter the lumen. The liver and the pancreas are extramural digestive glands (see Chapter 18, Digestive System III: Liver, Gallbladder, and Pancreas) that gready increase the secretory capacity of the digestive system. They deUver their secretions into the duodenum, the first part of the small intestine.

The lamina propria contains glands, va11els thai transpori absorbed substances. and components of the immune system. As noted, the mucosal glands extend into the lamina propria throughout the length of the alimentary canal. In addition, in several parts of the alimentary canal (e.g., the esophagus and anal canal), the lamina propria contains aggregations of mucuHecreting glands. In general, they lubricate the epithelial surface to protect the mucosa from mechanical and chemical injury. These glands are described below in rela~ tion to specific regions of the digestive tube. In segmenu of the digestive tract in which absorption occurs, principally the small and large intestines, the absorbed products of digestion diffUse into the blood and lymphatic vessels of the lamina propria for distribution. Typically, the blood capillaries are of the fenestrated type and collect most of the ab-sorbed metabolites. In the small intestine, lymphatic capillaries are numerous and receive some absorbed lipids and proteins. • The lymphatic tissues in the lamina propria function as an integrated immunologic barrier that protects against pathogens and other antigenic substances that could

potentially enter through the mucosa from the lumen of the alimentary canal. Lymphatic tissues present in the lamina propria are as follows: • Diffuse lymphatic tissue consisting of numerous lymphocytes and plasma cells located in the lamina propria and lymphocytes transiendy residing in the intercellular spaces of the epithelium • Lymphatic nodules with well-developed germinal centers • Eosinophils, macrophages, and sometimes neutrophils The diffuse lymphatic tissue and the lymphatic nodules are referred to as gut-associated lymphatic tissue (GALT). In the distal small intestine, the ileum, extensive: aggregates of nodules, called Payer's patches, occupy much of the lamina propria and submucosa. They tend to be located on the side of the tube opposite the attachment of the mesentery. Aggregated lymphatic nodules are also present in the appendix. The muscularis mucosae forms the boundary between mucosa and submucosa. The muscularis mucosae, the deepest portion of the mucosa, consists of smooth muscle cells arranged in an inner circular and outer longitudinal layer. Contraction of this muscle produces movement of the mucosa, fonning ridges and valleys that facilitate absorption and secretion. lh.is localized movement ofthe mucosa is independent ofthe peristaltic movement of the entire wall of the digestive tract.

Submucosa The submucosa consists of a dense irregular connective tissue layer containing blood and lymphatic vessels, a nerve plexus, and occasional glands. The submucosa contains the larger blood vessels that send branches to the mucosa, muscularis enema. and serosa. The submucosa also contains lymphatic vessels and a nerve plexus. The extensi:ve nerve network in the submucosa contains visceral sensory fibers of mainly sympathetic origin, p:uasy:mpathetic (terminal) ganglia, and preganglionic and postganglionic parasympathetic nerve fibers. The nerve cell bodies of parasympathetic ganglia and their postganglionic nerve fibers represent the enteric nervous system, the thil:d division of the autonomic nervous system. This system is primarily responsible for innervating the smooth muscle layers of the alimentary canal and can function totally independendy of the central nervous system. In the submucosa, the network of unmyelinated nerve fibers and ganglion cells constitute the submucosal plexus (also called Meissner's plexus). At; noted, glands occur occasionally in the submucosa in certain locations. For example, they are present in the esophagus and the initial portion of the duodenum. In histologic sections, the presence of these glands often aids in identifying the specific segment or region of the tract.

Muscularis Externa In most parts ofthe digestive tract, the muscularis ex:tema consists of two concentric and relatively thick layers of smooth muscle. The cells in the inner layer fonn a tight spiral, described as a circularly oriented layer; those in the outer layer form a loose spiral, described as a longitudinally

oriented layer. Located between the two muscle layers is a

hypertrophic pyloric stenosis. This condition occurs

thin connective tissue layer. Within this connective tissue lies the myenteric plexus (also called the Auerbach's plexus), containing nerve celt bodies (ganglion cells) of postganglionic parasympathetic neurons and neurons of the enteric nervous system as well as blood vessels and lymphatic vessels.

most commontv during the first 2 to 12 weeks of life and results in obstruction in flow of chvme into the duodenum, which causes projectile vomiting (without bile) after feeding. If untreated, it may lead to de· hvdration and hvpochloremic, hvpokalemic metabolic alkalosis. Hvpertrophv of the pyloric muscle can be diagnosed bv ultrasonography and is also easilv palpable as an •olive,. in the right upper quadrant of abdomen. Laparoscopic pvloromvotomv that involves transection of pyloric muscle without disruption of undertving mucosa remains the primary surgical treatment. • Ileocecal valva. Located at the junction of the small and large intestines, it prevents reflux of the contents of the colon with its high bacterial count into the distal ileum, which normally has a low bacterial count. • Internal anal sphincter. This, the most distally located sphincter, surrounds the anal canal and prevents passage of the feces into the anal canal from the undistended rectum.

Contractions of the muscularisexterna mix and propel the contents of the digel1ivetract. Contraction of the inner circular layer of the muscularis extema compresses and .mi:Jrt:s the contenu by constricting the lumen; contraction of the outer, longitudinal layer propels the contents by shortening the rube. The slow, rhythmic contraction of these muscle layers under the control of the enteric nervous system produces peristalsis (i.e., waves ofcontracti.on). Peristalsis is marked by constriction and shortening of the rube, which moves the contents through the intestinal tract. A few sites along the digestive tube exhibit variations in the muscularis extema. For example, in the wall of the proximal ponion of the esophagus (pharyngoesophageal sphincter) and around the anal canal (external anal sphincter), suiated muscle forms part of the muscularis externa. In the stomach, a third, obliquely oriented layer of smooth muscle is present deep into the circular layer. Finally, in the large intestine, part of the longitudinal smooth muscle layer is thickened to form three distinct, equally spaced longitudinal bands called tenlae coli. During contraction, the teniae facilitate shonening of the tube to move its contents.

The circular smooth muscle layer forms sphincters at specific locations along the digestive tract At several points along the digestive tract, the circular muscle layer is thickened to form sphincters or valves. From the oropharynx distally, these sttucwres include the following:

• Pharyngoesophageal sphincter. Actually, the lowest part of the cricopharyn~us muscle is physiologically referred to as the superior (upper) esophageal sphincter. It prevents the entry of air into the esophagus. • Inferior (lower) esophageal sphincter. As its name implies, this sphincter is loc:atc:d at the lower end ofthe esophagus; iu action is reinforced by the diaphragm that surrounds this pan of the esophagus as it passes into the abdominal cavity. It cn:ates a pressure c:l.ifk:rcnce between the esophagus and stomach that pmrenu reflux ofgastric contenu into the esophagus. Abnormal relaxation of this sphincter allows the acidic contents of the stomach to return (reflux) into the esophagus. If nottreeted, this condition mav progress to gastroesophageal reflux disease {GERD), characterized bv inflammation of the esophageal mucosa (reflux esophagitis), strictures, end difficulty in swallowing (dvsphegia) with accompanving chest pain. • Pyloric sphincter. Located at the junction of the pylorus ofthe stomach and duodenum (gasuoduodenal sphincter), this sphincter controls the release of chyme, the partially digested contents of the stomach, into the duodenum. Nitric oxide svnthase (NOS), which produces nitric oxide (NO}, is responsible for phvsiologic relaxation of the pvloric sphincter. Deficiency in NOS causes smooth muscle spasm of the pyloric sphincter and subsequent

Serosa and Adventitia Serosa or adventitia constitutes the outermost larer of the alimentarr canal. The serosa is a serous membrane consisting of a layer of simple squamous epithellwn, called the mesothelium, and a small amount of underlying connective tissue. It is equivalent to the visceral peritonewn described in gross anatomy. The serosa is the most superficial layer of those parts of the digestive tract that are suspended in the peritoneal cavity. As such, the serosa is continuous with both the mesentery and the lining of the abdominal cavity. Large blood and lymphatic vessels and nerve t:runks travd through the serosa (from and to the mesentery) to reach the wall of the digestive tract. Large amounts of adipose tissue can develop in the connective tissue of the serosa (and in the mesentery). Parts of the digestive traer do not possess a serosa. These include the thoracic part of the esophagus and portions of suuctures in the abdominal and pelvic cavities that are med to the cavity wall-the duodenwn, ascending and descending colon, rectum, and anal canal. These structures are attached to the abdominal and pelvic wall by connective tissue, the adventitia, which blends with the connective tissue of the wall.

•ESOPHAGUS The esophagus is a fixad muscular tuba that delivers food and liquid from the pharynx 1o the stomach. The esophagus courses through the neck and mediastinwn., where it is attached to adjacent structures by connective tissue. As it enters the abdominal cavity, it is free for a shon distance, approximately 1 to 2 em. The overall. length of the esophagus is about 25 em. On cross section (Fig. 17.2), the lumen in its normally collapsed state has a branched appearance because oflongitudinal folds. When a bolus of food passes through the esophagus, the lwnen expands without mucosal injury.

611

• m

(J)

0

-u

::I:

~

c(J)

612

t/)

::>

~ :::c c..

~ w

-•

FIGURE 17.2. Photomlaogf'lph of the esophagus. This low-magnification photomicrograph shows an hematoxylin and eosin (H&E)-stained section of the esophagus with its characteristically folded wall. giving the lumen an irregular appearance. The mucosa consists of a relatively thick stratified squamous epithelium. a thin layer of lamina propria containing occasional lymphatic nodules. and muscularis mucosae. Mucous glands are present in the submucosa; their ducts, which empty into the lumen of 1he esophagus, are not evident in this section. External to the submucosa in this part of the esophagus is a thick muscularis extema made up of an inner layer of circularly arranged smooth muscle and an outer layer of longitudinally arranged smooth muscle. The adventitia is seen just external to the muscularis externa. X8.

The mucosa that lines the length of the esophagus has a nonkeratinized stratified squamous epithelium (Fig. 17.3 and Plate 54. page 644). In many animals. however, the epithelium is keratinized, reflecting a coarse food diet. In humans, the surface cells may exhibit some keratohyalin pnules. but ketatinb:ation does not normally OCQLt. The underlying lamina propria is similar to the lamina propria throughout the alimentary tract; diffuse lymphatic: tissue is scattered throughout. and lymphatic: nodules are present. often in proximity to ducts of the esophageal mucous glands (see page 614). The deep layer of the mucosa, the muscularis mucosae, is composed of longitudinally organized smooth muscle that begins near the level of the cricoid cartilage. It is unusually thick in the proximal portion of the esophagus and presumably functions as an aid in swallowing. The submucosa consists of dense irregular connective tissue that contains the l~r blood and lymphatic vessels, nerve fibers, and ganglion cells. The nerve fibers and ganglion cells make up the submucosal plexus (Meissner's plexus). Glands are also present (see page 610). In addition, diffuse lymphatic tissue and lymphatic nodules are present mostly in the upper and lower parts of the esophagus where submucosal glands are more prevalent. The: muscularis extema consisu oftwo muscle layers. an inner circular layer and an outer longitudinal layer (Plate 54, page 644). It d.iffi:rs from the muscularis c::xtc:rna found in the rest of the digestive ttact in that the upper one·third is striated muscle:, a continuation of the muscle of the phar· y:nx. Striated muscle and smooth muscle bundles are mixed and interwoven in the muscularis externa of the .middle third

of the esophagus; the muscularis externa of the distal third consists only of smooth muscle, as in the rest of the digestive tract. A nerve plexus, the myenteric plexus (Auerbach's plexus), is present between the outer and inner muscle lay· ers. & in the submucosal plexus (Meissner's plexus). nerves and ganglion cells ate: present here. This plexus innervates the muscularis c::xterna and produces peristaltic activity. & noted, the esophagus is fixed to adjoining struaures throughout most of its length; thus. its outer layer is com· posed of adventitia. After entering the abdominal cavity. the short remainder of the tube is covered by serosa, the visceral peritoneum. Mucosal and submucosal glands afthe esophagus secrete mucus to lubricate and protect the luminal wall. Glands are present in the wall of the esophagus and are of two types. Both secrete mucus. but their locations differ. • Esophageal glands proper lie in the submucosa. These glands are scattered along the length of the esophagus but are somewhat more concentrated in the upper hal£ They are small. compound. tubuloalveolar glands (Fig. 17.4). The excretory duct is composed of stratified squamous epithdium and is usually conspicuous when present in a section because of its dilated appear.mce. • Esophageal cardiac glands are named for their simi· larity to the cardiac glands ofthe stomach and are found in the lamina propria of the mucosa. They are p.n:sent in the tenninal part ofthe esophagus and frequently. although not consistendy, in the begiruUng portion of the esophagus.

The mucus produced by the esophageal glands proper is slightly acidic and serves to lubricate the luminal wall. Because the secretion is relatively viscous, transient cysts often occur in the ducts. The esophageal cardiac glands produce neuttal

613

mucus. Those glands near the stomach tend to protect the esophagus from regurgitated gastric contents. Under certain conditions, however, they are not fully effective, and excessive reflux results in pyrosis, a condition more commonly known as heartburn. This condition may progress to

gastroesophageal reflux disease (GERD).

The muscle of the esophageal wall is innervated by bo1h autonomic and somatic nervous sptems. The striated musculature in the upper part of the esophagus is inne.rvated by somatic motor neurons of the vagus nerve, cranial nerve X (from the nucleus ambiguus). The smooth muscle of the lower part of the esophagus is innervated by visceral motor neurons of the vagus nerve (from the dorsal motor nucleus). These motor neurons synapse with pomynaptic neurons whose cell bodies are located in the wall of the esophagus. FIGURE 17.4. Photomicrograph of an esophageal submuCOHI gland. This photomicrograph shows a mucicarmine-stained section of the esophagus. An esophageal gland. deeply stained red by the carmine. and an adjacent excretory duct are seen in the submucosa. These small, compound, tubuloallleolar glands produce mucus that lubricates the epithelial surface of 1he esophagus. Note the stained mucus within the excretory duct. The remaining submucosa consists of dense irregular connective tissue. The inner layer of the muscularis externa lbottoml is composed of circularly arranged smooth muscle. X110.

•sTOMACH The stomach is an expanded part of the digestive tube that lies beneath the diaphragm. It receives the bolus of macerated food from the esophagus. Mixing and partial digestion of the food in the stomach by its gastric secretions produce a pulpy fluid mix called chyme. The chyme then passes into the small intestine for further digestion and absorption.

The stomach is divided histologically into 1hree regions based on the type of gland that each contains.

FIGURE 17.3. Photomicrograph of the esoph•a••• mucosa. This higher magnification photomicrograph shows the mucosa of the wall of the esophagus in an hematoxylin and eosin (H&El preparation. It consists of a stratified squamous epithelium. lamina propria, and muscularis mucosae. The boundary between the epithelium and lamina propria is distinct. although uneven, because of the connective tissue papillae. The basal layer of the epithelium stains intensely. appearing as a dark band because the basal cells are smaller and have a high nucleus-to-cytoplasm ratio. Note that the loose connective tissue of the lamina propria is very cellular, containing many lymphocytes. The deepest part of the mucosa is the muscularis mucosae. which is arranged in two layers (inner circular and outer longitudinal! similar in orientation to the muscularis externa. X240.

Gross anatomists subdivide the stomach into four regions. The cardia surrounds the esophageal orifice; the fundus lies above the level of a horizontal. line drawn through the esophageal (cardiac) orifice; the body lies bdow this line; and the pyloric antrum is the funnel-shaped region that leads into the pylorus, the distal, narrow sphincteric region between the stomach and duodenum. Histologists also subdivide the stomach but only into three regions (Fig. 17.5). These subdivisions are based not on location but on the types of glands that occur in the gastric mucosa. The histologic regions are as follows: • Cardiac region, the part near the esophageal orifice, which contains the cardiac glands (Fig. 17.6 and Plate 55, page 646) • Pyloric region, the part proximal to the pyloric sphincter, which contains the pyloric glands • Fundic region, the largest part of the stomach, which is situated between the cardia and pylorus and contains the fundic or gastric glands (see Fig. 17.6)

• (I)

0 3:: l'oo

n

::I:

or foveolae. They can be readily demonstrated with the scanning electron microscope (Fig. 17.7). The gastric glands open into the bottom of the gastric pits.

614 esophagus

Surface mucous cells line the inner surlace of the stomach and the gastric pita. The epithelium that lines the surface and the gastric pits of the stomach is simple columnar. The columnar cells are des· ignated surface mucous cells. Each cd1 possesses a large, apical cup of mucinogen granules, creating a glandular sheet of cells (Fig. 17.8). The mucous cup occupies most of the volume of the cell. It typically appears empty in rou· tine hematoxylin and eosin (H&E) sections because the mucinogen is lost in fixation and dehydration. When the mucinogen is preserved by appropriate fixation, however, the granules stain intensely with toluidine blue and with the periodic acid-schilf (PAS) procedure. The toluidine blue staining reflects the p~ence of many strongly anionic groups in the glycoprotein of the mucin, among which is bicarbonate. The nucleus and Golgi apparatus of the surfuce mucous cells are located below the mucous cup. The basal part of the cell contains small amounts of rough endoplasmic reticulum

cardiac region

-

pyloric region duodenum RGURE 17.5. PhotDgraph of • hiMIIiNd8d human stDmach with its hiltDiofic divisions. This photograph shows the mucosal surface of the posterior wall of the stomach. Numerous longitudinal gastric folds are evident. These folds or ru99e allow the stomach to distend as it fills. The histologic divisions of the stomadl differ from the anatomic division.The former is .based on the types of glands found in the mucosa. Histologically, the portion of the stomadl adjacent to the entrance of the esophagus is the cardiac region in which cardiac glands are located. A dashed line approximates its boundary. A slightly larger region lea~ ing toward the pyloric sphincter, the pyloric region, contains the pyloric glands. Another dashed line approximates boundary of the pyloric sphincter. The remainder of the stomach, the fundic region, is located between the cardiac and pyloric regions and contains the fundic (gastric) glands.

stomach

esophagus I

Gastric Mucosa Longitudinal submucosal folds, rugae, allow the stomach to distend when filled. The stomach has the same general sttuaural plan throughout, consisting of a mucosa, submucosa, muscularis externa, and serosa. Examination of the inner surface of the empty stomach reveals a number of longitudinal folds or ridges called rugae. They are prominent in the narrower regions of the stomach but poorly dcvdoped in the upper portion (see Fig. 17.5). When the stomach is fully distended, the rugae, composed of the mucosa and underlying submucosa, virtually disappear. The rugae do not alter total surface area; rather, they serve to accommodate expansion and filling of the stomach. A view of the stomach's surface with a hand lens shows that smaller regions of the mucosa are formed by grooves or shallow trenches that divide the stomach surface into bulging irregular areas called mamillated areas. These grooves provide a slighdy increased surface area for secretion. At higher magnification, numerous openings can be ob· served in the mucosal surface. These are the gastric pits

FIGURE 17.8. Photomicrograph of the esophagogaatric junction. This low-magnification photomicrograph shows the junction between the esophagus and stomach. At the esophagogastric junction, the stratified squamous epithelium of the esophagus ends abruptly, and the simple columnar epithelium of the stomach mucosa begins. The surface of the stomach contains numerous and relatively deep depressions called gastric pits that are formed by the surface epithelium. The glands in the vicinity of the esophagus, the cardiac glands. extend from the bottom of these pits. The fundic (gastric) glands similarly arise at the base of the gastric pits and are evident in the remaining part of the mucosa. Note the relatively thick muscularis externa. X40.

615

• (I)

0

FIGURE 17.7. Mucosal surface of the stomach. a. Scanning electron micrograph showing the mucosal surface of the stomach. The gastric pits contain secretory material, mostly mucus ls"ows). The surface mucus has been washed fJINSY to reveal the surface mucous cells. x 1,000. b. Higher magnification showing the apical surface of the surface mucous cells that line the stomach and gastric pits. Note the elongate polygonal shape of the cells. X3.000.

(rER) that may impart a light basophilia to the cytoplasm when observed in well-preserved specimens. Several mechanisms help protect the gastric mucosa from exogenous iniury and contribute to recovery of its functional integrity after damage.

The lining of the stomach does not function in an absorptive capacity. However, some water, salts, and lipid-soluble drugs may be absorbed. For instance, alcohol and certain drugs such as aspirin or nonsteroidal anti-inflammatory drugs (NSAIDs) enter the lamina propria by damaging the surface epithelium. Even small doses of aspirin suppress the production of protective prostaglandins by the gastric mucosa. In addition, aspirin's direct contact with the wall of the stomach interferes with the hydrophobic properties of the gastric mucosa.

The first line of protection from injury of gastric mucosa is the mucous secretion from the surface mucous cells. It is described as visible mucus becau.sc of its cloudy appearance and forms a thick. viscous, gel-like coat that adheres to the epithelial surface. It protects against abrasion from rougher components of the chyme. Additionally, its high bicar- Fundic Glands of the Gastric Mucosa bonate and potassium concentration protects the The fundic glands produce the gastric juice ofthe stomach. epitheliwn from the acidic: content of the gast:ric juice. The bicarbonate that makes the mucus alkaline is secreted The fundic glands, also called gastric glands, are present by the surface c:ells but is prevented from mixing rapidly with throughout the entire gastric mucosa except for the relathe contents of the gastric: lumen by its containment within tively small regions occupied by cardiac and pyloric glands. The fundic glands are simple, branched, tubular glands that the mucous coat. The second line of protection is related to regulation of extend from the bottom of the gastric pits to the muscularis the submucosal blood by a number of mediators including mucosae (see Fig. 17.8). Located between the gastric pit and prostaglandins (PGE.z), nitric: oxide (NO), and sensory neu· the gland below is a short segment known as the isthmus. ropeptides. Prostaglandins (PGE2 ) and nitric oxide (NO) The isthmus of the fundic gland is a site of stem cell location appear to play an important role in protecting the gastric mu· (stem cell niche) in which stem cells replicate and d.ifferenti· cosa. PGE,z stimulate sec:retion ofbic:arbonates and in~ase the ate. Cells destined to become mucous surface cells migrate thickness ofthe mucous layer with acc:ompanied vasodilatation upward in the gastric: pits to the stomach surface. Other cells in the lamina propria. Nittic oxide (NO) released from vascular migrate downward, maintaining the population of the fundic endothelium, sensory afferent nerves, and gastric epithdiwn gland epithelium. increases gastric mucosal blood flow, thus improving Typically. srn:ral glands open into a single gastric pit. Each the supply of nutrients to damaged areas of the gastric mucosa. gland. has a narrow, relatively long neck segment and. a shorter This ability of the gastric mucosa to optimize conditions for tis-- and wider base or fundic segment. The base of the gland sue repair after injury (independendy of the inhibition of acid usually divides into two and sometimes three branches that secretion) is referred to as gastric cytoprotection. become sUghtly coUed near the musc:ularis mucosae. The cells

3::

1} ::I:

LUMEN

616

GASTRIC PIT

..•

•• •

--

......_ \ ourfaco

/

mucous cells

~ ISTHMUS NECK

enteroendoa'fne -~.T

cell

·:

GASTRIC QLAND

entero- -endocrine cell

b

RGURE 17.8. Gastric glands. a. This photomietograph shows the fundic mucosa from an Alcian blue/periodic ac~Schiff (PAS) preparation to visualize mucus. Note that the surface epithelium invaginates to form gastric pits. The surface mucous cells and the cells lining the gastric pits are readily identified in this preparation because the neutral mucus within these cells is stained intensely. One of the gastric pits and its associated fundic gland are depicted by the dashed fines. This gland represents a simple branched 111bular gland (BI7DIIVS indicate the branching pattem). It extends from the bottom of the gastric pit to the muscularis mucosae. Note the segments of the gland: the short isthmus. the site of cell divisions; the relatively long nect; and a shorter and wider fundus. The mucous secretion of mucous ned< cells is different from that produced by the surface mucous cells as evidenced by the fighter magenta staining in this region of the gland. X320. b. Schematic diagram of a gastric gland, illustrating the relationship of the gland to the gastric pit. Note that the isthmus region contains dividing cells and undifferentiated cells; the neck region contains mucous ned:: cells. parietal cells. and enteroendocrine cells. including amine precursor uptake and decarboxylation (APUDI cells. Parietal cells are large. peaF-shapad acidophilic cells found throughout the gland. The fundus of the gland contains mainly chief cells, some parietal cells, and several types of enteroendocrine cells.

of the gastric glands produce gastric juice (about 2 Uday), which contains a variety ofsubstances. In addition to watz::r and electrolytes, gastric juice contains four major componenu: • Hydrochloric acid (HCI) in a concentration ranging from 150 to 160 mmolJL, which gives the gastric juice a low pH

(< 1.0 to 2.0). It is produced by parietal cells and initiates digestion of dietary protz::in (it promotes acid hydrolysis of substrates). It also converts inactive pepsinogen into the active enzyme pepsin. Because HCI is bacteriostatic, most of the bacteria entering the stomach with ingested food are

617 Achlorhydria is a chronic autoimmune disease characterized by the destruction of the gastric mucosa. Consequently, in the absence of parietal cells, the intrinsic factor is not secreted, thereby leading to pemicious anemia. Lack of intrinsic factor is the most common cause of vitamin B12 deftclency. However, other factors such as Gram-negative anaerobic bacterial overgrowth in the small intestine are associated with vitamin B12 deficiency. These bacteria bind to the vitamin B1rintrinsic factor complex, preventing its absorption. Parasitic tapeworm infections also produce clinical symptoms of pernicious anemia. Because the liver has extensive reserve stores of vitamin B12, the disease is often not recognized until long after significant changes in the gastric mucosa have taken place. Another cause of reduced secretion of intrinsic factor and subsequent pernicious anemia is the loss of gastric epithelium in partial or total gastrectomy. Loss of functional gastric epithelium also occurs in chronic or recurrent peptic ulcer disease (PUD). Often. even healed ulcerated regions produce insufficient intrinsic factor. Repeated loss of epithelium and consequent scarring of the gastric mucosa can significantly reduce the amount of functional mucosa. Histamine Hz receptor-antagonist dNgs such as ranitidine (Zantac) and cimetidine (Tagamet), which block attachment of histamine to its receptors in the gastric mucosa. suppress both acid and intrinsic factor production and have been used extensively in the treatment of peptic ulcers and gastroesophageal reflux disease (GERD). These drugs prevent further mucosal erosion and promote healing of the previously eroded surface. However, lon~term use can cause

destroyed. However, soma bacteria can adapt to the low pH of the gastric contents. Helk:obar:tw pylori contains large amounts of urease, the enzyme that hydrolyzes urea, in its cytoplasm and on its plasma membrane.This highly active enzyme creates a protective basic ..ammonia cloud" around the bacterium, allowing it to survive in the acidic environment of the stomach (Folder 17.1). • Pepsin, a potent proteolytic enzyme. It is formed from the conversion by HCl of pepsinogen, which is produced by the chief cells, when the pH is lower than 5. Pepsin hydrolyzes proteins into small peptides by splitting interior peptide bonds. Peptides are further digested into amino acids by enzymes in the small intestine. • Mucus, an acid·proa:ctive coating for the stomach secreted by several types of mucus-producing ce1ls. The mucus and bicarbonates trapped within the mucous layer maintain a neutral pH and contribute to the so-called physiologic gastric mucosa barrier. In addition, mucus serves as a ph}'5ical barrier between the cc:lls of the gastric mucosa and the ingested material in the lumen of the stomach. • Intrinsic factor, a glycoprotein secreted by parietal cells that binds to vitamin 8 12• It is essential for its absorption, which occurs in the distal part of the ileum. Lack of i ntri nsic factor leads to pemicious anemia and vitam in B12 deficiency (see Folder 17.1).

vitamin 8 12 deficiency. Recently. new proton pump inhibitors (e.g., omeprazole and lansoprazole) have been designed that inhibit H+11upled receptors and tyrosine-kinase activity. There is evidence that chromogranin A regulates biosynthesis of dens&COre secretory granules, whereas chromogranin B controls sorting and packaging of produced peptides into secretory vesicles. Table 17.1 lists important gastrointestinal hormones, their sites of origin, and their major functions. Neoplastic transformations of ONES cells are responsible for development of gastroenteropancreatlc (GEP) neuroendocrine tumors. These tumors represent rare neoplasms of the gastrointestinal tract and pancreas that often secrete hormonally active agents, causing distinct clinical syndromes. The appendix is the most common gastrointestinal site of origin for neuroendocrine tumors. The classical example is the carcinoid syndrome caused by the release of a variety of hormonally active substances by tumor cells. Sym~ toms include diarrhea (case by serotonin). episodic flushing. bronchoconstriction, and right-sided cardiac valve disease. Some enteroendocrine cells may be classifiable functionally as amine precursor uptake and decarboxylation (APUD) cells. They should not, however, be confused with the APUD cells that are derived from the embryonic neural crest and migrate to other sites in the body. APUD cells secrete a variety of regulator substances in tissues and OFgans, including the respiratory epithelium, adrenal medulla, islets of langerhans, thyroid gland lparafollicular cells), and pituitary gland. The enteroendocrine cells differentiate from the progeny of the same stem cells as all of the other epithelial cells of the digestive tract. lhe fact that two different (continues on page 620)

619

620 FUNCTIONAL CONSIDERATIONS: THE GASTROINTESTINAL ENDOCRINE SYSTEM (CONTINUED) cells may produce similar products should not imply that they have the same origin. Enteroendocrine cells produce not only gastrointestinal hormones such as gastrin. ghrelin. secretin. cholecystokinin (CCKJ. gastric inhibitory peptide (GIPJ. and motilin but also paracrine hormones. A paracrine hormone differs from an endocrine hormone in that it diffuses locally to its target cell instead of being carried by the bloodstream to a target cell. A well-known substance that appears to act as a paracrine hormone within the gastrointestinal tract and pancreas is somatostatin, which inhibits other gastrointestinal and pancreatic islet endocrine cells. In addition to the established gastrointestinal hormones. several gastrointestinal peptides have not been definitely classified as hormones or paracrine hormones.

that complexes with vitamin 8 12 in the stomach and duodenum, a step necessary for subsequent absorption of the vitamin in the ileum. Autoantibodies directed against intrinsic factor or parietal cells themselves lead to an intrinsic factor deficiency, resulting in malabsorption of vitamin 8,2 and pemicioua anemia (see Folder 17.1).

These peptides are designated

candidate or putative

hormones. Other locally active agents isolated from the gastrointestinal mucosa are neurotransmitters. These agents are released from nerve endings close to the target cell, usually the smooth muscle of the muscularis mucosae, the muscularis extema, or the tunica media of a blood vessel. Enteroendocrine cells can also secrete neurotransmitters that activate afferent neurons, sending signals to the CNS and enteric division of the autonomic nervous system. In addition to acetylcholine (not a peptide), peptides found in nerve fibers of the gastrointestinal tract are vasoactive intestinal peptide (VIP), bombesin, and enkephalins. Thus. a particular peptide may be produced by endocrine and paracrine cells and also be localized in nerve fibers.

Enteroendocrine cells secrete their products into either the lamina propria or underlying blood vassals. Enteroendocrine cella are found at every level ofthe fundic gland, although they tend to be somewhat more prevalent in the base (Folder 17.3). In general, two types of enteroendocrine cella can be distinguished throughout

LUMEN

cluniporter channel

carbonic

anhydrase-,...:-.;... action

uniporter channel

FIGURE 17.11. DiagFIIm of parietal cell hydrochloric aeid (HCI) synth•is. After parietal cell stimulation, several steps occur leading to the production of HCI. Carbon dioxide (C02) from the blood diffuses across the basement membrane into the cell to form H2C03. The H2C03 dissociates into W and HC03-. The reaction is catalyzed by carbonic anhydrase, which leads to the production of H+ ions in the cytoplasm, which are then transported across the membrane to the lumen of the intracellular canaliculus by a W/K+-ATPase proton pump. Simultaneously, K+ within the canaliculus is transported into the cell in exchange for the W ions. c1· ions are also transported from the cytoplasm of the parietal cell into the lumen of the canaliculus by cl- channels in the membrane. HCI is then formed from W and Cl-. The HC03 -/CI- anion channels maintain the normal concentration of both ions in the cell as well as Na+JK+·ATPase on the basolateral cell membrane.

lumen

621

{ secretory

vesicles

n

:z:

> ~

..

m

:II ~

c i5 m b

~

m

"CLOSED" CELL

~

m

1:

•

• (I)

d ~

1:; ::I:

"OPEN" CELL

C

FIGURE 17.1 2. Electron miCIOfraph and diagrams of enteroendocrine cells. a. This electron micrograph shows an example of a u closed w enteroendocrine cell. Arrowheads marie: 1he boundary between the enteroendocrine cell and the adjacent epithelial cells. At its base, the enteroendocrine cell rests on the basal lamina IBL). This cell does not extend to the epithelial or luminal surface. Numerous secretory vesicles (G) in the base of the cell are secreted in the direction of the BTTOWS across the BLand into the connective tissue ICn. En, endothelium of capillary; M. mitochondria; rER, rough endoplasmic reticulum; sER. smooth endoplasmic reticulum. b. This diagram of an enteroendocrine uclosedw cell is drawn to show that it does not reach the epithelial surface. The secretory vesicles are regularly lost during routine preparation. Because of the absence of other distinctive organelles, the nucleus appears to be surrounded by a small amount of clear cytoplasm in hematoxylin and eosin IH&E)-stained sections. c. The enteroendocrine Hopenw cell extends to the epithelial surface. Microvilli on the apical surface of these cells possess taste receptors and are able to detect sweet. bitter. and umami sensations. These cells serve as chemoreceptor cells. which monitor the environment on the surface of the epithelium. They are involved in regulation of gastrointestinal hormone secretion.

the gastrointestinal tract. Most represent small cells that rest on the basal lamina and do not always reach the lumen; they are known as enteroendocrine • closed• cells (Fig. 17.12a and band Plate 57, page 650). Some cells, however, have a thin cytoplasmic extension bearing microvilli that are exposed to the gland lumen (Fig. 17.12c); these are referred to as enteroendocrine •open• cells. Open cells serve as primary chemoreceptors that sample the contents of the gland lumen and release hormones based on the information obtained from those samples. Taste receptors, similar to those found in taste buds of the specialized oral mucosa (pages 570-573), detect sweet, bitter, and umami sensations and ate present on the free surface of open enteroendocrine cells. They belong to the TlR and T2R families of G protein-coupled receptors described in Chapter 16, Digestive System I: Oral Cavity and Associated Structures. Secretion from dosed cells,

however, is regulated by the luminal contents indirectly through neural and paracrine mechanisms. Electron micrographs reveal small membrane·bound secretory vesicles throughout the cytoplasm; however, these vesicles are typically lost in H&E preparations, and the cyto· plasm appears dear because of the lack of sufficient stainable material. Although enteroendocrine cells ate often difficult to identify because of their small size and lack. of distinc· tive staining. their dear cytoplasm sometimes stands out in contrast to adjacent chief or parietal cells, thus allowing their easy recognition. The names given to the enteroendocrine cdls in the older lite.tatute were based on their staining with salts of sllver and chromium (i.e., enterochromaffin cells, argentaffin cdls, and argyrophil cells). Such cells are currently identified and characterized by immunochemical staining for the more than 20 pep· tide and polypeptide hormones and hormone-like regulating

622

antrum

-

i

c

duodenum

I

------------------- 11 jejunum S

c

I

D..

