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Original Articles A comparative study of corneal epithelial cell surfaces utilizing the scanning electron microscope Clifford V. Harding,0 Mihir Bagchi, Allan Weinsieder, and Virginia Peters The mammalian corneal epithelium has been previously shown to have microprojections (microvilli, microplicae) which have been implicated in tear film retention in addition to other possible functions. In the present work, a comparative study of the corneal surface in several species of fish has been carried out with the scanning electron microscope in order to further probe the functional importance of these surface structures. The corneal microprojections in two elasmobranch species, the dogfish and skate (which have eyelids), are structurally very similar to one another, and resemble mammalian corneal surface microprojections. The five species of marine teleost (which do not have eyelids), on the other hand, show epithelial surface patterns consisting of long, curved surface ridges. Evidence of a material coating these ridges is presented. Individual cellular variations in surface coating material and structural patterns of the microprojections are reported. The possible roles of the corneal surface microprojections, in view of these comparative observations, is discussed. ous fine microprojections.1"11 It has been suggested that one function of these structures may be to assist in holding the tear film. The tear film forms an air-liquid interface which constitutes the main refractive surface of the mammalian eye. The tear film-holding role of the microprojections has, however, been brought into question.1-' ™ A comparative study of the topography of the external corneal surface in different species would appear to be useful in further analyzing the functional significance of the microprojections. In fish, because of the aquatic environment, there is a liquidliquid interface at the corneal surface. It would seem that under these circumstances a tear film would either be unnecessary or would be considerably different in its properties from that in the mammal. Consequently, the microprojections might be .he external surface of the mammalian cornea has been shown to consist of numerFrom the Kresge Eye Institute and Department of Anatomy, Wayne State University, School of Medicine, Detroit, Mich, and the Marine Biological Laboratory, Woods Hole, Mass. This study was supported by United States Atomic Energy Commission Contract AT( 11-1)2401, National Institutes of Health Grant EY 00362 (Oakland University), and National Institutes of Health Special Fellowship EY 55,624 from the National Eye Institute to MB, and EY 53483 to AW. Submitted for publication Feb. 28, 1974. Reprint requests: Dr. C. V. Harding, Kresge Eye Institute, Wayne State University School of Medicine, 540 E. Canfield Ave., Detroit, Mich. 48201. °1 dedicate this paper to the memory of Professor George K. Smelser who long realized the value of comparative studies in elucidating the mechanisms of the eye. 906 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932884/ on 08/03/2017 , Volume 13 Number 12 Corneal epithelial cell surfaces 907 Fig. 1. A, surface of dogfish corneal epithelium. The microprojections are approximately 0.2 /t wide and of variable length. xl2,000. B, dogfish corneal epithelial surface at lower magnification. Note cells of various size and texture. x2,000. C, skate corneal epithelial surface. The pattern of microprojections in the skate is similar to that seen in the dogfish. x2,000. either absent, or have a pattern different from that in the mammal. In the present study, the cornea! surfaces of seven species of marine fish were examined with the scanning electron microscope. This included two species of elasmobranch which have eyelids, and five species of marine teleost, which do not. The results are discussed in terms of the possible functional significance of the corneal surface microprojections. fj Materials and methods Two species of elasmobranch were used (the smooth dogfish, Mustelus canis; and the little skate, Raja erinacea); and five species of marine teleost (scup, Stenotomus chrysops; northern sea robin, Prionotus carolinm; summer flounder, Paralichthys dentatus; bluefish, Pomatornus saltatrix; and toadfish, Opsanus tau). The eyes were freshly dissected from healthy animals with no signs of ocular defects. The anterior portions of the eyes were fixed for 12 hours in 2 per cent glutaraldehyde in 0.1 M Na cacodylate made up in elasmobranch Ringer,111 or teleost Ringer,lfi or