Effect of Irritant Gases Upon Ciliary Activity

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THE EFFECT OF I H B . I T A B r T ' C I L I A H Y ACTIVITY

by X*ester V* Crslley

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, in the Department of Hygiene and Preventive Medicine, in the Graduate College of the State University of Iowa February, 1942

ProQuest Number: 10984054

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Th© author wishes to express his gratitude to Dr* Roland Hooks and Dr# Balph Heeren for their guidance and valuable assistance#

He also wishes to thank Professor

Gilford K # Barnes * Head of the Department of Hygiene and Preventive Medicine for his suggestions and constructive criticism* Professor «:rwin G* Gross, Head* Department of Pharmacology* Lewis J* Cralley and Dr* Clyde U# Berry for N|

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camphor and ©ue&Xyptol Into the nose by means of a nebulizer* Later the animals were killed and examinations were made to ascertain the extent of damage to the mucous membrance* Several devices have been constructed in the past for measurements of ciliary activity but, for various reasons, they have all been discarded*

Among these devices

may be mentioned that of Englemann (IQ).

A cylinder, free

to revolve on its axis, was placed in contact with the ciliated epithelium. This device was set up in such a manner as to make possible a permanent record of the rate of revolution of th© cylinder* Wymann (57) pinned the mucous membrance from th© roof of the mouth of th© frog onto a board*

Upon this was

placed a piece of akin cut from the throat of the frog, with th© inner surface adjacent to the cilia* of lead was then placed upon this skin*

A thin sheet

A pointer was

attached to the lead sheet to indicate th© distance through which th© sheet was moved* A oilloseribe, constructed by Dixon and Inchley (8) and later modified by inchley (25) consisted of an upright, free moving spindle supported upon bearings* Mucous membrane, cilia Inward, was partially wrapped around the spindle and the ends of the membrane

were fixed*

In

this way, the activity of the cilia gave rise to the move­ ment of the overlying mucus which in turn caused the spindle to rotate* A stroboscope was designed by Martius (37) to

measure the frequency of ciliary beats*

A revolving disc

with a central slit was inserted In the microscope between th© diaphragm and the source of light,

notation of this

disc gave rise to the stroboscopic effect* Effect of Gases upon Ciliated Epithelium Very little work has been done regarding the effect of irritant gases upon ciliary activity*

Most of

the investigators have limited the agents studied to those used In medication* Ernst (11) and Proetz (46) have found that nitrous oxide has no effect upon ciliary activity*

Carbon dioxide

and hydrogen In the pure state stop ciliary activity accord­ ing to Kuhne (29) and Englemann (10)* Bromine gas exerts a definite detrimental effect upon the cilia*

Hill (24), in a study made upon the excised

trachea of the horse, found that a concentration of one to five thousand stopped ciliary activity In six to seven seconds* There is disagreement as to the effect of chloro­ form and ether upon cilia*

Little or no effect was reported

by Proetz (46) and Hill (24).

However, in the studies made

by Ernst (11), dusts, introduced Into th© trachea of cats, were not removed during th© period of narcosis produced by chloroform or ether* A more complete list of the various physical and chemical agents and their effect upon cilia is given in the appendix*

Description of Apparatus

Construction Since a high humidity was used In this study, constant temperatures must be maintained to prevent con­ densation of water vapor upon the inner walls of th© apparatus*

The entire apparatus was enclosed in two incu­

bators as shown in Fig* 1*

The large glass chamber (D) was

located inside a constant temperature water bath which was kept at 29°C*, while th© remainder of the apparatus was placed in an air Incubator maintaining a temperature of 30-31°C*

Glass tubing led from the top of the glass gas

chamber (p) to the tissue chamber (B) which was situated under the microscope*

The valve system (c,d) produced a

unidirectional gas flow and the gas returned to the glass gas chamber (p) by glass tubing entering the bottom* end of the tissue chamber (B) was connected