>

ileum

colon

FIGURE 17.13. Gastrointestinal honnones. This schematic diagram shows the distribution of gastrointestinal peptide hormones produced by enteroendocrine cells in the alimentary canal. CCI(. cholecystokinin; GIP. gastric inhibitory peptide; VIP, vasoactive intestinal peptide.

agena that they secrete (a list of many ofthese agents and their actions is given in Fig. 17.13 and in Tables 17.1 and 17.2). With the aid of the TEM, at least 17 d.llferent types of entero~ endocrine cells have been described on the basis ofsize, shape, and density of their secretory vesicles.

Cardiac Glands of the Gastric Mucosa Cardiac glandaara campaaad af mucus~aacrating calls. Cardiae glands are limited to a narrow region of the stomach (the cardia) that surrounds the esophageal orifice.

TABLE 17.1

Their secretion, in combination with that of the esophageal cardiac glands, contributes to the gastric juice and hdps pro~ teet the esophageal epithelium against gastric reflux. The glands are tubular, somewhat tortuous, and occasionally branched (Fig. 17.14 and Plate 56, page 648). They are composed mainly of mucus~secreting cells, with occasional interspersed enteroendocrine cells. The mucus~secreting cells are similar in appearance to the cells of the esophageal cardiac glands. They have a flattened basal nucleus, and the apical cytoplasm is typically filled with mucin granules. A shon duct segment containing columnar cells with elongate nudei is interposed between the secretory portion of the gland and the shallow pits into which the glands sec.tm. The duct segment is the site at which the sur&cc: mucous cells and the gland cells are produced.

Pyloric Glands of tha Gastric Mucosa Pyloric gland cells are similar to surface mucous cells and help protect the pyloric mucosa. Pylorfc glands are located in the pyloric antrum (the part of the stomach between the fundus and the pylorus). They are branched, coiled, tubular glands (Plate 58, page 652). The lumen is relativdy wide, and the secretory cells arc similar in appearance to the surface mucous cells, suggesting a relatively viscous secretion. Enteroendocrine cells are found interspersed within the gland epithelium along with occasional parietal cells. The glands empty into deep gastric pits that occupy about half the thickness of the mucosa (Fig. 17.15).

Epithelial Cell Renewal in the Stomach Surface mucous calls are renewed approximately nary 3ta5days. The relatively short lifespan of the surface mucous cells, 3 to 5 days, is acwmmodated by mitotic activity in the isthmus, the na.rrow segment that lies between the gastric pit

Physiologic Actions of Gastrointestinal Honnonas Major Action

Honnone

Site of Synthesis

Stimulates

Gastrin

G cells in stomach

Gastric acid secretion

Ghrelin

Gr cells in stomach

GH secretion Appetite and perception of hunger

Lipid metabolism Fat utilization in adipose tissue

Cholecystokinin (CCK)

I cells in duodenum and jejunum

Gallbladder contraction Pancreatic enzyme secretion Pancreatic bicarbonate ion secretion Pancreatic growth

Gastric emptying

Secretin

S cells in duodenum

Pancreatic enzyme secretion Pancreatic bicarbonate ion secretion Pancreatic growth

Gastric acid secretion

Gastric inhibitory peptide (GIP)

K cells in duodenum and jejunum

Insulin release

Gastric acid secretion

Motllln

Mo cells in duodenum and jejunum

Gastric motility Intestinal motility

GH, growth hormone. Modified from Johnson LR, ed. Essential Medical Physiologv, 2nd ed. Philadelphia: Lippincott-Raven, 1998.

Inhibits

TABLE 17.2

Physiologic Actions of Additional Honnones in the Gastrointestinal Tract

623

Major Action Honnone C.ndidllt/1 honnon••

Site of Synthesis

Stimulates

Inhibits

Pancreatic polypeptide

PP cells in pancreas

Gastric emptying and gut motility

Pancreatic enzyme secretion Pancreatic bicarbonate secretion

>

PeptideYY

Lcells in ileum and colon

Electrolyte and water absorption in the colon

Gastric acid secretion Gastric emptying Food intake

..

Glucagon-like peptide-1 (GLP-1)

Lcells in ileum and colon

Insulin release

Gastric acid secretion Gastric emptying

c i5 m

Pttl'at:lin• honnon.. Somatostatin

D cells in mucosa throughout Gl tract

Histamine

Mucosa throughout Gl tract

Gastrin release Gastric acid secretion Release of other Gl hormones Gastric acid secretion

n

:z:

~

m

:II ~

~

m

~

m

1:

Nsu~n•honnon••

IIIII

Bombesin

Stomach

Gastrin release

Enkephalins

Mucosa and smooth muscle throughout Gl tract

Smooth muscle contraction

Intestinal secretion

Vasoactive inhibitory peptide (VIP)

Mucosa and smooth muscle throughout Gl tract

Pancreatic enzyme secretion Intestinal secretion

Smooth muscle contraction Sphincter contraction

Gl. gastrointestinal. Modified from Johnson LR. ed. Essential Medical Physiology, 2nd ed. Philadelphia: Lippincott-Raven. 1998.

FIGURE 17. t4. Photomicrograph of c.rdlac glanda. This ph~ tomicrograph shows the esophagogastric junction. Note the presence of the stratified squamous epithelium of the esophagus in the upper right corner of the micrograph. The cardiac glands are tubular, somewhat tortuous, and occasionally branched. They are composed mainly of mucus-secreting cells similar in appearance to the cells of the esophageal glands. Mucous secretion reaches the lumen of the gastric pit via a short duct segment containing columnar cells. X240.

FIGURE 17.11. Photomicrograph of pyloric gllnda. This ph~ tomicrograph shows a section of the wall of the pylorus. The pyloric glands are relatively straight for most of their length but are slightly coiled near the muscularis mucosae. The lumen is relatively wide, and the secretory cells are similar in appearance to the surface mucous cells, suggesting a relatively viscous secretion. They are restricted to the mucosa and empty into the gastric pits. The boundary between the pits and glands is, however, hard to ascertain in routine hematoxylin and eosin IH&El preparations. X120.

• (I)

0 s:

1:; ::I:

624 FUNCTIONAL CONSIDERATIONS: DIGESTIVE AND ABSORPTIVE FUNCTIONS OF ENTEROCYTES The plasma membrane of the microvilli of the enterocyte plays a role in digestion as well as absorption. Digestive enzymes are anchored in the plasma membrane, and their functional groups extend outward to become part of the glycocalyx. This arrangement brings the end products of digestion close to their site of absorption. Included among the enzymes are peptidases and disaccharidases. The plasma membrane of the apical microvilli also contains the enzyme enteropeptidate (enterokinase), which is particularly important in the duodenum, where it converts trypsinogen into trypsin. Trypsin can then continue to convert additional trypsinogen into trypsin, and trypsin converts several other pancreatic zymogens into active enzymes (Fig. F17.4.1). A summary of digestion and absorption of the three major nutrients is outlined in the following paragraphs. Carbohydrate final digestion is brought about by enzymes bound to the microvilli of the enterocytes (fig. F17.4.2l. Galactose, glucose, and fructose are absorbed directly into venous capillaries and conveyed to

salivary and panCnlalic

amyl-.

...... G Na+

en1erocyte

G Na+

Gt .+

aucraaa

GNf.( ' F

I ~'/ •

---

glucma

l

T Gt

)border

fruclau I"

capillary

FIGURE F17.4.2. Diagram showing the digestion and absorption of cai'Hhydrates by an entarocyta_ carbohy-

pancreatic zymogens (inactive proenzymes) chymDirypahlgan

proeiiiiUe

active enzymes trypsin

pi'DCirboxypaptklua B

propholphollpaae A,

pancreatic zymogen

chymot~WJ~in

elutue l:albaxypaptldua A l:albaxypaptldua B

PI'DCI~ptidue A

phoaphollp111111 At

~===~·active enzyme

trypsinogen

drates are delivered to the alimentary canal as monosaccharides (e.g., glucose, fructose, and galactose), disaccharides (e.g., sucrose, lactose, and maltose), and polysaccharides (e.g., glycogen and starch). Enzymes involved in digestion of carbohydrates are classified as salivary and pancreatic amylases. Further digestion is performed at the striated border of the enterocytes by enzymes breaking down oligosaccharides and polysaccharides into three basic monosacx:harides (glucose, galactose, and fructose(. Glucose and galactose are absorbed by the enterocyte via active transport using Na+-dependent glucose transporters {SGLT11. These transporters are localized at the apical cell membrane (circles with G and Ns+ labels). Fructose enters the cell via facilitated Na+-independent transport using GLUTS (gray circle with F label) and GLUT2 glucose transporters (orangs octagons with G:z /sbsl). The three absorbed monosacx:harides then pass through the basal membrane of the enterocyte, using GLUT2 glucose transporters, into the underlying capillaries of the portal circulation to reach their final destination in the liver.

enterocyte

FIGURE F17_4_ 1- Diagram showing ev.nts in th• amvatian of th• pm.alytic •nzym• of th• pane....._ The majority of pancreatic enzymes (p~ses) are secreted as inactive proenzymes. Their activation is triggered by the arrival of ci1yme into the duodenum. This stimulates the mucosal cells to release and activate the enterokinase (blue box) within the glycocalyx. The enterokinase activates trypsinogen, converting it into its active form, trypsin (green~- In turn, trypsin activates other pancreatic proenzymes (red box) into their active forms (pvrple box). The active proteases hydrolyze peptide bonds of proteins or polypeptides and reduce them to small peptides and amino acids.

the liver by the vessels of the hepatic portal system. Some infants and a larger percentage of adults cannot tolerate mille and unfermented milk products because of the absence of lactase. the disaccharidase that splits lactose into galactose and glucose. These individuals are unable to break down mille or milk products, which leads to bloating caused by the buildup of gas from the bacterial digestion of the unprocessed lactose and diarrhea. The condition is completely alleviated if lactose (milk sugar) is eliminated from the diet. For some individuals, milk intolerance may be also partially or completely alleviated by using lactose-reduced milk products or tablets of lactase (enzyme that digests lactose), which are available as overthe-counter drugs. (continues on psge 625)

625 FUNCTIONAL CONSIDERATIONS: DIGESTIVE AND ABSORPTIVE FUNCTIONS OF ENTEROCYTES (CONTINUED) Trlglycerldes are broken down into glycerol, monoglycerides, and long-, medium-, and short-chain fatty acids. These substances are emulsified by bile salts and pass into the apical portion of the enterocyte. Here, the glycerol and long-chain fatty acids are resynthesized into triglycerides. The resynthesized triglycerides appear first in apical vesicles of the sER (see Fig. 17.21), then in the Golgi (where they are converted into chylomicron•. small droplets of neutral fat), and finally in vesicles that discharge the chylomicrons into the intercellular space. Instead of being absorbed directly into venous capillaries, chylomicrons are conveyed away from the intestine via lymphatic vessels (lacteals) that penetrate into each villus. Chylomicron-rich lymph then drains into the thoracic duct, which flows into the venous blood system. When in the blood circulation, chylomicrons are rapidly disassembled, and their constituent lipids are utilized throughout the body. Short- and medium-chain fatty acids and glycerol cross the apical cell membrane and enter and leave the enterocyte exclusively via capillaries that lead to the portal vein and the liver. Protein digestion and absorption is shown in Figure F17.4.3. The major end products of protein digestion are amino acids (about 30%) and oligopeptides (about 70%), which are absorbed by enterocytes. The mechanism of amino acid absorption is conceptually identical to that of carbohydrates. The apical plasma membrane of the enterocytes bears at least four Na'"-amino acid cotransporters. The dipeptides and tripeptides are transported across the apical membrane into the cell cytoplasm by the H+-oligopeptide cotransporter (PepT1 ). Most of the dipeptides and tripeptides are then digested by cytoplasmic peptidases into free amino acids, which are subsequently transported through the basal membrane (without a need for cotransporterl into the underlying capillaries of the portal circulation. In one disorder of amino acid absorption (Hartnup's disease), free amino acids appear in the blood when dipeptides are fed to patients but not when free amino acids are fed. This supports the conclusion that dipeptides of certain amino acids are absorbed via the PepT1 cotransporter. which is involved in different pathways from those involved in absorption of free amino acids.

and the fundic gland (Fig. 17.16). 1he isthmus of the fundic

n :z:

,.

~ m ::a ~

~

D

C5 rn

< = m

~m 1:

enterocyte

-

•

(J)

?amino acid transporter

\

FIGURE F17.4.3. Diagram showing the digestion and absorption of protein by an entaracyta. Proteins entering the alimentary canal are completely digested into free amino acids (sa) and small dipeptide or tripeptide fragments. Protein digestion starts in the stomach with pepsin, which hydrolyzes proteins to large polypeptides. The next step occurs in the small intestine by the action of pancreatic proteolytic enzymes. The activation process is shown in Figure F17.4.1. Free amino acids are transported by four different amino acid Na+ cotransporters. The dipeptides and tripeptides are transported across the apical membrane into the cell by W oligopeptide cotransporters (PepT11. Most of the dipeptides and tripeptides are then broken down by cytoplasmic peptidases. and free amino acids are transported through the basal membrane into the underlying capillaries of the portal circulation.

gland cells, and enteroendocrine cells that constitute the gland contains a reservoir of tissue stem cells that undergo mi- gland epithdium. These cells have a relativdy long lifespan. totic activity, providing for continuous cdl renewal. Most of Parietal cells have the: longest l.ikspan, apprwd.matdy 150 the newly produced cells at this site become surface mucous to 200 days. Although parietal cells develop from the same cells. They migrate upward along the wall of the pit to the lu- undifferentiated stem cells, their lifespan is distinctly difminal surface of the stomach and are ultimatdy shed into the ferent. It has been hypothes.izcd. that parietal cells may have originated from a fungus called Neurospora t:nJssa that stomach lumen. previously existed in a symbiotic relationship with the cells of The cells of the fundic glands have a relatively long lifespan. the human stomach. The basis for this hypothesis is that the Other cells from the isthmus migrate down into the gastric human proton pump (H+JK+ -ATPase) found in parietal cells glands to give rise to the parietal cells, chief cells, mucous bears a strong genetic resemblance to proton pumps found

d

~

f) I

Gastric Submucosa

626

The submucosa is composed of a dense connective tissue containing variable amounts of adipose tissue and blood ves· sels as well as the nerve fibers and ganglion cells that compose the submucosal {Meissner's) plexus. The latter inner· vates the vessels of the submucosa and the smooth muscle of the muscularis mucosae.

w

z

~ !z

Gastric Muscularis Extema

...J

~

(/)

-•

FIGURE 17.16. Photomicrograph of a dividing cell In the lsthmgs of • pylolk gl•nd. The gastric pits in this photomicrograph were sectioned in a plane that is oblique tD the axis of 1he pit. Note that on 1his section, gastric pits (BITOW~ can be recognized as invaginations of surface epi1helium that are surrounded by lamina propria. The lamina propria is highly cellular because of the presence of large numbers of lymphocytes. X240. lnMt. This high magnification of the area indicated by the square shows a dividing cell in the isthmus. x580.

in this organism. The fungal DNA is thought to have been translocated and subscquendy incorporated into the nucleus of the stem ceUs, probably with the help of a virus. Chief and enteroendocrine cells are estimated to live fur about 60 to 90 days before they are replac:ed by new cells mi· grating downward from the isthmus. Mucous neck cells, in contrast, have a much shorter lifespan, approximately 6 days.

Lamina Propria and Muscularis Mucosae The lamina propria of the stomach is telativcly scant and restric:ted to the limited spaces surrounding the gastric pits and glands. The stroma is composed lar~ly of retic:ular fi. hers with associated fibroblasts and smooth muscle cells. Other components include cells of the immune system, namely, lymphocytes, plasma cells, macrophages, and some cosinophils. When inflammation occurs, as is ofu:n the case. neutrophils may also be prominent. Occ:asionallymphatic nodules are also ptescnt, usually intruding partially into the muscularis mucosae. The muscularis mucosae is composed of two relatively thin layers, usually arranged as an inner circular and outer longitudinal layer. In some regions, a third layer may be present; its orientation tends to be in a circular pattern. Thin strands of smooth muscle cells extend toward the surface in the lamina propria from the inner layer of the muscularis mucosae. These smooth muscle cells in the lamina propria are thought to help outflow of the gastric gland secretions.

The muscularis extema of the stomach is traditionally described as consisting ofan outer longitudinal layer, a middle circular layer, and an inner oblique layer. This description is somewhat misleading, as distinct layers may be difficult to dis· cern. As with other hollow, spheroidal organs (e.g., gallbladder, urinary bladder, and uterus), the smooth muscle of the mus· cularis externa of the stomach is somewhat more randomly oriented than the term l4ytr implies. Moreover, the longitu· dinallayer is absent from much of the anterior and posterior stomach surfaces, and the circular layer is poorly developed in the periesophageal region. The arrangement of the muscle lay· ers is functionally important, as it telates to its role in mixing chyme during the digestive process as well as to its ability to furce the partially digested contents into the small intestine. Groups of ganglion cells and bundles of unmyelinated nerve fibers are present between the muscle layers. Collectively, they represent the myenteric {Auerbach's) plexus, which provides innervation of the muscle layers.

Gastric Serosa The serosa of the stomach is as described above fur the alimentary canal in general. It is continuous with the parietal peritoneum of the abdominal cavity via the greater omentum and with visceral peritoneum of the liver at the lesser omentum. Otherwise, it exhibits no special features.

• SMALL INTESTINE The small intestine is the longest component of the diges· tive tract. measuring over 6 m, and is divided into three anatomic portions: • Duodenum (-25 em long) is the first, shortest, and widest part of the small intestine. It begins at the pylorus of the stomach and ends at the duodenojejunal junction (Plate 59, page 654). • Jejunum (-2.5 m long) begins at the duodenojejunal junction and constitutes the upper two-fifths of the small intestine. It gradually changes its morphologic characteristics to become the ileum (Plate 60, page 656). • Ileum (-3.5 m long) is a continuation of the jejunum and constitutes the lower three-fifths of the small intestine. It ends at the ileocecal junction, the union of the distal ileum and cecum (Plate 61, page 658).

The small intestine is the principal site for the digestion of food and absorption of the products of digestion. Chyme from the stomach enters the duodenum, where enzymes from the pancreas and bile from the liver are also delivered to continue the solubilization and digestion process. Enzymes, particularly disaccharidases and dipeptidascs, are

also located in the glycocalyx of the microvilli of the antaro· cytas, the intestinal absorptive calls. These enzymes contribute to the digestive process by completing the breakdown of most sugars and proteins to monosaccharides and amino acids, which are then absorbed (Folder 17.4). Water and electrolytes that reach the small intestine with the chyme and pancreatic and hepatic secretions are also reabsorbed in the small intestine, particularly in the distal portion. Plicae circulares, villi, and microvilli increase the absorp· tive surface area of the small intestine. The absorptive surface area of the small intestine is amplified by tissue and cell spc:cializarions of the submucosa and mucosa. • Plicae circulares (circular folds), also known as the valves of Kn-clmng, are permanent transverse folds that contain a core ofsubmucosa. Each circular fold is circularly arranged and c:nends about one-half to two-thirds of the way around the circumference of the lwnen (Fig. 17.17). The folds begin to appear about 5 to 6 em beyond the pylorus. They are most numerous in the distal part of the duodenum and the beginning of the jejunwn and become reduced in size and frequency in the middle of the ileum.

FIGURE 1 7.17. Photograph of the mucosal surface of the small lnteltlne. This photograph of a segment of a human jejunum shows the mucosal surface. The circular folds (plicae circularesl appear as a series of transversely oriented ridges that extend partially around the lumen. Consequently, some of the circular folds appear to end (or begin! at various sites along the luminal surface !arrows}. The entire mucosa has a velvety appearance because of the presence of villi.

• Villi are unique, finger-like and leaf-Uke projections of the mucosa that extend from the theoretical mucosal surface for0.5 to 1.5 mm into the lumen (Fig. 17.18).1heycompletdy cover the surface of the small intestine, giving it a velvety appearance when viewed with the unaided eye. • Microvilli of the enterocytes provide the major ampllfication of the luminal surf.u:e. Each cell possesses several thousand closely packed microvilll. They are visible in the light microscope and give the apical region of the cell a striated appearance, the so-called striated border. Enterocytes and their microvilli are described below. The villi and intestinal glands, along with the lamina propria, associated GALT, and muscularis mucosae, constitute the essential features of the small intlltinal mucosa. VIlli, as noted, are projections of the mucosa. They consist of a core ofloose connective tissue covered by a simple colwnnar epithdiw:n. The core of the villus is an extension of the lamina propria, which contains numerous fibroblasts, smooth muscle cdls, lymphocytes, plasma cells, eosinophils, macrophages, and a network of fenestrated blood capillaries located just beneath the epithdial basal lamina. In addition, the lamina propria of the villus contains a central, blind-ending lymphatic capillary. the lacteal (Fig. 17.19 and Plate 60, page 656). Smooth muscle cells derived from the muscularis mucosae extend into the villus and accompany the lacteal. These smooth muscle cells may account for the observation that villi contract and shorten intermittendy. an action that may force lymph from the lacteal into the lymphatic vessel network surrounding the muscularis mucosae. The intestinal glands, or crypts of Liabarkiihn, are simple tubular structures that extend from the muscularis mucosae through the thickness of the lamina propria, where they open onto the luminal surfuce of the intestine at the base of the villi (see Fig. 17.18). The glands are composed of a simple columnar epithdium that is continuous with the epithelium of the villi. As in the stomach, the lamina propria surrounds the intesti.~ nal glands and contains numerous cells of the immune system (lymphocytes, plasma cells, mast cells, macrophages, and eosin~ ophils), particularly in the villi. The lamina propria also con~ tains numerous nodules of lymphatic tiuua that represent a major component of the GALT. The nodules are particularly large and numerous in the ileum, where they are preferentially located on the side of the intestine opposite the mesenteric at~ tachment (Fig. 17.20). These nodular aggregations are known as aggregated nodules or Payer's patches. In gross spec~ imens, they appear as aggregates of white specks. The muscularis mucosae consists of two thin layers of smooth muscle cells, an inner circular and an outer longitudinal layer. As noted above, strands of smooth muscle cells extend from the muscularis mucosae into the lamina propria ofthe villi. At least five types of cells are found in intestinal mucosal epithelium. The mature cells of the intestinal epithdium are found both in the intestinal glands and on the surfuce of the villl. They include the following: • Enterocytes, whose primary function is absorption • Goblet cells, unicellular m.ucin·sec:reting glands

627

• (I)

~

r

~

m

~

z

m

628

w

z

lacteal

~ !z ...J

~

(/)

-• b muscularis mucosae

vein

FIGURE 17.18. Intestinal villi In the small Intestine. •· Scanning electron micrograph of the intestinal mucosa showing its villi. Note the openings {a/TOws) located between the bases of the villi that lead into the intestinal glands (crypts of Lieberkuhn). X800. b. This threedimensional diagram of the intestinal villi shows the continuity of the epithelium covering the villi with the epithelium lining the intestinal glands. Note blood vessels and the blind-ending lymphatic capillary, called e lacteal, in the core of the villus. Between the bases of the villi, the openings of the intestinal glands can be seen {aiTOwsl. Also, the small openings on 1he surface of the villi indicate the location of discharged goblet cells.

• Paneth cells, whose primary function is to maintain mucosal innate immunity by secreting antimicrobial substances • Enteroendocrine cells, which produce various para~ crine and endocrine hormones • M cells (microfold cells), specialized cells located in the epithelium that covers lymphatic nodules in the lamina propria

Enterocytas are absorptive cells specialized for the transport of substances from the lumen of the intestine to the circulatory system.

RGURE 17.18. Ph~mlcragreph of an lntltltlnll 'VIllus. The surface of the villus consists of columnar epithelial cells, chiefly enterocytes with a striated border. Also evident are goblet cells that can be readily identified by the presence of the apical mucous cup. Located beneath 1he epithelium is the highly cellular loose connective tissue, 1he lamina propria. The lamina propria contains large numbers of round cells, mostly lymphocytes. In addition, smooth muscle cells can be identified. A lymphatic capillary called a lacteal occupies the center of the villus. When 1he lacteal is dilated, as it is in 1his specimen, it is easily identified. X 160.

Enterocytes ~tall columnar c:ells with a basally positioned nuc:lcus (see Figs. 17.18 and 17.21). MicrovilU increase the apical surfac:e ~ as much as 600 times; they ~ recognized in the light microscope as forming a striated border on the luminal surface. Each microvillus has a core of vertic:ally oriented actin micro6lamcnts that an: anchored to villin located in the tip of the microvillus and that also attach to the microvillus plasma membrane by myosin I molecules. The actin microfilaments extend into the apical cytoplasm and insert into dte tenninal web, a network of ho.rizontally oriented contractile micro6laments that form a layer in the most apical cytoplasm and attach to the intracellular density associated with the zonula adherens. Conttaction of the terminal web causes dte microvilli to spread apart, thus increasing the space between them to allow more surface area o:posure for absorption to take place. In addition, contraction of the terminal web may aid in "closing"' the holes left in the epithelial sheet by exfoliation of aging cells. Entcro· cytes an: boWld to one another and to goblet, entc.roendocrinc, and odter c:eUs ofthe epithelium by junctional complm:s.

patdl••·

FIGURE 17.20. Photomicrograph of Peyer's This photomicrograph shows a longitudinal s&Ction through the wall of a human ileum. Note the extensive lymphatic nodules located in the mucosa and the section of a circular fold projecting into the lumen of the ileum. Lymphatic nodules within the Peyer's patch are primarily located within the lamina propria, although many extend into the submucosa. Thev are covered by the intestinal epithelium, which contains enterocytes, occasional goblet cells, and specialized antigen-transporting M cells. X40.

Tight junctions establish a barrier between the intestinal Iuman and the epithelial intercellular compartment. The tight junctions between the intestinal lumen and the connective tissue compartment of the body allow selective retention of substances absorbed by the enterocytes•.& noted in Chapter 5, the "'tightness" of these junctions can vary. In relatively impermeable tight junctions, as in the llewn and colon, active transport is required to move solutes across the battier. In simplest terms, active transport systems, for example, sodium pumps (Na+ IK+ -ATPase), located in the lateral plasma membrane, transiently reduce the cytoplasmic concent.tation ofNa+ by transporting sodium ions across the lateral plasma membr.me into the extracellular space below

the tight junction. This transport of Na+ creates a high intercellular Na+ concenaation, causing water from the cell to enter the intercellular space, reducing both the water and Na+ concenaations in the cell. Consequently, water and Na+ enter the cell at its apical swface, passing through the cell and exiting at the lateral plasma membrane as long as the sodiwn pump continues to function. Increased osmolarity in the intercellular space draws water into this space, establishing a hydrostatic pressure that drives Na+ and water across the basal lamina into the connective tissue. In epithelia with more permeable tight junctions, such as those in the duodenum and jejunwn, a sodiwn pwnp also creates law intrac:dlular Na+ concenttarlon. When the contenu that pass into the duodenum and jejunum are hypotonic, however, considerable absorption ofwater, along with additional Na+ and other small solutes, takes place directly across the tight junctions of the enterocytes into the intercellular spaces. 1h.is mechanism ofabsorption is referred to as solvent drag. Other transport mechanisms also increase the concenaations of specific substances, such as sugars, amino acids, and other solutes in the intercellular space. These substances then diffuse or Bow down their concentration gradients within the intercellular space to cross the epithelial basal lamina and enter the fenestrated capillaries in the lamina propria located immediately beneath the epitheliwn. Substances that are too large to enter the blood vessels, such as lipoprotein particles, enter the lymphatic lacteal. The lateral cell surface of the enterocytes exhibits elaborate, Battened cytoplasmic processes (plicae) that interdigitate with those of adjacent cells (see Fig. 5.24). These folds increase the lateral surface area of the cell, thus increasing the amount of plasma membrane containing transport enzymes. During active absorption, especially ofsolutes, water, and lipids, these lateral plication• separate, enlarging the intercellular compartment. The increased hydrostatic pressure from the accumulated solutes and solvenu causes a directional flaw through the basal lamina into the lamina propria (see Fig. 5.1). In addition to the membrane specializations associated with absorption and aansport, the enterocyte cytoplasm is also specialized for these functions. Elongated mitochondria that provide energy for transport are concentrated in the apical cytoplasm between the tenninal web and the nucleus. Tubules and cisternae of the smooth endoplasmic reticulum (sER), which are involved in the absorption of fatty acids and glycerol and in the resynthesis of neutral fat, are found in the apical cytoplasm beneath the tenninal web.

Enteracytes are also secretory cells, producing enzymes needed far terminal digestion and absorption as well as secretion of water and electrolytes. The secretory function of enterocytes, primarily the synthesis of glycoprotein enzymes that will be inserted into the apical plasma membrane, is represented morphologically by aligned stacks ofGolgi cisternae in the immediate supranuclear region and by the pn:sence of free ribosomes and rER lateral to the Golgi apparatus (see Fig. 17.21). Small secretory vesicles containing gl.ycoproteins destined for the cell surface are located in the apical cytoplasm, just below the terminal web, and along the lateral plasma rn.cmbr.mc:. Histochemical or autoradiographic methods are needed, however, to distinguish these secretory vesicles from c:ndocytotic vesicles or smalllysosomes.

629

• (I)

~

r

~

m

~

z

m

630

terminal web

w

z

~ !z ...J

~

(f)

-•

a

b

ABSORPnVE CELLS FIGURE 17.21. Diagrams of an en..rocytll in diffltt'11nt ph.... of absorption. a. This cell has a striated border on its apical suriaoe and junctional complexes that seal the lumen of the intestine from the lateral intercellular space. The characteristic complement of major organelles is depicted in 1he diagram. b. This cell shows the distribution of lipid during fat absorption as seen with the transmission electron microscopy (TEMI. Initially, lipids are seen in association with the microvilli of the striated border. Lipids are internalized and seen in vesicles of the smooth endoplasmic reticulum (sER) in the apical portion of the cell. The membrane-bounded lipid can be traced to the center of the cell. where many of the lipid-containing vesicles fuse. The lipid is then discharged into the intercellular space. The extracellular lipids, recognized as chylomicrons, pass beyond the basal lamina for further transport into lymphatic !green) andfor blood vessels {reo'). rER, rough endoplasmic reticulum.

The small intestine also secretes water and elc:ctrolytcs. This aa:i.vity occurs mainly in the cells within the intestinal glan.ds.

The secretion that occurs in these glands is thought to assist the process of digestion and absorption by maintaining an appropriate liquid state of the intestinal chyme. Under normal conditions, the absorption of fluid by the villus enterocyte is balanced by the secretion offluid by the gland entcrocyte.

Goblet cells represent unicellular glands that are interspersed among the other cells of the intestinal epithelium. As in other epithelia. goblet cells produce mucus. In the small intestine, goblet cells increase in number from the duodenum to the terminal part of the ileum. Also, as in other cpithdia, because watc:Noluble mucinogen is lost during preparation of routine H&.:E sections, the part of the cell that normally contains mucinogen granules appears empty. Examination with the TEM reveals a large accumulation of mucinogen granules in the apical cytoplasm that distends the apex of the cell and distorts the shape of neighboring cells (Fig. 17.22). With the apex ofthe cell containing a large accumulation ofmucinogen

granules. the basal portion of the cell resembles a narrow stem. This basal portion is intensely basophilic in histologic prepa· rations because it is occupied by a heterochromatic nucleus. e:x:tcnsive rER. and free ribosomes. Mitochondria are also con· centtated in the basal cytoplasm. The characte.ristic shape. with the apical accumulation ofgranules and the narrow basal stem, is responsible for the name of the cell, as in a glass "goblet."' An e:x:tcnsive array of flattened Golgi cisternae forms a wide cup around the newly formed mucinogen granules adjacent to the basal part of the cell (see Fig. 17.22a). The microvilli of goblet cells are restticted to a thin rim of cytoplasm (the theca) that surrounds the apical-lateral portion of the mucinogen granules. Microvilli are more obvious on the immature goblet cells in the deep one-halfof the intestinal gland.

Panath calls play a role in regulation of normal bacterial flora of 1he small intestine. Paneth cells are found in the bases of the intestinal glands. ('They are also occasionally found in the normal colon in small numbers; their number may inc.n:ase in certain pathologic

microvilli

631

• (I)

~

r

~

b FIGURE 17.22. Electron micrograph and diagram of a goblet cell. a. This electron micrograph shows the basal portion of a goblet cell depicted on 1he adjacent diagram. The cell rests on the basal lamina. The basal portion of the cell contains the nucleus, rough endoplasmic reticulum, and mitochondria. Just apical to the nucleus are extensive profiles of Golgi apparatus. As the mucous product aocumulates in the Golgi cisternae, they become enlarged (asterisks). The large mucinogen granules fill most of the apical portion of the cell and collectively constitute the Nmucous cupN seen in the light microscope. x15,000. b. This diagram shows the entire goblet cell. The boxed region on this diagram represents an area from which the adjacent electron micrograph was most likely obtained. The nucleus is located at the basal portion of the cell. The major portion of the cell is filled with mucinogen granules forming the mucous cup that is evident in the light microscope. At the base and lower sides of the mucous cup are flattened saccules of the large Golgi apparatus. Other organelles are distributed throughout the remaining cytoplasm, especially in the perinuclear cytoplasm in the base of the cell.

conditions.) They have a basophilic basal cytoplasm; a supra· nuclear Golgi apparatus; and large, intt:nsdy acidophilic, refra· ctile apical secretory vesicles. These vesicles allow their easy identification in routine histologic sections (Fig. 17.23). The sa· cretory vesicles contain the antibacterial enzyme lysozyme, a.-defensins, other glycoproteins, an arginine-rich protein (probably responsible for the intense acidophilia), and zinc. Lysozyme digests the cell walls of certain groups of bacte· ria. a.·Defenslns are homologs of peptides that function as mediators in cytotoxic CDS+T lymphocytes. Their antibacterial action and ability to phagocytose certain bacteria and protozoa suggest that Paneth cells play a role in regulating the normal bacterial flora of the small intestine.

Enteroendocrine cells in the small intestine produce nearly all of the same peptide honnonesasthey do in the stomach. Enteroendocrine cella in the small intestine resemble those that n:side in the stomach (see Fig. 17.12). The "closed cdls.. are c:oncenttatcd in the lower portion ofthe intestinal gland, whereas the "open cells"' can be found at all levels ofeach villus..Activation of wte receptors found on the apical cell membrane of "open cells" activates the G protein signaling cascade, resulting in the release of peptidcs that regulate a variety of gastroin~al

functions. These include pancreatic sec::retion, inducing digu· tion and absorption. and energy homeostasis by acting on neu· ra1 pathways of the brain-gut-adipose axis. Nearly all of the same peptide honnones identified in this ceU type in the stomach can be demonst:rated in the enteroendoc:rine cells ofthe intx:stine (see Table 17.1). Cholecystokinin (CCK), secretin, gastric inhibitory polypeptide (GIP), and motilin are the most ac-tive regulators of gastroinu:stinal physiology that are released in this portion of the gut (see Fig. 17.13). CCK and sec.tetin in· crease pancreatic and gallbladder activity and inhibit gastric secretory function and motility. GlP stimulates insulin n::lease in the pancreas, and motilin initiatc::s gastric and intestinal motility. Although other peptides produced by entt:rocndocrinc cells have been isolau:d, they are not considered hormones and are therefun: called candidate honnones (pagt: 623). Enteroendocrine cells also produce at least two honnones, somatDstatin and histamine, which act as paracrine honnones (see page 623) (i.e., hor· mones that have a local effect and do not circulate in the blood· stmun). ln addition, seve.tal. peptides are ~ by the nerve cells located in the submucosa and muscularis cx.tema. These peptides, called neurocrine honnones, are rep.resented by vasoactive intestinal peptide (VIP), bombes.in, and the enkepha· tins. The functions ofthese peptides are listc:d in Table 17.2.

m

~

z

m

as highly specialized antigen-transporting cells that relocate intact antigens from the intestinal lumen across the epithelial barrier. Antigens that reach the immune cells in this manner stimulate a response in the GALT that is described below.

