Download Unusual structure of rat conjunctival epithelium. Light and

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.
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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).
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STRUCTURE OF RAT CONJUNCTIVA / Serzer er ol.
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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.
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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-
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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
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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.
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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
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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,
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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-
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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.