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)

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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. By using this procedure, the mean epithelial cell den- half of the cornea was inverted, and both sides were sity and standard deviation were calculated, providing displayed. This allowed direct comparison and differen- the extent of variation in cell area and a measure of tiation between epithelial and endothelial surfaces. The polymegethism. mounted specimens were coated with 12–15 nm of gold- Measurements of the membrane elevations, including palladium in a Polaron sputter coater and placed in an finger-like processes (microvilli), short, <1 ␮m in length oven at 40°C overnight before being examined. (microplicae), and long, >1-␮m (microridges) folds, mi-

TABLE 1. (Continued)

CLASS Subclass Family Species Common name Size Habitat AMPHIBIA Lissamphidia Ambystomidae 170 mm TL, Aquatic (freshwater). Sedentary on bottom of lake. Feeds on Ambystoma mexicanum premetamorphic tubifex worm and has external gills Salamander (axolotl) REPTILIA Diapsida Crocodylidae 1.4 m in length Estuarine bodies of water in Northern Australia Crocodilus porosus Crocodile Agamidae 15 cm TL Terrestrial. Lives under low-lying rocks in forested regions of Ctenophorus ornatus southwestern Australia Ornate lizard Anapsida 10 cm TL hatchling Aquatic (marine). Predominantly aquatic but breathes at the Chelonidae surface and lays eggs on land Caretta caretta Loggerhead turtle AVES Ornithurae Phoenicopteridae Adult Long-legged scavenger for crustaceans, worms, and small animals Phoenicopterus chilensis in water. Graceful flyer, comfortable in water and on land Chilean flamingo Phoenicopterus ruber 13-year-old male Long-legged scavenger for crustaceans, worms, and small animals Carribean flamingo in water. Graceful flyer, comfortable in water and on land Dromaiidae 150 cm, 45 kg Terrestrial. Inhabits desert areas of Australia Dromaius novaehollandiae Emu Strigidae 25 cm, 420 g Arborial, relying on camouflage to avoid predation. Captures prey Bubo strix at twilight Barred owl MAMMALIA Theria (Infraclass Eutheria) Cetacea 536 km female Aquatic (marine). Pelagic aquatic mammal Pseudorca crassidens False killer whale Theria (Infraclass Metatheria) Phascolarctidae 9.2 kg Arboreal. Perches in eucalyptus trees Phascolarctos cinereus Australian koala

Cornea, Vol. 19, No. 2, 2000 CORNEAL EPITHELIUM IN VERTEBRATES 221

TABLE 2. Summary of the origin and histological preservation of corneal tissue of the specimens used in this study

Common name Origin Fixation Tiger shark Donated by R. Scoones, The University of Fixed in 10% formalin buffered with sea water Western Australia Black shark Challenger Cruise No. 94 (1992) by OTSB at Fixed and stored in Karnovsky EM fixative for 840 m 2 years Ratfish Collected by otter trawl net at Friday Harbor, Fixed and stored in Karnovsky EM fixative San Juan Island, Washington, U.S.A. Black bream Supplied by the Maritime Centre of T.A.F.E. in Fixed and stored in Karnovsky EM fixative Fremantle, W.A. Salamander fish Collected by seine net under permit in pools Fixed and stored in Karnovsky EM fixative for 10 km south of Northcliffe and 300 km south years of Perth, W.A. Yellow-eye mullet Collected by seine net in Swan River, W.A. Fixed and stored in Karnovsky EM fixative for 24 h Bar-tailed flathead Collected by seine net in Swan River, W.A. Fixed and stored in Karnovsky EM fixative for 24 h Yellow-tail trumpeter Collected by seine net in Swan River, W.A. Fixed and stored in Karnovsky EM fixative for 24 h Perth herring Collected by seine net in Swan River, W.A. Fixed and stored in Karnovsky EM fixative Great sculpin Collected by otter trawl net at Friday Harbor, Fixed and stored in Karnovsky EM fixative for San Juan Island, Washington, U.S.A. years Lobefin snailfish Collected by otter trawl net at Friday Harbor, Fixed and stored in Karnovsky EM fixative for San Juan Island, Washington, U.S.A. years Dover sole Collected by otter trawl net at Friday Harbor, Fixed and stored in Karnovsky EM fixative for San Juan Island, Washington, U.S.A. years Salamander (axolotl) Local suppliers in W.A. Fixed and stored in Karnovsky EM fixative for 2w Crocodile Donated by crocodile hatchery in Fremantle, Fixed and stored in Karnovsky EM fixative for W.A. 1mo Lizard Reared in the Department of Zoology, Fixed and stored in Karnovsky EM fixative for University of Western Australia 3w Loggerhead turtle Stranded off the Northwest shelf, Western Fixed in 10% formalin Australia Chilean flamingo Donated by M.P. Anderson and K.G. Osborn, Fixed and stored in formal saline for years San Diego Zoo Caribbean flamingo Donated by M.P. Anderson and K.G. Osborn, Fixed and stored in formal saline for years San Diego Zoo Emu Donated by emu farm in W.A. Fixed and stored in Karnovsky EM fixative for years Barred owl Donated by J.D. Pettigrew, The University of Fixed and stored in Karnovsky EM fixative for Queensland years False killer whale Eye donated by S. Ridgeway, San Diego Fixed and stored in Karnovsky EM fixative for years Australian koala Donated by C. Leigh, Department of Anatomy, Fixed in 3% glutaraldehyde, 3% University of Adelaide, South Australia paraformaldehyde in phosphate buffer and stored in fixative for 2 mo

