Experimental Type 2 Herpes Simplex Ophthalmitis in the Newborn Rat

Experimental type 2 herpes simplex ophthalmitis in the newborn rat

D. H. Percy, K. A. Galil, L. A. Hatch, L. B. Pancer, andj. P. Crawford

An animal model for the study of type 2 herpes simplex virus (HSV2) ophthalmitis is described. Wistar rats were inoculated intracerebrally with HSV2 at 0, 5, 10, 15, 20, or 30 days of age. Eyes and brain from all animals, whether they survived, or succumbed, with encephalitis, were collected for microscopic and virologic studies. Up to 20% or more of the HSV2- inoculated rats had lesions in the cornea, it veal tract, and lor retina. Herpetic heratitis occurred in a few animals while the eyelids were still fused, indicative of internal spread of HSV2. Intranuclear inclusions were observed, in corneal epithelium and neural retina, and herpesvirus particles were demonstrated in the cornea, iris, and retina. Lesions of the cornea and. iris were also visualized by scanning electron microscopy. Virus was isolated, from over 40% of the eyes tested. In general, titers of the virus in the eyes were less than those in the brains of HSV2- inoculated. rats. The newborn rat thus represents another animal model to study herpetic ophthalmitis. Unlike most studies, ocular lesions were produced by a route other than the usual topical or intraocular inoculation of the virus.

Key words: type 2 herpes simplex ophthalmitis, newborn rat, keratitis, uveitis, destructive retinitis

Heipes simplex virus (HSV) is the most infections with HSV2 classically affect the common cause of corneal opacities in devel- genital tract, and the virus has been impli- oped countries of the world.1 Although cor- cated as a factor in cervical cancer.2 Neonatal neal lesions are the most frequent manifesta- HSV2 infections also occur, either as a trans- tion of HSV infections of the eye, other areas placental infection or by exposure during may also be involved.1 The majority of cases passage through the infected birth canal.2' ;t of herpetic ophthalmitis are associated with Frequently, cases of neonatal HSV2 infec- type 1 HSV (HSV,). However, recently at- tions have concurrent ocular lesions. In one tention has been focused on the role of type 2 study, 51 of 297 cases of neonatal HSV2 in- HSV (HSV2) in diseases of the eye. Clinical fection had lesions of the eye such as keratitis, conjunctivitis, and chorioretinitis.4 Reti- nitis and chorioretinitis have been described From the Departments of Microbiology and Immu- in other reported cases of neonatal herpetic nology (D. H. P., L. B. P., J. P. C.) and Anatomy infections.5"7 Although some cases of con- (K. A. G.), University of Western Ontario, and the genital herpetic keratitis have been attrib- Department of Microbiology (L. A. H.), St. Joseph's 4 8 Hospital, London, Out., Canada. uted to HSV,, - all cases of neonatal HSV Supported by National Institutes of Health grant chorioretinitis from which viral isolates have 4 EY01833-01. been typed have been HSV2. Most patients Submitted for publication May 14, 1979. with ocular lesions due to HSV have had a Reprint requests: Dr. D. H. Percy, Department of Mi- concurrent encephalitis.4"7 crobiology and Immunology, Health Sciences Centre, University of Western Ontario, London, Ont., In addition to neonatal HSV2 infections Canada, N6A 5C1. of the eye, there have also been recorded

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Table I. Summary of histopathology and viral isolation studies

Lesions" Virus isolation6 Age at inoculation Survivors4 Cornea Uveal tract Retina Eye Brain

0 days 11/45 (24%) 3/24 4/27 3/27 6/11 11/14 (12.5%) (15.0%) (11.0%) (54.5%) (78.5%) 5 days 16/93 (17%) 11/72 26/74 20/73 6/10 30/35 (15.0%) (35.0%) (27.0%) (60.0%) (85.5%) 10 days 33/99 (34%) 10/86 15/86 12/86 19/39 4/12 (11.5%) (17.5%) (14.0%) (49.0%) (33.0%) 15 days 22/55 (40%) 0/20 4/31 10/32 4/18 7/7 (12.0%) (31.0%) (22.0%) (100%) 20 days 32/32 (100%) 1/22 4/23 4/23 — 1/11 (4.5%) (17.5%) (17.5%) (9.0%) 30 days 36/36 (100%) 2/36 8/36 3/36 0/3 1/4 (5.5%) (22.0%) (8.5%) (25%) Grand totals 151/360 (42%) 26/260 61/277 52/277 35/81° 54/83": (10%) (22%) (19%) (43%) (65%)

ANo. of inoculated rats that survived/No, of rats inoculated. "No. of animals with lesions/No, of animals examined histologically. cNo. of animals with positive isolates of HSV-2/No. of animals tested. "Positive HSVj isolations from eye ranged from 4 to 20 days pi. ^'Positive HSV2 isolations from brain ranged from 4 to 12 days pi.

