Scanning and transmission electron
microscopic observations of the
topographic anatomy of dendritic
lesions in the rabbit cornea
William H. Spencer and Thomas L. Hayes
Scanning electron microscopic observations of experimentally induced dendritic lesions in
rabbit corneas ivere correlated with those obtained with light and transmission electron microscopy of the same tissues. A sharply demarcated central zone of ulceration (zone A) toas
noted which contained many degenerating epithelial cells, possibly derived from the bordering cells and from the basal layers. These had a rounded configuration and averaged lOyin diameter. Transmission electron microscopy of these cells showed particles resembling
herpes simplex virus between cells and in their nuclei and cytoplasm. An adjacent zone of
epithelium (zone B) presented a depressed appearance with scalloping of individual cells. The
configuration of the outer margins of the lesions resembled that of the border behoeen zones
A and B. The linear branching pattern of the figure may be the restdt of spread of the virus
from cell to cell, modified by guiding factors such as temporary local cellular resistance to
infection or neuronal pathiuays.
Key words: corneal dendritic ulcer, herpes simplex virus, pathogenesis, corneal epithelium,
corneal stroma, histopathology, ultrastructure, light microscopy, scanning electron
microscope, rabbits.
similar in size and configuration to those
which occur spontaneously in human corneas infected with this virus. Considerable
morphologic variation has been observed,
but most figures are linear with numerous
side branches. The pathogenesis of the
characteristic branching pattern of these
figures has not been satisfactorily demonstrated anatomically, and remains unexplained. We wish to report our observations of the topographic anatomy of experimentally induced dendritic lesions in
the rabbit corneal epithelium with scanning, light, and transmission electron
microscopy.
hen a rabbit's corneal epithelium is
infected with herpes simplex virus, a series
of dendritic figures developes which appear
From the Department of Ophthalmology, and
the Francis I. Proctor Foundation for Research
in Ophthalmology, University of California, San
Francisco Medical Center, San Francisco, Calif.,
and the Donner Laboratory, Lawrence Radiation Laboratory, University of California, Berkeley, Calif.
This work was supported in part by USPHEY
00052 and by the United States Atomic Energy
Commission.
Manuscript submitted July 28, 1969; manuscript
accepted, Aug. 13, 1969.
183
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184 Spencer and Hayes
Fig. 1. For legend see opposite page.
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In oestigative Ophthalmology
March 1970
Volume 9
Number 3
Dendritic lesions in the rabbit cornea 185
Fig. 1. A, Survey view of 48 hour herpetic lesion. A broad irregular sharply demarcated central zone of ulceration (A) is observed. At its base the stroma is seen to be partially covered
with tiny rounded bodies (arrow). A zone of depressed epithelium (B) surrounds zone A.
(Scanning electron microscopy; xlOO.) B} The array of rounded bodies at the base of zone
A is one to two layers deep. They lie along the surface of the superficial stroma and are of
uniform size (average 10/i) and configuration. (Scanning electron microscopy; x300.) C, The
rounded bodies have an irregular wrinkled surface and appear to cohere. In some areas they
seem to adhere to the surface of the stroma. (Scanning electron microscopy; x 1,000.) D,
Higher power of bodies in area outlined by square in C. The wrinkled surface of each
rounded body interdigitates with that of its neighbors. Artifactitious shrinkage appears to
have partially disrupted zones of attachment (arrows). (Scanning electron microscopy;
xlO,000.)
Fig. 2. A, View of 48 hour herpetic lesion. Zone A varies in width. Its base is almost completely covered with rounded bodies. Several side branches have developed. The border of
zone A is sharply demarcated by the overhanging edge of zone B. The outer margins of the
lesion (C) can be faintly seen as an irregular discontinuous line which roughly parallels the
line demarcating the borders of zone A, but it is somewhat less angular in configuration. The
cut surface of the uninvolved corneal stroma (S) is apparent in the lower portion of the photograph. {Scanning electron microscopy; xlOO.) B, Some of the rounded bodies adhere to and
appear to arise from the overhanging edge of zone B (arrow). A bridge of partially degenerated epithelium is seen near the top of the figure. The surface epithelium in zone B has
an irregular scalloped appearance. (Scanning electron microscopy; x300.)