croholes, and other structures were made on photo- eost). There is also a considerable variation of cell den- graphic prints by using a magnifier and graticule. Mea- sities between members of each class. However, from surements were made of Ն10 examples of each surface Table 3, there does appear to be a general trend, in that feature, and a standard deviation calculated. Where there the cell densities of the aquatic vertebrates (with a mean were fewer than 10 examples of a particular feature, no of 17,602 ± 9,604 cells/mm2) are significantly greater (p than those of aerial or terrestrial (including (0.000018 ס standard deviation is given. Comparisons were made by using Student’s t test. amphibian) species (with a mean of 3,755 ± 2,067 cells/ mm2). An exception to this trend is the false killer whale, RESULTS Pseudorca crassidens, a marine mammal, which possesses a cell density of 2,211 ± 794 cells/mm2, similar to The corneal epithelial cell densities of the species in- that of the Australian koala. vestigated in this and other studies are shown in Table 3. Within the class Osteichthyes, there also appears to be In this study, there is a large range in cell densities from a strong relationship between epithelial cell density and 2,126 ± 713 cells/mm2 in the Australian koala Phasco- the type of water inhabited by each species. The species larctos cinereus (a marsupial), to 29,348 ± 12,917 cells/ that have a capacity to live in freshwater (for example, mm2 in the Dover sole, Microstomius pacificus (a tel- rivers or estuaries) have a mean cell density of 10,529 ±

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TABLE 3. Cell density and the diameter of microprojections or microholes for the vertebrates examined in this study and those previously published

CLASS Epithelial cell density Microvilli Microplicae Microridges Microholes Common name (cells/mm2) (nm) (nm) (nm) (nm) Source CHONDRICHTHYES Tiger shark (M) 8,286 ± 3,212 105 ± 10 This study Black shark (M) 14,492 ± 4,056 127 ± 19 136 ± 94 311 ± 102 This study Ratfish (M) Not measureda 70 ± 12 This study OSTEICHTHYES Trout (F) 2,309–3,567 8 Garfish (F) 9,560 7 Black bream (E) 5,999 ± 1,976 139 ± 29 This study Yellow-eye mullet (E, M) 10,943 ± 4,538 227 ± 18 This study Bar-tailed flathead (E, M) 11,171 ± 5,740 146 ± 17 This study Yellow-tail trumpeter (E, M) 13,453 ± 1,385b 166 ± 16 This study Sandlance (M) 18,080 ± 5,560 11 Perth herring (E, M) 19,639 ± 4,010 194 ± 27 This study Flounder (M) 19,870c 6 Salamander fish (F) 21,880 ± 4,200 123 ± 16 This study, 9 Scup (M) 21,894c 6 Great sculpin (M) 22,615 ± 4,800 141 ± 16 This study Lobefin snailfish (M) 28,229 ± 9,334 202 ± 28 This study Dover sole (M) 29,348 ± 12,917 203 ± 19 This study Searobin (M) 40,838c 6 AMPHIBIA Salamander (axolotl) 2,918 ± 1,753 161 ± 22 324 ± 76 This study REPTILIA Crocodile 2,283 ± 824 112 ± 6 This study Ornate lizard 5,095 ± 1,746b 82±6 111±9 96±13 Thisstudy Loggerhead turtle 4,191 ± 1,672 87 ± 24 This study AVES Chilean flamingo 5,944 ± 2,042b 96 ± 18 This study Caribbean flamingo 8,674 ± 3,034b 119 ± 28 This study Emu 5,250 ± 2,076 114 ± 5 This study Barred owl 5,351 ± 1,325 85 ± 5 This study MAMMALIA False killer whale 2,211 ± 794b 160d This study Australian koala 2,126 ± 713 101 ± 9 100 ± 9 This study New Zealand white rabbit 2,802 4 Dutch belt rabbit 1,898 19 Cat 1,306c 1 Human 2,525 33

