MICROSCOPY RESEARCH AND TECHNIQUE 33~296-319 (1996) Conjunctiva BARBARA A. NICHOLS Francis I . Proctor Foundation, University of California, San Francisco, California 94143 KEY WORDS External eye, Glycocalyx, Tear film, Mucus INTRODUCTION The conjunctiva is the mucous membrane that lines the inner surface of the eyelids and curves onto the anterior surface of the eyeball, where it extends to the cornea. It is continuous with the skin at the margin of the eyelids and with the cornea at the limbus (Wolff, 1976). Thus it forms a sac between the eyelids and the globe. Despite its small size, the conjunctiva plays a vital role in both vision and immunity. It physically protects the delicate structures of the eye by serving as a mechanical barrier to foreign substances, and it provides epithelium to cover corneal wounds (Kinoshita et al., 1983). Its goblet cells secrete mucus that forms the inner layer of the tear film and is essential for normal vision. The abundant blood supply of the conjunctiva delivers protective substances such as antibodies, complement, and white blood cells to the eye to combat infections and to remove dead or damaged tissue (Dawson, 1976, 1984). Lastly, the conjunctival, intestinal, bronchiolar, and other mucous membranes constitute the mucosal immune system (Chandler and Axelrod, 1980; Wolf and Bye, 1984), found in the regions of the body most commonly invaded by microorganisms. These topics will be discussed further in the following pages. When we began o u r study of the conjunctiva, our primary goal was to analyze the factors involved in the onset of an infection of the external eye. The surface of the epithelium, where invading microorganisms first impinge, had not yet been thoroughly investigated. We started by reexamining the structure of the normal epithelium and present here our observations of the structure of the conjunctiva, with emphasis on its surface. Of several species that we examined, the guinea pig most closely resembles man in Conjunctival structure. Therefore, conjunctiva from pathogen-free guinea pigs was used to illustrate this review. (See Technical Considerations.) STRUCTURE OF THE CONJUNCTIVA Although the conjunctiva is a continuum, stretching from lid margin to corneal limbus, for purposes of description it may be divided into three major areas: the palpebral conjunctiva lining the inner surfaces of the lids; the bulbar conjunctiva (Figs. 1-3) over the globe; and the superior and inferior fornices (Figs. 4-S), the cul-de-sacs formed by the junction of the palpebral and bulbar areas (Duke-Elder and Wybar, 1961). The conjunctiva is composed of two major layers of tissue, the epithelium and the underlying stroma (Figs. 4 and 9). The structure of the Conjunctival epithelium varies from one area of the orbit to another. The distribution 0 1996 WILEY-LISS, INC. of cell types is better appreciated by light rather than by electron microscopy because more extensive areas of tissue can be observed a t a glance. Most instructive are sections spanning the area from one lid margin to the other, such as those used by Latkovik and Nilsson (1979a) in studies of the guinea pig conjunctiva, and by Setzer et al. (1987) in studies of the rat conjunctiva. Several previous studies have provided detailed information about the structure of the conjunctiva. Spencer and Zimmerman (1985) have written an excellent, detailed review of the histology of human conjunctiva. Breitbach and Spitznas (1988) surveyed selected areas of the human conjunctiva by electron microscopy. Bock and Hanak (19711, Latkovic (1979a,b), and Latkovic and Nilsson (1979a,b) used electron microscopy to study the guinea pig conjunctiva, and the last four of these studies analyze in detail the variations in tissue ultrastructure in different areas of the conjunctiva. THE EPITHELIUM Although the Conjunctival epithelium varies in microscopic structure in different regions, it is classified as stratified columnar in man and the guinea pig based on the structure of the apical cells (Spencer and Zimmerman, 1985; Takakusaki, 1969). The surface layers actually vary gradually from columnar in the fornix (Fig. 4) and palpebral areas to cuboidal in the bulbar region (Fig. 11, and squamous near the lid margins and over lymphoid follicles (Fig. 9). The epithelium is generally three to six cells