some dynamic aspects of tissue structure in corneal epithelium*

7
SOME DYNAMIC ASPECTS OF TISSUE STRUCTURE IN CORNEAL EPITHELIUM* WlLHELM Bl New Some of the most essential factors in de- termining success or failure in corneal sur- gery are related to the complex problems of viability of tissue, of wound healing, and of compatibility of tissues. I do not have, be- fore this forum, to review the criteria which are applied in analyzing a given clinical situation from the diagnostic and prognostic viewpoint. As to the procedures and criteria which the pathologist applies, some of the preced- ing discussions have illuminated his methods of analysis. From the standpoint of classic pathology, the observed phenomena may be classified as degenerative, proliferative, and inflammatory ones in manifold combinations and successions and involving numerous tis- sue components. The preceding discussions have made it clear that the pathologist is no longer satis- fied with observing and classifying some final histologic picture. He also endeavors to re- construct the sequence of events and the role played by different tissue elements, by reviewing synoptically the individual his- tologic pictures of a clinical or experiment- ally produced succession of stages. The classic, predominantly descriptive approach is being replaced to an ever greater extent by an approach in which an attempt is made to interpret observations of histologic struc- ture in a dynamic way. In this approach, structural peculiarities of normal tissue and deviations from normal structure of cells and tissues are viewed as results of processes rather than as isolated static situations; this help's to break down the barriers between pathology and physiology and to obtain some insight into the action * From the Ayer Foundation Ophthalmic Re- search Laboratory, Manhattan Eye, Ear, and Throat Hospital. 7SCHKE, M . D . York mechanisms which lead to morphologic mani- festations. What appears to be of particular impor- tance, in this trend toward a dynamic inter- pretation of morphologic manifestations, is the fate of the single cell both as an in- dividual entity and as an integral part of an organized tissue. I should like here to illus- trate this approach and its implications for the understanding of both physiologic and pathologic phenomena in the tissue by a few examples taken from observations on corneal epithelium. While corneal epithelium may a priori ap- pear to be of relatively little interest in con- nection with corneal surgery, it is known from the work of other investigators to have great importance in the exchange of water 1 and of some essential metabolites 2 and thus in the physiology of the cornea. In addition, there is no obvious reason why similar prin- ciples and methods should not apply to the stroma as well; an approach which, in fact, has already been considered in some investi- gations on stroma cells. 3 OBSERVATIONS ON CORNEAL EPITHELIUM 1. The first example concerns the cellular damage due to ultraviolet irradiation and to mustard and related substances. With appro- priate doses of these agents, a peculiar mode of nuclear damage, nuclear fragmentation, develops within several hours and leads eventually to cell disintegration. This phenomenon has been previously described in detail. 4 - 5 Apart from the fact that with mustard the extent of this kind of cell damage is most marked in the basal layers of epithelium, while with ultraviolet it is more marked in the upper layers, the morphologic picture is very similar in both conditions, and so are

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Page 1: Some Dynamic Aspects of Tissue Structure in Corneal Epithelium*

SOME DYNAMIC ASPECTS OF TISSUE STRUCTURE IN CORNEAL EPITHELIUM*

WlLHELM Bl New

Some of the most essential factors in de­termining success or failure in corneal sur­gery are related to the complex problems of viability of tissue, of wound healing, and of compatibility of tissues. I do not have, be­fore this forum, to review the criteria which are applied in analyzing a given clinical situation from the diagnostic and prognostic viewpoint.

As to the procedures and criteria which the pathologist applies, some of the preced­ing discussions have illuminated his methods of analysis. From the standpoint of classic pathology, the observed phenomena may be classified as degenerative, proliferative, and inflammatory ones in manifold combinations and successions and involving numerous tis­sue components.

The preceding discussions have made it clear that the pathologist is no longer satis­fied with observing and classifying some final histologic picture. He also endeavors to re­construct the sequence of events and the role played by different tissue elements, by reviewing synoptically the individual his­tologic pictures of a clinical or experiment­ally produced succession of stages. The classic, predominantly descriptive approach is being replaced to an ever greater extent by an approach in which an attempt is made to interpret observations of histologic struc­ture in a dynamic way.