632

Intermediate cells constitute the amplifying compartment of the intestinal stem cell niche. lntemaediate cells constitute most of the cells found

w

z

~ !z

within the intestinal stem cell niche that is located in the lower half of the intestinal gland. These cells constitute the amplifying compamneot of the cells that are still capable of cell division and usually undergo one or two divisions before they become committed to differentiation into either absorptive or goblet cells. These cells have short, irregular microvilli with long core 6laments enending deep into the apical cytoplasm and numerous macular (desmosomal) junctions with adjacent cells. Small mucin-like secretory granules form a column in the center of the supranuclear cytoplasm. Intermediate cells that are committed to becoming goblet cells develop a small, rounded collection of secretory granules just beneath the apic:al plasma membrane; those that are committed to becoming absorptive cells lose the secretory granules and begin to show concentrations of mitochondria, rER., and ribosomes in the apic:al cytoplasm.

...J

~

(f)

-•

FIGURE 17.21 Photomlcrogl'lph of lntaltlnal 111ndl lhowlng Plneth calls. This photomicrograph shows the base of intestinal ijejunal) glands in an hematoxylin and eosin (H&E) preparation. The gland on the tight is sectioned longitudinally; the circular cross-sectional profile of another gland is seen on the left. Paneth cells are typically located in the base of the intestinal glands and are readily seen in the light microscope because of the intensive eosin staining of their vesicles. The lamina propria contains an abundance of plasma cells, lymphocytes, and other connective tissue cells. Note several lymphocytes in the epi1helium of the gland lan-ows). X240. Inset. This high magnification of the area indicated by the rectangle shows the characteristic basophilic cytoplasm in 1he basal portion of the cell and large accumulations of intensely staining, eosinophilic, refractile secretory vesicles in the apical portion of the cell. An arginine-rich protein found in the vesicles is probably responsible for the intense eosinophilic reaction. X680.

M cells convey microorganisms and other macromolecules from 1he intes1inallumen 1o Payer's patches. M cells are epithelial cells that overlie Payer's patches and other large lymphatic nodules; they differ significantly from the surrounding intestinal epithelial cells (Folder 17.5). M cells have a characteristic shape because each cell develops a deep pocket-like recess connected to the extracellular space. Dendritic cells, mac:rophages, and T and B lymphocytes reside in this space. Due to this unique shape, the basolateral cell surface of the M cell resides within a few microns of its apic:al surface, greatly reducing the distance that endocytic vesic:les must travel to cross the epithelial barrier. On their apical sur&.ce, M cells have microfolds rather than microvilli and a thin layer of gl.ycoc:alyx. The apical surface expresses an abundance of glycoprotein 2 (GP2) receptors that bind specific macromolecul.c:s and Gram-negative bacteria (e.g., Eschtriehia co/s). The substances bound to GP2 receptors are internalized in endocytic: vesicles and transported to the basolatetal cell sur&.ce of the pocket-like recess. Within the recess, the released contents are immediately transfio:rred to immune ceUs residing in this space. Thus, M ceUs function

GALT is prominent in the lamina propria of the small intestine. As noted above, the lamina propria of the digestive tract is heavily populated with elements of the immune system; approximately one·fourth of the mucosa consists of a loosely organized layer of lymphatic nodules, lymphocytes, maaophagcs, plasma cells, and eosinophils in the lamina propria (Plate 55, page 646). Lymphocytes are also loc:ated between epitheli.al cells. The GALT serves as an immunologic barrier throughout the length of the gasttointesrinal ttact. In cooperation with the overlying epithelial cells, particularly M cells, the lymphatic tissue samples the antigens in the epithelial intercellular spaces. Lymphocytes, macrophages, and other antigen·prcseoting cells process the antigens and migrate to lymphatic nodules in the l.amina propria where they undergo activation (see page 481), leading to antibody sec:rc:ti.on by newly differentiated plasma cells.

The mucosal surface is protected by immunoglobulinmedia1ed responses. The mucosal surface ofthe gut tube is constantlychallenged by the presence of ingested microorganisms (i.e., viruses, bacteria, parasites) and toxins, which after compromising the epithelial barrier, may cause infections or diseases. An example of a specific: defense mechanism is the immunoglobulin-mediated response using IgA, IgM, and IgE antibodies. Most of the plasma ceUs in the lamina propria of the intestine secrete dimeric: dlgA antibodies rather than the more common lgG; other plasma cells produce pentarneric: IgM and IgE (see page 593). Dimetic dlgA is composed of two monomeric lgA. subunits and a polypeptide J chain (see Fig. 16.28). Secreted dlgA molecules bind to the polymeric immunoglobulin receptor (plgR) located at the basal domain of the epithelial cells (Fig. 17.24). The plgR. receptor is a transmembrane glycoprotein (75 kDa) synthesized by enterocytes and expressed on the basal plasma membrane. The:

633 FUNCTIONAL CONSIDERATIONS: IMMUNE FUNCTIONS OF THE ALIMENTARY CANAL Immunologists have shown that the GALT not only responds to antigenic stimuli but also functions in a monitoring capacity. This function has been partially clarified for the lymphatic nodules of the intestinal tract. TheM cells that are part of the epithelium that cover Peyer's patches and lymphatic nodules have a distinctive surface that might be misinterpreted in sections as thick microvilli. The cells are readily identified with the scanning electron microscope because microfolds contrast sharply with the microvilli that constitute the striated border of the adjacent enterocytes. It has been shown with glycoprotein GP2 (molecular marker for M cells) that the M cells endocytose proteins and bacteria from the intestinal lumen. transport them in the vesicles through the cell, and discharge the contents by exocytosis into deep recesses that are continuous with the extracellular space (Fig. F17.5.1 l. Dendritic cells and lymphocytes within the deeply recessed extracellular space sample the luminal protein, including antigens, and

thus have the opportunity to stimulate development of specific antibodies against the antigens. The destination of these exposed lymphocytes has not yet been fully determined. Some remain within the local lymphatic tissue, but others may be destined for other sites in the body, such as the salivary and mammary glands. Recall that in the salivary glands, cells of the immune system (plasma cells) secrete lgA, which the glandular epithelium then converts into slgA. Some experimental observations suggest that antigen contact necessary for the production of lgA by plasma cells occurs in the lymphatic nodules of the intestines. Recent findings using GP2-deficient mice show that the interaction of GP2 with bacteria plays an important role in antigen-specific immune responses in Payer's patches. This discovery may lead to development not only of new oral vaccines for infectious diseases but also of the innovative treatment of tumors and inflammatory bowel diseases.

•s:

(J)

)>

r r

z

rrl

(J)

::!

z

m

lymphocyteS

absorptive - -

cells

maaophage--~~~---r

a FIGURE F17.5.1. Diagram ofM ct~lls covering the lymphatic nodule of the intestine. a. This diagrt~m shows the relationship of the M cells (microfold cells) and absorptive cells in the epithelium covering a lymphatic nodule. TheM cell is an epithelial cell that displays microfolds rt~ther than microvilli on its apical surface. It has deep recesses within which lymphocytes, macrophages, and processes of dendritic cells come close to the lumen of the small intestine. An intact antigen from the intestinal lumen is transferred across the thin layer of theM cell apical cytoplasm to lymphocytes and other antigen-presenting cells residing within the recesses. b. Scanning electron microgrt~ph of a Peyer's patch lymphatic nodule bulging into the lumen of the ileum. Note that the area of the follicle covered by M cells is surrounded by the finger-like projections of the intestinal villi. The surface of the M cells has a smooth appearance. The absence of absorptive cells and mucus-producing goblet cells in the area covered by M cells facilitates immunoreactions to antigens. X80. (Reprinted with permission from Owen RL. Johns AL. Epithelial cell specialization within human Peyer's patches: an ultrastructurt~l study of intestinal lymphoid follicles. Gastroenterology 1974;66:189-203.)

plgR--dlgA complex is then endocytosed and transported across the epithdium to the apical surface of the entc:rocyte (this type oftransport refers to as transcytosis). After the plgR-

dlgA complex reaches the apical surface, plgR is proteolytically cleaved and the exttacdlular part of the receptor that is bound to dlgA is released into the gut lumen (see Fig. 17.24). This cleaved extracellular binding domain of the receptor is

known as the secretory component (SC); secreted digA in association with the SC is known as secretory lgA (slgA). The release of slgA immunoglobulins is critical for proper

immunologic surveillance by the mucosal immune system. In the lumen, slgA binds to antigens, toxins, and microorganisms. Secretory lgA prevents the attachment and invasion of viruses and bacteria into the mucosa by

cells in the lamina propria (see pages 194-198), selectively sensitizing them to specific antigens derived from the lumen.

634

Submucosa A distinguishing characteristic of 1he duodenum is the presence of submucosal glands.

w

z

The submucosa consists of dense connective tissue and localized sites that contain aggregates of adipose cells. A conspicuous feature in the duodenum is the presence of

~ !z

submucosal glands, also called Brunner's glands. The branched, tubular submucosal glands of the duodenum have secretory cells with characteristics of both zymogen-secreting and mucus-secreting cells (Fig. 17.25). The secretion of these glands has a pH of8.1 to 9.3 and contains neutral and alkaline glycoproteins and bicarbonate ions. This highly alkaline secretion probably serves to protect the proximal small intestine by neutralizing the acid-containing chyme delivered to it. It also brings the intestinal contents close to the optimal pH for activation of pancreatic enzymes that are also delivered to the duodenum.

...J

~

(f)

-•

lamina

~

LAMINA PROPRIA

dlgA

:J c:hain

plasma cell

lgA FIGURE 17.24. Dla1ram of lmmuno1lobulln A (lgA) tecretlon and transport. A monomeric form of immunoglobulin A 1/gA) is synthesized by the plasma cell. lgAs are secreted into the lamina propria in a dimeric form as dlgA. Dimeric dlgA is composed of two monomeric lgA subunits and a polypeptide J chain also produced by the plasma cell. In the lamina propria, dlgA binds to the polymeric immunoglobulin receptor (pig!?} on the basal cell membrane of the enterocyte. The plgR-IgA complex enters the cell by endocytosis and is carried out within the endocytotic vesicles to the early endosome! compartment and then to 1he apical surface {a process called transcytosisl. Endocytic vesicles fuse with the apical plasma membrane. the plgR is proteolytically cleaved, and dlgA is released with the extracellular portion of the plgR receptor. This portion of the plgR remains with the lgA dimer and becomes the secretory component (SC) of the secretory lgA lslgA).

either inhibiting their motility, causing microbial aggregation, or masking pathogen adhesion sites on the epithelial surface. For example, slgA binds to a glycoprotein on the viral envelope of HIV, preventing its attachment, internalization, and subsequent replication in the cell. Secretory IgA. is the principal molecule of mucosal im· munity. However, IgM molecules utilize similar pathways of teceptor·mc:diated ttanscytosis to teach the mucosal surface. Some: of the IgE binds to the plasma membranes of mast

c

' .r-..:...

FIGURE 17.2&. Photomicrograph of Blunner'• gland• In the dllodenllm. This photomicrograph shows part of the duodenal wall in an hematoxylin and eosin IH&E) preparation. A distinctive feature of the duodenum is the presence of Brunner's glands. The dashed line marks 1he boundary between the villi and the typical intestinal glands !crypts of Lieberkuhnl. The latter extend to the muscularis mucosae. Beneath the mucosa is the submucosa. which contains Brunner's glands. These are branched tubular glands whose secretory component consists of columnar cells. The duct of the Brunner's gland opens into the lumen of the intestinal gland IarroW). X120.

Muscularis Externa

Epithelial Cell Renewal in the Small Intestine

cells in various stages of differentiation. A cell destined to become a goblet cell or absorptive cell usuilly undergoes several additional divisions after it leaves the pool of stem cells. The epithelial cells migr.tte upward in the intestinal gland onto the villus where they undergo apoptosis and slough off into the lumen. Autoradiographic stUdies have shown that the renewal time for absorptive and goblet cells in the human small intestine is 4 to 6 days. Enteroendocrine cells and Paneth cells are also derived from the stem cells at the base of the intestinal gland. Enteroend.ocrine cells appear to divide only once before differentiating. They migrate with the absorptive and goblet cells but at a slower rate. Paneth cells migr.tte downward and reside at the bottom of the inteUi.nal gland. They live for approximately 4 wub and are then replaced by differentiation of a nearby "committed" cell in the intestinal gland. Cells that are recognizable as Paneth cells no longer divide. As mentioned in Chapter 5, Epithelial TISSUe (page 160). expression oftranscription factor Math1 appears to determine the fate ofdifFerentiating cells in the intestinal stem cell niche. The cells committed to the secretory lineage (i.e., they will differentiate into goblet, enteroendocrine, and Paneth cells) have increased expression of Math1. Inhibition ofMathl expression characterizes the default developmental pathway into absotptive intestinal cells (enterocytes).

Mature cells of the intestinal epithelium are derived from a single stem cell population.

• LARGE INTESTINE

The muscularis extema consists of an inner layer of circularly arranged smooth muscle cells and an. outer layer of longitudinally arranged smooth muscle cells. The main components of the myenteric plexus (Auerbach's plexus) are located between these two muscle laye.rs (Fig. 17.26). Two kinds of muscular contraction occur in the small intestine. Local contractions displace intestinal contents both proximally and distally; this type of contraction is called segmentation. These contractions primarily involve the circular muscle layer. They serve to circulate the chyme locally; mixing it with digestive juices and moving it into contact with the mucosa for absorption. Peristalsis, the second type of contraction, involves coordinated action of both circular and longitudinal muscle layers and moves the intestinal contents distally.

Serosa The serosa of the parts of the small intestine that are located inttaperitoneally in the abdominal cavity corresponds to the general description at the beginning of the chapter.

Stem cells aze located in the base of the intestinal gland. This intestinal stem cell niche (zone of cell replication) is restricted to the lower one-half of the gland and contains highly prolife.rative intermediate cells (as previously explained) and

The large intestine comprises the cecum with its projecting vermiform appendix, the colon, the rectum. and the anal canal. The colon is further subdivided on the basis of iu anatomic locations into the ascending colon, tr.ms\le!Se colon,

FIGURE 17.26. Ele;tron micrograph of the myenterk (Auerbacfis) plexus. The plexus is located between the two smooth muscle ISMI layers of the muscularis extema. It consists of nerve cell bodies (CB) and an extensive network of nerve fibers (N). A satellite cell {SCI. also referred to as an enteric glial cell. is seen in proximity to the neuron cell bodies. These cells have structural and chemical features in common with glial cells of the CNS. BV. blood vessel. X3,800.

635

•> ~ z

m

~ ~

z

m

636

descending colon, and sigmoid colon. The four layers characteristic of the alimentary canal are present throughout. However, several distinctive features exist at the gross level (Fig. 17.27):

• Teniae coli represent thtc:e narrowed, thickened, equally spaced bands of the outer longitudinal layer of the: mus-w

z

~ !z w

(!)

~

-•

cularis e:x:terna. Thc:y ate: primarily visible: in the cecwn and colon and are absent in the rectum, anal canal, and vermiform appendix. • Haustra coli are visible sacculations between the teniac: coli on the: external surface: ofthe cecum and colon. • Omental appendices ate small, fatty projections ofthe serosa, observed on the outer surface: ofthe colon.

Mucosa The mucosa ofthe: large intestine has a "'smooth" surface:; neither plicae circulates nor villi are present. It contains numerous straight tubular intestinal glands (crypts oflieberkiihn) that extend through the full thickness of the mucosa (Fig. 17.28a). The glands consist ofsimple columnar epithelium, as does the intestinal surface: from which they invaginate. Examination of the luminal surf.u::c ofthe large intestine at the microscopic level reveals the openings ofthe glands, which are arranged in an orderly pattern (Fig. 17.28b).

The principal functions of 1he large intes1ine are reabsorption of electrolytes and water and elimination of undigested food and wasta. The primary function of the columnar absorptive cells is reabsorption ofwater and elec:trolytcs. The morphology ofthe absorptive cells of the large intestine is essentially identical to that of the enterocytes of the small intestine. ~absorption is

FIGURE 17.27. Photograph of thelaqelntHtlne. This photograph shows the outer (serosal) suriace {/effl and internal (mucosal) surface (right} of the transverse colon. On the outer surface, note the characteristic features of the large intestine: a distinctive smooth muscle band representing one of the three tenia& coli lTC ); haustra coli IHC ), the sacculations of the colon located between the teniae; and omental appendices (OA}. small peritoneal projections filled with fat. The smooth mucosal surface shows semilunar folds (arrows) formed in response to contractions of the muscularis externa. Compare the mucosal surface as shown here with that of the small intestine (Fig. 17.17}.

FIGURE 17.28 MuCOH of the large intestine. •· This photomicrograph of an hematoxylin and eosin (H&El preparation shows the mucosa and part of the submucosa. The surface epithelium is continuous with the straight, unbranched, tubular intestinal glands (crypts of Lieberkiihn). The openings of the glands at the intestinal surface are identifl&d latTOws). The epithelial cells consist principally of absorptive and goblet cells. As the absorptive cells are followed into the glands, they become fawer in number, whereas the goblet cells increase in number. The highly cellular lamina propria contains numerous lymphocytes and other cells of the immune system. b. Scanning electron micrograph of the human mucosal surface of the large intestine. The surface is divided into territories by clefts (arrows). Each territory contains 25 to 100 gland openings. x 140. (Reprinted with permission from Fenoglio CM, Richart RM, Kaye Gl. Comparative electron-microscopic features of normal, hyperplastic, and adenomatous human colonic epithelium. II. Variations in surface architecture found by scanning electron microscopy. Gastroenterology 1975;69:100-109.1

accomplished by the same Na+ JK+ ·activated ATPas~driven transport system as described for the small intestine. Elimination of semisolid to solid waste materials is fa.cili· tated by the large amounts of mucus secreted by the numer· ous goblet cells of the intestinal glands. Goblet cells are more numerous in the large intestine than in the small intestine (see Fig. 17.28a and Plate 62, page 660). They produce mucin that is secreted continuously to lubricate the bowd, facilitating the passage of the increasingly solid contents.

637

The mucosal epithelium of the large intestine contains the same call types es the small intestine except Paneth cells. which are nonnally absent in humans. Columnar absorptive cells predominate (4:1) over gob· let cells in most of the colon, although this is not always apparent in histologic sections (see Fig. 17.28a). The ratio decreases, however, approaching 1: 1, near the rectum, where the number of goblet cells increases. Although the absorp· tive cells secrete gl.ycocaiyx at a rapid rate (turnover time is 16 to 24 hours in humans), this layer has not been shown to contain digestive enzymes in the colon. As in the small int~ tine, however, Na+IK+·ATPase is abundant and is localized in the lateral plasma membranes of the absorptive cells. The intercellular space is often dilated, indicating active transport of fluid. Goblet cells may mature deep in the intestinal gland, even in the replicative zone (Fig. 17.29). They secrete mucus continuously, even to the point where they reach the lumi· nal surface. Here, at the surface, the secretion rate exceeds the synthesis rate, and "exhausted'" goblet cells appear in the epithelium. These cells are tall and thin and have a small num· her of mucinogen granules in the central apical cytoplasm. An infrequently observed cell type, the caveolated "tuft'" cell, has also been described in the colonic epithelium; however, this cell may be a form of exhausted goblet cell.

Epithelial Cell Renewal in the Large Intestine All intestinal epithelial cells in the large intestine derive from a single stem call population. As in the small intestine, all of the mucosal epithelial cells of the large intestine arise from stem cells located at the bottom of the intestinal gland. The lower third of the gland constitutes the intestinal stem cell niche, where newly genet· ated cells undergo two to three more divisions as they begin their migration up to the luminal surface, where they are shed about 5 days later. The intermediate cell types found in the lower third of the intestinal gland are identical to those seen in the small intestine. The turnover times of the epithdial cells of the large intes-tine are similar to those of the small intestine (i.e., about 6 days for absorptive cells and goblet cells and up to 4 weeks fur enteroendocrine cells). Senile epithelial cells that reach the mu· cosal surface undergo apoptosis and are shed into the lumen at the midpoint between two adjacent intestinal glands.

Lamina Propria Although the lamina propria ofthe large intestine contains the same basic components as the rest of the digestive tract, it

•

'· d RGURE 17.29. Electron micrograph of dividing goblet cells. This electron micrograph demonstrates that certain cells of the intestine continue to divide even after they have differentiated. Here, two goblet cells {GQ are shown dividing. Typically, dividing cells move away from the basal lamina toward the lumen. One of the goblet cells demonstrates mucinogen granules (Ml in its apical cytoplasm. The chromosomes (Q of the dividing cells are not surrounded by a nuclear membrane. Compare with the nuclei (NJ of the nondividing intestinal epithelial cells. The lumen of the gland (L} is on the right. CT. connective tissue; E, eosinophil. X5,000.

demonstrates some additional suuctural features and greater development of some others. These include the following: • Collagen table, which represents a thick layer of colla· gen and proteoglycans that lies between the basal lamina of the epithelium and that of the fenestrated absorptive venous capillaries. This layer is as much as 5 IJ.m thick in the normal human colon and can be up to three times

638

w

z

~ !z w

(!)

~

-•

that thickness in human hyperplastic colonic polyps. The Submucosa and Serosa collagen table participates in regulation of water and elec- The submucosa of the large intestine corresponds to the trolyte transport from the intercellular compartment of general description already given. Where the large intestine the epithelium to the vascular compartment. is directl.y in contact with other structures (as on much of its • Pericryptal fibroblast sheath, which constitutes a posterior surface), its outer layer is an adventitia; elsewhere, well-developed fibroblast popularl.on of regularly replicuing the outer layer is a typical serosa. cells. They divide inunediatdy beneath the base of the intestinal gland. adjacent to the stem cells found in the epithelium Cecum and Appendix (in both the large and small intestines). The fibroblasts then The cecum forms a blind pouch just distal to the ileoce· differentiate and migrate upward in parallel and synchrony cal valve; the appendix is a thin, finger·like extension of this with the epithelial cells. Although the ultimate fate of the pouch. The histology of the cecwn closely resembles that of pericrypCll fibroblast is unknown, most of these cells, after the rest of the colon; the appendix diffi::rs from it in hav· they reach the level of the luminal swface, Wee on the mor- ing a uniform layer of longitudinal muscle in the museu· phologic and histochemical charatteristics of macrophages. laris enema (Fig. 17.30 and Plate 63, page 662). The most Some evidence suggests that the macrophages of the core of conspicuous feature of the appendi.x is the large nwnber of the lamina propria in the large intestine may arise as a termi- lymphatic nodules that extend into the submucosa. In many nal differentiarlon of the periaypW fibroblasts. adults, the normal structure ofthe appendix is lost and the • GALT, which is continuous with that of the terminal appendage is filled with fibrous scar tissue. Blockage of ileum. In the large intestine, GALT is more extensively the opening between the appendix and the cecum, usually developed; large lymphatic nodules distort the regular due to scarring, buildup of thick mucus, or stool that enspacing of the intestinal glands and extend into the sub- ters the lumen of the appendix from the cecum, may cause mucosa. The extensive development of the immune sys- appendicitis (inflammation of the appendix). The appentem in the colon probably reflects the large number and dix is also a common site for carcinoid, a type of tumor variety of microorganisms and noxious end products of originating from enteroendocrine cells of lining mucosa metabolism normally present in the lumen. (see Folder 17.3). • Lymphatic vessels. In general, lymphatic vessels are not present in the core of the lamina propria between the intesti~ nal glands, and there are no vessels that extend toward the lu~ minal surface of the large intestine. However, using a selective marker for lymphatic epithelium, researchers have found o~ casional small lymphatic vessels at the bltses of the intestinal glands. These lymphatic vessels drain into the lymphatic network within the muscularis mucosae. The nen step in lymph drninage occurs in the lymphatic plexuses in the submurosa and muscularis externa before lymph leaves the wall of the large intestine and drains into the regional lymph nodes. To understand the clinical significance of the lymphatic pattam in the large intes1ine, sea Folder 17.6.

Muscularis Extema As noted. in the cecum and colon (the ascending, transverse. d~

scending. and sigmoid colons), the outer layer of 1fte musc:u· laril extema is, in part, condensed into prominent longitudinal bands of muscle, called teniae coli, which may be seen at the gross level (see Fig. 17.27). Between these bands, the longitudi~ nallayer forms an extremely thin sheet. In the rectum, anal canal, and vermiform appendix, the outer longitudinal layer ofsmooth muscle is a uniformly thick layer, as in the small intestine. Bundles of muscle from the teniae coli penetrate the inner, circular layer of muscle at irregular intervals along the length and circumference of the colon. These apparent discontinuities in the muscularis enema allow segments of the colon to contract independently, leading to the formation of haustra coIIi, sacculations of the colon wall. lhemuscularisex:temaofthelargeintestineproduc:estwomajor types of contr.letion: segmentation and peristalsis. Segmentation is local and does not result in the propulsion ofcontents. Peristalsis results in the distal mass movement of the colonic contents. Mass peristaltic movements occur ~uently; in healthy perso!U, they usually oa::ur once a day to empty the distal rolon.

.,. /7 FIGURE 17.30. Photomicrograph of a cross 11ctlon through the vannifonn appendix. The vermiform appendix displays the same four layers as those of the large intestine except that its diameter is smaller. Typically, lymphatic nodules are seen within the entire mucosa and usually extend into the submucosa. Note the distinct germinal centers within the lymphatic nodules. The muscularis externa is composed of a relatively thick circular layer and a much thinner outer longitudinal layer. The appendix is covered by a serosa that is continuous with the mesentery of the appendix (lower right}. X10.

FOLDER 17.&

CLINICAL CORRELATION: THE PATTERN OF LYMPH VESSEL DISTRIBUTION AND DISEASES OFTHE LARGE INTESTINE The normal absence of lymphatic drainage from the lamina propria of the large intestine was initially discovered using standard tetfmiques of analyzing tissue samples obtained from biopsies with the light and electron microscopy. A specific monoclonal antibody called 02-40 is currently being used to study the distribution of lymphatic vessels within the lamina propria that may be associated with several disease processes. 02-40 reacts with a 40 kDa 0-linked sialoglycoprotein expressed on the lymphatic endothelium. For instance, in the tfuonic superficial inflammation of the colon and rectum known as ulcerative colitis. the foFmation of granulation tissue is associated with proliferation of blood and lymphatic vessels within the lamina propria. Lymphangiogenesis (the growth of lymphatic vessels) in this disease is linked to the expression of vascular endothelial growth factors (VEGFs). The progress of treatment in ulceFative colitis can be monitored by biopsies. which show the disappearance of lymphatic vessels from the lamina propria. Conversely. an increased number of lymphatic vessels in the lamina propria signals the presence of active inflammation.

Discovery of the distribution of lymphatic vessels in the large intestine established the basis for the current management of adenomas (adenomatous polyps of the large intestine). These are intraepithelial neoplasms located on the mass of tissue that protrudes into the lumen of the large intestine (Fig. F17.6.1 ). The absence of lymphatic vessels from the lamina propria was important in understanding the slow rate of metastasis from certain colon cancers. Cancers that develop in large adenomatous colonic polyps may grow extensively within the epithelium and lamina propria before they gain access to the lymphatic vessels at the level of the muscularis mucosae. Because almost 50% of all adenomatous polyps of the large intestine are located in the rectum and sigmoid colon, they can be detected with rectosigmoidoscopy. As long as the lesion is confined to the mucosa, the endoscopic removal of such polyps is regarded as an adequate clinical treatment. However, the final therapeutic decision must be confirmed after careful microscopic examination of the resected specimen.

FIGURE F17.6.1. Adenomatoue polyp of tile lerge lntadne. •· This image shows a macroscopic view of a polyp (about 2 em in diameter) that was surgically removed from the large intestine during endoscopic colonoscopy. It has a characteristic bosselated surface (with rounded swellings) and a stalk bv which it attactees to the wall of the colon. b. This photomicrograph was obtained from the center of the polyp. At the tip of the polyp, note the repetitive pattern of tubules covered with neoplastic epithelial cells that have migrated and accumulated on the intestinal surface. The stalk in the center is continuous with the submucosa of the colon. Note also the normal simple columnar epithelium of the large intestine at the base of the stalk. (Reproduced from Mitros FA. Rubin E. The Gastrointestinal Tract. In: Rubin R. Strayer OS, eds. Rubin's Pathology: Clinicopathologic Foundations of Medicine, 5th ed. Baltimore: Lippincott Williams & Wilkins. 2008.)

called anal sinuses. The anal canal is divided into three Rectum and Anal Canal The rectum is the dilated distal portion of the alimentary zones according to the cb.uacter of the epithelial lining: canal. Its upper pan is distinguished from the rest of the large intestine by the presence of folds called transverse rectal folds. The mucosa of the rectum is similar to that of the rest of the distal colon, having straight, tubular intestinal glands with many goblet cells. The most distal portion of the alimentary canal is the anal canal. It has an average length of4 em and extends from the upper aspect ofthe pelvic diaphragm to the anus (Fig. 17.31). The upper part of the anal canal has longitudinal folds called anal column•. Depte$$ions between the anal columns are

• Colorectal zone, which is found in the upper third of the anal canal and contains simple columnar epithelium with c.haracteristia identical to that in the rectUm • Anal transitional zone (ATZ), which occupies the middle third of the anal canal. It represents a transition between the simple columnar epithelium of the rectal mucosa and the stratified squamous epithelium of the perianal skin. The ATZ possesses a stratified columnar epithelium interposed between the simple columnar epithelium and the stratified squamous epithelium, which

639

extends to the cutaneous zone of the anal canal (Fig. 17.32 and Plate 64, page 664). • Squamous zona, which is found in the lower third of the anal canal This zone is lined with stratified squamous epitheliwn that is continuous with that of the perineal skin.

640

w

z

~ !z

E

i

w

(!)

~

-• c

Cll

anal RGURE t7.3t. Drawing of the rectum and anal canal. The rectum and anal canal are the terminal portions of the large intestine. They are lined by the colo rectal mucosa that possesses a simple columnar epithelium containing mostly goblet cells and numerous anal glands. In the anal canal, the simple columnar epithelium undergoes transition into a stratifed columnar (or cuboidal) epithelium and then to a stratified squamous epithelium. This transition occurs in the area referred to as the anal transitional zone, which occupies the middle third of the anal canal between the colorectal mne and the squamous zone of the perianal skin.

In the anal canal, anal glands extend into the submucosa and even into the muscularis extcrna. These: branched, straight tubular glands sc:c.cete mucus onto the anal surface through ducts lined with stratified columnar epithelium. Sometimes the anal glands are surrounded by diffuse Jvmphatic tissue. They often lead to the formation of pathologic fistulas (an opening between the anal canal and the perianal skin). Large apocrine glands, the circumanal glands, are found in the skin surrounding the anal orifice. In some animals, the secretion of these glands acts as a sex attractant. Hair follicles and sebaceous glands are also found at this site. The submucosa of the anal columns contains the terminal ramifications of the superior rectal artery and the rectal venous plexus. Enlargements of these submucosal veins constitute intemal hemontaoids, which are related to elevated venous pressure in the portal circulation (portal hypertension). There are no teniae coli at the level of the recrum; the longitudinal layer ofthe muscularis cnerna forms a uniform sheet. The muscularis mucosae disappears at about the level of the ATZ, where the circular layer of the muscularis exte.ma thickens to form the intem.al anal sphincter. The external anal sphincter is formed by striated muscle of the pdvic floor.

FIGURE 17.32. Photomicrograph~ of tha anal c:anal. a. This photomicrograph shows a longitudinal section through the wall of the anal canal. Not& the three zones in the anal canal: the squamous zone (SOl) containing stratified squamous epithelium: the anal transitional zone IA1Z) containing stratified squamous, stratified cuboidal, or columnar epithelium and simple columnar epithelium of the rectal mucosa; and the colorectal zone {CRZ) containing only simple columnar epithelium like the rest of the colon. Note the anal valve that demarcates the transition between the Al2 and SOZ. The internal anal sphincter is derived from the thickening of the circular layer of the muscularis externa. A small portion of the external anal sphincter is seen subcutaneously. x 10. b. This high magnification of the area indicated by the rectsngle in a shows the area of the anal transitional zone. Note the abrupt transition between stratified cuboidal and simple columnar epithelium. The simple columnar epithelium of anal glands extends into the submucosa. These straight, mucus-secreting tubular glands are surrounded by diffuse lymphatic tissue. X200.

641 Colorectal cancer (colon or rectal cancer) is one of the major causes of canceF-related deaths in the United States. Almost 100,000 cases of colon cancer and 40,000 cases of rectal cancers are diagnosed in the United States each year, leading to more than 50,000 deaths. Colorectal cancer commonly occurs between the ages of 60 and 79 years in individuals with a low-fiber and high-fat diet. Most colorectal cancers (about 98%) are adenocarcinomas and begin as small, benign clumps of cells that arise from the glandular epithelium. They form adenomatous polyps, which typically can be detected by a sigmoidoscopy or colonoscopy. In microscopic examinations, the irregular intestinal glands are lined by one or more layers of dark-stained cancer cells with or without mucus production (Fig. F17.7.1 ). Colon cancers vary in distribution throughout the large intestine. Approximately 38% of cancers are found in the cecum and ascending colon, 38% in the transverse colon, 18% in the descending colon, and 8% in the sigmoid colon.

It is now thought that chromosomal instability associated with stepwise accumulation of mutations in protooncogenes and suppressor genes play a vital role in the development of colorectal cancer. Initially, when epithelial cells lose the APC tumor suppl'8880r gene, they develop small polyps. Next, mutation in the K-Ras protooncogene transforms the polyp into a benign adenoma. These cells further undergo mutation and/or deletion of the p53 tumor suppi'8Hor gene and DCC gene, thus leading to the development of the invasive form of adenocarcinoma. The second pathway leading to the development of colorectal cancer is caused by genetic lesions in DNA mismatch repair ganes in the epithelial cell of the colon. Colorectal cancer in its early stage usually produces general symptoms, such as changes in bawel movements, persistent constipation or diarrhea, rectal cramping, or rectal bleeding, which may be an indication of a developing malignancy. With earty detection, surgery, radiation, and/or chemotherapy can be effective treatments.

