Br. J. exp. Path. (I987) 68, 585-593

The histology of the eye after zosteriformspread of herpes simplex virus in the mouse

Charles Claoue, Tim Hodges, Terence Hillt, William Blytht and David EastyDepartment of Ophthalmology and tDepartment of Microbiology, University of Bristol, The Medical School,

University Walk, Bristol BS8 iTD, UK

Received for publication I 2 February I 98 7Accepted for publication 26 February I 98 7

Summary. A murine model of zosteriform spread of herpes simplex virus to the eye is reported.Viral antigens were demonstrated by a peroxidase-antiperoxidase technique in the trigeminalganglion, iris, ciliary body, and cornea. The histology suggested a spontaneous bacterialsuperinfection during the later stages of the herpetic disease of the cornea. The relevance tohuman disease is discussed.

Keywords: herpes simplex virus, keratouveitis, bacterial superinfection, viral antigens,peroxidase anti-peroxidase technique

Herpetic keratitis is a condition commonlyseen by ophthalmologists, and in manyinstances significant visual handicap resultsfrom corneal scarring and endothelialdecompensation. These cases require cornealtransplantation which is not always success-ful in terms of visual rehabilitation due torecurrent disease or graft rejection.The pathogenesis of recurrent herpetic

keratitis is poorly understood, and althoughshedding of virus can be induced in latentlyinfected animals (Willey et al. I984), there isno suitable animal model of recrudescentdisease of the eye. Furthermore, most pre-vious workers have used corneal inoculationof herpes simplex virus (HSV), a mode ofinfection which is not thought to occurfrequently in human herpetic eye disease.

Only recently has the significance of thefrequent occurrence of zosteriform spread inprimary infection with HSV been appreciated(Blyth et al. I984; Simmons & Nash I984;

Correspondence: Dr C. Claoue, Department ofSEI 7EH, UK.

Wildy & Gell I 98 5). Such spread permits theinduction of herpetic infection of the corneawithout first damaging the eye by directinoculation of virus. Thus, the use of miceinoculated with virus onto the skin of thesnout (an area within the ocular derma-tome) has some advantages over direct cor-neal inoculation, but does not fully recreatethe situation found in human disease(Shimeld et al. I985; Claoue I986).

Materials and methods

Mice. Four-week-old male inbred NIH strainmice bred in the Department of Microbiologywere used. Mice were kept on shreddednewspaper to avoid sawdust damage to theeye. Ocular examinations and inoculationwere carried out under anaesthesia pro-duced by intraperitoneal pentobarbitone.Eyes were examined using a Zeiss slit-lamp

Ophthalmology, St Thomas' Hospital, London,

585

biomicroscope; animals with abnormal eyeswere excluded before inoculation.

Inoculation. The left-hand side of the snoutwas shaved over an area approximately 6mm2 and then scarified io times with a 25gauge hypodermic needle through a io juldrop of I99 medium containing IO5 pfu. ofHSV strain SCi6 (Hill et al. I975). Stockvirus was titrated on VERO cells and stored at- 70°C. Control animals were inoculatedwith mock inoculum prepared from unin-fected VERO cells by the same method asused to make virus suspensions.

Histological methods. The left trigeminalganglion and the left eye were dissected free,fixed in io% formalin and embedded inparaffin wax. Since cervical dislocation occa-sionally caused hyphaemas, eyes were enuc-leated under deep general anaesthesia andthe mice killed imediately. Five micrometresections were cut for staining with haema-toxylin and eosin or a peroxidase-antiperoxi-dase (PAP) technique (adapted from Stern-berger et al. I970) against herpes simplexantigens. Antisera (Dakopatts a/s, Denmark)were: (i) rabbit immunoglobulin to HSVtype i (Maclntyre, Code BI I 4) diluted I: 90in phosphate buffered saline; (2) swineimmunoglobulins to rabbit immunoglobu-lins (Code ZI96) diluted I:40; and (3)soluble complexes of horseradish peroxidaseand rabbit anti-horseradish peroxidase (CodeZII3) diluted I:50. Controls includedmouse adrenals infected with HSV (Hill et al.I986) and specimens from which the pri-mary antibody was omitted. Sections wereviewed using a Leitz Laborlux K light micro-scope.