At the

an accurately

calibrated hypodermic syringe (G) which was used to control the volume and rat© of gas passing over the tissue*

This

served to simulate natural breathing as nearly as possible* The tissue chamber (B) consisted of a glass tube, whose diameter was 5 mm*, with a window cut Into the walls and covered by cellulose acetate sheeting* cover was tightly fastened by copper wiring*

This window The inside of

the tissue chamber was lined with paraffin to give a rectan­ gular cross chamber having an area of 7*7 sq* mm* (f)* microscope had a 1GX ocular and 32 mm* objective*

The

Light

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'Lm o t e ' L is h t S ource .

Th e

rmometer

S.

Te m p . Co n t r o l

Te m p . Co n t r o l

-T r a n s p a r e n t .S l o t i n

Fig.

Co v e r

Gl a s s T u b e

1

A - Valve B - ‘Tissue chamber C - Calibrated hypodermic syringe D - Class gas chamber M - Microscope a, b - Openings of gas chamber C, d - Valves (glass) e - Intake for humid air f - Paraffin lined tissue chamber (transverse view)

was directed upon the tissue chamber by the luelte rod* All connections were made glass to glass by rubber tubing* Small glass balls were used as valves (c and d) to direct the flow of the gas mixture* Since the volume of gas flowing over th© tissue is an important factor, it was necessary to ascertain the rate of flow of air in the trachea of the living rabbit and adjust th© value used in this study accordingly.

By meas­

urement , the tidal air of the rabbit (adult averaging 5 to 6 lbs.) was found to be approximately 12 cc* with an in­ spiration rat© of about 105 per minute* of the trachea averaged 5*5 mm*

The inside diameter

Therefore, 3*8 to 3*9 cc*

of gas pumped over the tissue in the tissue chamber at a rate of 105 strokes per minute by the calibrated hypodermic syringe corresponds to the rat© in natural breathing* Operation In starting the experiment, the hypodermic syringe was removed and the tissue chamber was connected to an aspirator to permit humid air to enter (valve opened to e) The air in the constant temperature water bath, having a relative humidity above 95, was passed through th© apparatus to sweep out the existing gas mixture*

Then, a definite

amount of pure gas was measured out in th© hypodermic syringe connected to the tissue chamber and circulated through the apparatus until th© existing mixture was uniform*

Th® gas

mixture was replaced after each experiment to avoid as much

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as passible a decrease In the gas concentration due to recirculation # immediately after death of the rabbit, produced by a blow at the base of the skull, the trachea was placed in Ringer,s solution and kept at 33° to 34°C« until used# The Hinger*s solution used bad a composition of 0*7 per cent sodium chloride, 0*03 per cent potassium chloride and 0«025 per cent calcium chloride*

Due to the increased

sensitivity of the cilia after death, the excised tissue was used no longer than 2 to

hours after death of the

rabbit« A section of trachea about 2 X 4 mm* was then placed in the tissue chamber at the angle necessary to reflect th© light from the lucit© rod to th© microscope* As viewed through the microscope, the cilia produced a succession of coordinated waves moving in one general direction at a rate of about 6 to 10 beats per seeond* The syringe was then operated manually to deliver 3*8 to 3*9 cc* per stroke at a rate of 105 per minute*

During

this time, the cilia were observed through the microscope to ascertain the time Interval when all the cilia In the field had stopped moving# When circulation of th© gas was stopped soon after cessation of activity, ciliary movement started up again within a fairly short period of time#

For this reason, it

was thought desirable to determine the time of exposure necessary to damage the cilia such that they would not

restart after circulation of the gas was discontinued* Other sections of trachea were then placed in the tissue chamber and exposed to various concentrations of gas for periods of 1, 3, 5 and 10 minutes and if there was no recovery in the tissue chamber after circulation of the gas had been stopped, the sections were removed to Ringer1s solution at 33-34°C* and were later examined for recovery* The mucus overlying the cilia is constantly being renewed*