•> :;g

G)

m

z

m z m

FIGURE F1 7. 7. 1. Macroacopic and microacopic feab11es of adenocarcinoma of the colon. a. This photograph shows a surgically resected segment of the colon containing an elevated and centrally ulcerated mass. b. This low-magnification photomicrograph shows a section of a tumor that was tak:en from a free margin of the lesion to show both 1he typical mucosa of the large intestine (/eft) and invasive adenocarcinoma (top left). The abrupt transition to adenocarcinoma is marked by a dashed line. Intestinal glands in 1he normal part of the epithelium are lined by a single layer of goblet and absorptive cells and occupy the entire thickness of the mucosa. In contrast. tissue invaded by the adenocarcinoma shows an irregular pattem of glands without the presence of mucous production. Cells and their nuclei are intensively stained with hematoxylin (hyperchromatic). Note that muscle fibers derived from the muscularis mucosa travel among colonic glands. X120. (Both images courtesy of Dr. Thomas C. Smyrlc.l

642

•• ...

6

>C!l 0

...J

~

:I:

•:E

s w

O~VI(W OF TH~ (OOPHAGU~ AND GMTROINTmTINAL TRAC.T

w ~

~

w

CJ

25

.. .-.: a: 1&.1

t:

c:::1:

u

-

• Extending from the esophagus to the anal canal, the alimentary canal is a hollow tube composed of four distinctive layers (&om the lumen going outwanl): mucosa, submucosa, muscularis extema, and serosa (when organ is covered by peritoneum) or adventitia (when organ is surrounded by connective tissue). • lhe mucosa is always associated with underlying lamina propria (loose connective tissue) and muscularis mucosae (smooth muscle layer). The type of mucosal epithelium varies &om region to region, as does the thickness of lamina propria and muscularis mucosae. • lhe submucosa consists ofdense irregular connective tissue containing blood and lymphatic vessels, nerve plexus, and occasional glands. • The muscularis extema mhres and propels the content of the canal. It consists of two layers ofsmooth muscle: The inner layer is circular and the outer layer is longitudinally oriented with myenteric nerve plexus between them. • The serosa or adventitia constitutes the outermost layer of the alimentary canal.

S!OPHAGU~

e lhe mucosa ofthe esophagus has nonkeratinized stratified squamous epithelium. The submucosa contains esophageal glands proper that lubricate and protect the mucosal surface. The muscularis extema is striated at its upper part and is gradually replaced by a smooth muscle layer in the lower part.

e At the asophagogastric junction, nonk.eratin.i.zed stratified squamous epithelium changes abruptly to simple columnar epithelium of the gastric mucosa. Esophageal cardiac glands are present in the lamina propria at this junction.

• The stomach has three histologic regions: cardiac surrounding the esophageal orifice, pyloric near the gastroduodenal junction, and fundic (anatomically occupied by fundus and body). • The mucosa of the fundic region forms a number of longi.rudinal folds (rugae). Surface mucous cells line the inner surf.lce of the stomach and the gastric pits, which are the openings into the branched fundic glands. Surfiu:e mucow cells produce an insoluble, viscous, gel-like coat that contains bi.cubonate ions to protm against physical and chemical injury of the gastric wall. • The fundic glands produce gastric juice containing four major components: hydrochloric acid (HCI), pepsin (proteolytic enzyme), intrinsic factor (for vitamin B12 absorption), and acid-protective mucus. • The epithelium of the fundic gland has five major cell types: mucous neck cells, which produce soluble and low-alkaline mucus secretions; parietal cells, which are responsible for the production of HQ within the lumen of their intracellular canalicular system; chief cells, which secrete pepsinogen; enteroendocrine cells, which produce small regulatory gastrointestinal and paracrine hormones; and stem cells, which are precursors to all fundic gland cells. • Mucous neck cells produce soluble and low-alkaline mucus secretions. • Parietal cells are large cells in the middle of the gland and are responsible for the production of HCI within the lumen of their intracellular canalicular system. They also secrete intrinsic factor. • Chief cells reside at the bottom of the fundic gland and secrete the protein pepsinogen. On contact with the low pH of gastric juice, pepsinogen is converted to pepsin, an active proteolytic enzyme. • Enteroendocrine cells are found at every level of the fundic gland. They produce small regulatory gastrointestinal and paracrine hormones. • Stem cells are precursors to all cells in the fundic gland and are located in the neck region of the gland. • Cardiac glands are entirely composed of mucus-sectrting cells with occasional interspersed enteroendocrine cells. • Pyloric glands are branched and. lined with cells resembling surface mucous cells and occasional enreroendocrine cells. --=,_..,.

643 ~MALLINTmriNE • The small Intestine is the longest component of the digestive tract and is divided into three anatomic regions: duodenum (with mucus-secreting Brunner's glands in the submucosa), jejunum, and ileum (with Peyer's patches in the submucosa). • The mucosa of the small intestine is lined by simple columnar epithelium, and its absorptive sur&ce is increased by the plicae circulares and villi. Simple tubular intestinal glands (or crypts) extend from the muscularis mucosae and open into the lumen at the base of the villi. • The intestinal mucosal epithelium has at least five types ofcells: enterocytes, which are absorptive cells specialized for the transport of substances from the lumen to the blood or lymphatic vessels; goblet cells, which are unicdlu· lar mucin·secreting glands interspersed among other cdls of the intestinal epithdium; Panath calls, which secrete antimicrobial substances (e.g., lysozyme, a·defensins); enteroendocrine cells, which produce various paracrine and endocrine gastrointestinal hormones; and M cells, which are specialized as antigen·ttansporting cells and cover lymphatic nodules in the lamina propria. • Cdls of the intestinal mucosal epithelium are found both in the intestinal glands and on the sur&ce of the villi, and their ratio changes depending on the region. • Entarocytas are absorptive cells specialized for the transport of substances from the lumen to the blood or lymphatic vessels. • Goblet cells are unicellular mucin-scctc:ting glands interspersed among other cdls of the intestinal epithdium. • Panath calls are found at the bases of the intest'mal glands, and their primary function is to secrete antimicrobial substances (e.g., lysozyme, a-dcfensins). • Enteroandocrine cells produce various paracrine and endocrine gastrointestinal hormones. • M calls {microfold cells) are specialized as antigen-uansporting cells. They cover lymphatic nodules in the lamina propria. ; • Stem cells are precursors to all cells in the intcsnnal · gland and are located ncar the bottom of the gland. • The muscularis axtema coordinates contractions of the inner circular and the outer longitudinal layers, produc· ing peristalsis that moves the intestinal contents distally. lhe autonomic myenteric plexus (Auerbach's plexus) innervates the muscularis externa.

J

- - - - • The large Intestine is composed of the cecum (with its projecting vannlfonn appendix), colon, rectum, and anal canal. The appendix has a large number oflymphatic nodules that ex.tend into the submucosa. • The mucosa of the Luge intestine contains numerous straight tubular intestinal glands (crypts of Lieberkiihn) that c:xtc:nd through the full thickness of this layer. The glands are lined by c:nterocytes (for resorption of water) and goblet cells (for lubrication). • The muscularis extema of the colon has its outer layer condensed inro three prominent longitudinal bands, the tanlae coli, which lead to formation of sacculations in the wall of the large intestine {hausua colli). • In the anal canal, simple columnar epithelium becomes stratified in the anal transitional zone (middle third of - - ----.1! the anal canal). lhe lower part of the anal canal is covered by stratified squamous epithelium that is continuous with the perineal skin.

•

:::c c;;

dr 0

(j')

-
-

...,__ _---! T4 + Ta

:r:

I-

•z

1-------.. . . .

~bloodbrain barrier

'\_ anter1or . Iobeof pitUitary ..

en

gland (adenohypophysis)

i 0

loll

z

-

Inhibitory

-

611!TIIllatory

ii!

g a

zw

... N

f5

t: ~

u

i growth

i metabolism

j development

j body tleat production

FIGURE 21.18. Production, tranaport, and reguldon of thyroid honnonee. Production ofT4 and T3 is regulated through a negative feedback system. The follicular cells of the thyroid gland predominately produce about 20 times moreT4 thanT3; however, T4 is converted in the peripheral organs (e.g., liver, kidney) to a more active form ofT3 . Approximately 99% ofT4 andT3 released from the thyroid gland into the circulation bind to specific plasma proteins. The remaining free (unbound)T4 andT3 exert negative feedback on the system and inhibit further release ofT4 andT3• This inhibition occurs at the level of the anterior lobe of the pituitary gland and the hypothalamus. At the pituitary level, T4 and T3 inhibit secretion ofTSH by thyrotropes. To elicit an inhibitory effact on the hypothalamus, both T4 and T3 need to cross the blood-brain barrier by utilizing the OATP thyroid hormone transporter expressed on the membrane of the endothelial cells. Increased concentration ofT4 andT3 reduces expression of OATP transporters as part of the negative feedback loop, thus decreasing the amount of available thyroid hormones in the brain. After crossing the blood-brain barrier, T4 andTa are transferred into neighboring astrocytes, whereT4 is converted toT3. Note thatTa is the predominant hormone that enters the neurons. T4 andT3 are also secreted into the cerebrospinal fluid and are taken up by the tanycytes (specialized ependymal cells) and astrocytes, where T4 is converted to T3. In addition to TRH, which also stimulates production of prolactin in lactotropes, the hypothalamus secretes somatostatin that has an inhibitory effect onTSH production bythyrotropes. The feedback. system is activated in response to low thyroid hormone levels in the blood or metabolic needs. In addition to chemical control mechanisms, a variety of nerve endings in the hypothalamus regulate secretion of TRH. For example, cold stress increases secretion ofTRH, whereas increased body temperature inhibits TRH secretion. CNS, central nervous system; TRH, thyrotropin-releasing hormone; TSH, thyroid-stimulating hormone (thyrotropin); OATP. organic anion transporting polypeptides.

One--third of circulating T4 is converted to T 3 in peripheral organs, such as the kidney, liver, and bean. T 3 is five times more potent than T4 and is mainly responsible fur biologic activity by binding to the thyroid nuclear receptors in the target cells. Transport acroa tha call membrana is assantial for thyroid honnona action and metabolism. Based on the biochemical structure of thyroid hormones, it was long thought that thyroid hormones can enter the cell by simple diffusion. However, it is now weD established that thyroid hormones are transported across cell membranes by several thyroid honnone transporter molecules. Within the CNS, T, and T4 are transported via the blood-brain

barrier to the nerve and glial cells by monocarboxylata transporter 8 (MCT8) and MCT10 as well as a family of organic anion transporting polypeptides (OATPs). For aam.ple, the OATPlCl transporter is exclusively expressed on the endothelial cells forming the blood-brain barrier and is responsible fur T 4 uptake into the brain. The MCf8 is also found in heart, kidney, liver, and skeletal muscle. Mutations in the MCTS gene cause severe psychomotor and intellectual disability associated with tligh serum T3 levels in affected male individuals, a condition known as Allan-Herndon-Dudley syndrome. Defective MCTB transporters are unable to transport T3 into nerve cells, which disrupt normal brain development. Because T3 is not utilized by nerve cells, excessive amounts of this

805 The most common symptom of thyroid disease is a goiter, the enlargement of the thyroid gland. It may indicate either hypothyroidism or hyperthyroidism. Hypothyroidism can be caused by insufficient dietary iodine (iodine-deficiency goiter, endemic goiter) or by one of several inherited autoimmune diseases, such as autoimmune thyroiditis (Hashimoto thyroiditis). Autoimmune thyroiditis is characterized by the presence of abnormal autoimmuneglobulins directed against thyroglobulin (TgAbl, thyroid peroxidase (TPOAb), and the TSH receptor (TSHAb). The results are thyroid cell apoptosis and follicular destruction. The low levels of circulating thyroid hormone stimulate release of excessive amounts ofTSH, which cause hypertrophy of the thyroid through synthesis of more thyroglobulin. Adult hypothyroidism, formerly called myxedema (due to the puffy appearance of the skin), is characterized by mental and physical sluggishness. The edema that occurs in the severe stages of hypothyroidism is caused by the accumulation of large amounts of hyaluronan in the extracellular matrix of the connective tissue of the dermis. In hyperthyroidism (toxic goiter or Graves disease}, excessive amounts of thyroid hormones are released into the circulation. Individuals with Graves disease have detectable levels of autoantibodies.

These abnormal immunoglobulins G (lgG) bind to the TSH receptors on the follicular cells and stimulate adenylate cyclase activity. As a result, increased levels of cAMP in follicular cells lead to continuous stimulation of the cells and increased thyroid hormone secretion. Because of negative feedbaclc, the levels ofTSH in the circulation are usually normal. However, under such stimulation. the thyroid gland undergoes hypertrophy, and thyroid hormone is secreted at abnormally high rates, causing increased metabolism. Most of the clinical features are associated with increased metabolic rate and increased sympathetic nerve activities. These include weight loss, excessive sweating, tachycardia, and nervousness. Noticeable signs include protrusion of the eyeballs and retraction of the eyelids, resulting from increased sympathetic activity and increased deposition of extracellular matrix in the adipose tissue located behind the eyeball (Fig. F21.4.1 a). The thyroid gland is enlarged. Microscopic features include the presence of columnar follicular cells lining the thyroid follicles. Because of the high utilization of colloid, the follicle tends to be depleted in the areas of contact with the apical surface of follicular cells (Fig. F21.4.1bl. Treatment for Graves disease is either surgery to remove the thyroid gland or radiotherapy by ingestion of radioactive iodine (131 1), which destroys most active follicular cells.

n :z:

,.. ~

m ::a

.. N

m 2

g §J

z

m 0

~

,.. z

OJ

• --i I

-
z 0

a FIGURE FZ1.4.1. HyperthJraidiam. •· A young woman with signs of hyperthyroidism. Note the enlarged mass on the neck and the typical ocular symptoms known as exophthalmos. b. Photomicrograph of a thyroid gland specimen from an individual with Graves disease. Due to the increased utilization of colloid, there is a lack of staining at the periphery of the colloid near the apical surface of the follicular cell. Note that the majority of the cells are columnar in shape. (Reprinted with permission from Rubin E. Gerstein F. Rubin R, et al. Rubin's Pathology. Clinicopathologic Foundations of Medicine, 4th ed. Baltimore: LippincottWilliams &Wilkins. 2005.)

806

hormone continue to circulate in the blood, causing signs and symptoms of thyroid homaone toxicity.

The triiodothyronine (T3) honnone is more biologically active than thyroxine lTc). Once T 5 and T 4 molcc:ulcs enter the cell, they interact with a spec:ific thyroid nuclear receptor that is similar to the nudear·initiated steroid signaling pathway (see Fig. 21.3b). T 5 binds to nuclear receptors much faster and with higher af. finity than T 4• thus T 3 is mo~ rapidly and biologically ac::tive than T 4• In addition, T 3 binds to mitochondria, increasing the production of ATI 1herefo~. biologic activity and metabolic effect of the thyroid hormone is largely determined by the intracellular concentration ofT3 • Several factors impact the intracellular concentration of T 3• These include serum concentration of circulating T 3, which depends on the conversion rate ofT4 to T 3 in the pe· ripheral organs; transport of thyroid hormones across the cell membrane by specialized thyroid hormone transporters; and the p~ence of iodothyronine deiodinase enzymes, which ac::tivate or inactivate thyroid hormones. For instance, two deiodinase enzymes called D 1 and D2 convert T4 to the mo~ active T 3, whereas the third enzyme called D 3 degrades T4 to the inac::tive form of rT3 (reverse T 3) and DIT. Both T 3 and T 4 ar:e deiodinatcd and dearninated in the target tissues, conjugakd in the liver, and then passed into the bile, where they are excreted into the intestine. Conjugated and free hormones ~also e:x:c~d by the kidney.

Thyroid hormones play an eaential role in normal fetal development. In humans, thyroid honnones are essential to normal

growth and development. In normal pregnancy, both T 3 and T 4 cross the placental bar:rier and are critical in the early stages of brain development. In addition, the fetal thyroid gland be-gins to function during the 14th week of gestation and also contributes additional thyroid hormones. Thyroid hormone deficiency during fetal development results in irreversible damage to the central nervous system (CNS), causing reduced numbers of neurons, defective myelination, and intellectual disability. If maternal thyroid deficiency is present before the development of the fetal thyroid gland, intellectual disability can be severe. Recent studies reveal that thyroid hormones also stimulate gene expression for GH in the somatotropes. Therefore, in addition to neural abnormalities, a generalized stunted body growth is typical. The combination of these two abnormalities is called congenital hypothyroidism.

• PARATHYROID GLANDS The parathyroid glands ~ small endocrine glands closely associated with the thyroid. They ~ ovoid, a few millimeters in diameter, and arranged in two pairs, constituting the superior and inferior parathyroid glands. They ar:e usually located in the connective tissue on the posterior surface of the lateral lobes of the thyroid gland (see Fig. 21.13). However, the number and location may vary. In 2% to 10% of individuals, additional glands ~ associakd with the thymus. Structurally, each parathyroid gland is sutTounded by a thin connective tissue capsule that separates it from the thyroid.

Septa extend from the capsule into the gland to divide it into poorly defined lobules and to separate the densely packed cords of cells. The connective tissue is more evident in the adult, with the development of fat cells that increase with age and ultimately constitute as much as 60% to 70% of the glandular mass. The glands receive their blood supply from the inferior thyroid arteries or from anastomoses between the superior and inferior thyroid arteries. Typical ofendocrine glands, rich networks of fenestrated blood capillaries and lymphatic capillaries surround the parenchyma of the parathyroids.

Parathyroid glands develop from the endodennal cells derived from the third and fourth pharyngeal pouches. Embryologically. the Inferior parathyroid glands (and the thymus) are derived from the superiorly located third pharyngeal pouch; the superior parathyroid glands (and ultimobranchial body) are derived from the fourth pharyngeal pouch. Initially, the inferior parathyroid glands descend with the thymus. Later, the inferior parathyroid glands separate from the thymus and come to lie below the superior parathyroid glands. Failure of these structures to separate results in the atypical association of the parathyroid glands with the thymus in the adult. The principal (chie0 cells diKerentiate during embryonic development and are functionally active in regulating fetal calcium metabolism. The oxyphil cells diff'erentiate later at puberty.

Principal cells and oxyphil cells constitute the epithelial cells of the parathyroid gland. • Principal (chief) cells, the more numerous of the parenchymal cells of the parathyroid (Fig. 21.19), are responsible for regulating the synthesis, storage, and secretion oflarge amounts of PTH. They are small, polygonal cells, with a diameter of 7 to 10 JLm and a centrally located nucleus. The pale-staining, slighdy acidophilic cytoplasm contains lipofuscin-containing vesicles, large accumulations of glycogen, and lipid droplets. Small, dense, membrane-limited vesicles seen with the TEM or after using special stains with the light microscope are thought to be the storage form of PTH. Principal cells can replicate when they are chronically stimulated by changes in blood calcium levels. • Oxyphil cells constitute a minor portion of the parenchymal cells and are not known to have a se

808

(/)

0

z

::5

(!)

~

w

a:

~

•I

i I

PTH appears to have a rather slow, long~term homeostatic action. Calcitonin, however, rapidly lowers blood calcium levels and has its peak effect in about 1 hour; therefore, it has a rapid, acute homeostatic action.

• ADRENAL GLANDS The adrenal (suprarenal) glands are paired organs located in the retroperitoneal space of the abdominal cavity. The right gland is flattened and triangular and the left gland is semilunar in shape. They are both embedded in the perirenal fat at the superior poles of the kidneys (Fig. 21.20). The adrenal glands secrete steroid hormones and catecholamines. The adrenal glands are covered with a thick connective tissue capsule from which trabeculae extend into the parenchyma, carrying blood vessels and nerves. The secretory paenchymal tissue is organized into two distinct regions (Fig. 21.21):

1&1

z

• The cortex is the steroid-sc:c:t:eting portion. It lies beneath the capsule and constitutes nearly 90% of the gland by weight. • The medulla is the catccholaminNecreting portion. It lies deep to the cortex and fonns the center of the gland.

z

Parenchymal calls of the cortex and medulla are of different embryologic origin.

1&1

Embryologically, the cortical cells originate from mesodermal mesenchyme, whereas the medulla originates from FIGURE 21.21. PhotomiCIOII'IPh of the adrenal gland. This lowpower micrograph of an H&E-stained specimen shows the full thickness of the adrenal gland with the cortex seen on both surfaces and a central region containing 1he medulla. Within the medulla are profiles of the central vein. Note that the deeper portion of the cortex stains darker than the outer portion, a reflection of the washec:klUt lipid in the zona glomerulose and outer region of 1he zona fasciculate. This section also includes a cross section of the adrenal vein, which is characterized by the longitudinally arranged bundles of smooth muscle in its wall. X20.

left superior suprarenal arteries

left inferior

suprarenal artery and vein

neural crest cells that migrate into the developing gland (Fig. 21.22). Although embryologically distinct, the two portions of the adrenal gland are functionally related (see below). The parenchymal cells of the adrenal cona: are controlled in part by the anterior lobe of the pituitary gland and function in regulating metabolism and maintaining normal electrolyte balance (Table 21.9).

Blood Supply left renal artery and vein

FIGURE 21.20.

Topogr~phy

and blood 1upply of the adrenal

Csupr~renal)

gland. This drawing shows the location of the left adrenal gland at the superior pole of the left kidney. The perirenal fat has been removed on this image to show blood supply to the organ. Note that the adrenal gland is supplied by three arteries. The middle suprarenal artery originates directly from the aorta, whereas the superior and inferior suprarenal arteries originate from the left inferior phrenic and left renal artery. respectively. Blood drains to the suprarenal vein. which on the left side empties into the left renal vein and on the right side directly to the inferior vena cava.

Each adrenal gland is supplied with blood by the superior, middle, and Inferior suprarenal arteries and drained by the suprarenal veins (see Fig. 21.20). On the left side, the suprarenal vein drains to the left renal vein, whereas on the right side, the suprarenal vein dJ:ains directly to the inferior vena cava. These vessels branch before entering the capsule to produce many small arteries that penet:J:ate the capsule. In the capsule, the arteries branch to give rise to three principal patterns of blood distribution (Fig. 21.23). The vessels form a system that consists of the following: • Capsular capillaries that supply the capsule • Fenestrated cortical sinusoidal capillaries that supply the cortex and then dr:ain into the fenesttated medullary capillary sinusoids

• Medullary arterioles that traverse the cortex, traveling within the trabeculae, and bring arterial blood to the medullary capillary sinusoids

neural crest

sympathetic ganglion (from cortical primordium neural crest) of fetal cortex /(from intermediate /

/

mesodenn)

~urogenital ridge ---~--dorsal mesentery

a

chromaffin cells of future : / medulla

..

(\

fetal

gut tube

cortex

cells

The medulla thus has a dual blood supply: arterial blood from the medullary arterioles and venous blood from the cortical sinusoidal capillaries that have already supplied the cortex. The venules that arise from the cortical and medullary sinusoids drain into the small adrenomedullary collecting veins that join ro fonu the large central adrenomedullary vein. which then drains directly as the suprarenal vein into the inferior vena cava on the right side and into the left renal vein on the left side (see Fig. 21.20). In hwnans, the central adrenomedullary vein and its aibutaries are wt.usual in that they have a tunica media containing conspicuous, longitudi.nalty oriented bundles of smooth muscle cells (Fig. 21.24). Synchronous cont.J:aetion of longitudi.nal smooth muscle bundles along the central adrenomedultary vein and its aibuwies cause the volume of the adrenal gland to decrease. This volume decrease enhances the efflux of hormones fi:om the adrenal medulla into the circulation. an action comparable to squeezing a wet sponge. Lymphatic vessels are present in the capsule and the connective tissue arowt.d the larger blood vessels in the gland. They also have been fowt.d in the parenchyma of the adrenal medulla. The lymphatic vessels have an important role in disaiburing chromogranin A. a secretory product of chromaffin cells. Chromogranin A is a 48 kDa inaacellular storage protein complex for epinephrine and norepinephrine and is also a precursor molecule for several regulatory peptides. including vasostatin, pancreastatin, catestatin. and parastatin. These peptides modulate the neuroendocrine function of the chromaffin cells (autocrine effect) and other cells in distant organs.

Cells of the Adrenal Medulla Chromaffin cells located in the adrenal medulla are innervated by presynaptic sympathetic neurons.

c

d FIGURE 21.22. Development of the adrenal gland. a. In this early stage, the cortex is shown developing from cells of the intermediate mesoderm, and the medulla is shown differentiating from cells in the neural crest and migrating from the neighboring sympathetic ganglion. The cells that form the fetal cortex originate from mesothelial cells located between the root of the dorsal mesentery and the developing urogenital ridges (future gonads). They divide and differentiate into fetal cortex cells. b. Mesodermal cells from the fetal cortex surround the cells of the developing medulla. Later. more mesenchymal cells arrive from the mesothelium of the posterior abdominal wall. They surround the original mass of cells containing the fetal cortex cells and chromaffin cells. These cells later give rise to the permanent cortex. c. At this stage (about 7 months of development!, the fetal cortex occupies about 70% of the cortex. The permanent cortex develops outside the fetal cortex. d. The fully developed adrenal cortex is visible at the age of 4 months. The permanent cortex replaces the fetal cortex. which at this age has completely disappeared. Note the fully developed zonation of the permanent cortex.

The central portion of the adrenal gland, the medulla, is composed of a parenchyma oflarge, pale--staining epithelioid cells called chromaffin cells (medullary calls), connective tissue, numerous sinusoidal blood capillaries, and nerves. The chromaffin cells are, in effect, modified neurons (Folder 21.5). Nwncrous myeUnated, presynaptic sympathetic netve fibers pass directly to the chromaffin cells of the medulla (sec Chapter 12, Netve TtSSue). When nerve impulses carried by the sympathetic fibers reach the catecholamine-secreting chromaffin cells, they release their secretory products. Therefore, chromaffin cells are considered the equivalent of postsynaptic neurons. However, they lack axonal processes. Experimental studies mreal that when chromaffin cells are grown in culture, they extend axon-Ukc processes. However, axonal growth can be inhibited by glucocorticoids-hormones secreted by the adrenal cortex. Thus, the hormones of the adrenal cortex exert control over the morphology of the chromaffin cells and prevent them from forming neural processes. Chromaffin cells therefore more closely resemble typical endocrine cells, in that their secretory product enters the bloodstream via the fenest.tated capillaries.

809

n

~

.. :II N

m

z

I II z

m

•~ ::D

m

~

r

G)

> z 0

(I)

810

TABLE 21.9 Honnones of the Adrenal Glands Honnone

Composition

Source

Major Functions

Steroid hormones (cholesterol derivatives)

Parenchymal cells of the zona glomerulosa

Aid in controlling electrolyte homeostasis (act on distal tubule of kidney to increase sodium reabsorption and decrease potassium reabsorption); function in maintaining the osmotic balance in the urine and in preventing serum acidosis

Steroid hormones (cholesterol derivatives)

Parenchymal cells of the zona fasciculate (and to a lesser extent of the zona reticularis)

Promote normal metabolism, particularly carbohydrate metabolism (increase rate of amino acid transport to live, promote removal of protein from skeletal muscle and its transport to liver, reduce rate of glucose metabolism by cells and stimulate glycogen synthesis by liver, stimulate mobilization of fats from storage deposits for energy use); provide resistance to stress; suppress inflammatory response and some allergic reactions

Steroid hormones (cholesterol derivatives)

Parenchymal cells of the zona reticularis (and to a lesser extent of the zona fasciculate)

As weak androgens, they induce develop-

Catecholamines (amino acid derivatives)

Chromaffin cells

Sympathomimetic (produce effects similar to those induced by the sympathetic division of the autonomic nervous system)•; increase heart rate, increase blood pressure, reduce blood flow to viscera and skin; stimulate conversion of glycogen to glucose; increase sweating; induce dilation of bronchioles; increase rate of respiration; decrease digestion; decrease enzyme production by digestive system glands; decrease urine production

Adrenal Cortex (/)

0

z

::5

Mineralocorticoid&: aldost&rona (95% of mineralocorticoid activity in aldosterone)

(.9 .....J

z 0

(I)

814

CIJ

0

z

:5 C!l ....J

us tubules open into the me testis by way of a straight tubule. Straight tubules are very short and are lined by Sertoli-like cells; no germ cell component is present. The connective wsue of the mediastinum is very dense but exhibia no other special featureS, nor is smooth muscle present. Adipose cell. (AC) and blood vwels (BV), particularly vcins of varying size, are present within the connea:ive tis5ue.

Note that bCCUJ&C of the aope ar which the straight tubule wu sectioned, it appean that the cuboidal epithelium cba=ri%.ing the me testis begiN on the upper side ofthe tubule before it is seen on the lower side of the tubule. The rete tesm (Rn collltituta an anutomosing sys· tt:m of channds that lead to the efferent ductula. The epithdlal alls l.i.n.mg the rete testis are sometime~ more tquamous than cuboidal or, occasionally, may be low columnar io appearance. Typically, they po•ICIII a single (primary) cilium; however, this is difficult to see in routine H&::E preparations.

ST. seminiferous tubules

At;. adipose cells

LC. Leydig cells

BV, blood vessels G, gonocytes

MT. mediastinum testis

TA. tunica albuginea

RT, rete testis

TR, tubulus rectus

867

868

PLATE 88 •

EFFERENT DUCTULES AND EPIDIDYMIS

The rate testis is connected via -20 efferent ductules (ductuli affsrentss; remnants of nephrons of the fetal mesonephric kidney) to the ductus epididymis. These are the first elements of the excurrent duct system of the male genital system. Most of the fluid secreted in the seminiferous tubules is reabsorbed in the efferent ductules. The muscular coat characteristic of the excurrent duct

system fi rat appears at the beg inning of the efferent ductules. The ductus epididymis is a highly coiled tube, 4 to 6 m long; sperm mature during their passage along its length, acquiring motility as wall as the ability to fertilize an egg. This maturation is also androgen dependent and involves changes in the sperm plasma membrane end addition to the glycocalyx of glycoprotein& secreted by the epididymal epithelial cells.

Efferent ductules, testis-epididymis, monkey, H&E, X60; inset X360.

tbey posaess (arrowhrd. imri). The basal surfilce of the duaule, in contrast, has a smooth contour (see figure below and. imd). Some of the cells, gen.eral.ly the tall columnar cdls, JI085ess cilia (Q (jmd). Wh~ tbe ciliated cells aid in moving the contents of the tubule toward the ~ididymil, the cells with the microvilli are largely n:spoiUiible fur absorbing ftuid from tbe lumen. In addition to the columnae and cuboidal cells, basal cells are also present; thus, the epithdium is designated pseudostratified columnar. The basal cells possess little cytoplasm and. presumably serve as stem cells. The efferent ductules possess a thin. layer of circularly arranged smooth muscle cells (SM, insd'). The muscle is close to the basal sur&ce of the epithelial cells, being s~arated from it by only a small amount of connective tissue (CT, imn). Because of this close association, the smooth muscle may be overlooked or misidentified as connective tissue. Smooth muscle facilitates movement ofluminal contenu of the duaule to the duaus epididymis.

B

About 12 to 20 efferent ductules leave the testis and serve as dtanneh from the rete testis to the ductus ~ididymis. Each of the effenmt duaules undergoes numerous $piral winding$ and convolution$ to fonn a group of wnlcal ruu~; together they coNtitute the initial part of the head of the epididymis. When cumined in a tissue section, the ductules ahibit a variety of irregular profiles due to their twisting and. turning. This is evident on the risht side of this micrograph. The epithelium that lines the efferent ductules is distinctive in that groups of tall columnar cdls alternate with groups of cuboidal cdls, giving the luminal surface an unevenly contoured appear.mce. Thus, small cup-like depressions are created where the epithelium contains groups of cuboidal or low columnae cells. Typically, these shorter c:el.ls exhibit a brush border-like apical surface because of the microvilli that

Epididymis, monkey, H&E, X180. The epididymis, by virtue of its shape, is divided into a head, body, and tail. The initial part of the head contains the ductus epldldymls, a single conwluted duct into which the efferent d.uctules open. 1he duct is, at first. highly convoluted but becomes less torruous in the body and tail. A sec-tion through the head of the epldldymls, as shown in figure above, cuts the ductus epididymis in numerous places, and as in the efferent duaules, cillferent-shaped profiles are observed. The epithelium contains two distinguishable c::dl types: tall columnar c:el.ls and basal cells simllar to those of the efferent ductules. The epitheltum is therefore also pseudostratified columnar. The columnar cells an: tallest in the head of the epididymis and diminish in height as the tail il reached. The free surface of tbe cell possesses stereocilia (SQ. These are ememdy long, branching microvilli. They evidendy adhere to each other during the preparation of the tissue to form the fine tapering structures tbat an: characteristically seen with the light microscope. The nuclei of the columnar cells an: elongated and. an: loc:ated a mod.e.ratc distance from the base of the cell. They an: readily

AT. adipose tissue

distinguished from the spherical nuclei of the basal cells that lie close to the basement memb~. Other wntplcuous features of tbe columnar cells include a very ~ supranuclear Golgi apparatus (not seen at tbe magnification offered here), pigment accumulatioDS (PJ, and numerous lysooomes, demonstrable witb appropriate techniques. Because of the unusual height of the wlumnar cells and. the torru~ osity of the duct. an uneven lumen appean in some sites; indeed, even ~islanm- of ~!thelium can be encountered in the lumen (see arrOWI, figure above). Such profiles an: accounted fOr by sharp turns in the duct when: tbe epithelial wall on one side of the duct is partially cut. For example, a cut in the plane of the tittubk-huJtti llmJW indicated in this figure would aeate such an isolated epithdial island. A thin. layer of smooth muscle circumscribes the duct and appears similar to that associated with the efferent duaules. In the terminal portion of the epididymis, however, the smooth muscle ac;quin:s a ~ thkkness, and longitudinal fibers are also praent. Beyond the smooth muscle coat. there is a small amount of connccrlve tissue (en that binds the loops of the duct together and. carries the blood vesseh (BV) and nerves.

P, pigment

BV, blood vessel

sc, stereocilia

C,cilia CT, connective tissue

SM, smooth muscle arrowhead (inset), brush border

arrows, Nislands~ of epithelium in the lumen

869

870

PLATE 89

SPERMATIC CORD AND DUCTUS DEFERENS

The ductus (vas) deferens continues from the duct of the epididymis as a thick-walled museu lar tube that leaves the scrotum and passes through the inguinal canal as a component of the spermatic cord. At the deep inguinal ring, it continues into the pelvis and, behind the urinary bladder, joins with the excretory duct from the seminal vesicle to form the ejaculatory dud. The ejaculatory duct then pierces the prostate gland and opens into the urethra. Mature sperm are stored in the terminal portion (tail) of the ductus epididymis. These sperm are forced into

the ductus deferens by intense contractions of the three smooth muscle layers of the ductus defarens following appropriate neural stimulation. Contraction of the smooth muscle of the ductus deferens continues the movement of the sperm through the ejaculatory duct into the urethra during the ejaculatory reflex. The seminal vesicles (see Plate 91 J are not storage sites for sperm but, rather, secrete a fructose-rich fluid that becomes part of the ejaculated semen. Fructose is the principal metabolic substrate for sperm.

