Recurrent Herpes Simplex After Corneal Transplantation in Rats

Transcription

1 Recurrent Herpes Simplex After Corneal Transplantation in Rats Susan M. Nicholls,* Carolyn Shimeld,* David L. Easty,\ and Terry J. Hill% Purpose. To ascertain the effect of trauma from surgery and rejection on the incidence and timing of recurrent herpes simplex virus (HSV) disease after corneal transplantation. To locate virus antigen and identify cells of the immune system infiltrating corneas with recurrent disease. Methods. PVG rats were inoculated on the cornea with HSV-1 McKrae. Recurrent disease was induced either by ultraviolet (UV)-irradiation of the cornea or by corneal transplantation. After corneal transplantation, animals shedding virus in the tear film were killed on days 1 to 4 of shedding. Eyes were fixed, embedded, sectioned, and stained for virus antigens, infiltrating cells, major histocompatibility complex class II, and adhesion molecule expression. Results. In the first 15 days after corneal transplantation, 8 of 91 rats shed virus, and between days 16 and 30, an additional 3 of 60 rats shed virus (12% of total rats, comparable to the percent that shed after UV irradiation). Shedding sometimes was accompanied by punctate epithelial lesions in the recipient cornea and stromal opacity. The rejection process itself did not induce or exacerbate recurrent disease. In all corneas examined from eyes that shed virus, antigen was found in several locations at the graft-host junction, sometimes in the absence of clinical signs of disease, and frequently it extended through the stroma to the endothelium. Granulocytes were the main infiltrating cell in areas of virus antigen. Conclusions. Corneal transplantation trauma is a stimulus to recurrent disease of similar potency to UV irradiation. The graft-host junction is an area in which virus spreads easily and can reach the endothelium readily. In humans, the incidence of recurrent disease at this location may be greater than has been recognized. Invest Ophthalmol Vis Sci. 1996; 37: vulinical studies have shown that there is a higherthan-average risk of graft failure in patients who undergo corneal transplantation for herpes simplex keratitis. 1 ' 2 The causes of failure are multifactorial and incompletely understood. Clinical lesions attributed to herpes simplex virus (HSV) after transplantation frequently occur at the graft margin but may not show the typical dendritic appearance. Moreover, virus isolation studies rarely are performed to confirm a clinical diagnosis. This, together with the fact that other signs of HSV disease, such as corneal edema and opacification, resemble those of immunologic rejection, of- From the *Departments of Ophthalmology and %Pathology and Microfriology, School of Medical Sciences, and the f Department of Ophthalmology, Bristol Eye Hospital, Bristol, United Kingdom. Submitted for publication July 17, 1995; revised October 30, 1995; accepted October 30, Proprietary interest category: N. Reprint requests: Susan Nicholls, Department of Ophthalmology, School of Medical Sciences, University Walk, Bristol, BS8 1TD, United Kingdom. ten makes the precise cause of failure difficult to establish. The higher risk of graft failure in patients who undergo corneal transplantation for herpes simplex keratitis largely results from a higher incidence of immunologic rejection. This may be caused by HSV-specific factors, or it may be merely an effect of the inflammation, manifest as vascularization and leukocyte infiltration; active inflammation of any cause has been shown to prejudice graft survival. 3 Clinical evidence of virus-specific factors includes the frequent observation that HSV recurrence is associated with rejection, 4 ' 5 implying that the immune response to reactivated virus might activate alloreactive cells incidentally, either locally or systemically. Such cross-stimulation by pathogens is well documented in autoimmune diseases such as arthritis, and it is reported that cytomegalovirus hepatitis may provoke liver graft rejection. 6 There also could be direct destruction of the endothelium after spread of the virus to this cell layer. 7 Alternatively, a Investigative Ophthalmology & Visual Science, February 1996, Vol. 37, No. 2 Copyright Association for Research in Vision and Ophthalmology 425

2 426 Investigative Ophthalmology 8c Visual Science, February 1996, Vol. 37, No. 2 rejection response could itself induce reactivation or provoke recurrent disease by creating more favorable conditions for virus replication in the cornea. We have addressed some of these questions using an experimental model of recurrent HSV for which inbred strains are available, together with monoclonal antibodies to identify infiltrating cells. We chose the rat rather than the mouse because corneal graft rejection in rats can be defined clearly 8 ; the larger eye makes grafting easier to perform, and more is known about the rejection response in different rat strains 9 than in mice. A suitable model has to satisfy three additional criteria: It should be possible to establish a high incidence of latent HSV infection in the trigeminal ganglion; the cornea should be relatively undamaged by the primary infection (i.e., corneal nerves should remain intact); and recurrent corneal HSV disease should be inducible. In a previous investigation of primary HSV disease in rats, 10 we showed that the PVG strain satisfied the first two criteria. After inoculation of the cornea with HSV-1 McKrae and resolution of the primary infection, corneas of all PVG rats were clinically normal (meaning that the sensory nerve supply remained intact), and latent infection was found in the majority of rats surviving the primary infection. In the current work, wefirstestablished that recurrent disease could be induced in the PVG strain by UV irradiation of the cornea, a known stimulus of HSV recurrence in human" 12 and mouse. 