thick, with polygonal cells forming intermediate and basal layers (Figs. 2, 4 and 6). The distribution of goblet cells varies among species (Setzer et al., 1987). In the guinea pig, goblet cells are present singly throughout the Conjunctival epithelium, except near the lid margins and the corneal limbus. They are most common in the fornix, where they are the predominant cell type (Fig. 4). Their localization is most vividly revealed in wholemounts (Kessing, 1968; Worgul et al., 1976) or surface impressions of the conjunctiva in which large areas of tissue are revealed (Lee et al., 1985). In tissue stained with periodic acid Schiff (PAS) stain to reveal mucus, the position of each goblet cell is demonstrated clearly (Kessing, 1968; Worgul et al., 1976). THE STROMA The epithelium is separated from the stroma by a typical basal lamina. The subepithelial layer, in which mononuclear leukocytes are commonly found, is often called the adenoid layer (Records, 1979; Sacks et al., Received March 16, 1992; accepted in revised form March 28, 1994. Address reprint requests to Barbara A. Nichols, Ph.D., University of California, Box 0944, San Francisco, CA 94143. Fig. 1. Superficial epithelial cells of the bulbar conjunctiva. The cells are endowed with abundant endoplasmic reticulum (er),Golgi complexes (G),and mitochondria (m), indicating an active metabolism. Note the extensive interdigitations of their lateral and basal surfaces (arrows). x 6,800. B E F G IN L M P b C cv Abbreviations basal cell d er epithelial cell fibroblast f Golgi complex gl intermediate cell m lymphocyte mf macrophage mt pericyte mu basal lamina mv t collagen coated vesicle v desmosome endoplasmic reticulum filaments glycocalyx mitochondrion microfilaments microtubule mucus microvillus tonofilaments digestive vacuoles 1986). The connective tissue of the stroma is interrupted by small blood vessels (Figs. 10 and 11) and lymphatic vessels (Figs. 12-14). Collagen (Fig. 151, scattered fibroblasts, mononuclear leukocytes, and other cells (Figs. 16-21) are also found in the stroma. Occasionally, leukocytes are seen traversing the junctions between endothelial cells on their journey into the stroma or overlying epithelium, where they are normally found in small numbers (Fig. 22; D y e r et al., 1983). Both epithelium and stroma are also popu- 298 B.A. NICHOLS Fig. 2. Micrograph showing that in the bulbar conjunctiva the basal cells (B)are elongated along the basal lamina (b) and contain many bundles of intermediate filaments. The cells (IN) of the intermediate layers are irregular in shape. x 14,000. Fig. 3. Part of a basal cell (B)in the bulbar conjunctiva showing several groups of intermediate filaments (f) at higher magnification. b, basal lamina; m, mitochondrion. x 38,400. CONJUNCTIVA Fig. 4. Low magnification view of the fornix, showing goblet cells engorged with mucous droplets. The epithelial cells (E)between them are slender, elongated, and inconspicuous. The inner surface of the epithelium, limited by a typical basal lamina (arrow), is irregular. 299 Small blood vessels and scattered cells lie just beneath the epithelium. Much of the remaining stroma is filled with collagen fibers (c). x 4,000. 300 B.A. NICHOLS Fig. 5. Higher magnificationview of the epithelium of the fornix. The numerous organelles in the cytoplasm suggest high metabolic activity. m, mitochondrion;er, endoplasmic reticulum; G, Golgi complex. x 25,800. CONJUNCTIVA Fig. 6. Basal cell (B) from a normal fornix showing that in this area of the conjunctiva, basal cells are polyhedral rather than flattened as in the bulbar conjunctiva. b, basal lamina; d, desmosome. x 12,000. 301 ing condensed chromosomes (arrows) and part of the mitotic spindle (arrowhead). Note that the desmosomes (d) remain intact. X 5,500. Fig. 8. Higher magnification of part of Figure 7, showing the centriole (arrow) and spindle microtubules (mt). x 25,000. Fig. 7. Dividing basal cell (B) from an inflamed conjunctiva, show- lated with occasional Langerhans cells (Bhan et al., 1982; Gillette et al., 1982; Rodrigues et al., 1981; Sacks et al., 1986)mast cells, eosinophils (Friedlaender et al., 1980), and plasma cells (Allensmith et al., 1976, 1978; Friedlaender et al., 1980). Aggregations of lymphocytes (Fig. 9) are common in the fornix, but may also appear in the bulbar or