Epithelial sheets from the cornea werefreed from the underlying stroma using theEDTA technique (Juhlin & Shelley I977).After removal of the epithelial sheets, theanterior segments, including the ciliarybody, iris diaphragm, and corneal stroma(iris sheet preparation) were removed undera binocular stereomicroscope. The epithelialsheets and the iris sheet preparations were

stained for herpes simplex antigens using thePAP technique (Shimeld et al. i986).

Results

Immediately after inoculation, mice were putinto groups. Mice were examined daily withthe slit lamp. Details of the clinical diseasehave been published elsewhere (ClaoueI 986; Claoue et al. I 98 7), but to summarizefrom the 6th day after inoculation the follow-ing were observed in the majority of animals:skin disease of the lids, pupillary mydriasis,iritis, and keratitis. The keratitis was fre-quently severe with an exudate on theanterior corneal surface, and has been calledexudative keratitis.

Trigeminal banglia. For the first 9 days afterinoculation, four coronal sections from thecentre of at least four ganglia per day wereexamined. Haematoxylin and eosin stainedsections appeared normal until day 3 whentwo of four specimens showed a lymphocyticinfiltration focally distributed amongst theneurones. By day 5, five of ten specimensshowed a similar infiltrate which appeared tobe centred on necrotic large clear neurones.On day 9, four of five specimens showed amore diffuse lymphocytic ganglionitis.On the first day after inoculation the PAP

technique revealed antigens apparently inSchwann cells in a small area of one gang-lion from a group of five. On day 5 all of ioganglia contained antigens in Schwann cellsand both large and small neurones (Fig. i).On days 8 and 9 respectively, two of four andtwo of five ganglia contained antigens.

Eyes. At least four eyes were available foreach of the first ten days following inocula-tion. There were no abnormalities until day5 when small foci of pyknotic cells in the iriswere present in four of nine globes. On day 6pyknotic cells were found in the stroma oftheciliary body in two of five eyes. However, onday 7 all of six globes showed a markedpolymorphonuclear kerato-irido-cyclitis,also seen on subsequent days (Fig. 2). On

586 C. Claoue et al.

Zosteriform spread of HSV to the eye

Fig. i. Section of trigeminal ganglion stained with PAP 5 days after inoculation of the snout with HSV.Viral antigens are present in glial cells (1) and large neurones (14). x 380.

Fig. 2. Section ofeye stained with haematoxylin and eosin 8 days after inoculation ofthe snout with HSV.Neutrophils are seen lining the endothelium (1). x 140.

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days 9 and Io, the keratitis appeared moresevere in seven of the eleven globes exam-ined, with deposition of fibrinopus on theanterior surface of an ulcerated and grosslythickened cornea. Neutrophils were foundthroughout the stroma and in the anteriorchamber, often lining the endothelium.Mononuclear cell were no longer seen in theiris. Descemet's membrane was invariablyintact. Two eyes showed a haemorrhagicirido-cyclitis, and there were limbal newvessels in the mid-stroma of two eyes. In oneeye, clumps ofcocci were seen superficially inthe fibrinopus.

Because PAP staining did not reveal HSVantigens in the cornea when four sectionsfrom the centre of the eyes were examined,we serially sectioned five, four, and eight eyesfrom days 4, 5 and 6 respectively. PAPstaining of every fifth slide (four contiguoussections per slide) revealed HSV antigens inthree corneas. One eye on day 5 showedstrong staining of three separated basalepithelial cells. On day 6, two eyes showed

plaques of viral antigen in the corneal epithe-lium. These plaques were wider in the super-ficial layers than in the basal cells, andsections at the periphery might have beeninterpreted as antigens only present in thesuperficial epithelium. Viral antigens werealso present in the anterior uvea in theseeyes.

Antigens were first detected in the base ofthe ciliary body (adjacent to the sclera) onday 5 in three of thirteen globes. Foci ofstaining were also present in the iris in allthree specimens (Fig. 3), and similar butmore extensive staining were also observedthe following day. On day 7, only one of teneyes contained antigen apparently limited tothe sphincter pupillae. Therefter no viralantigens were detected.By PAP staining of epithelial sheets (at

least eight specimens per day for the 9 dayspost-inoculation) plaques of cells containingviral antigens were seen in two of ten sheetson day 3. This number increased until day 5when nine of ten specimens showed viral

Fig. 3. Section ofeye stained with PAP 5 days after inoculation of the snout with HSV. Viral antigens arepresent in the iris and ciliary body. x 140.

