Outlines of histology. 9788024637433, 802463743X, 9788024637587, 8024637588

Table of contents :
Cover
Contents
Preface
Part I: The Cell
Cell membranes
Nucleus
Chromatin
Cell cycle
Mitosis
Ribosomes
Endoplasmic reticulum (ER)
Rough (granular) endoplasmic reticulum – RER (GER)
Smooth (agranular) endoplasmic reticulum – SER (AER)
Golgi complex (Golgi apparatus)
Mitochondria
Lysosomes
Peroxisomes (microbodies)
Nonmembranous organelles and cytoskeleton
Microtubules
Centrosome (diplosome)
Microfilaments
Intermediate filaments
Cell inclusions
Part II: The Tissues
A. Epithelial tissue
Apical surface of epithelia
Basement membrane
Cell adhesion
Junctional complex
Covering epithelia
Simple epithelia
Stratified epithelia
Glandular epithelia
Exocrine glands
Classification of exocrine glands according to shape
Glands according to the mechanism of secretion
Glands according to their secretory products
Myoepithelial cells
B. Connective tissue
Connective tissue proper
Cells
Extracellular matrix
Fibers
Ground substance
The types of connective tissue proper
Cartilage
Hyaline cartilage
Elastic cartilage
Fibrocartilage
Bone
Spongy (cancellous, trabecular) bone
Compact bone
Ossification
Intramembranous ossification
Endochondral ossification
Tooth
Development of the teeth
Histology of the tooth components
Enamel
Dentin
Cement
Periodontal membrane
The pulp
Alveolar bone and gingiva
Blood
Plasma
Erythrocytes
Leukocytes
Granulocytes
Agranulocytes
Thrombocytes
Hematopoiesis
C. Muscle tissue
Smooth muscle
Striated (sarcomeric) muscle
Skeletal muscle
Mechanism of contraction
Myosatellite cells
Cardiac muscle
Cardiac conducting system
Specialized myocardiocytes – myoendocrine cells
D. Nerve tissue
Neurons
Classification of neurons
Cytology of the neuron
Unmyelinated fibers
Myelinated fibers of CNS
Peripheral nerve
Synapses and a reflex arc
Sensory receptors
Free nerve endings
Meissner’s corpuscles
Pacinian corpuscles
Muscle spindles
Motor nerve endings
Conduction of nerve impulses
Neuroglia
Astrocytes
Oligodendrocytes
Microglia cells
Ependymal cells
Satellite cells
Meninges
Pia mater
Arachnoid
Dura mater
Blood-brain barrier
Cerebrospinal fluid
Figure captions
Literature recommended for further study

Citation preview

U UČ ČE EB BN N ÍÍ T TE EX XT TY Y U UN N II V VE ER RZ Z II T TY Y K KA AR R LL O OV VY Y V V P PR RA AZ ZE E

EXTY UNIVERZITY KARLOVY V PRAZE

COVER

OUTLINES ZÁKLADY HISTOLOGIE OF HISTOLOGY Jaroslav Slípka Jaroslav Slípka Zbyněk Tonar

KAROLINUM

Outlines of Histology Jaroslav Slípka Zbyněk Tonar

Reviewed by: Sarah Leupen, Department of Biological Sciences, University of Maryland Baltimore County, USA Ivan Varga, Institute of Histology and Embryology, Faculty of Medicine, Comenius University, Bratislava, Slovakia Published by Charles University Karolinum Press as a teaching text for the Faculty of Medicine in Pilsen Typesed by DTP Karolinum Press Second, revised edition © Charles University, 2017 Jaroslav Slípka – heirs, Zbyněk Tonar, 2017 ISBN 978-80-246-3743-3 ISBN 978-80-246-3758-7 (online : pdf)

Charles University Karolinum Press 2017 www.karolinum.cz [email protected]

CONTENTS

Cover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 PREFACE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 PART I: THE CELL. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nucleus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ribosomes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endoplasmic reticulum (ER). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rough (granular) endoplasmic reticulum – RER (GER). . . . . . . . . . . . . . . . . Smooth (agranular) endoplasmic reticulum – SER (AER). . . . . . . . . . . . . . . . Golgi complex (Golgi apparatus). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lysosomes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Peroxisomes (microbodies). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nonmembranous organelles and cytoskeleton. . . . . . . . . . . . . . . . . . . . . . . . . . . . Microtubules. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Centrosome (diplosome). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microfilaments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Intermediate filaments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell inclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8 10 14 15 19 20 23 24 24 26 26 27 28 29 29 29 29 30 31 31

PART II: THE TISSUES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Epithelial tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Apical surface of epithelia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Basement membrane. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Junctional complex. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Covering epithelia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

33 33 34 35 36 38 38

Simple epithelia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stratified epithelia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glandular epithelia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Exocrine glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classification of exocrine glands according to shape . . . . . . . . . . . . . Glands according to the mechanism of secretion . . . . . . . . . . . . . . . . Glands according to their secretory products. . . . . . . . . . . . . . . . . . . . Myoepithelial cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Connective tissue. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Connective tissue proper. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extracellular matrix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fibers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ground substance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The types of connective tissue proper . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cartilage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hyaline cartilage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Elastic cartilage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fibrocartilage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bone. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Spongy (cancellous, trabecular) bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . Compact bone. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ossification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Intramembranous ossification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endochondral ossification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tooth. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Development of the teeth. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Histology of the tooth components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enamel. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dentin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Periodontal membrane. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The pulp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alveolar bone and gingiva. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Plasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Erythrocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Leukocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Granulocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Agranulocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thrombocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hematopoiesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

38 39 40 40 41 42 42 44 44 45 45 49 49 51 52 54 54 55 56 56 58 58 60 61 62 65 65 68 68 70 71 72 72 73 73 74 74 75 75 78 80 82

C. Muscle tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Smooth muscle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Striated (sarcomeric) muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Skeletal muscle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanism of contraction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Myosatellite cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cardiac muscle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cardiac conducting system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Specialized myocardiocytes – myoendocrine cells. . . . . . . . . . . . . . . D. Nerve tissue. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neurons. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classification of neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytology of the neuron. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Unmyelinated fibers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Myelinated fibers of CNS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Peripheral nerve. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Synapses and a reflex arc. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sensory receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Free nerve endings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Meissner’s corpuscles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pacinian corpuscles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Muscle spindles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Motor nerve endings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conduction of nerve impulses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neuroglia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Astrocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oligodendrocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microglia cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ependymal cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Satellite cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Meninges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pia mater. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Arachnoid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dura mater. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Blood-brain barrier. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cerebrospinal fluid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

85 85 87 87 91 92 93 94 95 96 96 97 99 102 102 102 103 105 105 106 106 106 108 108 112 112 113 113 114 115 115 115 115 116 116 116

Figure captions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 Literature recommended for further study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

PREFACE

Histology is a science of the tissues which are formed by conglomeration of cells and extracellular matrix. We are always interested in understanding the origin, microscopic structure and function of these tissues. Contemporary general histology deals – in addition to the description of the fundamental unit of the tissue, the cell – with four kinds of basic tissues, presented in this textbook. These tissues participate in construction of various organs and organ systems. This textbook outlines the courses in general histology, given to the international students of general and dental medicine in the first year of their pregraduate studies at the Charles University, Faculty of Medicine in Pilsen. The first edition was prepared in 1994 and revised in 2004 by Prof. Dr. Jaroslav Slípka, DSc (1926–2013), who was an enthusiastic and inspiring researcher and teacher at the Department of Histology and Embryology. The second edition was updated in 2017 to reflect some of the advances in teaching of histology. Nevertheless, the illustrations and the concise concept of the book designed originally by prof. Slípka were kept. We recommend using this textbook for revising and summarizing the essential knowledge. For full color textbooks and atlases that are necessary for understanding the histological slides, see the literature recommended at the end. We wish all our students might enjoy the insight into the universe of cells and tissues the human body is made of. Welcome to the world of Histology! Zbyněk Tonar Pilsen, 2017

–7–

PART I: THE CELL

The cell is a basic integrated entity of all living organisms. It is a fundamental morphologic and physiologic unit, capable of multiplication, metabolism, growth, excitability and other specialized functions. The microscopic analysis of the fine structure and function of cells is referred to as cytology. There are two different structural types of cells: Prokaryotic cells with no nuclear membrane and no membranous organelles like in bacteria. Eukaryotic cells with a nuclear membrane and various membranous organelles. These cells can form assemblies, classified into four basic tissues in multicellular animals (Metazoa). The science of the morphologic and functional features of cells and tissues constitutes histology, the topic of this textbook. Shapes and dimensions of cells: Although the primary form of cells is rounded or spherical, during development of tissues cells become, depending on their function, squamous, cuboidal, columnar, pyramidal, spindle

Fig. 1 Shapes of cells –8–

shaped, star shaped, goblet shaped etc. The shape of cells is organized by an internal scaffolding of proteins known as the cytoskeleton. Those cells which have remained free, e.g. blood cells, retain their spherical form. Most of the cells of the human body range from 4–30 micrometers in diameter; the largest are the oocytes (150 µm in human). The red blood cell, which ranges around 7.5 µm in diameter, can be used as a rough measure of the size of the tissue cells seen in the same field. Composition of cells: The cell is formed by protoplasm which is composed of cytoplasm (or cytosol), the fluid matrix of the cell, and nucleoplasm, the matrix of the nucleus. Cytoplasm is composed of a colloid solution, contained by a cell (or plasma) membrane – plasmalemma. The cytoplasm contains many smaller elements – subcellular structures – called organelles which provide the framework for cellular activities. It contains also many of the essential enzymes and metabolites. In the nucleoplasm is the genetic material in the form of chromosomes. Chemical composition of cells: in addition to water and inorganic substances, cells contain four main classes of organic constituents: proteins, carbohydrates, fats (lipids) and

Fig. 2 The cell –9–

nucleic acids. The pH of the cytoplasm ranges between 7.0–7.4. However, in most histological staining methods, the cytoplasm of most cells appears as slightly acidophilic (e.g., stained pink to reddish by an acidic dye named eosin using the routine hematoxylin-eosin staining).

Cell membranes The term comprises not only the outer membrane surrounding each cell, i.e. plasmalemma, but also the membranes surrounding cellular organelles. The basic structure consists of a lipid bilayer containing specialized proteins in association with surface carbohydrates. The membrane lipids are of three types: phospholipids, cholesterol and glycolipids. The phospholipids are organized into a double layer of molecules, in which each molecule has an outer hydrophilic head and an inner hydrophobic chain. Cholesterol is inserted into the phospholipid bilayer and is present in about the same amounts as are phospholipids, and is responsible for the mechanical stability of the otherwise fluid membrane. Glycolipids with their associated sugars are exposed to the extracellular surface and are involved together with glycoproteins in intercellular communication as mediators of cellular interactions like adhesion and recognition. Membrane protein molecules can be classified according to their spatial relations to the membrane: Integral (intrinsic) membrane proteins of-

Fig. 3 Cell membrane – 10 –

ten span the whole lipid bilayer from one surface to another, and form a transmembrane channel for passing the ions through the membrane. Some integral membrane proteins are confined to the inner or outer part of the membrane only. The peripheral (extrinsic) membrane proteins are not fully embedded within the lipid bilayer and are more loosely attached to a membrane surface. Some membrane proteins are receptors, which allow cells to respond to external signals when binding a variety of signaling molecules (or ligands). Binding of signaling molecules (released by a signaling cell) to their receptors activates in the target cell an intracellular second messenger system, initiating a cascade of reactions that result in the required response. For example, a hormone (the first messenger) activates through a receptor a transmembrane protein – adenylate cyclase, which in cytoplasmic region catalysates the transformation of ATP to cyclic adenosine monophosphate (cAMP), i.e. the second messenger. Numerous membrane proteins bear polysaccharide chains and represent the glycoprotein molecules. The membrane carbohydrates cover the extracellular surface of the cell membrane like a sugar coating – the glycocalyx. The polysaccharide chains confer a certain surface specificity on every type

Fig. 4 Cellular processing of external signals – 11 –

Fig. 5 Membrane transport

of cell. The glycocalyx is directly involved in the recognition and adhesion of different cell types, particularly during morphogenesis. The carbohydrate chains can be demonstrated by staining with lectins (proteins extracted mostly from some plants). The role of the cell membrane lies not only in preserving the integrity of the cell, but also in cell-cell recognition and selective transport of molecules. Whereas the cell membrane is impermeable to most large molecules, it is permeable to some smaller molecules and ions, and so important for bringing needed material into the cell and releasing waste products. The membrane is equipped with active transport mechanisms to transfer various substances (e.g., glucose, amino acids) in the required direction. Small molecules are bound to an integral carrier protein which undergoes series of conformational (shape) changes to release the molecule on the cytoplasmic side. Some ions can be transported according to the ion gradient through the plasmalemma by ion selective channel proteins via a mechanism called facilitated diffusion. Another example is a sodium-potassium pump which utilizes energy from mitochondria to release the sodium ions from the cell and to pump potassium into the cell (see, e.g., nerve tissue). The cell can also ingest macromolecules from the extracellular space by invagination of the cell surface, termed endocytosis. The invaginated cell membrane encloses the incorporated material for further processing in an endocytotic vesicle (endosome). In the case of very small molecules, we specify the endocytotic process as pinocytosis (i.e. cell drinking) with the formation of pinocytotic vesicles. For ingestion of large particles (e.g. bacteria) the term phagocytosis (cell eating) is used. In such cases the cell – 12 –

extends cytoplasmic processes which engulf the material and ingest it in endosomes, called phagosomes. The reverse process is called exocytosis, in which the products produced in cells can be released. The membrane bound vesicles fuse with cell surface and discharge their content into the extracellular space. The invaginated cell membrane forms a so called coated pit, which bears surface receptors that bind specific extracellular ligands. The pit is covered on the cytosolic side by a coat protein – clathrin – in form of a lattice. The invaginated membrane then fuses to form an endocytic vesicle – endosome

Fig. 6 Exocytosis and endocytosis

Fig. 7 Transcytosis – 13 –

(pha­gosome). During the process of internalization the clathrin is shed and returns to the surface. Similar membrane invaginations, coated with another protein – caveolin – have been termed the caveoli. They are responsible for the transport of the substances in vesicles from one side of a flat cell (e.g., endothelium lining the blood and lymph vessels) to the opposite one. This process is called transcytosis. Their receptors can also play a role in intracellular signaling by triggering the intercellular messenger system (e.g., in smooth muscle).

Nucleus The nucleus is the largest membrane-limited, spherical or ovoid organelle, situated usually in the center of the cell (missing only in mature mammalian RBCs). Its diameter usually varies between 5–10 µm. It is composed of nucleoplasm, which contains chromatin, and the nucleolus. The nucleus is separated from the cytoplasm by the nuclear membrane (nucleolemma). The nuclear membrane is built by two concentric membranes, separated by a narrow space – the perinuclear space, which can be continuous with the rough endoplasmic reticulum. On the inner membrane are anchored some filamentous proteins – lamins, which form a sort of scaffolding of the nucleoplasm. The outer nuclear membrane can be associated with ribosomes. Both membranes fuse in numerous circular nuclear pores, which are not open, but bridged by a thin protein diaphragm, permeable to some molecules, e.g., to mRNA.

Fig. 8 Nucleus – 14 –

The nucleolus is a dense nonmembranous structure within the nucleus, seen during interphase only. It measures 1–3 µm. Active cells (embryonic, tumorous etc.) have usually larger or even multiple nucleoli. It stains basophilic, being rich in rRNA and proteins, and it disappears during cell division, but reappears in the telophase stage of mitosis. The nucleolus is rich in rRNA and protein, accumulated into ribosomal subunits, which are then transferred to cytosol through the nuclear pores. It consists of three distinct regions. The pale area – fibrillar center – surrounds the so called nucleolar organizer DNA regions. There the tips of 5 chromosomes are located and their genes (nucleolar organizers) code for rRNA. The dark thread-like structure, the dense fibrillar component, consists of primary transcripts of rRNA molecules beginning to form ribosomes. The granular regions – granular components – consist of maturing ribosomal subunits. Chromatin

There are two forms of chromatin: Heterochromatin is seen as condensed basophilic clusters of coarse granules, often adjacent to the nuclear membrane. It represents an inactive form of chromatin. Euchromatin represents actively transcribed DNA and appears as lightly stained areas of nucleoplasm. Its structure is seen in the electron microscope only. A molecule of deoxyribonucleic acid (DNA) is made up of two polynucleotide strands wound together in the form of a double helix. Each strand consists of alternating phosphate and deoxyribose sugar groups, and it has a nitrogenous base extending as a side chain from each sugar group into the double helix. There are two types of bases: purines (adenine and guanine), and the pyrimidines (cytosine and thymine). There is an obligatory pairing of the bases on one strand by hydrogen bonds with the bases on the other. Such complementary base pairing exists between adenine (A) and thymine (T), and between guanine (G) and cytosine (C). The building blocks of nucleic acids are the nucleotides, composed of bases linked to sugar and phosphate groups. DNA is the repository of genetic information. The information transfer from the nucleotide sequence of DNA to the amino acid sequence of a protein involves three forms of RNA – ribosomal (rRNA), transfer (tRNA) and messenger (mRNA). The molecule of ribonucleic acid (RNA) is single stranded and is not self-replicating, so that all forms of RNA are tran– 15 –

Fig. 9 Molecule of DNA

scribed from DNA. DNA serves as a template for a complementary strand of RNA during a process of transcription. RNA differs from DNA. The sugar ribose is present instead of deoxyribose and the base uracil (U) replaces thymine (T). Chromatin is formed mainly by coiled strands of DNA, wound around proteins – histones. The basic unit of chromatin is the nucleosome. Nucleosomes are regularly repeated globular structures, like beads on a string. The nucleosomes form a chromatin fiber of 30 nm in diameter. Coiled chromatin fibers surround the chromosome protein core during cell replication and are extensively condensed to form distinct chromosomes, structures that are visible with the light microscope. The mitotic chromosome possesses two arms, extending from its centromere. The adenine-thymine-rich regions of chromosomes produce a pattern of G bands (stained with Giemsa stain), unique for each chromosome and characteristic for each species. The units of heredity are located at specific regions on the DNA molecule and are called genes. Each gene repre– 16 –

Fig. 10 Chromatin fiber

sents a specific segment of the DNA molecule that codes for the synthesis of a particular protein. The number of chromosomes is specific for each species of living organism. The number and type of chromosomes in an individual is known as his or her karyotype. The human genome is made up of 46 chromosomes. This diploid number represents 23 homologous pairs of chromosomes. Of these 22 pairs are autosomes, and one pair represents sex chromosomes (heterochromosomes, allosomes). The germ cell has only 23 chromosomes

Fig. 11 Condensed chromosome visible and stained during mitosis – 17 –

(haploid number), from which are 22 autosomes, and one sex chromosome, which differs in female and in male germ cells. In females, each ovum always contains an X chromosome, while in males the spermatozoa are of two kinds – one carrying sex chromosome X and the other Y. The resulting sex of a newborn depends on the kind of sperm which fertilizes the ovum. A male somatic cell then possesses 44 autosomes and XY combination of sex chromosomes, whereas a female has also 44 autosomes, but XX sex chromosomes.

