M etabolic interactions between the

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1 The effect of the absence of corneal epithelium or endothelium on the stromal keratocytes Claes H. Dohlman, Antonio R. Gasset, and Jeannette Rose hitact calf corneas and calf corneas with epithelium and endothelium removed were incubated in vitro with inorganic sulfate- S5 S for 6 hours at 38 C. The incorporation of S5 S into the nondialyzable substances (essentially mucopolysaccharides) was measured. No significant difference was found between the 3S S incorporation of the denuded stroma and that of the intact cornea. Calf corneal stromas, swollen and normally hydrated, were similarly incubated in vitro. The siuelling per se had no effect on the 35 S incorporation. The corneal epithelium was removed from one eye of rabbits in vivo, and inorganic sulfate- S5 S was immediately injected intravenously. The absence of epithelium caused a more rapid diffusion of the sulfate- 35 S ion into the stroma than occurred in the control eye. Isotope incorporation, in the absence of epithelium, ivas lower near the anterior surface and slightly higher in the posterior stroma within the first 24 hours as compared with the normal cornea. When the endothelium toas removed from the rabbit cornea in vivo and sulfate- 35 S was administered intravenously, the results showed only a slight decrease in the rate of mucopolysaccharide synthesis in the stroma. Histologic studies were carried out on rabbit corneas from which the epithelium had been removed and kept away for up to 10 days. The stroma cells survived throughout the stroma unless undue trauma had occurred. Near the anterior surface, however, some of the keratocytes transformed into fibroblasts. Following removal of the endothelium, the corneas could be folloived histologically for up to 6 weeks. Here also, the keratocytes survived and the picture was similar when both epithelium and endothelium had been removed. Following removal of the endothelium, eosinophilic and metachromatic material accumulated in the anterior stroma and, eventually, in the basal cells of the epithelium, seemingly washed through the stroma to the corneal surface by a flow of fluid from the anterior chamber. It icas concluded that at least for a limited time the stromal cells can function independently of the epithelium and the endothelium. From the Department of Cornea Research, Institute of Biological and Medical Sciences, Retina Foundation, Boston, Mass. Supported, in part, by Public Health Service Research Grant NB and Public Health Service Training Grant NB-5518 from the National Institute of Neurological Diseases and Blindness, United States Public Health Service, and in part by the Massachusetts Lions Eye Research Fund, Inc. 520 M etabolic interactions between the corneal stroma and its surrounding cellular layers have been demonstrated by Herrmann and Hickman. 1 " 3 These authors ex- The biochemical data of this investigation were presented at the Sixth Conference on Ophthalmic Biochemistry, Howe Laboratory of Ophthalmology, Harvard University, Dedham, Mass., February, 1961.

2 Volume 7 Number 5 Absence of corneal epithelium 521 plored the carbohydrate metabolism in particular and found a definite pattern of exchange of metabolites between the epithelium and the stroma. Later, Herrmann 4 and Herrmann and Love 5 found that, in chicks, removal of the corneal epithelium practically abolishes the incorporation of glycine-^'c into the stroma in vitro. When Herrmann and Lebeau 0 later reinvestigated the problem, they found that actual absence of the epithelium was not the only factor involved, but also that the stroma cells became easily damaged during the removal procedure. With a more gentle technique of removal, glycine incorporation became near normal. Smelser, 7 ' s on the other hand, reported that if the epithelium is removed from the cornea of the living rat, the incorporation of inorganic sulfate- 3r> S into macromolecules of the stroma, presumably polysaccharides, is greatly diminished. The technique used was contact autoradiography of washed and dried corneas. Similarly, Langham and Pollack 9 found that if the epithelium or the endothelium was removed from the rabbit eye, the underlying stroma incorporates much less 35 S. Insertion of a very thin plastic membrane into the corneal stroma changed the autoradiographic picture in cross-section so that, in the absence of epithelium, only the region anterior to the membrane had a reduced 3fi S uptake. 0 Wortman 10 incubated excised rabbit and beef corneas in tissue culture solution containing inorganic sulfate- 3Fl S. By quantitative determination, he found that, in the absence of epithelium and endothelium, the uptake of 3f 'S was considerably diminished in the rabbit stroma, whereas the beef stroma was less affected. Praus and Obenberger 11 also used radioactive sulfate uptake as a measure of mucopolysaccharide synthesis in vitro. Beef corneas incubated for 20 hours showed a reduced uptake when denuded. When the incubation time was reduced to 6 hours, however, the difference between the denuded and the control corneas was negligible. 12 Recently, Sato 13 and Honda 1 ' 1 found that removal of either the epithelium or the endothelium had no significant effect on the incorporation of 35 S into the stroma of living rabbits. Knowledge of the interactions between the various layers of the cornea may ultimately explain why long-standing epithelial defects often lead to melting or scarring of the underlying stroma and why endothelial disturbance sooner or later results in deep scar formation in the adjacent stroma. The results of the studies cited are somewhat contradictory, and they cover only those events that occur within hours after the removal of the cellular layers. The purpose of the present investigation was to ascertain, by quantitative isotope determination and autoradiography, the immediate effect produced by the absence of epithelium or endothelium, or both, on the metabolic functions of the keratocytes. In addition, the eftect of prolonged absence of one or both of these limiting layers on the stroma was studied histologically. Material and methods Isotope experiments. In vitro experiments. Twenty fresh calf corneas and 50 cow corneas were used. The eyes (in pairs) were brought to the laboratory immediately after the animals had been slaughtered, and the corneas were excised with a fine scleral rim attached. In the first experiment, corneas with and without the limiting cellular layers were compared with regard to 35 S uptake. The epithelium and the endothelium (including Descemet's membrane) were gently removed with a knife from the cornea of one eye of each pair, avoiding undue pressure on the stroma. The cornea of the other eye, left intact, served as a control. All corneas were incubated in Sinims Z tissue culture solution containing inorganic sulfate- 3S S (1 fie per milliliter) for 6 hours at 38 C. Each group of 4 or 6 corneas was incubated in 50 ml. of the medium. The specimens were then rinsed and blotted. Hydration was determined by weighing the corneas in wet state and after drying in a desiccator over phosphorus pentoxide in vacuo at 60 C. for at least 24 hours. In the second experiment, designed to compare the 3r 'S uptake of the swollen corneal stroma with that of the unswollen stroma, the procedure was as follows: the epithelium and the endothelium (including Descemet's membrane) were re-