palpebra1 conjunctiva. These lymphocytic nodules are described clinically as “follicles.” The epithelium becomes thinner over the follicles and may consist of only a single layer of flattened, squamous-like cells. In the follicles, lymphocytes are interspersed with macrophages filled with large digestive vacuoles (Fig. 23). Such cells are often called “tingible body macrophages” because of their intense staining properties. The follicles sometimes contain germinal centers (Dawson, 1984) where lymphocytes are proliferating (Fig. 24). THE SURFACE OF THE CONJUNCTIVA The cells at the surface of the conjunctiva are covered by microvilli. Our studies (Nichols et al., 1983) have shown that there are regional structural variations among microvilli just as there are differences among the epithelial cells that bear them. Longer microvilli (300 nm; Figs. 25 and 26) are found on the columnar cells of the fornix, and progressively shorter ones on the bulbar epithelium. There are also microvilli on the cornea which is continuous with the conjunctiva. In the central cornea, the height of the microvilli (and also microplicae) diminishes to between 100 and 200 nm. Except for increasing the surface area of the epithelium, no function has yet been ascribed to the microvilli of either the conjunctiva or cornea. Cores of microfilaments are prominent in conjunctival microvilli, particularly in specimens prepared by freeze substitution (see below; Nichols et al., 1985). No labeling studies have yet been done to identify these filaments, but such studies may be important. Gipson and Anderson (1977) used heavy meromyosin labeling to identify actin filaments in the cores of corneal microvilli and in basal cells migrating centrally to cover wounded epithelium. Isolated intestinal brush borders with microvilli have been shown to contain actin and myosin. When treated with ATP and a divalent cation, Fig. 9. The epithelium over a lymphoid follicle is thin. Here it consists of two or three cell layers, but it sometimesconsists of a single layer. The cells are flattened,with their long axes parallel to the surface. The upper part of a follicle (arrow) lies just beneath a blood vessel. x 5,200. CONJUNCTIVA Fig. 10. Small blood vessel in the adenoid layer of the stroma, showing several endothelial cells (EN) forming the wall of the vessel. It is surrounded by pericytes (P)and a basal lamina (b). AROWSindicate erythrocytes. x 5,900. 303 304 B.A. NICHOLS Fig. 11. A small arteriole in the stroma, showing its endothelial cells (EN),elastic fibers (arrows),and smooth muscle cells (arrowheads). x 13,100. either calcium (Mooseker, 1976) or magnesium (Rodewald et al., 19761, the brush border microvilli are retracted into the terminal web. Similar movement of conjunctival microvilli might spread the tear film and assist in maintaining its even thickness, which is essential for normal vision. Madara and Pappenheimer (1987) also studied actomyosin in the intestinal epithelium and uncovered another function of it of potential interest with reference to the conjunctiva. In elegant in vitro experiments, they showed that activating the transport of glucose or amino acids into the intestinal epithelium produced condensation of the perijunctional actomyosin ring, with concomitant widening of lateral intercellular spaces and increased fluid absorption. THEGLYCOCALYX We next turned our attention to the glycocalyx, believed to be composed of glycoproteins present at the surfaces of all cells (Ito, 1974; Luft, 1976). As others have found previously (Holly and Lemp, 1977; Lee et al., 1981; Wells and Hazlett, 19841, we confirmed that ruthenium red produces thick deposits of precipitate over the surfaces of the microvilli (Fig. 25). This precipitate indicates that the cell coat contains a high concentration of polyanions (Luft, 1976), possibly sialic acid residues. However, we found that the intense staining reaction caused the filaments of the glycocalyx to aggregate thus obscuring their length and structure (Nichols et al., 1983). We found staining with tan- CONJUNCTIVA Fig. 12. Lymphatic vessel in the stroma. Its walls (arrow) are thinner and more irregular in shape than those of the blood vessel shown below (double arrow). L, lymphocyte. x 4,300. 