Fig. 12 Human male karyotype

Only one of the two XX sex chromosomes of the female somatic cell is activated during interphase. The other one is inactive and appears condensed as a clump of heterochromatin attached to the nuclear membrane in form of a Barr body. It can be observed in 20–40% of epithelial cells in smears taken from the oral mucosa or in form of a small drumstick attached to the nucleus of the neutrophilic granulocytes. – 18 –

Fig. 13 Barr body in a neutrophilic granulocyte of a female

Cell cycle The somatic cells exist in the states of division (mitosis) and nondivision (interphase). Interphase represents a longer period between mitotic division that begins immediately following telophase of the mitosis. It cannot be considered as a resting stage, because during this period of time the cell increases its size and replicates its genetic material.

Fig. 14 DNA replication and doubling of chromatids – 19 –

Fig. 15 Cell cycle and average duration of its phases

The two DNA strands unwind and separate and each strand serves as a template for the synthesis of new strand alongside it. Complementary base pairing occurs determined according the base sequence of preexisting strand. Two identical double-stranded DNA molecules are formed – one half persisting from the original one and the second half being synthesized anew. Interphase can be subdivided into three phases: During the G1 (gap) phase when the synthesis of material essential for DNA duplication begins, the nucleolus reappears and the centrioles begin their duplication. The next stage is represented by S phase (synthetic), when the genome is duplicated and the cell contains twice its usual complement of DNA in preparation for the mitosis. During the last stage of the interphase, the G2 phase, the RNA and proteins essential to cell division are synthesized, and energy is stored so that the cell is prepared for mitosis. Mitosis

Mitosis represents a short period in which the cell divides its nucleus and cytoplasm into two identical daughter cells. During mitosis the 46 chromosomes become highly condensed. In the cytoplasm spindle fibers appear and the centrioles split and migrate to opposite ends of the cell, where they act as organizing centers for the spindles. – 20 –

Mitosis is a continuous process, but can be conventionally divided into four main phases. During prophase the chromosomes become visible and split longitudinally except at the centromere. Each half, joined by the centromere, is known as a chromatid. Next to the centromere on each chromatid a new microtubule organizing center, the kinetochore, develops. The nucleolus disappears and the nuclear membrane breaks down. In the cytoplasm the spindle fibers stretch between the centrioles. The process of nuclear material division is called karyokinesis. During metaphase the chromosomes move accompanied in their migration by mitotic spindle microtubules. The chromosomes become maximally condensed and arrange themselves at the equator of the cell, in form of a star. In the next stage of anaphase the centromeres split and the sister chromatids become 92 separate chromosomes. The daughter chromosomes begin to separate from each other and move to opposite sides of the cell. Each daughter cell receives an identical set of 46 chromosomes, arranged on the cell poles in a double star pattern. In the last mitotic stage – telophase – the division of the cytoplasm occurs (cytokinesis). The nuclear membrane forms and the spindle fibers disintegrate and the nucleoli reappear. The 46 chromosomes of the daughter cells condense and the nucleus reforms. The chromosomes begin a cycle of decondensation and become dispersed throughout the nucleus in form of chromatin.

Fig. 16 Chromosomes during mitotic cell division

The central dogma of molecular biology: DNA → transcription → RNA → translation → protein The central dogma of molecular biology describes the process of expression of the genetic information stored in the DNA molecule and the – 21 –

Fig. 17 From genetic information to the protein

transfer of sequence information between information-carrying biopolymers. This process requires two steps. At first the genetic information has to be copied into messenger RNA (mRNA) in a process known as transcription. The information for the insertion of a specific amino acid in a protein is encoded in mRNA in a triplet of bases, a three-letter code, or codon. The mRNA passes into the cytoplasm, where the message has to be put into the language of the newly formed molecule of protein. This second step of the process, known as translation, requires an interaction of many components along with the mRNA, including the ribosomes, tRNA, amino acids, enzymes and energy sources. Ribosomes serve as workbenches upon which protein synthesis occurs. The transfer RNA (tRNA) contains a binding site for a specific type of amino acid and a three base segment known as anticodon. Within the ribosome the anticodon recognizes and binds to the complementary base sequence (codon) in mRNA, producing a polypeptide chain, which is released from the ribosome, e.g., to endoplasmic reticulum. The ultimate expression of this information in the phenotype are the proteins. – 22 –

Note: The central dogma was stated in 1958 by Francis Crick, a co-discoverer of the structure of DNA molecule. Although it remains a fundamental concept, there are now several known ways of transfer of biological sequential information, that are not covered by this dogma, such as reverse transcription, RNA replication, posttranslational modification, methylation of DNA, and others.

Ribosomes Ribosomes are smallest, basophilic, electron-dense cellular organelles (20–30 nm). They occur in great numbers free in the cytoplasm, or attached to the membranes, mostly of endoplasmic reticulum. The free ribosomes appear often in clusters, called polyribosomes or polysomes and synthesize proteins which are required for uses within the cell. The ribosomes attached to membranes are involved in synthesis of proteins that will be secreted out of the cell. The ribosomes are composed of ribosomal RNA (rRNA) and proteins, the rRNA being synthesized in nucleolus and protein in the cytoplasm. They appear in two different sized subunits, that associate to form a single ribosome (or monosome) during protein synthesis, and which leave the nucleus via nuclear pores.

Fig. 18 Ribosome – 23 –

The large subunit contains two rRNA molecules and more than 50 proteins; the small one consists of a molecule of rRNA (different than those in the large part) and more than 25–30 proteins. The small subunit contains a site that binds mRNA, and a decoding region that binds a tRNA molecule. The large subunit contains sites where amino acids are joined together and the exit point for the newly synthesized protein. The ribosomes are held together by a strand of messenger RNA, carrying the information of the amino acid sequence of proteins which should be synthesized during translation. This is the final stage of the directional flow of genetic information from DNA through RNA to protein, as stated by the central dogma of molecular biology.

Endoplasmic reticulum (ER) The endoplasmic reticulum is a system of membranous, mostly flattened channels, arranged into sheets within the cytoplasm. Their quantity depends on cellular metabolic requirements (it is absent in mature erythrocytes, but still present in erythroblasts). Cells which synthesize and secrete proteins contain vast amounts of ER. There are two specialized types of ER: the rough – RER (granular) and smooth – SER (agranular). Rough (granular) endoplasmic reticulum – RER (GER)

RER is well developed in protein secreting cells (e.g., exocrine pancreatic cells, fibroblasts, plasma cells etc.). Its rough-surfaced limiting membrane is studded with ribosomes, which become bound to the membrane of ER as soon as the protein synthesis begins. When the synthesis of a polypeptide chain begins, a part of the amino acid sequence – signal recognition particle – specifically recognizes the ER membrane and binds to a receptor in it (a so called docking protein). This signal portion also instructs the growing protein molecule to pass through this membrane into the lumen of RER. Finally, the attachment of sugar groups to most molecules that will become glycoproteins begins in the lumen of RER. The proteins are then delivered by transfer vesicles to the Golgi apparatus. After completion of peptide synthesis, the ribosome detaches from the docking protein and returns free into the cytoplasm. Due to the numbers of ribosomes the RER has a strong affinity for basic dyes and therefore cells specialized for synthesis – 24 –

of proteins have usually amounts of basophilic RER in their cytoplasm. In neurons, the RER is called Nissl bodies.

Fig. 19 Rough endoplasmic reticulum (RER) – 25 –

Smooth (agranular) endoplasmic reticulum – SER (AER)

The SER also forms a membranous (more tubular) network within the cell, but lacks associated ribosomes and is therefore called agranular. It is a vital cell membrane system, because it is the site of cell lipid synthesis, particularly membrane phospholipids. The lipid synthetic enzymes are located on the outer side of SER. The newly formed phospholipids from the lipid precursors are taken through the SER outer membrane bilayer by specific transport proteins (so called flippases). In muscle cells and fibers SER appears in the form of sarcoplasmic reticulum and it stores and releases intracellular calcium ions which regulate the muscle contraction. SER is also abundant in liver cells, where it is involved chemical modifications (biotransformation) of many molecules and also in metabolism of cholesterol and other lipids. In some endocrine organs (e.g., adrenal cortex, Leydig cells of testis, or cells of the ovarian corpus luteum) it is engaged in biosynthesis of steroid hormones.

Golgi complex (Golgi apparatus) This system of flattened and parallel-arranged cisterns, vesicles and vacuoles is situated usually around the nucleus (sometimes shifted towards the apical pole of the cell). It represents an intermediary between the endoplasmic reticulum and the rest of cell. It plays a central role in the process of secretion, being responsible for the chemical modification of secretory products. Equally important is the role in distribution and packing the products of ER into secretory vesicles and also packing hydrolytic enzymes into lysosomes. There are at least two structural and functional parts of Golgi: The nuclear-facing convex cis face receives transport vesicles from ER. On the opposite side of Golgi is the concave trans face, where the Golgi vacuoles accumulate. During transit of material through this Golgi complex glycosylation (adding saccharide side chains), sulphation, phosphorylation and partial proteolysis occur. The final contents of the vesicles can then be distributed to secretory granules, lysosomes, or the plasma membrane. The secretory vesicles which coalesce at the cell membrane can release their content extracellulary in a process of exocytosis. – 26 –

Mitochondria These are the “power plants of the cell”, because of their capability to transform the chemical energy stored in molecules of glucose, fatty acids and amino acids into the production of the energy-rich compound adenosine triphosphate (ATP) from its precursor adenosine diphosphate (ADP) by the process of oxidative phosphorylation. The energy stored in ATP can be liberated again by hydrolysis to ADP and can be applied to various energy-consuming processes in the cell. The ADP is then rephosphorylated to ATP. Mitochondria appear in variable numbers in each cell, but in great numbers in cells that require great amounts of energy (e.g., several thousands of mitochondria in in each liver cell). The mitochondria may be randomly distributed, or they may be concentrated at the sites of high energy utilization. They are usually of nearly spherical or ellipsoidal shape and about 0.5–2 µm long. They are believed to have evolved in eukaryotic cells as symbiotic prokaryotic organisms similar to bacteria. Each mitochondrion has its own DNA (mtDNA) and system for protein synthesis independent of the cell nucleus. Their genetic material is of maternal origin, i.e., derived from mitochondria present in ovum, with no paternal contribution. It is important to know that abnormal mitochondrial DNA can lead to defective cell functions and finally to developmental abnormalities of some organ systems, like muscle and nervous system.

Fig. 20 Mitochondrion

– 27 –

Mitochondria are enclosed by two membranes, separated by a narrow space. The outer membrane is porous and smooth in outline; the inner one is folded into series of extensions called cristae, which project into the interior and increase the whole surface area. The internal mitochondrial cavity is filled by mitochondrial matrix, which contains mtDNA, mtRNA (of all three types), ribosomes and proteins, as well as granules of calcium ions accumulations. The matrix is also the site of enzymes of the Krebs (citric acid) cycle and those concerned with protein and lipid synthesis. On the cristae many tiny globular structures are located – the inner membrane subunits. They consist of a globular head and a slender stalk and their enzymes are mostly involved in oxidative phosphorylation.

Lysosomes Lysosomes are membrane-limited organelles 0.25–0.5 µm in diameter, containing a number of hydrolytic enzymes, operating in an acid pH (acid hydrolases). They represent an intracellular digestive system, and their enzymes can break down all classes of macromolecules. These lytic enzymes are prevented by the enveloping lysosomal membrane from digesting the other cell organelles. The lysosomal enzymes are synthesized in the RER and packaged in small primary lysosomes in Golgi complex. The digestive process occurs in larger secondary lysosomes. The resulting nutrients then diffuse into cytoplasm, while the indigestible remnants remain in so called residual bodies (e.g., accumulation of pigment lipofuscin during ageing). The lysosomes play an important role in the process of phagocytosis, i.e., digestion of material taken up from the extracellular environment (known as heterophagy). The particle becomes at first engulfed by cell membrane in form of a phagocytic vesicle, which then enters the cytoplasm as socalled phagosome. Its membrane then fuses with the membrane of the primary lysosome which empties its enzymes in the phagocytic vesicle, starting the digestion in the newly created secondary vesicle. The residual bodies can be then extruded by exocytosis. In a similar process of autophagy the digested particles are of intracellular origin (organelles or parts of cytoplasm). This process occurs during reconstruction of the cytoplasmic content and is known in cells undergoing atrophy, or some hyperactive secretory cells. – 28 –

Peroxisomes (microbodies) These are small (0.5–1 µm) membrane-bound vesicles of spherical shape. They contain enzymes involved in lipid metabolism. The synthesis of peroxisomal enzymes occurs in RER like secretory proteins.

Nonmembranous organelles and cytoskeleton The cell contains, in addition to the membrane bound organelles, also many nonmembranous structures from which the ribosomes have been already described. In addition, the cytoplasm contains also a set of filamentous proteins in form of microfilaments, microtubules and intermediate filaments. These filamentous proteins are attached to cell membranes to form an internal three-dimensional scaffolding of the cell. They are responsible not only for the form and shape but also for the movement of the cell, for anchoring cells together, transport of material within the cytosol etc. The microtubules can be arranged in bundles and form a special organelle-like centriole, which plays an important role in the development of the microtubular network in dividing cells and in motile cilia. The detailed compositions of these structures follow in the next chapters. Microtubules

These are present in all cells as hollow, unbranched structures, composed of the protein tubulin. Each microtubule is 25 nm in diameter and is composed of protofilaments of alternating alpha and beta tubulin subunits. These subunits polymerize to form protofilaments which are arranged into groups of 13 forming the hollow tubule. They are supporting elements of the cell (analogous to the bones of the body), but they also participate in the movement of certain components inside the cell (organelles, neurotransmitters in axons, melanin in pigment cells etc.). Microtubules also represent part of some organelles, like cilia, flagella, and centrosomes. Centrosome (diplosome)

This is an organelle, present in both animal and plant cells, consisting of a pair of centrioles. It represents an organizing center for polymeriza– 29 –

tion of microtubules in both normal and dividing cells. The centrioles are cylindric structures (0.15 µm in diameter, 0.3–0.5 µm in length), composed of 9 sets of microtubule triplets. The microtubules in a triplet are closely stuck together, and the triplets are joined by protein links. In each pair the long axes of the centrioles are situated at right angles to one another. During mitotic cell division, a great number of microtubules arise and radiate from centrioles, situated on both poles, to the chromosomes and form the so called spindle apparatus. After cell division the microtubules disappear. Centrioles also serve as the basal bodies (kinetosomes) and site of origin of epithelial cilia, and spermatozoal flagella.

