Download A Comparative Study of Corneal Epithelial Cell Surfaces

Original Articles
A comparative study of corneal epithelial
cell surfaces utilizing the scanning
electron microscope
Clifford V. Harding,0 Mihir Bagchi, Allan Weinsieder, and Virginia Peters
The mammalian corneal epithelium has been previously shown to have microprojections (microvilli, microplicae) which have been implicated in tear film retention in addition to other possible functions. In the present work, a comparative study of the corneal surface in several species
of fish has been carried out with the scanning electron microscope in order to further probe
the functional importance of these surface structures. The corneal microprojections in two elasmobranch species, the dogfish and skate (which have eyelids), are structurally very similar
to one another, and resemble mammalian corneal surface microprojections. The five species of
marine teleost (which do not have eyelids), on the other hand, show epithelial surface patterns
consisting of long, curved surface ridges. Evidence of a material coating these ridges is presented. Individual cellular variations in surface coating material and structural patterns of the
microprojections are reported. The possible roles of the corneal surface microprojections, in
view of these comparative observations, is discussed.
ous fine microprojections.1"11 It has been
suggested that one function of these structures may be to assist in holding the tear
film. The tear film forms an air-liquid interface which constitutes the main refractive
surface of the mammalian eye. The tear
film-holding role of the microprojections
has, however, been brought into question.1-' ™
A comparative study of the topography of
the external corneal surface in different
species would appear to be useful in further analyzing the functional significance
of the microprojections. In fish, because of
the aquatic environment, there is a liquidliquid interface at the corneal surface. It
would seem that under these circumstances
a tear film would either be unnecessary or
would be considerably different in its properties from that in the mammal. Consequently, the microprojections might be
.he external surface of the mammalian
cornea has been shown to consist of numerFrom the Kresge Eye Institute and Department of
Anatomy, Wayne State University, School of Medicine, Detroit, Mich, and the Marine Biological
Laboratory, Woods Hole, Mass.
This study was supported by United States Atomic
Energy Commission Contract AT( 11-1)2401,
National Institutes of Health Grant EY 00362
(Oakland University), and National Institutes
of Health Special Fellowship EY 55,624 from
the National Eye Institute to MB, and EY
53483 to AW.
Submitted for publication Feb. 28, 1974.
Reprint requests: Dr. C. V. Harding, Kresge Eye
Institute, Wayne State University School of
Medicine, 540 E. Canfield Ave., Detroit, Mich.
48201.
°1 dedicate this paper to the memory of Professor
George K. Smelser who long realized the value
of comparative studies in elucidating the mechanisms of the eye.
906
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Number 12
Corneal epithelial cell surfaces
907
Fig. 1. A, surface of dogfish corneal epithelium. The microprojections are approximately 0.2
/t wide and of variable length. xl2,000. B, dogfish corneal epithelial surface at lower magnification. Note cells of various size and texture. x2,000. C, skate corneal epithelial surface.
The pattern of microprojections in the skate is similar to that seen in the dogfish. x2,000.
either absent, or have a pattern different
from that in the mammal. In the present
study, the cornea! surfaces of seven species
of marine fish were examined with the scanning electron microscope. This included
two species of elasmobranch which have
eyelids, and five species of marine teleost,
which do not. The results are discussed in
terms of the possible functional significance
of the corneal surface microprojections.
fj
Materials and methods
Two species of elasmobranch were used (the
smooth dogfish, Mustelus canis; and the little skate,
Raja erinacea); and five species of marine teleost
(scup, Stenotomus chrysops; northern sea robin,
Prionotus carolinm; summer flounder, Paralichthys
dentatus; bluefish, Pomatornus saltatrix; and toadfish, Opsanus tau). The eyes were freshly dissected from healthy animals with no signs of ocular
defects. The anterior portions of the eyes were
fixed for 12 hours in 2 per cent glutaraldehyde in
0.1 M Na cacodylate made up in elasmobranch
Ringer,111 or teleost Ringer,lfi or filtered sea water.
The tissues were postfixed in 1 per cent osmium
tetroxide in 0.1 M Na cacodylate buffer for one
Fig. 2. Scup corneal epithelial surface. A bold pattern of ridges is evident. The ridges are about 0.2
p. in width and many microns in length. x8,Q00.
hour, and washed several times in the 0.1 M Na
cacodylate buffer. The tissues were dehydrated
through either an acetone or alcohol series, and
passed through an acetone-Freon-TF (C«C]3Fs)
series (for the acetone-dehydrated material), or
an alcohol-Freon-TF series (for the alcohol-dehydrated material). The specimens were then dried
in a critical-point drying apparatus (Bomar SPC-
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Investigative Ophthalmology
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908 Harding et al.