C. Claoue et a].588

Zosteriform spread of HSV to the eye

Fig. 4. Corneal epithelial sheet stained with PAP 6 days after inoculation of the snout with HSV. Viralantigens are present in discrete groups of epithelial cells. x 70.

Fig. 5. Iris sheet preparation stained with PAP 6 days after inoculation of the snout with HSV. Viralantigens are present in the sphincter pupillae and adjoining tissue. x 380.

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antigens, before decreasing to seven often onday 6 (Fig. 4), and zero of eight on day 7.Plaques of antigen were distributed ingroups, and where staining occurred in morethan one quadrant, foci in different quad-rants were not necessarily of the same size.PAP staining of iris sheet preparations was

complicated by background staining of theremaining corneal stroma. However, withthe use of differential focussing to visualizeonly the plane of the iris, focal staining of theiris could be observed on day 5 (in one ofninespecimens), on day 6 (in two of ten speci-mens), and on day 7 (three often specimens).HSV antigens occurred as foci in both thedilator and sphincter pupillae. When thesphincter was involved, stain appeared to bein an arc corresponding to the sphincterfibres (Fig. 5). Such staining was never seenin eyes from mice given mock inoculum andprocessed in parallel with infected speci-mens.

Discussion

Previous attempts to develop animal modelsof herpetic stromal keratitis have involvedthe use of intrastromal injection of virus(Maudgal et al. I984) or viral antigens-(Meyers & Pettit I983), with the disadvan-tages of corneal trauma and probably anunnatural route of entry of antigen. Theroute whereby virus accedes to the eye afterinoculation on the snout may more closelyresemble human recurrent disease. Severalobservations support the hypothesis thatvirus is delivered to the eye via nerves as hasbeen shown in infection in the skin (Blyth etal. I984; Simmons & Nash I984) and in theeye (Shimeld et al. I985; Anderson & FieldI984; Claoue I986; Claoue et al. I987).Thus, as shown here, after snout inocula-tion, HSV antigens are detected in the trige-minal ganglion before they are found in theeye. Furthermore, there is a chronologicalprogression of infectious virus from theinoculation site on the snout, to the trigemi-nal ganglion, and then to the eye (Claoue etal. submitted).

The appearance of antigens in the trigemi-nal ganglion as early as 24 h after inocula-tion confirms other studies (Martin & DolivoI983; Itoyama et a]. I984) and is furtherevidence of spread of virus via nerves. Thelocalization of antigens to the corneal epithe-lium, iris and ciliary body is not surprisingsince all these structures are known to beinnervated by the trigeminal nerve, and PAPstaining of the retro-ocular orbital contentshas shown the presence of viral antigensonly in nerves, apparently in Schwann cells(unpublished observations).

Nerves to the anterior segment are pre-sumed to run in the suprachoroidal space asin man, and thus the sparing of the posteriorsegment suggests that virus is only shed atthe termini of nerves.The foci ofHSV antigens in corneal epithe-

lium and iris were extremely localized. Thiscontrasts with the generalized inflammatoryresponse, and suggests the production ofdiffusable initiators of inflammation.Although previously, corneal inoculation

of HSV has been known to result in iritis(Tullo et al. I983; Meyers-Elliott et al. I983),this could have been a reactive response tothe keratitis. After corneal inoculation in therabbit, the beneficial effect of antiviral agentssuggests that the uveitis is due to viralreplication (Maudgal et al. I984). There areno reports of viral antigens in the anterioruvea after corneal inoculation.Tokumaru and Wilentz (I975) have pre-

viously demonstrated mydriasis in rabbitsafter corneal inoculation of HSV. However,the site of viral infection was not investigatedexhaustively although HSV was culturedfrom the ciliary and superior cervical gang-lia; work confirmed by Martin et al. 1977).Shimeld et al. (I985b) have also culturedvirus from these sites after inoculation of themouse in the anterior chamber. Such anter-ior chamber inoculation has previously beendocumented to produce an iritis (KimuraI962; Martenet I966; Purohit et al. I980).The finding of viral antigens in the iris, withspread in the spincter pupillae after zosteri-form spread of HSV to the eye of the mouse

C. Claoue et al.590

Zosteriform spread of HSV to the eyeprovides a clinico-pathological correlate forthe iritis and mydriasis observed clinically(Claoue et al. I987).