In this approach, structural peculiarities of normal tissue and deviations from normal structure of cells and tissues are viewed as results of processes rather than as isolated static situations; this help's to break down the barriers between pathology and physiology and to obtain some insight into the action

* From the Ayer Foundation Ophthalmic Re­search Laboratory, Manhattan Eye, Ear, and Throat Hospital.

7SCHKE, M.D. York

mechanisms which lead to morphologic mani­festations.

What appears to be of particular impor­tance, in this trend toward a dynamic inter­pretation of morphologic manifestations, is the fate of the single cell both as an in­dividual entity and as an integral part of an organized tissue. I should like here to illus­trate this approach and its implications for the understanding of both physiologic and pathologic phenomena in the tissue by a few examples taken from observations on corneal epithelium.

While corneal epithelium may a priori ap­pear to be of relatively little interest in con­nection with corneal surgery, it is known from the work of other investigators to have great importance in the exchange of water1

and of some essential metabolites2 and thus in the physiology of the cornea. In addition, there is no obvious reason why similar prin­ciples and methods should not apply to the stroma as well; an approach which, in fact, has already been considered in some investi­gations on stroma cells.3

OBSERVATIONS ON CORNEAL EPITHELIUM

1. The first example concerns the cellular damage due to ultraviolet irradiation and to mustard and related substances. With appro­priate doses of these agents, a peculiar mode of nuclear damage, nuclear fragmentation, develops within several hours and leads eventually to cell disintegration. This phenomenon has been previously described in detail.4-5

Apart from the fact that with mustard the extent of this kind of cell damage is most marked in the basal layers of epithelium, while with ultraviolet it is more marked in the upper layers, the morphologic picture is very similar in both conditions, and so are

Page 2: Some Dynamic Aspects of Tissue Structure in Corneal Epithelium*

40 WILHELM BUSCHKE

Fig. 1 (Buschke). Effect of temperature on manifestation of nuclear fragmentation following exposure to ultraviolet rays. (A) Incubation for eight hours at 30°C. (B) Incubation for eight hours at 10°C. (Re­printed with permission from the Journal of Cellular and Comparative Physiology.)

Fig. 2 (Buschke). Effect of anaerobiosis on manifestation of nuclear fragmentation following exposure to ultraviolet rays. (A) Aerobic incubation for six hours at 38°C. (B) Anaerobic incubation for six hours at 38°C. (Reprinted with permission from the Journal of Cellular and Comparative Physiology.)

Page 3: Some Dynamic Aspects of Tissue Structure in Corneal Epithelium*

TISSUE STRUCTURE IN CORNEAL EPITHELIUM 41

some of the physiologic factors involved in the pathogenesis.

Nuclear fragmentation develops also in vi­tro; that is, in the corneal epithelium of enucleated eyes which had been exposed to either of these two agents and subsequently incubated in a moist chamber. It was thus possible to study the effect of some environ­mental variables in vitro.

Changes of temperature have a most pro­

nounced effect. At a temperature of 38°C, the pathologic manifestations develop after 4 to 6 hours, while morphologic integrity is well maintained for much longer periods of time at lower temperatures (fig. 1). The latent period is thus inversely related to temperature with a temperature coefficient (Qio) of about 3.5.

From the standpoint of the mechanisms involved, this is of particular significance in the case of ultraviolet-ray damage; for here no chemical agent is primarily added, the interaction of which with some compon­ent (s) of the tissue could conceivably re­quire higher temperature.

It is, therefore, to be postulated that met­abolic processes in the tissue itself are re­sponsible for the pathologic manifestation. Of what nature the mechanisms may be which, under normal conditions, prevent, counteract, or compensate those metabolic

processes, is unknown at the present time. But it may be considered as certain that, in the development of the damage, enzymes are involved which are part of the metabolic equipment of the cell itself.

It is conceivable and, in fact, quite likely that the same principle applies likewise to the mechanisms involved in the nuclear dam­age following exposure to mustard, and possibly also in the pathologic conditions of

cells and tissues due to a great number of other noxious agents which require a tem­perature-dependent latent period for mani­festation of the damage.

Nuclear disintegration, for example, which follows arrest of mitosis in metaphase under the influence of colchicine, has a simi­larly high temperature dependence.6 In the cases of ultraviolet irradiation and mustard, in addition, aerobiosis was found to be a requisite for the manifestation of nuclear fragmentation (fig. 2) .