Spennatic cord, human, H&E, X80.

contains nerves and some of the amaller blood vessels that supply the duct. In fact. some of these vessels can be aecn penetrating the outer longitudinal smooth mwcle layer (IZIUrislu). A unique feature of the spermatic cord is the presence of a plcrut~ of atypical veins (pamplnlfonn plexus) that arise from the spermatic veills. These vessels receive the blood from the testis. (The pampini· form plexus also receives tributaries &om the epididymis.) The plans is an anastomosing vascular network that constitutes the bulk of the spermatic cord. Portions of several of these vdm (BV) an: evident in the upper right of 6gure above along with a number of nerves (N). The u.nwual feature of the veins i5 their thick muscular wall that, at a glance, gives the appearance of an artery rather than a vein. Careful ~ination of these vessels (inset) shows that the bulk of the vessel wall is composed of two~ of smooth muscle-an outer circular layer SM(C) and an inner longirudinallayer SM(L).

A cross section through the ductus deferens and some of the vessels and nerves that accompany the dw:t in the spermatic cord is shown in this figure. The wall of the ductus deferens i5 enremely thick, mo.rtly because of the presence of a large amount of smooth muscle. The muscle eontr2Ct5 when the tis5ue is removed, cawing the mucosa to form longitudinal fuld.!:. For this ttuon, in histologic sections, the lumen (L) usually appean i.m:gular in cross section. The smooth muscle of the ductuB deferens ill arranged as a thick outer longitudinallayu (SM[L]), a thick middle circular ~r (SM[C]), and a thinner inner longitudinal layer (SM[Lj). Between the epithelium and the inner longirudinal smooth muscle layer there is a moderately thick cdlular layer of!oose connective tissue, the lamina propria (LPJ. The connective tissue immediately surrounding the ductus deferens

Ductus deferens, human, H&E, >. The surface of the CHary is visible on the right. Note the presence of two primary follicles (upper rightl. Tl, theca intema. X45.

883 ntECA IN'IERNA CELLS

dehydroeplandrosterone progesterone-_,..._. 17a..OH progesterone -t--.:.1

Q

v

FSH FSH receptor

@

7

side chain cleavage enzyme complex P 450 aromatase

G protein GRANULOSA CELLS

17~ estradiol

adenylyl cyclase

follicular fluid FIGURE 23.8. Syntheels of etCrvglllfln 1he ovadln follicle. Synthesis of estrogens in the ovary requires collaboration between theca interna and granulosa cells. Theca intema cells express both luteinizing hormone ILH} and low-density lipoprotein ILDL) receptors on their surface. LH stimulation of theca interns cells facilitate conversion of cholesterol (liberated from lactate dehydrogenase [LDHII to pregnenolone {P5) and then via further intermediates into androgens !androstenedione and testosterone). These androgens diffuse into neighboring granulosa cells where they are converted to estrogens by the enzyme P450 aromatase. Granulosa cells express follicle-stimulating hormone IFSHJ receptors and their activation by FSH is a primary stimulator of P450 aromatase activity. FSH also promotes the conversion af estrone to 17~radiol. ATP. adenosine triphosphate: cAMP. cyclic adenosine monophosphate.

production in the developing follicles. Neither granulosa nor capacity (conversion of androgens to estrogens) in the devel~ theca cells express the full c::omplement of enzymes needed for oping follicles occur in granulosa cells and are thus the only synthesis of estradiot the primary female sex honnone. Theca source ofestradiol in the follicular phase of the ovarian cycle. intema cells reside in a highly vascularized layer of the follicle and Estrogens secreted by granulosa cells stimulate their own pro~ express both LH and low-density lipoprotein (LDL) receptors liferation and thereby increase the size of the follicle. on their surf.u:.e. LDL ~tors in these cells expedite uptake of Aromatase inhibitors {Ais) are a class of drug used LDL molecules from which cholesterol is liberated and becomes in the treatment of estrogen-sensitive breast cancer. This the main substtate in steroid honnone synthesis (Fig. 23.8). type of breast cancer grows in response to estrogen. When stimulated by LH, theca interna cells facilitate con· Als bind to different sites on the aromatase enzyme and version of cholesterol to pregnenolone and then via further prevent the conversion of androgens to estrogens, thus intermediates to androgens (ie., dehydroepiandrosterone lowering the level of estrogen in the body and decreasing [DHEA], androstenediol, androstenedione, testosterone) (see the potential for growth of cancer cells. F'~g. 23.8). Due to the ladt of the enzyme P450 aromataae, the Increased estrogen levels from both follicular and systemic theca intema cells are not able to produce estrogens. In contrast,. sources are correlated with increased sensitization of gonad~ neighboring granulosa cells are equipped with P450 aromawe. otropes to gonadotropin·releasing hormone. A surge in the Thus, androgens secreted by theca intema cells enter granulosa release of LH (and a smaller surge of FSH) is induced in the cells where they are c::onverted in the cells' smooth endoplasmic adenohypophysis approximately 24 hours before ovula.tion. reticulum (sER) by P450 aromawe to estrogens in response In response to the LH surge, LH receptors are downregulated (desensitized), and granulosa cells no longer produce estro~ to FSH stimulation (see Fig. 23.8). FSH is the primary stimulator of P450 aromawe gene gens. Triggered by this surge, the first meiotic division of the expression in granulosa cells. However, not all granulosa cells primary oocyte resumes. This event ocxuu between 12 and have the same capacity to produce estrogens. The highest lev· 24 hours after the LH surge, resulting in the formation ofthe els ofaromatase activity are found in the peripheral cells near secondary oocyte and the first polar body. Both the granulosa the theca interna, whereas the lowest levels are in the cells and thecal cells then undergo luteini2ation and produce pro~ bordering the antrum. Almost all (99%) ofthe aromatization gesterone (see pages 88~87, section on the corpus luteum).

884 Polycystic ovary disease is a syndrome characterized by a variety of clinical signs and symptoms, including bilaterally enlarged ovaries with numerous follicular cysts, irregular menstrual periods, anovulation that may lead to infertility, obesity, excess hair growth on the face, acne, and oily skin. Morphologically, the ovaries resemble a small, white balloon filled with tightly packed marbles. Affected ovaries, often called oyster ovaries, have a smooth, pearlwhite surface but do not show surface scarring because no ovulations have occurred. Their appearance is attributable to the large number of fluid-filled follicular cysts and atrophic secondary follicles that lie beneath an unusually thick tunica albuginea. Although the pathogenesis of polycystic ovary syndrome (PCOS) is not clear, it may be related to a defect in the regulation of androgen biosynthesis that causes production of excessive amounts of androgens, which inhibits ovulation and causes the abnormal hair growth and acne that is often associated with PCOS. Insulin resistance also appears to play a role in PCOS. Treatments of PCOS are individualized based on a patient's desire for childbearing. For those wishing to have children, drugs that sensitize the body to insulin and promote weight loss may reduce insulin resistance and result in resumption of ovulation. If these measures fail, ovulationstimulating drugs and laparoscopic surgical procedures are implemented. In vitro fertilization may also be an option. For women who do not wish to have children, combined

Ovulation Ovulation is a hannana-madiatad praca11 resulting in lha release of the secondary oocyte. Ovulation is the process by which a secondary oocyte is released from the Graafian follicle. The follicle destined to ovulate in any menstrual cycle is recruin:d. from a cohort of several primary follicles in the first few days of the cycle. During ovulation, the oocyte traverses the entire follicular wall, including the germinal epithelium. A combination of hormonal ~ and enzymatic effecu is responsible for the actual release of the secondary oocyte, which OCCUl'S 14 days before the start of the nat menstrual cycle (i.e., on the 14th day ofa 28-day cycle). These factors include • increase in the volume and pressure of the follicular fluid; • enzymatic proteolysis of the follicular wall by activated plasminogen; • hormonally directed deposition of glycosaminoglycans between the oocyte-cumulus complex and the stratum granulosum; and • contraction of the smooth muscle fibers in the theca externa layer, triggered by prostaglandins.

Just before ovulation, blood flow stops in a small alCI. of the ovarian swfaa: overlying the bulging follicle. This alCI. of the germinal epithelium, known as the macula pellucid& or follicular IJI:igma, bcoomes el.evmd and then ruptun:s {Fig. 23.9a). The oocya:, surrounded by the corona radiaia and cdls of the cumulus oophorus, is released from the ruptured follicle. At the

contraception (containing both estrogen and progesterone) may regulate the menstrual cycle and reduce acne and abnormal hair growth. (Fig. F23 .1.1 ).

FIGURE F23.1.1. Polycya1lc ovary d....... This photomicrograph shows asection through the cortex of the ovary from an indMdual with polycystic ovary disease. Note the unusually thidc tunica albuginea (TAl that overlies numerous follicles. The thid) to simple columnar epithelium

Cervical glands, cervix, uterus, human, H&E, X500.

This figure shows, at high magnification, ponioru of the cervical gland identified in the rrdltllgk in the figure on the left. Note the tall epithelial celJs and the lighdy staining supranuclear cytoplasm, a rdlection of the mucin that dissolved out

BV, blood vessels

CC, cervical canal CEp, columnar epithelium Gl, cervical glands

character of the mucus secretion of its simple columnar epithelium vary at different times in the uterine cycle under the influence of ovarian hormones. At midcycle, there is a 10-fold increase in the amount of mucus produced; this mucus is thin and provides a favorable environment for sperm migration. At other times in the cycle, the mucus is thick and restricts the passage of sperm into the uterus. The myometrium forms the major thickness of the cervix. It consists of interweaving bundles of smooth muscle cells in an extensive, continuous network of fibrous connective tissue. section of the cervix is shown in these figures and that the actual spec-imen, as seen in a section, would present a similar image on the other side of the ceiVi.c:al tanal.) The mucosa (M~«) of the cervix differs according to the cavity it faces. The two lffllmgln in the uppn- figure delineate representative areas of the mucosa that are shown at higher magnification in the upptr right and mit/Jie right figures, respectivdy. The bottom figure emphasizes the nature of the cervical glands (Gl). The glands differ &om those of the uterus in that they branch extensively. They settete muCWI intu the cervlc:al tana1 that serves to lubricate the vagina.

seen in the vagina. In ether respecu, the epithelium has the same gen~ eral features as the wgina1 epithelium. Another similarity is that the c:pithdialiiiUfaa: of the ectocervbr. undergoes cyclical c.hanga .dmilar to those of the vagina in response to ovarian hormones. The mueo&a of the ectotervn, like that of the vagina, ~ devoid of glands.

(CEp) OCCUlS within the transfonnatlon zone (1Z) at the vaginal opening of the ceiVi.c:al canal (cxtemal os). The lower r«tll1tgge in the top f4t figure marks this site, lmown as the transformation r.one, which is shown at a higher magoific:arlon here. Note the abrupt change in the epithelium at the ttansformarlon zone as wdl as the large number of lymphocytes and blood vessels (BV) present in this region.

of the WI. during tissue preparation. The auwding and the cilange in shape of the nuclei (asterisk) seen at the uppn-Pfi.Tt of one of the glands in this figure are due to a tangential aat thtough the wall of the gland as It passed out of the plane of section. (It is not uncommon for cervic:al glands to develop into cysm as a result ofobstruction in the duct. Such cysm are rem~ to as nabothian cysts.)

Muc. mucosa 0•, ostium of the uterus SSEp, stratified squamous epithelium

TZ, transformation zone utert•k. tangential cut of the epithelial surface

933

934

PLATE 99

PLACENTA I

The placenta is a disc-shaped organ that serves fur the exchange of materials between the fetal and maternal circulations during pregnancy. It develops primarily from embryonic tissue, the chorion frondosum. One aide ofthe placenta is em bedded in the uterine wall at the basal plate. The other side faces the amniotic cavity that contains the fetus. After childbirth, the placenta separates from the wall of the uterus and is discharged along with the contiguous membranes of the amniotic cavity.

Placenta, human, H&E, X16.

B

A section extending from the amniotic swface into the substance of the placenta is shown here. This includes the amnion (A), the chorionic plate (CP), and the chorionic villi (CV). The amnion consists of a layer of simple cuboidal epitbdium and an underlying layer of connective tissue. The c:onneaive tissue ofthe amnion is continuous with the connective tissue of the cltorionic: plate as a ra:ult of their fusion at an earUer time. The plane offusion, however, is not evident in H&E secdona; the separation (mtnislu) in pam of this figure in the vicinity of the fusion is an artifact. The cltorionic plate is a thick connective tissue mass that contains the ramifications of the umbilical arteries and vein. These vessels (B'!1>) do not have the distinct organizational features characteristic ofarteries

Placenta, human, H&E, X70; inset X370.

B

The maternal 5ide of the plac:enta is mown in this figure. The basal plate (BP) is on the right silk of the illustration. This is the part of the uterus to whic:h the chorionic villi anchor. Along with the usual connec:rlve tiaiJue dements, the basal plate containa apccialized c:clls c:alled decidual cells (DC).

A. amnion BP, basal plate BVp, blood vessels in chorionic plate

The umbilical cord connects the fetus to the placenta. It contains two arteries that carry blood from the fetus to the placenta and a vein that returns blood from the placenta to the fetus. The umbilical arteries have thick muscular walla. These are arranged as two layers, an inner longitudinal layer and an outer circular layer. Elastic lamellae are poorly developed in these vessels and, indeed, may be absent. The umbilical vein is similar to the arteries, also having a thick muscular wall arranged as an inner longitudinal and an outer circular layer.

and vans; rather. they reaemble the V!:SSC1s of the umbilical cord. Although their identification as blood vessels is relatively simple, it is diffic:ult to distinguish which vessels ate branches of an umbilical arwy and which are tributaries of the vein. The main substance of the placenta consists of chorionic villi of diff'erent sizes (sec Plate 100). These emerge from the chorionic plate as large stem villi that branch into increasingly smaller villi. Branches of the umbilical arteries and vein (BVv, in the figure below) enter the stem villi and ramify through the branching villou.s network. Some villi atend fmm the c:borionic plate to the maternal side of the pla.a:nta and make contact with the maternal tissue; these are called anchoring villi. Other villi, the free villi, aimply arborize within the substanc.c of the placenta without anchoring onto the maternal aide.

The same c:clls are shown at higher magnification in the inm. Decidual c:clls are usually found in clusters and have an epithelial appearance. Because of these features, they are easily identified. Septa fmm the basal plate atend into the portion of the pla.a:nta that oontains the chorionic villi. The septa do not oontain the branches of the umbilical vessel!; and, on this basU, can fttquendy be distinguished from stem villi or their branches.

BVY, blood vessels in chorionic villi CP, chorionic plate CV, chorionic villi

DC, decidual cells

utertsks, separation that is actually an artifact

935

936

PLATE 100 •

PLACENTA II

As the embryo develops, the invasive activity of the syncytiotrophoblast erodes the maternal capillaries and anastomoses them with tha trophoblast lacunae, forming the maternal blood sinusoids. These communicate with each other and form a single blood compartment, lined by syncytiotrophoblasts, called the intervillous space. At the end of the second week of development, cytotrophoblast cells form primary chorionic villi. They project into the maternal blood space. In the third week of development, invasion of the extra embryonic mesenchyma into the primary chorionic villi creates secondary chorionic villi. At the end of the third week, core mesenchyma dif· farentiates into connective tissue and blood vessels that connect with the embryonic circulation. These tertiary chorionic villi constitute functional units for exchange of gases, nutrients, and waste products between maternal and fetal circulation without direct contact with each other. This separation of fetal and maternal blood is referred

to as the placental barrier. Each tertiary villus consists of a connective tissue core surrounded by two distinct layers of trophoblast-derived cells. The outermost layer consists of the syncytiotrophoblast; immediately beneath it is a layer of cytotrophoblast cells. Starting at the fourth month, these layers become very thin to facilitate the exchange of products across the placental barrier. The thinning of the wall of the villus is due to the loss of the inner, cytotrophoblastic layer. At this stage, the syncytiotrophoblast forms numerous trophoblastic buds that resemble the primary chorionic villi; however, the cytotrophoblast and the connective tissue grow vary rapidly into these structures, transforming them into tertiary villi. At term, the placental banter consists of the syncytiotrophoblast&; a spare, thin (or discontinuous), inner cytotrophoblast layer; the basal lamina of the trophoblast; the connective tissue of the villus; the basal lamina of the endothelium; and the endothelium of the fetal placental capillary in the tertiary villus.

Tertiary chorionic villi, placenta, full-term, human, H&E, X280.

syncytiotrophoblast layer appears relatively free ofnuclei (11m1W1). TheR stretches of the syncytiotrophoblast lll2Y be so attenuated in places that the villous sur&ce appears devoid of a covering. The syncytiotrophoblast contains microvilli that project into the intelVillous space. lD. wdl-prr:served specimellB, they may appear as a saiatr:d border (see nun bdow). The cytotrophoblast consistll of an irregular layer of mononucleatccl ceJis that lies beneath the syncytiotrophoblast. In immature placentas, the cytotrophoblasts fOrm an almost complete layer of cells. lD. this full-term placenta. only occasional cytotrophoblast cells (C) can be discerned. Most of the ceUs within the core of the villus are typical connective tissue fibrob1uts and endothelial cells. Other cells have a visible amount of cytoplasm that surrounds the nucleus. Thae are considered to be fr:ta.l placental antigen-presenting ceUs or placental maaophagcs (PM) historically known as Hofbauer cells.

ThU photomiaugraph shows a sea:lon through the intervillous5pac:e of the plac:enta at tenn. It includes chorionic L...-...L.L.---' villi ( CV) of different sizes and the surrounding intervillous space (IS). The connective tissue of the villi contains branches and tributariea of the umbilical vein ( UV) and arteries. The intelVillous space usually contains maternal blood (only a few maternal blood ceJis are seen here). The outermost layer of each chorionic villus derives from the fusion of cytotrophoblast ce.lls. This layer, known as the syncytiotrophoblast (S), bas no in.wcellular boundaries, and its nuclei are rather evenly distributed. giving this layer an appearance similar to that of cuboidal epithelium. In some areas, nuclei are gathc:rr:d ill clusters funning syncytial knots (SK}; in other n:gions, the

Secondary chorionic villi, placenta, midterm, human, H&E, X320; inset X640. ThU micrograph 5hows the secondary chorionic villi in the third week of embryonk: development. These vUU are composed of a mesenchymal core (MC) surrounded by two distinct layus of the trophoblast. Secondary villi have a much

Tertiary chorionic villi, placenta, full-term, human, H&E, X320. ThU higher magnification photomicrograph mows a croas sea:ion through Immature chorionic tertiary villi surrounded by the intervillous space (IS). Ax this stage. chorionic villi are growing by proliferatio.n of their core mesenchyme, syncytiotrophobla.u (S), and fr:ta.l endotbdial c:dls. Note a discontinued

C, cytotrophoblast cells CV, chorionic villi IS, intervillous space

larger number of cytotrophoblast cells (C) than the mature tertiary villi and form an almost complete layer of cdls immediately deep to the syncytiotrophoblast (S) (see inset). The syncytiotrophoblast not only coven the surface of the chorionic villi but also e:nends into the chorionic plate. Maternal red blood cells are present in the intervillous space.

layer of cytotrophoblast c:dls (C). The syncytiotrophoblast surrounding the chorionic villus (cmter t~Jthe i""'ft) forms syncytial knots (SK), which are prcsent in the £Ull.term mature placenta. They represent aggregation of syncytiotrophobla.n nuclei on the surface of ma· ture terminal viiiL In addition to fibrob1uts, several fr:ta.l placental antigen-presenting cells (placental macrophages) (PM) can be identified by the amount of cytopl:wn IIUtmunding their nuclei.

MC, mesenchymal core PM, placental macrophages S, syncytiotrophoblast

SIC. syncytial knot UV, umbilical vein

937

938

PLATE 101 • VAGINA The vagina is the fibromuscular tube of the female reproductive tract that leads to the exterior of the body. The wall ofthe vagina consists of three layers: a mucosa, a muscularis, and an adventitia. The epithelium of the mucosa is nonkeratinized stratified squamous. It undergoes changes that correspond to the ovarian cycle. The amount of glycogen stored in the epithelial cells increases under the influence of estrogen, whereas the rate of desquamation increases under the influence of progesterone. The glycogen liberated from the desquamated cells is fermented by lactobacilli vaginalis, producing lactic acid that acidifies the vaginal surface and inhibits colonization by yeasts and potentially harmful bacteria.

Yaglna, human, H&E, X90.

The vagina has certain histologic similarities to the proximal portion of the alimentary canal but is distinguished by the following features: The epithelium does not keratinize, and except for the deepeat layers, the cells appear to be empty in routine H&E sections; the mucosa contains neither glands nor a muscularis mucosae; and the muscle is smooth and not well ordered. This can be contrasted with the oral cavity, pharynx, and upper part of the esophagus in which the muscle is striated. The more distal portion of the esophagus, which contains smooth muscle, can be distinguished easily from the vagina because it has a muscularis mucosae.

granules may be found in the superficial cells. keralinization does not in human vaginal epithelium. Thus, nuclei can be observed throughout the entire thickness of the epithdium despite the fact that the cytopl:wn of most of the cells above the basal layers appears empty. These cells are normally filled with large clcposits of glycogen that is lost in the pruc:esses of fixation and embedding of the tissue. The ~ outlines a portion of the epithelium and oonneaive ti8sue papillae that is examined at higher magnification below. The musr;ular layer of the vaginal wall consists ofsmooth murde arranged io two ill-clc:6.nc:d layers. The outer layer is generally said m be longirudinallyarrangc:d (SML), and the inner layer is generally said m be circularly~ (SM'C), but the fibCIS are more wually organized as interlacing bundles sunounclcd by con· nectivc tissue. Many blood vessels (BV) are seen in the a~nnective tissue. Ocau'

The mu(;05a of the vagina a~rui.m of a stratified squamous epithelium (~) and an underlying fibrous a~n­ nectivc tissue (CT) that often appeai!l more cdlulac than other fibrous a~nnective tissue. The bouwlary ~ the two is readily identified because ofthe conspiruous staining of the closely packed small cclJs of the basal layer (B) of the cpith.el.ium. ConnectM: tissue papillae: project into the undmiclc of the epithelium, giving the ~ithdial-amnect:M: tissue junction an llllCVCil appearance. The papillae may be cut obliqudy or in cross section and thus may appear as connective tissue islands (IITTDUIS) within the lower portion of the epithelium. The epithelium is char.tcteristically thick. and although keratohyalin

Mucosa, vagina, human, H&E, x 110. 1hi5 is a higher magnification of the c:pithdium (~) that includes the area outlined by the rratmgk in the "}lpn"6gure

(rumc:d 90"). The obliquely cut and cross-sectioned portions

Mucosa, vagina, human, H&E, X225. 1hU b a higher magnification micrograph of the basal portion of the epithelium (Ep) be~ connective msue papillae. Note the regularity and dense packing of the basal epithelial cells. They are the stem cells for the strat:ili.c:d squamous epithdium. Daughter cells of these cells migrare toward the

Muscularis, vagina, human, H&E, X125. This higher magnification micrograph of the smooth mw. de of the vaginal wall emphasizes the irregularity of the ac· rangement of the muscle bundles. At the right edge of the figure is a bundle of smooth muscle cut in a longitudinal

B, basal layer of vaginal epithelium BV, blood vessels CT. connective tissue Ep, epithelium

of a~nncctive tissue papillae that appear as connective tissue islands in the epith.el.ium are more clearly seen here (lltTOWt), in some instanc:c:s, outlined by the swrounding closely packed cells of the basal epithelial cell layer. Note, again, that the epithelial cells even at the surface still retain their nuclei and there is no evidence of ker:atinization. surf.u:e and begin to accumulate glycogen and become less regularly arranged as they move toward the surface. The highly cellular connective tissue (en immc:diately beneath the basal layer (B) of the epithelium typically cont:ains many lymphocytes (L). The number of lymphocytes varies with the stage of the ovarian cycle. Lymphocytes invade the epithellum around the time of menstruation and appear along with the epithelial cells in vaginal smears. section (SML). Adjacent to this is a bundle of smooth muscle cut in cross section (SMC). This bundle abuts on a longirudinally sectioned lymphatic vessel (LV). To the left of the lymphatic vessel i8 another longitudinal bundle of smooth muscle (SML). A valve (WI) is seen in the lymphatic vessel. A small vein (V) is present in the circular smooth muscle close to the lymphatic veael.

L.lymphocytes LV, lymphatic vessel SMC, smooth muscle, cross section SML. smooth muscle, longitudinal section

V,vein

V., valve in lymphatic vessel arrows, connective tissue islands in epithelium

939

940

PLATE 102

MAMMARY GLAND INACTIVE STAGE

The mammary glands are branched tubuloalveolar glands that develop from epidermis and coma to lie in the subcutaneous tissue (superficial faacia). They begin to develop at puberty in the female but do not reach a fully functional

Mammary gland, inactive stage, human, H&E, xao. This figure is a section through an inactive gland. The parenchyma is sparse and. coosists mainly of duct dements. Seft:tal duca (D) are .hown i.n the «11kT of the field. A small lumen can be seen In eadl. The dua. are surrounded by a

Mammary g~and, inactive stage, human, H&E, X200; Inset X400. Addidonal detalb are evident at higher magnification. In di&tinguish1ng between the loose and dente connetdve t~ sue, m:a.ll that both emacellular and cellular ~ show diffi:rcllCCI that are evident in both the figwe and the irud. Note the thicker collagenoua fibcn in the dense connective tissue in contralt to the much thinner fibets of the loose connective tissue. The loose connective tissue (CF[L]) cootairu f.ar mo.re cells per unit area and a greater variety of cell types. This figwe shows a cluster of lymphocytel (L) and. at .still higher magnification {iltltt), plasma cell• (P) and. individual lymphocytes (L). Both plasma cells and lymphocytes are cells with a rounded shape, but plasma cells are larger and show mo.re cymplum. In addition, .regions of plasma cdl cytoplasm display ba10philla. FJongate nuclei in •pindle-.haped cells belong to fibroblasa.

A. adipocytes CTID). dense connectMt tissue loose connective tissue

crew.

D. O.X:ts

L. lymphocytes M. IT'IYOBPittleial cells

state until after pregnancy. Tht glands also develop In the male at puberty; the development is limited. however, and the glands usually remain in a stabilized state.

loose con.nmlve tissue (see CF[L], in figure below), and ~. the

ducts and surrounding con.nmlve dssu.e r.onrutu~ a lobule. Two terminal dua lobular uniu (mLCI) are bracketed i.n thi.s figure. Beyond the lobular unit, the connective O.UC is more dense (CF[D]) and contains adipoqta (A). The two types of connective tiNues can be di.dnguilhed at the low magnification of thi1 figwe.

In contralt, although the cell types in the dense connective tiNue may a.I.o be diverse. a simple aamination of equal areas of loose and dense connective tissue will, by far. show fewer cells in the dense connective tiuue. Chara.c:terilldcally. the daue connective tissue contains numerous aggregates ofadipocytes (A). The epithdial cdls within the .resting lobular units are regatded as being chidly duct elements (D). Usually, alveoli are not found; their preausors, however, are represented as cellular thickenings of the duct wall. The epithelium of the resting lobule is cuboidal; in a.ddi~ tion, myoepithelial cells are present. RHx!lmination of the inm showt a thickening of the epithelium in one location, presumably the precursor of an alveolu., and myoepithelial cells (M) at the bale of the epltbellwn. A$ elsewhere, the myoepithelial cells are on the epithelial side of the basement membrane. During pregnancy, the gland. begin to prolimte. This can be thought of as a dual process in which ducts proliferate and alveoli grow from the ducts.

P, plasma cells TDLU, terminal o.x:t lobular unit

941

942

PLATE 103 MAMMARY GLAND, LATE PROLIFERATIVE AND LACTATING STAGES Mammary glands exhibit a number of changes during pregnancy in preparation for lactation. Lymphocytes and plasma cells infiltrate the loose connective tissue as the glandular tissue develops. As the cells of the glandular portion proliferate by mitotic division, the ducts branch and alveoli begin to develop at their growing ends. Alveolar development becomes most prominent in the later stages of pregnancy, and accumulation of secretory product takes place in the alveoli. At the same time, lymphocytes and plasma cells become prominent in the loose connective tissue of the developing lobules. Myoepithelial cells proliferate between the base of the epithelial cells and the basal lamina in both the alveolar and the ductal portion of the glands. They are most prominent in the larger ducts. Both merocrine end apocrine secretion are involved in the production of milk. The protein component is synthesized, concentrated, and secreted by exocytosis in a

manner typical for protein secretion. The lipid component begins as droplets in the cytoplasm that coalesce into large droplets in the apical cytoplasm of the alveolar cells and cause the apical plasma membrane to bulge into the alveolar lumen. The droplets are surrounded by a thin layer of cytoplasm and are enveloped in plasma membrane as they are released. The initial secretion in the first days after birth is called colostrum. This premilk is an alkaline secretion with a higher protein, vitamin A, sodium, and chloride content than milk and a lower lipid, carbohydrate, and potassium content. Considerable amounts of antibodies sre contained in colostrum, and these provide the newborn with passive immunity to many antigens. The antibodies are produced by the plasma cells in the stroma of the breast and are carried across the glandular cells in a manner similar to that for secretory lgA in the salivary glands and intestine. A few days after parturition, the secretion of colostrum stops and lipid-rich milk is produced.

Mammary gland, late proliferative stage, human, H&E, X90; inset X560.

The epithelium of the intralobular ducts is similar in appearance to the alveolar epithelium. The cclli of both components an: seaetory. The al~ veoli as wdl as the intralobular ducts consist ofa single layer ofcuboidal epithelial cdls subtcndcd by myoepithdial cdls. Often, what appear to be several alveoli are seen merging with one another (lllteriJu). Sw:h profiles represent alveolar units opening into a duct. Interlobular ducts (D) are easy to identify as they are surrounded by dense connective tissue. In one instance, an intralobular duct can be seen emptying into an interlobular duct (4m!W). The imrt shows the seaetory c:pithcllum at a muclt higher magnification. Note that it is a simple columnar epithelium. The nucleus of a myaepithelial call (M) 15 seen at the base of the epithelium. Generally, these cclli are difficult to recognize. Also, as noted above. numerous plasma telb (P) and lymphocytes (Ly) are present in the loose coDDective tiasue of the lobule.

Whereas the development of the duct elements in the mammary gland occurs during the urty proliferative stage, the development of the alveolar elements becomes oowpicuous in the late proliferative stage. This figure shows the tennlnal duct lobular units (TDLU) a1 the late proUferative stage. Individual lobular units are separated by narrow, dense cannective tissue septa (S). The connective tissue within the lobular unit is a typical loose connective tissue thal is now more cdlular, containing mostly plasma telb and lymphoc:ytes. The alveoli are wdl developed, and many exhibit precipitated seaetory product. Ram of the alveoli is joined to a duct, although that rdation$hip can be dUfu:ult to identify.

Mammary gland, lactating stage, human, methyl green-osmium, X90; inset X700. The specimen shown here is from a lactating mammary gland. It i& aim.ilar in appearance to the gland at the . lm prolifctarlve stage but diffets mainly to the extent that the alveoli are more uniform in appearance and their lumina are larger. As in the late proliferative stage. several alveoli can be seen merging with one another (llStnilks). The use of osmium in this specimen stains the lipid component of the secretion. The insrt reveals the lipid droplets within the epithelial cell cytoplasm as well as lipid that has been secreted

D, interlobular duct Ly, lymphocyte M, myoepithelial cell

into the lumen of the alveolus. The lipid first appcan as small droplets within the epithelial cells. These droplets become larger and ultimately are secreted into the alveolar lumen along with milk proteins. The milk proteins are present in small VliCU.Oles in the apical part of the cell but cannot be seen by light microscopic methods. They are secreted by em~ cytosis. The lipid droplets, in conl:ra.!;t, are large and surrounded by the apical cell membrane as they are pinched off m enter the: lumen; thu.oi, it is an apocrine secretion. Sc:vl:ral intertobular ducts (D) are evident. One of these ducts reveals a small branch, an ending intralobular dutt (amiWS) joining the interlobular duct.

P, plasma cell S, connective tissue septa TDW, terminal duct lobular unit

arrows, union of intralobular duct with interlobular duct asterisks, sites of merging alveoli

943

EYE OVERVIEW OF THE EYE I 944 GENERAL STRUCTURE OF THE EYE /944 Layers of the Eye I 944 Chambers of the Eye /946 Development of the Eye /946

MICROSCOPIC STRUCTURE OF THE EYE /947 Corneoscleral Coat /947 Vascular Coat (Uvea)/951 Retina /956 Crystalline Lens /967 Vitreous Body /969

ACCESSORY STRUCTURES OF THE EYE /969

Folder 24.1 Clinical Correlation: Glaucoma /954 Folder 24.2 Clinical Correlation: Retinal Detachment /955 Folder 24.3 Clinical Correlation: Age-Related Macular Degeneration /956 Folder 24.4 Clinical Correlation: Clinical Imaging of the Retina /961 Folder 24.5 Clinical Correlation: Color Blindness /964 Folder 24.8 Clinical Correlation: Conjunctivitis /968

HISTOLDGY 101/ !f12

~

cortex located in the occipital lobes processes the d..ifferences between the two images to create the perception of depth. The aye is a complex sensory organ that provides the sense The final image is then projected onto the visual cortex. In of sight. In many ways, the eye is similar to a digital camera. addition, other complex neural mechanisms coordinate eye: Like the optical system of a camera. the comaa and lens of movements, enabling refinements in the perception of depth the eye capture and automatically focus light, whereas the iris and distance. Therefore, the way in which we see the world automatically adjwts the diameter of the pupil to differences around us largely depends on impulses processed within the: in illumination. The light detector in a digital camera. the retina and analysis and interpretation of these impulses by charge-coupled device (CCD), consists ofclosely spaced pho- theCNS. todiodes that capture, collect, and convert the light image into a series of electrical impulses. Similarly, the photoreceptor • GENERAL STRUCTURE OF THE EYE calls in the retina of the eye detect light intensity and color (wavelengths of visible light that are reflected by different The eye measures approximately 25 mm in diameter. It is sus-objects) and encode these parameters into electrical impulses pended in the bony orbital socket by six extrinsic muscles that for ttansrnission to the brain via the optic: nerve. The retina control its movement. A thick layer ofadipose tissue partially has other capabilities beyond those of a CCD: It can extract surrounds and cushions the eye as it moves within the orbit. and modify speciJic impulses from the visual image before The: exttaocular muscles are coordinated so that the eyes move sending them to the central nervous system (CNS). symmetrically around their own central axes. In other ways, the optical system of the eye is far more elaborate and complex than a camera. For example, the eye Layers of the Eye is able to track moving objects with coordinated eye move- The wall of the eya consists af three concentric layers or ments. The eye can also protect, maintain, self-repair, and coats. clean its transparent optical system. The eyeball is composed of three concentric structural layers Because the eyes are paited and spatially separated, two (Fig. 24.1): slightly different and overlapping views (visual fields) are sent to the brain. The brain integrates these two slightly different • The comaoaclaral coat, the outer or fibrous layer, includes the sclera, the white portion, and the c:omea, images from each eye into a single three dimensionaii3D) image in a process called stereopsis. The primary visual the transparent portion.