Fig. 21 Centrosome and centriole

Microfilaments

These are slender rods about 7 nm in diameter, made up of the protein actin in interaction with myosin. Actin is a globular protein (G-actin) which polymerizes to form filaments (F-actin). In only half of cells does actin appear in the form of filaments. The polymerization is under the control of changes in calcium level, and the degree of polymerization is regulated by various actin-binding proteins. The peripheral zone just beneath the plasmalemma is rich in actin microfilaments, forming a sort of a cell cortex. It is composed of a meshwork of actin and so called actin-linking proteins (e.g., filamin, spectrin, ankyrin etc.). – 30 –

Fig. 22 Actin filament as a part of the contractile apparatus

The microfilaments can form also bundles like in microvilli where the actin is associated with fimbrin and fascin. In all cells actin microfilaments interact with a protein myosin to generate the motile forces. The microfilaments of the muscle cells contain in addition to actin also troponin and tropomyosin. The interactions of these molecules will be described in the description of muscle tissue. Intermediate filaments

In addition to the thin (actin) and thick (myosin) filaments cells also contain intermediate sized cytoskeletal filaments of about 10–12 nm thickness, anchored to transmembrane proteins at special sites on the cell membrane. They are made of proteins which vary between different cell types and functions. In epithelial cells there are cytokeratins, forming usually a tough outer layer (it is the main constituent protein of hair and nails). In muscle tissue (smooth and striated) there is desmin. In tissues of mesenchymal origin (e.g., embryonic undifferentiated cells) are characteristic vimentin filaments. Neurofilaments form a part (next to microtubules and microfilaments) of the neuron cytoskeleton. They also differ from the glial filaments (glial fibrillary acidic protein – GFA), found in astrocytes. The nucleus of all cells contains nuclear lamin, found on the inner side of the nuclear membrane.

Cell inclusions These are the accumulations of metabolites or deposits of varied nature. Proteins are stored in glandular cells as secretory granules and are released – 31 –

periodically by exocytosis. Carbohydrates are stored in the form of the polysaccharide glycogen, seen in the electron microscope as coarse electron dense particles mainly in hepatocytes and muscle cells. Fats (lipids) are stored as non-membrane-bound vacuoles which appear as large clear spaces mostly in the cytoplasm of adipose tissue cells. The next category of cytoplasmic inclusions are the pigments. The red iron-containing pigment hemoglobin in RBCs transports oxygen around the body. In RBCs destroyed by macrophages hemoglobin becomes degraded to two other pigments – brown hemosiderin (contains iron) and yellowish bilirubin. The brown to black pigment of the skin, hair, eye and substantia nigra of the brain is melanin. In tissues from elderly persons appears, particularly in nerve, heart muscle and liver cells, a golden-brown pigment lipofuscin. This “wear and tear” pigment resists digestion by lysosomal enzymes and accumulates in the form of residual bodies in the cytoplasm during ageing.

– 32 –

PART II: THE TISSUES

The tissues are assemblies of cells that share both their embryonic origin and their morphological characteristics, and usually perform similar functions. The term “Tissue” was first used in human anatomy by French surgeon Marie Francois Xavier Bichat (1771–1802). In his books “Traité des membranes” (1800) and “Anatomie générale” (1801) he classified and described, without using a microscope, 21 tissues as basic morphological and physiological units of an organism, as well as a subject of pathological processes. At the present time, we know that despite its complexity the human body is composed of only four basic types of tissue: A. Epithelial tissue B. Connective tissue C. Muscle tissue D. Nerve tissue

A. Epithelial tissue Origin: all three germ layers, i.e. ectoderm, endoderm and mesoderm Function: protection and secretion, absorption, respiration, excretion, reception Morphology: Epithelium is composed of closely aggregated cells (with nearly no intercellular substance). Epithelia line internal and external surfaces of the body or they are organized into structures of higher order (tubules, acini, trabeculae, etc.). Each cell, then, has polarity: one surface, apical, is directed to the external surface or lumen of a hollow organ, and the opposite surface, basal, is oriented toward the underlying connective tissue (basement membrane). Apical and basal surfaces often differ in their morphology and physiology. – 33 –

Apical surface of epithelia

Microvilli – projections of the cytoplasm which enlarge the surface of the cell. They appear mostly in cells responsible for absorption. They contain actin microfilaments anchored in a terminal web (actin + myosin). At the top of the villi is a glycoprotein glycocalyx. Accumulation of microvilli is called a brush border. Stereocilia – a variety of microvilli. These are long and branching finger-like nonmotile microvilli (not cilia!) in epididymis and in inner ear. Unlike kinocilia, stereocilia do not have axonemes. Kinocilia – these are motile processes 2–10 µm in length and 0.5 µm in diameter. They possess a core of 9 pairs of microtubules surrounding two central tubules (so called axoneme). The walls of two peripheral microtubules in a doublet share 2–3 common protofilaments. The doublet tubules are made of tubulin, while arms that extend from subfiber A are of the protein dynein (a molecular motor that transforms chemical energy into mechanical movement). Among the doublets are links composed of another protein – nexin. The central pair of separated tubules is enclosed in a central sheath and linked to the doublets by radial spokes. The energy-dependent movements of cilia and flagella are induced by sliding of adjacent doublets within the axoneme. The energy required for

Fig. 23 Microvilli – 34 –

Fig. 24 Stereocilia and kinocilia

the sliding mechanism of axoneme bending is released from ATP through the enzymatic activity of dynein (ATPase). Basement membrane

is situated beneath epithelia (i.e., on the basal side) and formed by the basal and reticular lamina. The basal lamina consists of a clear layer (also called lamina lucida; 15 nm thickness, contains the multiadhesive glyco– 35 –

protein laminin) and a dense layer (lamina densa; 30–300 nm thick network of very fine fibrils). In places where epithelial cells are in contact with connective tissue, the basal lamina lies on the reticular lamina formed by reticular and collagenous fibers.

Fig. 25 Basement membrane

Cell adhesion

The cell contacts created by cell junctions occur not only between epithelial cells, but also in non-epithelial cells, enabling cell-cell communication (see, e.g., cardiac muscle of smooth muscle). This intercellular contact is enabled by the cell adhesion molecules. There are two main groups of adhesion molecules: 1. Calcium dependent molecules, including cadherins and selectins 2. Calcium independent molecules – these include the immunoglobulin superfamily and integrins Cadherins play a major role in cell adhesion and differentiation, establishing a link between the internal cytoskeleton of a cell and the exterior of another cell. There are about 40 different cadherins. E-cadherin (epithelial cadherin) appears along the lateral cell surfaces to mediate cell-cell attachments in epithelial layers. They form dimers which bind to dimers of the same class of cadherins in the opposite cell membrane (i.e. homophilic interaction). N-cadherin is found in the CNS and in striated muscles. Selectins are bound to carbohydrates, namely to a specific oligosaccharide attached to a protein or a lipid. They belong to the lectin family and participate in the migration of leukocytes from the blood stream into surrounding tissue to reach the sites of inflammation and enable the homing mechanism, i.e., to permit the T-lymphocytes to “home” in lymph nodes. – 36 –

There are three major classes of selectins: P-selectin found in platelets, E-selectin on activated endothelial cells and L-selectin on leukocytes. Ig superfamily proteins mediate both homophilic and heterophilic interactions, i.e., they can bind to identical molecules on another cell or to other members of the family. Their cell adhesion molecule (CAM) is folded into immunoglobulin-like domains. The best known is ICAM – i.e., intercellular cell adhesion molecule, which is expressed on the endothelial surface and facilitates the transendothelial migration of leukocytes from the bloodstream into the tissues. Others are VCAM – vascular cell adhesion molecule, and NCAM – neural cell adhesion molecule. The surface marker CD4 of T-helper lymphocytes also belongs to this Ig-superfamily. Integrins are mainly involved in cell-extracellular matrix interactions. Almost every cell expresses one or several integrins. The cytoplasmic domain of integrins is linked to actin filaments (through connecting proteins), while the extracellular one binds to laminin and fibronectin (components of basement membrane), which interact with various collagen types. The relationship between integrin and extracellular matrix is important for cell migration during embryogenesis. This process can be disrupted by small peptides – disintegrins.

Fig. 26 Junctional complex – 37 –

Junctional complex The boundary between adjacent epithelial cells is formed by tight junctions (zonula occludens) which form a belt around the cell and usually just below them (more basal) are adherens junctions (zonula adherens) with fine filaments which form a belt around the cell. Another important protein complex at the cell-cell junctions is the desmosome (macula adherens) – not in belts but in plaques – which are sites of local cell adhesion and of attachment of the cytoskeleton (tonofilaments of cytokeratin). The last component is the gap junction or nexus – this appears in plaques and mediates flow of current between cells through connexon protein molecules arranged in hexamers passing through both cell membranes. Covering epithelia

form a tissue with a protective function for external and internal surfaces of the body. As in all epithelial tissues, blood vessels are absent; however, nerve endings are present. Simple epithelia These form one layer of cells lying on a basement membrane. Simple squamous epithelium is made up of one layer of flattened cells. E.g., glomerular (Bowman’s) capsule of kidney. A modification of the simple squamous epithelium is represented by endothelium (lining of the whole circulatory system) and mesothelium (lining of the body cavities). These are called false or secondary epithelia because of their mesenchymal origin. Simple cuboidal epithelium is made of cells like paving stones. E.g., thyroid follicles, sweat glands, intralobular ducts of salivary glands. Simple columnar epithelium (cylindrical) is made of tall, prismatic cells. The nucleus is usually closer to the basal part of the cell. E.g., alimentary canal (stomach to rectum), uterus, oviduct. Pseudostratified – a variety of simple columnar epithelium. A single layer of cells which rest on the basement membrane but only the tallest reach the surface. Cells are mostly ciliated. E.g., nasal cavity, trachea.

Fig. 27 Simple epithelia – 38 –

Stratified epithelia The cells are arranged in multiple layers, named according the form of cells of the superficial layer. Stratified squamous – basal layer is columnar, then polyhedral and to the surface more and more flattened, i.e., squamous. They appear in two types: a) Stratified squamous keratinized: the upper horny layer contains dead squamous cells (stratum corneum and stratum desquamans or disjunctum), e.g., epidermis. b) Stratified squamous non-keratinized: with no horny layer on the surface. E.g. upper part of digestive system (mouth cavity to cardia of stomach), vagina, cornea.

Fig. 28 Stratified squamous epithelia

Fig. 29 Stratified columnar epithelium

Stratified columnar – a very rare type. Deepest layer formed by cuboidal cells, then come polyhedral and on the surface columnar cells. E.g., conjunctiva. – 39 –

Fig. 30 Transitional epithelium (urothelium) in contracted and relaxed shape

Transitional – a variety of stratified epithelium. Allows deformation in contractions and stretching. Superficial layer formed by “umbrella” cells (often with two nuclei). Most of the cells are in touch with the basal membrane, but the surface umbrella cells are losing this connection, thus making a separate layer. Appears in urinary passages. Glandular epithelia

The glands can be divided into exocrine which possess ducts and secrete onto an epithelial surface, and endocrine glands which are ductless and secrete hormones into the bloodstream (they will be described in the microanatomy part). Exocrine glands These consist of cells producing secretions (proteins, lipids, glycoproteins). The process of protein secretion proceeds in three steps. 1. Ingestion – amino acids from the bloodstream enter into the cytoplasm. 2. Synthesis – association of the amino acids with tRNA and their transport to the ribosomes which move along the mRNA reading in codons (3 nucleotides) the instructions (message from DNA). Amino acids are inserted into the protein molecule being developed on the ribosome. The ribosomes are attached to the membrane of endoplasmic reticulum in which the newly formed protein molecules are released. Steps 1–2 are common steps of protein synthesis in all cells. In glands, the following third step is added. 3. Extrusion – the secretory product is transferred from RER to the Golgi complex and in secretory vesicles moves to the surface and is extruded by the process of exocytosis. The secretory portion may be composed of one cell type – unicellular glands (such as goblet cells in the respiratory epithelium and in the intestine), or many cells organized into structures of various shapes – multicellular glands. – 40 –

Classification of exocrine glands according to shape According the shape of the secretory units, the exocrine glands can be categorized as tubular (the secretory portion is a straight or coiled tube), or alveolar (also named acinar, the secretory portion is a wide outpouching, having a shape of a flask or grape). An alveolus is very similar to an acinus in its external shape, but alveoli have a wider inner lumen than acini. If the tube ends in a sac-like dilation or if the gland contains tubules and alveoli at the same time, the gland is tuboalveolar. If the duct is unbranched, the gland is called simple, even if the single duct sometimes receives secretions from several secretory units (which is called a simple branched gland then). If the duct is branched into a system or hierarchically arranged ducts, the gland is compound. For example: • simple alveolar glands do not occur in human • simple acinar glands are, e.g., paraurethral glands outpouching from the urethra • straight simple tubular glands are, e.g., intestinal glands of the small and large intestine (also known as the crypts of Lieberkühn) • simple coiled tubular glands are the eccrine sweat glands of the skin • simple branched tubular glands are, e.g., gastric glands (in the body and the fundus of the stomach), or endometrial glands (in the uterine mucosa) • simple branched acinar glands are, e.g., sebaceous glands of the skin • compound tubular glands are the submucosal glands of Brunner in duodenum;

Fig. 31 Types of exocrine glands according to shape – 41 –

• compound acinar gland is, e.g., the parotid gland or the exocrine portion of the pancreas • compound tuboalveolar (tubuloacinar) glands are, e.g., the sublingual and the submandibular salivary glands. Glands according to the mechanism of secretion 1. Merocrine (eccrine) glands: The secretory product is released in small quantities by repeated exocytosis and the shape of the cell does not change substantially. E.g., sweat glands, parotid, pancreas. 2. Apocrine glands: first, the apical portion of the cell accumulates the secretion and then it is lost with the secretory product. E.g., large axillary sweat glands. In mammary glands, the milk lipids are secreted by the apocrine secretion, the proteins (casein) by the merocrine way. 3. Holocrine glands: The secretory product is accumulated in the cytoplasm and the secretion process results in a complete breakdown of the dying cells. E.g., sebaceous glands.

Fig. 32 Patterns of secretion

Glands according to their secretory products 1. Serous – the cell body is basophilic due to the abundant rough endoplasmic reticulum. The cells produce watery secretions with proteins. A serous cell contains rounded nucleus, abundant RER and zymogen granules (i.e. secretory vesicles with temporarily inactive enzymes). E.g., parotid gland, exocrine portion of pancreas. 2. Mucous – the cell cytoplasm is pale. The cells produce viscous mucus. The nucleus is flattened on the cell base, the cytoplasm filled by mucus-containing vesicles that have a high polysaccharide content and therefore in routine methods stain only weakly or not at all. E.g., submucosal duodenal glands of Brunner. – 42 –

3. Seromucous (mixed) glands. Secretory units are composed from mucous cells in tubular part of the gland. The serous cells are arranged in acini, or they form a halfmoon-shaped cap of the acinus, named serous demilune (of Gianuzzi). E.g., submandibular gland (mostly serous) and sublingual gland (mostly mucous). Also the nasal cavity, larynx, trachea and bronchi contain seromucous glands

Fig. 33 Serous and mucous cells

The ducts of compound glands, such as the parotid, submandibular or sublingual glands, have the following parts: 1. Intercalated ducts are lined by squamous or cuboidal cells. Each intercalated duct drains a separate secretory unit. 2. Several intercalated ducts merge into intralobular ducts, that are still surrounded by secretory units. 3. They open into interlobular striated ducts lined by columnar cells with striations in the basal part, caused by infolding of the basal plasmalemma and by elongated mitochondria (ion transporting cells). The striation is not visible in pancreas. 4. These ducts join into interlobular ducts, the lining of which is stratified cuboidal becomes finally stratified. Interlobular ducts merge into one or several major excretory ducts. – 43 –

Fig. 34 Seromucous gland in a section (left) and surface view (right)

Myoepithelial cells These embrace the glandular alveoli or ducts like a basket and that is why they are also called basket cells. They are situated between the secretory or ductal cells and basement membrane. The cells contain actin as well as myosin filaments and can contract and help to release the secretory products. The myoepithelial cells are interconnected by gap junctions and also contain cytokeratin filaments which prove their epithelial character.

B. Connective tissue Origin: mesenchyme Function: connects cells, tissues and organs, supports the body, provides nutrition and defense Morphology: Connective tissue consists of cells and extracellular matrix composed of protein fibers and ground substance. According to the consistency of the ground substance four chief types of connective tissue can be recognized: – 44 –

1. Connective tissue proper 2. Cartilage 3. Bone 4. Blood, lymph and tissue fluid Connective tissue proper

Its main appearance corresponds to its function: to connect skin to the underlying structures and to fill spaces between tissues and organs. Possesses a large amount of intercellular matrix in which many extracellular fibers and bundles of fibers are embedded. There are relatively few cells, which can be either fixed cells, that develop from the mesenchyme in the connective tissue itself, or wandering cells, that develop from mesenchymal hemopoietic stem cells elsewhere in the body and migrate into connective tissue proper by way of the bloodstream. Cells 1. Fibroblasts represent the mother cells of connective tissue. The term fibroblast is used for active stages with ability to proliferate and to produce significant amounts of extracellular matrix. Less active and resting

Fig. 35 Fibroblast and fibrocyte – 45 –

forms are called fibrocytes. The cells are usually spindle-shaped, with an ovoid and pale nucleus with one or two nucleoli, rich rough endoplasmic reticulum, Golgi complex and elongated mitochondria. They do not have phagocytic properties. Fibroblasts synthesize collagen, reticular and elastic fibers, as well as ground substance. Some modified fibroblasts mostly in wound sites contain bundles of actin filaments and dense bodies similar to smooth muscle cells. These cells, called myofibroblasts, are implicated in wound contraction. Other fibroblast-like cells are represented by reticular cells. 2. Reticular cells, which are similar to fibroblasts but differ from them by their star-shaped form. They provide the framework of the lymphoid and hemopoietic organs. The long processes contain tonofilaments and are joined with the neighboring cell processes by desmosomes. They produce reticular fibers which remain in close contact with them. They contain free ribosomes, smooth endoplasmic reticulum and Golgi complex.