305 Fig. 14. Higher magnification view of the wall of a lymphatic vessel, showing the infoldings (bracket) between its endothelial cells. x 24,300. Fig. 13. Higher magnification of the endothelial cell body (EN) shown in Figure 12. x 14,500. nic acid to be more useful, as it clearly revealed a filamentous glycocalyx on the microvilli (Fig. 26).Such a glycocalyx is seen in relatively few tissues, but has been intensively studied on the microvilli of intestinal brush borders (Moog, 1981). The filaments of the glycocalyx had a nearly uniform length of about 300 nm over the entire ocular surface, from the conjunctiva to the central cornea. They fanned outward from the mi- crovilli and reached adjacent microvilli, so that the glycocalyx formed a continuum over the ocular surface. In some areas, the origin of the filaments at the membranes of the microvilli could be discerned (Nichols et al., 1983). It may be instructive to use the intestinal glycocalyx as a model for similar filamentous projections on other epithelia. In the intestine, the prominent filaments are 306 B.A. NICHOLS Fig. 15. Collagen in the stroma, well-stained because the specimen was prepared with a quaternary ammonium compound in the fixative. x 21,700. an external coating over the surface of the eye in our study (Nichols et al., 1983) and in others (Holly and Lemp, 1977; Lee et al., 1981; Wells and Hazlett, 19841, it was uncertain whether the staining revealed the true extent of the mucous layer. It seemed likely that at least some mucus had been lost during tissue preparation. Therefore, we used additional techniques to demonstrate the mucus. We knew that quaternary ammonium compounds precipitate glycoproteins and form very insoluble complexes. We chose two of these compounds, cetylpyridinium chloride (CPC) and hexadecyltrimethylammonium bromide (HTAB), and added them to our fixatives (Nichols et al., 1985). We found that the mucus was precipitated as it was discharged from the goblet cells (Fig. 27) and that it spread over the surface of the corDEMONSTRATION OF THE MUCOUS LAYER nea and conjunctiva to form a relatively smooth layer. OF THE TEAR FILM We then intensified the demonstration of the mucus by When we previously analyzed the cell coat of the staining it with tannic acid (Fig. 281, a stain for the conjunctiva we had hoped to elucidate the localization similar glycoproteins of the cell coat (Simionescu and of mucus, which forms the inner layer of the tear film. Simionescu, 1976). With these methods, we found that Remnants of mucus had been seen on the ocular sur- the mucus measured as much as 0.6 micrometers over face by scanning electron microscopy (Greiner et al., the cornea and as much as two micrometers over the 1977; Hazlett et al., 1981; Pfister, 1975) and by trans- conjunctiva. These methods are not ideal for demonstrating mumission electron microscopy of tissue treated with carbohydrate stains (Wells and Hazlett, 1984; Wells et al., cus, because mucus is extracellular and thus prone to 1988). Although ruthenium red staining had revealed loss or redistribution. We confirmed our results, how- believed to be carbohydrate side chains of glycoprotein enzymes that are embedded in the microvillar membrane. The enzymes are maltase, sucrase, lactase, aminopeptidases, and other enzymes that hydrolyze nutrients before transport into the epithelial cells that bear them. The protein moieties are integral membrane proteins, not adsorbed to the surface (Ito, 1974; Moog, 1981). Future studies of the ocular glycocalyx may uncover similar enzymatic functions operating locally. Meanwhile, the glycocalyx of the conjunctiva is believed to perform the important function of binding mucus to the conjunctival surface by means of noncovalent bonding between the similar carbohydrate components of the mucus and the glycocalyx (Holly and Lemp, 1977). CONJUNCTIVA 307 Fig. 16. Lymphocyte (L) and fibroblast (F) in the stroma. c, collagen. x 14,100. ever, by another method, quick-freezing and freeze substitution (Nichols et al., 1985).This method involves freezing at the temperature of liquid helium (-272"C), which retains the mucus in place, and fixation a t low temperature (-8O"C), which preserves the tissue and also the mucus in its native position. We found that the results with freeze substitution substantiated our results with the quaternary ammonium compounds in that the mucus was up to one micrometer