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Unusual Structure of Rat Conjunctiva! Epithelium Light and Electron Microscopy Pauletre Y. Setzer, Barbara A. Nichols, and Chandler R. Dawson The conjunctiva of the adult Sprague-Dawley rat was studied by light microscopy of 3 Mm glycol methacrylate sections of whole eyes with lids and by electron microscopy of conjunctiva from the lower fornix. Rat conjunctiva is unique among species studied. All the superficial epithelial cells are squamous cells rather than polyhedral or columnar cells. Furthermore, the goblet cells are aggregated into clusters rather than distributed randomly throughout the epithelium. These clusters are not found at the lid margin or limbus, but are present in the palpebral and bulbar conjunctivae and achieve maximal size and number near the fornix. The stratified squamous epithelium is typical, composed of a layer of basal cells, an intermediate zone of wing cells, and an upper zone of several layers of squamous cells. Dividing cells are seen only in the basal layer. Occasional mononuclear leukocytes are found in the basal and intermediate layers. The goblet cell clusters are largely composed of columnar cells. Goblet cells predominate, but there are also occasional tuft cells, characterized by thick microvilli at their apices. Basal cells form only an incomplete layer beneath the columnar cells, which in places span the entire epithelium. The conjunctiva of the adult rat has few cells with potential for immunological activity. It does not contain appreciable numbers of plasma cells, and lymphoid follicles are absent. Invest Ophthalmol Yis> Sci 27:531-537, 1987 Although it is of obvious importance in the eye's defense against infection, the conjunctiva has received scant attention. We have studied the structure and functions of normal conjunctiva, using the guinea pig as a source of material.1"3 Studying the rat, we were suprised when our initial specimens revealed a conjunctival structure fundamentally different from that of the other rodent species we had seen. This difference prompted us to undertake a more detailed morphological study of rat conjunctiva. Previous studies in which rat conjunctiva was examined microscopically4"7 were not focused on structure and did not indicate the tissue's distinctive organization. This may have resulted from the orientation of sections or differences between Sprague-Dawley rats and the Wistar4'5 and Fischer6'7 strains used in those studies. outbred Sprague-Dawley rats (retired breeders) were examined. Eyes from two rats were processed for light microscopy (lm); those from two others for electron microscopy (em). The other three rats each provided one eye for lm and one for em. Rats were killed by intraperitoneal injection of 500 mg/kg of sodium pentobarbital (Somlethol, Med Tech, Inc., Elwood, KS). The eyes were fixed immediately, some by vascular perfusion and others by topical fixation in situ. Thefixativesused were 3% glutaraldehyde (3% G) or mixtures of 1 % paraformaldehyde- 3% glutaraldhyde (PG)8 or 5% glutaraldehyde- 2% acrolein (GA), each in 0.1 M sodium cacodylate-HCl, pH 7.4. Allfixativesgave good results. Eyes for lm were bisected equatorially or vertically and fixed an additional 3 hr. Pieces of fornix for em were removed, post-fixed in 1% osmium tetroxide, and stained in Kellenberger's uranyl acetate.9 All samples were dehydrated in ethanols and embedded in DupontSorvall resin (Fisher Scientific, Pittsburgh, PA) for lm or Epon 812 for em. For lm, bisected eyes were cut horizontally or vertically in 3-fim thick sections using a JB-4 microtome. Every eighth section was kept and stained in Lee's stain,10 so that mounted sections were separated by 24nm intervals. The specimens were photographed with a Zeiss photomicroscope using Kodak Panatomic-X film. Some prints were joined to form a montage of the conjunctiva from lid margin to limbus. Thin sections were cut on a Sorvall MT-5000 ultramicrotome, Materials and Methods Use of animals in this study conformed to the guidelines established in the ARVO Resolution on Use of Animals in Research. The eyes of seven adult male From the Francis I. Proctor Foundation, University of California, San Francisco, San Francisco, California. Supported by grants EY-03858 and EY-02162 from the National Eye Institute and an unrestricted grant from Research to Prevent Blindness, Inc. Submitted for publication: January 30, 1986. Reprint requests: Paulette Y. Setzer, Proctor Foundation, 95 Kirkham, Box 0988, University of California, San Francisco CA 94122. 