13 Further groups of rats then underwent transplantation to determine whether the trauma of surgery induced recurrent disease, whether rejection induced recurrent disease, and whether previous HSV infection of the cornea predisposed animals to rejection. Eyes showing recurrent disease after transplantation were examined histologically for virus antigen, adhesion molecule expression, and infiltrating cells. METHODS Rats Specific pathogen-free female rats, PVG (RT1 C ), Lewis (LEW) (RT1 1 ), and DA (RTF) were purchased from Harlan Olac (Oxford, United Kingdom). RT1 is the rat major histocompatibility complex (MHC). All experiments adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Inoculation With Virus Eight- to 9-week-old PVG rats were anesthetized by intramuscular injection of 0.5 ml/kg fentanyl-fluanisone (Hypnorm; Janssen, Oxford, UK) in one hind limb and 2.5 mg/kg diazepam (Valium; Roche, Welwyn Garden City, UK) in the other. Ten microliters of medium 199 containing 4 X 10 4 plaque-forming units HSV-1 strain McKrae or mock inoculum were placed on the right cornea. Corneas were then scarified lightly using a 26-gauge needle. In total, 10 scarification marks were made through the virus suspension in two directions at right angles, extending to within 1 mm of the limbus. This was designed to maximize the number of nerve endings and, therefore, the number of neurons that might become infected. Care was taken not to penetrate to the corneal stroma. Induction of Recurrent Disease by Ultraviolet Irradiation At least 1 month after the primary infection had resolved, rats were anesthetized with half the dose of Hypnorm and Valium used for virus inoculation. The right cornea was irradiated for 105 seconds with a lamp (Hanovia, Slough, UK) of peak emission 4.02 mj/cm 2 per second. Induction of Recurrent Disease by Corneal Transplantation Rats were anesthetized as they were for virus inoculation. Transplant surgery was performed, as previously described, 14 at least 1 month after the primary disease had resolved. A 3-mm diameter central button was removed from the right cornea and was replaced with a 3.5-mm corneal button removed from the donor cornea. The graft was secured with eight or nine interrupted sutures, which were not removed. Chloramphenicol ointment was applied topically to the recipient eye immediately after surgery and then daily for 1 week. Technical failure occurred in 3 of 91 animals with iris prolapse, severe synechiae, or cataracts, and they were excluded from the analysis and killed. Clinical Examination and Isolation of Virus Immediately before UV irradiation or transplantation, rats were anesthetized and examined, using a slit lamp, for signs of spontaneous recurrent HSV eye disease and for virus in the tear film. Virus was detected by washing the eye of the anesthetized animal with 50 fj,\ of medium 199 and inoculating the eye washings onto Vero cell monolayers. These monolayers were examined for viral cytopathic effect after incubation for 2 days at 37 C. After UV irradiation or corneal transplantation, rats were examined daily in a similar manner for clinical signs of recurrent disease and graft rejection and for virus in the tear film. Control, latently infected animals that had not received a reactivating stimulus similarly were monitored. Steroid Treatment From days 2 to 11 after UV irradiation and after transplant surgery in some animals, 15 (A of 0.1% dexamethasone eye drops were applied once daily to the transplanted cornea. This was intended to maximize virus yield, and it was sufficient to delay, but not to prevent, rejection. The same treatment was begun at

3 Recurrent Herpes Simplex in Rats 427 the time of rejection of each allograft, and it continued until the end of the study period, again to maximize virus yield. An isograft performed on the same day as each allograft that showed rejection received equivalent steroid treatment. Control, latently infected animals that did not receive a reactivating stimulus also were treated with steroid. Fixation and Embedding of Tissue Animals were killed, and right eyes and cervical lymph nodes were removed and fixed for immunoperoxidase staining as previously described. 15 Periodate-lysineparaformaldehyde containing 0.25% paraformaldehyde was injected gently into the vitreous. Whole eyes were fixed overnight in PLP, after which they were bisected sagitally and lenses were removed. The two halves of each eye were dehydrated and embedded under vacuum in low-temperature paraffin wax at 53 C; blocks were stored at 20 C. For antibody staining, they were removed and allowed to warm to room temperature, and serial 6-//m sections were cut and transferred to glass slides. Immunoperoxidase Staining ABC Staining for Cell Surface Molecules. Infiltrating cells, MHC class II expression, and adhesion molecule expression were detected by the ABC method using the following monoclonal antibodies: OX34 (CD2 T cells), OX19 (CD5 T cells), W3/25 (CD4 helper T cells, some macrophages and dendritic cells), OX8 (CD8a cytotoxic T cells and natural killer cells), OX39 (CD25 interleukin-2 receptor on T and B blasts), OX33 (CD45 on B cells), OX6 (MHC class II), ED2 (tissue macrophages), HIS 48 (granulocytes), 1A29 (CD54 ICAM-1), and WT.3 (CD18 LFA-10). Antibodies were obtained from Seralab (Crawley Down, UK), except for ED2 (Serotec, Oxford, UK) and HIS 48 (PharMingen, San Diego, CA). Rehydrated sections were incubated with 0.3% hydrogen peroxide in phosphate-buffered saline to block endogenous peroxidase activity, then with 1.5% normal horse serum to block