thick over the cornea (Fig. 29). Freeze substitution also had the advantage of preserving mucous droplets better than conventional fixation (Fig. 30). The mucus varied in depth over the conjunctiva (Figs. 31 and 32)because of the irregularity of the epithelium; it was as much as seven micrometers deep over depressions, particularly in the lower fornix, where the tear fluid pools. Our results also show that the mucus and the glycocalyx overlap in distribution (Figs. 31 and 32). This finding supports the hypothesis that they are bound to each other by means of weak bonds linking their similar glycoproteins. We consider that our results are only an approximation of the thickness of the mucous layer because of its extracellular distribution and proclivity for loss or movement. We conclude nonetheless that the mucus layer is of considerable thickness over the surface of the eye, and is much thicker than formerly appreciated. THE FUNCTION OF THE MUCOUS LAYER The tear film is the first structure that refracts light entering the eye (Holly and Lemp, 1977).Therefore, its even thickness and smooth, uninterrupted distribution over the cornea are essential for normal vision. Mucus secreted by the goblet cells forms the innermost layer of the tear film. Mucus is hydrophilic and serves to reduce the surface tension of the tear film, thereby en- 308 B.A. NICHOLS Fig. 17. Macrophage (M) in the stroma. x 15,000. hancing its spread (Holly and Lemp, 1971). It also lubricates and moistens the ocular surface, traps foreign particles, and facilitates gas exchange across the tear film. Evidence for the derivation of mucus from goblet cells of the conjunctiva was obtained by Moore and Tiffany (19791, who used immunofluorescence to demonstrate a protein of human ocular mucus in goblet cells. Deficiencies of goblet cell function are found in serious cicatricial ocular disorders such as StevensJohnson syndrome and ocular pemphigoid (Ralph, 1975; Holly and Lemp, 1977), indicating the need for properly functioning goblet cells in the healthy conjunctiva. A deficiency of ocular mucus such as that caused by insufficient vitamin A (Holly and Lemp, 1977; Mishima, 1965; Ralph, 1975) may interrupt the continuity of the tear film, causing dry spots on the cornea and damage that may lead to corneal melting. Cytochemical investigations have shown that subsurface vesicles in upper conjunctival epithelial cells contain glycoproteins similar to those on the conjunctival surface (Greiner et al., 1985; Wells et al., 1988).It has been suggested that such glycoproteins contribute to the mucous layer. Openings of the vesicles to the surface have been shown by electron microscopy, but whether these vesicles are being released or internalized was not evaluated in those studies with tracers. However, it was shown in another investigation by means of the f luid-phase tracer horseradish peroxidase that similar vesicles traveled inward from the conjunctival surface (Stock et al., 1987). It would not be surprising if such incoming pinocytic vesicles contained glycoproteins like those of the tear film. THE INFLAMED CONJUNCTIVA The architecture of the conjunctival epithelium changes greatly when the conjunctiva becomes infected or inflamed (Carroll and Kuwabara, 1968). The intercellular spaces widen and become filled with fluid. When the tissue is edematous, the cells are held together by the desmosomes that connect them (Figs. 33 and 34) and by prominent tonofilaments. The vessels in the conjunctival stroma dilate during acute infection and polymorphonuclear leukocytes (PMN) are commonly seen attached to their walls. The epithelium is filled with many PMN beginning as soon as four hours after the onset of infection. In more chronic infections, mononuclear leukocytes increase in number and outnumber PMN in both the epithelium and the stroma. THE CONJUNCTIVA IN HOST DEFENSE The conjunctiva participates in nonspecific host defenses by removing foreign particles, including bacteria, that impinge upon its surface. Zimianski et al. CONJUNCTIVA Fig. 18. Polymorphonuclear leukocyte in the stroma. x 13,000. Fig. 19. Eosinophilic leukocyte in the stroma. x 10,800. Inset: Higher magnification of an eosinophilic granule showing its crystalline core. x 25,900. (1974) showed the phagocytosis of the bacterium Listeria monocytogenes by