cases of keratoconjunctivitis due to HSV2 in Canada) were acquired in late gestation and housed adults.9"11 In one study of herpetic infections in conventional shoebox type cages throughout the of the eye, 13% of the isolates were HSV2. study. After parturition, suckling rats were inoculated with either the MS strain of HSV (originally Several of the patients with HSV2 infection 2 had unusually severe disease, with lesions obtained from Dr. A. J. Nahmias, Emory Univer- such as keratoconjunctivitis and stromal in- sity, Atlanta, Ga.) or the 075/72 strain isolated from a patient with herpetic vulvovaginitis.18 The volvement.10 Others have reported similar titer of stock virus was 1000 tissue culture infec- findings." Experimental HSV infections have tious doses (TCID ) per milliliter. Rats were in- revealed a similar pattern. Oh and his co- 50 12 14 oculated intracranially at 0, 5, 10, 15, 20, or 30 workers " compared the pathogenicity of days of age with stock virus or in dilutions of 1:10, HSVi and HSV2 in primary cultures of rabbit 1:100, or 1:1000 in Medium 199 (Grand Island 12 13- H cornea and in intact rabbits. In general, Biological Co., Burlington, Out., Canada). Titers lesions were more severe with HSV2. In view of virus varied from 0.0125 to 50 TCID5O per in- of the frequent occurrence of ocular lesions in oculum, depending on the age of rats at the time of 4 neonatal HSV2 infection, the apparent in- inoculation. Control littermates in each category crease in cases of genital herpes,15 and the received equal volumes of diluent. The intersect- comparative severity of ocular infections due ing point between the medial canthi and the mid- to HSV ,10- n it is an infection of considerable line of the calvarium were used as external land- 2 marks for inoculation procedures.17 A total of 420 public health importance. In this communi- newborn rats were used in this study, including cation we report on studies of the eye in 278 inoculated with the 075/72 strain, 82 rats in- suckling rats inoculated intracerebrally with oculated with the MS strain, and 60 control ani- HSV2. Aspects investigated included age, mals. susceptibility, incidence and distribution of Necropsy and tissue collection. Necropsies ocular lesions, viral isolation, and ultrastruc- were performed on all animals. Rats clinically af- tural findings. fected died or were killed in extremis by chloroform inhalation. Animals inoculated at 0 to 20 days of age that survived were killed at 30 days and rats Materials and methods inoculated at 30 days were killed at 10, 15, or 20 Virus and inoculation procedures. Pregnant days post inoculation (pi) by chloroform inhalation. Wistar rats (Woodlyn Laboratories, Guelph, Out., At necropsy, tissues from infected and control

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Table II. Comparison of HSV2 isolations from brain and eye of same animals Comparison of titersA of vims isolated Age at No. positive/ inoculation Tissue No. tested <60 60 600 6000 60,000

0 days" Eye 4/8 3 1 Brain 7/8 2 4 1 5 days (J Eye 4/5 1 2 Brain 5/5 1 1 2 1 10 days" Eye 6/8 4 1 1 Brain 5/8 2 3 15 daysK Eye 3/7 1 1 Brain 111 4 3 Total positive Eye 17/28 Brain 24/28

AInfectious viral particles/gram of tissue.

"Eye: positive HSV2 isolations ranged from 4 to 8 days pi; brain: positive HSV2 isolations ranged from 4 to 12 days pi. (Tissues evaluated ranged from 4 to 12 days pi.) c Eye and brain: positive HSV2 isolations ranged from 4 to 8 days pi. (Tissues evaluated ranged from 4 to 8 days pi). D Eye: positive HSV2 isolations ranged from 4 to 20 days pi; brain: positive HSV2 isolations ranged from 4 to 6 days pi. (Tissues evaluated ranged from 4 to 20 clays pi). K Eye and brain: positive HSV2 isolations ranged from 4 to 6 days pi. (Tissues evaluated ranged from 4 to 15 days pi).

animals were collected as follows. Portions of brain Results and eyes for virology were removed aseptically Clinical observations. At approximately 2 and stored in screw-cap vials at —70° C. Virus to 8 days pi, many HSV2-inoculated animals culture, deration, and typing were performed as showed neurologic signs, frequently with previously described. ui~18 fatal termination. Rats inoculated with HSV Light and electron microscopy. Eyes and se- 2 lected optic nerves were collected for light and at 0, 5, or 10 days of age were particularly electron microscopy. Portions of brain were fixed susceptible to clinical disease, whereas ani- and prepared for electron microscopy as previ- mals inoculated at an older age frequently ously described.17 Tissues were removed imme- survived with little clinical evidence of dis- diately after death and immersed in 3% glutaral- ease (Table I). In a small percentage of inocu- dehyde in sodium cacodylate buffer containing lated animals, typical herpetic vesicles were 0.5% calcium chloride. Following fixation, eyes observed, particularly around the head and were halved along the meridional plane and exam- back regions. These were usually associated ined under a dissecting microscope for evidence of with detectable neurologic signs except in a focal hemorrhage or other abnormalities. Fixed, few rats which survived following inoculation minced tissues were washed in cacodylate buff- at 30 days of age. er, post-fixed in osmium tetroxide, dehydrated through ascending concentrations of ethanol, trans- Virus isolation and titration studies. Of the ferred to propylene oxide, and embedded in Epon viral isolation studies from eyes and brain tis- 812 or Spurr's medium. Thick sections were cut sue of HSV-inoculated animals, 43% of the with glass knives, and thin sections for transmis- eyes and 65% of brain specimens tested were sion electron microscopy (TEM) were cut with a positive for virus (Table I). The earliest that diamond knife and examined in a Zeiss EM 9S HSV2 was isolated from eyes was at 4 days pi electron microscope. In selected cases, portions of in rats inoculated at birth or at 5, 10, or 15 retina, iris, and cornea were processed for scan- days of age. In viral typing studies, all isolates ning electron microscopy (SEM). The critical were identified as HSV . Viral titers in the point drying technique19 was employed. Speci- 2 eye ranged from <60 to 6000 infectious viral mens were examined in a Hitachi scanning electron microscope (Model HHS 2R). Portions of particles per gram of tissue and in brain tis- brain from all animals and selected eyes were fixed sue from 60 to 60,000 infectious viral parti- by immersion in Bouin's fluid, embedded in cles per gram. Ten eyes from control rats paraffin, sectioned, and stained with hematoxylin were negative for virus. A comparison of viral and eosin. titers in the eye and brain was made in a few