nonspecific binding of the secondary antibody. They were next incubated sequentially with monoclonal antibody (overnight at 4 C) and biotinylated horse antimouse immunoglobulin G (preadsorbed with rat serum; Vector Labs, Peterborough, UK), followed by avidin-biotinylated horseradish peroxidase complex (ABC; Vector Labs) and diaminobenzidine (DAB: Sigma, Poole, UK) with 0.01% hydrogen peroxide. On all staining runs, sections incubated with an inappropriate primary monoclonal antibody, OX21 (against human complement factor I), were included as negative controls, and sections of lymph node incubated with the appropriate monoclonal antibodies were included as positive controls. Slides were lightly counterstained with hematoxylin, dehydrated, cleared, and mounted in Histomount (National Diagnostics, Atlanta, GA). Peroxidase-Antiperoxidase Staining for Herpes Simplex Virus Antigens. Sections were rehydrated, and endogenous peroxidase activity was blocked with hydrogen peroxide as above. Nonspecific binding of the secondary antibody was blocked with 20% normal swine serum. Sections were then incubated in a polyclonal anti-hsv antibody raised in rabbit (Dako, High Wycombe, UK) at a dilution of 1 in 500 overnight at 4 C. This was followed sequentially by swine anti-rabbit secondary antibody, horseradish peroxidase-rabbitantihorseradish peroxidase complex, and diaminobenzidine-hydrogen peroxide. Included in each staining run were negative control sections incubated in normal rabbit serum in place of the primary antibody and positive control sections consisting of corneas with known primary HSV lesions incubated with the HSV antiserum. Double staining for HSV antigens and cell surface molecules was achieved by first staining with the appropriate monoclonal antibodies for cell surface molecules according to the protocol delineated in the subsection, ABC Staining for Cell Surface Molecules. After the addition of the DAB, HSV antigens were visualized as described, except that the endogenous peroxidase blocking step was omitted, the primary antibody was applied at a dilution of 1:300 for 1 hour at room temperature, and Vectastain VIP (Vector Labs) was substituted for DAB as the chromogen, yielding a purple endproduct. Slides were lighdy counterstained with hematoxylin. Statistical Analysis The effect of transplantation trauma on recurrent disease was determined by using chi-square analysis. RESULTS Recurrent Disease After Ultraviolet Irradiation Corneas of five uninfected PVG rats were UV irradiated to determine the damage attributable to radiation alone. At 24 hours after irradiation, there was focal loss of epithelium; by clinical examination, this appeared to have been replaced after 2 or 3 days. Mild vessel ingress and stromal opacity were induced in the central area of the cornea, but they resolved within 7 to 10 days. Corneas of 17 additional PVG rats were inoculated with virus. Three months after the primary infection subsided, corneas were UV irradiated, treated with steroid eye drops, and examined daily for 15 days for clinical signs of disease and virus in the tear film. Two animals shed virus, beginning on days 7 and 10 after irradiation, respectively, and in each animal, two or three characteristic HSV lesions developed in one area of the corneal epithelium (Fig. la).

4 428 Investigative Ophthalmology & Visual Science, February 1996, Vol. 37, No. 2 two that had previously shown recurrent disease, were again irradiated and examined for recurrent disease. A third rat shed virus, beginning on day 7, and developed epithelial lesions and opacity similar to that seen in the other two animals. The overall incidence of virus shedding and recurrent disease after UV irradiation was 12%. Epithelial lesions and shedding lasted 3 to 7 days (Table 1). FIGURE i. Recurrent epithelial lesions {arrows) in PVG rat corneas, (a) Punctate lesions 11 days after ultraviolet irradiation, with cell infiltration into die underlying stroma, on the second day of virus shedding (rat 2 in Table 1). The two bright areas near center are lamp reflections, (b) Severe lesions in the recipient cornea of an allograft, 10 days after transplantation, on the second day of virus shedding (rat 10 in Table 1 and Figs. 2a to 2c). There is focal loss of epithelium at the margin in the area of the lower suture, (c) Lesions near the graft-host junction of an isograft 13 days after transplantation (rat 4 in Table 1). The pupil is dilated by mydriatic. Severe opacity gradually developed in this cornea,. Lesions were similar (i.e., punctate) to those seen in primary infection, occasionally coalescing to form a more dendritic shape, and they were followed by vessel growth toward the site of lesions and corneal opacification. Opacity persisted for approximately 2 weeks and resolved during the next 2 weeks, and vessels regressed. Six weeks after the first UV irradiation, the corneas of nine of these rats, including one of the Recurrent Disease After Corneal Transplantation After it was established that recurrent disease could be induced by UV irradiation, corneal transplantation was performed on latendy infected rats. One hundred three PVG rats that had been inoculated with virus at least 2 months earlier were divided into three groups. One group received a syngeneic corneal transplant, a second group (DA or LEW) underwent allogeneic corneal transplantation, and the third group did not undergo transplantation. Some animals in each group were given topical steroid after the operation. They were monitored for signs of recurrent disease and rejection either for 15 days or for at least 30 days (to allow rejection to occur). Effect of Transplantation Trauma and Rejection on Recurrent Disease. Virus was shed in the tear film of 8 of 91 