3 522 Dohlman, Gasset, and Rose Investigative Ophthalmology October 1968 moved from the freshly excised corneas. Each stroma was placed in a dialysis bag and immersed in one of two beakers containing 50 ml. of Simms Z solution and sodium sulfate- 35 S (2 (ic per miniliter); to one of the beakers, dextran had been added to a final concentration of 7 per cent. Bodi beakers were slowly shaken at 4 C. for 24 hours to allow the isotope to diffuse into the tissue and swelling to take place whenever possible. The beakers were then transferred to 38 C. and slowly shaken at this temperature for 6 hours. Hydration was determined as described. In both experiments, the 35 S incorporation was determined as the dialyzable and the nondialyzable radioactive material. The dried tissues were placed in moistened dialysis bags and dialyzed against 100 ml. of 0.1 per cent unlabeled sodium sulfate at 4 C, with slow shaking for 24 hours. The sulfate in the outside dialysis solution was precipitated with barium. The tissues were then hydrolyzed with 6N HC1 at 100 C. for 18 hours, and the isotope was precipitated as barium sulfate. Counting was carried out with an end-window G-M tube at infinite thickness of the precipitate. In vivo experiments. Adult albino rabbits, weighing 2 to 3 kilograms, were anesthetized with Nembutal. REMOVAL OF EPITHELIUM: The epithelium, including the limbal fringe, was gently removed with a knife from one eye. The completeness of the removal was checked with fluorescein and, on one occasion, was checked histologically. Immediately after the epithelium had been removed, 3.0 me. of carrier-free inorganic sulfate- 35 S was injected intravenously into each rabbit. The animals were killed at different intervals, ranging from 15 minutes to 24 hours after injection, and the epithelium was removed from the control cornea. Both corneas were then rapidly cut out, and the hydration was determined by weighing the tissue in wet and dry states. The dialyzable and nondialyzable 35 S were assayed in the manner described in the preceding section. To compare the uptake of 35 S in the central and peripheral regions, a central 7 mm. disc was trephined out of the excised cornea, and the radioactivity in the disc and in the remaining part, both in the cornea denuded of epithelium and in the control cornea, was assayed. In other experiments, the anterior portion of the corneal stroma, both in the cornea denuded of epithelium and in the control cornea, was separated from the posterior portion by blunt dissection, and the 35 S uptake was determined. REMOVAL OF ENDOTHELIUM: A curved spatula was inserted through a small scleral incision, and the endothelium was scraped off in two or three sweeps. Sulfate- 3F> S (3.0 me.) was given intravenously, but not until 24 hours after scraping in order to allow the edema to develop fully. At different intervals, ranging from 15 minutes to 24 hours after injection, the rabbits were killed, and the dialyzable and nondialyzable 3B S were determined in both the scraped and the control corneas. Autoradiography. The temporal half of the corneal epithelium of each eye in two adult albino rabbits was removed with a knife. Inorganic sulfate- 35 S (3.0 me.) was given intravenously immediately afterward. The rabbits were killed 24 hours later, and the corneas were stained with fluorescein. The area denuded of epithelium was carefully mapped, and the remaining epithelium was removed. After sutures had been inserted to mark the position of the denuded area, the corneas were excised, and the endothelium was removed. The stromas were then dialyzed against 100 ml. of 0.1 per cent unlabeled sodium sulfate solution containing 7 per cent dextran (to prevent swelling) at 4 C. for 24 hours, and the tissues were dried. A dried rabbit cornea is 80 to 100 ii thick. The subsequent procedure was similar to that described by Smelser." Very thin Saran Wrap was placed on both sides of the tissue, and Kodak Dental Film DF7 was pressed against both sides of it. The exposure time was 40 days (the half-life of 3r> S is 87 days). The films were removed from the two sides and developed and fixed simultaneously. Comparison of the density of blackening in the autoradiographs indicated the difference in incorporated 3r 'S. Histology. A histologic study of the effects on the cellular constituents of the corneal stroma produced by the absence of either the epithelial or the endothelial layer, or both layers, was carried out on 42 adult albino rabbit eyes in vivo. Removal of epithelium. Ophthaine (0.5 per cent, E. R. Squibb & Sons) was dropped onto the corneas of both eyes 10 minutes before general anesthesia with sodium pentobarbital (Nembutal). Initial removal of the entire epithelium was performed on bodi corneas of the same animal with a Bard-Parker scalpel and blade under Fig. 1. A and B indicate the right and left corneas, respectively, of the same rabbit; the epithelium has been removed from both. The epithelium was removed daily from one eye (C); it was allowed to regenerate in the other eye (D). The dotted lines indicate the areas excised for histologic study.

