Cornea 19(2): 218–230, 2000. © 2000 Lippincott Williams & Wilkins, Inc., Philadelphia A Comparative SEM Study of the Vertebrate Corneal Epithelium Shaun P. Collin, M.Sc. (Melb), Ph.D.(Qld), and H. Barry Collin, A.M., Ph.D.(Melb), D.Sc. (NSW), F.R.C.Path. (Lond) Purpose: The anterior surface of the cornea of mammals, in- The cornea of all vertebrates is an essential structure cluding humans, has numerous folds in the anterior epithelial for clear vision, providing a smooth optical surface and a cell membranes in the form of microvilli and microplicae. The role of these surface irregularities may be to increase cell- protective goggle to ensure a focused image on the surface area and therefore aid in intra- and extracellular move- retina. However, the cornea, and particularly the epithe- ment of nutritional and waste products across the cell mem- lium, of species other than humans and other mammals branes in addition to stabilizing the corneal tear film. The aim has received little attention. of this study was to investigate and compare the nature of these In humans (1,2), some other primates (1), and mam- corneal-surface features in various vertebrate classes residing in different environments. Methods. The anterior corneal sur- mals including the cat, dog, rat (1), guinea pig (3) and faces of various vertebrates were investigated by using field rabbit (1,4,5), the surface of the corneal epithelial cells is emission scanning electron microscopy. Cell areas were ana- covered with microvilli and/or microplicae. Similarly, lyzed by using image-analysis software. Results. Representa- some aquatic vertebrates, such as some elasmobranchs tive species were examined from all the vertebrate classes, with the exception of the Cephalaspidomorphi. The mean epithelial (6) and some freshwater teleosts (7,8) have been shown cell density of aquatic vertebrates (17,602 ± 9,604 cells/mm2) to possess corneal microplicae. In contrast, a freshwater than that of aerial and terrestrial teleost (9) and several marine teleosts (6,10,11) possess (0.000018 ס is greater (p vertebrate species, including amphibians (3,755 ± 2,067 cells/ a regular pattern of microridges covering the anterior 2 mm ). Similarly, the mean epithelial cell density for the marine corneal surface. (0.0015 ס vertebrates (22,553 ± 8,878 cells/mm2) is greater (p than that of the freshwater and estuarine species (10,529 ± Several functions have been attributed to the micro- 5,341 cells/mm2). The anterior corneal surfaces of all species villi and/or microplicae observed in humans and other examined were found to show a variety of cell-surface struc- mammals. These include increasing the surface area of tures. Microvilli are predominant in reptiles, birds, and mam- the superficial cells to maximize the absorbance of oxy- mals; microridges appear to be characteristic of the Osteich- gen and nutrients (12) and to stabilize the corneal tear thyes; and microholes were observed only in the Chondrich- thyes. Conclusion. The function of these morphologic film (1). In air, a tear film is essential for clear vision. It variations in surface structure appear to be correlated with the constitutes the major refracting surface of the eye in the range of ecologic environments (marine, aerial, and terrestrial) production of a focused retinal image and prevents dry- occupied by each species, corneal phylogeny, and the demands ing of the superficial cells of the corneal epithelium. placed on the cornea to ensure clear vision. In aquatic vertebrates, the corneal surface contributes Key Words: Cornea—Microplicae—Microvilli—Micro- ridges—Epithelium—SEM. less to the refractive power of the eye because of the smaller difference between the refractive indices of the cornea [e.g., 1.3719 at 486 nm for the freshwater teleost, Ambloplites rupestris (13) and sea water, 1.340 (14)] compared with the cornea and air. Hence, the mainte- Submitted December 17, 1998. Revision received April 29, 1999. nance of a smooth corneal surface may be less critical. Accepted May 3, 1999. However, the corneas of some aquatic teleosts are cov- From the Department of Zoology, The University of Western Aus- ered with a mucous coating (6,9). The role of this “tear tralia, Nedlands, Western Australia (S.P.C.); and Department of Op- tometry and Visual Sciences, The University of Melbourne, Parkville, film” in relation to both retinal image formation and the Victoria (H.B.C.), Australia. maintenance of a healthy epithelium is unknown. Address correspondence and reprint requests to Dr. S.P. Collin, De- The corneas of two representatives of the amphibia partment of Zoology, The University of Western Australia, Nedlands 6907, Western Australia, Australia. E-mail: [email protected]. have been described previously. The corneal surface of edu.au the frog, Rana pipiens, is covered with microprojections 218 CORNEAL EPITHELIUM IN VERTEBRATES 219 (15), whereas the corneal surface of the salamander, source of each specimen, and its habitat also are shown. Triturus cristatus cristatus, is differentiated