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\\~ 4 Fig. 1. Section of Epon-embedded cornea from rat Fig. 2. Cornea from same animal examined by D31-3 inoculated intracerebrally with HSV2 at 5 SEM. Early lesions with characteristic rounding days of age and killed in extremis at 5 days pi. up of cells on the corneal surface. Lesions of this Note the ballooning degeneration of the corneal type later progressed to crateri form ulcers that epithelial cells, the presence of intranuclear in- were readily seen by SEM. (x 14,400.) clusions (arrow), and the sparing of the underlying corneal stroma. The eyelids of" the suckling rat are quent in rats inoculated at 0, 5, or 10 days of still fused at this age, excluding the possibility of age (Table I). Heipetic keratitis was observed topical exposure. (Toluidine blue; X640.) in a few young animals inoculated intracerebrally while eyelids were still fused, indicat- selected animals that had succumbed to the ing that the invasion of the cornea by the infection. In general, viral titers in the eye virus was via internal spread rather than by were lower than the titers of virus from the topical inoculation. Lesions frequently were brain tissue of the same HSV2-inoculated rats confined to the corneal epithelium. Balloon- (Table II). ing degeneration of epithelial cells, the pres- Histopathology and ultrastructural stud- ence of intranuclear inclusion bodies, and the ies. The earliest that lesions were detected separation of superficial epithelial cells from histologically was at 3 to 4 days pi in animals adjacent cells were common features (Fig. 1). inoculated at birth or at 5, 10, or 15 days of On SEM, corneal lesions varied from distinct age. On light microscopy, ocular lesions were rounding of individual affected epithelial observed in the cornea, uveal tract, and ret- cells in HSV2 infections interpreted to be of ina (Table I). In general, lesions were de- recent onset, to crateriform ulcers with exca- structive in nature, characterized by acute vation to Bowman's membrane in lesions in- necrosis, sometimes with features such as terpreted to be of some duration. In early concurrent leukocytic infiltration, focal hem- lesions, affected epithelial cells were round- orrhage, and the presence of intranuclear ined, irregular, and raised above the contours clusion bodies. of adjacent corneal epithelial cells inter- Cornea. Corneal lesions were most fre- preted to be normal morphologically (Fig. 2).

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Fig. 3. Section of cornea from the same rat exam- Fig. 4. Cornea from rat D31-6, inoculated in- ined by TEM. Note the dissociation of adjacent tracerebrally with HSV2 at 5 days and euthanized corneal epithelial cells, undulations of the nuclear at 5 days pi. There are numerous polymoiphonu- membrane, and the presence of viral particles in clear (p) and mononuclear cells in the corneal the nuclei (arrowheads). (X9400.) stroma, whereas the overlying basal epithelial cells (upper right) appear to be intact. (x3600.) By TEM the morphology of virus-infected mal cells frequently had irregular nuclear epithelial cells varied considerably. Some outlines, margination of nuclear chromatin, cells had rounded cytoplasmic outlines but and numerous viral particles within the nu- intact cytoplasmic membranes, frequently cleus (Fig. 5). In addition, typical herpes- with discernible virus particles in the nu- virus particles were observed in the cyto- cleus. Severely damaged corneal epithelial plasm of some infected cells and interspersed cells were frequently separated from adjacent in stromal lamellae. In the specimens exam- cells, with fragmentation of the cytoplasmic ined, viral particles were not observed in membrane and degeneration of organelles in corneal endothelial cells. the cytoplasm. The nuclear outline was fre- Uveal tract. Lesions of the uveal tract oc- quently enlarged and irregular, with mar- curred in all areas including the choroid, gination and fragmentation of the nuclear ciliary body, and iris. They were observed in chromatin. Frequently large numbers of typ- animals of all age groups after HSV2 inocula- ical herpesvirus particles were present in de- tion (Table I). generate nuclei (Fig. 3). In some animals Ciliary body and iris. By light microscopy, with corneal lesions, there was stromal in- lesions in this area varied from those primar- volvement characterized by intercellular ily inflammatory in nature to lesions which edema and degeneration of keratocytes, fre- were destructive, characterized by necrosis, quently accompanied by polymorphonuclear exudation of proteinaceous material, and leu- and mononuclear cell infiltration (Fig. 4). kocytic infiltration. The distribution and in- Evidence of viral replication within kerato- tensity of lesions varied, but virtually all cytes was also observed. HSV-infected stro- areas of the iris and ciliary body appeared to