4 Volume 7 Number 5 Absence of corneal epithelium 523 a Zeiss operating microscope. When the duration of the study was longer than 24 hours, the central cornea of the right eye was kept free of epithelium by daily removal of the cells as they regenerated at the periphery (Fig. 1). Thus the area overlying the central stroma was not subjected to repeated scraping. The epithelium was allowed to regenerate over the contralateral eye; hence the contralateral eye of each pair served as a control. The eyes were enucleated after 6, 12, 19, and 24 hours, and 3, 5, and 10 days. In 6 eyes, the epithelium was removed once from only a small central area 3 to 4 mm. in diameter; the corneas were excised 1, 2, and 3 days later. In another 7 cases, the lids were kept closed between the daily removals of epithelium to determine whether any differences would occur in the appearance of the stromal cells when the influence of evaporation was eliminated. The corneas were excised after 5 and 10 days. Removal of endothelium. The endothelium was gently removed in vivo under the Zeiss operating microscope, as already described. A 0.1 ml. solution of sodium heparinate (1 mg.) was injected into the anterior chamber to prevent fibrin formation. The eyes were enucleated after 12 and 24 hours, 3 and 5 days, 1, 2, 3, 5, and 6 weeks. Removal of both epithelium and endothelium. When both of these lavers were removed, the endothelium was scraped once, with the technique described, and the epithelium was removed daily. The corneas were excised after 24 hours, 3, 5, and 10 days. In control experiments, it was found that neither the dropping of Ophthaine onto the cornea nor the injection of sodium heparinate into the anterior chamber had any histologically detectable effect on the stromal cells. Fixation and staining. The enucleated eyes were fixed in 0.5 per cent cetylpyridinium chloride in 10 per cent formalin. Three days after fixation, a wide strip from the central area, including the peripheral margin, was excised and embedded in paraffin. The perpendicular sections were stained with hematoxylin and eosin or with 0.1 per cent toluidine blue in acetate buffer adjusted to ph 4.0. Results Isotope experiments. Accuracy of methods. experiments were carried out to check the dialysis and hydrolysis procedures. In five separate experiments, frozen corneas (cells destroyed) containing inorganic sulfate- 35 S were dialyzed. After dialysis, 0.85 per cent Table I. 3fi S incorporation in calf corneas in vitro with and without the limiting cellular layers Corneal tissue Stroma (4) Intact (4) Stroma (6) Intact (6) Hydration (mg. ILO/mg. dry tissue tot.) Dialyzable 35 S (c.p.m./mg. H.O) 1,762 ± 70 1,908 ± 172 Nondialyzable S5 S (c.p.m./mg. dry tissue wt.) 6,884 ± 808 7,888 ± ± 85 1,140 ± 184 1,053 ± 300 1,036 ± 333 The results of two separate experiments, with the standard error of the mean, are given. The number of corneas in each group is indicated in parentheses. Table II. Effect of swelling on 35 S incorporation in isolated cow comeal stromas in vitro Stroma Hydration (mg. HsO/mg. dry tissue wt.) Dialyzable S5 S (c.p.m./mg. H 2 O) Nondialyzable si S (c.p.m./mg. dry tissue wt.) Swollen (10) (10) Swollen ( 9) ( 9) Swollen ( 6) ( 6) 5.2 ± ± ± ± ± ,229 ± 121 1,286 ± 125 1,638 ± 625 1,552 ± 388 1,165 ± 107 1,162 ± 90 2,239 ± 441 1,506 ± 288 4,489 ± 1,622 2,903 ± 572 1,193 ± 420 2,249 ± 396 The results of three separate experiments, with the standard error of the mean, are given. The number of stromas in each group is indicated in parentheses.