The standard deviation for the cell density data may represent a measure of polymegethism. Where there is no measurement provided, this feature did not exist in the samples examined. a The density of microvilli made cell borders difficult to see and precluded measurement of cell density. b Indicates that <10 cells were suitable for measurement of cell area. c Cell densities calculated from micrographs presented in previously published studies. d This is an estimate as accurate measurement was not possible. E, estuarine; F, freshwater; M, marine.

-mean width of the microvilli (96 ± 17 nm) is signifi ס cells/mm2, which is significantly lower (p 5,341 ס than that for the purely marine species, with a cantly less than both the microplicae (123 ± 25 nm; p (0.0034 The .(0.0004 ס mean of 22,553 ± 8,878 cells/mm2. However, the excep- 0.04) and the microridges (158 ± 44; p in (0.03 ס tion to this trend is the salamander fish, Lepidogalaxias microridges also are significantly greater (p salamandroides, which possesses a cell density of width than the microplicae. ∼21,880 cells/mm2. Although this species lives in freshwater, it spends appreciable amounts of time estivating in Chondrichthyes shallow, peaty swamps characterized by a low pH of 4.3, a high dissolved organic content, and high levels of urea Within the representatives of the class Chondrich- (17). thyes, the shape of the superficial epithelial cells varies. The dimensions of the microvilli, microplicae, micro- The surface of the cornea of the tiger shark, Galeocerdo ridges, and/or microholes for all of the species studied cuvier (Elasmobranchii) comprises generally straight- are also shown in Table 3. There are no apparent differ- sided polygonal cells (Fig. 1a) covered with a moderately ences in the dimensions of any of these specific features dense array of microplicae (Fig. 1b). In contrast, the in relation to taxonomic position or habitat. However, the epithelial cells of the black shark, D. licha, (Elasmo-

Cornea, Vol. 19, No. 2, 2000 CORNEAL EPITHELIUM IN VERTEBRATES 223

FIG. 1. Surface features in the Chondrichthyes. Low-power (a) and high-power (b) scanning electron micrographs of the surface of the corneal epithelium in the tiger shark, Galeocerdo cuvier, showing the polygonal cells and microplicae. c: High-power micrograph of the dense array of microholes surrounded by microvilli and microplicae in the black shark, Dalatias licha. d: High power micrograph of the surface microvilli in the ratfish, Hydrolagus colliei. branchii) are generally polygonal with occasional croridges appear to be somewhat random, although rounded cells. On the surface, the predominant feature is sometimes grouped in parallel bundles (Fig. 2a and b). In the presence of numerous, small holes (microholes) sur- contrast, those of the lobefin snailfish, Polypera greenii rounded by fine short microvilli and microplicae (Fig. (Fig. 2c and d), the Dover sole, Microstomius pacificus 1c). The epithelial cell surface of the ratfish, Hydrolagus (Fig. 2e and f), the salamander fish, Lepidogalaxias sala- colliei (Holocephali), is covered by densely packed mi- mandroides (Fig. 3a and b), the bar-tailed flathead, crovilli (Fig. 1d), which are markedly different from Platycephalus endrachtensis (Fig. 3c and d), the yellow- those of the other two species of Chondrichthyes. Al- eye mullet, Aldrichetta forsteri (Fig. 4a and b), the yel- though there appear to be some gaps between the micro- low-tail trumpeter, Amniabata caudavittatus, the Perth villi, these are not thought to be microholes as seen in the herring, Nematolosa vlaminghi, and the black bream, black shark, D. licha. Acanthopagrus butcheri (Fig. 4c and d) show a greater tendency to have increased numbers of microridges lying parallel to the cell borders. In the bar-tailed flathead, P. Osteichthyes endrachtensis, holes or pits were occasionally seen (Fig. ∼ ␮ The cell borders of the nine species of Osteichthyes 3c). These varied in size up to 2 m in diameter and examined are difficult to observe. However, the approxi- were situated at the junctions between three and some- mate cell shapes can be determined from the complex times four cells. patterns of microridges covering each epithelial cell. The microridges are often continuous, never crossing Amphibia from one cell to the next, and are frequently oriented parallel to each other and to the cell border. The cells are In the sole representative of the Amphibia examined, generally rounded and polygonal in shape without cor- the salamander or axolotl, Ambystoma mexicanum, the ners. corneal epithelial cells are mostly polygonal, although a Although similar, the patterns of microridges for all few are rounded. However, there is a marked variation in species of Osteichthyes studied are different. In the great cell size (Fig. 5a). All cells are covered with a dense sculpin, Myoxocephalus polyacanthocephalus, the mi- pattern of microplicae, and the majority of cells have