Since the mucus in the excised tissue under ex­

posure to gas cannot be renewed, the exposure never exceed­ ed 10 minutes* Controls showed no decrease in clliary activity after exposure to the circulated humid air, free from any irritant gas, for more than 15 minutes*

In these studies,

at least three experiments were carried out for each ex­ posure* Active gases as nitrogen dioxide and chlorine react with water and thus th© concentration may be decreased under the prevailing conditions of these experiments*

Chlorine

introduced in a concentration of 50 ppm* showed a decrease of approximately 30-35 per cent within 1/2 hour (using the o-tolidine test}*

Sulfur dioxide did not show any appre­

ciable decrease (iodine-thiosulfate method)*

In order to

minimi?;© the above factor, the experiment was carried out immediately after the gas was introduced and thoroughly mixed* In addition to the above factor, there is a

possible error of t

10 per cent In preparation of the

lower gas concentrations* The gases were prepared as follows: 1*

Ammonia by gently heating ammonium hydroxide (c*P«)

2*

Hydrogen sulfide by ferrous sulfIda and hydro­ chloric acid (C *P•)

3*

Formaldehyde by heating paraformaldehyde

4*

Sulfur dioxide by sodium sulfite (C«P») and hydrochloric acid ( c * P * )

5*

Chlorine by hydrochloric acid (c*P*) and potassium permanganate

6*

MItrogen dioxide by concentrated nitric acid (C*P*) and copper (electrolytic)

7*

Hydrogen chloride by sodium chloride (G*P*) and concentrated sulfuric acid (C*P.) RESULTS In the preliminary experiments it was observed

that the sensitivity of the tissue varied considerably with the time ^tiich had elapsed since Its removal from the animal* This tendency was toward a marked increase In sensivity ?/Ith the lapse of time*

Therefore, it was necessary, before

proceeding with the next problem to measure the degree of this Increased sensitivity to Irritant gases*

Accordirgly,

the response of 50 ppm* of sulfur dioxide for time intervals up to 7 1/2 hours after removal from the animal was measured* The Ringer’s solution used was frequently renewed during the course of the Investigation* In excised tissue, tested 1/2 hour follov/ing

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removal from th© rabbit, exposure to a concentration of 50 ppta* sulfur dioxide caused cessation of ciliary activity in 80 seconds*. On the other hand, tissue tested 7 1/2 hours after its removal from th© rabbit, using the same concentra­ tion of sulfur dioxide, showing cessation of ciliary activity within 15 seconds (Flg*2)« Previous to testing, the tissue was kept in Hinger’s solution at 34°C* However, this more rapid response may not be due entirely to an Increased sensitivity*

After submersion of

the tissue in Hinger’s solution for 30 minutes, a large part of the mucous covering lias been swept away*

While

there is still a mucous covering after 7 1/2 hours, It Is probably much less than that existing after 30 minutes sub­ mersion*

This may account in part for the quickened response. The effect of various concentrations of hydrogen

sulfide, ammonia, nitrogen dioxide (containing small amounts of nitric oxide), hydrogen chloride, sulfur dioxide, chlorine and formaldehyde upon the rat© of motion of the cilia in the excised trachea of th© rabbit is shown In Fig. 3.

The

experimental values varied only slightly and the average value was plotted in the graph* (Detailed data are given In the appendix*) The pH resulting from the dissolution of th© gas in th© aqueous layer covering the cilia may be a factor in stopping ciliary movement*

In fact, the results of this

Investigation as shown in FlgwS, suggest th© importance of this factor As is further Indicated by the experiments of

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360

120

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tO 20 30 40 SO SECO NDS N E C E S S A R Y FOR C E SS A TIO N OF A C T IV IT Y

Fig* 2 Increased Response of Ciliary Activity with. Tlxn© (Minutes) after Death.

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