rats in the first 15 days after transplantation (three allografts and five isografts; Tables 1 and 2), whereas none was shed in the control untransplanted rats (Table 2) (P= 0.046). Incidence of recurrence (9%) and duration of virus shedding during this period were similar to those seen after UV irradiation. Between days 15 and 30, virus additionally was shed from 3 of 60 corneas (two isografts and one allograft), bringing the total to 11 of 91 (12%). Virus recurred in four allografts and seven isografts. Allografts consisted of 2 of 10 DA grafts and 2 of 26 LEW grafts. In 3 of 11 corneas that shed virus, recurrent lesions of the type seen after UV irradiation arose in the recipient cornea. None was seen solely within the donor cornea. Lesions usually were small, punctate, and close to the graft margin, although they could be extensive (Fig. lb). They were seen occasionally in the absence of virus shedding (data not shown). Focal epithelial loss in the absence of typical lesions was recorded adjacent to or near the graft margin in 4 of 11 corneas that shed virus. In 2 of 3 corneas that shed virus at a later stage (between days 15 and 30), epithelial loss was noted in the donor cornea, but in one of these, the loss also coincided with rejection. In 3 of 11 corneas, no clinical abnormality was seen at the time of virus shedding, although virus antigen was found at the graft-host junction in corneas examined histologically (see next section). Rejected grafts became vascularized, edematous, and opaque. The day of rejection was defined as the day when moderate or severe opacity developed in a

5 Recurrent Herpes Simplex in Rats 429 T3 s in 3 s <U ai ( 2 13 H 03 O U O 81 i i O. U 1 I H O m o o 0)0 OM O 00 oo QHH 2AA oo o o in O A O CO o A A oo oo 1 I I I A A noto m o i i TO TO TO ra ra ra n b Sobo SoSohoboo o o oooos 3 ' : j -s c II c > 3 3 % S - S previously clear donor cornea. Without topical steroid, this occurred between days 10 and 14 after transplantation (DA-to-PVG strain combination). Steroid treatment from days 2 to 11 (LEW-to-PVG and DA-to- PVG combinations) delayed rejection until after day 17. By day 30, 24 of 26 allografts had been rejected and were monitored for at least 10 days after rejection. Syngeneic grafts remained clear except for those in which disease recurred; in these, focal opacity and vascularization developed in the area surrounding lesions. One such syngeneic graft (Fig. lc) eventually became completely opaque, and this only partially resolved. Of the four recipients of allografts that shed virus, shedding occurred in three before the onset of rejection. Immunohistologic Examination of Corneas With Recurrent Disease. Four animals that shed virus were killed, one on each of the first 4 days of shedding (10, 11, or 12 days after transplantation; Table 1), and their corneas were examined for infiltrating cells and virus antigens (two allografts and two isografts). Three latendy infected animals that did not shed virus also were killed on days 10, 11, and 12 after transplantation, and their corneas were examined. All seven animals had been treated with steroid. Serial sections of one eye processed on the second day of virus shedding, together with one eye from which virus had not been shed, were stained in sequence either for one of the cell surface antigens or for HSV antigens. Thus, each antigen was visualized every 14th slide (each slide contained three sections). Sections of the remaining eyes were stained by the double-staining technique for one of the cell surface antigens, as well as for HSV antigens, so that the presence or absence of HSV was established on all sections. If lesions on the recipient cornea had been identified clinically (2 of 4), antigen was found at these locations (Fig. 2a). It was present only in the epithelium, and large numbers of neutrophils-his 48 + granulocytes underlay these lesions by day 2 (Figs. 2a, 2b). Granulocytes appeared in the lesion itself. However, the most striking feature of the histologic study was the extent of virus antigen at the graft-host junction, where it was found in all four corneas. At this location, as stated previously, virus antigen was not associated with characteristic clinical epithelial lesions, although epithelial loss sometimes was noted, both by clinical examination and histology (Fig. 2c). In some sections, antigen at the junction was confined to the epithelium, but it frequendy was present in the stroma (Figs. 2c to 2f), sometimes in its entire thickness, and it even spread into the endothelium (Fig. 2d). On the first day of virus shedding, antigen was found in only two separate locations at the junction (estimated to be approximately 1 mm apart). On days 2, 3, and 4, it was present at the junction in three or four quadrants of the cornea and was most

6 430 Investigative Ophthalmology & Visual Science, February 1996, Vol. 37, No. 2 TABLE 2. Incidence of Virus Shedding in Eyewashings After Corneal Transplantation Treatment Days 1-15 Days Isograft 1/19 Isograft + steroid 4/36 Allograft 1/10 Allograft + steroid 2/26 Total 8/91 (9) None (control) 0/27 (0) Steroid (control) 0/33 (0) Values are number with virus/total (%). ND = not done. ND 2/31 ND 1/29 3/60 (5) ND ND extensive on day 3 in a cornea in which no clinical signs of disease had been seen (Fig. 3). No virus antigen was seen in the three transplanted corneas from which virus had not been shed. Granulocytes were clustered at sites of virus antigen at the junction (Fig. 2f), as well as in lesions in the recipient cornea (Fig. 2b), and they were sparse in corneas lacking virus. ED2 + macrophages were present near lesions, particularly at the junction, but these were not clustered around antigen (Fig. 2g), and they were seen in