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Investigative Ophthalmology
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186 Spencer and Hayes
Fig. 3. A small, presumably early, lesion exhibits a "Y" configuration with beadlike depressions
at the extremities of the incipient narrow zone A. Zone B is quite prominent and appears
depressed. The outer limits of the figure (C) resemble the configuration of the central zone
to some degree. The edge of another lesion is visible at the upper left of the photograph.
(Scanning electron microscopy; xlOO.)
Materials and methods
New Zealand white rabbits were infected with
the PH strain of herpes simplex virus by gently
abrading the corneal epithelial surface with a
rounded, sterile, platinum spatula in a crosshatched pattern (10 horizontal and 10 vertical
strokes). A suspension of virus particles was then
dropped on the corneal surface, the lids held
closed momentarily, and the rabbit returned to
the cage. Within 24 hours, punctate epithelial
lesions developed. Typical branching dendritic
figures were visible with the slit lamp by 36
hours. The animals were killed at 48 hours, at
which time each cornea exhibited multiple (7 to
12) dendritic lesions. The corneas were removed
by trephination immediately after death and fixed
in fresh cold 3 per cent glutaraldehyde solution.
Portions of each cornea were postfixed in osmium
and stained en bloc with uranyl acetate and lead
citrate"1 for electron microscopic sections.* Aral°The Siemens Elmiskop 1A Electron Microscope was
utilized for examination of these tissues.
dite-embedded sections cut at l(i and stained with
Richardson's stain2 were utilized for light microscopic examination. The remainder of each cornea
was air dried and coated with a thin conducting
layer of platinum-palladium alloy, deposited at
normal incidence in a vacuum evaporator. The
tissues were then placed on the stage of the
scanning electron microscope*1 with the slide at
a 45 degree angle with the scanning electron
beam. The instrument was operated in the secondary electron mode at an accelerating potential of
25 kV. and specimen current of 3xlO- n amps.
Photographs of the display cathode ray tube were
made with Type 42 Polaroid roll film.
Results
Four corneas containing 14 dendritic
figures were studied with the scanning
*The microscope used in this study was a modified Japan
Electron Optics Laboratory Co. Scanning Electron Microscope, Model JSM-1.
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Dendritic lesions in the rabbit cornea 187
Fig. 4. A typical linear herpetic lesion is shown. Zones A and B are of variable width and
branch at the right side of the photograph. The configuration of the outer edge of the lesion
is less angular than that of zone A. (Scanning election microscopy; xlOO.)
electron microscope. Although minor differences in size and pattern were noted,
the basic architecture was similar in all.
Each figure was composed of an irregular
linear central zone of ulceration of varying
width (A) with shorter side branches. In
some of the figures the ulceration extended
through the entire thickness of the epithelium, partially exposing the underlying
stroma (Fig. 1, A), but in most, almost the
entire stromal surface was covered by a
great number of spherical bodies, each
presenting a wrinkled surface and averaging about IOIJL in diameter (Fig. 1, B, C,
and D). The size and configuration of
these bodies varied only slightly from one
area to another. They were present in
every dendritic figure studied and appeared to be arising from the epithelium
bordering zone A (Fig. 2, B). The margins
of zone A were sharply demarcated and
often presented an overhanging edge composed of several layers of epithelial cells.
The surface layer of epithelium over a relatively broad bordering zone (B) appeared
depressed, as if its underlying support
had been partially lost (Figs. 2, A, 3, and
4). Individual surface cells within this zone
presented a scalloped or partially collapsed
appearance (Fig. 5). Surrounding the entire figure, at the junction between zone
B and the normal-appearing adjacent epithelium, a discontinuous line (C) could be
seen (Figs. 2, A, 3, and 4). In many of the
dendrites the configuration of line C resembled that of the sharp border between
zones A and B.