2. The healing of epithelial wounds may serve as a second example illustrating dy­namic aspects of tissue structure. The most widely used approach to a study of epithelial wound healing is that in which measurable changes in the size of a defect following injury are recorded over a period of time and under exposure to various agents. In order to reduce the number of

Fig. 3 (Buschke). Temperature dependence of posttraumatic cell movements in corneal epithelium. (A) Fresh pin-prick injury. (B) Similar lesion following three hours' incubation at 38° C. (C) Similar lesion following eight hours' incubation at 21° C. (Reprinted with permission from the Bulletin of The Johns Hopkins Hospital.)

Page 4: Some Dynamic Aspects of Tissue Structure in Corneal Epithelium*

42 WILHELM BUSCHKE

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Fig. 4 (Buschke). Inhibitory effect of cyanide on the posttraumatic cell movements in epithelium. Incubation for three hours at 38°C. in (A) Phosphate buffer, pH 7.4, and (B) M/1,000 cyanide in phosphate buffer pH 7.4. (Reprinted with permission from the Bulletin of The Johns Hopkins Hospital)

Fig. 5 (Buschke). Pseudopodial cell movements in corneal epithelium in healing of pin-prick injury. (Reprinted with permission from the Archives of Ophthalmology.)

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TISSUE STRUCTURE IN CORNEAL EPITHELIUM 43

variable factors in the experimental ap­proach, a study of the cellular events taking place in the surrounding of very small in­juries was initiated, and an attempt was made to use these morphologic observations on the cellular level in assaying the effects of variables applied over shorter periods of time.7 It was hoped to reveal with this approach some of the dynamic aspects, and

Fig. 6 (Buschke). Control sheet of corneal epi­thelium following incubation of cornea for 16 hours at 38°C. No loss of cohesion.

thus of the mechanisms, involved in healing of epithelial wounds.

Pin-prick injuries to corneal epithelium heal with cell movements of the surround­ing epithelia, and in this process pseudopo-dial cell extensions play an important role8

(fig. 5). 3. A study of the variables which would

interfere with these cell movements revealed that this phenomenon is highly temperature dependent (Q10 = 5)7 and is susceptible to interference by anaerobiosis,7 cyanide, iodo-

Fig. 7 (Buschke). Loss of intercellular cohesion in sheet of corneal epithelium following incubation for one hour with 2.5 mg. percent chymotrypsin.

Fig. 8 (Buschke). Loss of intercellular cohesion following incubation of epithelium for two hours with 2.5 mg. percent chymotrypsin.

acetate,7 and other sulfhydryl binding sub­stances,9 as well as to that by local anes­thetics1 (figs. 3 and 4) .

Page 6: Some Dynamic Aspects of Tissue Structure in Corneal Epithelium*

44 WILHELM

Fig. 9 (Buschke). Loss of intercellular cohesion following incubation of cornea with M/7 fluoride for six hours at 38°C. (Reprinted with permis­sion from the Journal of Cellular and Comparative Physiology.)

In addition, however, this approach re­vealed some differences in the mode of move­ment of the individual cells which were de­pendent on the size and shape of the injury, and on the location of the cell in the multi-layered epithelium. These differences sug­gested that intercellular cohesion may be an important variable affecting the mode of cell movement.8

A study of factors involved in tissue con­tinuity or cell cohesion provided another oc­casion to apply dynamic viewpoints to the study of structure in the tissue. Among the most potent agents which interfered with tissue continuity and brought about isola­tion of epithelia from each other in a mor­phologically well-preserved state, were some proteolytic enzymes, notably chymotrypsin and trypsin10 (figs. 6 and 8) .

It was suspected, therefore, that intrinsic proteolytic enzymes of the tissue itself might be responsible for physiologic and pathologic variations of cell cohesion. Under conditions of rest and in a normal organized tissue like that of corneal epithelium, the effect of in­trinsic proteolytic activity may not manifest

BUSCHKE

Fig. 10 (Buschke). Loss of intercellular cohesion following incubation of cornea with M/100 iodo-acetamide for six hours at 38° C.

itself in an overall loss of cohesion, because of restraining or compensating metabolic processes.