similar numbers in control corneas in which virus was absent. Abundant HSV expression in the stroma at the junction (Figs. 2c, 2e, 2f) suggested that infiltrating cells and keratocytes were infected. Indeed, some cells were double stained for the granulocyte antigen and HSV. T cells recognized by OX19 (CD2 + ) were absent (Fig. 2e), and few cells recognized by OX8, W3/25, and OX34 (CD5 + ) antibodies were at the site of lesions. However, by the fourth day of virus shedding, large numbers of these cells, together with cells expressing MHC class II, were infiltrating recipient corneas, compared with those of virus-negative animals. Above areas of heavy infiltration, the corneal epithelium expressed class II (RT1B) antigen. Some infiltrating cells expressed LFA-1/?; these were particularly abundant in areas of virus antigen and, thus, probably were granulocytes. By day 4, ICAM-1 was expressed strongly on the endothelium of limbal blood vessels and vessels invading infected corneas, but not on such vessels in corneas lacking virus (Fig. 2h). ICAM-1 was expressed on the basal corneal epithelium of all corneas examined, but was more extensive in corneas with virus. In corneas lacking virus, it was confined mainly to the vicinity of sutures. A few of the cells infiltrating the recipient cornea expressed the interleukin-2 receptor. Effect of Previous Corneal Herpes Simplex Virus Infection on the Incidence and Timing of Rejection Twenty PVG rats were inoculated with HSV, and nine were inoculated with mock inoculum. After 2 months, the mock-inoculated rats and 10 virus-inoculated rats received DA allografts. The remaining 10 virus-inoculated rats were given syngeneic grafts. No topical steroid was applied. Syngenic grafts remained clear after FIGURE 2. Histology of corneas with recurrent disease after corneal transplantation, r = recipient cornea. All sections were lightly counterstained with hematoxylin. Bar = 50 ^m in a, b, d to h; bar = 200 //m in c. (a) Herpes simplex virus (HSV) antigens (brown) in the recipient cornea of the allograft shown in Fig. lb (rat 10 in Table 1, second day of virus shedding). Neutrophils-granulocytes underlie the antigen, which is restricted to the epithelium, (b) Section adjacent to a showing HIS 48 + granulocytes (brown) underlying and within the HSV lesion, (c) Section of an HSV lesion at the graft-host junction of the cornea in a and b. HSV antigens (brown) are in the recipient epithelium, which is peeling off. At the junction, the epithelium is missing, and HSV antigens extend down to the endothelium. (d) HSV antigens (dark purple) in cells resembling endothelial cells on Descemet's membrane of the donor side of the graft-host junction on the third day of virus shedding (allograft 11 days after transplantation, rat 11 in Table 1). (e) Double stain for HSV antigens (dark purple) and OX19 + T cells at the graft-host junction of the cornea in d. HSV antigens extend through the stroma to the endothelium (no lesions were identified at clinical examination). T cells, which would be stained brown, are absent, (f) Double stain of section near e showing HSV antigens in the same locations (dark purple; arrows show examples) and granulocytes (brown) densely clustered in areas of virus antigen, (g) Double stain for HSV antigens (purple) and ED2 + cells (macrophages, brown) in the vicinity of two sutures in a different area of the graft-host junction of the cornea shown in e and f. Macrophages are not clustered around antigen, (h) (left) ICAM-1 expression (brown) on the basal corneal epithelium and on die endothelium of blood vessels (arrows) and cells invading the recipient cornea on the fourth day of virus shedding (isograft, 10 days after transplantation, rat 6 in Table 1). (right) Lack of ICAM-1 on epithelium, blood vessels (arrows), and cells in the recipient cornea of an isograft on day 10 that had no histologic or clinical signs of recurrent disease.

7 » Recurrent Herpes Simplex in Rats 431 :x>.v b - ' ; ' ~ 0 0 _ f.r. o ; "'J r \

8 432 Investigative Ophthalmology & Visual Science, February 1996, Vol. 37, No. 2 the initial edema had resolved. One of the latently infected rats with an allograft shed virus on days 8 and 9 after transplantation, before rejection, without clinical signs of HSV disease. Rejection of allografts occurred between days 10 and 14 (median day 13) in animals that previously had been infected widi HSV and between days 11 and 13 (median day 12) in those that had not. Thus, there was no difference between the two groups in the incidence of rejection or time to rejection. DISCUSSION In our previous study, which evaluated primary disease and latent infection in four strains of rat after inoculation of the cornea, we found that the PVG strain was the only one suitable for further studies on recurrent disease. The others were susceptible both to spread of virus within the nervous system and to severe eye disease. 10 In the current experiments, we have shown that in the PVG strain, recurrent HSV can be induced by UV irradiation of the cornea and by corneal transplantation. However, the incidence of recurrent disease was considerably lower than it was after UV irradiation of NIH mice 13 (12% compared to as much as 56%). Our previous experiments 10 showed a correlation between resistance of the PVG strain to HSV (compared with other rat strains) and low levels of virus replication and spread within the trigeminal ganglion during the primary infection. Moreover, the incidence of latent infection in the ophthalmic division of the PVG ganglion (as detected by co-cultivation of the ganglion with Vero cells) averaged 61 %, 10 lower than that in NIH mice. Therefore, the low incidence of recurrent disease in rats after UV irradiation and transplantation may have occurred because fewer neurons in the ophthalmic division of the trigeminal ganglion became latendy infected than in mice, or because the number of genome copies per cell was lower. It was not possible to increase the incidence of latent infection with a higher virus inoculum because the current dose of 4 X 10 4 plaque-forming units was lethal to a high proportion of rats (25% to 50% l0 ). The incidence and pattern of virus shedding during recurrent disease after corneal transplantation was clearly similar to that induced by UV irradiation. It took at least 1 week for recurrent lesions to develop after either stimulus, compared with 2 to 5 days in mice after UV irradiation. 