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FIG. 2. Surface features in the Osteichthyes. Low-power (a) and high-power (b) micrographs of the complex pattern of microridges covering the epithelial surface of the great sculpin, Myoxocephalus polyacanthocephalus. Low- and high-power micrographs of the microridge patterns in the lobefin snailfish, Polypera greenii (c, d) and the Dover sole, Microstomius pacificus (e, f). myriad of microholes lying between the microplicae (Fig. However, a few cells also show some microplicae and 5a and b). long microridges (Fig. 6c and d).

Reptilia Aves

The surface of the cornea of the crocodile, Crocodilus The corneal surface of both the Chilean flamingo, porosus, is covered in polygonal cells with straight sides, Phoenicopterus chilensis (Fig. 7a) and the Caribbean fla- which show considerable variation in size (Fig. 6a). The mingo, P. ruber, have relatively straight-sided polygonal cells possess numerous microvilli and occasional micro- cells covered by rather short microvilli (Fig. 7b). The plicae, each of which shows variation in density (Fig. surface cells of the flightless emu, Dromaius novaehol- 6b). The epithelial cells of both the ornate lizard, landiae (Fig. 7c and d), are similar to those of the Ctenophorus ornatus, and the loggerhead turtle, Caretta flighted barred owl, Bubo strix (Fig. 7e and f), where caretta, also are polygonal and somewhat irregular in most cells are hexagonal or pentagonal and are covered shape. Most cells are covered with numerous microvilli. with numerous microvilli. However, a few of the epithe-

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FIG. 3. Surface features in the Osteichthyes. High- and low-power micrographs of the surface structure of the salamander fish, Lepidogalaxias salamandroides (a, b) and the bar-tailed flathead, Platycephalus endrachtensis (c, d). Note the small holes or pits located at various cell junctions in the flathead. lial cells of the barred owl possess very few microvilli, all representatives of the aquatic classes, Chondrichthyes 2 which appear to resemble microplicae (Fig. 7f). and Osteichthyes (17,602 ± 9,604 cells/mm ) is signifi- greater than that for the other (0.000018 ס cantly (p classes, Amphibia, Reptilia, Aves, and Mammalia (3,755 Mammalia ± 2,067 cells/mm2). The inclusion of the false killer whale, P. crassidens, The borders of the corneal cells of the false killer in the nonaquatic group may seem inappropriate. How- whale, Pseudorca crassidens, were difficult to discern ever, optically, the whale is believed to have reverted to accurately and appeared somewhat rounded. Numerous short microvilli covered the cell surface. The epithelial the aquatic type, “perfectly so in the Odontoceti” (18). cells of the Australian koala, Phascolarctos cinereus, (a Similarly, the amphibians, including the salamander or marsupial), are predominantly polygonal, although axolotl, A. mexicanum, which is aquatic as a larva and sometimes appear rounded with a wide range of shapes terrestrial as an adult, and reptiles that have ventured into and sizes (Fig. 8a). In contrast to the false killer whale, water, such as the crocodile, C. porosus, and the logger- the surface of some cells is covered with microvilli, head turtle, C. caretta, do not have epithelial cell densi- whereas others have microridges (Fig. 8b). ties consistent with an aquatic origin. However, the adult salamander is terrestrial, and the two species of reptiles have also “gone back into the water” after developing “a DISCUSSION full panoply of terrestrial ocular accessories” (18). This concept is supported by the findings of low epithelial cell Cell Density densities, particularly for the false killer whale, P. cras- The vertebrates described show a wide range of cor- sidens, the premetamorphic salamander, and the croco- neal epithelial cell densities from 2,126 cells/mm2 in the dile, C. porosus, which are all similar to those of mam- Australian koala, P. cinereus (a marsupial), to 29,348 mals such as the Australian koala, P. cinereus. cells/mm2 in the Dover sole, M. pacificus (a teleost). A comparison of the mean cell density (10,529 ± However, as the effects of the various fixatives are un- 5,341 cells/mm2) for the freshwater and estuarine tele- known, the specific densities may not be accurate. A osts with that of the marine species (22,553 ± 8,878 ס comparison of the cell densities shows that the mean of cells/mm2) also shows a significant difference (p

