Moderation of Herpetic Stromal Keratitis by Basic Fibroblast Growth Factor

Exp. Eye Res. (1996), 63, 1–8

Moderation of Herpetic Stromal Keratitis by Basic Fibroblast Growth Factor D O R I T G A M U Sa, A M A L I A R O M A N Oa, M E N A C H E M R U B I N S T E I Nb    N A P H T A L I S A V I O Na* a

Maurice and Gabriela Goldschleger Eye Research Institute, Sackler Faculty of Medicine, Tel-Aviv University ; and b Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel (Received Cleveland 3 August 1995 and accepted in revised form 14 November 1995) The effect of basic fibroblast growth factor (bFGF) on the evolution of herpes simplex virus (HSV) infection in eyes of rabbits was investigated. Rabbit eyes were infected with HSV-1 by a non-invasive inoculation and treated for 7 days with an eye drop solution containing either bovine bFGF (50 ng ; three times daily), or bFGF diluent as control. The treatment started 2 hr, 24 hr or 96 hr post-inoculation (p.i.). Follow-up of clinical disease parameters, such as conjunctivitis, epithelial keratitis, stromal disease, corneal neovascularization and of viral isolation continued for 17 days. The most significant difference between bFGF and control treatments was observed in the development of stromal keratitis. The incidence of stromal disease in the bFGF treated group (2}16 eyes) was significantly lower than in the control group (11}12 eyes) (P ¯ 0±0001), when bFGF was administered 2 hr or 24 hr p.i. The severity of the disease developed in the bFGF treated eyes was also milder than in the control eyes (determined by serial slit-lamp clinical examinations and by histologic sections). Such effect was not demonstrated if the treatment started 96 hr p.i. The same duration of viral shedding was obtained with bFGF treated eyes (2 hr, 24 hr, or 96 hr p.i.) and control eyes. Neither HSV-1-infected, nor sham-inoculated bFGF-treated eyes demonstrated increased neovascularization of the cornea, as compared with the corresponding vehicletreated control eyes. This study demonstrates that bFGF treatment (starting 2–24 hr p.i.) decreased the occurrence and severity of herpetic stromal keratitis, without subsequent aggravation of corneal vascularization. This beneficial anti-inflammatory effect of bFGF may have future application in the treatment of the most devastating stage of herpetic corneal infection. # 1996 Academic Press Limited Key words : herpes simplex virus ; stromal keratitis ; basic fibroblast growth factor ; cornea ; inflammation.

1. Introduction Ocular herpes simplex virus (HSV) infections, a leading cause of corneal blindness around the world (Cloaue, Menage and Easty, 1988 ; Feng, 1990 ; Liesegang, 1991), present a major therapeutic challenge in ophthalmology. The infection is usually mild and self-limited. However, it is prone to recurrences, which may lead to stromal inflammation, resulting in corneal scarring (McGill, 1987 ; Wilhelmus, 1987 ; Cloaue, Menage and Easty, 1988). Moderation of herpetic damage to corneal stroma is, therefore, of considerable value in preserving visual acuity. The benefits of the conservative treatment of HSV-induced stromal disease by a combination of antiviral and steroid therapy are subject to controversy (McGill, 1987 ; O’Brien and Taylor, 1991), and the optimal management of herpes simplex stromal keratitis is still to be found. The effect of various HSV strains on the severity of stromal keratitis (Tullo et al., 1987 ; Grau, Visalli and Brandt, 1989 ; Hendricks and Tumpey, 1990) and the host’s immunopathologic reaction to herpetic antigens in the stroma have been extensively studied (Foster, * For correspondence at : Goldschleger Eye Research Institute, Sheba Medical Centre, 52621 Tel-Hashomer, Israel.