Fig. 36 Reticular cell

Fig. 37 Adipocytes

3. Fat cells, also named adipocytes, form white and brown adipose tissue. White (or yellow) adipose tissue forms about 10% of the whole body weight. This consists of unilocular adipocytes, spherical in shape and – 46 –

filled with one large fat droplet (not seen in the slide, because it has been dissolved by the histological techniques). The cell has a signet ring shape with a flattened and eccentric nucleus in a very thin layer of cytoplasm. There is only a little smooth ER and filamentous mitochondria. Brown adipose tissue appears mostly in hibernating animals whereas in other animals and in humans it is rare and located in interscapular region and around the kidney, adrenals, aorta and neck. It is present in large amounts in newborns and in adults it is gradually reduced. The cells are multilocular and contain small droplets of lipids and a lot of mitochondria which can produce 100 times more heat than other cells (under the influence of norepinephrine). 4. Pigment cells, melanocytes, are of ectomesenchymal origin (i.e., derived from neural crest). They are located usually at the dermo-epidermal junction and enter between the basal cells of epidermis. They can have various shapes and become branched with long cytoplasmic extensions that ramify between the cells of deep epidermal layers (not connected with them by desmosomes). They synthesize the pigment melanin in membranous vesicles (parts of Golgi complex) called melanosomes. The mature melanin granules are ellipsoid and can be transferred into epidermal cells (keratinocytes). Although the number of melanocytes is comparable among various human populations, their activity and amounts of melanosomes may vary significantly, thus contributing to the skin pigmentation.

Fig. 38 Melanocyte and mastocyte – 47 –

5. Mastocytes (mast cells, heparinocytes) – the precursors of these cells come from the mesenchymal cells of the bone marrow. Their name comes from German word “Mast” which means fatten. They appear in loose connective tissue along the vessels, intestine, thyroid gland etc. They are large oval cells filled with many secretory granules, a small nucleus, RER and Golgi complex. Granules contain heparin (sulphated glycosaminoglycan – an anticoagulation factor) and histamine – a mediator of inflammation (increases the permeability of vessels and allows the plasma proteins, e.g. immunoglobulins – to leak from them into the tissues). The granules show a special staining property – metachromasia – the granules are stained in a different color from the color of the dye (e.g. toluidine blue stains purple instead of blue). They very much resemble the basophils. They possess receptors for IgE on the cell membrane and play a role in allergic reactions. 6. Macrophages – these cells, also known as tissue histiocytes, are related to bone marrow precursors which mature into monocytes. The monocytes migrate from the blood circulation in connective tissue and there they can mature under the influence of immunoglobulins or the helper type of T-lymphocyte into macrophages. They are usually round to oval in shape and have an irregular and eccentrically placed, dark-staining nucleus, well developed Golgi complex, RER and a number of phagosomes and lysosomes. Their main function is phagocytosis. The antigen, phagocyted by a macrophage (antigen presenting cell), becomes stored

Fig. 39 Macrophage and plasmocyte – 48 –

within a phagocytic vesicle. This vesicle fuses with a lysosome to become a phagosome. The lysosomal hydrolytic enzymes break down the antigen into small peptide fragments which bind to MHC molecules (major histocompatibility complex) inserted in the phagosome membrane. The phagosome fuses with the plasma membrane and the peptide-MHC is exposed to T-lymphocyte, which then secretes interleukins. Interleukins bind to B-lymphocytes and they increase their number (after clonal divisions) and differentiate into plasma cells. 7. Plasma cells (plasmocytes) are the descendants of mesenchyme-derived B-lym­pho­cytes that migrated in connective tissue. They are rounded in shape, the nucleus is spherical, lies eccentrically and its chromatin shows a “cart-wheel” effect (or clockface appearance), the nucleolus is large. The basophilic cytoplasm is full of RER, ribosomes and Golgi complex, mitochondria and secretory vesicles, all of which are critical for plasma cells’ function, which is to synthesize and secrete the immunoglobulins (humoral antibodies of all classes e.g. IgM, IgG, IgA, IgE etc.). 8. Some more originally mesenchymal cells are related to the loose connective tissue, like endothelial cells (lining of circulatory system which we discussed already in the chapter on Epithelial tissue) or pericytes (perivascular cells which shall be mentioned in microanatomy of vessels). In loose connective tissue also various types of blood cells can be found. Extracellular matrix This consists of fibers and an amorphous ground substance. Fibers

There are three main types of protein fibers: 1. Collagen fibers – they appear white, unbranched, wide, wavy and stain pink with eosin. They are firm and resist stretch. The fibers swell in acids and alkalis, and when boiled they dissolve and yield gelatin (glue). They can be digested by pepsin but resist trypsin. In transmission electron microscope, the fibers appear striated (cross striation) and show axial periodicity. They can be 1–20 µm thick. The thickness depends on the amount of fibrils which form a collagen bundle. The fibrils are conjoined with a protein substance and are about 0.3–0.5 µm thick. They consist of protofibrils (or microfibrils – 40 nm – 49 –

in diameter), containing tropocollagen molecules. Each molecule measures 280 nm in length and 1.5 nm in width, and it extends beyond its neighbor by one quarter of its length. The overlapping regions cause the axial periodicity. The collagen fibril formation is a self-assembly process that occurs in the extracellular matrix.

Fig. 40 Assembly of a collagen microfibril, a fiber and a bundle of fibers

Collagen is the most abundant protein of our body (30% of the whole body’s protein content). It is composed of three principal amino acids: glycine, proline, and hydroxyproline. There are many types of collagen – the most important are these five:  Type I – produced by fibroblasts and osteoblasts, forms collagen fibers, appears in loose and dense connective tissue and bones.  Type II – produced by chondrocytes and appears in fibrils in hyaline and elastic cartilage.  Type III – produced by fibroblasts, smooth muscle cells and endothelial cells, forms reticular fibers, appears in loose connective tissue, arteries, liver, spleen, kidney, lung etc.  Type IV – produced by epithelial and endothelial cells, muscle cells and Schwann cells. Forms the basal laminae and basement membranes.  Type V – not well known – produced probably by fibroblasts and appears in fetal membranes and bone. 2. Reticular fibers are fine and delicate fibers which branch and form a supporting network. They are argyrophilic (stain by silver) and appear around the vessels, nerves, muscles, adipose cells, alveoli, in basement membranes, lymph nodes, spleen, glands etc. They do not swell in acids and when boiled they do not give gelatin. Most fibers in fetal connective tissues and in healing wounds are of the reticular type, considered as immature collagen fibers and called precollagenous, and they are grad– 50 –

ually replaced by collagen fibers. Reticular fibers are made up of narrow bundles of fibrils with the periodic structure typical of collagen. They contain more hexoses than in collagen (this is the reason for being able to reduce silver cations to black atomic silver). Although reticular fibers are considered a separate type of fiber, they are actually composed of type III collagen. 3. Elastic fibers are yellow, long, thin, homogenous, non fibrilar and branched fibers but also membranes. They are 0.5–4 µm thick. They appear, e.g., in yellow ligaments, vagina, elastic cartilage, and in blood vessels, where they also occur in form of fenestrated membranes. They can be stained by orcein or resorcin-fuchsin. They can not be digested by trypsin but elastin can be hydrolyzed by pancreatic elastase, they do not yield gelatin and are resistant against acids and alkali. They consist of amorphous protein elastin in the central region, surrounded by microfibrils (structural glycoprotein subfibrils of 10 nm). In addition to glycine and proline elastin contains also desmosine and valine amino acids, which form hydrophobic domains that are responsible for elastic coiling of the molecules.

Fig. 41 Reticular fibers and elastic fibers

Ground substance The amorphous jelly-like substance in which the cells and fibers are embedded. It is derived from connective tissue cells and its consistency is liquid – a loose gel. Chemically it is a protein-polysaccharide complex, i.e. – 51 –

glycoprotein, formerly called mucopolysaccharide. There are two groups of glycoproteins: 1. Glycosaminoglycans are complex substances of a carbohydrate nature consisting of a hexosamine + uronic acid combined with protein. Two main kinds are: Hyaluronic acid consists of glucosamine + glucuronic acid with about 2% protein only. Forms intercellular substance of fetal and adult tissues (umbilical cord, vitreous humor of the eye, synovial fluid and cartilage). Represents a barrier against bacteria (however, some bacteria posses enzyme hyaluronidase that depolymerizes the acid, thus facilitating the spreading of infection through the extracellular matrix). Sulphuric esters of glycosaminoglycans The most known is chondroitin sulfate – consists of galactosamine + glucuronic acid with about 20% protein. Appears in cartilage, bone, cornea, skin, notochord etc. Similar are: dermatan sulphate (skin, tendon), heparan sulphate (lung, liver, basal laminae) and keratan sulphate (cornea, intervertebral discs). 2. Structural glycoproteins are predominately protein to which carbohydrates are attached. They are responsible for cell adhesion. Fibronectin is synthesized by fibroblasts and epithelial cells. Binds cells, collagen and glycosaminoglycans. Laminin appears in basal laminae. Responsible for adhesion of epithelial cells to basal lamina. Chondronectin appears in cartilage. Responsible for adhesion of chondrocytes to collagen. The types of connective tissue proper 1. Loose connective tissue (the cells of the ground substance predominate) Mesenchyme – nonspecialized embryonic connective tissue contains mesenchymal cells and reticular fibers. Mucoid connective tissue – star-shaped fibroblasts and jelly-like intercellular matrix. Umbilical cord (Wharton jelly), dental pulp, iris. Collagenous connective tissue – fills spaces between tissues and organs. Consists of fibroblasts, fibrocytes and all wandering types of cells, collagen and elastic fibers. Appears in mucous membranes and in submucous layers. Adipose tissue – adipocytes and reticular fibers. White adipose tissue appears around the kidney, adrenal, mesentery, deep layers of skin (pan– 52 –

Fig. 42 Mesenchyme and loose connective tissue

niculus adiposus). Brown adipose tissue in fetuses, newborns and infants. Also present in hibernating animals. 2. Dense connective tissue (the fibers predominate) Irregular – collagen fibers form a felt structure (rare elastic and reticular fibers). Appears in dermis and fibrous capsule. Regular – including tendons, where collagen fibers form primary bundles (between are fibroblasts). Aponeuroses have the same structure but in flat sheets. Ligaments are elastic connective tissue (elastic and collagen fibers, appear in vertebral column, e.g., yellow ligaments).

Fig. 43 Regular dense collagenous (left) and elastic (right) connective tissue – 53 –

Cartilage

Cartilage is a semirigid supporting tissue with a firm intercellular matrix. Related to its supporting function it is closely associated with bones which mostly develop on the basis of a cartilaginous model. Most of the fetal skeleton is formed from cartilage. Genetically (mesenchymal origin) and morphologically it corresponds to the connective tissue. Boiled cartilage yields glue. Cartilage consists of cells (chondrocytes) and intercellular matrix (ground substance and fibers). Mature cartilage lacks blood vessels. There are three slightly different subtypes of cartilage. Hyaline cartilage It is of white appearance in the fresh state. It forms most of the fetal skeleton. In postnatal life: articular cartilages of joints, costal cartilages (ventral ends of ribs), cartilages of the nose, larynx, trachea, bronchi. Hyaline cartilage occurs usually in plates covered with a vascular fibrous membrane, i.e. perichondrium (missing in the articular cartilage in joints). Perichondrium contains fibroblasts, collagen fibers and many vessels which do not penetrate into the matrix. The inner part of the perichondrium forms a chondrogenic layer with chondroblasts which produce intercellular matrix. The chondroblasts buried in cartilage matrix mature into chondrocytes. The daughter cells remain in matrix cavities – lacunae – and form cell nests – isogenous groups. The wall of a lacuna thickens in a more basophilic capsule which represents a deposit of chondroitin sulphate produced by the chondrocytes. In addition to proteoglycans the chondrocytes produce type II collagen and chondronectin. They contain protrusions and organelles for protein synthesis (RER). The saccharides and sulphates are incorporated in Golgi complex where they are combined with proteins to form mucopolysaccharides of the matrix. The chondrocytes are nourished by fluid exchange with matrix (from blood vessels in perichondrium). The intercellular matrix stains with basic dyes. It is formed by chondromucoproteins (mucoprotein + chondroitin-sulphate). It contains a dense network of fine collagen fibers. They can not be seen in normal slides because they have the same refractive index as the ground substance, but can be demonstrated by silver impregnation or by digesting the tissue with trypsin. – 54 –

Fig. 44 Hyaline cartilage and chondrocyte

Elastic cartilage is adapted to repeated bending. It appears in epiglottis and external ear. It resembles hyaline cartilage but contains in addition to collagen fibers an amount of elastic fibers (can be demonstrated by orcein or resorcin-fuchsin). There is perichondrium on the surface and the chondroblasts and chodrocytes produce all components of the matrix, including elastin. The cells are embedded in lacunae and do not form so frequently, as in hyaline cartilage, small isogenous groups. – 55 –

Fibrocartilage This tissue is transitional in form between dense connective tissue and hyaline cartilage. Appears in places of tendon insertions, pubic symphysis, intervertebral discs and intraarticular menisci. It possesses no perichondrium, and contains small amount of matrix among abundant and thick bundles of collagen fibers, which form irregular bundles or are arranged in parallel and sandwich the chondrocytes.

Fig. 45 Fibrous cartilage

Bone

Bone represents a variety of connective tissue, the ground substance of which is impregnated with calcium salts (97% of the calcium in the whole body). Apart from the rare fibrous bone which appears in dental cement and bony tuberosities only, most of the skeleton is formed by lamellar (Haversian) bone, where layers of osteocytes are found between the lamellated bone matrix. Therefore, lamellar bone is made up of layers (lamellae, plates) of calcified interstitial substance. The bone cells, osteocytes, are situated in lacunae, connected in a continuous system by bone canaliculi. Osteocytes send their processes which are joined to those of other osteocytes via gap junctions. They possess reduced rough endoplasmic reticulum and Golgi complex and condensed chromatin. The bone matrix contains calcium and phosphorus in the form of hydrated hydroxyapatite [Ca10(PO4)6(OH)2].nH2O crystals and amorphous calcium phosphate Ca3(PO4)2. The organic substance is of type I collagen and – 56 –

Fig. 46 Osteocytes

glycoprotein ground substance. The collagen fibers run in parallel, oriented in neighboring lamellae in different directions. All bones are lined on both external and internal surfaces by dense connective tissue – periosteum and endosteum, both containing osteogenic cells. Both epiphyses of a long bone are covered by an articular hyaline cartilage (with no perichondrium). Two kinds of lamellar bone exist, namely the spongy bone and the compact bone.

Fig. 47 Long and flat bone – 57 –

Spongy (cancellous, trabecular) bone It consists of bony trabeculae and spicules with large spaces among them (which contain bone marrow). This type of bone appears in epiphysis of long bones, diploe of flat bones, in ribs and vertebral bodies.

Fig. 48 Spongy bone

Compact bone Compact bone forms dense bony tissue with lacunae and narrow canaliculi (which contain osteocytes with processes). In the compact bone the lamellae are concentrically arranged to form osteons (Haversian systems). The diaphysis (and lateral part of epiphysis) is covered by periosteum which consists of an outer fibrous layer (collagen fibers and fibroblasts) and inner osteogenic layer (spindle shaped osteoprogenitor cells, which develop in osteoblasts). Periosteum becomes fixed to bone by strong collagenous fibers which penetrate into the bone – Sharpey’s fibers. The inner surface of bone marrow cavity is lined by thin connective tissue which contains one layer of osteoprogenitor cells – endosteum. The function of these membranes is nutrition (penetration of blood vessels into the bone through Volkmann’s canals) and production of new osteoblasts for growth and repair of bone. The bone lamellae underneath periosteum and endosteum are arranged concentrically, forming outer and inner circumferential lamellae. Between these peripheral lamellae many osteons (Haversian systems) are situated. These systems are formed by 4–20 concentric lamellae which contain the osteocytes. These cells are interconnected by their processes, through – 58 –

Fig. 49 Compact bone

the canaliculi. In the center of each system a Haversian canal contains blood and lymphatic vessels, nerves and some connective tissue. Among the Haversian systems are irregular (triangular) groups of parallel lamellae called interstitial lamellae, as remnants of destroyed systems during the growth of the bone.