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FIG. 4. Surface features in the Osteichthyes. Two more examples of the diversity of microridge patterns in the yellow-eye mullet, Aldrichetta forsteri (a, b) and the black bream, Acanthopagrus butcheri (c, d).

0.0034). Thus there appears to be a progressive de- manderfish is unique in that it lives in freshwater ponds crease in epithelial cell density from the marine dwell- of pH 4.3, which contain large amounts of tannin (20) ers through the freshwater and estuarine species to and high levels of dissolved organic matter. These ponds the terrestrial animals; the mammals possess a particu- dry out in summer, and the fish estivate in moist burrows larly low cell density (mean of 2,129 cell/mm2). Al- for Յ6 months, where the microenvironment may be though there are few published reports of epithelial cell closer to the relatively high osmotic concentration of sea densities, they do appear to support this concept (Ta- water because of high levels of excreted urea (17). Thus ble 3). its environment is sometimes similar to that of marine In contrast to the majority of these data, Collin and dwellers with respect to the increased osmotic effects on Collin (9) claimed a cell density of ∼21,880 cells/mm2 the external surface of the cornea. Hence our present for the freshwater salamanderfish, Lepidogalaxias sala- findings are consistent with previous reports and confirm mandroides. This appears anomalous. However, the sala- that there is a clear reduction in epithelial cell density

FIG. 5. Surface features in the Amphibia. a: Low-power scanning electron micrograph showing the polygonal cells of the salamander, Ambystoma mexicanum. b: High-power micrograph of the junction of four epithelial cells illustrating the polymorphic appearance (microholes and microplicae) in the surface structure of some cells.

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FIG. 6. Surface features in the Reptilia. Low-power (a) and high-power (b) micrographs of the anterior surface of the epithelium in the crocodile, Crocodilus porosus. Note the straight- sided polygonal cells, which are sculptured into a dense pattern of microvilli. c, d: High- power micrographs of the epithelial surfaces of the ornate lizard, Ctenophorus ornatus (c) and the loggerhead turtle, Caretta caretta (d) showing a mixture of microvilli, microplicae, and microridges. from marine teleosts to terrestrial animals, culminating in Microholes mammals. However, a source of error or variation in cell density The presence of holes (microholes) between the mi- arising from topographic variation across the cornea, crovilli/microplicae on the corneal surface of the black which is not assessed in this study, must also be taken shark, D. licha (an elasmobranch), is an interesting find- into account. Doughty and Fong (21) found that the su- ing. The microholes (311 ± 102 nm) are distinctly dif- perficial corneal epithelial cells of the pigmented rabbit ferent in both size and shape from the holes or pits that are progressively larger toward the periphery, with an are occasionally seen at the junctions between three and apparently symmetric change across the inferior-nasal sometimes four cells on the corneal surface of the bar-tail and superior-temporal corneal surface. In partial confir- flathead, P. endrachtensis (Յ2 ␮m) and the peripheral mation of this, Collin and Collin (11) found the cell cornea of the sandlance, L. fasciatus (∼1.6 ␮m) (11), and density across the surface of the cornea of the sandlance, the large pits or holes (Յ10 ␮m in diameter), which are L. fasciatus, was greater in the center (21,475 ± 4,750 found in the central areas of superficial corneal cells in cells/mm2) than in the periphery (14,785 ± 3,630 cells/ mammals, and may be part of the normal epithelial cell mm2). destruction and replacement (1). However, it is not An additional correction factor should also be applied known if the holes found in the bar-tail flathead are to these findings to obtain an estimate of the in vivo cell central or peripheral. density because no attempt was made to assess the de- Although not shown in Fig. 3a, holes have also been gree of shrinkage in our study. With similar processing described in the corneal surface of the estivating sala- techniques, Doughty (4) reported a tissue contraction of manderfish, L. salamandroides (9). They are usually 35.8 ± 1.2% as a result of fixation and critical-point present at the junctions of three epithelial cells and repre- drying, which could be applied to the findings presented sent the external pores of the goblet cells present in the in Fig. 3. However, in this study, several fixatives were cornea (9). The only other vertebrates known to possess used, and different degrees of shrinkage may have goblet cells in the epithelium are the adult hagfish, Ep- occurred. tatratus stouti (S.P. Collin and H.B. Collin, unpublished