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Fig. 5. Section of cornea! stroma from same animal Fig. 6. Iris from rat D4I-B9 inoculated in- as in Fig. 4. Viral particles (arrowheads) are pres- tracerebrally with HSV2 at 30 days which survived ent in both the nucleus and cytoplasm of kerato- and was killed at 15 days pi. Acute iritis, with cyte. Damage to cell appears to be minimal at this hypertrophy and separation of epithelilal cells stage. (X 10,440.) Inset, Higher magnification from iris stroma (arrow) and hypercellularity of from another area of corneal stroma in same stroma! tissue. (Toluidine blue; x544.) animal illustrating typical heipesvirus particles. (X32.000.) On TEM, destructive lesions were characterized by necrosis of epithelial and stro- be susceptible to infection with HSV2. Both mal cells, leukocytic infiltration, intercellular the endothelial cells of the anterior surface edema, and the presence of homogeneous ma- and the epithelial cells of the posterior sur- terial and cellular debris in reactive areas. face of the iris were sometimes reactive. Herpesvirus particles were observed within Cells were enlarged and rounded, with sep- nuclei of epithelial and stromal cells, as aggre- aration from the underlying iris stroma (Fig. gates in the cell cytoplasm, and in remnants of 6). Intranuclear inclusions were occasionally cellular debris (Fig. 8). Occasionally virus observed. Limbal vessels were often packed particles were observed within the cytoplasm with inflammatory cells. On examination by of phagocytic cells, particularly neutrophils SEM, the typical undulations of the surfaces (Fig. 9). These findings were interpreted to be of the iris and the outlines of individual cells due to phagocytosis of debris and virus parti- were discernible in control and in nonaf- cles in reactive areas, rather than a result of fected, inoculated rats. On the other hand, in HSV2 replication in these polymorphonuclear animals with lesions of the iris, inflammatory cells. exudate was comprised of amorphous strands Choroid. Choroiditis in association with of material, and usually large numbers of leu- retinal lesions was frequently observed. Oc- kocytes were adherent to the surface of the casionally there were inflammatory lesions of iris (Fig. 7). A sprinkling of erythrocytes was the choroid interpreted to be several days in occasionally present in these areas. duration, characterized by marked cellular

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Fig. 7. SEM photomicrograph of iris from rat D41-B4 inoculated with HSV2 at 30 days and killed at 10 days pi. Note the typical crypts and folds of the anterior surface of the iris (arrows), and the tnflammatoiy exudate composed of leukocytes and strands of amorphous material and debris. (X4080.)

infiltration in the choriocapillaries. Mono- within nuclei of pigment epithelial cells in- nuclear cells, particularly lymphocytes and dicative of HSV2 replication (Fig. 12). . plasma cells, were observed (Fig. 10). Hy- Neural retina. Retinal lesions were ob- pertrophied pigment epithelial cells and served in rats following HSV2 inoculation at macrophages were present in reactive foci ad- all ages from 0 to 30 days but were most fre- jacent to retinal lesions, particularly in rats quently observed in rats inoculated at 15 days with an acute destructive retinitis. Virus par- (Table I). Foci of hemorrhage associated with ticles were not visualized in samples of cho- destruction were readily seen through the roid examined by electron microscopy. retina in portions of the eye examined by Retina a dissecting microscope and in embedded, Pigment epithelium. Separation of the neu- stained tissues viewed with the light micro- ral retina from the underlying pigment epi- scope. On light microscopy, retinal lesions in thelium was observed in a few animals with general, were destructive in nature, varied erythrocytes and mononuclear and polymor- from focal to segmental in distribution, and phonuclear cells present in areas of separa- frequently involved several or all layers of the tion, Phagocytic cells, including reactive retina, with no particular predilection for pigment epithelial cells, contained degener- geographic location in the eye or layer of the ate organelles, cellular debris, and occasion- retina observed. Destructive lesions were ally identifiable remnants of degenerate pho- characterized by karyorrhexis and karyolysis, toreceptor cells (Fig. 11). Occasionally large and in some cases, leukocytic infiltration. In- numbers of viral particles were observed tranuclear inclusions were sometimes ob-