5 524 Dohlman, Gasset, and Rose Investigative Ophthalmology October 1968 of the isotope remained, instead of the calculated 0.4 per cent. This very small error was ignored in subsequent experiments. In a second control series, the possible influence of the amount of tissue on hydrolysis was investigated. Eleven samples of dry corneas, weighing from 2 to 48 milligrams, were hydrolyzed together with identical amounts of sulfate- 35 S. The average weight of the barium sulfate precipitate was mg. (S.E.M.), whereas the average count was 1,747 ± 21.1 c.p.m. (S.E.M.). Thus, the weight of the tissue samples did not influence the results in the range below 48 mg. In vitro experiments with bovine corneas. Table I shows the 35 S incorporation in calf corneas incubated in vitro. In each pair of eyes, one intact cornea was compared with the isolated stroma from the contralateral eye. In one of the two series, the concentration of nondialyzable 3fi S was approximately the same in both the intact cornea and the denuded stroma. In the other series, it was slightly lower in the isolated stroma. This difference was not statistically significant, however. Other experiments gave similar results. To determine whether swelling of the corneal stroma per se influences 35 S incorporation, one isolated stroma from each pair of cow eyes was allowed to swell and the hydration of the other stroma was kept as close as possible to the normal. The data from three such experiments are given in Table II. The concentration of dialyzable 35 S was similar in the swollen and unswollen tissues. Moreover, there was no significant difference between the nondialyz- Table III. Effect of absence of epithelium on 33 S incorporation in rabbit corneal stromas in vivo Time (hours) Cornea Hydration (mg. HsO/mg. dry tissue tot.) Dialyzable 35 S c.p.m./mg. H;O Nondialyzable S5 S c.p.m./mg. dry tissue tot i ,540 2, , , ,885 1, , ,212 2, ,236 1, ,985 1, ,302 3, ,

6 Volume 7 Number 5 Absence of corneal epithelium 525 able 3r 'S of the swollen and unswollen stromas. In vivo experiments with rabbit corneas. Table III is a summary of the results obtained on 11 rabbits in which the corneal epithelium had been removed from one eye and sulfate- Hfi S injected intravenously. The denuded corneal stroma swelled to some extent (a hydration of 3.4 mg. H-.0 per milligram dry tissue weight is normal according to Duane 15 ). The concentration of dialyzable 35 S was, in most instances, higher in the cornea denuded of epithelium than in the control. Moreover, the concentration of nondialyzable 35 S was generally higher in the denuded cornea. Table IV shows the effect of the absence of epithelium on the concentration of 35 S in the central and peripheral regions of denuded and intact corneas. In the 3 rabbits of this series, there were no significant differences between the concentrations of nondialyzable 3F> S in the central and peripheral regions of either eye. The concentration of inorganic sulfate- 35 S in the tissue was not determined in these experiments. In another series of rabbits, however, a comparison of the anterior and posterior regions of corneas denuded of epithelium showed considerable differences (Table V). In all three animals, the specific activity of the anterior region was considerably lower than that of the posterior region of the denuded cornea. The average of the ratios of the specific activities of the anterior and posterior regions was found to be 0.5 for the denuded cornea and 1.2 for the control. The effect of the absence of endothelium on 35 S incorporation is shown in Table VI. Since the corneas had been allowed to swell for 24 hours before the injection of :Hn S, there was considerable hydration of the denuded corneas. The amount of dialyzable 3F 'S per milligram of HX) was, in all instances, considerably higher in the denuded cornea than in the control. The amount of nondialyzable 3S S was also higher in the denuded cornea, but the difference was less marked. Autoradiography. The results of contact autoradiography of the anterior and posterior sides of a rabbit cornea from which the temporal half of the epithelium had Table IV. Effect of absence of epithelium on 35 S incorporation in the central and peripheral regions of the rabbit cornea in vivo Rabbit No cornea Nondialyzable 35 S (c.p.vi./mg. dry tissue wt.) Central Peripheral 3,096 3,259 4,035 3,527 1,920 2,179 Central Peripheral Intact cornea Nondialyzable 35 S (c.p.m./mg. dry tissue wt.) Central Peripheral 3,795 3,049 3,263 3,019 2, Central Peripheral Table V. Effect of absence of epithelium on 3r> S incorporation in the anterior and posterior regions of the rabbit cornea in vivo Rabbit No. cornea Nondialyzable 35 S (c.p.m./mg. drij tissue wt.) Anterior Posterior 2,400 1,500 2,200 4,600 4,400 3,800 Anterior Posterior Intact cornea Nondialyzable 35 S (c.p.m./mg. dry tissue wt.) Anterior Posterior 3,900 3,200 3,800 2,600 3,400 2,900 Anterior Posterior