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FIG. 7. Surface features in the Aves. Series of low- and high-power micrographs showing the diversity of epithelial cell shape and the dense aggregations of microvilli in the Chilean flamingo, Phoenicopterus ruber (a, b), the emu, Dromaius novaehollandiae (c, d), and the barred owl, Bubo strix (e, f).

data), and the larval stage of the sea lamprey, Petromy- uncertain if this is due to the loss of goblet cells within zon marinus (22,23). the corneal epithelium. The holes that appear in the cornea of the black shark, The function of the goblet cells and the production of D. licha, are similar to those described by Dickson et al. a mucous coating may be protective as suggested for the (23). They reported that the outer stratified epithelium of salamanderfish, L. salamandroides, a burrowing teleost the outer dermal cornea of the larval (ammocoete) stage with goblet cells in the cornea (9) and the burrowing of the anadromous sea lamprey, P. marinus, was com- ammocoete of the sea lamprey, P. marinus (23), in ad- posed of tall columnar goblet cells with elongated apical dition to aiding clear vision. Similarly, the microholes in microvilli, between which lie mucus-filled channels the marine black shark, D. licha, may comprise goblet opening onto the epithelial surface. With maturity, the cell openings, but this remains to be confirmed by trans- mucus-producing cells disappear from the cornea. This is mission electron microscopy. Interestingly, although the consistent with our finding of microholes in the cornea of function of the large holes between the cells of the bar- the premetamorphic salamander, A. mexicanum, which tail flathead (Fig. 3c and d) are unknown, this sedentary disappear after induced metamorphosis (S.P. Collin and teleost also burrows under the sand (Table 1). Similarly, H.B. Collin, unpublished data), although, at present, it is the sandlance, L. fasciatus, which has holes between the

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FIG. 8. Surface features in the Mammalia. Low-power (a) and high-power (b) micrographs of the polymorphic appearance of the epithelial surface of the Australian koala, Phasco- larctos cinereus, showing both microvilli and microridges. epithelial cells of the peripheral cornea, dives into the corhynchus mykiss [an actinopterygian (8)], and the sand (10). However, goblet cells are not found in the Florida garfish, Lepisosteus platyrhincus [an actinopte- cornea of the sandlance (10). rygian (7)], all of which have microplicae. The presence of microplicae and microholes in the Microridges arranged in fingerprint-like patterns are premetamorphic salamander or axolotl, A. mexicanum, is similar to those found in the cornea, conjunctiva, and of particular interest due to its progression from an head region of the sandlance, L. fasciatus (11). It is im- aquatic to a terrestrial environment during development. portant to note that microridges also are present over the In the premetamorphic stage, the microholes may be epithelium in a number of other sensory regions in tele- goblet cell openings to the surface, as discussed earlier osts. Microridges are present surrounding the neuromasts for a number of other species, and the microplicae may of the lateral line receptors in the cichlid, Cichlasoma be an adaptation to its aquatic habitat. Interestingly, the nigrofasciatum (25), the electroreceptors in the weakly microholes disappear in the postmetamorphic stage electric fish, Eigenmannia lineata (26), and the chemo- where the surface structure is composed entirely of mi- sensory cells of the roach, Rutilus rutilus, and the sand croplicae and microvilli, indicative of its new terrestrial goby, Pomatoschistus minutus (27). Emphasizing the environment (S.P. Collin and H.B. Collin, unpublished ubiquitous nature of this epithelial surface pattern, they data). also are found in the nasal epithelium of the sandlance, L. fasciatus (11), the olfactory epithelium of the flying fish, Cheilopogon agoo (28), on the oral mucosa of the carp, Microridges Cyprinus carpio (29,30) and the taste buds of the sea The presence of a well-developed array of microridges robin, Prionotus carolinus (31). appears to be a characteristic of the Osteichthyes (teleosts). In addition to the five estuarine and three marine Microvilli species described here, microridges have also been described in the scup, Stenotomus chrysops, the northern The observation that the corneal surfaces of the Chil- sea robin, Prionotus carolinus, the summer flounder, ean flamingo, Phoenopterus chilensis, the Caribbean fla- Paralichthyes dentatus, the bluefish, Pomatomus sal- mingo, P. ruber, the emu, Dromaius novaehollandiae, tatrix, the toadfish, Opsanus tau (6), and the sandlance, and the barred owl, Bubo strix, are densely populated Limnichthyes fasciatus (10,11), all of which are marine. with microvilli is new. Considering the expected increase Of the freshwater varieties studied, only the salamander- in evaporation when a bird is in flight or running swiftly, fish, L. salamandroides, reported here, and previously the microvilli may stabilize the corneal tear film (1). described by Collin and Collin (9), possesses microridges. However, this species is peculiar in that it has the The Function of Microprojections capacity to estivate in acidic, sundried, mud pools with a high osmotic concentration (24). Microprojections may maximize the absorbance of To date, the only member of the Osteichthyes in which oxygen (12) and nutrients. In air, they may stabilize the microridges do not occur appears to be the freshwater corneal tear film (1), which is essential for clear vision. Australian lungfish, Neoceratodus forsteri (a sarcopte- However, in aquatic animals, the role of the micropro- rygian), which has microvilli (S.P. Collin and H.B. Col- jections in the maintenance of a mucous layer and of lin, unpublished data), the freshwater rainbow trout, On- vision is uncertain. Actin filaments play a part in the