Fig. 50 Osteon – 59 –

Fig. 51 Section of compact bone showing the lamellas of an osteon

The matrix of the lamellae, i.e. intercellular substance of bone, is impregnated with insoluble calcium salts (amorphous calcium sulfate, hydroxyapatite crystals). This inorganic part forms about 50% of the whole matrix. Organic matter contains twice as much collagen (type I) than in the matrix of a hyaline cartilage. The collagen fibers are oriented in parallel in each lamella, but in neighboring lamellae in different directions, to enable the bone to withstand bending, twisting etc. The amorphous ground substance contains glycosaminoglycans (chondroitin sulfate and keratan sulphate). There is a difference between calcification (deposition of calcium salts in any tissue) and ossification (process of bone tissue formation). Ossification Ossification means the process of bone histogenesis. Some bones develop directly from mesenchyme (i.e., embryonic connective tissue). This process is named intramembranous ossification. In that way the bones of face, cranial vault, definitive mandible (embryonic lower jaw is formed by Meckel’s cartilage which does not ossify) and clavicles develop. But the majority of bones develop on the basis of a cartilaginous model, which becomes replaced by bony tissue – endochondral ossification. The histogenic process is very similar: the transformation of embryonic connective tissue i.e., mesenchyme into the bone by the activity of osteoblasts. – 60 –

Intramembranous ossification The primitive connective tissue becomes richly vascularized, the cells multiply and form reticular and collagen fibers. The young fibroblasts increase in size, and form clusters (bone blastema). They differentiate, become polyhedral and arranged in rows, and they are transformed in osteoblasts. These cells are basophilic, with GER and Golgi apparatus. They produce an enzyme-alkaline phosphatase which hydrolyses calcium phosphoric ester (circulating in the blood) to form free salts – calcium phosphate. The original matrix has been replaced by a new interstitial substance which is only slightly calcified – osteoid. The osteoblasts divide and their daughter cells become embedded in the ground substance – these are the osteocytes (less basophilic, less GER and Golgi apparatus). In that way the first trabecules of primary bone have been formed.

Fig. 52 Intramembranous ossification

Fig. 53 Primary bone trabeculae – 61 –

These trabecules are rapidly absorbed by the activity of multinucleated cells of macrophage type – osteoclasts. They are motile, branched, and slightly eosinophilic with many lysosomes. Most probably develop by fusion of monocytes and they secrete proteolytic enzymes responsible for the bone resorption. The cavities produced by this resorption are called Howship’s lacunae. Simultaneously new bone lamellae are laid down by the activity of the next wave of neighboring osteoblasts. Endochondral ossification This is the process in which the hyaline cartilage degenerates to be replaced by bone. It has several stages. The whole process and histological changes during the endochondral ossification can be summarized as follows: 1. Swelling of cartilage cells in the center of the cartilaginous structure (due to lack of nutrition) and arrangement of the chondrocytes into rows. 2. Development of the collar bone around the diaphysis (intramembranous ossification on the basis of connective tissue of the perichondrium). 3. Calcification of the cartilage matrix – formation of the primary ossification center. 4. Invasion of a vessel with mesenchymal cells from the perichondral (now remodelled into periosteal) tissue. 5. Resorption and excavation of the calcified cartilage by the chondroclasts (now becoming osteoclasts, i.e. macrophages of vessel mesenchyme origin) and formation of the primitive marrow cavity. 6. The branches of vessels continue to phagocytose (along a line of erosion) the degenerated cartilaginous cells situated in the zone of hypertrophy and calcification. These cells are replaced from the zone of proliferation and maturation, which develop from the resting cartilage in the epiphyseal plate. 7. Mesenchymal cells of the vessel develop into the cells of primitive marrow and are then transformed into osteoblasts. 8. Osteoblasts lay down the ground tissue (osteoid) in which their daughter cells become embedded as osteocytes. 9. Destruction of this newly formed primary bone by osteoclasts and excavation of Howship’s resorption lacunae (see intramembranous ossification), around which the circularly arranged lamellae develop (osteons). – 62 –

Fig. 54 Principles of endochondral ossification

In an epiphyseal growth plate, the following zones of endochondral ossification can be distinguished: 1. The resting zone of normal hyaline cartilage 2. The proliferation zone of rapidly dividing chondrocytes, which are arranged into columns. 3. The hypertrophic zone of enlarged chondrocytes – 63 –

4. The calcification zone, where the cartilage matrix is provisionally calcified by mineral salts. 5. The erosion zone, where the chondroclasts destroy the cartilage, thus making space for new bone tissue 6. The ossification zone, where primary bone trabeculae are formed by osteoblasts.

Fig. 55 Zones of endochondral ossification (upper scheme) and cross section showing the penetration of blood vessels into an ossification center (lower scheme) – 64 –

Tooth

The tooth represents a complex structure which consists in addition to the soft pulp of three different hard tissues: dentin, enamel and cement. Even though the enamel does not correspond to the criteria of the connective tissue (because of its ectodermal origin), we present in this chapter the description of all these integrated tissues together. To understand the structure of the tooth we have to describe at least the principles of its development. Development of the teeth The first sign of tooth primordium appears at about 6 week of pregnancy as a thickening of the oral ectodermal epithelium which proliferates and forms a bud. This tooth bud is accompanied by the condensation of the surrounding mesenchymal cells (i.e. ectomesenchyme, of neural crest origin). Therefore, the epithelial-mesenchymal interactions are the most important processes of tooth development, which can be characterized as a history of interactions between ectodermal epithelial cells, from which the enamel-depositing ameloblasts develop, and neural crest-derived ectomesenchymal cells, from which dentin-depositing odontoblasts develop. These processes are under the control of specific (MSX) homeobox genes, which then activate a cascade of molecular changes, leading to the expression of various adhesive molecules (syndecan, tenascin, fibronectin, growth factors etc.) involved in early tooth development. The proliferation of the oral epithelium forms a continuous horseshoe-shaped dental lamina in both jaws. Shortly after its formation, the dental lamina shows on each side of the jaw five regions of intensive mitot-

Fig. 56 Primordia of two dentitions – 65 –

ic activity, representing the future primordia of the deciduous teeth (2 incisors, 1 canine, and 2 molars). The first bud stage is followed by the cap stage. The ectodermal part forms the enamel organ in which the stratified epithelium becomes subdivided into flattened outer and high inner enamel epithelium (preameloblasts). The epithelial cells between these layers become more and more reticulated. The underlying mesenchyme cells are condensed in the dental papilla, with the cells on its surface, forming a layer of preodontoblasts.

Fig. 57 Early stages of tooth development

The enamel organ becomes surrounded by the condensed mesenchyme, called the dental follicle (sac). On the labial side of each enamel organ the remainder of the dental lamina produces solid epithelial buds of permanent teeth (10–12 week). These are the 20 counterparts of the deciduous teeth. The dental lamina then extends gradually backwards and the remaining 3 germs of permanent teeth are formed (the primordia of the last two molars are not formed until after birth). During the next bell stage the inner epithelium of the enamel organ differentiates into ameloblasts which deposit the enamel matrix. The production of enamel occurs only in the future crown region under the influence of the stellate reticulum which fills the gap between the both enamel epi– 66 –

Fig. 58 Bell stage of tooth development

Fig. 59 Enamel organ

thelia. Ameloblasts project on their base into conical extensions – Tomes’ processes (with numerous secretory granules) which are sites of secretion of the enamel matrix. This matrix is composed of unique enamel proteins which direct the mineralization of enamel into the hardest tissue in the – 67 –

body. After the end of secretory phase, the ameloblasts regulate the maturation of enamel, and they degenerate during tooth eruption. The preodontoblasts of the dental papilla differentiate into odontoblasts, which secrete procollagen, which then matures in collagen fibrils of predentin. Mineralization of these fibrils results in formation of dentin. Dentin resembles bone in its composition although the histological appearance is different, as the secretory odontoblasts do not get incorporated into the dentin matrix. Each odontoblast sends its cytoplasmic process – Tomes’ fiber, which becomes embedded in dentin and contributes to the formation of dentin tubules. Odontoblast cell bodies remain as a confluent cell layer between the dentin and the cells of dental pulp. At this stage also the nerve fibers enter the papilla. After the enamel organ has formed the enamel of the crown, both sheets of its enamel epithelium grow down into the future root area. They form a two-layered epithelium because they are not separated by stellated epithelium, which cannot therefore perform its enamel inductive potency in the root region; instead, the inner enamel epithelium has retained its general potency to initiate the differentiation of the dentin forming odontoblasts on the pulp surface. This epithelial root sheath (of Hertwig) is a simple tubular structure in case of a tooth with a single root. In multi-rooted teeth this simple tube becomes subdivided into separate tubes, depending on the number of roots. This is achieved by the inward growth of horizontally directed processes of epithelium from the epithelial root sheath, which meet, fuse and produce separate, epithelial surrounded parts of the subdivided root pulp with odontoblasts. The epithelial sheath breaks gradually up (the remnants of it are known even long after tooth eruption as rests of Malassez) and the newly formed dentin induces the differentiation of cementoblasts from the surrounding inner layer of the dental follicle. These cementoblasts form then the cement – the fibrous bone covering the root. The middle layer of the follicle develops in the periodontal ligament and the outer one in the alveolar bone. Histology of the tooth components Enamel This is the most highly calcified and hardest tissue of the body. It consists almost entirely of calcium salts (95% hydroxyapatite) and only 0.5 % comprises organic substances, produced by ectodermal ameloblasts (pro– 68 –

teins amelogenin and enamelin). The enamel consists of some million slightly flattened hexagonal rods or prisms, oriented perpendicular to the dentine surface, and having a wavy arrangement. The bends of the rods in two neighboring zones cross one another. The crossings of groups of rods appear as light and dark lines – the Hunter-Schreger bands. They are curved with the convexity rootwards and occupy about two thirds of the enamel thickness. Other bands – incremental lines (of Retzius) – represent the former outline of the enamel at succeeding stages of its formation. The free surface of the enamel is covered by a thin primary enamel cuticle (named also Nasmyth’s membrane), which represents the final product of ameloblasts.

Fig. 60 The tooth – 69 –

Fig. 61 Formation and structure of the enamel

Dentin Forms the bulk of the tooth substance and gives the basic shape to each tooth, and is yellowish in color. It is less hard than enamel, but harder than either bone or cement. It consists of an organic (20% – most of it collagen) and an inorganic (80%, mostly hydroxyapatite crystals) part. It has a radially striated appearance because of minute canals, the dentinal tubules, which radiate from the pulp cavity, curving and branching toward the periphery. The layer of dentin immediately surrounding each tubule is more refractive and is called the lamina limitans (also sheath of Neumann). The tubules are penetrated by cytoplasmic processes (Tomes’ fibers) from the odontoblasts, situated outside the dentin on the surface of the pulp. The odontoblastic process with some smaller lateral branches extends through the unmineralized predentin before it enters into tubular channels in the mineralized dentin. The calcifying inorganic element of the dentin appears in the organic matrix as globules (calcospherites) which fuse to form a homogenous substance. In some areas near the amelo-dentinal junction the globules may not fuse and the organic matrix remains uncalcified. It forms mainly in the crown region a layer of interglobular dentin, which appears black in dried sections as so called interglobular dentin spaces (lacunes of Czermak). – 70 –

Fig. 62 Odontoblasts

Fig. 63 Dentin

A similar layer of granular appearance can be seen immediately beneath the cement and is known as Tomes’ granular layer. Cement The cement covers the whole root of a tooth as a thin layer of a fibrous bone. It is yellowish in color and less hard than dentin. It is composed of an – 71 –

organic matrix and of an inorganic element. The organic matrix consists of collagenous fibers embedded in an amorphous cementing substance which contains acid mucopolysaccharides. The inorganic element is represented by calcium salts in the form of apatite molecules. It is deposited as submicroscopic crystallites in the cementing substance among collagenous fibers. Two types of cement are recognized depending on the presence or absence of cells – acellular (primary) and cellular (secondary) cement, deposited in layers. Deposition of cement continues throughout life. Acellular cement forms the innermost layer of cement and consists of layers of collagen fibers (Sharpey’s fibers) formed by the fibroblasts of the periodontal membrane. The cellular cement is mostly found at the apical region of the root. The cells – cementocytes – are irregularly distributed. They lie in spaces – lacunae – like the osteocytes in bone. The processes of cementocytes penetrate in fine canaliculi and often anastomose with those of other cells. In both acellular and cellular cement incremental lines run roughly parallel with the root surface and represent intervals between successive depositions of cement. Periodontal membrane Consists of bundles of collagenous fibers which pass from the cement to the periosteum of the tooth alveolus, to adjacent teeth or into the gingival tissue surrounding the tooth. These fibers are known as the principal fibers of the periodontal membrane and are bound together by a cementing substance. They can be divided into groups according to the direction in which they run or according to their function. Oblique fibers are most common and run obliquely inward to the root. In the region of the neck the fibers run horizontally and form a cervical group. From the neck of the tooth a group of fibers pass to the very rim of the socket and are known as the alveolar crest fibers. Another group of fibers also run from the neck radially into the overlying gum as gingival fibers. The pulp It occupies the central cavity of each tooth and consists of loose connective tissue, on the surface of which is a layer of the highly differentiated cells – odontoblasts. They are tall columnar cells with an oval nucleus on their base, rich granular endoplasmic reticulum, Golgi complex and mitochondria. Each odontoblast has a long cytoplasmic process (Tomes’ fiber) which passes into the tubule of the predentin and traverses the whole thick– 72 –

ness of the dentin. Immediately beneath the odontoblast layer is a cell-free zone (basal zone of Weil). The subodontoblastic pulp contains spindle shaped fibroblasts, smaller undifferentiated mesenchyme cells, as well as macrophages. In the ground substance of the pulp containing glycosaminoglycans are fine and irregularly arranged collagen and fibrils. The thicker fibers, which pass the odontoblastic layer (mostly during the dentinogenesis stage), and mingle with the thin fibrils of the pulp, are known as the Korff fibers. We can also find reticular fibers in the vicinity of cells, but no elastic fibers. The whole pulp is highly vascularized and innervated. The nerves and vessels enter the apical foramen together and form numerous branches in the pulp. Some nerve fibers lose their myelin sheath and form underneath predentin a marginal plexus, branches of which, sensitive to pain, can enter the dentinal tubules for some distance. Alveolar bone and gingiva Alveolar bone is that part of the facial skeleton of upper and lower jaws which forms the alveoli and is in immediate contact with the periodontal membrane. It is made up of a surface cortical layer of dense bone and interior zone of cancellous bone. It differs from the typical bone because the collagen fibers are not arranged in the lamellar pattern, but appear in bundles which penetrate from the bone through the periodontal membrane into cement. The gingiva is a mucous membrane bound to periosteum of the jaws. It is covered by stratified squamous epithelium, which is attached to the crown by means of a cuticle which forms the epithelial attachment. It represents the union of the mouth epithelium with the epithelial derivative enamel. Between the enamel and the epithelium is a small deepening – the gingival sulcus. Blood

Blood consists of a fluid called plasma in which float the formed elements of the blood. The formed elements are: erythrocytes, leukocytes and blood platelets. Blood forms about 7–9% of body weight, which corresponds to a volume of approx. 5–6 litres in an average adult. Its pH is regulated to stay within the narrow range between 7.36 and 7.44. – 73 –

The proportion of blood occupied by formed blood cells is called hematocrit (HCT, or packed cell volume, PCV) and is normally about 45%. The remaining 55% of blood volume is occupied by the plasma. Plasma Plasma is the liquid extracellular material that transports the nutrients and products of metabolism. It contains about 90% water and 7% proteins (albumins, globulins, fibrinogen) and the rest include inorganic salts, lipoproteins, vitamins and hormones. Plasma that lacks coagulation factors is called serum. Erythrocytes Erythrocytes, i.e. red blood cells (RBCs) are actually not complete cells – during development in humans and in all mammals (not in other vertebrates!) the nuclei are lost. Most of the other organelles are lost as well, which results in no protein synthesis capability. The absence of mitochondria means that the erythrocytes must use anaerobic methods to produce ATP. The RBC count in adults ranges between 3.8–5.2 million/1 microliter, i.e. per 1 mm3 in female, and 4.0–5.8 million/1 mm3 in men. There are approximately 25 billion RBCs in the whole body and their total surface is about 3 500 m2. In a newborn there are about 6.8 million/microliter (within one week this falls back to 6 million), in an 11- year-old 5.5 million. They are of biconcave shape of 7.2–7.8 µm in diameter, and about 2.6 thick in the periphery and 0.8 µm thick in the center. In fresh conditions, the upper range of the diameter is true, but in a peripheral blood smear on histological slides, the diameter observed is slightly smaller. In hypotonic solution RBCs swell and undergo hemolysis (isotonic solution is approx. 0.85% NaCl). RBCs greater than 9 µm are called macrocytes, less than 6 µm are microcytes.