0014–4835}96}070001­07 $18.00}0

1989 ; Hendricks and Tumpey, 1990 ; Pepose, 1991). The mechanism of HSV progression from corneal epithelium to the stromal layer and the nature of the entire complex of factors involved in chronic stromal inflammation, however, are still not completely understood. Our previous research in which the influence of epidermal growth factor (EGF) on the evolution of herpetic ocular infection was studied, demonstrated aggravation of stromal keratitis by local treatment of rabbits eyes with murine EGF (Romano et al., 1994). In this study, the potential role of bFGF in modulating the severity of herpetic stromal disease was examined using the model of HSV-1 keratitis in rabbits. It was previously demonstrated that bFGF in topical application penetrates deepithelized rabbit cornea and accumulates in basal lamina, stroma and endothelium (Fredj-Reygrobellet et al., 1989). As HSV corneal infection is characterized by epithelial lesions, we assumed that bFGF would penetrate and might exert its effect in the areas of epithelial defects. 2. Materials and Methods Virus HSV-1 strain (R-555) was isolated from corneal lesions of a patient with bilateral recurrent herpetic # 1996 Academic Press Limited

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dendritic and stromal keratitis, propagated in VERO (African monkey kidney) cells and identified as type 1 by direct immunofluorescent staining, utilizing monoclonal antibodies for HSV-1 and HSV-2 (Syva Co., Palo Alto, CA, U.S.A.). The virus was purified by banding on 12–52 % (w}w) sucrose gradients and resuspended in Dubelcco’s modified Eagles medium (DMEM) with 2 % fetal bovine serum (FBS). A virus preparation with a titer of 10' plaque-forming units (as determined by plaque assay on VERO cells) was used in this study.

Animal Inoculation and Treatment Twenty adult albino rabbits, weighing 2±5–3 kg were used in compliance with the ARVO resolution on the Use of Animals in Research. The rabbits were examined by slit-lamp biomicroscopy before inoculation. None of the rabbits demonstrated prior corneal defects. HSV inoculation was performed by the following non-invasive procedure : 50 µl of the stock suspension were applied locally to the inferior cul de sac, the eyelids were then closed and gently massaged. This procedure was repeated four times for each inoculated eye over a total period of 5 min. Sham infected eyes were inoculated with DMEM with 2 % FBS (by the above procedure). This technique was adopted in order to avoid any mechanical damage and}or direct viral inoculation to the corneal stroma, as may result from the use of scarification procedure.

bFGF Highly purified bFGF was prepared from bovine brain and characterized as previously described (Gospodarowitcz et al., 1984). The bFGF was determined by sodium-dodecyl-sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) as a single band with a molecular weight of 18 kDa. The activity of bFGF was tested in vitro on bovine vascular endothelial cells and maximal proliferation was observed at a concentration of 1 ng ml−". A bFGF solution of 1 µg ml−" in phosphate buffered saline (PBS), supplemented with 1 % heat-inactivated rabbit serum (HI RS) was prepared and used as topical eye drops (50 µl per application). This dose (50 ng) is within the previously reported range of bFGF concentrations used in several experimental corneal wound healing models (FredjReygrobellet et al., 1987 ; Landshman et al., 1987 ; Knighton, Phillips and Feigel, 1990 ; Rieck et al., 1992). Single application of the "#&I-bFGF (23 ng) on denuded rabbit cornea results in early fixation (10 min) of bFGF, followed by a second accumulation in the anterior segment 8 hr after the application, and then gradual decrease for 48 hr. Our treatment management included therefore three applications of bFGF a day in order to continuously maintain a high level of bFGF in the anterior segment.

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Experimental Design Rabbits were randomly divided into four groups as follows : group A, 12 HSV-1-infected eyes were treated with 50 µl of bFGF (1 µg ml−"), three times daily, starting 2 hr p.i., for 7 days ; group B (control), 12 HSV-1 infected eyes received 50 µl PBS with 1 % HI RS (bFGF diluent) ; groups C and D, four eyes in each group were infected as described above, bFGF treatment starting after 24 hr and 96 hr, respectively. Sham-inoculated eyes received either bFGF treatment (four eyes) or the diluent (four eyes). The number of eyes in groups A and B is the sum of two independent experiments. Disease Scoring Rabbit eyes were examined daily for the first 7 days p.i. and on alternate days for the next 10 days. Slitlamp observations were made by the same investigator, and confirmed by an unbiased experienced ophthalmologist on days 9 and 17. Conjunctivitis was scored as follows : 0, no pathology ; 1, mild hyperemia and secretion ; 2, moderate hyperemia and mild purulent discharge ; 3, moderate discharge ; and 4, severe discharge or eye totally shut. Epithelial keratitis was scored after corneal staining with fluorescein : 0, no pathology ; 1, ! 25 % of the cornea involved ; 2, 25–50 % of the cornea involved ; 3, 50–75 % of the cornea involved ; and 4, " 75 % of the cornea involved. Stromal disease was scored : 0, no pathology ; 1, detectable corneal edema, iris details clearly visible ; 2, gross corneal edema, iris details still distinct ; 3, pupillary border no longer distinct ; and 4, opaque cornea, anterior chamber structures not visible. Vascularization was scored : 1, ! 25 % of the cornea involved ; 2, 25–50 % involved ; and 3, " 50 % of the cornea involved. Data were reported as mean disease score for each group of rabbits, on each day of observation. HSV-1 Isolation from Tears Tears were collected on the examination days on sterile cotton swabs and titrated by plaque formation on VERO cells. Neutralizing Antibodies Rabbit serum was collected before inoculation and at the end of the experiment (day 17 p.i.). HSV-1 neutralizing titers were determined by the plaque reduction assay (Tokumaru, 1969). Histologic Examination A randomly selected rabbit in each group was killed by an i.v. overdose of pentobarbitone sodium 6, 9 and 17 days after initiation of the experiment. The eyes

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were enucleated, fixed and stained with hematoxylin and eosin. The evaluation was performed in a masked fashion by an unbiased observer. Statistics Significant differences in the disease scoring were determined by day after day Chi square analysis. The Fisher’s exact test was used to determine significant differences in the proportion of eyes that developed each of the evaluated parameters of herpetic ocular disease (on a daily basis). 3. Results Time Course of the Disease Rabbit eyes were infected with HSV-1 (2¬10& pfu per eye) by a non-invasive inoculation. Analysis of the various disease parameters was performed during the 17 days p.i. period (Fig. 1). 2.00 (A)

Mean disease score

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F. 2. Histology of HSV-infected rabbit cornea treated with vehicle (A) or bFGF (B) 6 days p.i. Note loss of polarity of basal epithelial cells and the inflammatory cell infiltration of the superficial stromal layer underlying an epithelial lesion in the vehicle-treated eye (A), vs. relatively well conserved structural morphology of the corneal epithelium and the absence of stromal keratitis in the bFGF-treated eye (B). Hematoxylin and eosin stain, magnification : ¬100.

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Mean disease score

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F. 1. Development of ocular herpetic disease—a time course. Rabbit eyes were infected with HSV-1 (2¬10& pfu) by a non-invasive inoculation and treated with vehicle (*) or with bFGF (+), starting 2 hr p.i. Results are reported as mean disease scores of epithelial keratitis (A) and stromal keratitis (B). * P ! 0±05 ; ** P ! 0±005.

In the virus-infected eyes (both vehicle control and bFGF treated), signs of conjunctival involvement were first apparent between days 2 and 3 p.i. reaching a peak by days 4–5 and gradually subsiding by day 17. Epithelial keratitis [Fig. 1(A)] developed in all infected eyes during the follow-up period. In most of the eyes, the disease was first evident between days 3 and 4 p.i. A significant difference in the development of stromal keratitis between treatment groups A and B was observed : while 11}12 of the vehicle treated eyes demonstrated stromal disease during the follow-up period, only 2}12 of the bFGF treated eyes demonstrated stromal involvement (P ! 0±0006 by twotailed Fisher exact test). The severity of the vehicle treated eyes ranged from moderate to severe keratitis, and two eyes demonstrated corneal abscess with endophthalmitis. When the occurrence and intensity of stromal disease were evaluated on the basis of day by day analysis, both parameters were significantly lower (P ! 0±05) in the bFGF treated eyes (group A) on days 7–15 p.i. than in the vehicle treated eyes (group B) [Fig. 1(B)]. On day 17 analysis of the above

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Histopathologic Findings

F. 3. Histology of HSV-infected rabbit cornea treated with vehicle (A) or bFGF (B) 9 days p.i. Both sections demonstrated similar epithelial defects. Note mild, diffuse inflammatory response in the stroma of bFGF-treated eye (B), vs. superficial and deep acute keratitis in the vehicletreated eye (A). Hematoxylin and eosin stain, magnification : ¬100.

The difference in corneal pathology between the two treatment groups was first apparent on day 6 p.i. (Fig. 2). Vehicle-treated eyes demonstrated a marked epitheliopathy with underlying superficial stromal keratitis. Epitheliopathy manifested itself by loss of polarity of basal cells of the corneal epithelium, cloudy swelling of the cytoplasm of deep and superficial epithelial cells and rounding up with falling out of some of the epithelial cells [Fig. 2(A)]. Although basal epithelium of bFGF treated eyes was edematous, the loss of polarity was mild, corneal epithelium maintained its normal structure, and no evidence of stromal keratitis could be demonstrated [Fig. 2(B)]. These differences became clearly evident 9 days after virus inoculation (Fig. 3) : while the bFGF treated group demonstrated a mild stromal inflammation [Fig. 3(B)], the vehicle treated corneas presented moderate superficial and deep stromal keratitis [Fig. 3(A)]. At the end of the follow-up period (17 days) the placebo treated corneas still demonstrated a considerable stromal inflammation [Fig. 4(A)], while the bFGF treated eyes showed a clear cornea [Fig. 4(B)]. The effect of bFGF on the development of stromal disease, when administrated at later time points p.i., was studied in groups C and D. Treatment was

parameters yielded P ¯ 0±086 and P ¯ 0±0821, respectively. Vascularization of the cornea was first evident on day 6 or 7 p.i. and culminated on days 11 to 13, followed by a slight decline towards the end of the experiment. Vascularization scores were also generally smaller in the bFGF treated group, though a statistically significant difference between the above groups could not be demonstrated. None of the sham inoculated rabbit eyes (whether treated with placebo or bFGF), showed signs of conjunctivitis, keratitis or corneal vascularization during the follow-up period. Virus Isolation and Neutralizing Antibodies Positive viral isolations in tears were first detected on day 1 p.i. and disappeared by day 11 in both bFGF and placebo treated eyes. There was no significant difference in the mean duration of virus shedding between the two groups : 7±9 days (placebo) and 8±2 days (bFGF). The titers of HSV-1 neutralizing antibodies were also similar between the two treatment groups : 1}2048 (bFGF) and 1}2048–1}4096 (vehicle control).

F. 4. Histology of HSV-infected rabbit cornea treated with vehicle (A) or bFGF (B) 17 days p.i. (A) : Flattened epithelium, superficial and deep keratitis in the placebo treated eye (B) : Flattened epithelium, in the bFGF treated eye, the corneal stroma does not appear inflamed. Hematoxylin and eosin stain, magnification : ¬100.

MODERATION OF HERPETIC KERATITIS BY bFGF

initiated 24 hr (group C) or 96 hr (group D) postinoculation, and the follow-up was as described above. None of the eyes (0}4) treated with bFGF 24 hr p.i. developed stromal keratitis. However, when treatment was delayed until day 4 after the HSV infection, the development of stromal keratitis in all eyes was evident (4}4) and similar to the control group. 4. Discussion The results of this study show, for the first time, the beneficial effect of bFGF treatment on herpetic infection in the in vivo experimental model of HSV-1 keratits in rabbits. bFGF attenuated stromal disease as demonstrated both by clinical (Fig. 1) and by histologic (Figs 2–4) evaluations. The protective effect of bFGF was still evident when the treatment commenced within 24 hr p.i. but not at 96 hr p.i. The ability of bFGF to attenuate the herpetic stromal keratitis is comparable with other treatment modalities in experimental models of HSV keratitis. Passive immunization of mice with anti-glycoprotein D (anti-gD) monoclonal antibodies provided protection against stromal but not against epithelial keratitis (Inoue et al., 1992), and was effective when administered for up to 24 hr after virus inoculation (Inoue et al., 1992) with no significant effect on HSV clearance from the eye (Laush et al., 1989). Furthermore, local antiviral treatment of rabbit stromal keratitis by trifluorothymidine and vidarabine monophosphate was also most effective when treatment was initiated on day 1 p.i. (McNeill and Kaufman, 1979). Therefore, it is suggested that moderation of stromal inflammation within 24 hr p.i. reflects the existence of a critical time-point in the dynamics of disease progression from epithelium to the stromal layer and of subsequent involvement of immune response. The mechanism by which bFGF attenuates stromal keratitis is not clear. However, recent controversy concerning the involvement of bFGF cellular receptor in HSV infection is well documented (Baird et al., 1990 ; Kaner et al., 1990 ; Lycke et al., 1991 ; Dix, Hurst and Keane, 1992 ; Mirda et al., 1992). Current data indicate that bFGF inhibits HSV infection by competitive binding to low-affinity binding sites, consisting of cell surface heparan sulfate, but not to the high-affinity FGF receptor (WuDunn and Spear, 1989 ; Lycke et al., 1991 ; Mirda et al., 1992). Our observations probably did not result from direct inhibition of HSV binding by bFGF for the following reasons : (i) the concentration of bFGF used in our study was much lower than the comparable in vitro concentrations (5–100 n) used by Kaner et al. (1990) and Mirda et al. (1992) ; (ii) while bFGF reduced the incidence and severity of stromal inflammation, the course of conjunctival and epithelial diseases was not significantly affected ; (iii) viral shedding in the tears of infected rabbit eyes and serum neutralizing antibodies titers were similar in the bFGF

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treated and control animals ; and (iv) no protective effect of bFGF on HSV replication was seen in cultured human corneal fibroblasts using bFGF concentration equivalent to our in vitro experiment (unpublished data). Furthermore, topical heparin treatment promoted the healing of epithelial ulcers of experimental HSV-1 keratitis without any beneficial effect on HSV ocular shedding or on the development of stromal disease (Rieck et al., 1994). The most prominent effect of bFGF treatment in our study was moderation of the inflammatory response in corneal stroma. This observation is in concert with a previously reported anti-inflammatory effect of bFGF in another experimental model. Minipigs eyes perfused with PBS demonstrated marked inflammatory reaction, identified by a macrophage specific antibody. The addition of bFGF (but not of denaturated bFGF) to the perfusion solution diminished this inflammatory response (Soubrane et al., 1994). The results of the present research and of our previous study (Romano et al., 1994) demonstrate a remarkable effect of growth factors on the evolution of herpetic keratitis. It is of interest to note that the major impact of EGF and bFGF was on the development of stromal inflammation (whereas EGF aggravated the disease, bFGF diminished stromal damage). The possible mechanisms involved in the effects of these two growth factors on the evolution of HSV-1 induced stromal keratitis remains to be investigated. It is possible that the already known differences in the effect of EGF and bFGF on the proliferative response of corneal epithelium, keratocytes and stromal wound healing (Gospadorowicz et al., 1977 ; Woost et al., 1985 ; Tripathi, Kwait and Tripathi, 1990), may also exert differential influence on the permissiveness of corneal cells to HSV infection. It was also previously suggested that these two cytokines may differ in their effects on immune-mediated processes (Meyers-Elliott et al., 1981 ; Tripathi, Raja and Tripathi, 1990). Whether the decrease in the inflammatory response of the cornea observed by us in HSV-1 infected rabbit eyes treated by bFGF is due to inhibition of macrophage infiltration (Soubrane et al., 1994) or through the recently reported regulatory processes of ocular cytokines (Li and Tseng, 1994 ; Rosenbaum, 1993) is still unclear and requires further investigation. In spite of two limitations inherent to our study : only one HSV-1 strain was used and the relatively small number of infected eyes treated at later time points of the disease (24 hr p.i., 96 hr p.i.), the moderation of HSV-induced inflammatory damage to corneal stroma by bFGF is evident. No adverse effect of bFGF on the cornea (such as excessive vascularization), either in the infected or in the sham-inoculated eyes could be demonstrated. The very selective and stable binding of bFGF to the injured cornea (FredjReygrobellet et al., 1989) and the results of our study indicate that bFGF treatment of experimental HSV infections and other immune-mediated processes

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should be further investigated. Better understanding of the complex dialogues between cellular growth factors and inflammatory cytokines may be highly beneficial in minimizing the deleterious consequences of corneal inflammatory diseases.

Acknowledgements The authors wish to thank Professor Y. R. Barishak for pathology expertise and A. Lusky (M.Sc.) for her assistance in statistical analysis. This work is in partial fulfillment of the requirements for the Ph.D. degree of D. Gamus from the Sackler Faculty at Tel-Aviv University.

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isolates of herpes simplex virus causing different forms of keratitis in man. Curr. Eye Res. 6, 33–8. Wilhelmus, K. R. (1987). Diagnosis and management of stromal keratitis. Cornea 6, 286–9. Woost, P. G., Brighwell, J., Eiferman, R. A. and Schultz, G. S. (1985). Effect of growth factors with dexamethasone on healing of rabbit corneal stromal incisions. Exp. Eye Res. 40, 47–60. WuDunn, D. and Spear, P. G. (1989). Initial interaction of herpes simplex virus with cells is binding to heparan sulfate. J. Virol. 63, 52–8.