Cornea, Vol. 19, No. 2, 2000 230 S.P. COLLIN AND H.B. COLLIN formation of microridges (30), which impart epithelial 11. Collin SP, Collin HB. The head and eye of the sandlance, Lim- rigidity or plasticity to the corneal surface (32). Long nichthyes fasciatus: a field emission scanning electron microscopy study. Clin Exp Optom 1997;80:133–8. microridges organized in a highly regular pattern are 12. Beuerman RW, Pedrosa L. Ultrastructure of the human cornea. more stable than short ridges (30). The foldings of mi- Microsc Res Tech 1996;33:320–35. croridges cause an increase in the surface area for attach- 13. Kro¨ger RHH. Methods to estimate dispersion in vertebrate ocular media. J Opt Soc Am 1992;9:1486–90. ment of mucus and may also function to anchor the lu- 14. Jerlov NG. Marine optics. Amsterdam: Elsevier, 1976. bricating and cushioning layer of mucus, which protects 15. Kaltenbach JC, Harding CV, Susan S. Surface ultrastructure of the the underlying plasmalemma from abrasion (29). This is cornea and adjacent epidermis during metamorphosis of Rana pipiens: a scanning electron microscopic study. J Morphol 1980;166: particularly true for the various burrowing species. 323–35. Apart from the fact that the potential increase in cell- 16. Margaritis LH, Politof TK, Koliopoulos JX. Quantitative and com- membrane area is greatest with microvilli, which are parative ultrastructure of the vertebrate cornea: 1. Urodele amphibia. Tissue Cell 1976;8:591–602. mostly found in corneas in air, and least with micro- 17. Pusey BJ. The effect of starvation on oxygen consumption and ridges, which appear to be confined to species inhabiting nitrogen excretion in Lepidogalaxias salamandroides (Mees). J environments with a high osmotic concentration (usually Comp Physiol B 1986;156:701–5. 18. Walls GL. The vertebrate eye and its adaptive radiation. Bloom- marine), the specific functions of each of the three mi- field Hills, MI: The Cranbrook Institute of Science, 1942. croprojections described here are still uncertain. 19. Doughty MJ. Acute effects of chlorobutanol- or benzalkonium chloride-containing artificial tears on the surface features of rabbit Acknowledgment: We thank Michael Archer of the Depart- corneal epithelial cells. Optom Vis Sci 1994;71:562–72. ment of Zoology, The University of Western Australia, and 20. Christensen P. The distribution of Lepidogalaxias salamandroides Brendon Griffin of the Centre for Microscopy and Microanal- and other small freshwater fishes in the lower south-west of West- ern Australia. J R Soc West Aust 1982;65:131–41. ysis, The University of Western Australia, for technical assis- 21. Doughty MJ, Fong WK. Topographical differences in cell area at tance. Dr. S.P. Collin is currently an ARC QE II Research the surface of the corneal epithelium of the pigmented rabbit. Curr Fellow. This work was funded by Australian Research Council Eye Res 1992;11:1129–36. and National Health and Medical Research Council grants to 22. Van Horn DL, Edelhauser HF, Schultz RO. Ultrastructure of the Dr. S.P. Collin. primary spectacle and cornea of the sea lamprey. J Ultrastruct Res 1969;26:454–64. 23. Dickson DH, Graves DA, Moyles MR. Corneal splitting in the REFERENCES developing lamprey Petromyzon marinus L. eye. Am J Anat 1982; 165:83–98. 