Fig. 64 Erythrocyte – 74 –

RBCs contain 60% water and 33% conjugated protein – hemoglobin, the remainder consists of other proteins and lipids. Hemoglobin (15 g Hb per 100 ml in average) is a chromoprotein which consists of globin (a sulphur-containing protein) and heme (an iron-containing pigment – feroprotoporphyrin). It is able to combine with oxygen to form oxyhemoglobin and to release oxygen in tissues – reduced hemoglobin (carbaminohemoglobin). The RBC plasmalemma contains about 40% lipids, 50% proteins and 10% carbohydrates. Half of the proteins are integral proteins. On the external surface are oligosaccharides which determine the blood groups. The life span is about 100–120 days. The old RBCs are removed by macrophages of the spleen, liver and bone marrow. The young RBCs which contain remnants of some organelles (but no nucleus!) are called reticulocytes (about 1% of all RBCs in circulation). They remain in blood circulation for 24 hours. If they contain remnants of nucleus (DNA), they are called Howel-Jolly bodies (1–2 granules). Leukocytes The white blood cells (WBCs) are classified into 2 groups according the presence and type of granules in their cytoplasm: granulocytes (polymorphonuclear leukocytes) which possess specific granules and agranulocytes (mononuclear leukocytes) with no specific granules. Granulocytes

Fig. 65 Neutrophilic, eosinophilic, and basophilic granulocytes White blood cell differential count (all WBCs=100%) Granulocytes

Agranulocytes

Neutrophils 45–70%

Lymphocytes 20–45%

Eosinophils 0–5%

Monocytes 2–10%

Basophils 0–2%

– 75 –

Neutrophils Count: 45–70% of WBC Size: 12–15 micrometers in diameter Life span: in blood several hours, in connective tissue 1–4 days. They develop in myeloid tissue of bone marrow and at maturity are released in the blood circulation. Nucleus: consists of 2–5 lobes, connected with delicate strands, contains coarse chromatin densely packed, stains deeply blue with basic dyes. No nucleolus is visible. In female a drumstick-like appendix on one of the lobes (Barr-body, inactivated X-chromosome) appears in about 3% of nuclei of neutrophils. There are 3 lobes in average – Hynek’s nuclear figure = 2.7 (e.g., number of lobes in 100 neutrophils divided by 100). The Arneth formula is a useful concept used for assessing the average age of the population of neutrophils in an individual. The neutrophils are arranged according the number of their lobes from immature nonsegmented “bands” to overmatured 5 lobes. A left shift means prevalence of young neutrophils (less than 3 lobes) and often occurs as a part of reaction to bacterial infection. A right shift denotes prevalence of older neutrophils (more than 3 lobes) in circulation and may indicate e.g., insufficient formation of new cells in the bone marrow. Arneth formula Nuclear lobes

1

 2

 3

 4

5

[%] of neutrophils

5

35

41

17

2



left shift

← mean nuclear figure (2.7) →

right shift

Cytoplasm: occupies more space than nucleus and contains a few ribosomes, GER, mitochondria. Two kinds of granules: 1. Fine specific granules of lavender (pink) color. They are neither acidophil nor basophil and contain alkaline phosphatase 2. Larger azurophilic granules of purple color. They are primary lysosomes and form about one third of all granules. Function: First line and fast defense against antigens. Neutrophils can move and migrate during the inflammation process from vessels into tis– 76 –

sue due to chemical attraction (chemotaxis). They are microphages, they phagocytose bacteria and destroy the microorganisms by hydrolytic enzyme named lysozyme. Thus the contribute to formation of pus, which consists of dead neutrophils, macrophages, microorganisms and cell debris. Eosinophils (oxyphils, acidophils) Count: 0–5% of WBC Size: 12–15 micrometers in diameter (slightly larger than neutrophils) Life span: about 8–17 days Nucleus: two lobes connected with a thin chromatin bridge. Chromatin is not so densely packed as in neutrophils.

Fig. 66 Eosinophilic granule (0.3–1.0 µm)

Cytoplasm: apart from a few mitochondria, contains large eosinophilic granules (about 200 in one cell). Granules are lens-shaped – with a centrally situated crystalloid core (containing basic parasite-killing proteins), surrounded by externum (containing acid phosphatase). Function: they are attracted (amoeboid movement) to sites where antigens and antibodies react together and phagocytose antigen-antibody complexes. They neutralize foreign proteins and are found in allergic reactions and parasitic diseases (eosinophilia). Basophils Count: 0.5–1% of WBC Size: 10–12 micrometers (the smallest granulocyte) Nucleus: irregular in shape (irregular lobes or S-shaped) much less intensively colored than neutrophils and eosinophils. Cytoplasm: large blue granules stain metachromatically (like in mast cells). They contain heparin and histamine. – 77 –

Function: They liberate heparin and histamine in response to an antigen. They also defend against parasites. They are involved in most inflammatory reactions, including allergic reactions (like eosinophils). Agranulocytes Lymphocytes Count: 20–45% of WBC Size: They are smallest elements within WBC. Classification is very difficult because their function does not always correspond with their morphological differences. We divide them into two groups: small lymphocytes (6–8 µm) and large lymphocytes (8–18 µm). In the circulation the small lymphocytes prevail (90%) – the large lymphocyte form is only 10% of those in circulation. Small lymphocyte – rounded cell nearly entirely occupied by the nucleus (dense chromatin). The nucleolus can be demonstrated in electron microscope only. Cytoplasm is reduced and forms a narrow rim around the nucleus. It contains a few mitochondria, ribosomes, poor GER and Golgi apparatus, one or two azurophilic granules (lysosomes). Large lymphocyte – can be considered to be a lymphoblast. It is a mother cell of the small lymphocytes and can also develop from the small lymphocytes by dedifferentiation after antigenic stimulation. Such a lymph-

Fig. 67 Lymphoblast – 78 –

oblast is able to differentiate into a specific immunocompetent lymphocyte (i.e. immunocyte). These large lymphocytes occur mostly in lymph nodes and lymph follicles. Functional and developmental characteristic: Lymphocytes form various subpopulations which play different roles in immune defense of the organism. The lymphoblasts develop in the bone marrow from the multipotent hemocytoblast (mesenchymal origin) and pass into the blood circulation and mature in lymphoid tissue to be so-called immunocompetent cells. This maturation process proceeds in two directions: T-lymphocytes and cellular immune response Lymphoblasts pass from bone marrow into thymus – the place where – “Thymus dependent – i.e. T-lymphocytes” mature. They pass through circulation into lymph nodes (paracortical region) – then pass again into circulation and if they meet an antigen, they return to a lymph node and dedifferentiate into a lymphoblast which undergoes mitosis and produces a clone of activated lymphocytes (immunocytes). These lymphocytes migrate in place of antigenic stimulation and form subpopulations which can be identified by the presence of different surface markers (cluster of differentiation, CD): • T-helper lymphocytes (Th cells) – (surface marker CD4) secrete various chemical messengers = interleukins, by which they help other lymphocytes to perform their functions (e.g. activation of Tc and B cells, maturing monocytes into macrophages etc.) • T-cytotoxic lymphocytes (Tc cell, surface marker CD8). They are activated by TH cells for killing virus infected cells and tumor cells • T-regulatory lymphocytes (Treg cell) mostly suppress immune reactions against self-antigens by switching off the immune response. • T-memory lymphocytes (Tm cell) – remain in lymph node and can respond to an antigen repeated in later life (immunological memory). B-lymphocytes and humoral immune response In addition to the thymus lymphocytes mature in birds in a specialized lymph organ, situated in the cloaca and called “bursa of Fabricius” (Bursa-dependent = B-lymphocytes). In mammals the bursa-equivalent has not been found and the B-lymphocytes undergo maturation directly in bone-marrow. They are then activated in the lymph nodes, but in the cortical region (follicles) and in spleen. Only few enter the circulation and upon – 79 –

meeting an antigen they return and mature (after stimulation by TH cells) in plasma cells which produce antibodies = immunoglobulins of various classes (IgG, IgA, IgM, IgD, IgE). In addition to these plasma cells also B-memory cells are produced. Natural killer cells (NKC) Next to the B- and T-lymphocytes, there exist also cells (non-specific) without the properties of T and B cells. These are “natural killer cells” (NKC) and they are not thymus or bursa dependent and mature in the bone marrow and lymph organs. Their activity is stimulated and regulated by the interleukins of T-helper cells. NKC play an important role in anti-tumor activity (by production of a substance “perforin” which kills the tumor cells perforating their plasma membrane). Distribution of lymphocytes in the human body Only 2% of all lymphocytes can be found in the blood circulation in the relation: T-Ly=75%, B-Ly=15%, 0 (NKC)=10%. The majority of lymphocytes reside in the lymph nodes (40%), further the tissues (18%), spleen (15%), thymus 10%), bone marrow (10%) and intestine (5%). Lifespan can be very long (approx. 300 days). Monocytes Count: 2–10% of all WBC Size: 12–20 µm (largest WBC) Nucleus: large ovoid, kidney-shaped (horseshoe-shaped, sometimes with deep indentations), chromatin not so dense as in lymphocytes, usually two visible nucleoli Cytoplasm: basophilic, few azurophilic granules (lysosomes), few RER, polyribosomes Monocytes are motile cells (pseudopodia) – in the blood circulation they appear in their juvenile forms, when entering the tissues, they can differentiate into macrophages. Their lifespan comprises about 3 days. Thrombocytes Count: 150 000–400 000 in mm3 Size: 2–4 µm Thrombocytes (“blood platelets”) are ovoid, biconvex, non-nucleated bodies. They are fragments of a giant megakaryocyte. – 80 –

Fig. 68 Cooperation between cellular and humoral immune response The T-lymphocytes recirculate between lymph nodes and tissue and may differentiate into T-helpers (Th), T-cytotoxic (Tc), T-regulatory (Treg) or T-memory (Tm) lymphocytes. The B-lymphocytes in the lymph nodes and lymphoid follicles differentiate in plasma cells and B-memory (Bm) lymphocytes.

Fig. 69 Monocyte – 81 –

Fig. 70 Thrombocyte

Their lifespan is about 4–12 days. The ground substance is light blue stained – hyalomere – in its marginal part are the filaments and a system of microtubules which open into invaginations of plasma membrane (through which the platelet factors are released). The membrane is covered by a cell coat (glycosaminoglycans which enable the platelet adhesion). The central part is a dense blue granulomere (chromomere). It contains in addition to some mitochondria various granules: Alpha-granules contain platelet-specific proteins (e.g. platelet factor). Dense granules contain calcium ions and serotonin. Lambda-granules contain lysosomal enzymes. Function: Serotonin starts the contraction of the damaged vessel. The platelet factor reacts with some plasma substances to form thromboplastin. Thromboplastin reacts with prothrombin (a plasma protein) to form thrombin. Thrombin reacts with fibrinogen (another plasma protein) giving rise to a polymer fibrin, which then forms the thrombus (blood clot). The thrombus as a result of blood coagulation contains also RBCs, WBCs, and thrombocytes. Hematopoiesis The highly specialized blood cells with relatively short lifespans have to be continuously replaced by new populations of cells, produced in hemopoietic tissues of mesenchymal origin. Blood cell development – hematopoiesis (or hemopoiesis) occurs in various stages during human ontogenesis: 1. Mesoblastic period: (1st–3rd embryonic month) Hematopoiesis starts extraembryonically in the primary mesoderm (the source of mesenchyme) which is situated on the endodermal yolk sac wall, connecting stalk and chorionic plate, and later in the first embryonic mesenchyme, derived from the germ layers. – 82 –

The mesenchyme condenses into “blood islands”. Their mesenchymal cells are called “angioblasts” and differentiate on the periphery into endothelial cells and in the center into proerythroblasts and myeloblasts.

Fig. 71 Blood islands

2. Hepatolienal period: (2nd–8th fetal month) Starting from the second month the mesenchymal part of the liver and from the fourth month also the spleen serve as producers of most types of blood stem cells (proerythroblasts, myeloblasts, megakaryoblasts - and from the 3rd month also lymphoblasts). 3. Medullary period: Depends on ossification and consequently on the development of the bone marrow. Even though the first ossification process starts in the clavicle very early (end of 2nd month!), true hematopoiesis begins in bone marrow approximately during the 5th month of development and continues even into postnatal life, when the red bone marrow represents the predominant hemopoietic tissue, from which all kinds of blood cells develop in a process, named accordingly: erythropoiesis, leukopoiesis, lymphopoiesis, monopoiesis, and megakaryopoiesis (thrombopoiesis). According the monophyletic theory, all the blood elements develop from a pluripotent hemopoietic stem cell – hemocytoblast. The process of differentiation and maturation of all kinds of blood cells is shown in the presented diagram, from which is seen that e.g. during erythroblast maturation the formation of hemoglobin starts and at the same time the degeneration and extrusion of the nucleus occurs. During leukopoiesis the formation of specific granules and segmentation of nucleus etc. occur. – 83 –

Fig. 72 Hematopoiesis Parameter

Reference limits

Units

Peripheral blood smear

 

 

Red blood cell (RBC) count (in male)

4.0 mil.–5.8 mil.

/mm3

Red blood cell count (in female)

3.8 mil.–5.2 mil.

/mm3

Hemoglobin (in male)

130–160

g/l

Hemoglobin (in female)

120–160

g/l

Hematocrit (HCT) (in male)

0.40–0.50

–

Hematocrit (in female)

0.35–0.47

–

Mean corpuscular volume (MCV) = HCT/RBC

82–98

fl

Red blood cell distribution width (RDW, variability of RBC size)

10–15.2

%

Mean corpuscular hemoglobin (MCH)

28–34

pg

Mean corpuscular hemoglobin concentration (MCHC)

320–360

g/l

Reticulocyte count

0.5–2.5 %

–

– 84 –

White blood cells (WBC, leukocytes)

4000–10000

/mm3

Platelets (thrombocytes)

150000–400000

/mm3

White blood cell differential count

 

 

Lymphocytes

20–45%

–

Monocytes

2–10%

–

Segmented neutrophilic granulocytes (PMN, polymorphonuclears)

45–70%

–

Nonsegmented neutrophilic granulocytes (bands, stabs)

0–4%

–

Eosinophilic granulocytes

0–5%

–

Basophilic granulocytes

0–2%

–

C. Muscle tissue Origin: mesenchyme Function: generating force by contraction, i.e. shortening and thickening of cells (fibers); creating (in some cells only) and propagating (spreading) electric action potentials Morphology: there are two main types: smooth and striated muscle. In smooth muscle, the intracellular contractile proteins are not organized in a periodic manner. In striated muscle, the contractile proteins are arranged in repeating units named sarcomeres and the cells and fibers exhibit cross-striations at the light microscope level. The striated (sarcomeric) muscle tissue occurs in two forms with different microscopic and physiologic properties: skeletal muscle and cardiac muscle. Smooth muscle

Distribution: The uterus comprises the biggest accumulation, the muscle layers of the wall of the digestive tube (from the middle part of the esophagus to the anus), the muscle layers of the bronchi and bronchioles, arteries, veins, large lymphatics, arrector pili muscle, areola of the mammary gland, scrotum, iris (ectomesenchymal origin!), ductus deferens. Smooth muscle cells are independent of direct voluntary control, though the rate and strength of contractions are modulated by the autonomic nervous system as they are innervated by autonomic nerve system. – 85 –

Fig. 73 Bundle of smooth muscle cells connected with gap junctions

Morphology: Smooth muscle consists of 20–400 µm long spindle shaped cells. These cells are non-striated and may be packed in bundles by a network of reticular fibers. The cells are invested by a thick extracellular coating (basal lamina) which together with the gap junctions enable coordinated contraction (e.g. peristalsis in the intestine). Among the cells and around their bundles also a little connective tissue with collagen fibers, blood vessels and nerves can occur. The plasmalemma which (together with the surrounding basal lamina) in muscle is called sarcolemma contains many small inpocketings, called caveoli (pinocytic vesicles important for Ca intake). These cavities increase the cell surface and have the same function as the T-tubules of striated muscles (see below). The caveoli are often continuous with microtubules. The cytoplasm, i.e. sarcoplasm, contains only one elongated, rod shaped and centrally situated nucleus. Next to it there appear some long slender mitochondria, a small Golgi complex, few cisterns of granular endoplasmic reticulum and clusters of free ribosomes. The contractile elements are present in the form of actin and myosin filaments, forming a fine network. These proteins are inserted into focal densities. These sarcolemma and sarcoplasma dense bodies represent an equivalent of the Z disc of striated muscle. With contraction the cells change their shape. The thin filaments are of actin and tropomyosin (troponin of striated muscle is absent) and they interact with thicker myosin filaments by a sliding mechanism (similar to that in striated muscles). This mechanism is based on phosphorylation of – 86 –

Fig. 74 Smooth muscle cell

myosin. The Ca ions must react with a calcium binding protein – calmodulin – to activate myosin kinase, responsible for myosin phosphorylation. The phosphorylated myosin is able to interact with actin, thus generating the contraction force.

Fig. 75 Myocyte contraction

Striated (sarcomeric) muscle

can be divided into skeletal and cardiac muscle. Skeletal muscle Skeletal muscle is under voluntary control and is attached to the skeleton or to the skin (facial muscles). It is composed of long, cylindrical and multinucleated syncytial fibers, grouped in fascicles. The fibers can be – 87 –

from 10 µm to 400 mm in length. The whole muscle is enclosed by a dense connective tissue sheath named epimysium, from which collagenous septa surround the fascicles, thus forming an envelope named perimysium. A delicate layer of reticular connective tissue named endomysium passes among the individual muscle fibers. In these connective tissue septa pass the blood vessels (rich capillary network) and nerves. Each muscle fiber is enclosed by a  delicate membrane  – sarcolemma – (which includes also an extracellular glycoprotein coating and basal lamina). The sarcoplasm (cytoplasmic matrix) contains numerous nuclei (sometimes several hundred!) and a large number of myofibrils, arranged in groups in Cohnheim’s fields (these are seen in cross section after histological processing, separated by various amount of myoglobin). Each muscle fiber contains a number of nuclei, which are positioned under the

Fig. 76 Skeletal muscle fascicles

Fig. 77 Myofibrils grouped in Cohnheim’s fields, peripheral position of nucleus – 88 –

sarcolemma. Near each nucleus is a small Golgi apparatus and there are many mitochondria mostly under the sarcolemma. The myofibrils run in parallel longitudinally and are cross-striated. The dark staining bands are doubly refractile or optically anisotropic A-bands, the light bands are optically isotropic – I-bands. Each I-band is halved by a transverse line Z-line. Each A-band is crossed by a M-line. The structural and functional unit between two successive Z-lines is called sarcomere and is approx. 2.5 µm long. Ultrastructure: A myofibril represents a bundle of 2 types of filaments – thin and thick – running symmetrically and parallel to the fibrillar axis. The Z-line has the form of a zigzag line in which the thin filaments are anchored. These thin, i.e. actin filaments form the I-band and are about 1 µm long and 8 nm wide. The thin filaments are composed of 3 proteins: actin, tropomyosin, and troponin. The main component is actin – a twisted double filamentous polymer which carries thin and short filamentous molecules of tropomyosin on which three small troponin subunits are attached.