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ganglion cell, bipolar cell, and photoreceptor cell layers (Fig. 15). Viral particles were also observed in cell processes and cellular debris (Fig. 14). In one animal with retinal lesions which was inoculated at 15 days and killed in extremis at 8 days pi, there were macrophages with electron-dense globules in the cytoplasm scattered in all layers of the retina. These were interpreted to be reactive pigment epithelial cells in the process of migra- tion to an area of retinal damage. Incidence of residual retinal lesions. In three animals that survived following intracerebral inoculation of HSV2 at 15 days and were examined at 15 days pi, there were focal to segmental residual lesions scattered in the retina, which were characterized by segmental reduction in width, incomplete or irregular delineation of individual layers, scattering and intermingling of bipolar cells and photoreceptor cells in the same layer, and infiltration with lymphocytes and plasma Fig. 8. Section of iris stronia from animal D31-6, cells (Figs. 16 to 18). Separation of the retina examined at 5 days pi. Cell contains numerous from pigment epithelium and undulations of nonenveloped (arrowheads) and enveloped (V) the photoreceptor cell layer were observed in viral particles. (X 16,400.) one of these animals. Ocular changes were attributed to primary retinal damage with served, particularly in the bipolar cell layer. HSV2 resulting in degeneration and reduc- Other changes observed included distortion tion of normal cells in all layers, proliferation of the retinal architecture, presence of pig- of glial cells, loss of normal orientation, fold- ment-bearing cells in the retina, hemor- ing, reduction in width of the retina, and loss rhage, retinal undulations, and separation of of delineation of the individual layers. the retina from the adjacent pigment epithe- Optic nerves. In selected optic nerves ex- lium. On SEM, examination of the retina at amined microscopically from rats examined sites of focal hemorrhage from the vitreous at 0, 5, or 10 days of age, including animals surface revealed a raised area, sometimes with retinal damage, lesions were not ob- with free erythrocytes lying on the vitreous served. On the other hand, focal optic neu- surface of the retina. Examination of the cut ritis was observed in five of 12 animals follow- surface of the retina occasionally revealed ing HSV2 inoculation at 15 days. In two rats, abnormal architecture and the presence of lesions were observed in the optic nerves but free erythrocytes, but accurate interpretation not in the retina; two others had lesions in was frequently hampered by tissue or prep- the retina but not in the optic nerve, and aration artifacts. three animals had lesions in both the optic Ultrastructural studies of HSV2-infected nerve and retina. In one of these animals, retina by TEM revealed, in most cases, a de- retinal lesions were confined primarily to the structive retinitis. Intercellular edema, ne- ganglion cell layer, suggesting that retinal in- crosis, fragmentation of cells, and cellular vasions by the virus may have occurred via infiltration were features frequently observed the optic nerve. In one animal with optic (Figs. 13 and 14). Typical herpesvirus par- neuritis examined by TEM, degenerating ticles were observed in nuclei of cells in the myelin sheaths and infiltrating mononuclear

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Fig. 9. Another section from iris stronia of same animal as shown in Fig. 8. Note the cellular debris and the typical herpesvirus particles (arrowheads) scattered in the cellular debris and in cytoplasmic vesicles of an infiltrating polymorphonuclear cells (P). (x 16,920.)

and polymorphonuclear cells but no viral Discussion particles were observed. Nahmias et al.4 reported an incidence of Brain. In those rats that succumbed to the eye lesions in 17% of 297 cases of spontane- disease, lesions were frequently observed at ous neonatal HSV infections in infants. Of the the genera] site of inoculation in the thalamus 51 cases with ocular involvement, 29 had a and hippocampal regions, but all areas of the concurrent encephalitis, and none of the brain appeared to be susceptible to the virus. cases of disseminated herpetic infection with There was an acute necrotizing meningoen- no involvement of the CNS had lesions in the cephalitis involving both grey and white mat- eye, emphasizing the frequent association of ter, and intranuclear inclusions were ob- neonatal herpetic ophthalmitis with a concur- served. In some animals inoculated at 15 or rent encephalitis. In our experimental HSV2 more days that survived, central nervous sys- studies, virtually all rats with ocular lesions tem (CNS) lesions were primarily inflamma- had demonstrable lesions in the CNS. This tory in nature and are described elsewhere.l7 was an expected finding, since our animals When animals were grouped according to age were inoculated intracerebrally with the at inoculation (0 to 30 days), the percentage virus in order to produce ocular lesions. We of inoculated rats with lesions in the brain have, however, observed lesions in newborn ranged from 46%to to 78%, a twofold or more rats inoculated intraperitoneally with HSV2. increase compared with the incidence of ocu- However, the incidence of lesions in the eye lar lesions in these same rats. Virtually all of (and CNS) of animals inoculated by this route those animals with herpetic ophthalmitis had was less than with intracerebrally inoculated typical HSV2 lesions in the brain. rats. In another experiment, we have studied