13 They were, however, similar in appearance to those that developed in mice, and there were similar amounts of virus shed in the tear film. Corneal disease occurred with and without topical steroid treatment. The incidence and severity of disease was higher with steroid, but the low incidence of reactivation precluded full evaluation of the effect of this treatment. Because of the low incidence of recurrent disease, only four eyes were examined histologically. Examination was confined to the early period of disease to locate virus antigen and to identify the leukocytes most likely to be involved in eliminating virus. As in humans, recurrent lesions most frequendy were located close to the graft margin. Lesions in the recipient cornea were of the typical punctate type seen after UV irradiation, whereas those at the margin attributed to HSV were manifest only as focal loss of epithelium. Dyes were not applied to identify ulcers more clearly because this might reduce virus yield, alter die pathology, or both. It was interesting that virus in the recipient always was restricted to the epithelium, which was also true of primary disease in this strain. 10 This may reflect the effectiveness of the barrier provided by the basement membrane after egress of virus from nerve endings in the epithelium. In contrast, at the grafthost junction, virus clearly was able to spread to all levels of the cornea and, unexpectedly, was found in many locations around its circumference from an early stage, i.e., from the second day of virus shedding onward. Granulocytes were the only cell type clustered in large numbers around virus antigen, wherever antigen was found. Macrophages, though present, were found in equal numbers in corneas that had not shed virus. Therefore, their presence was probably a response to the trauma of the surgery or to the sutures rather than to the virus. Thus, it appears that granulocytes may play a major role in the early elimination of recurrent virus. However, the large number of T cells infiltrating the recipient cornea by day 4, some of which expressed the interleukin-2 receptor, would be available to eliminate persisting virus or virus present at earlier time points in more peripheral areas of the cornea. Unfortunately, too few eyes were available for histologic examination to establish the initial sites of virus replication at the graft-host junction (i.e., die epithelium or the stroma) or the precise mechanisms of spread. By the time recurrent lesions occurred (on days 10 to 12 after transplantation), the peripheral termini of stromal nerves severed during surgery may have regenerated sufficiendy to have grown back into die epithelium at the graft-host junction. This has been shown to occur rapidly in the rabbit. 16 Virus initially would be shed into the epithelium, and penetration to the deeper layers might be facilitated by defects in the epithelial basement membrane at the junction. Alternatively, initial release of virus may have occurred from severed nerve endings remaining in the stroma. Wherever the virus initially appears, it is clear that there is extensive replication within the stroma at the donor-recipient junction, sometimes in the absence of clear epithelial ulceration (as shown in Figs. 2d, 2e), and sometimes extending to the endothelium. However, this infection apparently does not spread far into recipient or donor cornea. The rapid appearance

9 Recurrent Herpes Simplex in Rats 433 Epithelium 0 Epithelium and stroma []] Stroma Epithelium, stroma and endothelium FIGURE 3. Map of the three layers of the cornea at the grafthost junction of an allograft in which no herpes simplex virus (HSV) lesions were seen at clinical examination, showing the presence or absence of HSV antigens (third day of virus shedding, 11 days after transplantation; cornea from rat 11 in Table 1 and Figs. 2d to 2g). of virus at several locations around the circumference of the cornea at the junction and the appearance of lesions in several locations after UV irradiation suggests that virus reactivated in several neurons. However, lateral spread around the circumference of the graft also may have occurred through the large numbers of infiltrating granulocytes (some of which expressed virus antigens at sites of disease) or through proliferating and migrating keratocytes, which would be involved in the repair and remodeling process at the junction. Inflammation caused by the sutures may have exacerbated spread of virus. Whatever the mechanisms involved, it is clear from these observations that the junction is a region in which release, spread, or both, of the virus may be facilitated. The fact that lesions at the junction were so widespread and were not recognized easily by clinical examination supports the idea that HSV recurrence may occur in humans without being recognized. Because the rejection response of the PVG strain to LEW and DA corneal grafts is relatively vigorous, these donor strains were chosen specifically to test the effect of the immune response to the transplant on the incidence and severity of recurrent disease. The findings that a similar incidence and pattern of recurrent disease occurred in allografts and isografts and that 3 of 4 reactivation events in allografts occurred by day 10 (the earliest time point that clinical rejection occurred) showed that the trauma of the transplant operation, rather than the rejection response, was the cause of recurrence. There is clinical evidence in humans that HSV recurrence induces rejection. 