It is now known from other tissues, how­ever, that the proteins of the tissue, even the structural ones and those considered as most stable, are actually in a steady flux of break­down and resynthesis.11

In applying this line of thought to the problem of intercellular cohesion, the as­sumption was made that such a steady state of processes of synthesis and breakdown would also prevail in respect to proteins involved in cell cohesion. It appeared likely that met­abolic energy would be required for the processes of protein synthesis. Interference with certain energy-producing or energy-transferring metabolic pathways in the tis­sue might, therefore, be expected to let intrinsic proteolysis manifest itself in loss of cell cohesion.

Exposure of corneas to fluoride, iodoace-tate, iodoacetamide, 2,4-dinitrophenol, 4,6-dinitrocresol, and to some local anesthetics. with incubation at body temperature foi several hours lead to a marked loss of inter­cellular cohesion12 (figs. 9 and 10). It is oi interest that some of these agents are knowr

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TISSUE STRUCTURE IN CORNEAL EPITHELIUM 45

primarily as inhibitors of glycolytic or lism may be responsible for physiologic and phosphorylative pathways. Under otherwise pathologic variations in cell cohesion. similar conditions, anaerobiosis and inter- These examples may serve to illustrate ference with oxidative pathways, such as the possibilities inherent in an approach exposure to cyanide or malonate, had no which stresses the dynamic state of tissue effect on intercellular cohesion. and cell structure. From the practical stand-

Much further work will be required to point, they demonstrate the possibility of obtain more insight into the mechanisms of modifying and directing by environmental cell cohesion. The observations to date, how- agents the course of events which are ever, appear to be compatible with the causally related to the maintenance and idea that processes of breakdown and re- reestablishment of functional and morpho-synthesis of the proteins concerned in inter- logic integrity of tissue in prophylactic and cellular cohesion play some role and therapeutic procedures. that factors regulating the extent of these 210 East 64th Street (21). two opposite phenomena of tissue metabo-

REFERENCES

1. Cogan, D. G.: Clinical physiology of the cornea: The interrelationship of corneal turgescence, epithelial edema, bullous keratopathy, and interstitial vascularization. Am. J. Ophth., 32:625-633, 1949.

2. Herrmann, H., and Hickman, F. H.: Further experiments on corneal metabolism in respect to glucose and lactic acid. Bull. Johns Hopkins Hosp., 82 :260-272,1948.

3. Friedenwald, J. S.: Note on karyolysis of the corneal stroma cells. Bull. Johns Hopkins Hosp., 82:178-181,1948.

4. Buschke, W., Friedenwald, J. S., and Moses, S. G.: Effects of ultraviolet irradiation on corneal epithelium: Mitosis, nuclear fragmentation, post-traumatic cell movements, loss of tissue cohesion. J. Cell. & Comp. Physiol., 26:147-164,1945.

5. Friedenwald, J. S., and Buschke, W.: Nuclear fragmentation produced by mustard and nitrogen mustards in corneal epithelium. Bull. Johns Hopkins Hosp., 82:161-177,1948.

6. Friedenwald, J. S., Buschke, W., and Moses, S. G.: Comparison of the effects of mustard, ultra­violet, and X-radiation, and of colchicine on the cornea. Bull. Johns Hopkins Hosp., 82:312-325, 1948.

7. Friedenwald, J. S., and Buschke, W.: The influence of some experimental variables on the epi­thelial movements in the healing of corneal wounds. J. Cell. & Comp. Physiol., 23 :95-107, 1944.

8. Buschke, W.: Morphologic changes in cells of corneal epithelium in wound healing. Arch. Ophth., 41:306-316, 1949.

9. : Experimentelle Studien zur Patho-Physiologie des Hornhautepithels: Zellbewegungen bei der Wundheilung, Zellteilung, Mitosehermung und andere Kernphaenomene. Ophthalmologica, 118: 407,1949.

10. : Studies on intercellular cohesion in corneal epithelium: Methods: Effects of proteolytic enzymes, salts, hydrogen-ion concentration, and polar-nonpolar substances. J. Cell. & Comp., Physiol., 33:145,1949.

11. Schoenheimer, R.: The Dynamic State of Body Constituents. Cambridge, Mass., Harvard, 1949. 12. Buschke, W.: Effects of metabolic poisons and of some other agents on intercellular cohesion in

corneal epithelium. Am. J. Ophth., 33:59,1950.