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Fig. 10. Choroid stroma of rat D34-3A which was Fig. 11. Section of retina from animal inoculated at inoculated intracerebrally witli HSV2 at 10 days, 15 days and euthanized at 5 days pi. Focus of acute survived, and was killed at 20 days pi. Cellular retinitis illustrating cleft of separation between infiltrate is comprised of lymphocytes and plasma photoreceptor cells and pigment epithelial cell cells fa). (X4320.) layers. Reactive pigment epithelial cell within cleft contains pigment granules (g) and phagocy- the eyes of 44 suckling rats following the in- tized outer segments (os) and has engulfed a nu- oculation of HSV directly into the vitreous cleus (n), probably that of a degenerate photore- 2 ceptor cell. (x7380.) cavity. We observed fewer lesions in the cornea (8%) and iris (7%) than in rats inoculated intracerebrally with the virus. On the other taneous disease in infants, HSV2 ophthalmitis hand, the incidence (and type) of HSV2- may be either bilateral or unilateral in distri- induced lesions observed in the retina were bution. similar in both groups {D. H. Percy, unpub- Corneal lesions due to HSV2 infection have lished data, 1979). been described in rabbits following topical Patterns of disease observed in the eye in administration of the virus.13' l4- 20 However, spontaneous HSV infection in infants in- to our knowledge, this is the first report cluded conjunctivitis, keratitis, and chorio- of experimental herpetic keratitis following retinitis. To date, all reported cases of neo- HSV2 inoculation via a route other than local natal HSV chorioretinitis in which the virus administration. Hollenberg et al.20 inocu- was typed were HSV2. In general, ocular lated the corneas of rabbits with HSVi or manifestations in human cases occurred ei- HSV2 and studied the sequence of events by ther as the first manifestation of HSV infec- light microscopy, TEM, and SEM. They tion or after skin, CNS, or systemic involve- noted a characteristic rounding of corneal ment had been observed.4 In our study, the epithelial cells in animals inoculated with earliest that ocular lesions were noted histo- either type, but observed that the edges of logically was in rats examined at 3 or 4 days the HSV2 lesions were raised above the cor- pi, depending on the age of the rats at inocu- neal surface, whereas the HSVj-infected cor- lation. In our experiments, as in the spon- neal lesions were flattened in this area. In our

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Fig. 12. Pigment epithelial cell from animal D24-3 inoculated intracerebrally with HSV2 at 10 days, and euthanized at 4 days pi. Numerous herpesvirus particles (arrowhead) are present in the nucleus. Destructive lesions were present in the adjacent neural retina. (X 17,860.)

Fig. 13. Section of bipolar cell region of retina from rat D33-8 inoculated at 15 days and euthanized at 8 days pi. Note the electron-dense cellular debris (arrows) and nuclear changes varying from pycnosis to karyorrhexis in degenerating bipolar cells. (X5040.)

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Fig. 14. Ganglion cell layer region of retina from Fig. 15. Bipolar cell of retina from rat D24-3 in- rat D32-7 inoculated at 15 days and euthanized at oculated with HSV2 at 10 days and euthanized at 4 10 days pi. Destructive rerinitis is characterized days pi. Note the undulations of the nuclear mem- by edema, fragmentation of cells, and a scattering brane and the numerous enveloped viral particles of electron dense cellular material. Herpesvirus (V) in the cytoplasm, (x 16,400.) particles (arrowhead) are present in the cytoplasm of a degenerate ganglion cell. (x9400.) than to HSVj.10' u- 1:J- 14 Although viral particles have been observed in the corneal HSV2 study, the rounding of infected epithe- stroma in cases of HSV keratitis in man, it lial cells, presence of intranuclear inclusions, may be difficult to demonstrate virus in this and the raised periphery of the corneal le- area during the acute clinical phase of stromal sions were similar to previous findings. Of disease. One theory is that stromal disease interest was the presence of corneal lesions in probably represents the host's immune re- our intracerebrally inoculated rats that oc- sponse to viral antigen subsequent to viral curred in several animals while the eyelids replication in the stromal cells or to diffusion were still fused. The eyes of the suckling rat of antigen from epithelial lesions.1 In view of normally opened at 13 days of age.21 Thus, in the presence of virus in our cases with these animals, the virus must spread to the stromal involvement and the short course of cornea via a route other than topical applica- the disease, lesions in this study are attrib- tion, such as neurogenous or hematogenous uted to primary HSV2 infection. dissemination. Uveal tract lesions have been frequently The presence of virus within keratocytes of observed in HSV infections, and primary the corneal stroma as demonstrated ty TEM herpetic uveitis may be produced locally by 22 in our studies indicates that HSV2 may read- direct intraocular inoculation of HSV, but ily replicate in the corneal stromal cells of the also as a blood-borne infection following in- newborn rat. Deep stromal involvement ap- travenous inoculation of the virus in rab- pears to be more common in experimental bits,23 Although the immune response of the and naturally occurring keratitis due to HSV2 host appears to play an important role in de-

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Fig. 16. Section of dysplastic retina from animal Fig. 17. Section of retina adjacent to vitreous sur- 32-9a inoculated at 15 days which survived and face of rat D32-10 inoculated intracerebrally with was killed at 15 days pi. Pigment epithelial cells HSV2 at 15 days and killed 15 days pi. Chronic (PE) are hypertrophied and hyperplastic, and retinitis is characterized by hypertrophy of en- there is intermingling of the photoreceptor ce\\(p) dothelial cells (E) of retinal vessel and infiltration and bipolar cell (B) layers in the neural retina. by lymphopoietic cells (L) and macrophages (M). Note prominent, thick-walled retinal arterioles in Note the prominent perivascular cells (po). the ganglion cell layer. (Toluidine blue; X432.) (X3420.)