45 Unfortunately, because of the low incidence of recurrent disease, it was not possible to investigate this in the current study. However, we tested whether a previous HSV infection of the cornea could predispose a subsequent allograft to rejection by comparing the timing of rejection in latendy infected and mock-inoculated animals. We found no difference in survival between the two groups, despite histologic evidence of leukocyte accumulation in the tarsal conjunctivae of rats that had previous corneal HSV infection (data not shown). The overall pattern of recurrent disease after transplantation, including the location of virus within the cornea, is consistent with the hypothesis that trauma to neurons in this case, the severing of their peripheral termini during surgery caused reactivation of virus in the trigeminal ganglion and its release from nerve endings in the cornea. Although in the mouse model we have evidence that the ganglion is the most likely source of recurrent virus (unpublished data, 1995), we cannot exclude the possibility that virus could originate in the cornea. It has been shown that virus capable of producing disease is present in human eye bank corneas, 17 and we found HSV DNA by polymerase chain reaction in 2 of 5 rat corneal buttons removed at the time of transplantation (data not shown). However, 15 corneas from latendy infected animals transplanted to uninfected recipients failed to elicit HSV disease in recipients. Evidence of infection was sought by clinical examination of corneas, by testing for latent infection by co-cultivation, and by neutralizing antibody assay (data not shown). Furthermore, Openshaw et al 18 were unable to elicit disease in uninfected rabbits by transplanting corneas containing HSV DNA. Various stimuli that involve damage to nerves, including neurectomy, 19 are known to cause recurrent disease in humans and virus reactivation in mouse sensory ganglia. 20 ' 21 Excimer laser photokeratectomy causes virus shedding in mice. 22 In rabbits, trauma to corneal nerves (including nerve section 23 and penetrating keratoplasty) M causes virus shedding and recurrent corneal disease. In latently infected rabbits, spontaneous shedding of virus is relatively common (15.7% for the McKrae strain of virus 25 ). There was no spontaneous shedding in untransplanted controls in the current experiments, and a review of our accumulated data on untransplanted rats showed the incidence of such shedding to be only 0.1% (1 of 1041 tear samples tested). We have, therefore, now shown that transplantation can cause recurrent disease in a species in which spontaneous shedding is rare. If, as has been suggested, a crucial stimulus for

10 434 Investigative Ophthalmology & Visual Science, February 1996, Vol. 37, No. 2 reactivation is stress to the neuron, 26 there might be increased risk for reactivation after transplantation for as long as sensory neurites regenerate (respond to injury) within the cornea. This may account for the sporadic virus shedding from transplanted corneas in rats after the immediate postoperative period from days 15 to 30 (daily monitoring for virus shedding usually was not continued after day 30). Studies 5 ' 27 " 29 have shown convincingly that topical antiviral therapy, particularly in die first few months after transplantation, when the risk of recurrence and rejection is highest, significantly enhances the chance of graft survival. Our results, particularly the extensive subclinical virus activity we observed at the graft-host junction, support this therapeutic strategy and die use of aggressive antiviral therapy when rejection episodes occur. It is clear from our studies and from those in humans reported by others that virus antigen 7 and infiltrating cells 30 can be present in herpetic corneas without obvious signs of clinical disease. Moreover, infectious virus has been isolated from such corneas after 5- to 13-day periods of in vitro culture 31 and from the tears of humans and rabbits, also in the absence of clinical disease. 32 It may be of additional benefit to give antiviral drugs systemically during these high-risk periods. This would increase drug levels in the anterior chamber and iris and would be more likely to result in the achievement of therapeutic levels within the trigeminal ganglion, thereby preventing virus reaching the eye. One study 33 in rabbits and a recent small prospective human study 34 using oral acyclovir showed that this treatment does reduce HSV recurrence significandy and does reduce the incidence of corneal graft failure. The experimental value of diis rat model of recurrent disease is limited by the low level of recurrence. However, we have shown that transplantation is a stimulus of reactivation in a species in which spontaneous shedding of virus rarely occurs. Moreover, it has revealed for the first time in an experimental situation the susceptibility of the graft-host junction to recurrent disease. At this location, there may be extensive virus replication throughout the thickness of the cornea and around its circumference without clinical signs of disease. This supports the idea that HSV recurs in human corneal transplants more often than is realized. Key Words corneal transplantation, herpes simplex keratitis, immunocytochemistry, rat, ultraviolet light Acknowledgments The authors thank Amina Benylles for technical assistance and Esther Grinfeld for performing the polymerase chain reaction analysis of herpes simplex virus DNA referred to in the discussion. References 1. Epstein RJ, Seedor JA, Dreizen NG, et al. Penetrating keratoplasty for herpes simplex keratitis and keratoconus: Allograft rejection and survival. Ophthalmology. 