into micro- Two of the three species of Chondrichthyes, the black villi (16). There do not appear to be any reports of in- shark, Dalatias licha, and the ratfish, Hydrolagus colliei, vestigations of the anterior corneal surface of birds, rep- and all of the nine representatives of the Osteichthyes tiles, or marsupial mammals. were anesthetized and killed by using methane sulfonate The aim of this study was to examine the corneal salt (MS222; 1:2,000). All animals were killed in accor- epithelial surface of a variety of vertebrate species and to dance with the ethical guidelines of the National Health correlate these findings with the environmental demands and Medical Research Council of Australia. After placed on each species. The relationship between corneal enucleation, the corneas of these species were dissected surface structure and function also is examined in the free and fixed in Karnovsky’s fixative (2% paraformal- context of vertebrate phylogeny. dehyde, 2.5% glutaraldehyde, 0.1 M sodium cacodylate buffer, 2% sucrose, and 0.1% calcium chloride at pH 7.2) MATERIALS AND METHODS and rinsed in 0.1 M sodium cacodylate buffer. This pro- The vertebrates examined in this study are listed in cedure has been found to give excellent fixation for both Table 1. Details of their taxonomic classification, the freshwater (8,9) and marine (10) teleosts. TABLE 1. Summary of the taxonomic position of all the vertebrates examined in this study CLASS Subclass Family Species Common name Size Habitat CHONDRICHTHYES Elasmobranchii Charcharhinidae 2 m TL Aquatic (marine). Scavenger feeding on fish, crustaceans, Galeocerdo cuvier cephalopods, birds, mammals, and reptiles Tiger shark Squalidae 160 cm TL Aquatic (marine). Benthic to mesopelagic. Found between 200 and Dalatias licha 1000 m Black shark Holocephali 41.0 TL Aquatic (marine). Deep-sea species with large eyes. Pelagic or Chimaeridae benthopelagic feeding on small invertebrates and fishes Hydrolagus colliei Ratfish OSTEICHTHYES Actinopterygii Sparidae 12 cm TL Aquatic (river and estuarine). Frequently found over sand and Acanthopagrus butcheri eel-grass beds. Feeds on crustaceans and mollusks Black bream Lepidogalaxidae 4.5 cm TL Aquatic (freshwater). Aestivating fish which burrows into mud and Lepidogalaxias salamandroides leaf litter to avoid dessication over summer Salamander fish Mugilidae 26.0 cm TL Aquatic (marine and estuarine). Omnivorous, benthopelagic (free Aldrichetta forsteri swimming just above the bottom) Yellow-eye mullet Platycephalidae 30.0 cm TL Aquatic (marine and estuarine). Omnivorous, lie-in-wait benthic Platycephalus endrachtensis predator, frequently found beneath the sand Bar-tailed flathead Teraponidae 25.0 cm TL Aquatic (marine and estuarine). Schools over sand and weed Amniabata caudavittatus bottom, able to tolerate fresh and salt water Yellow-tail trumpeter Clupeidae 25.0 cm TL Aquatic (marine and estuarine). Possesses adipose eyelids over Nematalosa vlaminghi front and back of eye. Central cornea not covered by eyelids Perth herring Cottidae 25.0 cm TL Aquatic (marine). Benthic lie-in-wait predator inhabiting the cold Myoxocephalus waters of the northern Pacific Ocean. Carnivorous, often found polyacanthocephalus above sand and a sluggish swimmer capable of quick darting Great sculpin escapes Cyclopteridae 15.0 cm TL Aquatic (marine). Small benthic soft-bodied species, which has a Polypera greenii suctorial disk on the underside of the body Lobefin snailfish Pleuronectidae 30.0 cm TL Aquatic (marine). Benthic lie-in-wait predator inhabiting the cold Microstomius pacificus waters of the northern Pacific Ocean. Often found camouflaged Dover sole beneath the sand All specimens are adults unless otherwise stated. (continues) Cornea, Vol. 19, No. 2, 2000 220 S.P. COLLIN AND H.B. COLLIN All other specimens were obtained opportunistically. The corneal surfaces were examined by using a Joel The range of fixatives is listed in Table 2. All corneas FSEM (field emission scanning electron microscope) were rinsed in 0.9% saline to minimize precipitation or with an accelerating voltage of 3 kV. Results were re- other effects due to the presence of unexpected salts, in corded both on 35-mm film and digitally. The areas of the fixatives or storage solutions, before being placed in individual epithelial cells, which were predominantly two changes of 0.1 M sodium cacodylate buffer. All from the central cornea, were obtained by digital analysis specimens were postfixed in 0.1% osmium tetroxide in of the computer images by using Image Slave software 0.1 M cacodylate buffer followed by dehydration in a (Optimas, Adept Electronic Solutions, Australia). The graded series of alcohols. Specimens were critical-point number of cells measured for each species varied from 5 dried in a Polaron critical-point dryer and mounted on to 37 cells. Species in which fewer than 10 measurable 10-mm aluminum stubs with double-sided tape. Each cells were obtained are indicated by an asterisk in Table specimen was oriented and/or hemisected so that one 3.