the conjunctival epithelium, and Latkovic and Nilsson (1979~) traced the removal of inert particles from this tissue using latex spheres as markers. Latkovic’s (1985) detection of acid phosphatase in phagocytic vacuoles in the conjunctival ep- 309 Fig. 20. Mast cell in the stroma. x 10.900. Fig. 21. Plasma cell in an infected conjunctiva. x 12,300. ithelium suggests digestive activity in this tissue. Pinocytosis has also been revealed cytochemically in conjunctiva stimulated with the protein horseradish peroxidase (Stock et al., 1987). No transport of the tracer into the stroma was documented. However, Chait (1950) has evidence of protein absorption across 310 B.A. NICHOLS Fig. 22. A lymphocyte (L)is at the junction between two endothelial cells (EN) apparently leaving the circulation to enter the stroma. X 7,500. the human conjunctiva, suggesting that some route through the epithelium is accessible to large molecules. The conjunctiva also plays a role in more specific host defenses as a part of the mucosal immune system. When stimulated with antigen, mucous membranes have in common the potential to secrete IgA and other antibodies locally, and to produce lymphoblasts that migrate to other mucosae, where they mature into T and B lymphocytes and antigen-specific plasma cells (Wolf and Bye, 1984). Specific antibodies to proteins applied to rabbit conjunctiva have been found in the circulation by Hall and Pribnow (1981). They also detected lymphocytes producing these specific antibodies in the conjunctiva and other tissues of the body. Thus, stimulation of the conjunctiva with antigen may have far-reaching specific effects throughout the body. IgA, the predominant antibody found in tears, has been localized at the ocular surface (Hazlett et al., 1981). In the intestinal mucosa, which has been extensively analyzed, Owen (1977) has proposed that an antigenic stimulus gains access to antibody-forming cells in Peyer’s patches via “M cells” that lie directly over lymphoid cells. Our results and those of Latkovic (1979a) show that the conjunctival epithelium of the guinea pig thins markedly to one or two layers of flattened cells over a lymphoid follicle. It has been suggested that M cells may be among these (Latkovic, 1989) but no distinguishing features of M cells have yet been defined in the flattened conjunctival cells. Using the Toxoplasma dye test, we have demonstrated titers of antibody as high as 1:64,000 in the blood of guinea pigs infected with parasites via the conjunctiva, when the lacrimal puncta were occluded (Skorich et al., 1988). The parasites multiplied in successively deeper layers of the epithelium, and after 48 hours they crossed the basal lamina and reached the stroma. However, it is possible that these highly invasive parasites, which can disrupt cellular membranes, may have traveled from the conjunctiva to other sites in the body, possibly via the circulation. Nonetheless, analysis of the lymphoid follicles of the infected animals showed lymphoblasts in division (Fig. 24; Skorich et al., unpublished results), which might have been stimulated by the infection of the overlying tissues. This hypothesis awaits confirmation. Research to date shows that the conjunctiva, although diminutive in size, is complex and contributes significantly to the overall well-being of an animal. Additional research may well uncover further essential functions of this multifaceted tissue. TECHNICAL CONSIDERATIONS The conjunctiva is a thin, flexible membrane so delicate that it is difficult to dissect without pulling and stretching it. As a result, preservation of the conjunctiva for morphological studies is best obtained by fixation in situ, before dissection, by flooding the tissue with fixative for 15-30 minutes. Hence, experimental animals are used for the most detailed morphological and experimental studies. It is important to note, however, that the conjunctiva in some species differs structurally from the human conjunctiva (Setzer et al., 1987; Nichols, unpublished results). The guinea pig CONJUNCTIVA Fig. 23. The cells in a follicle are mostly lymphocytes (L),but conspicuous macrophages (M)full of digestive vacuoles (v) are interspersed among them. X 3,700. 