• OVERVIEW OF THE EYE

944

CORNEOSCLERALCOAT sclera

945

cornea VASCULAR COAT (UVEA) choroid ciliary body iris

comea

RE11NA

neural retina nonphotosensitive part photosensitive part retinal pigment epithelium (RPE) C>

\'~··-

nonphotosensitive part of retina

meningeal coverings

retinal pigment

photosensldve part of retina FIGURE 24.1. Schematic diagram of the layers of the eye. The wall of the eyeball is organized in three separate concentric layers: an outer supporting fibrous layer, the corneoscleral coat; a middle vascular coat or uvea; and an inner layer consisting of the retina. Note that the retina has two layers: neural retina lyello~ and a retinal pigment epithelium lotange). The photosensitive and nonphotosensitive parts of the neural retina occupy different regions of the (loJe. The photosensitive part of the retina is found in the posterior part of the 6tf9 and terminates anteriorly along the ora serrate. The nonphotosensitive region of the retina is located anterior to the ora serrate and lines the inner aspect of the ciliary body and the posterior surface of the iris. The vitreous body !partially removed! occupies considerable space within the eyeball. epithelium

m z m

~

~

:D

c

Q c

:D

m

.,0 -1 ::I:

m

~

m

• The vascular coat. the middle layer. or uvea, includes the choroid and the stroma of the ciliary body and Iris. • The retina, the inner layer, includes an outer pigment epithdiwn. the inner neural retina. and the epitheliwn of the ciliary body and iris. The new:al retina is continuous with the cenual nervous system through the optic nerve.

The corneoscleral coat consists of the transparent cornea and the white opaque sclera. The comea covers the anterior one-sixth of the eye (see Fig. 24.1). In this window-like region. the swface of the eye h2s a prominence or convexity. The comea is continuous with the sclera [Gr. sit/nos, hard]. The sclera is composed of dense fibrous connective tissue that provides attachment for the extrinsic muscles of the eye. The corneoscleral coat encloses the inner two layers except where it is peneuared. by the optic nerve. The sclera constitutes the .,white" of the eye. In children, it has a slightly blue tint because of its thinness; in elderly people, it is yellowish because of the accumulation of lipofuscin in its stromal cells. A noticeable feature of patients with Jaundice is yellow discoloration of the sclera (sderal Icterus) caused by a high level of circulating bilirubin.

The uvea consists principally of the choroid. the vascular layer that provides nutrients to the retina. Blood vessels and melanin pigment give the choroid an intense dark brown color. The pigment absorbs scattered and re:flectcd light to minimize: glare within the eye. The choroid contains numerous venous plexuses and layers of capillaries and is firmly attached to the retina (see Fig. 24.1). The anterior rim of the uveal layer continues forward, where it forms the sttoma of the ciliary body and iris.

The c111ary body is a ring-like thickening that extends inward just posterior ro the level of the comeoscleral junction. Within the ciUary body is the ciliary muscle. a smooth muscle that is responsible for lens accommodation. Contraction of the ciliary muscle changes the shape of the lens. which enables it to bring light rays from different distances to focus on the retina. The Iris is a contractile diaphragm that extends over the anterior surfuce of the lens. It also contains smooth muscle and mdanin-containing pigment cells scattered in the connective tissue. The pup11 is the cenual circular aperture of the iris. It appears black because one looks through the lens toward the heavily pigmented back of the eye. In the process of adaptation. the iris contracts or expands, changing the size of the pupil in response to the amount oftight that passes through the lens to reach the retina.

The retina consists of two components: the neural retina and pigment epithelium. The retina is a thin, deli.cate layer (see Fig. 24.1) consisting of two components: • The neural retina is the inner layer that contains lightsensitive receptors and complex neuronal networks. • The retinal pigment epithelium (RPE) is the outer layer composed of simple cuboidal melanin-containing cells. Externally, the retina rests on the choroid; intcmally. it is associated with the vitteous body. The newal retina consists largely of photoreceptor cells, called retinal rods and cones, and intemeurons. Visual information encoded by the rods and cones is sent to the brain via impulses conveyed along the optic nerve.

946

Chambers of the Eye The layers of the eye and the lens serve as boundaries for three chambers within the eye. The chambers of the eye are as follows: • The anterior chamber is the space between the cornea and the iris. • The posterior chamber is the space between the posterior surface of the iris and the anterior surface of the lens. • The vitreous chamber is the space between the posterior surface of the lens and the neural retina (Fig. 24.2). The cornea, the anterior and posterior chambers, and their contents constitute the anterior segment of the eye. The vitreous chamber, visual retina, RPE, posterior sclera, and uvea constitute the posterior segment.

• The vitreous body is composed of a transparent gel-like substance that 611s the vitreous chamber. It acts as a "shock absorber" that protects the &agile retina during rapid eye movement and helps to maintain the shape of the eye. The vitreous body is almost 99% water with soluble proteins, hyaluronan, glycoprotein&, widdy dispersed collagen fibrils, and traces of other insoluble proteins. The ftuid component of the vitreous body is called the vitreous humor. The comea is the chief.tdiactive dement of the eye. It has a .tdiactive index of 1.376 (air has a ~ index of 1.0). The lens is second in impottanc:e to the cornea in the .tdi:action of light .rays. Because of its dasticity, the shape of the lens can undergo slight ch.angt:s in ~nse to the tension of the cili.aJ.y muscle. These changes are important in accommodation for properfucusingonncarobjects.The aqueous humor and vitreous body have only minor roles in n:fraction. However, the aqueous humor plays an important role in providing nuttients to two avascular structures, the lens and cornea. In addition to transmi.t· ting light, the vitreous body helps maintain the position of the lens and hdps b:cp the neu.ral retina in contact with the RPE.

The refractile media components of the eye alter the light path to focus it on the retina. As light rays pass through the components of the eye, they are refracted. Refraction focuses the light rays on the photoreceptor cells of the retina. Four transparent components of Development of the Eye the eye, called the refractile (or dioptric} media, alter the To appreciate the unusual structUral and functional relationships in the eye. it is helpful to understand how it forms in the embryo. path of the light rays: The tissues of the eye are derived from neuroectodenn, • The comea is the anterior window of the eye. surface ectodenn. and mesodtnn. • The aqueous humor is the watery B.uid located in the By the 22nd day of devdopment, the ayes are evident as shalanterior and posterior chambers. • The lens is a transparent, crystalline, biconcave structure low grooves-the optic sulci or optic grooves-located in suspended from the inner surface of the ciliary body by a the neural folds at the cranial end of the embryo. Ar. the neuring of radially oriented fibers, the zonule of Zinn. .ral tube closes, the paired grooves form outpocketings called

superior rectus muscle

retinal arterioles andvenulee optic disc

vitreous body (cut) inferior rectus muscle FIGURE 24.2. Schematic diagram illustrating the intemal structures of the human eye. This diagram shows the relationship between

the layers of the eye and internal structures. The lens is suspended between the edges of the ciliary bodV. Note the posterior chamber of the eye, which is a narrow space between the anterior surface of the lens and posterior surface of the iris. It communicates through the pupil with the larger anterior chamber that is bordered by the iris and the cornea. These spaces are filled with the aqueous humor produced by the ciliary body. The large cavity posterior to the lens. the vitreous chamber, is filled with a transparent jelly-like substance called the vitreous body. In this figure, most of the vitreous body has been removed to illustrate the distribution of the central retinal vessels on the surface of the retina. The other layers of the eyeball and the attachment of two of the extraocular muscles to the sclera are also shown.

forebrain

lens placode

a outer layer of optic cup opUcct.~p

lumen of optic stalk invaginating lens vesicle

7

choroid

fissure Inner layer of

b

optlcct.~p

neural retina

.I

optic nerve

\

hyaloid artery vitreous outer vascular body layer

inner vascular layer

c

FIGURE 24.3. Schematic drawlnglllultratlng the development of the eye. •· Forebrain and developing optic vesicles as seen in a

double-layered optic cup (Fig. 24.3b).The inner layer becomes the neural retina. The outer layer becomes the RPE. The mesenchyme surrounding the optic cup gives rise to the sclera. Invagination of the centtal region of each lens placode results in the formation of the lens vesicle. By the fifth week of development, the lens vesicle loses contact with the surf.r.ce ectoderm and comes to lie in the mouth of the optic cup. After the lens vesicle detaches from the surface ectoderm, this same site again thickens to form the corneal epithelium. Mesenchymal cells from the periphery then give rise to the co meal endothelium and the comeal stroma. Grooves containing blood vessels derived from mesenchyme devdop along the inferior surface of each optic cup and stalk. Called the choroid fissums, the grooves enable the hyaloid artery to reach the inner chamber of the eye. 1b.is artery and its branches supply the inner chamber of the optic cup, lens vesicle, and mesenchyme within the optic cup. The hyaloid vein returns blood &om these structures. The distal portions of the hyaloid vessels degenerate, but the proximal portions remain as the cen· tral retinal artery and central retinal vein. By the end of the seventh week, the edges of the choroid fissure fuse and a round opening, the future pupil, forms over the lens vesicle. The outer layer of the optic cup forms a single layer of pigmented cells (Fig. 24.3c). Pigmentation begins at the end of the fifth week. The inner layer undergoes a complex di.lferentiation into the nine layers of the neural retina. The photoreceptor cells (rods and cones) as well as the bipolar, amacrine, and ganglion cells and nerve fibers are present by the seventh month. The macular depression, a futUre site of fovea central.is, begins to develop during the eighth month and is not complete until about 6 months after birth. During the third month. growth of the optic cup gives rise to the ciliary body and the futUre iris. which forms a double row of epithelium in front of the lens. The mesoderm located external to this region becomes the stroma of the ciliary body and iris. Both epithelial layers of the iris become pigmented. In the ciliary body, however, only the outer layer is pigmented. At birth, the iris is light blue in fair~skinned people because pigment is usually not presenL The dilator and sphincter pu~ pillary muscles develop during the sinh month as derivatives of the neuroectoderm of the outer layer of the optic cup. The embryonic origins of the individual eye structures are summarized in Table 24.1.

• MICROSCOPIC STRUCTURE OF THE EYE The three layers of the eye-the comeoscleral coat. the vascular coat, and the min...-are in turn composed of complex molecular layers and structures that reflect their var· ious functions.

4-mm embryo. b. Bilayered optic cup and invaginating lens vesicle as seen in a 7.5-mm embryo. The optic stalk connects the developing eye to the brain. c. The eye as seen in a 15-week. fetus. All the layers of the eye are established, and the hyaloid artery traverses the vitreous body from the optic disc to the posterior surface of the lens.

Comeoscleral Coat

optic vesicles (Fig. 24.3a). k c:ach optic vesicle grows later· ally. the connection to the foreb.tain becomes constricted into an optic stalk. and the overlying surface ectoderm thickens and forms a lens placode. These events are followed by concomitant invagination of the optic vesicles and the lens placodes. The invagination of the optic vesicle results in the formation of a

The cornea consists of five layers: 1hree cellular layers and two noncellular layers. The transparent comea (see Figs. 24.1 and 24.2) is only 0.5 rwn thick at its center and about 1 mm thick peripherally. It consists of three cellular layers that are distinct in both appearance and origin. These layers are separated by two important membranes that appear homogeneous when

947

3::

n

~

U>

n

0

iJ

n

~ :JJ

c

~:JJ 0,

m

~

m

~

m

948

TABLE24.1

UJ

ru

Source

Derivative

Surface ectoderm

Lens Epithelium of the cornea, conjunctiva, and lacrimal gland and its drainage system

Neural ectoderm

Vitreous body (derived partly from neural ectoderm of the optic cup and partly from mesenchyme) Epithelium of the retina, iris, and ciliary body Sphincter pupillae and dilator papillae muscles Optic nerve

Mesoderm

Sclera Stroma of the cornea, ciliary body, iris, and choroids Extraocular muscles Eyelids (except epithelium and conjunctival Hyaloid system (most of which degenerates before birth) Coverings of the optic nerve Connective tissue and blood vessels of the eye, bony orbit, and vitreous body

UJ

:I: ILL

0

UJ

Embryonic Origins of the Individual Structures of the Eye

a:

:::;1

t:::J a:

I-

V)

u c:::

0

u r.n

0 a: u ~

w

> w ~

a: w

viewed in the light microscope. Thus, the five layers of the comaa seen in a transverse section are the following:

The cornaalepilllalium is a nonkeratinizad stratified squamous epithelium.

• • • • •

The corneal epithelium (Fig. 24.4) represents nonkaratinized stratified squamous epithelium that consists of apprmimately five layers of cells and measures about 50 ~m in average thickness. It is continuous with the conjunctival epithdium that overlies tb.e adjacent sclera. The epithelial

Corneal epithelium Bowman's membrane (anterior basement membrane) Corneal stroma Dascamat's membrana (posterior basement membrane) Corneal endothelium

c==

:::r::::

u

-__.,. -

-~

FIGURE 24.4. Photomicrograph of the comaa. a. This photomicrograph of a section through the full thickness of the cornea shows the corneal stroma and the two corneal surfaces covered by different types of epithelia. The corneal stroma does not contain blood or lymphatic vessels. x 140. b. A higher magnification of the anterior surface of the comea showing the corneal stroma covered by a stratified squamous (corneal) epithelium. The basal cells that rest on Bowman's membrane, which is a homogeneous condensed layer of corneal stroma, are low columnar in contrast to the squamous surface cells. Note that one of the surface cells is in the process of desquamation (atTOw). X280. c. A higher magnification photomicrograph of the posterior surface of the cornea covered by a thin layer of simple squamous epithelium (corneal endothelium!. These cells are in direct contact with the aqueous humor of the anterior chamber of the eye. Note the very thick Descemet's membrane (basal lamina) of the corneal endothelial cells. x280.

cells adhere to neighboring cells via desmosomes that are present on short interdigitating processes. Like other stratified epithelia, such as that of the skin, the cells proliferate from a basal layer and become squamous at the surf.tce. The basal cells are low columnar with round, ovoid nuclei; the surface cells acquire a squamous or discoid shape, and their nuclei are flattened and pyknotic (Fig. 24.4b). As the cells migrate to the surface, the cytoplasmic organelles gradually disappear, indicating a progressive decline in metabolic activity. The corneal epithelium has a remarkable regenerative capacity with a turnover time of approximately 7 days. The acwai stem cells for the corneal epithelium, called comeolimbal stem cells, reside at the comeoscleral limbus, the junction of the cornea and sclera. The microenvironment of this stem cell niche is important in maintaining the stem cell population. It also acu as a barrier that prevent& migration of conjunctiwl epithelial cells to the corneal surface. The corneolimbal stem cells may be partially or totally depleted by disease or extensive injury, resulting in abnormalities of the corneal surface that lead to conjunctivalization of the cornea, which is characterized by vascularization, appearance of goblet cells, and an irregular and unstable epithelium. These changes cause ocular discomfort and reduced vision. Minor injuries of the corneal surface heal rapidly by inducing stem cell proliferation and migration of cells from the corneoscleral limbus to fill the defect. Numerous free nerve endings in the corneal epithelium provide it with extreme sensitivity to touch. Stimulation of these nerves (e.g., by small foreign bodies) elicit& blinking of the eyelids, flow of tears, and, sometimes, severe pain. Microvilli present on the surface epithelial cells help retain the tear film over the entire corneal surface. Drying of the corneal surface may cause ulceration. DNA in corneal epithelial cells is protected from UV light damage by nuclear ferritin. Despite constant exposure of the corneal epithelium to W light, cancer of the corneal epithelium is extremely rare. Unlike the epidermis, which is also exposed to W light, melanin is not present as a defense mechanism in corneal epithelium. The presence of melanin in the cornea would diminish light transmission. Instead, it has recendy been shown that corneal epithelial cell nuclei contain ferritin, an iron-storage protein. Experimental studies with avian corneas have shown that nuclear ferritin protects the DNA in the corneal epithelial cells from free radical damage caused by UV light exposure. Bowman's membrane is a homogeneous-appearing layer on which the corneal epithelium rests. Bowman's membrane (anterior basement membrane) is a homogeneous, &indy fibrillar lamina that is approximately 8 to 10 p.m. thick. It Ues between the corneal epitheUurn and the underlying corneal stroma and ends abruptly at the corneosclcrallimbus. The collagen fibrils ofBowman's mem· brane have a diameter of about 18 run and are randomly oriented. Bowman's membrane imparts some strength to the cornea, but more significantly, it acts as a barrier to the spread of infections. It does not regenerate. Therefore, if damaged, an opaque scar forms that may impair vision. In addition, changes in Bowman's membrane are associated with recurrent corneal erosions.

The camaal stroma constitutes 90% of the corneal thickness. The corneal stroma, also called substantia propria, is composed of about 60 thin lamellae. Each lamella consists of parallel bundles of collagen fibrUs. Located between the lamellae are nearly complete sheets of slender, flattened fibroblasts. The collagen fibrils measure approximately 23 nm in diameter and as long as 1 em in length and are arranged at approximately right angles to those in adjacent lamellae (Fig. 24.5). 'The ground substance of cornea contains small leucine-rich proteoglycans (SLRPs), which comprise sulfuted glycosaminoglycaD.9--C:hidly, keratan sulfute proteoglycan (lumican) and chondroitin sulfate proteoglycan (decorin). Lumican regulates the nonnal collagen fibril assembly in the cornea and is critical to the development of a highly o.rganb:ed collagenous mattix. It is believed that the unifonn spacing of collagen fibrils and lamellae, as wdl as the orthogonal all'8y of the lamellae (alternating layers at right angles), is responsible for the transparency of the cornea. Proteoglycans (lumian), along with type V collagen, regulate the precise diameter and spacing ofthe collagen fibrils maint:aini.ng corneal clarity. Comeal swelling after injury to the epithelium or endothelium disrupts this precise array and leads to translucency or opacity of the cornea. Lumican is overexpressed during the wound-healing process following corneal injury. Normally, the cornea contains no blood vessels or pigments. During an inflammatory response involving the cornea, large numbers of neutrophils and lymphocytes migrate from blood vessels of the corneoscleral limbus and penetrate the stromal lamellae. Dacamet's membrana is an unusually thick basal lamina. Descemet's membrane (posterior basement membrane) is the basal lamina of corneal endothelial cells. It is intensely positive to periodic acid-SchifF (PAS) and can be as thick

FIGURE 24.1. Electron micrograph of the comMI stroma. This electron micrograph shows parts of three lamellae and a portion of a corneal fibroblast (Cf} between two of the lamellae. Note that the collagen fibrils in adjacent lamellae are oriented at right angles to one another. X16,700.

949

3::

n

~

U>

n

0

iJ

n

~ :JJ

c

~:JJ 0,

m

~

m

~

m

950

w

~

w

::c ~

LL.

0

w

a:

=> ~ => a:

t; 0

a. 0

u (f) 0

a: u ~

as 10 JJ.m. Descemet's membrane has a felt~like appearance and consists of an interwoven meshwork of fibers and pores. It separates the corneal endothelium from the adjacent corneal stroma. Unlike Bowman's membrane, Descemet's membrane readily regenerates after injury. It is produced continuously but slowly thickens with age. Descemet's membrane extends peripherally beneath the sclera as a trabecular meshwork forming the pectinate ligament. Strands from the pectinate ligament penetrate the ciliary muscle and sclera and may help to maintain the nor~ mal curvature of the cornea by exerting tension on Descemet's membrane.

The corneal endothelium provides for metabolic exchange between the cornea and aqueous humor. The corneal endothelium is a single layer of squamous ce11s covering the surface of the cornea that faces the anterior chamber (Fig. 24.4c). The cells are joined by well...cfevdoped zonulae adherentes, relatively leaky zonulae ocdudentes, and desmosomes. Virtually. all of the metabolic exchanges of the cornea occur across the endothe.Uwn. The endothelial cdls contain many mitochondria and vesicles and an extensive rough-surfaced endoplasmic reticulwn (rER) and Golgi apparatus. They demonstrate endocytotic activity and are engaged in active transport. Na+IK.+ -activated ATPase is located on the lateral plasma membrane. Transparency of the cornea requires precise regulation of the water content of the stroma. Physical or metabolic damage to the endothelium leads to rapid comeaI swelling and, if the damage is severe, corneal opacity. Restoration of endothelial integrity is usually followed by deturgescence (dehydration necessary to maintain the transparency}, although corneas can swell beyond their ability for self-repair. Such swelling can result in permanent focal opacities caused by aggregation of collagen fibrils in the swollen cornea. Essential sulfated glycosaminoglycans that normally separate the corneal collagen fibers are extracted from the swollen cornea. Human corneal endothelium has a limited prollfel'atlve capacity. Severely damaged endothelium can be repaired only by transplantation of a donor cornea. Recent studies indicate that the periphery of the cornea represents a regenerative zone of the corneal endothelial cells. However, soon after corneal transplantation, endothelial cells exhibit contact inhibition when exposed to the extracellular matrix of Descemet's membrane. The discovery that inhibitory factors released by Descemet's membrane prevent proliferation of endothelial cells has focused current corneal research on reversal or prevention of this inhibition with exogenous growth factors.

The sclera is an opaque layer thet consists predominantly of dense connective tissue. The sclera is a thick fibrous layer containing flat collagen bundles that pass in various directions and in planes parallel to its sur&ce. Both the collagen bundles and fibrils that form them are irregular in diameter and arrangement. Interspersed between the collagen bundles arc: fine networks of dastic fi. hers and a moderate amount ofground substance:. Fibroblasts are scattered among these fibers (Plate 107, page 980). The: opacity of the sclera, like that of other dense connective tissues, is primarily attributable: to the irregularity of its

structure. The sclera is pierced by blood vessels, nerves, and the optic nerve (see Fig. 24.2). It is 1 mm thick posteriorly, 0.3 to 0.4 mm thick at its equator, and 0.7 mm thick at the comeoscleral margin or limbus. The sclera is divided into three rather ill-defined layers: • The episcleral layer {episclera), the external layer, is the loose connective tissue adjacent to the periorbital fat. • The substantia propria (sclera proper, also called Tenon's capsule) is the investing fascia of the eye and is composed of a dense network of thick collagen fibers. • The suprachoroid lamina (lamina fusee), the inner as~t of the sclera, is located adjacent to the choroid and contains thinner collagen fibers and elastic fibers as well as fibroblasts, melanocytes, macrophages, and other connec:-tive tissue cells. In addition, the episcleral space (Tenon's space) is located between the episcleral layer and subsmntia propria of the sclera. This space and the surrounding periorbital fat allow the eye to rotate fi:edywithin the orbit. The tendons of the extraocular muscles attach to the substantia propria of the sclera.

The comeoaclarallimbus is the transitional zona between the cornea and sclera that contains cornaolimbal stem cells. At the junction of the comea and sclera (Fig. 24.6 and Plate 107, page 980), Bowman's membrane ends abrupdy. The overlying epithelium at this site thickens from the 5 cell layers of the cornea to the 10 to 12 cell layers of the conjunctiva. The sur&ce of the Umbus is composed of two distinct types of epithelial cells: One type constitutes the conjunctival cells and the other constitutes the corneal epithelial cells. The basal layer of the limbus contains the comeolimbal stem cells that generate and maintain the corneal epithelium. These cells proliferate, differentiate, and migrate to the surface of the limbus and then toward the center of the cornea to replace damaged epithelial cells. As mentioned previously, this movement of cells at the corneosc:lerallimbus also creates a barrier that prevents conjunctival epitheUum from migrating onto the cornea. At this junction, the corneal lamellae become less regular as they merge with the obUque bundles of collagen fibers of the sclera. An abrupt transition from the avascular cornea to the well-vascularized sclera also oc:c:urs here. The limbus region, specifically, the iridocomeal angle, contains the apparatus for the outflow of aqueous hwnor (Fig. 24.7). In the stromal layer, endothelium-Uned chan· nds called the trabecular meshwork (or spaces of Fontana) merge to form the scleral venous sinus (canal of Schlemm). This sinus encircles the eye (sec Figs. 24.6 and 24.7). The aqueous humor is produced by the ciliary pro· cc:sses that border the lens in the posterior chamber of the eye. The fluid passes from the posterior chamber into the anterior chamber through the valve-Ulcc potential opening between the iris and lens. The :fluid then passes through the openings in the trabecular meshwork in the limbus region as it con· tinucs its course to enter the scleral venous sinus. Collecting vessels in the sclera, called aqueous veins because they con· vey aqueous humor instead of blood, transport the aqueous humor to (blood) veins located in the sclera. Changes in the iridocorneal angle may lead to blockage in the drainage of aqueous humor, causing glaucoma (see Folder 24.1).

D~MK:emi'Tielmerbms::mbmn}cornea cornea

sphincter m. } pigment layer

951

iris

., ............ m.

ciliary muscle ciliary process

3::

n

~

U>

n

0

""'0

n

~ :JJ

ganglion cells bipolar cells photoreceptOr cells

c

~:JJ 0,

m

~

m

FIGURE 24.6. Sch1mtrtlc dlagf'lm of the stru;tuf'l of the eye. This drawing shows a horizontal section of the eyeball with color-coded layers of its wall. Upplr inset. Enlargement of the anterior and posterior chambers is shown in more detail. Note the location of the iridocorneal angle and canal of Schlemm (scleral venous sinus), which drains the aqueous humor from the anterior chamber of the eye. Lower iRHt. Typical organization of the cells and nerve fibers of the fovea.

The iridocorneal angle can be visualized during eye examination using a gonioscope, a specialized optical device that uses mirrors or prisms to reflect the light from the iridocorneal angle into the direction of the observer. In conjunction with a slit lamp or operating microscope, the ophthalmologist can examine this region to monitor various eye conditions associated with glaucoma. The iridocorneal angle can be also visualized using the ultrasound biomicroscopy (UBM). This high-resolution imaging technique utilizes a high-frequency ultrasound transducer to visualize the narrowed iridocorneal angle in primary angle-closure glaucoma.

Vascular Coat (Uvea) The iris, the most anterior part of the vascular coat, forms a contractile diaphragm in front of the lens. The iris arises from the anterior border of the cUiary body (sec Fig. 24.7) and is attached to the sclera about 2 nun posterior to the cornc:oscleral junction. The pupil is the central aperture of this th.i.n disc. The iris is pushed slighdy furward as it changes in size in response to light intensity. It consists of highly vascu· 1arized connc:ctive tissue stroma that is covered on its posterior surf.u:e by highly pigmented cells, the posterior pigment epithelium (Fig. 24.8). The basal lamina of these cells f.u:es the posterior chamber of the eye. The degree of pigmentation is so great that neither the nucleus nor character of the cytoplasm

can be seen in the light microscope. Located bc:nc:ath this layer is a layer of myoepithelial cells. the anterior pigment myoepithelium. The apical (posterior) portions ofthese myo· epithelial cells are laden with melanin granules, which dfectively obscure their boundaries with the adjacent posterior pigment epithelial cells. The basal (anterior) portions of myoepithelial cells possess processes containing contractile elements that ex· tend radially and collc:c:tively make up the dilator pupillae muscle of the iris. The contrac:tile processes are enclosed by a basal lamina that separates them from the adjacent stroma. Constriction of the pupil is produced by smooth muscle cells located in the stroma of the iris near the pupillary margin of the iris. These circumfeR:ntially oriented cells collectively compose the sphincter pupillae muscle. The anterior surface of the iris reveals numerous ridges and grooves that can be sc:en in clinical examination with the ophthalmoscope. When this surface is examined in the light microscope, it appears as a discontinuous layer of fibroblasts and melanocytes. The number of melanocytes in the stroma is responsible fur variation in eye color. The function of these pigment-containing cells in the iris is to absorb light rays. If there are few melanocytes in the stroma, the color of the iris is derived from light reflected from the pigment present in the cells of the iris's posterior surface, giving it a blue appearance. With increasing amounts of pigment present in the stroma, the iris color changes from blue to shades of greenish blue, gray, and, finally, brown.

!!;! m

952

w

it w

::r::

1-

u..

0

w

a:

::>

t>::> a:

t> (.)

a_

0

u (/) 0

a:

u

~

FIGURE 24.7. Photomlcrogf'lph of tile clll1ry body 1nd lrldocome•l•ngle. This photomicrograph of the human eye shows the anterior portion of the ciliary body and parts of the iris and sclers. The inner surface of the ciliary body forms radially arranged, ridge-shaped elevations, the ciliary processes, to which the zonu/sr fibers are anchored. The ciliary body contains the ciliary muscle, connective tissue with blood vessels of the vascular coat and the ciliary epithelium, which is responsible for 1he production of aqueous humor. Anterior to the ciliary body, between the iris and the cornea, is the iridoccmesl angle. The scleral venous sinus (canal of Schtemm) is located in close proximity to this angle and drains the aqueous humor to regulate intraocular pressure. X 120. The inset shows that the ciliary epithelium consists of two layers, the outer pigmented layer and the inner nonpigmented layer. x480.

neurotransmitter of the parasympathetic nervous system (it innervates the sphincter pupillae muscle); the addition of atropine blocks muscarinic acetylcholine receptors, temporally blocking the action of the sphincter muscle, and leaving the pupil wide open and unreactive to light originating from ophthalmoscope. The ciliary body is the thickened anterior portion of the vascular coat and is located between the iris and choroid. • The sphincter pupillae muscle, a circular band of The ciliary body extends about 6 mm from the root of the smooth muscle cells (Plate 106, page 978), is innervated iris posterolaterally to the ora seJT&ta (see Fig. 24.2). As seen by pamsympathetic nerves carried in the oculomotor nerve from behind. the late.tal. edge of the ora serrata bears 17 to (cran.ial nerve III) and is responsible for reducing pupillary 34 grooves or crenulations. These grooves mark the anterior size in response to bright light. Failure of the pupil to re- limit of both the retina and the choroid. The anterior third of spond when light is shined into the eye-"'pup11 fixed the ciUary body has approximately 75 radial ridges or ciliary and dilated" -is an important clinical sign showing processes (see Fig. 24.7). The fibers ofthe zonule arise from the grooves between the ciliary processes. lack of nerve or brain function. The layers of the ciliary body are similar to those of the • The dilator pupillae muscle is a thin sheet of radially iris and consist of a stroma and. an epithelium. The stroma is oriented contractile processes of pigmented. myoepithedivided into two layers: lial cells constituting the anterior pigment epithelium of the iris. This muscle is innervated by sympathetic nerves • An outer layer of smooth muscle, the ciliary muscle, from the superior cervical ganglion and. is responsible for makes up the bulk of the ciliary body. • An inner vascular region extends into me ciliary processes. increasing pupillary size in response to dim light.

The sphincter pupillae is innervated by parasympathetic nerves; the dilator pupillae muscle is under sympathetic nerve control. The size of the pupil is contrOlled by contraction of the sphincter pupillae and dilator pupillae muscles. The process ofadaptation (increasing or decreasing the size ofthe pupil) ensures that only the appropriate amount of light enters the eye. Two muscles are actively involved in adaptation:

Just before ophthalmoscopic examination, mydriatic agents such as atropine are given as eye drops to cause dilation of the pupil. Acetylcholine (ACh) is the

The epithelial layer covering the internal surface of the dUary body is a direct continuation of the two layers of the retinal epithelium (see Fig. 24.1).

953 stromal melanocytes

3::

n

~

(/)

(')

0

""'0

n

(/)

-1 ::0

c(') -1

c

::0

m

0

"'TI

-1

FIGURE 24.8. Structure of the Iris. a. This schematic diagram shows the layers of the iris. Note that the pigmented epithelial cells are reflected as occurs at the pupillary margin of the iris.The two layers of pigmented epithelial cells are in contact with the dilator pupillae muscle. The incomplete layer of fibroblasts and stromal melanocytes is indicated on 1he anterior surface of the iris. b. Photomicrograph of the iris showing the histologic features of this structure. The lens, which lies posterior to the iris, is included for orientation. The iris is composed of a connectill9 tissue stroma covered on its posterior surface by the posterior pigment epi1helium. The basal lamina !not visible) faces the posterior chamber of the eye. Because of intense pigmentation. 1he histologic features of these cells are not discernible. Just anterior to these cells is the anterior pigment myoepithelium layer !the dashed line separates the two layers). Note that the posterior portion of the myoepithelial cells contains melanin, whereas the anterior portion contains contractile elements forming the dilator pupillae muscle of the iris. The sphincter pupillae muscle is evident in the stroma. The color of the iris depends on the number of stromal melanocytes scattered throughout the connective tissue stroma. At the bottom. note the presence of the lens. X570.

The ciliary muscle is organized into three functional

portions or groups of smooth muscle fibers. The smooth muscle of the ciliuy body has its origin in the scleral spur. a ridge-like projection on the inner surface of the sclera at the corneoscleral junction. The muscle fibers spread out in several directions and are classified into three functional groups on the basis of their direction and insertion: • The meridional (or longitudinal) portion consists of the outer muscle fibers that pass posteriorly into the stroma of the choroid. These fibers function chiefly in stretchlng the choroid. It also may help open the iridocorneal angle and facilitate drainage of the aqueous humor. • The radial (or oblique) portion consists of deeper mus-cle fiber bundles that radiate in a &n-Uke fashion to insen in the ciliary body. Its contraction causes the lens to :flatten and thus focus for distant vision. • The circular (or sphincteric) portion consists of inner muscle fiber bundles oriented in a circular pattern that forms a sphincter. It reduces the tension on the lens, causing the lens to accommodate for near vision.

Examination of a histologic: preparation does not dearly reveal the arrangement of the musc:le fibers. Rather. the organizational grouping is based on microdissection techniques.

Ciliary processes are ridge-like extensions of the ciliary body from which zonular fibers emerge and extend to the lens. Ciliary processes are thickenings of the inner wscular region of the ciliary body. They are continuous with the vascular layers of the choroid. Scattered macrophages containing melanin pigment granules and elastic fibers are present in these processes (Plate 106, page 978). The processes and the ciliary body are covered by a double layer of columnar epithelial cells, the ciliary epithelium. which was originally derived from the two layers of the optic cup. The ciliary epithelium has three principal functions: • Secretion of aqueous humor • Participation in the blood-aqueous barrier (part of the blood-ocular barrier) • Secretion and anchoring of the zonular fibers that form the suspensory ligament of the lens

:::c:

m m -< m

954

UJ

ru

UJ

:I: ILL

0 UJ

a:

:::;1

t:::J a:

I-

V)

u c:::

0

u r.n

0 a: u ~

w

> w ~

a: w

ti:

c

:::r::::

u

Glaucoma is a clinical condition resulting from increased intraocular pressure over a sustained period of time. It can be caused by excessive secretion of aqueous humor or impedance of the drainage of aqueous humor from the anterior chamber. The internal tissues of the eye, particularly the retina, are nourished by the diffusion of oxygen and nutrients from the intraocular vessels. Blood flows normally through these vessels (including the capillaries and veins) when the hydrostatic pressure within the vessels exceeds the intraocular pressure. If the drainage of the aqueous humor is impeded, the intraocular pressure increases because the layers of the eye do not allow the wall to expand. This increased pressure interferes with normal retinal nourishment and function, causing the retinal nerve fiber layer to atrophy (Fig. F24.1. 1). There are two major types of glaucoma: • Open-angle glaucoma is the most common type of

Carbonic anhydrase Inhibitors, which were used in the past to decrease the production of aqueous humor, have largely been replaced by prostaglandin analogs that have fewer systemic side effects. There are two main types of laser surgery to treat glaucoma. They facilitate drainage of aqueous humor from the iridocorneal angle. Laser trabaculoplasty utilizes a laser beam to induce focal scarring of the trabecular meshwork. This results in mechanical stretching of the surrounding untreated regions of the meshwork:, which facilitates drainage of the aqueous humor. Trabeculoplasty is often used in open-angle glaucoma when medications are not effective or cause intolerable side effects. lridotomy is used in patients with angle-closure glaucoma. The laser beam incises a small opening at the base of the iris, which widens the iridocorneal angle to allow better drainage of aqueous humor.

glaucoma and the leading cause of blindness among adults. The removal of aqueous humor is obstructed because of reduced flow through the trabecular meshwork: of the iridocorneal angle into the scleral venous sinus (canal of Schlemm).