Herpetic iritis in humans has beenreported to produce a mydriasis (Water-worth I967), a situation analogous to thatseen in the mouse after zosteriform spread.Furthermore, HSV has been cultured fromthe aqueous humor of a patient with uveitis(Pavan-Langston & Brockhurst I969) andthe model described here may be ofrelevancein the study of this condition.Our finding of a lymphocytic inflamma-

tory response in the trigeminal ganglionconfirms the work of Cook and Stevens(I973; I983), but contrasts strongly withthe ocular polymorphonuclear infiltrate.-Thefinding that the predominant cell type in thecornea is the polymorphonuclear neutrophilwas initially surprising, since work by Met-calf (Metcalf & Gregory I 980; Metcalf I 982)had suggested that stromal disease in themouse would be mediated by lymphocytes.However, Campbell et al. (I980) have de-scribed stromal keratitis in which the neutro-phil is the predominant cell after intra-stromal injection of type 2 HSV in the rabbit.Moreover, Meyers-Elliott and Chitjian(I98I) have reported neutrophil-mediatedpathogenesis of stromal keratitis after cor-neal inoculation of HSVi in the rabbit.The histology of the eyes after day 8 is

strongly reminiscent of a purulent keratitis;an interpretation supported by the finding ofcocci in the fibrinopurulent exudate. Indeed,we have direct evidence from bacterial cul-tures (Claoue et al., submitted) that themajority of eyes do suffer a bacterial superin-fection after day 8. Moreover, the clinicalpicture of exudative keratitis correlates wellwith the isolation of significant numbers ofpathogenic bacteria (usually Staphylococcusaureus) and the incidence of such exudativekeratitis is almost abolished by the use ofappropriate antibiotics (Claoue et al., submit-ted).

Microbial superinfection of human her-petic keratitis, is well recognized, although arare complication (Jones I959; Dohlman &

Zucker I965; Nozik & Smolin I970; Ben-Tovim et al. 1974; Dawson & Togni 1976;Jones et al. 1977; Wilhelmus et al. I98I;Wilhelmus I 982; Nissenkorn & Wood I982;Boisjoly et al. I983). The effect on vision isusually devastating (Wilhelmus I982). Inview of this, it is perhaps surprising thatthere appear to have been no previouslaboratory studies of this phenomenon. Themodel reported here is suitable for furtherstudies; we have already shown that ade-quate doses of appropriate antibiotics canprevent such bacterial superinfection(Claoue et al., submitted), a point hithertobelieve not possible (Boisjoly et al. I983).

Preliminary experiments using antibiotictreatment suggest that even without bac-terial superinfection the predominantinflammatory cell is still the neutrophil.Whether neutrophils infiltrate the corneasolely as a non-specific inflammatory re-sponse to cell damage caused by HSV, orwhether an immunological (i.e. antigen spe-cific) mechanism is involved (Rouse I985) isbeing investigated.

Zosteriform spread allows a short intervalbetween inoculation and virus entering theeye. Thus, whilst an immune response toHSV becomes detectable at about the timethat virus reaches the eye (reviewed byWildy & Gell I985), this will not be the sameas that prevailing at the time of humanrecrudescent disease where a period ofweeksor months may elapse between the primaryinfection and first eye disease. The significantadvantage of zosteriform spread is that virusis not inoculated into the eye directly but vianerves from the trigeminal ganglion, a situa-tion which is also thought to occur in humanrecurrent disease.The initial keratouveitis is likely to be due

to direct virus invasion because of the pres-ence of viral antigens before bacterial super-infection. Furthermore, it is likely that theviral keratitis produces an environment con-ducive to bacterial superinfection. We arenot aware of any previously described modelof a mixed bacterial and herpes viral keratitisand suggest that the model described here

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rq2 C. Claoue et al.could be useful in defining the pathology andtherapy for this condition.

Acknowledgements

This work was financed by the WellcomeTrust. We are grateful to Dr S. Lewkowicz-Moss and Mrs C. Shimeld for sharing theirtechnical expertise, and to Miss K. StevensonFRCS for useful discussion.

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