531 Downloaded From: http://iovs.arvojournals.org/ on 05/08/2017 502 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / March 1987 Vol. 28 Fig. 1. A montage of light micrographs at very low power shows the enire upper lid from lid margin (left) to cornea (lower right) in a 3 /im-thick vertical section of a rat eye fixed in PG and stained in Lee's stain. The boxed areas are shown in greater detail in Figs. 2-6 (X23). Figs. 2-6. Light micrographs of selected areas of the rat conjunctiva (X145). Fig. 2. The thickened stratified squamous epithelium of the lid margin (left) becomes thinner in the tarsal conjunctiva (right). Fig. 3. A portion of tarsal conjunctiva exhibits small goblet cell clusters distally (left) and larger ones proximally (right). Fig. 4. Palpebral conjunctiva approaching the fornix displays numerous, large clusters of goblet cells. Fig. 5. A portion of the folded fornix with closelyspaced goblet cell clusters. Fig. 6. Goblet cell clusters are seen infrequently in the bulbar conjunctiva, and there are none at the limbus (bottom center). Downloaded From: http://iovs.arvojournals.org/ on 05/08/2017 STRUCTURE OF RAT CONJUNCTIVA / Serzer er ol. No. 3 533 Fig. 7. Goblet cell cluster* in a 3 /im-thick vertical section from conjunctiva near the fornix are separated by short segments of stratified squamous epithlium. Many goblet cells extend from the basal lamina to the conjunctival surface, but since they are not so tall as the adjacent multilayered squamous epithelium, they appear to lie in depressions (X400). Fig. 8. A portion of rat fornix cut 3 jim-thick and parallel to the surface of the conjunctiva demonstrates the discoid shapes of the goblet cell clusters (X330). Fig. 9. Dividing cells are seen in the basal layer of stratified squamous epithelium of rat conjunctiva. This photomicrograph is of a toluidine blue-stained, 1 ^in-thick Epon section of tissue that was perfused in 3% G and processed for electron microscopy (X960). Fig. 10. A light micrograph of the nictitating membrane with its collagen core from a 3 /im-thick horizontal section of a rat eye fixed in GA and stained in Lee*s stain. Near the free edge, the membrane is covered with stratified squamous epithelium, but small clusters of goblet cells can be seen in the proximal epithelium of both the outer (above) and inner surfaces (X40). stained with uranyl acetate and lead citrate, and examined in a Siemens 1A electron microscope operating at 80 kv. Results Light microscopy of the rat conjunctiva revealed a highly structured tissue consisting of stratified squamous epithelium punctuated for most of its extent by clusters of goblet cells (Figs. 1-7). The typical squamous epithelium is composed of a basal cell layer, an intermediate zone of overlapping wing cells, and several layers of squamous cells. The goblet cells are not as tall as the multilayered squamous epithelium, so that the goblet cell clusters form depressions in the surrounding tissue (Figs. 3 and 7). When sectioned parallel to the surface of the epithelium, the clusters were seen to be disc-shaped (Fig. 8). Mitotic figures were seen occasionally in the basal layer of the squamous epithelium (Fig. 9), but rarely in the goblet cell clusters. Occasional mononuclear leukocytes appear as isolated cells in the basal layer and intermediate zone. A survey of the rat conjunctiva from lid margin to limbus (Figs. 1 -6) revealed regional variations that were essentially the same for the upper and lower lids. At the lid margins, the stratified squamous epithelium was considerably thickened (Fig. 2), but the neighboring palpebral conjunctiva exhibited the five- to seven-layered squamous epithelium seen in most of the conjunctiva. Downloaded From: http://iovs.arvojournals.org/ on 05/08/2017 The inner portion of the palpebral conjunctiva contained clusters of goblet cells (Fig. 3). Closer to the lid margin, these clusters were small; the number and size of goblet cells in the clusters increased progressively in the palpebral epithelium approaching the fornix (Fig. 4), which