layed and recurrent herpetic uveitis, cer- served in one fatal case of herpetic en- tainly HSV inoculation may produce uveitis cephalitis and retinitis in an adult.25 The ul- soon after intravitreal inoculation which is at- tras tructural studies of the iris by SEM were tributable directly to primary viral damage.22 a useful procedure. We were able to demon- This appears to be the case in our HSV2 stud- strate the extent of involvement over rela- ies in rats with acute iritis, in which damage tively large areas of iris, and this technique was observed soon after inoculation and was also clearly delineated changes such as the associated with local replication of HSV2. In invasion by inflammatory cells. The single one report from a case of acute herpetic illustrated SEM photomicrograph was se- uveitis and deep keratitis in an adult, large lected from many representative specimens numbers of herpesvirus particles were ob- from both normal and affected areas. Hans- served in pigment epithelial cells and dilator son26 has described the normal architecture muscle and stromal cells of the iris, indicative of the iris in the rat examined by SEM. On of the diverse tropism and ability of HSV to the other hand, SEM studies of the retina replicate in a variety of cell types in the iris.24 were hampered by fixation and processing The presence of typical herpesvirus parti- artifacts, making accurate interpretation of cles in the cytoplasm of polymoiphonuclear lesions difficult at best. cells indicate that this may be one means of In our studies of rats with acute lesions in virus spread following phagocytosis by these the retina due to HSV2, virtually all layers of cells. Macrophages containing virus were ob- the retina appeared to support the replication

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Fig. 18. Section oi dysplastic retina from rat D32-9a. There are identifiable bipolar cells (B), structures inteipreted to be remnants of outer segments (arrow), and an infiltrating pigment epithelial cell (PE). (X8100.)

of the virus. Intranuclear aggregations of likely that hematogenous spread played a virus were observed in ganglion cell, bipolar role in our studies. Lesions were widespread cell, and photoreceptor cell layers of the ret- in the eye including all layers of the retina, ina and in pigment epithelial cells. The pigment epithelium, and iris, and we were ultrastructural findings have been described unable to correlate lesions in the retina with in two cases of HSV encephalitis with ret- lesions in the optic nerve. There is evidence initis. In both the adult case25 and an 18- that HSV may be disseminated experimen- month-old infant,27 lesions were destructive tally to the eye as a blood-borne infection. in nature, and intranuclear viral particles Uveitis has been produced in rabbits inocu- were observed in the retina of these patients. lated intravenously with HSV.23 Further- There has been speculation concerning the more, Brick and Oh,29 inoculated suckling

route of invasion by the virus in cases of rabbits subcutaneously with HSV2J and the herpetic retinitis. Some reports have pro- virus was subsequently isolated from eyes posed that spread may occur by the optic and from blood mononuclear cells, indicative nerve.5' 28 Cibis et al.,27 however, studied a of blood-borne dissemination. It has been case of fatal HSV encephalitis with retinitis in shown that following intracerebral inocula- an 18-month-old infant and suggested that tion in mice (even with a very small volume the virus migrated to the eye as a hematoge- of inoculum) the majority of the material is nous infection. They observed foci of retinal dispersed via the arachnoid villi into the sys- necrosis centered around blood vessels, in- temic circulation.30 Thus, in addition to pos- tranuclear inclusions in endothelial cells, and sible spread of virus to the eye following ini- foci of retinal hemorrhage.27 Similarly, it is tial replication in the CNS, hematogenous