Fig. 3. A, scup corneal epithelial surface. x2,000. B, scup corneal endothelial surface. x2,000.
900, utilizing Freon 13, CClF a , as a transitional
fluid) and mounted on specimen holders.10"19 At
this point the specimens were examined briefly
with a Zeiss light microscope equipped with an
epiillumination attachment fitted with a 40x long
working distance objective. This was found to be
very useful in screening specimens (for cell loss,
etc.) prior to examination with the scanning electron microscope. The specimens were coated in a
vacuum evaporator with carbon and with gold,
and examined with a Jeolco JSM-U3 scanning
electron microscope.
Results
Elasmobranchs. Fig. 1, A shows, at high
magnification, a portion of the surface of
a dogfish corneal epithelial cell. A microprojection pattern not unlike that seen in
mammalian corneas is evident. The microprojections are approximately 0.2 p. wide
and of variable length. Fig. 1, B shows a
portion of a dogfish corneal surface at lower
magnification. Cells of various "size" and
"texture," due to some variations in the pattern of microprojections from cell-to-cell,
are evident. Fig. 1, C shows, at relatively
low magnification, a portion of a skate corneal surface. The pattern of microprojections in the skate is similar to that seen
in the dogfish.
Teleosts. Fig. 2 shows, at high magnifica-
tion, a portion of the corneal surface of a
scup. The surface of one whole cell appears
toward the left of the picture. Portions of
several other cells appear in the field. A
bold pattern of ridges is evident. They are
approximately 0.2 JX in width, and many
microns in length. It is clear from Fig. 2
that the pattern seen here is distinctly different from that seen in the dogfish and
skate. Fig. 3} A, at lower magnification,
shows the overall cellular arrangement better. For comparison with the epithelial cells
shown in Fig. 3, A, Fig. 3, B shows the
endothelial surface of a scup cornea. The
scup corneal endothelium has the typical
appearance of corneal endothelium as described in the mammal.20
In some areas of the preparations, a
"coating material" covers some of the cells
completely or partially. This is evident in
Fig. 4, A. One cell (at the upper right portion of the field) is almost completely covered. The other cells are partially covered.
In another field of the same preparation,
shown in Fig. 4, B, the ridges appear to be
partially coated with strands of material
which extend out between the ridges. In
some cases the strands appear to have
broken and retracted to bead-like deposits
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Fig. 4. A, scup corneal epithelial surface. Note coating material covers some of the cells completely or partially. x5,00O. B, scup corneal epithelial surface. Note: ridges appear to be
partially coated with coating material and strands of this material extend out between the
ridges. x6,000.
Fig. 5. A, flounder corneal epithelial surface. Note bold, long ridge pattern. x2,000. B, flounder
corneal epithelial surface. Tlie cell at the bottom has more coating material than surrounding
cells. xlO,000.
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910 Harding et al.
Fig. 6. Toad fish corneal epithelial surface. x3,000.
on the sides of the ridges. Here again, as
in the cells in Fig. 4, A, the amount of
coating material varies from cell to cell.
The cell in the lower right in Fig. 4, B
has relatively few strands; whereas the cell
at the left has many. Others have an intermediate amount. The amount of coating
material appears to change abruptly at cell
borders. Three other species (flounder, toadfish, and sea robin) also show evidence of
coating material in the form of these strands.
Fig. 5, A shows, under low magnification,
a field of corneal epithelial cells from the
flounder. The bold, long-ridge pattern similar to that seen in the scup is evident. This
field is relatively free of coating material,
however, other areas containing varying
amounts of coating material were observed
(see Fig. 5, B), The cell at the lower right
in Fig. 5, B has more coating material than
the surrounding cells. The grooves between
the ridges are filled, but the ridges still
stand out. At the periphery of this cell
(near the center of the photograph), the
grooves are not completely filled, but many
strands, bridging the gaps between the
ridges, are evident.
Fig. 6 shows a corneal epithelial preparation from the toadfish. Once again, the
long-ridge pattern is evident. Although this
pattern is similar to that seen in the scup
and flounder, there appears to be a species
specificity to the pattern. Other cells
showed evidence of coating material
(strands).