was richly endowed with goblet cells (Fig. 5). In palpebral conjunctiva near the fornix, the clusters sometimes had more than 10 goblet cells across their diameters (Fig. 4). In the fornix itself, the clusters were somewhat smaller (Fig. 5), but were very close together (Fig. 8). Approaching the limbus, the goblet cell clusters decreased in size and number; very few were seen in the bulbar and none in the limbal conjunctiva (Fig. 6). The stratified squamous epithelium of the limbal area superficially resembled the corneal epithelium, except that the conjunctiva was somewhat thinner and had a less smooth surface (Fig. 6). However, the conjunctival basal cells were small and angular, while those of the cornea were larger and cuboidal. The surface of the nictitating membrane that is exposed when the membrane is extended over the eye was composed of uninterrupted stratified squamous epithelium, as was the distal portion of the inner surface, which slides across the globe. The proximal portions of both surfaces, however, possessed intermittent small clusters of goblet cells (Fig. 10). The sections spanning entire eyes at 24-jim intervals revealed no structure comparable to the lymphoid fol- 534 INVESTIGATIVE OPHTHALMOLOGY & VI5UAL SCIENCE / March 1987 Vol. 28 Figs. 11-12. Electron micrographs of rat fornix sectioned perpendicularly to the epithelium. Fig. 11. A segment of stratified squamous epithelium from tissue fixed in GA. The basal, intermediate, and squamous cell zones form the tissue from the basal lamina (bottom) to the conjunctiva! surface (top) (XI0,000). licle (or "lymphoid nodule") of guinea pigs'' or the lymphoid aggregates of rabbits. 1213 The stratified squamous cells composing the epithelial portion of the rat conjunctiva were ultrastructurally typical (Fig. 11). Electron microscopy of the goblet cell clusters, however, revealed some interesting features: 1) The basal cells formed an incomplete layer beneath the clusters, so that the columnar cells often spanned the entire distance from the basal lamina to the surface (Fig. 12); 2) Tuft cells were present in the goblet cell clusters. Positioned either centrally or peripherally in the goblet cell clusters, the tuft cells equalled the goblet cells in height (20 /xm) but were more slender (diameter Downloaded From: http://iovs.arvojournals.org/ on 05/08/2017 about 4 nm) and had narrow apices (Fig. 12). The cells' tufted appearance was due to their thick apical microvilli, which were at least twice the diameter (280 nm) of microvilli of any other rat conjunctival cells. The microvilli contained cores of microfilaments, arranged in parallel arrays, which extended deeply into the cytoplasm (Fig. 12). Favorable sections showed long tubules and vesicles between the apical microfilament bundles; 3) Those intraepithelial mononuclear leukocytes that were identified by lm and em were all lymphocytes or macrophages. The lack of desmosomes or other junctional connections with the epithelial cells surrounding them suggested that these cells were pres- STRUCTURE OF RAT CONJUNCTIVA / Serzer er QI. No. 3 535 Fig. 12. The edge of a goblet cell cluster in a PG-fixed fomix shows two goblet cells (Gb), filled with mucous droplets, flanking a tuft cell (T) with its distinctive, thick microvilli (X9,000). ent in the rat conjunctiva as migrants. No plasma cells were seen. Discussion Rat conjunctiva differs from that of other mammals in two major features. First, superficial epithelial cells throughout the rat conjunctiva are squamous cells. Second, the goblet cells are aggregated into clusters. The structure of tissue from several areas of the conjunctiva has now been examined in three species. Many Downloaded From: http://iovs.arvojournals.org/ on 05/08/2017 workers14 19 have reported on the microscopic structure of human conjunctiva! epithelium. Latkovic, separately11'20 and with Nilsson,21'22 has comprehensively examined the ultrastructure of several regions of the guinea pig conjunctiva. These investigations have shown that human and guinea pig conjunctivae are structurally similar. In both species, the superficial conjunctival cells are squamous near the lid margin,l5il9>21 cylindrical in the fornix and tarsus," 1719 ' 21 and polyhedral in the bulbar zone.' M 4 J 9 The superficial cells of the guinea pig perilimbal