One-micron sections obtained through
the base of zone A, and studied with the
light microscope, showed several degenerating epithelial cells averaging about 15jU
in diameter lying along the surface of the
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188 Spencer and Hayes
Investigative Ophthalmology
March 1970
Fig. 5. Individual surface cells in zone B (arrows) appear scalloped and depressed. The
overhanging edge of zone B is sharply demarcated. (Scanning electron microscopy; x300.)
superficial stroma (Fig. 6, A and B). On
examination of ultrathin sections with the
transmission electron microscope, these
cells were observed to vary somewhat in
configuration, but many had a rounded
profile (Figs. 7 and 8). Structures which
were morphologically compatible with
virus particles were observed in the nucleus and cytoplasm of these degenerating
cells. Similar structures were noted in the
cells bordering zone A and in the intercellular space between adjacent bordering
cells (Fig. 9).
Discussion
The spherical bodies which occupy the
base of the area of ulceration appear to
be exposed, degenerating, epithelial cells
in part derived from the basal layers (Fig.
7). Centrally, they appear to represent the
residua of the initial phases of infection
with newly formed spherical bodies (de-
generating cells) developing from the
overhanging edge of the epithelium, bordering the zone of ulceration (Fig. 2, B).
Their wrinkled surface may represent distortion induced by air drying3; however, it
is likely that the wrinkling has been modified in some way by the viral infection,
since the adjacent surface cells appear
smooth. It is also possible that the wrinkles
represent the scanning election microscopic
appearance of the cytoplasmic projections
noted in the well-fixed transmission electron microscopic sections (Figs. 7 and 8).
It is our impresison that zone A is the
zone which is seen with the slit lamp as
an area of staining when fluorescein is
applied to these lesions. Zone B may possibly represent the zone of epitheliolysis*
which is readily removed with debridement clinically, and which has been shown
to stain differentially with vital stains.5 The
presence of structures morphologically
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Dendritic lesions in the rabbit cornea 189
50
Fig. 6. A, Light microscope section (1/f thick) through a branched herpetic lesion. Two areas
of epithelial ulceration are seen (arrows 1 and 2) which are believed to correspond to zone
A in the scanning electron microscope photographs. At the base of area 1, rounded epithelial
cells are observed lying along the stromal surface. The adjacent epithelium is irregularly distorted and is presumed to correspond to zone B of the scanning electron microscope photographs. (Richardson's stain; x320.) B, Higher power of area 1 noted in A. The rounded
epithelial cells at the base of the ulcer adhere to each other and to the surface of the stroma.
These cells are believed to correspond to the rounded bodies observed in this area with the
scanning electron microscope. (Richardson's stain; xl,280.)
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10)1
ML J O B
7. Transmission electron microscope thin section through the base of an area of epithelial ulceration (zone A). The distorted degenerating basai cells
re to the surface of the underlying stroma and to adjacent cells. The surface of each cell exhibits many cytoplasmic projections. (Uranyl acetate and
citrate; x3,000.)
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H1
8. Transmission electron microscope thin section showing a typical rounded degenerating cell in zone A. Particles resembling virus are observed in the
orted nucleus (arrow 1). Coated particles are also observed in the extranuclear portions of neighboring cells (arrow 2). Cytoplasmic projections cut in a
ety of planes are observed along the surface of the cells. (Uranyl acetate and lead citrate; xl.2,000.)
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192 Spencer and Hayes
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March 1970
Fig. 9. Transmission electron microscope thin section at the junction of several cells in zone
B. Particles resembling virus (arrows) are observed in the intercellular space and nuclei in
this field. (Uranyl acetate and lead citrate; x32,000.)