The sea robin also exhibits the longridge structure similar to that seen in the
other marine teleosts, although it does apparently have its own species specificity
(Fig. 7, A). Fig. 7, B shows the sea robin
ridge pattern at higher magnification. Other
cells showed varying amounts of coating
material. In some cases the grooves were
filled, in others, strands were evident.
In the bluefish, the corneal epithelium
seemed to be almost completely coated.
However, areas were found in which cell
outlines could be distinctly seen. In a number of cases, although the cells were heavily
coated, a ridge pattern could be discerned.
Discussion
A number of investigators7'iM1 have described the external surface of the mammalian cornea as seen with the scanning
election microscope. Microvilli and ridgelike structures (microplicae) have been
described on the epithelial cell surfaces.
The earlier work of Jakusr' and others2'fi|!)
utilizing the transmission electron microscope have also shown evidence of microvilli on the corneal epithelial surface. In
mammals, these projections may play a role
in holding the tear film. However, Ehlers1has stated, relative to the mammalian precorneal tear film: ". . . that an aqueous film
of the thickness found could purely hydrodynamically be stable or almost stable in
the vertical position. Thus it is not necessary to assume that a supporting structure,
e.g., a gel structure or a mechanical support, should be present." Maurice-1 has also
questioned the role of the microprojections
in maintaining the tear film. The possible
assistance of the microprojections in holding the mammalian tear film, therefore,
would apppear debatable.
Evidence has been presented recently11
that the ridges (microplicae) on the surface of some mammalian corneas may be,
in fact, microvilli which are lying flat on
the cell surface. The microprojections seen
on the elasmobranch corneas might also be
interpreted as microvilli. However, the
ridges seen in the five species of marine
teleost are difficult to interpret in this way.
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Corneal epithelial cell surfaces 911
Fig. 7. A, sea-robin corneal epithelial surface. x4,000. B, sea robin corneal epithelial surface.
xlO,000.
Their great length and pattern of airangement make it seem unlikely. A transmission
electron microscope (TEM) study of the
corneas in two species of marine teleost indicate that the ridge pattern is formed by
protrusions of the cell surface. The TEM
study also revealed the presence of coating
material. Preliminary investigations of a
fresh water teleost (the dark moor goldfish) show that the corneal projections are
not similar to those in the marine teleosts,
but resemble more the dogfish and skate
corneas.2a Other functions of the projections
have been suggested.7
If the arrangements of the microprojections should differ among closely related
species as they do for the species reported
here, the scanning electron microscopic
study of fish corneas may be helpful in
making specific species identifications.
The fact that two fish (the dogfish and
skate) have structures very similar to those
seen in the mammal is perhaps of some
theoretical interest.211 It might be expected
that in an aquatic environment, a tear film
with the properties of mammalian tears
would be rapidly washed away and would,
therefore, be ineffective, for example, in
providing a stable optical surface or protecting the corneal surface. However, relatively viscous secretions on the corneas of
dogfish and skate might provide a stable
surface which could be maintained as a
smooth surface through the action of the
eyelids. In this case, the surface structures
on the elasmobranch cornea may play a
role similar to that proposed for the mammal (assisting in holding a precorneal film),
even though there is no tear film as seen
in the mammal.
The evidence for a surface coating material is more striking in the marine teleosts
than in the elasmobranchs or mammals.
The marine teleosts do not have eyelids to
help protect the surface of the cornea. The
coating material would have to be relatively stable (e.g., highly viscous or semisolid) in order to be retained. This might
account for the fact that the coating material shows up more distinctly in the marine teleosts than in the elasmobranchs.
Little is known about the chemical nature of the coating material, its function, or
the site of secretion. It presumably serves
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912 Harding et al.
as a protective coating for the cornea (particularly in the teleosts), and may play an
optical role by providing a smooth refractive surface. The variation in amount of
coating material from cell-to-cell, with distinct lines of demarcation at cell borders
suggests that cells at different stages of differentiation or senescence are present.
There are several possible interpretations.
For example, a newly surfacing epithelial
cell may require time to accumulate coating
material; or cells of different ages may have
different affinities for the coating material.
This latter possibility could result in a differential removal during the preparation
procedures of the coating material from
some cells and not others. In any case, a
variation in surface properties, perhaps as
a function of cellular age, is suggested by
the reported observations.
In addition to the possible role of holding a coating material on the surface of
the cornea, the microprojections also greatly increase the surface area, which should
assist in the processes of diffusion and active transport.
Finally, the possible optical role of the
ridges themselves has not been discussed.
The possibility that their refractive or reflective characteristics may play a special
role in vision in the marine teleosts should
be considered.24
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