conjunctiva, however, 536 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / March 1987 are polyhedral,22 while those of the human are usually squamous.15'19 Wanko,18 reporting on the fine structure of only this area, pronounced rat conjunctiva similar to human. In fact, the unvarying stratified squamous epithelium of rat conjunctiva is in striking contrast to the diversity of cell shapes seen in the same tissue in humans and guinea pigs. Goblet cells in the rat conjunctiva are always found in clusters, with their tops below the level of the surrounding superficial epithelial cells. Aggregation of all goblet cells in pits or depressions has not been reported for any other species, although similar aggregations occur in two small regions of the human conjunctiva. Kessing,23 in his analysis of flat-mounted human conjunctivae stained for mucins, has shown that clusters of goblet cells (which he called "mucous crypts") occur on the semilunar fold and in the inferior fornical area of most human specimens. However, the goblet cells of all other areas of the human conjunctiva are solitary. In flat-mounted conjunctivae like those used by Kessing, each goblet cell appears as a dot. Such preparations provide a "map" from which numbers and distribution of goblet cells can be readily determined. Unfortunately, photographs of flat-mounted preparations of conjunctivae of other species have not yet been published. However, studies of several areas of the guinea pig conjunctiva using transmission em11'21 have yielded information on the presence and relative numbers of goblet cells in different parts of the tissue. The distribution of goblet cells in the guinea pig conjunctiva is similar to that in the rat, except that the rat goblet cells are clustered, whereas those of the guinea pig are solitary. In the guinea pig, goblet cells are found in the palpebral zone 20 and fornix," but rarely in the bulbar conjunctiva." Latkovic and Nilsson found no goblet cells in the perilimbal zone of the guinea pig conjunctiva.21 Until now, tuft cells have been reported in the conjunctiva of only the cow.24 However, they appear quite consistently in epithelia of the gastrointestinal tract and its derivatives in rodents, dogs, humans (see Wattel and Geuze's review25), and domestic ruminants. 24 These cells have been reported under a variety of names, including "caveolated cells"26 and "fibrillovesicular cells,"25'27 but Isomaki's28 original designation, "tuft" cell, is both brief and descriptive. Although the function of these cells has been the subject of speculation,25 it remains unknown. Some lymphocytes and macrophages are interspersed among the basal and wing cells of the rat conjunctiva, but otherwise, cells with potential immunological activity are absent. Rat conjunctiva lacks the numerous plasma cells observed in human conjunctivae29 and the aggregated lymphoid cells found in rabbits 1213 and guinea pigs." Not surprisingly, rat con- Downloaded From: http://iovs.arvojournals.org/ on 05/08/2017 Vol. 28 junctivae are reported to be immunologically inactive.30 This study shows that the rat conjunctiva is strikingly different from that of other mammals. Its dissimilarity even to conjunctivae of other rodents indicates that one cannot assume uniformity of conjunctival structure, organization, or immunology in all mammals. Key words: conjunctiva, epithelium, microscopy, goblet cells, tuft cells Acknowledgments The authors thank Use Sauerwald and the Department of Stomatology at U.C.S.F. for allowing us generous use of the JB-4 microtome and Steve Parente for the fine photographic reproductions. References 1. Malaty R, Nichols B, Schachter J, Togni B, and Dawson C: Stimulation of peroxidase by chlamydial infection: cytochemistry of guinea pig conjunctival epithelium. Infect Immun 25:417, 1979. 2. Nichols B, Dawson CR, and Togni B: Surface features of the conjunctiva and cornea. Invest Ophthalmol Vis Sci 24:570, 1983. 3. Nichols BA, Chiappino ML, and Dawson CR: Demonstration of the mucous layer of the tear film by electron microscopy. Invest Ophthalmol Vis Sci 26:464, 1985. 4. Iwata T: Cytochemical studies on endogenous peroxidase in conjunctival and corneal epithelial cells. Invest Ophthalmol 15: 297, 1976. 