Apoptosis as a mechanism for tissue damage in murine coronavirus induced retinopathy

Purpose: The coronavirus, MHV, infection in BALB/c mice is a biphasic disease which culminates in a retinal degeneration. This model is characterized by a genetic predisposition associated with immunopathologic and autoimmune factors. In this study we evaluate apoptosis as a mechanism of retinal cell damage. Methods: BALB/c mice were inoculated by the intravitreal route with 10^4,5 TCID₅₀ / 5 ul of MHV, JHM strain or with media. At varying times after inoculation, eyes were removed and fixed in 10% formalin for H&E staining and in situ Apoptosis analysis or in glutaraldehyde for electron microscopy. Apoptosis was detected by two methods: (1) terminal dUTP nick end labeling (TUNEL) in situ detection of DNA fragmentation and (2) electron microscopic evaluation for morphologic changes. Results: A total of 35 eyes from virus infected mice, 10 eyes from mock injected mice and 12 eyes from untreated mice were obtained at 1, 3, 6, 10, 20, 41 and 62 days after inoculation. The retinas from untreated mice contained 1 to 3 TUNEL positive (apoptotic) cells while retinas from mock injected mice contained 0.5 to 6.5 apoptotic cells. In contrast, the retinas from virus infected mice contained 3.6 to 39.8 apoptotic cells. The number of apoptotic cells seen in the virus infected retinas is low at day 1 and 3, reaches a maximum at day 6 to 10, remains elevated at day 20, returning to normal levels after day 40. The difference between the apoptotic events in the virus infected retina in comparison to the control retina is P < 0.01 (paired T test). Electron microscopic analysis revealed condensed chromatin, membrane blebbing and apoptotic bodies. Conclusion: These studies demonstrate that apoptosis may be one of the mechanisms involved in virus induced retinal tissue damage.