7 526 Dohhnan, Gasset, and Rose iuije Ophthalmology October ~ * --.U.»t.W..fT-JA Fig. 2. Autoradiographs of the anterior surface (left) and posterior.surface (right) of a rabbit stroma. The temporal half of the epithelium was removed in vivo, and the animal was injected immediately thereafter with inorganic sulfate- :is S. The anterior radioautograph of the area without epithelium was less dense than the area with epithelium. The result in the posterior radioautograph was the opposite. The dividing line between the intact and scraped areas is faintly visible. This probable distribution within the stroma is illustrated schematically (top). been removed are shown in Fig. 2. The autoradiograph of the anterior side shows less blackening over the area without epithelium than over the intact area; the autoradiograph of the posterior side shows the opposite pattern. The posterior stroma underlying the denuded area shows more uptake of as S than the other half, where the epithelium was intact. The dividing line between the two areas can be faintly seen and corresponds to the epithelial edge on the anterior side. The periphery, in particular, shows marked uptake of 3fl S in the area without epithelium. The other three corneas gave similar results. Histology. Absence of epithelium. The integrity of the lamellar structure was not disrupted by the removal of the epithelial layer, even when mild edema was present. After 6 hours, there was no loss of keratocytes in the stroma. The majority of the stromal cells appeared to be slightly hypertrophic in that they were larger and less basophilic than the normal keratocytes. The hypertrophic cells, as well as the normal keratocytes present, contained metachromatic red granules, indicating the presence of mucopolysaccharides in the cells. 10 Some atypical cells were found in the superficial stroma. These cells were small and ovoid, with blunt ends and no cytoplasmic processes. They were intensely basophilic, and frequently metachromatic red granules were absent. The structure, staining, and location of the cells suggested that they were young fibroblasts in an early stage of transformation from keratocytes. At the periphery, numerous polymorphonuclear leukocytes had accumulated in the anterior lamellae, but they had not infiltrated the central or posterior regions. The appearance of the stroma was essentially the same after 12, 19, and 24 hours. The main difference was in the number of

8 Volume 7 Number 5 Absence of corneal epithelium 527 Fig. 3. Epithelium absent 24 hours. An occasional kerntocyte was lost or had changed into a young fibroblast, but there was no loss of cells in the anterior lamellae. (xloo.) Fig. 4. An example of loss of keratocytes after 5 clays clue to excessive trauma produced by scraping the anterior surface. (xloo.) keratocytes in. the anterior lamellae. In some cases, no loss of cells occurred (Fig. 3); but in others, the anterior 10 to 20 per cent of the total stroma was lacking in cells of any kind. The most anterior of the cells, in each instance, had the appearance of young fibroblasts; the remaining cells were normal keratocytes. Not until 24 hours had elapsed did leukocytes reach the central region, but when an acellular zone was present, they infiltrated below it. After 3 days, on the average, one fifth

9 528 Dohlman, Gasset, and Rose Iiwextigatioe Ophthalmology October 1968 of the stroma, in both the denuded and the control corneas, was devoid of cells. The thickness of the acellular area varied considerably. Thus, after 5 days, two denuded corneas showed an extreme loss of cells (Fig. 4); other denuded corneas, and the control corneas, showed minimal cell loss at this time. The location and characteristics of the fibroblasts had not changed, and the remainder of the stroma was normal. After 10 days, there was no acellular zone in either the denuded or the control corneas. In the denuded corneas, about one fifth of the anterior stroma was populated by fibroblasts (Fig. 5). There were more fibroblasts in the central region than in the peripheral. They showed a homogeneous cytoplasm and several nucleoli, with less intense basophilia, and some of them were elongated and possessed cytoplasmic processes (Fig. 6). The remainder of the stroma contained normal keratocytes. In the control corneas, fibroblasts of the same structure as those seen in the denuded corneas were present, but they were fewer in number. Many leukocytes were evident anteriorly, some of them extending into the central region. In those animals from which only the central epithelium had been removed and in those whose lids had been kept closed, the cellular changes in the stroma were essentially the same as the changes described. Absence of endothelium. Following the removal of the endothelium, no endothelial cells were seen in any of the corneas for up to 6 weeks. Descemet's membrane was present; occasionally it was interrupted in a small area, but comparisons of the overlying stroma showed that this caused no significant difference. Absence of endothelium initiated a rapid onset of stromal edema, judged from the disruption of the lamellar structure (Fig. 7). After 2 weeks, the swelling began to subside, and after 5 and 6 weeks, the lamellar structure was almost normal despite the fact that no regenerating endothelium could be found. Epithelial edema was evidenced by the presence of small, occasional bullae 24 hours after removal of the endothelium. By Fig. 5. Epithelium absent 10 clays. Prolonged absence of epithelium did not result in absence of stromal cells. The cells in the anterior lamellae are fibroblasts. (xloo.)