filtered sea water. The tissues were postfixed in 1 per cent osmium tetroxide in 0.1 M Na cacodylate buffer for one Fig. 2. Scup corneal epithelial surface. A bold pattern of ridges is evident. The ridges are about 0.2 p. in width and many microns in length. x8,Q00. hour, and washed several times in the 0.1 M Na cacodylate buffer. The tissues were dehydrated through either an acetone or alcohol series, and passed through an acetone-Freon-TF (C«C]3Fs) series (for the acetone-dehydrated material), or an alcohol-Freon-TF series (for the alcohol-dehydrated material). The specimens were then dried in a critical-point drying apparatus (Bomar SPC- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932884/ on 08/03/2017 Investigative Ophthalmology December 1974 908 Harding et al. Fig. 3. A, scup corneal epithelial surface. x2,000. B, scup corneal endothelial surface. x2,000. 900, utilizing Freon 13, CClF a , as a transitional fluid) and mounted on specimen holders.10"19 At this point the specimens were examined briefly with a Zeiss light microscope equipped with an epiillumination attachment fitted with a 40x long working distance objective. This was found to be very useful in screening specimens (for cell loss, etc.) prior to examination with the scanning electron microscope. The specimens were coated in a vacuum evaporator with carbon and with gold, and examined with a Jeolco JSM-U3 scanning electron microscope. Results Elasmobranchs. Fig. 1, A shows, at high magnification, a portion of the surface of a dogfish corneal epithelial cell. A microprojection pattern not unlike that seen in mammalian corneas is evident. The microprojections are approximately 0.2 p. wide and of variable length. Fig. 1, B shows a portion of a dogfish corneal surface at lower magnification. Cells of various "size" and "texture," due to some variations in the pattern of microprojections from cell-to-cell, are evident. Fig. 1, C shows, at relatively low magnification, a portion of a skate corneal surface. The pattern of microprojections in the skate is similar to that seen in the dogfish. Teleosts. Fig. 2 shows, at high magnifica- tion, a portion of the corneal surface of a scup. The surface of one whole cell appears toward the left of the picture. Portions of several other cells appear in the field. A bold pattern of ridges is evident. They are approximately 0.2 JX in width, and many microns in length. It is clear from Fig. 2 that the pattern seen here is distinctly different from that seen in the dogfish and skate. Fig. 3} A, at lower magnification, shows the overall cellular arrangement better. For comparison with the epithelial cells shown in Fig. 3, A, Fig. 3, B shows the endothelial surface of a scup cornea. The scup corneal endothelium has the typical appearance of corneal endothelium as described in the mammal.20 In some areas of the preparations, a "coating material" covers some of the cells completely or partially. This is evident in Fig. 4, A. One cell (at the upper right portion of the field) is almost completely covered. The other cells are partially covered. In another field of the same preparation, shown in Fig. 4, B, the ridges appear to be partially coated with strands of material which extend out between the ridges. In some cases the strands appear to have broken and retracted to bead-like deposits Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932884/ on 08/03/2017 Volume 13 Number 12 Corneal epithelial cell surfaces Fig. 4. A, scup corneal epithelial surface. Note coating material covers some of the cells completely or partially. x5,00O. B, scup corneal epithelial surface. Note: ridges appear to be partially coated with coating material and strands of this material extend out between the ridges. x6,000. Fig. 5. A, flounder corneal epithelial surface. Note bold, long ridge pattern. x2,000. B, flounder corneal epithelial surface. Tlie cell at the bottom has more coating material than surrounding cells. xlO,000. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932884/ on 08/03/2017 909 Investigative Ophthalmology December 1974 910 Harding et al. Fig. 6. Toad fish corneal epithelial surface. x3,000. on the sides of the ridges. Here again, as in the cells in Fig. 4, A, the amount of coating material varies from cell to cell. The cell in the lower right in Fig. 4, B has relatively few strands; whereas the cell at the left has many. Others have an intermediate amount. The amount of coating material appears to change abruptly at cell borders. Three other species (flounder, toadfish, and sea robin) also show evidence of coating material in the form of these strands. Fig. 5, A shows, under low magnification, a field of corneal epithelial cells from the flounder. The bold, long-ridge pattern similar to that seen in the scup is evident. This field is relatively free of coating material, however, other areas containing varying amounts of coating material were observed (see Fig. 5, B), The cell at the lower right in Fig. 5, B has more coating material than the surrounding cells. The grooves between the ridges are filled, but the ridges still stand out. At the periphery of this cell (near the center of the photograph), the grooves are not completely filled, but many strands, bridging the gaps between the ridges, are evident. Fig. 6 shows a corneal epithelial preparation from the toadfish. Once again, the long-ridge pattern is evident. Although this pattern is similar to that seen in the scup and flounder, there appears to be a species specificity to the pattern. Other cells showed evidence of coating material (strands). The sea robin also exhibits the longridge structure similar to that seen in the other marine teleosts, although it does apparently have its own species specificity (Fig. 7, A). Fig. 7, B shows the sea robin ridge pattern at higher magnification. Other cells showed varying amounts of coating material. In some cases the grooves were filled, in others, strands were evident. In the bluefish, the corneal epithelium seemed to be almost completely coated. However, areas were found in which cell outlines could be distinctly seen. In a number of cases, although the cells were heavily coated, a ridge pattern could be discerned. Discussion A number of investigators7'iM1 have described the external surface of the mammalian cornea as seen with the scanning election microscope. Microvilli and ridgelike structures (microplicae) have been described on the epithelial cell surfaces. The earlier work of Jakusr' and others2'fi|!) utilizing the transmission electron microscope have also shown evidence of microvilli on the corneal epithelial surface. In mammals, these projections may play a role in holding the tear film. However, Ehlers1has stated, relative to the mammalian precorneal tear film: ". . . that an aqueous film of the thickness found could purely hydrodynamically be stable or almost stable in the vertical position. Thus it is not necessary to assume that a supporting structure, e.g., a gel structure or a mechanical support, should be present." Maurice-1 has also questioned the role of the microprojections in maintaining the tear film. The possible assistance of the microprojections in holding the mammalian tear film, therefore, would apppear debatable. Evidence has been presented recently11 that the ridges (microplicae) on the surface of some mammalian corneas may be, in fact, microvilli which are lying flat on the cell surface. The microprojections seen on the elasmobranch corneas might also be interpreted as microvilli. However, the ridges seen in the five species of marine teleost are difficult to interpret in this way. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932884/ on 08/03/2017 Volume 13 Number 12 Corneal epithelial cell surfaces 911 Fig. 7. A, sea-robin corneal epithelial surface. x4,000. B, sea robin corneal epithelial surface. xlO,000. Their great length and pattern of airangement make it seem unlikely. A transmission electron microscope (TEM) study of the corneas in two species of marine teleost indicate that the ridge pattern is formed by protrusions of the cell surface. The TEM study also revealed the presence of coating material. Preliminary investigations of a fresh water teleost (the dark moor goldfish) show that the corneal projections are not similar to those in the marine teleosts, but resemble more the dogfish and skate corneas.2a Other functions of the projections have been suggested.7 If the arrangements of the microprojections should differ among closely related species as they do for the species reported here, the scanning electron microscopic study of fish corneas may be helpful in making specific species identifications. The fact that two fish (the dogfish and skate) have structures very similar to those seen in the mammal is perhaps of some theoretical interest.211 It might be expected that in an aquatic environment, a tear film with the properties of mammalian tears would be rapidly washed away and would, therefore, be ineffective, for example, in providing a stable optical surface or protecting the corneal surface. However, relatively viscous secretions on the corneas of dogfish and skate might