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The corneas of latently infected mice were UV irradiated to induce reactivation of herpes simplex virus type 1 (HSV-1) in the trigeminal ganglion (TG). On days 1 to 4 after irradiation, TG were removed, serially sectioned and double stained to identify immune cells and virus antigens. Virus antigen was detected in small numbers (most commonly one) of neurons per ganglion as early as day 1, confirming the rapidity of reactivation and the neuron as the likely site of this event. The immune response was also rapid and effective since virus antigen was identified in immune cells at day 1 and by day 4 all samples were negative. The predominant infiltrating cells on days 1 and 2, when virus antigen was present and being cleared, were T cells, both CD4 + and CD8 ÷. Later, large numbers of B cells appeared, suggesting that local antibody production may also be involved in controlling the reactivated infection. The observations suggest that a significant proportion of reactivation events do not result in disease of the eye or shedding of virus in the tear film. However, they also suggest that as little as one reactivating neuron in the ganglion may be sufficient to lead to such disease and/or shedding.

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We describe a method for immunohistochemical localization of T-cells, CD4+ T-cells, CD8+ T-cells, B-cells, activated lymphocytes, major histocompatibility complex (MHC) class II antigens, macrophages, dendritic cells, and granulocytes in rat and mouse tissue fixed in periodate-lysine-paraformaldehyde (PLP) and embedded in paraffin. Rat and mouse spleen and eyes were fixed in PLP for 18-24 hr, rapidly dehydrated, infiltrated under vacuum with paraffin at 54 degrees C, sectioned, and stained with appropriate monoclonal antibodies (MAbs). Sections of PLP-fixed, paraffin-embedded spleen were compared with acetone-fixed frozen spleen sections with respect to morphology and staining quality. Nine of 10 MAbs to rat antigens and eight of nine MAbs to mouse antigens stained paraffin sections equally or more intensely than frozen sections. The two MAbs that showed weaker staining still gave good staining on paraffin sections. Paraffin-embedded rat and mouse eyes were easier to section serially than frozen eyes, showed superior morphology, and individually stained cells were readily identified. Therefore, a combination of PLP fixation and low-temperature paraffin embedding permits detection of the major types of immune cell in rat and mouse tissues while maintaining good morphology, particularly in diseased, damaged, or delicate tissues.

Mice were passively immunized with serum containing antibodies to herpes simplex virus type 1 (HSV-1) before inoculation on the cornea with HSV-1 strain McKrae. After such immunization most mice survived and most had normal eyes. When primary infection had subsided, mice with normal eyes were selected and treated with cyclophosphamide, dexamethasone and UV irradiation of the inoculated eye or UV irradiation alone, to reactivate latent virus. After either treatment mice developed signs of recurrent infection (virus in eyewashings and recurrent corneal and/or lid disease). The incidence of such signs was 17/33 (52%) in mice receiving immunosuppressive drugs and UV irradiation and 19/32 (59%) in mice given UV irradiation alone. In mice treated with either stimulus dendritic or geographic ulceration of the cornea was seen. These closely resembled the herpetic lesions seen in humans. There was good correlation between the pattern and distribution of recurrent corneal disease and the distribution of cells containing virus antigens in corneal epithelial sheets. Again, as in humans, the induction of recurrent infection was found to correlate poorly with a rise in the level of serum neutralizing antibody. In mice treated with UV irradiation alone corneal ulcers healed and the eyes returned to normal. By contrast, in mice given immunosuppressive drugs and UV irradiation, the ulceration became more severe and the eyes became opaque and vascularized. The use of passive immunization has greatly improved our previously reported model of recurrent herpetic eye disease since it has increased the incidence of mice suitable for the induction of recurrent infection and has increased the incidence of such infection.

SUMMARYIn both non-immune and immune mice infected with herpes simplex virus the incidence of latent infection of the trigeminal ganglion was related to the severity of ocular virus infection. During primary infection, virus was shown to travel via the ophthalmic part of the ganglion to reach the brainstem, from where centrifugal spread resulted in latent infection of neurones in the trigeminal ganglion which did not serve the site of inoculation. Primary infection also resulted in latent infection of the superior cervical ganglion. Shedding of virus occurred rarely in the tears of animals which had recovered from primary disease. In immune mice, spread of virus resulted in a much lower incidence of latent infection and that occurred only in ophthalmic neurones.

The spread of herpes simplex virus type 1 (HSV-1) during primary ocular infection and after reactivation of latent infection in the trigeminal ganglion (TG) was examined in the mouse using a genetically modified virus containing the lacZ reporter gene under the control of the immediate-early 110 promoter. Whole tissue mounts of the eye and lids, their sensory nerves, and TG with the attached dorsal root entry zone (DRE) into the central nervous system (CNS) were stained for ␤-galactosidase. Sixteen hours after inoculation of the cornea by scarification, staining was found in the scarified epithelium of the cornea and in the unscarified conjunctiva.