10 Volume 7 Number 5 Absence of corneal epithelium 529 the third day, the bullae had become larger and more frequent, and metachromatic red granules were present within the cytoplasm of the basal epithelial cells. By the seventh day, the bullae were of gross size, and the accumulation of red granules had increased (Fig. 8). After 2 weeks, there were only a few small, intermittent bullae, and there was no evidence of red granules. After 3, 5, and 6 weeks, the epithelium appeared to be normal. As early as 12 hours after the endothelium had been scraped, an intense eosinophilic band of stain was present in the stroma immediately below the epithelium, and an intense metachromatic band was located beneath it. By the third day, the eosinophilic band had disappeared, and the metachromatic band was located directly underneath the epithelium. By the seventh day, the metachromasia had become more diffuse, and after 2 weeks, the staining was normal. After 12 and 24 hours, most of the keratocytes located in the anterior third of the stroma still appeared normal. In stromas exhibiting an intense metachromatic band of stain, the stromal cells within the band contained many more intense red metachromatic granules. In the posterior stroma, the cells resembled normal keratocytes in configuration, but they were slightly larger and less basophilic. By the third day, the disruption of the fibers had distorted the cell configuration, but both metachromatic and basophilic staining indicated that these cells were forms of keratocytes. Some were obviously of the hypertrophic type. By the seventh day, there were fewer normal cells in the anterior third of the stroma. After 2 and 3 weeks, the keratocytes were less distorted. After 5 and 6 weeks, the structure of the lamellae was nearly normal, and most of the cells were typical, normal keratocytes (Fig. 9). Only in the anterior stroma were numerous young fibroblasts observed. These cells were nongranular and intensely basophilic, and they possessed several nucleoli. When the endothelium had been absent for 3 or more days, a linear arrangement of fibroblasts appeared directly anterior to Fig. 6. Epithelium absent 10 clays. The fibroblasts in the superficial stronia have developed nucleoli and cytoplasmic processes. (xloo.)

11 530 Dohlman, Gasset, and Rose Investigative Ophthalmology October 1968 Fig. 7. Enclothelium absent 24 hours. The resulting edema caused a moderate disruption of the lamellae but no apparent loss of stromal cells. (xloo.) Descemet's membrane (Fig. 8). These cells were elongated, very basophilic, and nongranular, and they seldom contained metachromatic red granules. Absence of epithelium and endothelium. Removal of both layers resulted in the same disiaiption of the lamellar structure as was observed when only the endothelium was absent. There was no loss of cells in the posterior stroma, and the only deviation from the normal cell structure was the appearance of an occasional hypertrophic cell after 24 hours. The principal change occurred in the keratocytes of the anterior lamellae. These keratocytes resembled the fibroblasts observed when the epithelium alone was removed. Nucleoli appeared in the fibroblasts as early as the third day. The fibro- Fiij. 8. Endothelium absent 7 days. In spite of stromal edema with resultant distortion of the cells, there was no massive cell death in the stroma. A row of fibroblasts can be seen in the stroma just anterior to Descemet's membrane. (xloo.) (x200. In excessive edema, the tissue tends to shrink considerably during processing.) blasts increased in number over the extended period; there were many more at 10 days than at 3 days. Moreover, there was no loss of cells in the anterior stroma at any time. Discussion The absence of the comeal epithelium in rabbits in vivo caused the denuded stroma to incorporate more sulfate- 35 S into nondialyzable compounds (presumably mucopolysaccharides) than did the intact control cornea. The concentration of the inorganic precursor i.e., the labeled sulfate ion was also higher in the denuded cornea (Table III). An exact correction for the increased specific activity of the in-

12 Volume 7 Number 5 Absence of cornea! epithelium 531 Fig. 9. Endothelium absent 5 weeks. Prolonged absence resulted in no loss of keratocytes. (xloo.) organic sulfate pool, at any given time, cannot be made on the basis of the present findings. It seems, however, that the concentrations of dialyzable and nondialyzable afi S increased in about the same proportions in the denuded and control corneas, which would indicate that the rate of polysaccharide synthesis is approximately equal in stromas in vivo with and without epithelium during the first 24 hours. The localization of * r 'S incorporation was altered, however, after removal of the epithelium. The labeling showed a moderate decrease in the anterior stroma, with a curious increase in the posterior layers (Fig. 2 and Table V). Autoradiography gives the impression of a great difference between the labeling of areas covered and not covered by epithelium, whereas the results of quantitative determinations indicate only a moderate change. The reduction in Sfl S uptake in the surface layers of the denuded stroma may have been caused by the unavoidable slight trauma produced by the removal of the epithelium. The histologic picture supports this assumption. The absence of endothelium in rabbits in vivo resulted in a higher 3r> S incorporation in the denuded stroma than in the control. The concentration of dialyzable 35 S, however, was considerably higher in the scraped corneas than it was in the control corneas. If an approximate correction is made for this difference in precursorspecific activity, the polysaccharide synthesis seems slightly lower in the cornea without endothelium than it is in the intact cornea. The experiments were carried out during the second day after scraping of the endothelium, when the edema of the cornea was marked the water content having increased 2Vfe to 4 times (Table VI). The slightly decreased metabolic activity of the rabbit stromal cells in vivo after removal of the endothelium may be the result of trauma rather than the effect of swelling. However, incubation of swollen and unswollen bovine stromas with sulfate- 35 S in vitro showed no significant difference in * 5 S incoi-poration (Table II). Therefore, edema per se does not seem to affect the metabolic activity of the stromal cells. In the comeal stroma, inorganic sulfate- 35 S is incorporated mainly into sulfated polysaccharides. 17 ~ 10 Even though this sulfate incorporation measures only one synthetic mechanism of the stromal cells, it is likely that it mirrors the over-all function of the cells. From the results of the isotope experiments presented here, it seems that removal of either the epithelium or the endothelium had little immediate influence on the metabolic activity of the stromal cells. Interestingly, when two thin impermeable membranes were inserted parallel to each other in the cat corneal