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dissemination of virus to the eye could occur volvement in neonatal heipes simplex virus infec- immediately following intracerebral inoculation. Arch Ophthalmol 82:169, 1969. 8. Hutchison DS, Smith RE, and Haughton PB: Con- tion of HSV2. genital herpetic keratitis. Arch Ophthalmol 93:70, Of interest were the chronic retinal lesions 1975. observed in three rats that survived following 9. Oh JO, Kimura SJ, et al.: Oculogenital transmission HSV2 inoculation at 15 days of age. Cogan et of type 2 herpes simplex virus in adults. Surv al.5 reported on a fatal case of HSV en- Ophthalmol 21:106, 1976. cephalitis with necrotizing retinopathy in an 10. Hanna L, Ostler HB, and Keshishyan H: Observed relationship between herpetic lesions and antigenic infant that developed fever and convulsions type of herpesvirus hominis. Surv Ophthalmol at 3 weeks and died at 24 weeks of age. There 21:110, 1976. was a bilateral destructive retinitis accom- 11. Neuman-Haefelin D, Sundmacher R, Wochnik G, panied by hemorrhage with loss of normal et al.: Heipes simplex virus types 1 and 2 in ocular architecture, massive metaplasia of neural disease. Arch Ophthalmol 96:64, 1978. 12. Oh JO: Type 1 and type 2 heipes simplex virus in retina, and pigment epithelium. They sug- corneal cell cultures. Surv Ophthalmol 21:160, gested that the selective necrotizing reti- 1976. nopathy might be due to an immune process 13. Oh JO, Moschini GB, Okumoto M, and Stevens T: rather than to a direct infection with the Ocular pathogenicity of type 1 and 2 herpesvirus virus. Our findings in three rats that survived hominis in rabbits. Infect Immun 5:412, 1972. 14. Stevens TR and Oh JO: Comparison of type 1 and 2 following intracerebral inoculation of HSV2 at herpesvirus hominis infections of rabbit eyes. II. 15 days were similar in some respects to this Histopathologic and virologic studies. Arch Oph- case report. In our animals there were areas thalmol 90:477, 1973. of retinal destruction and sclerosis with con- 15. Werner GH: Laboratory models of heipes virus current metaplasia of adjacent pigment epi- infection. J Antimicrob Chemother 3(Suppl. A):71, 1977. thelial cells. However, we interpreted these 16. Percy DH and Hatch LA: Experimental infection changes to be sequelle to primary viral dam- with heipes simplex virus type 2 in newborn rats: age with subsequent inflammatory cell infil- effect of treatment with iododeoxyuridine and cyto- tration, scarring, metaplasia, and dystrophy sine arabinoside. J Infect Dis 132:256, 1975. of affected retinal tissue. 17. Crawford JP, Percy DH, and Hatch LA: Experimen- tal encephalitis in the newborn rat due to heipes The excellent technical assistance of Philip Floyd, simplex virus type 2. Exp Mol Pathol 31:44, 1979. Elaine Hunter, and J. R. Hewett is gratefully ac- 18. Nahmias AJ, Del Buono I, Pipkin J, et al.: Rapid knowledged. identification and typing of herpes simplex virus types 1 and 2 by direct immunofluorescent tech- REFERENCES nique. Appl Microbiol 22:455, 1971. 1. Davvson CR and Togni B: Herpes simplex eye in- 19. Cohen AL: Critical point drying. In: Principles and fections: clinical manifestations, pathogenesis and Techniques of Scanning Electron Microscopy. Bio- management. Surv Ophthalmol 21:121, 1976. logical Applications, Hayat MA, editor, vol 1, New 2. Nahmias AJ and Roizman B: Infection with herpes- York, 1974, Van Nostrand Reinhold Company, p. simplex viruses 1 and 2. N Engl J Med 289:667, 719, 44. 781, 1973. 20. Hollenberg MJ, Wilkie JS, Hudson JB, et al.: Le- 3. Florman A, Gershon AA, Blackett PR, et al.: In- sions produced by human herpesviruses 1 and 2. trauterine infections with herpes simplex virus. Re- Arch Ophthalmol 94:127, 1976. sultant congenital malformations. JAMA 225:129, 21. Lane-Petter W: The laboratory rat. In: The UFAW 1973. Handbook on the Care and Management of Labora- 4. Nahmias AJ, Visintine AM, Caldwell DR, and Wil- tory Animals. Edinburgh, 1972, Churchill Living- son LA: Eye infections with herpes simplex viruses stone, p. 205. in neonates. Surv Ophthalmol 21:100, 1976. 22. Oh J: Primary and secondary herpes simplex uveitis 5. Cogan DG, Kuwabara T, Young GF, et al.: Heipes in rabbits. Surv Ophthalmol 21:178, 1976. simplex retinopathy in an infant. Arch Ophthalmol 23. Sery TW and Foster L: Viremia induction of herpes 72:641, 1964. simplex uveitis. Ophthalmol Res 9:171, 1977. 6. Young GF, Knox DL, and Dodge PR: Necrotizing 24. Witmer R and Iwamoto T: Electron microscope ob- encephalitis and chorioretinitis in a young infant. servations of herpes-like particles in the iris. Arch Arch Neurol 13:15, 1965. Ophthalmol 79:331, 1968. 7. Hagler VVS, Walters DV, Nahmias AJ: Ocular in- 25. Minckler DS, McLean EB, Shaw CM et al.: Her-

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pesvirus hominis encephalitis and retinitis. Arch 29. Brick DC and Oh J: Ocular changes associated with Ophthalmol 94:89, 1976. dissemination of type 2 herpes simplex virus from 26. Hansson H-A: Ultrastructure of the surface of the skin infections in newborn rabbits. INVEST OPII- iris in the rat eye. Z Zellforsch 110:192, 1970. THALMOL VISUAL SCI 17(ARVO Suppl.):274, 1978. 27. Cibis CVV, Flynn JT, and Davis B: Heipes simplex 30. Cairns HJF: Intracerebral inoculation of mice: fate retinitis. Arch Ophthalmol 96:299, 1978. of the inoculum. Nature 166:910, 1950. 28. Johnson BL and Wisotzkey HM: Neuroretinitis associated with heipes simplex encephalitis in an adult. Am J Ophthalmol 83:481, 1977.

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