provide a stable surface which could be maintained as a smooth surface through the action of the eyelids. In this case, the surface structures on the elasmobranch cornea may play a role similar to that proposed for the mammal (assisting in holding a precorneal film), even though there is no tear film as seen in the mammal. The evidence for a surface coating material is more striking in the marine teleosts than in the elasmobranchs or mammals. The marine teleosts do not have eyelids to help protect the surface of the cornea. The coating material would have to be relatively stable (e.g., highly viscous or semisolid) in order to be retained. This might account for the fact that the coating material shows up more distinctly in the marine teleosts than in the elasmobranchs. Little is known about the chemical nature of the coating material, its function, or the site of secretion. It presumably serves Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932884/ on 08/03/2017 Investigative Ophthalmology December 1974 912 Harding et al. as a protective coating for the cornea (particularly in the teleosts), and may play an optical role by providing a smooth refractive surface. The variation in amount of coating material from cell-to-cell, with distinct lines of demarcation at cell borders suggests that cells at different stages of differentiation or senescence are present. There are several possible interpretations. For example, a newly surfacing epithelial cell may require time to accumulate coating material; or cells of different ages may have different affinities for the coating material. This latter possibility could result in a differential removal during the preparation procedures of the coating material from some cells and not others. In any case, a variation in surface properties, perhaps as a function of cellular age, is suggested by the reported observations. In addition to the possible role of holding a coating material on the surface of the cornea, the microprojections also greatly increase the surface area, which should assist in the processes of diffusion and active transport. Finally, the possible optical role of the ridges themselves has not been discussed. The possibility that their refractive or reflective characteristics may play a special role in vision in the marine teleosts should be considered.24 REFERENCES 1. Sheldon, H.: An electron microscope study of the epithelium in the normal mature and immature mouse cornea, J. Biophys. Biochem. Cytol. 2: 253, 1956. 2. Pedlar, C : The fine structure of the corneal epithelium, Exp. Eye Res. 1: 286, 1962. 3. Kaye, G. I., and Pappas, G. D.: Studies on the cornea, J. Cell Biol. 12: 457, 1962. 4. Payrau, P., Poulegren, R., and Jaure, J. 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Ehlers, N.: The precorneal film, biomicroscopical, histological, and chemical investigations, Acta Ophthalmol. 43 (suppl. 81): 101, 1965. 13. Lemp, M. A., Holly, F. J., Iwata, S., et al.: The precorneal tear film, Arch. Ophthalmol. 83: 89, 1970. 14. Chemical Handbook, Marine Biological Laboratory, Woods Hole, Mass. 15. Forster, R. P., and Taggart, J. V.: Teleost Ringer, J. Cell Comp. Physiol. 36: 251, .1950. 16. Smith, M. S., and Fincke, E. M.: Criticalpoint drying of soft biological material for the scanning electron microscope, INVEST. OPHTHALMOL. 11: 127, 1972. 17. Worthen, D. M., and Wickham, M. G.: Scanning electron microscopy tissue preparation, INVEST. OPHTHALMOL. 11: 133, 1972. 18. Wickham, M. G., and Worthen, D. M.: Scanning and transmission electron microscopy on the same tissue sample, Stain Technol. 48: 63, 1973. 19. Harding, C. V., Harding, D., Peters, V., et al.: Epithelial cell surfaces and the basement membrane in the ocular lens studied with the scanning electron microscope, Exp. Eye Res. 16: 1, 1973. 20. Polack, F. M.: Scanning electron microscopy of host-graft endothelial junction in corneal graft reaction, Am. J. Ophthalmol. 73: 704, 1972. 21. Maurice, D. M.: The cornea and sclera, in: The Eye, Ed. 2, Davson, H., editor: New York, 1969, Academic Press, vol. 1, p. 495. 22. Harding, C. V., Bagchi, M., Weinsieder, A., et al.: Corneal epithelial cell surface in elasmobranchs and teleosts as seen with the scanning electron microscope, Biol. Bull. 165: 438, 1973. 23. Smelser, G. K., and Chen, D. K.: A comparative study of the structure and hydration properties of corneas adapted to air and aquatic environment, Acta XVII Congr. Ophthalmol. 17: 490, 1954. 24. Cowden, J.: Personal communication. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932884/ on 08/03/2017