13 532 Dohlman, Gasset, and Rose Investigative Ophthalmology October 1968 Table VI. Effect of absence of endothelium on 35 S incorporation in rabbit comeal stromas in vivo Time (hours) y Cornea Hydration (mg. HtO/mg. dry tissue wt.) 'Value uncertain because of low count. Dialyzable 35 S c.p.m./mg , ,193 2, , , , Nondialyzable 35 S c.p.m./mg. dry tissue wt ,985 1,697 2,385 2, stroma, Langham and Pollack 9 found, by autoradiography of cross-sections, that the 35 S incorporation was normal throughout the stroma. Thus, separation of a part of the stroma from the limiting cellular layers did not seem to modify its ability to incorporate inorganic sulfate- 35 S. In other similar autoradiographic experiments by the same authors, 9 however, removal of either the epithelium or the endothelium resulted in a markedly decreased labeling in the portion of the stroma between the denuded surface and the intralamellar membrane. These latter findings are in some contrast to ours. Wortman 20 showed that an extract from beef comeal epithelium could be used to incorporate inorganic sulfate- 35 S into chondroitin sulfate. This finding does not necessarily implicate the epithelial cells as the source of the stromal polysaccharides or of their immediate sulfate precursor 3'-phosphoadenosine 5'-phosphosulfate. It seems more likely that the stromal cells contain all of the necessary enzymes for polysaccharide synthesis, and the results on isolated stromas in vitro presented here and in earlier studies 10 13> 14> 19 ' support this view. 35 S uptake was considered to be impractical if the effect of denudation was to be followed for many days. A sizable accumulation of fibroblasts after a week would increase the sulfate esterification in an unpredictable manner, thus rendering the results nonrepresentative of the stromal cell population as a whole. 21 Histology was therefore considered to be the safest technique for evaluation after the first 2 days. It was immediately apparent from the histologic appearance of the corneas which were denuded of epithelium that the keratocytes were sensitive to trauma, as has been reported by Herrmann. 0 Unless precautions were taken, the anterior one third or more of the stroma became devoid of cells within 24 hours after removal of the epithelium. It is possible, however, that dehydration of the superficial stroma may also have been a factor. With minimal trauma and with protection against exposure, there was little or no cell loss in the anterior stroma. The keratocytes near the surface, however, were transformed into fibroblast-like cells. It is possible that this morphologic change was still a result of trauma rather than an interruption of a metabolic relationship between epithelial and stromal cells. This assumption is supported by the fact that the