311 312 B.A. NICHOLS Fig. 24. Lymphoblasts in division (arrows) are common in germinal centers of follicles when the conjunctiva is infected. x 3,700. CONJUNCTIVA Fig. 25. Microvilli (mv) at the surface of the normal conjunctiva stained with dense deposits of ruthenium red. x 77,200.(Reproduced from Nichols et al., 1983, with permission of J.B. Lippincott Company.) 313 Fig. 26. Similar microvilli (mv) on the conjunctiva after 8 .aining with tannic acid. The fine filaments of the glycocalyx are clearly defined with this more delicate procedure. x 79,400.(&produced from Nichols et al., 1983,with permission of J.B. Lippincott Company.) Fig. 27. Conjunctiva prepared with cetylpyridinium chloride (CPC) added to the fixative. CPC, a quaternary ammonium compound, precipitated the mucus (mu) as it was discharged from the goblet cells. x 40,500. (Reproduced from Nichols et al., 1985,with permission of J.B. Lippincott Company.) 314 B.A. NICHOLS Fig. 28. Conjunctival tissue prepared with fixative containing CPC,then stained with tannic acid. The precipitated mucus (mu) is shown clearly by the dense stain. mv, microvillus. x 16,500.(Reproduced from Nichols et al., 1985,with permission of J.B. Lippincott Company.) Fig. 29. Freeze substitution of the cornea showed that the mucus (mu) measured a s much as one micrometer in depth. x 34,000. (Reproduced from Nichols et al., 1985,with permission of J.B. Lippincott Company.) Fig. 30. Freeze substitution of the conjunctiva preserved the membranes (arrows) of the mucous droplets in the goblet cells better than did several routine fixations. x 28,700. 315 CONJUNCTIVA Fig. 31. Freeze substitution of the conjunctiva,showing that the microfilaments (mf) of the microvilli (mv) extended well into the apical cytoplasm. The overlapping of the glycoealyx (gl) and the mucus (mu) is well shown with this technique. x 41,500. Insek Coated vesicles (arrow) were commonly seen at the plasmalemma and in the cytoplasm of the epithelial cells. x 49,100. conjunctiva has proved to be a suitable model for investigation. The tissues shown here in illustrations not previously published (Figs. 1-24, 30, 31, 33 and 34) were fixed with an ice-cold mixture of 3% glutaraldehyde and 2% acrolein in 0.1 M sodium cacodylate-HC1 buffer, pH 7.4.The fixative was dropped onto the eye immediately after death, before dissection, and was left in place for 15-30 minutes, with renewal as needed to prevent drying of the tissues. This initial fixation preserved and hardened the tissue so that mechanical damage during dissection was minimal. The specimen was then removed from the orbit, and fixation was continued for 2-4 hours in the same mixture on ice. The tissue was postfixed in 2% osmium tetroxide in acetateveronal buffer for 2 hours at 4"C, stained in block with uranyl acetate (Farquhar and Palade, 1965) and flatembedded in Epon. The methods for specialized techniques such as freeze substitution, tannic acid staining, and treatment with cetylpyridinium chloride and hexadecyltrimethylammonium bromide are cited in the references (Nichols et al., 1983, 1985; Setzer et al., 1987; Skorich et al., 1988). ACKNOWLEDGMENTS I am particularly grateful for the outstanding technical assistance of Mary Louise Chiappino without whom this work could not have been completed. I also thank Steve Parente for the excellent photographic reproductions and Barbara Poetter for editorial assistance. I am also indebted to Chandler R. Dawson and Steven L. Wissig for their careful reviews of the text. I owe special thanks to my husband, Richard M. Eakin, for advice and encouragement of all sorts during the preparation of the manuscript. Last but not least, I acknowledge and greatly appreciate the indispensable support of these projects by the National Eye Institute 316 B.A. NICHOLS Fig. 32. Freeze substitution of the conjunctiva provided confirmation of the localization of the mucous layer. In this area of the fornix, it measured two micrometers deep. The glycocalyx is shown a t the arrow. G, Golgi complex; cv, coated vesicle; m, mitochondrion; mu, mucus. x 36,400. Inset:Higher magnification of a microvillus, showing its core of microfilaments (f). x 82,700. (Reproduced from Nichols et al., 1985, with permission of J.B. Lippincott Company.) CONJUNCTIVA Fig. 33. When the conjunctivais inflamed or infected, the intercellular spaces are widened by edema and filled with fluid. 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