5. Iwata T, Ohkawa K, and Uyama M: The fine structural localization of peroxidase activity in goblet cells of the conjunctival epithelium of rats. Invest Ophthalmol 15:40, 1976. 6. McMaster PRB, Aronson SB, and Bedford MJ: Mechanisms of the host response in the eye. IV. The anterior eye in germ-free animals. Arch Ophthalmol 77:392, 1967. 7. Pu Z, Pierce NF, Silverstein AM, and Prendergast RA: Conjunctival immunity: compared effects of ocular or intestinal immunization in rats. Invest Ophthalmol Vis Sci 24:1411, 1983. 8. Karnovsky MJ: A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J Cell Biol 27:137a, 1965. 9. Kellenberger E, Ryter A, and Sechaud J: Electron microscope study of DNA-containing plasms. II. Vegetative and mature phage DNA as compared with normal bacterial nucleoids in different physiological states. J Biophys Biochem Cytol 4:671, 1958. 10. Bennett HS, Wyrick AD, Lee SW, and McNeil JH Jr. Science and art in preparing tissues embedded in plastic for light microscopy, with special reference to glycol methacrylate, glass knives and simple stains. Stain Technol 51:71, 1976. 11. Latkovic S: The ultrastructure of the normal conjunctival epithelium of the guinea pig. III. The bulbar zone, the zone of the fornix and the supranodular zone. Acta Ophthalmol 57:305, 1979a. 12. Axelrod AJ and Chandler JW: Morphologic characteristics of conjunctival lymphoid tissue in the rabbit. In Immunology and Immunopathology of the Eye, Silverstein AM and O'Connor GR, editors. New York, Masson, 1979, pp. 292-301. 13. Franklin RM and Remus LE: Conjunctival-associated lymphoid tissue: Evidence for a role in the secretory immune system. Invest Ophthalmol Vis Sci 25:181, 1984. 14. Carroll JM and Kuwabara T: Ocular pemphigus. An electron No. 3 15. 16. 17. 18. 19. 20. 21. 22. STRUCTURE OF RAT CONJUNCTIVA / Serzer er ol. microscopic study of the conjunctival and corneal epithelium. Arch Ophthalmol 80:683, 1968. Duke-Elder S and Wybar KG System of Ophthalmology, Vol. II. London, Henry Kimpton, 1961, pp. 543-545. Hogan MJ, Alvarado JA, and Weddell JE: Histology of the Human Eye. Philadelphia, WB Saunders, 1971, p. 128. Takakusaki I: Fine structure of the human palpebral conjunctiva with special reference to the pathological changes in vernal conjunctivitis. Arch Histol Jap 30:247, 1969. Wanko T, Lloyd BJ Jr, and Matthews J: The fine structure of human conjunctiva in the perilimbal zone. Invest Ophthalmol 3:285, 1964. Wolff E: Anatomy of the Eye and Orbit. London, HK Lewis and Co, Ltd, 1961, pp. 185-189. Latkovic S: The ultrastructure of the normal conjunctival epithelium of the guinea pig. IV. The palpebral and the perimarginal zones. Acta Ophthalmol 57:321, 1979b. Latkovic S and Nilsson SEG: The ultrastructure of the normal conjunctival epithelium of the guinea pig. I. The basal and intermediate layers of the perilimbal zone. Acta Ophthalmol 57: 106, 1979a. Latkovic S and Nilsson SEG: The ultrastructure of the normal conjunctival epithelium of the guinea pig. II. The superficial layer of the perilimbal zone. Acta Ophthalmol 57:123, 1979b. Downloaded From: http://iovs.arvojournals.org/ on 05/08/2017 537 23. Kessing SV: Mucous gland system of the conjunctiva. Acta Ophthalmol (Suppl) 95:1, 1968. 24. von Weyrauch KD: Ultrastructure of the tuftcell in some epithelia of the domestic ruminants. Anat Anz 146:141, 1979. 25. Wattel W and Geuze JJ: The cells of the rat gastric groove and cardia. An ultrastructural and carbohydrate histochemical study, with special reference to the fibrillovesicular cells. Cell Tissue Res 186:375, 1978. 26. Nabeyama A and Leblond CP: "Caveolated cells" characterized by deep surface invaginations and abundant filaments in mouse gastro-intestinal epithelia. Am J Anat 140:147, 1974. 27. Hammond JB and LaDeur L: Fibrillovesicular cells in the fundic glands of the canine stomach: evidence for a new cell type. Anat Rec 161:393, 1968. 28. Isomaki AM: A new cell type (tuft cell) in the gastrointestinal mucosa of the rat. A transmission and scanning electron microscopic study. Acta Pathol Microbiol Scand Sect A (Suppl) 240, 1973. 29. Allansmith MR, Kajiyama G, Abelson MB, and Simon MA: Plasma cell content of main and accessory lacrimal glands and conjunctiva. Am J Ophthalmol 82:819, 1976. 30. Gudmundsson OG, Sullivan DA, Bloch KJ, and Allansmith MR: The ocular secretory immune system of the rat. Exp Eye Res 40:231, 1985.