14 Volume 7 Number 5 Absence of corneal epithelium 533 control corneas, in which the epithelium had been allowed to grow back immediately, still showed the same fibroblast-like cells in the surface layers. The fact that the absence of epithelium in an otherwise normal stroma causes no widespread disturbance among the stromal cells is in marked contrast to the events that occur during wound healing of the stroma. Dunnington and Weimar- 2 have shown that, in the absence of epithelium, the transformation of keratocytes into fibroblasts is retarded. In another study on the tensile strength of corneal wounds, it has been found that the absence of epithelium markedly delays the build-up of wound strength. 23 Thus, it seems that an intact stroma can tolerate removal of the epithelium much better than a wounded one. When the endothelium was scraped off, no noticeable cell destruction followed only such changes in the size and basophilia of the keratocytes as could be explained by massive stromal edema. There was no evidence to indicate that the endothelium means more to the keratocytes than a barrier to influx of fluid. The eosinophilic and metachromatic bands which appeared in the anterior stroma and the metachromatic granules which eventually moved into the basal epithelium are of considerable interest. The movement of these bands gave the impression that, after removal of the endothelium, a flow of fluid from the anterior chamber to the corneal surface washes along materials containing mucopolysaccharides and glycoproteins until these substances are trapped behind and in the epithelium. As a matter of fact, a slow flow, due to evaporation from the surface, has been demonstrated in the normal rabbit cornea.-' When the endothelium is damaged or removed, the rate of flow would be expected to increase. There is evidence of increased flow in human corneas with bullous keratopathy, since an intralamellar membrane causes dehydration of the anterior stroma and more edema accumulates posteriorly. 2r> Whether or not the accumulation of eosinophilic and metachromatic material under the epithelium has any particular significance in the picture of corneal edema cannot be stated with certainty at this time. Another peculiarity following the removal of the endothelium was the string of fibroblasts which appeared on the anterior side of Descemet's membrane at the height of the stromal edema. One may wonder whether this phenomenon has any relation to the scar formation which often occurs just anterior to Descemet's membrane in patients with an edematous corneal graft. When the over-all result of this investigation is compared with earlier studies, it agrees with those who found little immediate metabolic damage following removal of the epithelium or endothelium. The histologic changes were also small and when the epithelium was removed limited to the anterior layers of the stroma. Since the factor of mechanical damage during removal cannot be excluded, one has to conclude that the keratocytes can survive for some time, at least even in the absence of the epithelium or the endothelium. REFERENCES 1. Herrmann, H., and Hickman, F. H.: Exploratory studies on corneal metabolism, Bull. Johns Hopkins Hosp. 82: 225, Herrmann, H., and Hickman, F. H.: Further experiments on corneal metabolism in respect to glucose and lactic acid, Bull. Johns Hopkins Hosp. 82: 260, Herrmann, H., and Hickman, F. H.: The consumption of pyruvate, acetoin, acetate and butyrate by the cornea, Bull. Johns Hopkins Hosp. 82: 273, Herrmann, H.: Protein synthesis and tissue integrity in the cornea of the developing chick embryo, Proc. Nat. Acad. Sc. 43: 1007, Herrmann, H., and Love, D. S.: Autoradiographic demonstration of the effect of the corneal epithelium on amino acid incorporation into the insoluble constituents of the corneal stroma, J. Biophys. & Biochem. Cytol. 6: 135, Herrmann, H., and Lebeau, P. L.: ATP level, cell injury, and apparent epitheliumstroma interaction in the cornea, J. Cell Biol. 25: 465, 1962.

15 534 Dohlman, Gasset, and Rose Investigative Ophthalmology October Smelser, G. K.: The importance of the epithelium in the synthesis of the sulfated ground substances in normal connective tissue, Tr. New York Acad. Sc. 21: 575, Smelser, C. K.: Role of the epithelium in incorporation of sulphate in the corneal connective tissue, in Duke-Elder, W. S., and Perkins, E. S., editors: The transparency of the cornea, Oxford, 1960, Blackwell Scientific Publications, p Langham, M. E., and Pollack, I. P.: cited in Langham, M. E.: The interrelationship of metabolism and deturgescence of the living cornea, INVEST. OPHTH. 1: 187, Wortman, B.: Metabolism of sulfate by beef and rabbit cornea, Am. J. Physiol. 198: 779, Praus, R., and Obenberger, J.: Importance of epithelium in the incorporation of radioactive sulphate into the acid mucopolysaccharides of isolated corneae, Cs. Oftalmologie 20: 13, 1964 (in Czech). 12. Praus, R.: Unpublished data. 13. Sato, K.: Studies on the swelling and the uptake of radioactive sulfate in the rabbit corneal stroma after removal of epithelium and endothelium, Jap. J. Ophth. 9: 93, Honda, N.: Studies of the corneal metabolism by a radioactive isotope. I. Incorporation of S :i5 sulfate into the mucopolysaccharide of the rabbit cornea under various fundamental conditions, Folia Ophth. Jap. 16: 675, Duane, T. D.: Steady state of corneal hydration, Am. J. Ophth. 32 (II): 203, Kitano, S.: Cytoplasmic granules of the corneal stroma cell, INVEST. OPHTH. 5: 277, Dohlman, C. H.: Incorporation of radioactive sulfate into the rabbit eye, Acta ophth. 35: 115, Wortman, B., and Strominger, J. L.: Incorporation of inorganic sulfate-s 35 into sulfated mucopolysaccharides of cornea in vitro, Am. J. Ophth. 44 (II): 291, Dohlman, C. H., Wortman, B., and Hultman, S.: Incorporation of sulfate- 3a S, N-acetylglucosamine-l- 1J C, glucose-l-"c, and galactose- 1-1J C into calf and beef corneal glycosaminoglycans, INVEST. OPHTH. 4: 867, Wortman, B.: Enzymic sulfation of corneal mucopolysaccharides by beef cornea epithelial extract, J. Biol. Chem. 236: 974, Dohlman, C. H.: On the metabolism of the corneal graft, Acta ophth. 35: 303, Dunnington, J. H., and Weimar, V.: Influence of the epithelium on the healing of corneal incisions, Am. J. Ophth. 45 (II): 89, Gasset, A., and Dohlman, C. H.: The tensile strength of corneal wounds, Arch. Ophth. In press. 24. Mishima, S., and Maurice, D. M.: The effect of normal evaporation on the eye, Exper. Eye Res. 1: 46, Brown, S. I., and Dohlman, C. H.: A buried corneal prosthesis, Arch. Ophth. 70: 736, 1963.