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similar to viral particles of herpes simplex
within cells in zone B as seen with the
transmission electron microscope suggests
that the area of infection extends beyond
the area of fluorescein staining. Just how
far it extends cannot be stated with certainty. The depressed appearance of the
surface cells in zone B as seen with the
scanning electron microscope may be interpreted as an indication of loss of cellular support due to partial collapse of the
underlying layers. This may have been
produced either by the destructive action
of the virus on the underlying cells, or
possibly by the well-documented tendency
of the deeper epithelial cells to slide
toward the center of a defect as a part of
the healing process.6'7
If we assume that line C represents the
advancing edge of the figure, it is of some
interest to speculate about the manner in
which the figure develops. From our observations of several dendritic figures, we
have concluded that the configuration of
Dendritic lesions in the rabbit cornea 193
line C tends to duplicate that of the borders of zone A. In our transmission electron
microscopic sections we have observed
particles resembling herpes virus within
epithelial cells and in the extracellular
space between adjacent cells. A similar
distribution has been noted by Tanaka and
Kimura8 and by Dawson and associates.9
Presumably, therefore, at least one route
of spread of the virus is from epithelial
cell to epithelial cell. A careful examination of line C at a point of branching suggests the possibility that the infection
spreads centrifugally from cell to cell in a
manner similar to that diagrammed in
Fig. 10, extending in a pattern which is
reminiscent of the surface ripples on water
which spread outward from the point of
impact of a pebble. Presumably the factors
which predetermine the general outline of
the figure are established early in its development, with subsequent minor variations in shape as it enlarges. It is possible
that some modifying guiding factor, such
Fig. 10. Diagrammatic representation of a possible mode of extension of the viral infection
which assumes that the initial outline of the figure is established by a modifying, guiding
factor (such as the distribution of nerve fibers in the superficial cell layers) with subsequent
centrifugal spread from cell to cell. The outline of the lesion becomes progressively less angular as it enlarges. Factors not taken into account in the model, such as multiple cell layers and
variation in cell survival time, would modify this representation.
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194 Spencer and Hayes
Fig. 11. Diagrammatic representation of a possible mode of extension of the viral infection
which assumes that the infection spreads from cell to cell and branches at points where one
cell or cell group temporarily resists infection. (Shaded area represents infected cells. Arrows
indicate direction of spread.)
as the distribution of nerve fibers in the
superficial cell layers, serves as a primary
pathway for spread of the virus and is
responsible for the linear configuration and
the initial tendency to branching.10 One
may also postulate that no neuronal directionalizing factor exists and that following
the initial infection of a single cell or
group of cells the infection spreads from
cell to cell, branching at points where one
cell or cell group temporarily resists infection (Fig. 11). This explanation, however, would not account for the tendency
of many herpetic lesions to be linear in
configuration.
The techniques used in preparing the
specimens probably introduce several
structural artifacts. Air drying from an
aqueous solution, in particular, alters the
structure of individual cells, but it is unlikely that the over-all topography of the
dendritic figuers is significantly affected
by this procedure. Artifacts are not always
to be avoided. Many artifacts, such as the
staining processes of the light microscope,
have proved to be most valuable in study-
ing biological systems. It is often more
useful to obtain an image of a system containing artifacts (e.g., stain) than an image
without artifacts that more nearly represents the cell or tissue in the living state.
The surface tension experienced during
air drying causes the surfaces to be pressed
down upon the underlying cellular components. These components are now visible
in the scanning electron microscope as
variations in height of the collapsed surface. The production of such an artifact
can be useful in partial identification of
subsurface detail while still using the
scanning electron microscope in the secondary electron surface mode. A similar
method of collapsing blood cells in order
to identify interior content has been suggested.11 With respect to the general question of artifact, our aim is to try to improve
our total understanding of the biological
system rather than to limit ourselves only
to the preservation of the in vivo morphologyThe authors wish to express their appreciation
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Dendritic lesions in the rabbit cornea 195
to Mr. Masao Okumoto, Miss Betty Cassiman, and
Mrs. Irmgaard Wood for their technical assistance.
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