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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. The cells appear to be held together at discrete points by desmosomes (arrows). x
10,500.
317
318
B.A. NICHOLS
Langerhans cells in normal and diseased conjunctiva. Am. J. Ophthalmol., 94:205-212.
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