• Angle-closure glaucoma (acute glaucoma} is less common and is characterized by a narrowed iridocorneal angle that obstructs inflow of the aqueous humor into the scleral venous sinus. Usually it is associated with a sudden, painful, complete blockage of the scleral venous sinus and can result in permanent blindness if not treated promptly. Visual deficits associated with glaucoma include blurring of vision and impaired dark: adaptation (symptoms that indicate loss of normal retinal function) and halos around lights (a symptom indicating corneal endothelial damage). If the condition is not treated, the retina will be permanently damaged, and blindness will occur. Treatment is directed toward lowering the intraocular pressure by decreasing the rate of production of aqueous humor or eliminating the cause of the obstruction of normal drainage. Topical prostaglandin analogs (i.e., latanoprost, bimatoprost, travoprost) are the first line of treatment of open-angle glaucoma. They are very effective in reducing intraocular pressure by increasing the drainage of aqueous humor into the canal of Schlemm.

The inner cdl layer of the ciliary epithelium has a basal lamina facing the posterior and vitreous chambers. The cells in this layer are nonpigmented. The cell layer that has its basal lamina facing the connective tissue stroma of the ciliary body is hcavUy pigmented and is dircctl.y continuous with the pigmented epithelial layer of the retina. The double-layered ciliary epithelium continues over the iris, where it becomes the posterior pigmented epithelium and anterior pigmented myoepithelium. The zonular fibers extend from the basal lamina of the nonpigmented epithelial cells of the ciliary processes and insert into the leas capsule (the thickened basal lamina of the lens).

FIGURE F24.1.1. Glaucoma. This image shovvs a view of the fundus of the left fY'f9 in a patient with advanced glaucoma. As aresult of the increased intraocular pressure, retinal nerve fibers undergo atrophy and shrink in size. Note a pale optic disc in the center of the image with a less pronounced rim due to atrophy of nerve fibers. Enlargement of the optic nerve cup (central area of the optic disci is also visible and a characteristic finding for glaucoma. Compare this image to a normal retina in Fig. 24.15. (Courtesy of Dr. Renzo A. Zaldivar.)

The blood-aqueous barrier separates the interior environment of the aye from the blood entering the ciliary body. The cells of the nonpigmented layer have all the characteristics ofa fluid-transporting epithelium, including complex cell-to-cell junctions with a well-developed zonula occludens, extensive lateral and basal plications, and localization ofNa+I K+ -ATPase in the lateral plasma membrane. In addition, they have an elaborate rER and Golgi complex consistent with their role in secretion ofzonular fibers. Tight junctions (zonulae occludentcs) between the nonpigmented ciliary epithelial cells are responsible for maintaining the blood-aqueous barrier. This barrier restricts free diffusion across the ciliary

epithelium to maintain the unique environment of the aqueous humor, which is quite different from that of blood vessels and stroma of the ciliary body. The blood-aqueow barrier contributes to the nutrition and function of the cornea and the lens. Disruption of the blood-aqueous barrier may be observed in ocular inflammation, intraocular surgery, trauma, or vascular diseases. The aqueous humor becomes cloudy due to leakage of plasma proteins (fibrinogen) and migration of inflammatory cells from the stroma of the ciliary body and iris into the posterior and anterior chambers of the eye.

The cells of the pigmented layer have a less d.cvc:loped junctional zone and often exhibit large, irregular lateral intercellular spaces. Both desmosomes and gap junctions hold together the apical surfaces of the two cell layers, creating discontinuous "luminal" spaces called ciliary channels.

The aqueous humor is derived from plasma and maintains intraocular pressure. The aqueous humor is secreted by the double-layered ciliary epithelium and originates from blood capillaries. It is simUar in ionic composition to plasma but contains less than 0.1% protein (compared to 7% protein in plasma). The main functions of the aqueow humor arc to maintain intraocular pressure and to provide nutrients and remove metabolites from the avascular tissues of the cornea and lens. The aqueow humor passes &om the ciliary body toward the lens, and then between the iris and lens, before

it reaches the anterior chamber of the eye (sec Fig. 24.6). In the anterior chamber of the eye, the aqueous humor passes laterally to the angle formed between the cornea and iris. Here, it penetrates the tissues of the limbw as it enters the labyrinthine spaces of the limbus's trabecular meshwork in the iridocorneal angle and finally reaches the canal of Schlemm, which communicates with the veins of the sclera (see Folder 24.1). Nonnal turnover of the aqueous humor in the human eye is approximately once every 1.5 to 2 hours.

955

The choroid is the portion of the vascular coat that lias deep to the retina. The choroid is a dark brown vascular sheet only 0.25 mm thick posteriorly and 0.1 mm thick anteriorly. It lies between the sclera and retina (sec Fig. 24.1). Two layers can be identified in the choroid:

• Choriocapillary layer, an inner vascular layer • Bruch's membrane, a thin, amorphous hyaline membrane

s: (")

:D

0

(/)

(")

0

];!

The choroid is attached firmly to the sclera at the margin of the optic nerve. A potential space, the perichoroidal space (between the sclera and retina), is traversed by thin, ribbon-like branching lamellae or strands that pass &om the sclera to the choroid. These lamellae originate &om the suprachoroid lamina (lamina fusca) and consist of large, flat melanocytes scattered between connective tissue elements, including collagen and elastic fibers, fibroblasts,

(")

~

:D

c ~ c

:D

m 0

'T1

-1

I

m m -< m

A potential space exists in the retina as a vestige of the space between the apical surfaces of the two epithelial layers of the optic cup. If this space expands, the neural retina separates from the retinal pigment epithelium (APE), which remains attached to the choroid layer. This condition is called retinal detachment. As a result of retinal detachment, the photoreceptor cells are no longer supplied by nutrients from the underlying vessels in the choriocapillary plexus of the choroid. Clinical symptoms of retinal detachment include visual sensations commonly described as a ushower of pepper" or floaters. These are caused by red blood cells extravasated from the capillary vessels that have been injured during the retinal tear or detachment. In addition, some individuals describe sudden flashes of light as well as a uwebn or uveilu in front of the eye in conjunction with the onset of floaters. A detached retina can be observed and diagnosed during ophthalmoscopic eye examination (Fig. F24.2.1). Another common retinal condition occurs with aging. As tha vitreous body ages (in the sixth and seventh decades of life), it tends to shrink and pull 8Wf!o( from the neural retina, which causes single or multiple tears in the neural retina. If not repositioned quickly, the detached area of the retina will undergo necrosis, resulting in blindness. An argon laser is often used to repair retinal detachment by photocoagulating the edges of the detachment and

producing scar tissue. This method prevents the retina from further detachment and facilitates the repositioning of photoreceptor calls.

FIGURE F24.2.1. R811nal datechment. This image shows a view of the fundus of the right f1V9 in a patient with retinal detachment. The central retinal vessels emerging from the optic disc are in focus, but in the Bf98 of the rstinal detachment they appear to be out of focus. Because the area of retinal detachment is elevated (note multiple ridges and shadowsl, it is located anterior to the plane of focus of the ophthalmoscope. (Courtesy of Dr. Renzo A. Zaldivar.)

956

LLJ

~

LLJ

:I: ILl..

0

LLJ

c:

::J

tl

::J

c:

1-CIJ

u

a:

0

u (/.) 0

0::

u

~ LLI

~

•a: N

w

t:

C'(

::c

Age-related macular degeneration (ARMD) is the most common cause of blindness in older individuals. Although the cause of this disease is still unknown, evidence suggests both genetic and environmental (UV irradiation, drugs) components. The disease causes loss of central vision, although peripheral vision remains unaffected. Two forms of ARMD are recognized: a dry (atrophic, nonexudative) form and a wet (exudative, neevascular) form. The latter is considered a complication of the first. Dry ARMD is the most common form 190% of all cases) and involves degenerative lesions localized in the area of the macula lutea. The degenerative lesions include drusen, which are focal thickenings of Bruch's membrane; atrophy; depigmentation of the APE; and obliteration of capillaries in the underlying choroid layer. These changes lead to deterioration of the overlying photosensitive retina, resulting in the formation of blind spots in the visual field IFig. F24.3.1J. WetARMD is a complication of dry ARMD caused by neovascularization of blind spots of the retina in the large drusen. These newly formed, thin, fragile vassals frequently leak and produce exudates and hemorrhages in the space just beneath the retina, resulting in fibrosis and scarring. These changes are responsible for the progressive loss of central vision over a short time. The treatment of wet ARMD includes conventional laser photocoagulation therapy and pharmacologic therapy with intravitreal injection of ranibizumab, a vascular endothelial growth factor

(VEGF) Inhibitor. Other surgical methods, such as macular translocation, have been recently introduced. In this procedure, the retina is detached, translocated, and reattached in a new location, away from the choroid neovascular tissue. Conventional laser treatment is then applied to destroy pathologic vessels without destroying central vision.

FIGURE F24.3.1. PhotGgreph depleting the vl1ual field In Individual• with age-related macular daganeretlon. Note that

central vision is absent because of the changes in the macula region of the retina. To maximize their remaining vision, individuals with this condition are instructed to use eccentric fixation of their eyes.

u

macrophages, lymphocytes, plasma cells, and mast cells. The lamellae pass inward to surroU.tld the vessels in the remainder of the choroid layer. Free smooth muscle cc:lls, not associated with blood vessels, are present in this tissue. Lymphatic channels called epichoroid lymph spaces, long and shon posterior ciliary vessds, and nerves on their way to the front of the eye are also present in the suprachoroid lamina. Most of the blood vessels decrease in size as they approach the retina. The largest vessels continue forward beyond the ora serrata into the ciliary body. These vessels can be seen with an ophthalmoscope. The large vessels are mostly veins that course in whorls before passing obliquely through the sclera as vortex veins. The inner layer of vessels, arranged in a single plane, is called the choriocapillary layer. The vessels of this layer provide nutrients to the cells of the retina. The fenestrated capillaries have lumina that an: large and irregular in shape. In the region of the fovea, the choriocapillary layer is thicker, and the capillary network is denser. This layer ends at the ora serrata. Bruch's membrane, also called the lamina vitrea, measures 1 to 4 tJ.m in thickness and lies between the choriocapillary layer and the pigment epithelium of the retina. It runs from the optic nerve to the ora serrata, where it undergoes modifications before continuing into the ciliary body. Bruch's membrane is a thin, amorphous refractile layer. The transmission electron microscope (TEM) reveals that it consists of a multtlaminar sheet containing a center layer of

elastic and collagen fibers. Five different layers are identified in Bruch's membrane: • The basal lamina of the endothelial cells of the choriocapillarylayt:r • A layer of collagen fibers approximately 0.5 tJ.m thick • A layer of dastic fibers approximatdy 2 tJ.m thick • A second layer ofcollagen fibers (thus forming a "'sandwich" around the intervening elastic tissue layer) • The basal lamina of the retinal pigment epithdial cells At the om scrrata. the collagenous and elastic layen disappear into the ciliaty sttoma, and Bruch's membrane becomes continuous with the basal lamina of the RPE of the ciliary body.

Retina Tha ratina rapresants tha innannast layar of tha aya. The retina, derived from the inner and outer layers of the optic cup, is the innermost of the three concentric layers of the eye (see Fig. 24.1). It consists of two basic layers: • The neural retina or retina proper is the inner layer that contains the photoreceptor cells. • The retinal pigment epithelium (RPE) is the outer layer that rests on and is firmly attached through the Bruch's membrane to the choriocapillary layer of the choroid. A potential space exists between the two layers of the retina. The two layers may be separated mechanically in

the preparation of histologic specimens. Separation of the layers, "'retinal detachment" (see Folder 24.2), also occurs in the living state because of eye disease or trauma. In the neural retina, two regions or portions that diffi::r in fimction are recognized: • The nonphotoaensitive region (nonvisual part), located anterior to the ora serrata, lines the inner aspect of the ciliary body and the posterior surface of the iris (this portion ofthe retina is described in the sections on the iris and ciliary body). • The photosensitive region (optic pan) lines the inner surface of the eye posterior to the ora serrata except where it is pierced by the optic nerve (see Fig. 24.1). The site where the optic nerve joins the retina is called the optic disc or optic papilla. Because the optic d.isc is devoid of photoreceptor cells, it is a blind spot in the visual field. The fovea centralis is a shallow depression located about 2.5 nun late.ral to the optic d.isc. It is the area of greatest visual acuity. The visual axis of the eye passes through the fovea. A yellowpigmented zone called the macula lutea surrounds the fovea. In relative terms, the fovea is the region ofthe retina that contains

the highest concentration and most precisely ordered ~ ment of visual elements. The region of the retina surrounding the macula lutea may be affected in older individuals by age-related macular degeneration (see Folder 24.3).

957

Layers of the Retina Tan layers of calls and their pracassas constitute the retina. Before discussing the ten layers of the retina, it is importp ant to identify the types of cells found there. This identiDcap tion will aid in understanding the fimctional relationships of the ceUs. Studies of the retina in primates have identified at least 15 types of neurons that form at least 38 different types of synapses. For convenience, neurons and supporting cells can be classified into four groups of cells {Fig. 24.9): • Photoreceptor cells-the retinal rods and cones • Conducting neurons-bipolar neurons and ganglion cells • Association neurons and others-horizontal. centrifugal, interplexiform. and amacrine neurons • Supporting (neuroglial) cells-Muller's cells. microglial cells, and astrocytes

3::

n

~

U>

n

0

iJ

n

~ :JJ

c incident

~:JJ 0,

m

light

innerlimiting ~ /

membrane

-

·········-······················layer of optic

'\.

nerve fibers

~

m

~

m

Inner nuclear

layer

outer plexiform layer outer nuclear layer

MAJOR CELLS OF RETINA

FIGURE 24.9. Schematic drawing and photomicrograph of thelaye,. of the Ntlne. On the basis of histologic features that are evident in the photomicrograph on right, the retina can be divided into ten layers. The layers correspond to the diagram on left. which shows the distribution of major cells of the retina. Note that light enters the retina and passes through its inner layers before reaching the photoreceptors of the rods and cones that are closely associated with the retinal pigment epithelium. Also, the interrelationship between the bipolar neurons and ganglion cells that carry electrical impulses from the retina to the brain is clearly visible. Bruch's membrane (lamina vitrea} separates the inner layer of the vascular coat (choroid) from the retinal pigment epithelium. X440.

958

w

~

w

::c

The specific arrangement and associations of the nuclei and processes of these cells form ten retinal layers that can be seen with the light microscope. The layers of the retina can also be imaged and examined in living individuals using spectral domain optical coherence tomography {see Folder 24.4}. The ten layers of the retina, from outside inward, are as follows (see Fig. 24.9): 1.

~

LL.

0

w

2.

=> ~ => a:

3.

a:

t;

4.

0 a..

5.

0

u (f) 0

a: u ~

6. 7. 8.

9. 10.

eosin (H&E) preparation. Because the lipofuscin pigment is fluorescent, it can be clearly seen in the ultraviolet fluorescent microscope. A supranuclear Golgi apparatus and an extensive network of smooth·surfaced endoplasmic reticulum (sER) sur· round the melanin granules and residual bodies that are present in the cytoplasm. The RPE serves several important functions, including the following: Retinal pigment epithelium (RPE)-the outer layer of the retina. actually not part of the neural retina but • It absorbs light passing through the neural retina to prevent reflection and resultant glare. intimately associated with it • It isolates the retinal cells from blood·borne substances. Layer of rods and cones-contains the outer and It serves as a major component of the blood-retina inner segments of photo.teceptor cells barrier via tight junctions between RPE cells. Outer limiting membrane-the apical boundary of • It participates in restoring photosensitivity to visual Muller's cells pigments that were dissociated in response to light. The Outer nuclear layer-contains the cell bodies (nuclei) metabolic apparatus for visu.al pigment resynthesis is of retinal rods and cones present in the RPE cells. Outer plexiform layer-contains the processes of .tetinal rods and cones and processes of the horizontal, • It phagocytoses and diapo~es of membranous discs from the rods and cones ofthe retinal photoreceptor cells. amacrine, and bipolar cells that connect to them Inner nuclear layer-contains the cell bodies (nuclei) The rods and cones of the photoreceptor call (layer 2) ofhorizontal, amacrine, bipolar, and Miiller's cells extend from the outer layer of the neural ratina to the Inner plexiform layer-contains the procenes ofhori· pigment epithelium. zontal, amacrine, bipolar, and ganglion cells that connect The rods and cones are the outer segments of photoreceptor to each other cells whose nuclei form the outer nuclear layer of the retina Ganglion cell layer-contains the cell bodies (nuclei) (Figs. 24.9 and 24.1 0). The light that reaches the photoreceptor of ganglion cells Layer of optic nerve fibers-contains processes of ganglion cells that lead from the retina to the brain Inner limiting membrane-composed of the basal lamina of Miiller's cells

Each of the layers is more fully described in the following sections (see corresponding numbers). The cells of the retinal pigment epithelium (layer 1) have extensions that surround the processes of the rods and cones. The RPE is a single layer of cuboidal cells about 14 JJ.m wide and 10 to 14 JJ.m r:all. The cells rest on Bruch's membrane of the choroid layer. The pigment cells are tlillest in the fovea and adjacent regions, which account for the darker color of this region. Adjacent RPE cells are connected by a jwtctional complex consisting ofgap junctions and elaborate zonulae occludentes and adherentes. This junctional compla is the site of the blood-retina barrier. This barrier malces the retinal vessels impermeable to molecules larger than 20 to 30 kDa. The pigment cells have cylindrical sheaths on their apical surface that are associated with, but do not directly cont:act, the tip of the photoreceptor processes of the adjacent rod and cone cells. Compla cytoplasmic processes project for a short distance between the photoreceptor cells of the rods and cones. Nwnerous dongated melanin granules, unlike those found elsewhere in the eye, are present in many of these processes. They aggregate on the side ofthe cell nearest the rods and cones and are the most prominent fearure of the cells. The nucleus with its many convoluted infoldings is located near the basal plasma membrane adjacent to Bruch's membrane. The cells also contain material phagocytosed from the p~ cesses of the photoreceptor cells in the form of lamellar debris (lipofuscin) contained in residual bodies or phagosomes. These lipofuscin granules reside in the basal cytoplasm ofthe RPE cell and are relatively difficult to detect in routine hematoxylin and

Golgi-apparatus mitochondria

\

I

retinal pigment epithelial cells FIGURE 24.10. Schematic diagram of the ultranucture of rod and cone cell•. The outer segments of the rods and cones are closely associated with the adjacent pigment epithelium.

959

3::

n

180

'EE

160 140

!' •'Cii'

120

......

~

U>

n

0

""'0

n

i~ 100

.. w

1.5

~~

I

i.

~ :JJ

0

Vca

c

a.

80

~:JJ 0,

Cll

'C

.5

60 40

:a

20 0 _ _ _Cones _ _ _. . 7(1' 6(1'

Cones 1(1'

20° 30° 40" 50" 60" 70" 80"

center cit fovea

nasal part of retina

distance from visual axis (degraes) FIGURE 24..11. Dlmtbu'don of rods and cones In die human eye. This graph shows the density of rods and cones per mm 2 across the retina. The peak number of cones occurs in the fovea centralis. where it reaches approximately 150,000 cones}mm2 • Rod density peaks about 20° from the visual axis and is roughly the same as that of cones. Rods density decreases toward the periphery of the retina. Note that there are no photoreceptors at the optic disc.

cells must first pass through all of the internal layers of the neural retina. The rods and cones are arranged in a palisade manner; therefore, in the light microscope, they appear as vertical striations. The retina contains approximately 120 million rods and 7 million cones. They are not disuibuted equally throughout the photosensitive part of the retina. The highest density of cones is detected in the fovea centralis, which corresponds to the highest visual acuity and best color vision (Fig. 24.11). The highest density of rods is outside the fovea cenualis, and their density steadily decreases toward the periphery of the retina. RDd.s are not present in the fovea cenualis nor at the optic disc, which is devoid of any photoreceptors (see Fig. 24.11). The rods are about 2 1J..tn thick and 50 ~m long (ranging from about 60 ~m at the fovea to 40 IJ.m peripherally). The cones v;u:y in length from 85 IJ.m at the fovea to 25 .,._m at the periphery of the retina.

Rods are sensitive in low light and produce black-andwhite images; cones are less sensitive in low light and produce color images. Functionally, the rods are more sensitive to light and are the receptors used during periods of low Ught intensity (e.g., at dusk or at night). The rod pigments have a maximum

~

m

5(1' 40" 30" 20" 10° 0"

temporal part of retina

m

absorption at 496 nm of visual specmun, and the image provided is one composed of gray tones (a "black-and-white picture"). In contrast, the cones exist in three classes: L, M, and S (long-, middle-, and short-wavelength sensitive, respectively) that cannot be distinguished morphologically. They are less sensitive to low light but more sensitive to red, green, and blue regions of the visual spectrum. Each class of cones contains a different visual pigment molecule that is activated by the absorption of llght at the blue (420 nm), green (531 nm), and red (588 nm) ranges in the color spectrum. Cones provide a visual image composed of color by mixing the appropriate proportion of red, green, and blue light. For a description of different types of color blindness, see Folder 24.5. Each rod and cone photoreceptor consists of three parts: • The outer segment of the photoreceptor is toughly cylindrical or conical (hence, the descriptive name rod or cone). This portion of the photoreceptor is intimately related to microvilli projecting from the adjacent pigment epithelial cells. • The connecting stalk contains a cilium composed of nine peripheral microrubule doublets extending from a basal body. The connecting stalk appears as the constricted

~

m

960

w

~

w

::c ~

LL.

0

w

a:

=> ~ => a:

t;

0 a..

0

u (f) 0

a: u ~

FIGURE 24.12. Electron micrograph• of portion• of the Inner and outer eegmente of conM and rode. a. This electron micrograph shows the junction between the inner and outer segments of the rod cell. The outer segments contain the horizontally flattened discs. The plane of this section passes through the connecting stalk and cilium. A centriole, a cilium and its basal body, and a calyceal process are identified. x32,000. b. Another electron micrograph shows a similar section of a cone cell. The interior of the discs in the outer segment of the cone is continuous wi1h the extracellular space (arrows). X32,000. (Courtesy of Dr. Toichiro Kuwabara.l

region of the cell that joins the inner to the outer segment. In this region, a thin, tapering process called the calyceal process extends from the distal end of the inner segment to surround the proximal portion of the outer segment (see Fig. 24.10). • The inner segment is divided into an outer ellipsoid and an inner myoid portion. This segment contains a typical complement of organelles associated with a cell that actively synthesize proteins. A prominent Golgi ap· paratus, rER. and free ribosomes are concent.tated in the myoid region. Mitochondria are most numerous in the dlipsoid region. Mi.crotubules are distributed throughout the inner segment. In the outer ellipsoid portion, cross-striated fibrous rootlets may extend from the basal body among the mitochondria.

The outer segment is the site of photosensitivity, and the inner segment contains the metabolic machinery that supports the activity of the photoreceptor cells. The outer segment is considered a highly modified cilium because it is joined to the inner segment by a short connecting stalk containing a basal body (Fig. 24.12a). With the TEM, 600 to 1,000 regularly spaced horizon· tal membranous discs an:: seen in the outer segment (Fig. 24.12). In rods, these discs are membrane-bounded structures measuring about 2 J1.m. in diameter. They are en· closed within the plasma membrane of the outer segment (see Fig. 24.12a). The parallel membranes of the discs are about 6 nm thick and are continuous at their ends. The cen· tral enclosed space is about 8 nm across. In both rods and cones, the membranous discs are formed from repetitive

961 The standard ophthalmoscopic examination of the eye has been recently supplemented by a new examination technique that utilizes spectral domain optical coherence tomography (SD ocn. This noninvasive and noncontact examination is not only useful in visualizing the retinal surface but it also provides a high-resolution cross-sectional image of the retina in vivo. All histologic layers of the retina can be easily differentiated with SD OCT (Fig. F24.4.1). and they can be objectively measured for tissue thickness and change. SD OCT technology is based on comparisons of spectral characteristics of the reflected light beam from the retina with those of the reference beam. For this purpose, an infrared laser beam (approximately 840 nm wavelength with 50 nm bandwidth) is used that is able to produce images at 5-J.Lm resolution. The laser beam passes through the structures of the eye and is partially absorbed and partially reflected depending on tissue

characteristics. The reflected light is detected by a multichannel spectrometer, and the interference pattern is compared to the reference beam using complex computer algorithms. The spectral differences are used to construct the cross-sectional (line) scans as shown in Figure F24.4.1 or the three-dimensional images of the retina as shown in Figure F24.4.2. Introduced in the 1990s, the SD OCT has revolutionized the management and diagnosis of many eye diseases. SD OCT established itself as an imaging modality of choice in glaucoma (measurement of optic nerve and retinal nerve fiber layer) and retinal diseases. It is used for the early and accurate detection of macular

degeneration, retinal detachment, macular holea, epiretinal membranes, and optic disc pits and for detection of fluid accumulation within the retina that occurs in conditions such as diabetic retinopathy, cystoid macular edema, and central serous choroidopathy.

s:

n

:D

0

(J)

n 0

];!

n ~

:D

c ~ c

::c

m 0

"T1

-1

I

m

~

m

r------------------,I

I I I

I I

I

I

---vitreous body·---· --Inner llmldng membrane-.......___

-.._laYIIr of optic nerve fibers-

~oulllr plexifonn layer-

.__--outer nuclear layer-- -outer limiting membrane__......._ _ rods and~

c::::::_retinal pigmlllllepilhelium~ ~lamina vhrea/

----choroid----:

FIGURE F.24.4.1. Spectl"81 Domain Optical Coherence 1bmogl"8phy (SD OCTI cron-Hctlonal (llnel Image of the retina In a hHithy eve. The upper image represents a normal cross-sectional image of the retina containing fovea and optic disc on the right side

of the image. The optically transparent vitreous body is invisible and appears as the black: region in the upper part of the image. Hyperand hyporeflective bands of retinal tissue correspond to the histologic layers of the retina. Note the photoreceptor layer containing rods and cones as well the retinal pigment epithelium are well defined and are separated from the choroid layer containing blood vessels. {Courtesy of Drs. Andrew J. Barlcmeier and Denise M. Lewison). (continues on page 962)

962

LLJ

~

LLJ

:I: ILl..

0

LLJ

c:::

::J

tl

::J

c:::

1-CIJ

u

a:

0

u

CIJ

0

0::

u

~ LLI

~

•a: N

w

t:

FIGURE F24.4.2. Spectral Domain Optical Coher.nce Tomography (SD OCTI three-dimensional Image of the nrllna of a healthy right eye. The scan area is approximately 12 x 9 mm in size and includes a portion of the optic disc (on /eM and fovea (on righO. A three-dimensional dataset is acquired from four scans (two vertical and two horizontal), which is then processed with a motioncorrection technology (MCTI algorithm. The MCT algorithm analyzes and compares the vascular pattern in each of the scans and reduces artifacts and image distortions associated with f1V9 movement. This image has two parts. The upper false-rolor image (optical densities are coded in different colors) shows the surface and thickness of all layers of the retina and represents a motion-corrected, threedimensional volume rendering of the entire data set. The lower grayscale vascular map image (optical densities are coded in gray scale} is a two-dimensional image created by summing all the pixels in each column. It is curved to match the curvature of the eye. The letters S (for superior) andT (for temporal) on the eye orientation icon in the lower right corner provide reference to the positioning of the scan in the patient's eye. (Image courtesy of Pravin Dugal, MD, Phoenix. Arizona.).

C'(

::c

u

transverse infolding of the plasma membrane in the region of ofcones are photopsins. 1he chromophore of rods is a vitathe outer segment near the cilium. Auto.radiographic studies min A--derived carotenoid called retinal. Thus, an adequate have demonstrated that rods form new discs by infolding of intake of vitamin A is essential for normal vision. Prothe plasma membrane throughout their life span. Discs are longed dietary deficiency of vitaminA leads to the inability funned in cones in a similar manner but are not replaced on to see in dim light (night blindness). a regular bam. The interior of the discs of cones is continuous with the Rod discs lose their continuity with the plasma membrane extracellular space. &om which they are derived soon afttt they are formed. They then pass like a stack of plates, proximally to distally, along 1he basic difference in the structure: of the rod and cone the length of the cylindrical portion of the outer segment discs-that is, continuity with the plasma membrane-is until they are eventually shed and phagocytosed by the pig- correlated with the sllghdy different means by which the ment epithdial cells. Thus, each rod disc is a membrane- visual pigments are renewed in rods and cones. Newly synenclosed compartment within the cytoplasm. Discs within thesized rhodopsin is incorporated into the: membrane: of the: the cones retain their continuity with the plasma membrane rod disc as the disc is being formed at the base of the outer segment. It then takes several days for the disc to teach the (Fig. 24.12b). tip of the: outer segment. In contrast, although visual proteins Rod calls contain the visual pigment rhodopsin; cone calls are constandy produced in retinal cones, the proteins are contain tha visual pigmant iodopsin. incorporated into cone discs located anywhere in the outer Rhodopsin (also called visual purple) is a 39 kDa pro- segment. tein in rod cells that initiates the visual stimulus when it is Vision is a procau by which light striking 1ha retina is bleached by light. Rhodopsin is present in globular form on converted into alactrical impulsas that are transmitted 1a the outer surface of the lipid bilayer (on the cytoplasmic side) the brain. of the membranous discs. In the: cone: cells, the visual pigment protein on the: membranous discs is the photopigment 1he impulses produced by light reaching the photoreceptor cells Iodopsin. Each cone cell is specialized to respond maximally are conveyed to the brain by an elaborate: network of nerves. The to one: of three: colors: red, green, or blue. Both rhodopsin conversion of the incident light into electrical nerve impulses is and iodopsin contain a membrane-bound subunit called an called visual processing and involves several steps: opsin and a second small light-absorbing component called a chromophora. The: opsin of rods is scotopsin; the opsins

• A photochemical reaction occurs in the outer segment of the: rods and cones. In the: dark. rhodop1in molecules

contain a chromophore called retinal in its isometric form of 11-cis-retinal. When rods are exposed to light, the 11-ds-retinal 1mdergoes conformational change from a bent to a more linear molecule called all-transofetinal. The conversion of 11-ds-retinal to all-mm.r-retinal activates opsin, which results in the release of all-tran.r--retinal into the rod's cytoplasm (a reaction called bleaching). • The activated opsin interacts with a G-protein called transducin, which subsequendy activates phosphodiesterase that breaks down cyclic guanosine mono· phosphate (cGMP). In the dark, high levels of cGMP molecules produced in the photoreceptor cells by guanylyl cyclase are bound to the cytoplasmic surface of cGMP-gated Na+ channels, causing them to stay open. Steady influx of Na+ into the cells results in depolarization of the plasma membrane and continuous release of the neurotransmitter (glutamate) at the synaptic j1mction with the bipolar neurons (Fig. 24.13). • A decrease in the concentration of cGMP within the cytoplasm of the inner segment of the photoreceptor ceUs is due to the action of phosphodiesterase. Dissociation

of cGMP from Na+ channels effectively closes the channels and reduces influx of Na+ into the cell, resulting in hyperpolarization of the plasma membrane. The hyperpolarization causes a decrease of glutamate secretion at the synapses with bipolar cells, which is detected and conveyed as electrical impulses (see Fig. 24.13).

963

Released retinal from opsin is converted back to its origi· nal conformation in the RPE cells and MUller's cells. After release, all-tram-retinal is converted to all-tram-retinol in the cytoplasm of rods and cones and then transported to the cytoplasm of RPE cells (from rods) or both RPE ceHs and Mutter's ceHs (from cones). The energy for this process is provided by the mitochondria located in the inner segment of these photoreceptors. Both Muller's ce1ts and RPE cells participate in a multistep conversion of all-mtns-retinol to 11-ds-retinal, which is transported back to the photoreceptor celts for resynthesis of rhodopsin. The retinal pigment epithelium-specific protein 65 kDa (RPE85) is involved in this conversion; thus, the visual cycle can. begin again.

continuous release of neurotransmitter in synapses with bipolar cells

3::

n

~

(/)

(')

0

-o

n

(/)

-1 ::0

c(')

decrease of neurotransmitter release proportional to amount of light

-1

c

::0

m

glutamate

0

II

-1

:::c:

m m -< m

depolarization of plasma

hyperpolarization of plasma

membrane (-40 mV)

membrane (-70 mV)

'tcGMP

J.cGMP

light

Na+ channels cloeed • • ~MP-.._.

......

,...._

•

,(" "

activated :_----,.__ (bleached) opsin '

r

I

phosphodiesterase

-· --- ~

•.

00+ ~Ko

~0 Np.+ oO 00

oo

~

L,~~j

retinal pigment epithelium (RPE) cell

all·flans-

a

retinal

conwrslon

11-a.

retinal

b

RGURE 24.1 3. Schematic dlqram of vllual pro telling In 1he photoreceptor cell. e. In the dark, high levels of cGM P generated by guanytvl cyclase are present in the cytoplasm of the rod. Some of the cGMP molecules are bound to the cytoplasmic surface of cGMP-gat&d Na+ mannels, causing them to stay open and resulting in continuous influx of Na+ and depolarization of the plasma membrane. This results in a steady release of glutamate, a neurotransmitter, in the synaptic junctions with bipolar neurons. Also in the dark, rhodopsin molecules that contain 11·cisretinal are inactive. b. After exposure to light, 11-ci9-fetinal undergoes conformational mange to all-t18rnHetinal. This conversion activates opsin {a reaction called bleaching) and releases all-tr.:m~retinal into the rod's cytoplasm. The activated opsin interacts with G-protein, which subsequently activates phosphodiesterase that breaks dCMin cGMP. effectively lowering the concentration of cGMP in the cell. In this condition, cGMP molecules dissociate from Na+ thannels, resulting in their closing and hyperpolarization of the plasma membrane. This results in a decrease of glutamate secretion, whid1 is detected by the bipolar neurons and conveyed as electrical impulses to the brain. The released retinal from opsin is converted to its Ol'iginal conformation in retinal pigment epithelial IRPE) cells by the RPE65 enzymatic complex and is recycled to the photoreceptor cell. cGMP. cyclic guanosine monophosphate; GDP. guanosine diphosphate; GMP, guanosine monophosphate; GTP, guanosine triphosphate; RPE, retinal pigment epithelium.

964

LU

~

LU

:I: ILL

0

LU

c:

::J

b

::J

c:

1-CIJ

u

a: 0

u en 0

0::

u

~

LLI

~

•a: N

LLI

t: C'(

::c u

In individuals with normal color vision, the three primary colors (red, green, and blue) are combined to achieve the full spectrum of color vision. These individuals are called trichromats and possess three independent channels for conveying color information that are derived from three different classes of cones (L-red-sensitive; M--green sensitive; and &-blue-sensitive). Approximately 90% of trichromats can apperceive any given color from impulses generated in all three classes of cones. Some individuals have an impairment of normal color vision, which occurs when one of the cones is altered in its spectral sensitivity. For example. about 6% of trichromats matches colors with an unusual proportion of red and green. These individuals are called anomalous trichromats. Color blindness is a condition in which individuals are missing or have a defect in a specific class of cones. True color-blind individuals are dichromats and have a defect either in either the L, M, or S cones. In this condition, the affected cones are completely missing. Dichromats can only distinguish different colors by matching the impulses generated by the two remaining normal classes of cones. Three major types of color blindness have been identified:

blindness, affecting about 5% of the male population. It is also a sex-linked disorder because the genes encoding M cone photoreceptor proteins are located in the same region of the X chromosome as the genes for L cones. Similar to protanopia, red and green are the main problem colors (see Fig. F24.5.1). • Tritanopia is characterized as a defect affecting the short-wavelengthS cones responsible for blue vision (see Fig. F24.5.1 ). The defect is autosomal and involves mutation of a single gene encoding Scone photoreceptor proteins that reside on chromosome 7. This color blindness occurs very rarely (1 in 10,000) and affects women and men equally.