1. Pfister RR. The normal surface of the corneal epithelium: a scan- 24. Pusey BJ. The life history of the Shannon mud minnow Lepido- ning electron microscopic study. Invest Ophthalmol 1973;12:654– galaxias salamandroides (Mees) with special reference to aestiva- 68. tion. Honours Thesis. The University of Western Australia, Ned- 2. Lohman LE, Rao GN, Tripathi RC, Tripathi BJ, Aquavella JV. In lands, Australia, 1981. vivo specular microscopy of edematous corneal epithelium with 25. Webb JF. Neuromast morphology and lateral line trunk canal on- light and scanning electron microscopy. Ophthalmology 1982;89: togeny in two species of cichlids: an SEM study. J Morphol 1989; 621–9. 202:53–68. 3. Nichols B, Dawson CR, Togni B. Surface features of the conjunc- 26. Vischer HA. The development of lateral-line receptors in Eigen- tiva and cornea. Invest Ophthalmol Vis Sci 1983;24:570–6. mannia (Teleostei, Gymnotiformes) 1: the mechanoreceptive lat- 4. Doughty MJ. Analysis of areas and shapes of cells on the corneal eral-line system. Brain Behav Evol 1989;33:205–22. surface of the albino rabbit by scanning electron microscopy. Curr 27. Whitear M. Solitary chemosensory cells. In: Hara TJ, ed. Fish Eye Res 1990;9:295–305. chemoreception. New York: Chapman & Hall, 1992:103–25. 5. Doughty MJ. Scanning electron microscopy study of cell dimen- 28. Yamamoto M. Comparative morphology of the peripheral olfac- sions of rabbit corneal epithelium surface. Cornea 1991;10:149– tory organ in teleosts. In: Hara TJ, ed. Chemoreception in fishes. 55. Vol 8. New York: Elsevier Scientific Publishing, 1982:39–59. 6. Harding CV, Bagchi M, Weinsieder A, Peters V. A comparative 29. Uehara K, Miyoshi M, Miyoshi S. Microridges of oral mucosal study of corneal epithelial cell surfaces utilizing the scanning elec- epithelium in carp, Cyprinus carpio. Cell Tissue Res 1988;251: tron microscope. Invest Ophthalmol 1974;13:906–12. 547–53. 7. Collin SP, Collin HB. The visual system of the Florida garfish, 30. Uehara K, Miyoshi M, Miyoshi S. Cytoskeleton in microridges of Lepisosteus platyrhincus (Ginglymodi): II. cornea and lens. Brain the oral mucosal epithelium in the carp, Cyprinus carpio. Anat Rec Behav Evol 1993;42:98–115. 1991;230:164–8. 8. Doughty MJ, Bergmanson JPG, Blocker Y. Impact of glutaralde- 31. Finger TE. Evolution of taste and solitary chemoreceptor cell sys- hyde versus glutaraldehyde-formaldehyde fixative on cell organi- tems. Brain Behav Evol 1997;50:234–43. sation in fish corneal epithelium. Tissue Cell 1995;27:701–12. 32. Zeiske E, Theise B, Breucker H. Structure, development and evo- 9. Collin HB, Collin SP. The fine structure of the cornea of the lutionary aspects of the peripheral olfactory system. In: Hara TJ, salamander fish, Lepidogalaxias salamandroides (Lepidogalaxi- ed. Fish chemoreception. London: Chapman & Hall, 1992:13–39. idae, Teleostei). Cornea 1996;15:414–26. 33. Lemp MA, Gold JB. The effects of extended wear hydrophilic 10. Collin HB, Collin SP. The cornea of the sand lance, Limnichthyes contact lenses on the human corneal epithelium. Am J Ophthalmol fasciatus (Creeiidae). Cornea 1988;7:190–203. 1986;101:274–7.

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