Fig. 78 Skeletal muscle fiber – 89 –

Fig. 79 Thin myofilament composed of actin and associated proteins

Fig. 80 Sarcomere

The thick, i.e. myosin filaments form the A-band, are 1.6 µm long and 15 nm wide and are arranged in parallel among thin filaments in form of a comb. Each rod consists of hundreds of myosin molecules. The heads of these molecules appear as tiny outgrowths. The thick filaments are interconnected in the middle by M-line (contains creatine kinase, important for supply of ATP). During muscle contraction the actin filaments slide in between the myosin filaments and so the whole sarcomere shortens. The ends of thin filaments reach nearer to the opposite ones and form between them a lighter zone, the H-band which becomes very narrow in full contraction and widens during relaxation of the muscle fiber. The intake of Ca ions occurs through the transverse tubule system, consisting of a tubular invagination of the sarcolemma named T-tubule which encircles the myofibrils at the level of the A-I junction. Adjacent to both sides of each T-tubule are situated the terminal (transverse) cisternae of the sarcoplasmic reticulum and they form together with – 90 –

Fig. 81 Contraction of a sarcomere in striated muscle

the T-tubule a triad. The T-tubule is not a part of sarcoplasmic reticulum. The terminal cisternae continue in a branching system of the longitudinal sarcotubules of sarcoplasmic reticulum, which contains the Ca ions. Calcium is released into myofibrils, binds to troponin, which exposes myosin-binding sites on actin, allowing cross bridges between actin and myosin to form.

Fig. 82 Ultrastructure of a skeletal muscle fiber

Mechanism of contraction In normal unstimulated conditions the troponin and tropomyosin molecules prevent the actin molecu­les from interacting with myosin. Both regulatory proteins lose their protective function in presence of Ca ions and actin and myosin interact with each other. (The Ca ions bind to the troponin, the tropomyosin molecule is pushed deeper in action helix and the binding site of actin is exposed to myosin). The myosin heads contain an ATPase. – 91 –

Fig. 83 Interaction between actin and myosin filaments

During the relaxed state, the ATPase hydrolyzes ATP to ADP and inorganic phosphate (Pi). Once myosin is bound to actin, the Pi molecule is released and the power stroke occurs, pulling actin relative to myosin. ADP then is released and a new ATP molecule can bind. The cycle continues as long as Ca++ is available Myosatellite cells Multinucleated skeletal muscle fibers cannot divide but can grow larger in response to muscle activity due to lengthening and increasing their myofibrils, i.e. not to addition of new muscle fibers. But in case of e.g. injury they can be replaced by new muscle fibers, which differentiate from satellite (myosatellite) cells. These are unipotential stem cells, closely attached to the surface of the muscle fiber, and covered by the same basal lamina. They are mitotically quiet in the adult, but in case of necessity they proliferate and become a source of new myoblasts (muscle cell precursors), which undergo cell divisions before they can fuse with existing myofibers. However in case of massively damaged muscle fibers the muscle becomes replaced by a fibrous tissue.

Fig. 84 Myosatellite cell – 92 –

Cardiac muscle forms the middle layer of the heart wall, i.e. the myocardium. Some cardiac muscle cells are also present in the walls of the pulmonary vein and superior vena cava. Even though it is striated, it differs from skeletal muscle by its involuntary activity and by some additional morphological features. In contrast to skeletal muscle it is not formed by long multinucleated fibers, but by single cardiac muscle cells which are joined end to end by cell junction to form fibers. These cardiac muscle fibers branch and anastomose, forming a special sort of syncytium, which enables spreading of a contraction wave. The endomysium among the muscle fibers is a very vascular loose connective tissue, containing many blood capillaries and lymphatic vessels. The cells are about 15 µm in diameter and 80–150 µm in length and possess one or two centrally located pale nuclei. There are pigment granules (lipofuscin) near the poles of a nucleus, as well as a small Golgi complex. The abundant mitochondria are situated mostly in rows among the myofibrils. These myofibrils are striated but they are not arranged in Cohnheim’s fields, are more delicate and spread throughout the whole fiber. The border between two adjacent cells is formed by the intercalated discs. It is a darkly stained transverse line across the fiber which represents a junctional complex. It is usually steplike arranged. In the transverse portion (fascia adherens) the actin filaments are anchored in the form of desmosomes (macula adherens). The lateral portion of the disc contains a gap junction (nexus) which allows the ions to pass through the junction.

Fig. 85 Cardiac muscle – 93 –

Fig. 86 Intercalated disc

The sarcolemma is penetrated by T-tubules at the level of the Z-line, the sarcoplasmic reticulum is not so well developed, and transversal cisterns are missing – we speak, instead of triads, about dyads (1 tubule + 1 cistern). Cardiac conducting system In addition to the cardiac muscle cells whose function is contraction, there exists a specialized system for initiating impulses (“pacemaker”), and to conduct them through the heart. This system consists of the sinoatrial

Fig. 87 Cardiac conducting system – 94 –

Fig. 88 Purkinje fibers

node, the atrioventricular node, situated beneath the endocardium (see location on the diagram) and atrioventricular bundle (bundle of His). The cells of the cardiac conducting system retain the ability of embryonic cardiac muscle cells to generate action potentials spontaneously. Although all cardiac muscle cells generate action potentials, the firing rate of the sinoatrial controls the heart rate under normal conditions. The bundle of His enters the fibrous portion of the interventricular septum from the A-V node and divides into two branches, distributed to right and left ventricles. The structure of the nodes is represented by specialized cells, smaller than ordinary cardiac muscle cells, arranged in a network embedded in dense connective tissue. The cells of the atrioventricular bundle conduct the wave of depolarization and are formed by specialized conducting muscle fibers, called Purkinje (Purkyně’s) fibers (Jan Ev. Purkyně was a famous Czech scientist, 1787–1869). Purkinje fibers are situated deep in the endocardium and are much larger then cardiomyocytes and have one or two nuclei in a clear central mass of sarcoplasm (which contains much glycogen), which possess many mitochondria and only few striated myofibrils, arranged peripherally. The intercalated discs are not typical, but there are also desmosomes along the cell boundaries between adjacent fibers. Specialized myocardiocytes – myoendocrine cells Among the cardiac muscle cells of the right atrium appear specialized cardiac myocytes – myoendocrine cells. They possess many osmiophilic (i.e., staining with osmium salts) granules mostly in the perinuclear Golgi region, but also in rows between myofibrils. Their function is endocrine, they produce the hormone atrial natriuretic peptide (cardiodilatin – regulation of blood pressure). They are rarely found also in the interventricular septum. – 95 –

Fig. 89 Myoendocrine cell in atrial myocardium

D. Nerve tissue Origin: ectoderm Function: reception of stimuli from environment, formation and conduction of nerve impulses, storage and processing of information, coordination of other body functions, secretion of neurohormones. Morphology: Nerve tissue contains two major groups of cells: the neurons and the neuroglia. Neurons

The morphological and functional unit of nerve tissue is a neuron. The neuron consists of a cell body, (also called perikaryon, or soma) and processes. The processes are of two kinds: 1. Dendrites, which form the receptor portion of the neuron, and are specialized in receiving stimuli from the environment (or other neurons) and conducting them toward the cell body (afferent conduction), and 2. Axon (or neurite) which is always single and conducts impulses away from the cell body (efferent conduction). The distal (i.e. effector) portion of an axon is branched into terminal arborizations, ending by axon terminals (knob-like dilatations, fr. boutons terminaux) which form the synapses, in which an impulse is transmitted to the next neuron or effector. – 96 –

Fig. 90 Neuron

Classification of neurons The central nervous system contains about 100 billion neurons. They can be classified according to the number of their processes. Whereas there is always only a single axon, the dendrites can vary in the number: 1. The embryonic neuroblasts represent a cell without processes and can be considered as apolar neurons. 2. During early development the first process gradually appears – it is an axon. To such unipolar neurons also olfactory axons can be with some reservation added (unmyelinated axons of the olfactory cells with no typical dendrites). – 97 –

3. Neurons which possess a single axon and single dendrite are bipolar neurons. They appear as intermediate neurons, e.g. in the retina of the eye, but also in the ganglia of the vestibulocochlear nerve (VIII.), as well as in the cerebrospinal ganglia of lower vertebrates (fishes). 4. From the bipolar neurons the pseudounipolar neurons developed by gradual fusing of the dendrite and axon. They are situated in spinal ganglia of higher vertebrates (including human). This sensory neuron possesses only single process, which then divides into two branches. They can be functionally considered as dendrite and axon (both are myelinated) and the stimuli do not pass through the cell body, which has a trophic function only. 5. Most neurons possess more than one dendrite – these are multipolar neurons. Typical star-shaped neurons belong mostly to motor neurons (e.g. situated in anterior horns of spinal cord), possess numerous branched dendrites capable of receiving stimuli from many other neu-

Fig. 91 Types of neurons – 98 –

rons, and a single axon, conducting the stimulus to the effector (muscle, glands). They can be also pyramid-shaped (motor brain cortex), or pear shaped (Purkinje) cells of cerebellum). Cytology of the neuron The cell body (also called perikaryon, or soma) is large, usually starshaped, containing a centrally located large, pale and vesicular nucleus with a conspicuous deeply staining nucleolus (similar to that of an oocyte). There is no centrosome – that is why once a nerve cell has been differentiated, it loses the ability to divide. Golgi complex is located in the form of cisterns around the nucleus like a wreath. (This apparatus was discovered by Camillo Golgi just in the nerve cell body in 1898). Numerous mitochondria witness the heavy energy demands of the neuron.

Fig. 92 Cell body of a neuron – 99 –

Nissl bodies in the form of large basophilic granular areas consist of rough endoplasmic reticulum and polyribosomes and are sites of active protein synthesis. In exhausted neurons there is a temporary reduction of Nissl bodies (tigrolysis or chromatolysis). Nissl bodies also extend into dendrites but are absent from the axon hillock. The neurofibrils are composed of neurofilaments (diameter 10 nm) and neurotubules (microtubules – 24 nm), which also extend into all processes. The granules of a brown, age dependent pigment, called lipofuscin are found in the axon hillock. In some CNS ganglia black pigment granules of neuromelanin can also be observed. Dendrites are afferent processes sometimes with rich branching which enlarges the area of reception of nerve impulses. Arborization and dendritic spines enable contacts with terminal arborizations of other neurons (there can be thousands of synapses). The larger dendrites contain most of the same organelles as the cell body. The axon is usually the largest and the longest process of the neuron, reaching up to 100 cm in length (e.g., in motor neurons innervating the foot muscles) with a constant diameter along its course. It is covered by axolemma and it can branch in axon collaterals and at the end it ramifies into terminal arborizations. Axoplasm contains no Nissl bodies or ribosomes (proteins are supplied from the cell body). It contains neurofilaments and neurotubules which are protein tubular fibers running parallel to longitudinal axis of the axon. There are also microfilaments built of the protein actin – these can run crosswise to be fixed to the axolemma. In addition to the filaments the mitochondria, neurotransmitter vesicles and lysosomes, as well as axoplasmic reticulum cisterns (of unknown function) can also be observed.

Fig. 93 Axon sheaths – 100 –

Fig. 94 Development of myelin sheath

The axon leaves the cell body at an axon hillock from which the bundles of neurofilaments and neurotubules continue into the initial segment. It is the naked part of the axon in which the action potential starts. After the axon hillock the axon of myelinated fibers becomes invested by a segmented myelin sheath, interrupted at regular intervals by myelin-free gaps – nodes of Ranvier. The part between two sequential nodes is called an internodal segment and is covered by one oligodendrocyte glia cell (in CNS) or Schwann glia cell (in PNS), which is responsible for production of myelin. During development of a PNS neuron the Schwann cell wraps round the axon and rotates around it and gradually forms thinner and thinner layers of pulled over cell membrane of the Schwann cell. The resulting myelin sheath consists therefore of mainly lipoprotein turns (from few to 50) of plasmalemma and a little cytoplasm, which projects near the node (paranodal zone) in little tongues (loops), bent up to contact the axon.

Fig. 95 Node of Ranvier – 101 –

Near the paranodal zone the myelin incisures (clefts) can be seen. These cone-shaped figures are the result of dehiscence and loss of the plasmalemma layers in oblique convergent lines during myelination. Unmyelinated fibers In addition to the myelinated fibers, many fibers of peripheral and central nervous system as well as most of those in autonomic nervous system are unmyelinated (grey or Remak fibers). In those nerves as many as nine axons may be enfolded by each Schwann cell. The myelin sheath is absent and therefore also Ranvier’s nodes are not present (i.e. non-segmented fibers).

Fig. 96 Grey fibers in PNS (one Schwann cell encloses multiple fibers)

Myelinated fibers of CNS In the white matter of the CNS the fibers (axons) are also enclosed within the myelin sheath but instead of Schwann cells, there are oligodendrocytes which are responsible for production of myelin. The difference between them is that whereas Schwann cells build the myelin on one single axon only, the oligodendrocyte sends more processes which wrap several axons to build the myelin of white matter. Even though the Ranvier’s nodes occur, the myelin clefts in the CNS myelinated fibers are absent. Peripheral nerve

The nerve is formed by fibers, running in bundles, covered by a connective tissue coat. The myelin-ensheathed axons with Schwann cells are enveloped by a thin layer of connective tissue forming endoneurium. A bundle of axons is covered by flat cells of perineurium and the whole nerve is enveloped by a fibrous coat of dense connective tissue – the epineurium. – 102 –

Fig. 97 Oligodendrocyte providing a glial sheath and a myelin sheath in CNS

Fig. 98 Peripheral nerve

Synapses and a reflex arc

The contact among neurons or between a neuron and effector tissue (muscle, gland etc.) is realized through a special arrangement – the synapse. The interneuronal contact can occur in various sites of the neuron. The most common is the axo-dendritic synapse, i.e. the synapse of end-branches of terminal arborizations with the dendrites of the next neuron. Another type is the axo-somatic synapse. In this case the axon contacts the cell body (soma) of another neuron directly. An axon can form a syn– 103 –

Fig. 99 Synapses

apse only at an unmyelinated site. The dendrites or cell body of one neuron can be contacted by few axonal branches, but some of them (e.g. pyramidal cells) by many – and form as many as 10 000 synapses. Nerve fibers can carry the information from a receptor inside or outside of the body to the center (centripetally) – such fibers are afferent i.e. senso-

Fig. 100 Reflex arc – 104 –

ry. From the CNS the impulses are carried by efferent i.e. motor fibers to the effector organs. Simple contacts of both kinds of these nerve fibers are represented by the so called reflex arc. The afferent sensory neuron starts in this case on the periphery in the skin by its free endings of dendrites and passes through spinal ganglion (cell body of the pseudounipolar cells), from which its axon continues into dorsal root of the spinal cord gray matter. In the ventral horns the stimulus is transmitted (an axo-dendritic synapse) to the dendrites of an efferent multipolar neuron and passes via its axon to the effector organ (usually muscle). Sensory receptors

The peripheral end of the afferent neuron, where the sensory pathway starts, serves as a receptor, which can respond to various stimuli from the external environment or internal situation of the body and its parts. Detection of cutaneous sensation occurs by variably specialized nerve endings, the most important of which are: Free nerve endings These are formed by nonmyelinated fibers (dendrites) branched in the deep layers of epidermis. They detect pain and temperature (nociceptors and thermoreceptors). On the palms and soles the free endings can be attached to special basal epidermal cells with a lobulated nucleus and numerous granules, known as Merkel cells (mechanoreceptors – sensation of touch).

Fig. 101 Free nerve endings and Merkel cell – 105 –

Meissner’s corpuscles These are encapsulated nerve endings confined to the dermal papillae. Their capsule consists of fibroblasts and collagen fibers surrounding an inner core of spirally arranged Schwann cells and nerve terminals. They are situated vertically in the dermal papillae and are most numerous on the feet and hands. They are considered as the most sensitive mechanoreceptors detecting touch.