• Protanopia is characterized as a defect affecting the long-wavelength L cones responsible for red vision. The genes encoding L cone photoreceptor proteins are located on the X chromosome; therefore, protanopia is a sex-linked disorder affecting mainly males (1 % of the male population). These individuals have difficulty distinguishing between blue and green as well as red and green colors; thus, this color vision deficiency is a serious risk factor in driving (Fig. F24.5.1). • Deuteranopia is characterized as a defect affecting the middle-wavelength M cones responsible for green vision. Deuteranopia is the most common form of color

Pralanopla, color vision with loss of L cones (loss of red vision)

Narmal color vision wtth all three L, M, and Scones

Deulelwlopla, color vision with loss of M cones (loss of (1eel1 vision)

Trllllnapla, color vision with loss or S cones (loss of blue vision) FIGURE F24.&.1. Color bllndneu. This chart shows the si*

color spectrum in normal color vision and in individuals with the three types of color blindness.

Miiller's cells end at the base of the inner segments of the receptors, they mark the location of this layer. 1hus, the supporting processes ofMiiller's cells, on which the rods and cones rest, are pierced by the inner and outer segments of the photoreceptor cells. This layer is thought to be a metabolic barrier that restricts the passage oflarge molecules into the inner layers ofthe retina. The outer nuclear layer (4) contains the nuclei of the Discs are shed from both rods and cones. retinal rods and cones. In rods, after a period of sleep, a burst of disc shedding The tegion of the rod cytoplasm that contains the nucleus occurs as light :first enters the eye. The time of disc shedding is separamJ from the inner segment by a tapering process of in cones is more variable. The shedding of discs in cones also the cytoplasm. In cones, the nuclei are located close to the enables the receptors to eliminate superfluous membrane. outer segments, and no tapering is seen. The cone nuclei stain Although not fully understood, the shedding process in cones lighdy and are larger and more oval than rod nuclei. Rod also alters the size of the discs so that the conical form is main- nuclei are surrounded by only a thin rim of cytoplasm. In tained as discs are released from the distal end of the cone. contrast, a relatively thick investment ofcytoplasm surrounds The outer limiting membrane (layar 3) is farmed by a row of the cone nuclei (see Fig. 24.10). zanulaeadharentas between MOIIar"s calls. The outer plaxHom layer (5) is fonnad by the procaaas of 1he outer limiting membrane is not a true membrane. It the photoreceptor cells and naurons. is a row of zonulae adhercntes that attaches the apical ends of The outer plexifonn layer is formed by the processes of Miiller's cells (i.e., the end that faces the pigment epithelium) retinal rods and cones and the processes of horizontal, interto each other and to the rods and cones (see Fig. 24.9). Because plexiform, amacrine, and bipolar cells. The processes allow During normal functioning of the photoreceptor cells, the membranous d.iscs of the outer segment are shed and phagocytosed by the pigment epithelial cells (Fig. 24.14). It is estimated that each of these cells is capable of phagocytosing and disposing of about 7,500 discs per day. 1he discs are con~ standy turning over, and the production of new discs must equal the rate of disc shedding.

965

3::

n

~

U>

n

0

""'0

:~'D o

n

~-~-~

~

FIGURE 24.14. Electron micrograph of the rdnal pigment epithelium In 111oclatlon wtth the outer •eamenb of rodl and conet. Retinal pigment epithelial cells (RPE) contain numerous elongated melanin granules that are aggregated in the apical portion of the cell, where the microvilli extend from the surface toward the outer segments of the rod and cone cells. The retinal pigment epithelial cells contain numerous mitochondria and phsgosomes. The srraw indicates the location of the junctional complex between two adjacent cells. x20,000. (Courtesy of Dr. Toichiro Kuwabara.l

:JJ

c

~:JJ 0,

m

~

the electrical coupling of photoreceptor cells to these specialized interneurons via synapses. A thin process extends from the region of the nucleus of each rod or cone to an inner expanded portion with several lateral processes. The expanded portion is called a spherule in a rod and a pedicle in a cone. Normally, many photoreceptor cells converge onto one bipolar cell and form interconnecting neural networks. Cones located in the fovea, however, synapse with a single bipolar cell. The fovea is also unique in that the compactness of the inner neural layers of the retina causes the photoreceptor cells to be oriented obliquely. Horizontal cell dendritic processes synapse with photoreceptor cells throughout the retina and further contribute to the elaborate neuronal connections in this layer.

The inner nuclear layer(&) consista ofthe nuclei of horizontal, amacrine, bipolar. interplexifonn, and Miillefs cells. MUller's cells fonn the sca1folding for the entire retina. Their processes invest the other cells of the retina so completely that they fill most of the enracellular space. The basal and apical ends of MUller's cells form the inner and outer limiting membranes, respectively. Microvilli extending from their apical border lie between the photoreceptor cells of the rods and cones. Capillaries from the retinal vessels extend only to this layer. The rods and cones carry out their metabolic exchanges with extracellular fluids transported across the blood-retina barrier of the RPE. The four types of conducting cells-bipolar, horizontal, interplexiform, and amacrine-found in this layer have distinct orientations (see Fig. 24.9): • Bipolar cells and their processes extend to both the inner and outer plexiform layer. In the peripheral regions

of the retina, the axons of bipolar cells pass to the inner plexiform layer where they synapse with several ganp glion cells. Through these connections, the bipolar cells establish communication with multiple cells in each layer except in the fovea, where they may synapse only with a single ganglion cell to provide greater visual acuity in this region. • Horizontal cells and their processes extend to the outer plexiform layer where they intermingle with processes of bipolar cells. The cells have synaptic connections with rod spherules, cone pedicles, and bipolar cells. This electrical coupling ofcells is thought to affect the functional threshold between rods and cones and bipolar cells. • Amacrine cells' processes pass inward, contributing to a complex interconnection of cells. Their processes branch extensively to provide sites of synaptic connections with axonal endings of bipolar cells and dendrites of ganglion cells. Besides bipolar and ganglion cells, the amacrine cells synapse in the inner plexiform layer with interple:x:ifonn and other amacrine cells (see Pig. 24.9). • lnterplexiform cells and their processes have synapses in both inner and outer plexiform layers. These cells convey impulses from the inner plexiform to the outer plexifonn layer.

The inner plaxifonn layer (7) consists of a complex array of intermingled neuronal call processes. The inner plexiform layer consists of synaptic connec· tions between axons of the bipolar neurons and dendrites of ganglion cells. It also contains synapses between inter· mingling processes of amacrine celts and bipolar neurons,

m

~

m

966

w

it w

::r::

ILl-

0

w

a:

::>

t>::> a:

t> (.)

a_

0

(.) (/)

0

a:

u

~

ganglion cells, and interplexiform neurons. The course of these processes is parallel to the inner limiting membrane, thus giving the appearance of horizontal striations to this layer (see Fig. 24.9).

The ganglion cell layer (8) consists of the cell bodies of large multipolar neurons. The cell bodies oftarge multipolar nerve cells, measuring as much as 30 ~m in diameter, constitute the ganglion cell layer. These nerve cells have lighdy staining round nuclei with prominent nucleoli and Nissl bodies in their cytoplasm. An axonal process emerges from the rounded cell body, passes into the nerve fiber layer, and then enters the optic nerve. The dendrites extend from the opposite end of the cell to ramify in the inner plexiform layer. In the peripheral regions of the retina, a single ganglion cell may synapse with one hundred bipolar cells. In marked contrast, in the macular region surrounding the fovea, the bipolar cells are smaller (some authors refer to them as midga bipolar cells), and they tend to make one-to-one connections with ganglion cells. Over most of the retina, the ganglion cells are only a single layer of cells. At the macula, however, they are piled as many as eight deep, although they are absent over the fovea itself. Scattered among the ganglion cells are small neuroglial cells with densely staining nuclei (see Fig. 24.9).

The layer of optic nerve fibers (9) contains axons of the ganglion cells. The axonal processes of the ganglion cells form a :flattened layer running pazallel to the retinal surface. This layer increases in depth as the axons converge at the optic disc (Fig. 24.15). The axons are thin, nonmyelinated processes measuring as much as 5 JJ.m in diameter (see Fig. 24.9). The retinal vessds, including the superficial capillary network, are primarily located in this layer.

The inner limiting membrana (layer 10) consists of a basal

lamina separating the retina from the vitreous body. The inner limiting membrane forms the innermost boundary of the retina. It serves as the basal lamina of MUller's cells (see Fig. 24.9). In younger individuals, reflections from the internal limiting membrana produce a retinal•heen that is seen during ophthalmoscopic examination of the aya. In older individuals, a samitranslucent sheet of cells and extracellular matrix can be formed on the inner surface of the retina in conjunction with the inner limiting membrane. This condition is called epiretinal membrane (ERM) or macular pucker and is responsible for variable clinical symptoms, including optical distortion and blurred vision. ERM is initially formed by calls from within the ratina (RPE cells, Muller's cells, and astrocyte&) that begin proliferating and migrating onto the surface oftha internal limiting membrana. Later, the membrane is infiltrated by macrophages, fibroblasts, and myofibroblasts. To prevent damage to the underlying retina, surgical removal of the ERM may be performed.

Specialized Regions of the Retina The fovea (fovea centralls) appears as a small (1.5 mm in diameter), shallow depression located at the posterior

RGURE 24.1 S. Normal vlaw of the fundus In ophthalmoscopic Q11minlltion of the right eye. The site where the axons converge to form the optic nerve is called the optic disc. Because the optic disc is devoid of photoreceptor cells, it is a blind spot in the visual field. From the center of the optic nerve {clinically called the optic centrsl retinal vessels emerge. The artery divides into upper and lower branches. each of which further divides into nasal and temporal branches (note the nasal and temporal directions on the image!. Veins have a similar pattem of tributaries. Approximately 17° or 2.5 times optic disc diameters lateral to the disc, the slightly oval-shaped. bloodvessel·free. and pigmented area represents the macula lutes. The foves centrslis, a shallow depression in the center of the mscufa lutes, is also visible. {Courtesy of Dr. Renzo A. Zaldivar.)

cum.

pole of the visual axis of the eye. Its central region, known as the foveola, is about 200 JA-M ia diameter (see Fig. 24.15). Except for the photoreceptor layer, most of the layers of the retina are marb:dly reduced or absent in this region (see Fig. 24.6). Here, the photoreceptor is composed entirely of cones (approximately 4,000) that are longer and more slender and rod-like than they are elsewhere. The fovea is the area of the retina specialized for discrimination of details and color vision. The ratio between cones and ganglion cells is dose to 1:1. Retinal vessels are absent in the fovea, allowing light to pass unobstructed into the cones' outer segments. The adjacent pigment epithelial cells and choriocapillaris are also thickened in this region. The macula lutea is the area surrounding the fovea and is approximately 5.5 mm in diameter. It is yellowish because of the presence of yellow pigment (xanthophyll). The mac· ula lutea contains approximately 17,000 cones and gains rods at its periphery. Retinal vessels are also absent in this region. Here, the retinal cells and their processes, especially the ganglion cells, are heaped up on the sides of the fovea so that light may pass unimpeded to this most sensitive area of the retina.

Vessels of the Retina The central retinal artery and central retinal vein, the vessels that can be seen and assessed with an ophthalmoscope, pass through the center of the optic nerve to enter the bulb of the eye at the optic disc (see Fig. 24.2 and pages 946-947, the section on the development of the eye). The central retinal artery provides nutrients to the inner retinal layers. The artery branches immediately into upper and lower branches, each of which divides again into nasal and temporal branches

(see Fig. 24.15). Veins undergo a similar pattern of branching. The vessels initially lie between the vitreous body and inner limiting membrane. As the vessels pass laterally, they also move deeper within the inner retinal layers. Branches from these vessels form a capillary plexus that reaches the inner nuclear layer and therefore provides nutrients to the inner retinal layers (layers 6 to 10; see pages 965-966). Nutrients to the remaining layers (layers 1 to 5) are provided by diffusion from the vascular choriocapillary layer of the choroid. The branches of the central retinal artery do not anastomose and therefore are classified as anatomic end arteries. Evaluation ofthe retinal vessels and appearance of the optic disc during ophthalmoscopy not only gives important information on the state of the eye but also may reveal early clinical signs of a number of conditions, including increased intracranial pressure, hypertension, glaucoma, and diabetes.

Crystalline Lens The lens is a transparent, biconvex structure that has no ves-sels or nerves and is almost totally devoid of connective tissue except for an enveloping capsule of basal lamina. It is suspended between the edges of the ciliary body by the zonular fibers. The pull of the zonular fibers keeps the lens in a Battened condition. Release of tension causes the lens to widen

or accommodate to bend light rays originating close to the eye so that they focus on the retina. The lens has three principal components (Fig. 24.16): • The lens capsule is a thick basal lamina that surrounds the outer surface of the lens. It originates as the basal lamina of the embryonic lens vesicle. The anterior part of the capsule is thick, measuring approximately 10 to 20 J.Lm, and is produced by the anterior lens cells. The posterior part of the capsule is much thinner, measuring approximately 5 to 10 J.Lm. The lens capsule, composed primarily of type IV collagen and proteoglycans, is elastic. It is thickest at the equator where the zonular fibers attach to it. • The subcapsular epithelium is derived from the epithelial cells of the anterior part of the embryonic lens vesicle. It represents a single cuboidal layer of lens epithelial cells present only on the anterior sur&ce of the lens. The epithelial c::ells ofthe posterior part ofthe vesicle elongate anteriorly and form the primary lens fibers that 6U the cavity of the optic vesicle. • Secondary lens fibers (lens fiber cells) are fonned at the periphery near the lens equator. Here, epithelial cells proliferate and migrate along the posterior lens capsule to differentiate into mature lens fiber cells. In the center of the lens, epithelial cells are quiescent. As lens

967

3::

n

~

U>

n

0

""'0

n

~ :JJ

c

~:JJ 0,

m

~

m

~

m

subcapsular--:--

epithelium

LIGHT ) PATH

I a FIGURE 24.11. Stru;tu,. of thelen1. a. This schematic drawing of the lens suspended from ciliary processes by zonular fibers indicates its structural components. Note that the capsule of the lens is formed by the basal lamina of the lens fibers and the subcapsular epithelium located on the anterior surface of the lens. A strip of capsule was removed on this drawing to show underlying epithelium. Also note the location of the germinal zone (yellow) at the lens equator, where cells divide and diffarentiate into the lens fiber cells. The organelle-free center of the lens is occupied by the lens nucleus. b. This high-magnification photomicrograph of the germinal zone of the lens (near its equator) shows the active process of lens fiber formation from the subcapsular epithelium. Note the thick lens capsule and the underlying layer of nuclei of lens fibers during their differentiation. The mature lens fibers do not possess nuclei. x570.

968 Conjunctivitis, otherwise known as pinkeye, is an inL1J

~

L1J

:I: ILl..

0

L1J

c:::

::J

tl

::J

c:::

1-CIJ

u

a:

0

u

CIJ

0

0::

u

~ LLI

~

•a: N

w

t:

C'(

::c

u

flammation of the conjunctiva. It may be localized in either the palpebral conjunctiva or bulbar conjunctiva. Individuals may present with relatively nonspecific symptoms and signs that include redness, irritation, and watery discharge from the eye (Fig. F24.6.1). The symptoms can also mimic a foreign body sensation. Extended use of contact lenses can cause an allergic or bacterial conjunctivitis and may be the first sign of more serious ocular disease (i.e., corneal ulcer). In general, symptoms that last less than 4 weeks are classified as acute conjunctivitis, and those extending for a longer period are referred to as chronic conjunctivitis. Acute conjunctivitis is most commonly caused by bacteria; a variety of viruses, including HIV. varicella-zoster virus N'ZVJ. and herpes simplex virus (HSV); or allergic reactions. Bacterial conjunctivitis often causes an opaque purulent discharge containing white cells and desquamated epithelial cells. On eye examination, the purulent discharge and conjunctival papillae help to differentiate between bacterial and viral etiology. Viral conjunctivitis is most common in adults. Clinically, it presents as a diffuse pinkness of the conjunctiva with particularly numerous lymphoid follicles on the palpebral conjunctiva, often accompanied by enlarged preauricular lymph nodes. Viral conjunctivitis is very contagious and usually associated with a recent upper respiratory infection. Patients need to be advised to avoid touching their eyes, to wash their hands frequently, and to avoid sharing towels and washcloths. Bacterial conjunctivitis is usually treated with antibiotic eye drops or ointments. For viral conjunctivitis, no antimicrobial therapy is needed. However, conservative

fiber cells differentiate, they undergo massive elongation and lose all of their organelles, including nuclei, forming the organelle-free zone. Gap junctions connect the cuboidal cells of the subcapsular epithellum. 1hey have few cytoplasmic organelles and

stain faindy. The apical region of the cell is directed toward the internal aspect of the lens and the lens fibers, with which they form junctional complexes. 1he lens increases in size during normal growth and then continues to produce new lens fibers at an ever-decreasing rate throughout life. The new lens fibers develop from the subcapsular epithelial cells located near the equator (see Fig. 24.16) arc: laid down peripherally as concentric lamellae in an onion-like arrangement. Cells in this region increase in height and then differentiate into lens fibers. As the lens fibers develop, they become highly elongated and appear as thin, flattened structures. They lose their nuclei and other organelles as they become filled with proteins called crystalllns. Mature lens fibers attain a length of 7 to 10 mm, a width of 8 to 10 IJ.m, and a thickness of 2 IJ.m. In the adult lens, only lens fibers in the outermost region maintain their nuclei and organelles. Near the center, in the lens nucleus, the fibers are compressed and

management with artificial tears to k:eep the eye lubricated may relieve symptoms. For severe cases, topical corticosteroid drops may be prescribed to reduce the discomfort of inflammation. However, prolonged use of corticosteroid drops increases the risk of side effects. Antibiotic drops may also be used for treatment of secondary infections. Viral conjunctivitis usually resolves within 3 weeks. However. in worst cases. it may take more than a month.

FIGURE F24.&.1. ConJunctivitis. This photograph of the lower part of the eyeball with reflected lower eyelid shows an infected conjunctiva. The enlarged blood vessels of the conjunctiva are responsible for moderate redness of the eye with conjunctival swelling. Moderately, clear Un allergic conjunctivitis) or purulent (in bacterial conjunctivitis) discharge is visible. (Courtesy of Dr. Renzo A. Zaldivar.)

condensed to such a degree that individual fibers are impossible to recognize. The lens nucleus is an organelle-free zone and is composed of primary lens fiber cells laid down during embryonic and fetal development. The lens fibers are joined at their apical and basal ends by specialized junctions called sutures. Despite its density and protein content, the lens is normally transparent (see Fig. 24.16). The high density of lens fibers makes it difficult to obtain routine histologic sections of the lens that are free from artifacts.

Changes in the lens are aaociatad with aging. With increasing age, the lens gradually loses its elasticity and ability to accommodate. This condition, called presbyopia, usually occurs in the fourth decade of life. It is easily corrected by wearing reading glasses or using a magnifying lens. Loss of transparency of the lens or its capsule is also a relatively common condition associated with aging. This condition, called cataract, may be caused by conformational changes or cross-linking of proteins. The development of a cataract may also be related to disease processes, metabolic or hereditary conditions, trauma, or exposure to a deleterious agent

(such as ultraviolet radiation). Cataracts that significantly impair vision can usually be corrected surgically by removing the lens and replacing it with a plastic lens implanted in the posterior chamber.

• ACCESSORY STRUCTURES OF THE EYE

969

The primary functions of the eyelids are to cover, protect. and lubricate the eyes. Vitreous Body The eyelids represent folds of modified skin containing The vitreous body is the transparent jelly-like substance highly modified epidermal appendages to cover, protect, that fills the vitreous chamber in the pos1erior segment of and lubricate the anterior portions of the eyes. The anterior the eye. surface of the eyelid is covered by thin skin, and its posteThe: vitreous body is loosely attached to the SU.tTound- rior surface is lined by a specialized mucous membrane, the ing structures, including the: iMc:r limiting membrane conjunctiva. The skin of the eyelids is loose and elastic to of the: retina. The main portion of the: vitreous body is a accommodate their movement. Within each eyelid is a flexhomogeneous gel containing approximately 99% water ible support, the tarsal plata, consisting of dense fibrous (the vitreous humor), collagen, gl.ycosaminoglycans (prin- and elastic tissue. In the upper eyelid, the lower free edge of cipally hyaluronan), and a small population of cells called the tarsal plate e:nends to the lid margin, and its superior hyalocytes. These cells are believed to be responsible for border serves for the attachment of smooth muscle fibers of synthesis of collagen fibrils and glycosaminoglycans. Hyalo- the superior tarsal muscle (of Muller). The undersurface cytes in routine hematoxylin and eosin (H&E) preparations of the tarsal plate is covered by the conjunctiva (Fig. 24.17). are difficult to visualize. Often, they exhibit a well-developed The striated orbicularis oculi muscle, a facial expression rER and Golgi apparatus. Fibroblasts and tissue macroph.ages muscle, forms a thin oval sheet of circularly oriented skeletal are sometimes seen in the periphery ofthe: vitreous body. The muscle fibers overlying the tarsal plate. In addition, the conhyaloid canal (or Cloquet's canal), which is not always nective tissue of the upper eyelid contains tendon fibers of visible, runs through the center of the vitreous body from the the levator palpebrae superioris mu1cla that open the optic disc to the posterior lens capsule. It is the remnant of eyelid (see Fig. 24.17). A mucocutaneous junction between eyelid skin and conjunctiva occurs near the lid margin. the pathway of the hyaloid artery of the developing eye.

~

()

rn

(I)

0

~

~

;D

c ~ c

;D

rn ,

0

-I

:::c:

m m -< m lacrimal gland

orbicularis levator palpebrae oculi musc:l\suporioris musc:lo

aCCG$$0ry lacrimal

/gland (of K~ause)

~~=~~L14--apocrlne

glands

of eyelids

sebaceous glands of eyelashes (of zeis)

(of Moll)

a

b

FIGURE 24.17.. Structun~ of the eyelid. a. This schematic drawing of the eyelid shows the skin. associated skin appendages, muscles, tendons, connective tissue, and conjunctiva. Note the distribution of multiple small glands associated with the eyelid and observe the reflection of the palpebral conjunctiva in the fornix of the lacrimal sac to become the bulbar conjunctiva. b. Photomicrograph of a sagittal section of the eyelid stained with picric acid for better visualization of epithelial components of the skin and the numerous glands. In this preparation, muscle tissue (i.e., orbicularis oculi muscle) stains yellovv. and the epithelial cells of skin, conjunctiva. and glandular epithelium are green. Note the presence of the numerous glands within the eyelid. The tsrsal (Meibomian} gland is the largest gland, and it is located within the dense connective tissue of the tarsal plates. This sebaceous gland secretes into ducts opening onto the eyelids. x20. lnHt. Higher magnification of a tarsal gland from the boxed area. showing the typical structure of a holocrine gland. X60.

970

w

~

w

::c

1-

u...

0

V)

w

a:

~::::> ~ ~

0V) V)

w u

~

The eyelashes emerge from the most anterior edge of the lid margin. They are short, stiff, curved hairs and may occur in double or uiple row.s. The lashes on the .same eyelid margin may have different lengths and diameters.

secretory ducts

I

common canaliculus

The conjunctiva lines the space between the inner surface of the eyelids and the anterior surface of the eye without covering the cornea. The conjunctiva is a thin, uansparent mucous membrane that cnends from the corneosderal limbus located on the peripheral margin of the cornea across the sclera (bulbar conjunctiva) and covers the internal surface of the eyelids (palpebral conjunctiva). It consists ofa stratified columnar epithelium containing numerous goblet cells and rests on a lamina propria composed of loose connective tissue. The goblet cells secrete a component of the tears that bathe the eye. Conjunctivitis, an inflammation of the conjunctiva that is commonly called pinkeye, is characterized by redness, irritation, and watering ofthe eyes. For more clinical information about this condition, see Folder 24.6. Secretions from modified glands in the eyelid provide additional protection to the eye. In addition to eccrine sweat glands, which discharge their secretions directly onto the skin, the eyelid contains four other major types of glands (see Fig. 24.17): • The: tarsal glands (Meibomian glands), long sebaceous glands embedded in the tarsal plates, appear as vertical yellow sueaks in the tissue deep in the conjunc-tiva. Their elongated ducts open at the lid margin behind rows of eyelashes. About 25 tarsal glands are present in the upper eyelid, and 20 are present in the lower eyelid. The sebaceous secretion of the tarsal glands produces an oily layer on the surface of the tear film that retards the evaporation of the normal tear layer. Blockage of the tarsal gland secretion leeds to chalazion (tarsal gland lipogranuloma), en inflammation of the tarsal gland. It presents as a painless cyst usually on the upper eyelid that disappears after a few months without therapeutic intervention. • Sebaceous glands of eyelashes (glands of Zeis) are small, modified sebaceous glands that are connected with and empty their secretion into the follicles of the eyelashes. Bacterial infection of these sebaceous glands causes a stye (also celled a hordeolum), a painful tenderness and redness of the affected area of the eyelid. • Apocrine glands of eyelashes (glands of Moll) are small sweat glands with unbranched sinuous tubules that begin as a simple spiral. • Accessory lacrimal glands are compound serous tubuloalveolar glands that have distended lumina. They are located on the inner surf.tce of the upper eyelids (glands of WoHring) and in the fornix of the lacrimal sac (glands of Krause). All glands of the human eyelid are innervated by neu· rons of the autonomic nervous system, and their secretion is syncluonized with the lacrimal glands by a common neurotransmitter, vasoactive intestinal polypeptide (VIP).

/-moJsae

~

nasolacrimal duct inferior nasal meatus inferior nasal concha

nasal cavity RGURE 24.18. Schematic diagram of the eye and lacrimal •ppanatua. This drawing shows the location of the lacrimal gland and components of the lacrimal apparatus. which drains the lacrimal fluid into the nasal cavity.

The lacrimal gland produces tears that moisten the comea and flow to the nasolacrimal duct. Tears are produced by the lacrimal glands and, to a lesser degree, by the accessory lacrimal glands. The lacrimal gland is located beneath the conjunctiva on the upper lateral side of the orbit (Fig. 24.18). The lacrimal gland consists of sev· eral separate lobules of tubuloacinar serous glands. The acini have large lumina lined with columnar cells. Myoepithelial cells, located below the epithelial cells within the basal lamina, aid in the release of teats (Fig. 24.19). Approximately 12 ducts drain from the lacrimal gland into the reflection

of conjunctiva just beneath the upper eyelid, known as the

fomix of tha lacrimal sac. Tears drain from the eye through lacrimal puncta, the small openings of the lacrimal canaliculi, located at the medial angle. The upper and lower canaliculi join to form the common canaliculus, which opens into the Iacdmal sac. The sac is continuous with the nasolacrimal duct, which opens into the nasal cavity below the inferior nasal conchae. A pseudostratified ciliated epithelium lines the lacrimal sac and the nasolacrimal duct. Dacryocystitis is an inflammation of the lacrimal sac that is frequently caused by an obstruction of the nasolacrimal duct. It can be acute, chronic, or congenital. It usually affects older individuals and is most often secondary to stenosis (narrowing) of the lacrimal canaliculi.

Tears protect the comeal epithelium and contain antibac· terialand UV·protective agents. Tears keep the conjunctiva and corneal epithdium moist and wash foreign material from the eye as they B.ow across the corneal surface toward the medial angle ofthe eye (see Fig. 24.18). The thin film of tears covering the corneal surface is not homogeneous but a mixture of products secreted by the

lacrimal glands, the accessory lacrimal glands, the goblet cells of the conjunctiva, and the tarsal glands of the eyelid. The tear film contains proteins (tear albumins and lactoferrin), enzymes (lysozyme), lipids, metabolites, electrolytes, and medications, the latter secreted during therapy. The tear cationic protein Iactoferrin increases the activity of various natural anti.m.icrobial agents, such as lysozyme.

The eye is moved within the orbit by coordinated comraction of extraocular muscln. Six. muscles of the eyeball (also called extraocular or extrinsic muscles) attach to each eye. These are the medial, late.ral, superior, and inferior rectus muscles and the superior and inferior oblique muscles. The superior oblique muscle is innervated by the trochlear nerve (cranial nerve IV). The lateral rectus muscle is innervated by the abducens nerve (cranial nerve VI). All of the remaining extraocular muscles are innervated by the oculomotor nerve (cranial The combined, precisely controlled action of these nerve muscles allows vertical, lateral, and rotational movement of the eye. Normally, the actions of the muscles of both eyes are coordinated so that the eyes move in parallel (called

nn.

conjugate gaze).

971

972

....

0

OVERVlEW OF THE EVE .

.red s ecialized sensory organ that provides the sense of sight.

• The ~e 1S a pf the' p are derived from neuroectodenn {retina), surface - - - '!!:,;:~es{l:ns, cor:eal epithelium), and mesodenn {sclera. corneal stroma.

w ------------------

•

vascular coat~.

~

posed of three st:fUCtlll'lll layers: the outer comeosclenl com· • of the ,.,.,.nman:nt cornea and UJ.C ..L_ whi --1-.·. te opaque 5UC£" (flblous) coat conslStlilg • -:-rfth ch id -:1:~- b.....l.· and. iris; and the the middle vascular coat consiSttng o e oro , ............, Vt.JT• inner layer, the ~--· fo ~L-- -'---hers· the f threttna. d th l serve as bounu.ules r uua: [;IDI.lll •

e The

ili

a::

~

cy-156 Actin-bundling ptoteW, 66, 121 .Acdn-capplng proa:lnr, 67 Actin cross-llnking proldna, 67 ktln..tepcndent cndncytods, 38 .Acdn &lament(•), 29-30,65--68, 103-109 ab.bormalidcs In, 68, 76/ in anchorinj;junaions, 135, 140, 142 in cardiac muscle, 355 cdl adhesion molccules and, 139-140

charactcrima of. 77, distribution of. 65, 67 fOcal adlu:sions and, 155, 155-156 fimctioiiS of. 68 Ill. hair cells of lntemlll ear, 990 Ill. mlaovlll1, 121, 122 ml12111 (pointzd) end of; 6~6, 67 in musde tissue, 336 in cx:dudlng juru:dons, 135 in mu:odut dear zone, 24.4 in platdcr ruuaural zone:, 310, 311 pillS (barbed} end of; 65---'6, 67 polymerization of, 66, 67 iD salivary glands, 583-590 iD Scrtoli cdlt. 847, 848, 848 iD slWetal muscle, 341,341-343 iD slWetal muscle contw:tion, 34>-347, 346 iD &mall interti.De, 628 Ill. .unootb. mUlde, 191, 1%, 196/.359, 583-590 In smnoth muadc: conmaion, 35!J, 361,362 Ill. apleen, 507 in ~cllla, 121-122, 125 a:nnlnal web of, 68, 121, 122, 628 ttc:admHllng drcct of; 66, 122 Actin &lamenwevering proldns, ~ ktin-lndepcndcnt en~, 38 Actin motor ptoteins, 67 Action pot=tial. 387,405,991 Activated libroblam, 191 Activated lymphocytes. 307 &rive osteoblut., 240 &rive olltecK:Wts, 23!) Active transport, 36, 36 Active zones, of IIJ'IIIPSCS• 391 A.:tomyao:in cross-bridge cycle, 345-347, 346, 351 Acute boacwial rhinao:inlllldt, 715/ Acute conjunaividr, 968/ Acute gta.u:oma. 954f Acuu: lnllarnmatory dcm.ydlnatlng polyrac&uloncuropathy; 397/ Acute laryngilir. 709--710 Aqlarion-stimulating protein (ASP), 280t

Aqlglucosylccramide, m Adaptation, iD vil.ion, 945 Adaptin, 39, 40 .Adazonal plasma membrane, 395, 3!)7 ADCC. Su Antibody-dependent cdl-medlated cytotcD:iclty

Adducln, 67

Adcncx:an::bwmu, 120/.641/ Adcnohypophyds. &t Anterior lobe of pituitary

gland Adenoid., 492, 567 Adcnomu, 591/.639/ Adcnoslnc dipholphatc (ADP), 311 Adenosine ttipho.!phawc (ATPasc), 7. 8, 35, 56 in bile canaliculi. 679 calcium (Ca'+)-actimed pump, 361 iD ciliary movement, 126-128 coppa (Wdson), 668 in skdeta!IIUIIde. :m-339, 343. 346-34a in IIIDOOth m111cle, 363 Adcnoalne uiphoaphw: (ATP), 59-60, 61 in =In polymerization, 66 in 6w ttahlport, 389 in neurosccn:mry vesicles, 794 in skelelal ttuudc, 338, 346. 346-347 in IIIDOOth m111cle, 361-363 in cb.crmogencsif. 283

Adcnoalru: uiphoaphatc (ATP)IADP czchange protein, 60,61 Adcnoalne uipho.!phatc (ATP)-dcpcndent calcium pumps,36l Adcnoalne uipho.!phatc (ATP) synthase, 35,60 A.denylatc cydase/cycliG ade.noiinc mooophosphate (cAMP) system, 786 ADH. Sn Anddlurmc hnrmoru: Adhaian molecules, 133, 139-143, 140, 149 c:ndodtd.lal, 299, 324., 442-443, 442/. 497--498 neqttaphlJ, 299 Adipoblam (llpoblaoa), 275-276, 276 Adipoo:ytc(l) (adipose cdl), 171, 190, 199, 274, 288p---289p on bone marrow smear, 330p-33lp brown, 276. 280-282, 284-285, 288p-289p di1£uentialion oE 27>-276, 276. 282 atemallamina of. 276 lipid mass in, 277 mature, 276, 276 in obesity, 274

obesity aru:l mctabollsm of; 281/ 276-277. 277, 278 synthalo and occmlo.b by. 275, 275, 280t, 288p traDsdlfferentladan .,£ 276, 284-285 whltr:, 275-277, 276, 284-285, 288p---289p Adipoo:ytc(l), basal lamina in, 149 Adipokif!QI, 275, 275, 288p Adiponcctin,275.280t Adipopb.ilin, 280J Adipose tiuue, 199, 274-287, 288p-289p iD bone DW.row, 275, 324-325 brown, 274,279-284,282, 288p-289p in clerm!s, 288p, 529 u endocrine otga!t, 274 fearum of, 284t glucocortia>id. In, 813 in mammary gland., 275 of mammary &fa.bd.o, !JO!) mobillzadan ot 279 in obesity, 274 PET .KaDDing and, 280, 284, 285/ rcgularion of, 277-279, 283-284 ruucture o£ 276-277' 277 sttucll1l