1987;94: Ficker LA, Kirkness CM, Rice NSC, McG Steele AD. Longterm prognosis for corneal grafting in herpes simplex keratitis. Eye. 1988; 2: Williams KA, Roder D, Esterman A, Muehlberg SM, Coster DJ on behalf of all contributing surgeons. Factors predictive of corneal graft survival: Report from the Australian Corneal Graft Registry. Ophthalmology. 1992;99: Cobo LM, Coster DJ, Rice NSC, Jones BR. Prognosis and management of corneal transplantation for herpetic keratitis. Arch Ophthalmol. 1980; 98: Ficker LA, Kirkness CM, Rice NSC, McG Steele AD. The changing management and improved prognosis for corneal grafting in herpes simplex keratitis. Ophthalmology. 1989;96: Manez R, White LT, Linden P, et al. The influence of HLA matching on cytomegalovirus hepatitis and chronic rejection after liver transplantation. Transplantation. 1993; 55: Holbach LM, Font RL, Naumann GOH. Herpes simplex stromal and endothelial keratitis: Granulomatous cell reactions at the level of Descemet's membrane, the stroma, and Bowman's layer. Ophthalmology. 1990;97: Williams KA, Coster DJ. Penetrating corneal transplantation in the inbred rat: A new model. Invest Ophthalmol Vis Sci. 1985;26: Katami M, Madden PW, White DJ, Watson PG, Kamada N. The extent of immunological privilege of orthotopic corneal grafts in the inbred rat. Transplantation. 1989; 48: Nicholls SM, Benylles A, Shimeld C, Easty DL, Hill TJ. Ocular infection with herpes simplex virus in several strains of rat. Invest Ophthalmol Vis Sci. 1994; 35: Spruance SL. Pathogenesis of herpes simplex labialis: Experimental induction of lesions with UV light. J Clin Microbiol. 1985; 22: Perna JJ, Mannix ML, Rooney JF, Notkins AL, Straus SE. Reactivation of herpes simplex virus infection by ultraviolet light: A human model. J Am Acad Dermatol. 1987; 17: Shimeld C, Hill TJ, Blyth WA, Easty DL. An improved model of recurrent herpetic eye disease in mice. Curr Eye Res. 1989;8: Nicholls SM, Bradley BA, Easty DL. Effect of mismatches for major histocompatibility complex and minor antigens on corneal graft rejection. Invest Ophthalmol Vis Sci. 1991;32: WhitelandJL, Nicholls SM, Shimeld C, Easty DL, Williams NA, Hill TJ. Immunohistochemical detection of T-cell subsets and other leukocytes in paraffin-embedded rat and mouse tissue with monoclonal antibodies. JHistochem Cytochem. 1995;43: Rozsa AJ, Guss RB, Beuerman RW. Neural remodeling

11 Recurrent Herpes Simplex in Rats 435 following experimental surgery of the rabbit cornea. Invest Ophthalmol Vis Sri. 1983; 24: Cleator GM, Klapper PE, Dennett C, et al. Corneal donor infection by herpes simplex virus: Herpes simplex virus DNA in donor corneas. Cornea. 1994; 13: Openshaw H, McNeill JI, Lin XH, Niland J, Cantin EM. Herpes simplex virus DNA in normal corneas: Persistence without viral shedding from ganglia. JMed Virol. 1995; 46: Carton CA, Kilbourne ED. Activation of latent herpes by trigeminal sensory root section. N Engl J Med. 1952;246: Walz MA, Price RW, Notkins AL. Latent infection with herpes simplex virus type 1 and 2: Viral reactivation in vivo after neurectomy. Science. 1974; 184: McLennan JL, Darby G. Herpes simplex virus latency: The cellular location of virus in dorsal root ganglia and the fate of the infected cell following virus activation. JGen Virol. 1980;51: Pepose JS, Laycock KA, Miller JK, et al. Reactivation of latent herpes simplex virus by excimer laser photokeratectomy. Am J Ophthalmol. 1992; 114: Beyer CF, Hill JM, Reidy JJ, Beuerman RW. Corneal nerve disruption reactivates virus in rabbits latently infected with HSV-1. Invest Ophthalmol Vis Sri. 1990; 31: Beyer CF, Arens MQ, Hill JM, Rose BT, Hill GA, Lin DTC. Penetrating keratoplasty in rabbits induces latent HSV-1 reactivation when corticosteroids are used. CurrEyeRes. 1989;8: Hill JM, Rayfield MA, Haruta Y. Strain specificity of spontaneous and adrenergically induced HSV-1 ocular reactivation in latently infected rabbits. Curr Eye Res. 1987; 6: Fraser NW, Valyi-Nagy T. Viral, neuronal and immune factors which may influence herpes simplex virus (HSV) latency and reactivation. Microb Pathog. 1993; 15: Foster CS, Duncan J. Penetrating keratoplasty for herpes simplex keratitis. Am JOphthalmol. 1981;92: Witmer R. Results of keratoplasty in metaherpetic keratitis. In: Sundmacher R, ed. Herpetische Augenerkrankungen. Miinchen: Bergmann Verlag; 1981: Moyes AL, Sugar A, Musch DC, Barnes RD. Antiviral therapy after penetrating keratoplasty for herpes simplex keratitis. Arch Ophthalmol. 1994; 112: Knobel H, Hinzpeter EN, Naumann GOH. Keratoplastik bei Herpes corneae: Vergleich zwischen klinischem und histopathalogischem Befund an 100 Augen. In: Sundmacher R, ed. Herpetische Augenerkrankungen. Miinchen: Bergmann Verlag; 1981: In German. 31. Shimeld C, Tullo AB, Easty DL, Thomsitt J. Isolation of herpes simplex virus from the cornea in chronic stromal keratitis. BrJ Ophthalmol. 1982; 66: Kaufman HE, Brown DC, Ellison ED. Recurrent herpes simplex in rabbit and man. Srience. 1967; 156: Beyer CF, Arens MQ, Hill GA, Rose BT, Beyer LR, Schanzlin DJ. Oral acydovir reduces the incidence of recurrent herpes simplex keratitis in rabbits after penetrating keratoplasty. Arch Ophthalmol. 1989; 107: Barney NP, Foster CS. A prospective randomized trial of oral acyclovir after penetrating keratopasty for herpes simplex keratitis. Cornea. 1994; 13: