Original Articles. Experimental disciform edema and necrotizing keratitis in the rabbit. Joseph F. Metcalf, James I. McNeill, and Herbert E.

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1 Original Artiles Experimental disiform edema and nerotizing keratitis in the rabbit Joseph F. Metalf, James I. MNeill, and Herbert E. Kaufman The development of experimental disiform edema and nerotizing keratitis in the orneas of rabbits following intrastromal inoulation with the RE strain of herpes simplex virus is desribed. Following an initial episode of onjuntivitis and epithelial keratitis, a mild, entrally loalized, stromal edema developed on the fifth day. Stromal edema, opaifiation, and neovasularization of the ornea reahed maximum severity on the seventh to twentyseond day, and began to fade in most eyes thereafter. On the twenty-ninth day most orneas have attained a resolved state haraterized by subepithelial granular opaities. Several eyes were observed whih developed entral nerotizing keratitis. Marked similarities between the animal model and human herpeti stromal keratitis were apparent. Histologial observations show that early nerotizing keratitis in the rabbit is haraterized by an infiltration of plasma ells and lymphoytes in the limbus, with polymorphonulear leukoytes, lymphoytes, and marophages in the entral ornea. Key words: herpes simplex virus, animal model (rabbit), ornea (rabbit), biomirosopy, histology, herpeti keratitis. H,.erpes simplex virus (HS) auses natural disease only in man, but a wide variety of laboratory animals and ell ultures are readily infeted experimentally. 1 The ornea of the rabbit has proved to be an exellent model system in whih to study From the Department of Ophthalmology, College of Mediine, University of Florida. Supported in part by NIH Grants EY-158, EY-33, and EY-7 from the National Eye Institute. Presented in part at the Fall Meeting of the Assoiation for Researh in ision and Ophthalmology, Atlanti Setion, Nov., 1975, Duke University Eye Center. Submitted for publiation Feb. 25, Reprint requests: Dr. J. F. Metalf, Department of Ophthalmology, College of Mediine, Box J-284, J. Hillis Miller Health Center, Gainesville Fla the indution of herpeti keratitis, 2 " 1 and evaluate antiviral drugs for use in treating human patients. 5 ' li Appliation of a suspension of HS to the orneas of rabbits results in the appearane of puntate lesions whih are soon followed by the development of dendriti and geographi ulers and edema of the epithelium. 7 ' s The virus may subsequently invade the stroma and endothelium of the ornea as well as the anterior hamber. The dissemination of the virus in these tissues has been desribed. <J11 A delayed ompliation of herpeti keratitis in human beings is a deep stromal disease with disiform edema. 12 The milder form of the disease, haraterized by loalized edema and opaifiation, may persist for 2 to 3 months and then heal with

2 98 Metalf, MNeill, and Kaufman Investigative Ophthalmology Deember 1976 Table I Fig. 1. Intrastromal injetion in eye of rabbit. The injeted fluid lears within 24 hours without residual sarring exept the needle trat. little residual sarring. More severe forms of stromal keratitis are frequently seen with dense heesy opaities in the orneal stroma. This type of lesion is aompanied by nerosis of stromal ollagen fibers leading to the formation of permanent sarring. asularization of the ornea is assoiated with the nerotizing keratitis. 13 Histologially, stromal keratitis is haraterized by an infiltration of polymorphonulear leukoytes and marophages. This initial inflammatory reation is replaed by an invasion of lymphoytes and plasma ells, with sattered marophages. 13 Severe uveitis, hypopyons, irregular kerati preipitates, and seondary glauoma are ompliations of herpeti keratitis that may be observed during the ourse of the disease. 12 " 14 Animal models of herpeti stromal keratitis have long been sought for the purpose of studying the etiology of the disease and development of an appropriate therapeuti program. Williams and assoiates 15 desribed the indution of experimental disiform keratitis by several strains of HS, but these viruses have either been lost or no longer have the apaity to indue the disease in the rabbit ornea. Stromal edema resembling that seen in herpeti keratitis has also been demonstrated in guinea pigs and rabbits following the orneal injetion of soluble HS antigens into previously Group A D E Rabbit OS. * v* * * Eye O.D. * * * v* C = Human embryoni kidney ell lysate. = HS strain RE in ell lysate. = No injetion. * = irus suspension heated at 6 C. for 6 minutes. sensitized animals. 18 ' 17 However, these experimental models do not show the nerotizing keratitis with destrution of orneal stroma that leads to blindness in human stromal herpes. 12 Sery and assoiates 13 ' 19 were unsuessful in induing disiform edema or nerotizing keratitis with several widely divergent strains of HS. However, Irvine and Kimura- have reported that the RE strain of Herpesvirus hominis has a strong propensity for induing stromal keratitis in rabbits without inurring a high mortality from enephalitis. In this report we desribe biomirosopi and histologial observations showing that the RE strain of HS provides a simple, reproduible model of disiform edema and nerotizing keratitis, presenting a linial syndrome similar to the stromal disease observed in human patients. Materials and methods irus strain. The RE strain of HS was obtained from Dr. Chandler R. Dawson (San Franiso) and stored at -7 C. Prior to use, the virus was passed through human embryoni kid-

3 olume 15 Number 12 Disiform edema and nerotizing keratitis DAYS POST INFECTION Fig. 2. Inidene and duration of epithelial keratitis, disiform edema, and nerotizing keratitis in a rabbit model. ney ells (Mirobiologial Assoiates, In., Bethesda, Md.) and harvested when the ells were 75 per ent infeted as determined by observation of ytopathi effet. The infeted ell ulture was frozen at -7 C. overnight, then thawed and dispensed into vials ontaining 1 ml. eah of infeted ell lysate. The titer of the virus suspension, plated on tube ulture of human embryoni kidney ells, was 1 3 plaque-forming units per milliliter. A ulture of human embryoni kidney ells without virus was treated similarly, and a vial of the virus suspension was heated at 6 C. for 6 minutes, for injetion of ontrol animals. Inoulation of rabbit orneas. New Zealand White rabbits weighing 2 to 3 kg. were used. The animals were given intramusular injetions of hlorpromazine (Thorazine) (25 mg. per kilogram) 1 hour prior to inoulation of the orneas. Following a loal appliation of proparaaine (Ophthaine), the virus suspension, or ell lysate, was injeted into the orneal stroma with a 27- gauge needle attahed to a tuberulin syringe (Fig. 1). The injetion produed a bleb 3 to 4 mm. in diameter within the stroma. The average volume of fluid injeted was.2 ml. Five groups of four rabbits were injeted as shown in Table I. Biomirosopy. Biomirosopi observations were made on eah eye at regular intervals over a period of 63 days. A Mentor slit-lamp biomirosope with white illumination was used to evaluate stromal edema, and observe the endothelium, lens, and iris. Fluoresein was plaed on the ornea and observations were made with blue light to desribe epithelial lesions. Histology. Four additional rabbits with early nerotizing keratitis were sarified at 3 weeks postinfetion. The orneas were fixed in 4 per ent formaldehyde in phosphate buffer, embedded in paraffin, setioned, and stained with hematoxylin and eosin. Results Biomirosopi observations. The uninjeted orneas of animals whih reeived virus in the ontralateral eye remained lear throughout the 63 day period of observation. All of the orneas whih were injeted with ell suspension or heat-killed virus progressed through normally expeted healing stages for an intrastromal injetion, with no unusual inflammation and no permanent sequelae exept small injetion sars. Observations of the orneas of animals whih reeived intrastromal injetions of the RE strain of HS (16 eyes) are desribed for representative days of observation. The inidene and duration of orneal disease, without regard to severity, are shown graphially in Fig. 2. Following an initial episode of puntate and dendriti

4 982 Metalj, MNeill, and Kaufman Investigative Ophthalmology Deember 1976 Fig. 3. Charateristi disiform edema and haze (seventh day). Note the absene of onjuntivitis. Fig. 4. Early nerotizing keratitis in the rabbit, showing entral orneal opaifiation with neovasularization (eighteenth day). Fig. 5. Chroni stromal nerotizing keratitis (healed) resulting in permanent orneal opaity. keratitis whih rapidly lear, a high inidene of loalized stromal edema appears whih is maintained for about 2 weeks, between the seventh and twenty-seond day. Although the stromal edema resolves in most eyes, nerotizing keratitis develops in about 25 per ent of the inflamed eyes, resulting in permanent sarring and vasularization of the ornea. Stromal edema was graded as mild if slight edema with or without early infiltration was observed; as moderate if gross louding, edema, and infiltration were present with the pupillary border seen indistintly; and as severe if the ornea was totally opaque, with edema and infiltration. Day 1. The orneas were lear with little evidene of inflammation exept needle trats. Day 3. A sant serous or muopurulent onjuntivitis was present, and a diffuse, early (irregular), puntate keratitis with a few small dendriti figures was noted. Day 5. A sant to moderate muopurulent onjuntivitis was present. A moderate keratitis with a few dendriti figures, and the first mild, entral stromal edema were observed. Day 7. Both the onjuntivitis and epithelial keratitis were resolving, and entral stromal edema was learly evident in most eyes (Fig. 3). Day 1. The puntate and dendriti keratitis was leared, but some epithelial edema was present. A moderate to severe entral stromal edema was learly evident in most eyes. A small hypopyon was observed in one eye. Day 12, A moderate to severe entral stromal edema was present in all of the eyes exept three, in whih the edema was mild. A mild to moderate epithelial edema was also seen in four eyes. Day 15. A mild to moderate stromal edema was observed. The epithelial edema was leared. The first peripheral neovasularization was noted in three eyes. Two orneas were observed in whih the stromal disease had resolved to subepithelial granular opaities.

5 olume 15 Number 12 Disiform edema and nerotizing keratitis 983 «* Fig. 6. Aumulation of plasma ells and lymphoytes at the limbus. Day 18. Three eyes were observed with advaning peripheral vasularization nearing the entral third of the ornea (Fig. 4). A mild to moderate stromal edema was evident in most eyes. Two eyes with endothelial kerati preipitates were observed. Day 22. The stromal edema appeared to be fading in most eyes, and the neovasularization was unhanged. About one-third of the eyes showed the subepithelial granular opaities harateristi of the resolving state. One eye with kerati preipitates was still present. Day 29. Two eyes were observed with entral stromal nerosis. Neovasularization extended to the neroti area. In the remaining eyes the vasularity was stable or fading and the stromal edema was mild. About one-half of these eyes were in the resolved state with subepithelial granular opaities. Day 35. Eyes with stromal nerosis were unhanged. The remaining eyes were in the resolved state haraterized by subepithelial granular opaities in the entral ornea, but were otherwise lear. Day 63. Several eyes were lear, but most remaining eyes showed the finely granular subepithelial opaities desribed earlier. An example of hroni stromal nerotizing keratitis (healed) is shown in Fig. 5. Histologial observations. The orneas of rabbits sarified when early stromal nerosis was present (21 days) showed a heavy aumulation of plasma ells and lymphoytes at the limbus (Fig. 6). In the entral areas of the ornea two types of inflammatory reation were found. Large subepithelial foi of neroti stroma were found, ontaining a ellular infiltrate omposed of a mixed population of lymphoytes and marophages with sattered polymorphonulear (heterophili) leukoytes and plasma ells (Fig. 7). Other foi were found in whih the infiltrating ells were almost entirely omposed of polymorphonulear leukoytes (Fig. 8). Many of these ells appeared to be in various stages of disintegration, onsistent with the role of polymorphonulear leukoytes in releasing proteolyti enzymes after death. Inflammatory ells were also found sattered throughout the stroma.

6 984 Metalf, MNeill, and Kaufman Investigative Ophthalmology Deember 1976 t ^ Fig. 7. Area of entral stroma infiltrated with a mixed population of inflammatory ells, inluding lymphoytes and marophages. m* C *»9 - # Fig. 8. Area ot entral stroma showing an infiltration ot polymorphonulear leukoytes.

7 olume 15 Number 12 Disiform edema and nerotizing keratitis 985 Disussion The biomirosopi and histologial observations desribed in this report indiate that injetion of the RE strain of HS into the orneal stroma of the rabbit eye leads to the development of a harateristi herpeti stromal keratitis syndrome, very similar to that desribed by oular pathologists. The appearane of typial kerati endothelial preipitates and an oasional hypopyon and neovasular invasion of the ornea are all linial symptoms observed in human patients with stromal herpes. 12 " 14 The failure of the disease to resolve in some eyes, whih beame neroti and ulerative instead, is suggestive of nerotizing keratitis as seen in man. The presene of heterophils, plasma ells, marophages, and lymphoytes in the orneas of experimental animals indiates that a omplex inflammatory reation is assoiated with the indution of herpeti stromal keratitis in the rabbit. Further definition of histologial and pathologial events during the early stages of the disease, studies not possible in human patients, ould provide a better understanding of the pathogenesis of this blinding disease and may lead to improved linial management. REFERENCES 1. Darlington, R. W., and Granoff, A.: Repliation biologial aspets. In Kaplan, A. S., editor: The Herpesviruses, New York, 1973, Aademi Press, In., p Irvine, A. R., and Kimura, S. J.: Experimental stromal herpes simplex keratitis in rabbits, Arh. Ophthalmol. 78: 654, an Horn, D. L., Edelhauser, H. F., and Shultz, R. O.: Experimental herpes simplex keratitis, Arh. Ophthalmol. 84: 67, Tanaka, N., and Togni, B.: Further investigations by eletron mirosopy of herpes simplex virus in the orneal stroma of rabbits with experimental keratitis, Exp. Eye Res. 7: 142, Kaufman, H. E., Nesburn, A. B., and Maloney, E. D.: IDU therapy of herpes simplex, Arh. Ophthalmol. 67: 583, Sugar, J. arnell, E., Centifanto, Y., et al.: Trifluorothymidine treatment of herpeti iritis in rabbits and oular penetration, INEST. OPHTHALMOL. 12: 532, Spener, W. H., and Hayes, T. L.: Sanning and transmission eletron mirosopi observations of the topographi anatomy of dendriti lesions in the rabbit ornea, INEST. OPHTHALMOL. 9: 183, Hoffmann, F., Dumitresu, L., Haase, J., et al.: Keratitis dendritia unter dem Raster- Elektronenmikroskop, Graefes Arh. Klin. Exp. Ophthalmol. 193: 153, Oh, J. O.: Endothelial lesions of rabbit ornea produed by herpes simplex virus, INEST. OPHTHALMOL. 9: 196, Kaufman, H. E., Kanai, A., and Ellison, E. D.: Herpeti iritis: demonstration of virus in the anterior hamber by fluoresent antibody tehniques and eletron mirosopy, Am. J. Ophthalmol. 71: 465, Maloney, E. D. and Kaufman, H. E.: Dissemination of orneal herpes simplex, IN- EST. OPHTHALMOL. 4: 872, Pettit, T. H.: Herpes simplex keratitis: urrent onepts. In Beker, B., and Burde, R. M., editors: Current Conepts in Ophthalmology, St. Louis, 1969, The C.. Mosby Company, vol. 2, p Hogan, M. J., and Zimmerman, L. E.: Ophthalmi Pathology, Philadelphia, 1962, W. B. Saunders Co., p Yanoff, M., and Fine, B. S.: Oular Pathology, Hagerstown, Md., 1975, Harper & Row, Publishers, p Williams, L. E., Nesburn, A. B., and Kaufman, H. E.: Experimental indution of disiform keratitis, Arh. Ophthalmol. 73: 112, Swyers, J. S., Laush, R. N., and Kaufman, H. E.: Corneal hypersensitivity to herpes simplex, Br. J. Ophthalmol. 51: 843, Meyers, R. L., and Pettit, T. H.: The pathogenesis of orneal inflammation due to herpes simplex virus. I. Corneal hypersensitivity in the rabbit, J. Immunol. Ill: 131, Sery, T. W., Rihman, M. W., and Nagy, R. M.: Experimental disiform keratitis. I. Immune response of the ornea to herpes simplex virus, J. Allergy 38: 338, Sery, T. W., Nagy, R. M., and Nazario, R.: Experimental disiform keratitis. II. Loal orneal hypersensitivity to a highly virulent strain of herpes simplex, Ophthalmol. Res. 4: 99, 1972.