Fig. 102 Meissner’s corpuscle in the dermal papilla below the epidermis

Pacinian corpuscles These are large mechanoreceptors (up to 1 mm) situated mostly in the deep dermis of the digits or subcutaneous fat, as well as in the periosteum, mesenteries etc. where they detect pressure and vibration. Their capsule consists of many concentrically arranged lamellae which are modified Schwann cells separated from each other by fluid filled spaces. The lamellae surround centrally situated unmyelinated nerve terminals. Muscle spindles These form special sense organs situated between the muscle fibers in perimysium. They are about 1.5 mm long and consist of a connective tissue – 106 –

Fig. 103 Pacinian corpuscle

Fig. 104 Muscle spindle

capsule, surrounding some specialized myofibers (intrafusal fibers). These fibers lose their cross-striation in their equatorial region in which many nuclei accumulate. Sensory nerve fibers penetrate the capsule and their unmyelinated dendrites encircle the intrafusal fibers and register stretch and tension. Each of the intrafusal fibers also receives a motor innervation in the form of motor end plates. Similar structures are found in tendons near the insertion of muscle fibers – Golgi tendon organs. In these organs the connective tissue sheath encapsulates bundles of collagen fibers, surrounded by sensory nerve endings which respond to stretch. – 107 –

Motor nerve endings The efferent motor neurons usually terminate in a small branched structure – a motor end plate. The terminal axon loses its myelin, covered only by the Schwann cell forms a close irregular contact with the muscle fiber. It is irregular because the sarcolemma is folded in clefts and ridges, called junctional folds, in the places of contact. Between the axolemma and sarcolemma is a narrow intercellular gap – the synaptic cleft. The axon terminals contain numerous synaptic vesicles with the neurotransmitter acetylcholine. The signal is transmitted from axon to muscle by this neurotransmitter which initiates a wave of depolarization of the muscle fiber. Meanwhile the acetylcholine is hydrolyzed by the enzyme cholinesterase present in the synaptic cleft.

Fig. 105 Motor end plate

Conduction of nerve impulses

The conduction of a nerve impulse depends on the content of sodiumand potassium ions in the fluid inside and outside the neuron. The sodium-ion is a sodium atom, which has one negative electron given away so that it carries a positive electric charge. There is a ten times higher concentration of sodium-ions in extracellular fluid than inside the neuron. On the other hand the potassium concentration is 20–40 times higher inside than outside the neuron. The axolemma is only slightly permeable to both types – 108 –

Fig. 106 The Na+/K+ ATPase and ion channels in a membrane of a neuron

of ions, but significantly more to potassium, – so that the sodium-ions diffuse slowly into neuron and on the contrary the potassium-ions leave the neuron at a somewhat faster rate. The concentration balance must be maintained by special sodium-potassium-pumps (protein molecules), which continuously pump out sodium as they pump in potassium. This provides the concentration gradients that drive diffusion of these ions both at rest (mainly potassium, diffusion of which creates the resting membrane potential of approximately -70mV) and during the action potential (mainly driven by sodium diffusion, as described below). Excitation causes opening of channels of voltage-regulated integral proteins, the sodium enters into the cell and the axon becomes depolarized – action potential. Later, on the contrary, the potassium ions leave the axon and the resting potential becomes again restored. In myelinated fibers depolarization occurs in Ranvier’s nodes only – in this case the nerve impulse has to jump from one node to the next one – saltatory conduction – and the action potential is conducted approximately 100× faster than in nonmyelinated fibers.

Fig. 107 Saltatory conduction of action potential between two Ranvier nodes of a myelinated fiber – 109 –

Nerve signals are of electric nature while they are spread lengthways along the nerve fiber. At the moment they reach the end of it, they must jump over the synaptic cleft in a chemical way, i.e. with the help of a transmitter substance. Circulation of a neurotransmitter starts in the vicinity of the nucleus and it travels in a rapid transport to the end of the nerve fiber. As soon as the nerve excitation reaches the synapse, the calcium channels open and calcium enters the presynaptic bulb and helps to fuse the vesicles among themselves and with the presynaptic axolemma. The transport vesicles join the axolemma and evacuate their content (exocytosis). The neurotransmitter molecules traverse the synaptic cleft and join the receptors of the postsynaptic membrane and transmit the nerve excitation, i.e. causes the increased permeability of the postsynaptic membrane to ions, usually sodium, and its depolarization and generation of active potential in the next neuron. The neurotransmitter is either enzymatically degraded in the synaptic cleft (e.g., acetylcholine is degraded by cholinesterase) or taken back up into the presynaptic cell to be used again. After endocytosis, the vesicles containing the synaptic cleft fluid with remaining transmitter molecules become shifted back into the cell body where they are split by lysosomes and their content can contribute to the formation of new vesicles. The presynaptic terminals resemble an endocrine gland, and the chemical transmission is a modified form of hormone secretion. There are varie-

Fig. 108 Synapse – 110 –

ties of small molecules (e.g. acetylcholine, norepinephrine, glycine, serotonin, dopamine etc.) which can serve as transmitters and so can also various peptides.

Fig. 109 Neurotransmitter circulation – 111 –

Neuroglia

In addition to the nervous elements proper, the nervous system contains numerous non-neural support cells – so called neuroglia cells. These are situated either in CNS, like macroglia (astrocytes and oligodendrocytes) and microglia and ependymal cells, as well as in peripheral NS like the already mentioned Schwann cells and satellite cells. These cells are also of ectodermal origin (except mesenchymal microglia) and develop from the spongioblasts or the lining of the primitive neural tube. In early ontogeny they guide the migration of developing nerve cells. Astrocytes The astrocytes provide structural support for nerve tissue. These starshaped cells are the largest of the glial cells. They possess oval, pale nuclei and long cytoplasmic processes (named pedicles) with expanded tips that adhere to the vessels. These perivascular feet can induce changes in the endothelium so that it acts as a barrier to diffusion between blood and brain. Astrocytes also regulate the potassium level, altered by neuronal activity and transport it to the brain surface and cerebrospinal fluid. Other processes form a layer under the pia mater and separate it from the nerve cells. Astrocytes, unlike neurons, can undergo mitosis and participate in repair of injured CNS by their proliferation and formation of a glia scar.

Fig. 110 Astrocytes – 112 –

Fig. 111 Oligodendrocyte and microglia

There are two subtypes of astrocytes: Fibrous astrocytes are most evident in the white matter and have few long, not very branched processes, which contain rich bundles of cytoskeletal intermediate filaments (glial fibrillary acidic protein). Protoplasmic astrocytes are present in gray matter and possess many branched and relatively short processes. Oligodendrocytes They are smaller with few slender processes, situated in rows along nerve fibers. They are the myelin-forming cells of the CNS and are analogous to the Schwann cells which form the myelin of peripheral nerves. In contrast to a Schwann cell which produces the myelin by rotation around one axon only, one oligodendrocyte wraps its processes spirally around several neurons (see the diagram on preceding pages). Microglia cells They do not belong in fact to the glial elements because of their mesenchymal origin. That is why they are sometimes called mesoglia. Functionally they can be considered as specialized immune cells in the CNS, as specialized macrophages. They are tiny cells with rod-shaped nuclei and comparatively long ramifying spiny processes. They form about 10% of – 113 –

the neuroglia and increase in size in damaged CNS and become actively motile and phagocytic. Ependymal cells They are epithelially arranged in a layer called ependyma, which lines the cavities of the brain (ventricles) and the central canal of the spinal cord. Ependyma consists of two cell types: Ependymal cells are cuboidal cells linked by desmosomes with apical microvilli and cilia and abundant mitochondria, a small oval basal nucleus, Golgi complex and GER. The basal part does not lie on basement membrane and is in contact with astrocytic processes. Specialized ependymal cells – tanycytes – are found in the floor of the 3 ventricle. The basal process of tanycytes extends and forms end feet on a blood vessel. They are attached to each other and to the ependymal cells by tight junctions. Modified ependymal cells cover, as cuboid to columnar epithelium-like lining, the choroid plexus, which represents folds of pia mater. They secrete cerebrospinal fluid (daily 600–700 ml).

Fig. 112 Ependyma – 114 –

Fig. 113 Satellite cells surrounding a pseudounipolar neuron in a spinal ganglion

Satellite cells They are support cells which surround the cell bodies of craniospinal as well as autonomic ganglia. They are small cuboidal cells, resembling Schwann cells and forming a layer around the craniospinal ganglia and an incomplete layer around the autonomic and intramural ganglia. Meninges

The central nervous system is wrapped up in three protective coats – the meninges: Pia mater The innermost layer of meninges, covered by squamous cells of mesenchymal origin. The delicate membrane composed of loose connective tissue also accompanies the vessels and lines their perivascular space up to their transition in the capillaries. The pia does not adhere to the surface of nerve tissue because of close contact with processes of astrocytes. Arachnoid Middle layer of meninges separated from the dura mater by a thin subdural space. It is connective tissue, containing collagen fibers and covered by a layer of cells as in the pia mater. Under the roof is an irregular system of trabeculae – the subarachnoid space, filled with cerebrospinal fluid. In some places the arachnoid projects into the dural sinuses in form of – 115 –

Fig. 114 Meninges

arachnoid villi. There the cerebrospinal fluid is resorbed into the blood of the venous sinuses. Dura mater The outermost layer is made of dense connective tissue, continuous with the periosteum. It contains collagen fibers with some elastic fibers. The internal surface of dura is covered by simple squamous epithelium of mesenchymal origin. This kind of epithelium also covers the dura in spinal cord. Blood-brain barrier

The CNS is protected from toxic and other dangerous compounds that may get into the blood stream by a functional barrier, which results from the reduced permeability of the blood capillaries of nerve tissue. The lining endothelial cells are not fenestrated and are joined by continuous tight junctions. The capillaries are in addition surrounded by a thick basal lamina and covered by processes of astrocytes. Cerebrospinal fluid

Cerebrospinal fluid is considered as “cushioning fluid” of CNS and fills the brain vesicles, spinal central canal and the subarachnoid space. It repre– 116 –

sents a modified tissue fluid which can contain a few leukocytes. It is produced by choroid plexuses of brain vesicles (derived from ependyma) and passes through openings in the roof of the hindbrain to the subarachnoid space, and becomes resorbed into the blood from arachnoid villi. The CSF also drains the tissue fluid of the brain.

– 117 –

FIGURE CAPTIONS

Fig.   1 Shapes of cells 8 Fig.   2 The cell 9 Fig.   3 Cell membrane 10 Fig.   4 Cellular processing of external signals 11 Fig.   5 Membrane transport 12 Fig.   6 Exocytosis and endocytosis 13 Fig.   7 Transcytosis 13 Fig.   8 Nucleus 14 Fig.   9 Molecule of DNA 16 Fig. 10 Chromatin fiber 17 Fig. 11 Condensed chromosome visible and stained during mitosis 17 Fig. 12 Human male karyotype 18 Fig. 13 Barr body in a neutrophilic granulocyte of a female 19 Fig. 14 DNA replication and doubling of chromatids 19 Fig. 15 Cell cycle and average duration of its phases 20 Fig. 16 Chromosomes during mitotic cell division 21 Fig. 17 From genetic information to the protein 22 Fig. 18 Ribosome 23 Fig. 19 Rough endoplasmic reticulum (RER) 25 Fig. 20 Mitochondrion 27 Fig. 21 Centrosome and centriole 30 Fig. 22 Actin filament as a part of the contractile apparatus 31 Fig. 23 Microvilli 34 Fig. 24 Stereocilia and kinocilia 35 Fig. 25 Basement membrane 36 Fig. 26 Junctional complex 37 Fig. 27 Simple epithelia 38 Fig. 28 Stratified squamous epithelia 39 Fig. 29 Stratified columnar epithelium 39 Fig. 30 Transitional epithelium (urothelium) in contracted and relaxed shape 40 Fig. 31 Types of exocrine glands according to shape 41 – 118 –

Fig. 32 Patterns of secretion 42 Fig. 33 Serous and mucous cells 43 Fig. 34 Seromucous gland in a section (left) and surface view (right) 44 Fig. 35 Fibroblast and fibrocyte 45 Fig. 36 Reticular cell 46 Fig. 37 Adipocytes 46 Fig. 38 Melanocyte and mastocyte 47 Fig. 39 Macrophage and plasmocyte 48 Fig. 40 Assembly of a collagen microfibril, a fiber and a bundle of fibers 50 Fig. 41 Reticular fibers and elastic fibers 51 Fig. 42 Mesenchyme and loose connective tissue 53 Fig. 43 Regular dense collagenous (left) and elastic (right) connective tissue 53 Fig. 44 Hyaline cartilage and chondrocyte 55 Fig. 45 Fibrous cartilage 56 Fig. 46 Osteocytes 57 Fig. 47 Long and flat bone 57 Fig. 48 Spongy bone 58 Fig. 49 Compact bone 59 Fig. 50 Osteon 59 Fig. 51 Section of compact bone showing the lamellas of an osteon 60 Fig. 52 Intramembranous ossification 61 Fig. 53 Primary bone trabeculae 61 Fig. 54 Principles of endochondral ossification 63 Fig. 55 Zones of endochondral ossification (upper scheme) and cross section showing the penetration of blood vessels into an ossification center (lower scheme) 64 Fig. 56 Primordia of two dentitions 65 Fig. 57 Early stages of tooth development 66 Fig. 58 Bell stage of tooth development 67 Fig. 59 Enamel organ 67 Fig. 60 The tooth 69 Fig. 61 Formation and structure of the enamel 70 Fig. 62 Odontoblasts 71 Fig. 63 Dentin 71 Fig. 64 Erythrocyte 74 Fig. 65 Neutrophilic, eosinophilic, and basophilic granulocytes 75 Fig. 66 Eosinophilic granule (0.3–1.0 µm) 77 Fig. 67 Lymphoblast 78 Fig. 68 Cooperation between cellular and humoral immune response The T-lymphocytes recirculate between lymph nodes and tissue and may differentiate into T-helpers (Th), T-cytotoxic (Tc), T-regulatory (Treg) or T-memory (Tm) lymphocytes. The B-lymphocytes in the lymph – 119 –

nodes and lymphoid follicles differentiate in plasma cells and B-memory (Bm) lymphocytes. 81 Fig.   69 Monocyte 81 Fig.   70 Thrombocyte 82 Fig.   71 Blood islands 83 Fig.   72 Hematopoiesis 84 Fig.   73 Bundle of smooth muscle cells connected with gap junctions 86 Fig.   74 Smooth muscle cell 87 Fig.   75 Myocyte contraction 87 Fig.   76 Skeletal muscle fascicles 88 Fig.   77 Myofibrils grouped in Cohnheim’s fields, peripheral position of nucleus 88 Fig.   78 Skeletal muscle fiber 89 Fig.   79 Thin myofilament composed of actin and associated proteins 90 Fig.   80 Sarcomere 90 Fig.   81 Contraction of a sarcomere in striated muscle 91 Fig.   82 Ultrastructure of a skeletal muscle fiber 91 Fig.   83 Interaction between actin and myosin filaments 92 Fig.   84 Myosatellite cell 92 Fig.   85 Cardiac muscle 93 Fig.   86 Intercalated disc 94 Fig.   87 Cardiac conducting system 94 Fig.   88 Purkinje fibers 95 Fig.   89 Myoendocrine cell in atrial myocardium 96 Fig.   90 Neuron 97 Fig.   91 Types of neurons 98 Fig.   92 Cell body of a neuron 99 Fig.   93 Axon sheaths 100 Fig.   94 Development of myelin sheath 101 Fig.   95 Node of Ranvier 101 Fig.   96 Grey fibers in PNS (one Schwann cell encloses multiple fibers) 102 Fig.   97 Oligodendrocyte providing a glial sheath and a myelin sheath in CNS 103 Fig.   98 Peripheral nerve 103 Fig.   99 Synapses 104 Fig. 100 Reflex arc 104 Fig. 101 Free nerve endings and Merkel cell 105 Fig. 102 Meissner’s corpuscle in the dermal papilla below the epidermis 106 Fig. 103 Pacinian corpuscle 107 Fig. 104 Muscle spindle 107 Fig. 105 Motor end plate 108 Fig. 106 The Na+/K+ ATPase and ion channels in a membrane of a neuron 109 Fig. 107 Saltatory conduction of action potential between two Ranvier nodes of a myelinated fiber 109 – 120 –

Fig. 108 Synapse Fig. 109 Neurotransmitter circulation Fig. 110 Astrocytes Fig. 111 Oligodendrocyte and microglia Fig. 112 Ependyma Fig. 113 Satellite cells surrounding a pseudounipolar neuron in a spinal ganglion Fig. 114 Meninges

110 111 112 113 114 115 116

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LITERATURE RECOMMENDED FOR FURTHER STUDY

Mescher A.L Junqueira’s Basic Histology. Text & atlas. 12th edition. McGraw Hill, New York, 2010. Ovalle W., Nahirney P.: Netter´s Essential Histology, 2nd edition, Elsevier, Philadelphia, 2013. Gartner L.P., Hiatt J.L.: Color Atlas and Text of Histology. 6th edition. Wolters Kluwer, Baltimore, 2014. Ross M., Pawlina W.: Histology: a Text and Atlas with Correlated Cell and Molecular Biology. 6th edition. Wolters Kluwer, Baltimore, 2014. Young B., Woodford P., O´Dowd G.: Wheater´s Functional Histology. A text and colour atlas. 6th edition, Churchill Livingstone, Philadelphia, 2013. Ovalle W., Nahirney P.: Netter´s Histology Flash Card Updated Edition, 1st edition, Elsevier, Philadelphia, 2013. Lee L.: Lippincott’s Pocket Histology. Wolters Kluwer, Baltimore, 2014. Lowe J.S., Anderson P.G. Human Histology, 4th Edition. Elsevier, Philadelphia, 2015. Berkovitz, B.K.B. – Holand, G.R.: Colour Atlas and Textbook of Oral Anatomy,Histology and Embryology 3rd Edition, Elsevier Science, Philadelphia, 2005. Vaňkhara P., Sedláčková M., Lauschová I., Čech S., Hampl A. Guide to General Histology and Microscopic Anatomy. Masaryk University, Brno, 2017.

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