Scanning and transmission electron microscopic observations of the topographic anatomy of dendritic lesions in the rabbit cornea

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1 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. 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 and by the United States Atomic Energy Commission. Manuscript submitted July 28, 1969; manuscript accepted, Aug. 13, 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.

2 184 Spencer and Hayes In oestigative Ophthalmology March 1970 Fig. 1. For legend see opposite page.

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.

) 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.

3 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.)

186 Spencer and Hayes Investigative Ophthalmology March 1970 Fig. 3.

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.

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

4 186 Spencer and Hayes Investigative Ophthalmology March 1970 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 The Siemens Elmiskop 1A Electron Microscope was utilized for examination of these tissues. 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.* Araldite-embedded sections cut at l(i and stained with Richardson's stain 2 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.

Volume 9 Number 3 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.

Although minor differences in size and pattern were noted, the basic architecture was similar in all.

In some of the figures the ulceration extended through the entire thickness of the epithelium, partially exposing the underlying stroma (Fig.

5 Volume 9 Number 3 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

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.

Similar structures were noted in the cells bordering zone A and in the intercellular space between adjacent bordering cells (Fig. 9).

6 188 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 (degenerating 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 drying 3 ; 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

Volume 9 Number 3 Dendritic lesions in the rabbit cornea 189 50 Fig. 6. A, Light microscope section (1/f thick) through a branched herpetic lesion.

7 Volume 9 Number 3 Dendritic lesions in the rabbit cornea 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.)

8 10)1 JOB. Transmission electron microscope thin section through the base of an area of epithelial ulceration (zone A). The distorted degenerating basai cells e to the surface of the underlying stroma and to adjacent cells. The surface of each cell exhibits many cytoplasmic projections. (Uranyl acetate and itrate; x3,000.)

9 H1 8. Transmission electron microscope thin section showing a typical rounded degenerating cell in zone A. Particles resembling virus are observed in the rted nucleus (arrow 1). Coated particles are also observed in the extranuclear portions of neighboring cells (arrow 2). Cytoplasmic projections cut in a ty of planes are observed along the surface of the cells. (Uranyl acetate and lead citrate; xl.2,000.)

192 Spencer and Hayes Investigative Ophthalmology

Particles resembling virus (arrows) are observed

10 192 Spencer and Hayes Investigative Ophthalmology 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.)

Volume 9 Number 3 Dendritic lesions in the rabbit cornea 193 similar to viral particles of herpes simplex within cells in zone B as seen with the transmission electron microscope suggests that the

11 Volume 9 Number 3 Dendritic lesions in the rabbit cornea 193 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 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 Kimura 8 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.

194 Spencer and Hayes Investigative Ophthalmology March 1970 Fig. 11.

temporarily resists infection. (Shaded area represents infected cells. Arrows indicate direction of spread.

12 194 Spencer and Hayes Investigative Ophthalmology March 1970 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 studying 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 morphol- ogy- The authors wish to express their appreciation

Volume 9 Number 3 Dendritic lesions in the rabbit cornea 195 to Mr. Masao Okumoto, Miss Betty Cassiman, and Mrs. Irmgaard Wood for their technical assistance. REFERENCES 1. Karnovsky, M. J.

13 Volume 9 Number 3 Dendritic lesions in the rabbit cornea 195 to Mr. Masao Okumoto, Miss Betty Cassiman, and Mrs. Irmgaard Wood for their technical assistance. REFERENCES 1. Karnovsky, M. J.: The ultrastructural basis of capillary permeability studied with peroxidase as a tracer, J. Cell. Biol. 35: 213, Reynolds, L. W.: The use of lead citrate at high Ph as an electron opaque stain in electron microscopy, J. Cell. Biol. 17: 208, Barber, V. C, and Boyde, A.: Scanning electron microscopic studies of cilia, Z. Zellforschung 84: 269, Duke-Elder, S.: System of ophthalmology, in Diseases of the outer eye. Part I. Conjunctiva, London, 1965, Henry Kimpton, vol VonReuss, A.: Kiinstliche Doppelfarbung oberflachlicher hornhautdefekte, Arch f. Ophthal. Leip. 78: , Buschke, W., Friedenwald, J. S., and Moses, S. G.: Effect of ultraviolet irradiation of corneal epithelium: Mitosis, nuclear fragmentation, post-traumatic cell movements, loss of tissue cohesion, J. Cell. Comp. Physiol. 26: , Sigelman, S., and Friedenwald, J. S.: Mitotic and wound-healing activities of corneal epithelium. Effect of sensory denervation, Arch. Ophthal. 52: 46, Tanaka, N., and Kimura, S. J.: Localization of herpes simplex antigen and virus in the corneal stroma of experimental herpetic keratitis, Arch. Ophthal. 78: 68, Dawson, C. R., Togni, B., and Moore, T. E. Jr.: Structural changes in chronic herpetic keratitis studied by light and electron microscopy, Arch. Ophthal. 79: 740, Dawson, C. R., Togni, B., and Thygeson, P.: Herpes simplex virus particles in the nerves of rabbit corneas after epithelial inoculation, Nature 211: 316, McDonald, L. W., and Hayes, T. L.: Correlation of scanning electron microscope and light microcope images of individual cells in human blood clots, Exp. Molec. Path. 10: 186, 1969.

Scanning electron microscopy of corneal wound healing in the rabbit. Barrett G. Haik and Marilyn L. Zimny

Scanning electron microscopy of corneal wound healing in the rabbit Barrett G. Haik and Marilyn L. Zimny Corneal lesions 7.5 mm. in diameter were made with an ocidar trephine in rabbits. The time periods