Uninfected Neuronal Cells in the Retina Become Apoptotic following Supraciliary Inoculation of MCMV

Abstract

Abstract: : Purpose: Our previous results showed that (a) retinal cells of BALB/c mice infected with MCMV via the supraciliary route became apoptotic and (b) most of the apoptotic cells did not appear to be MCMV-infected. The aims of this study were to determine which cells in the retina become apoptotic. Methods: Immunosuppressed (IS) (2mg methylprednisolone acetate per mouse, i.m. every 4 days) or non-IS BALB/c mice were inoculated with 5 x 10₃ PFU of k181 via the supraciliary route. At intervals after infection, mice were sacrificed. MCMV-infected cells, apoptotic cells, glial cells, RPE cells, leukocytes were identified in frozen sections of ocular tissue by detection of MCMV early antigen (EA), TUNEL assay, GFAP, RPE-65 and CD45 respectively. Double staining of TUNEL (FITC labeled) and MCMV EA, RPE-65, GFAP, or CD45 (Texas red or PE labeled) was used to determine which cells were apoptotic. Results: Many MCMV infected cells and apoptotic cells were observed in the inner retina of IS mice. Apoptotic cells were usually observed in proximity to MCMV infected cells, but only few of the EA positive cells were apoptotic. Most MCMV infected retinal cells were TUNEL negative and conversely, most TUNEL positive cells were not MCMV infected. Early in infection (≤day 7 p.i.), most of the apoptotic cells were not glial cells whereas late in infection (10-14 days p.i.), some glial cells were apoptotic. On day 7 p.i., 2.6% of apoptotic retinal cells were glial cells and at day 10 p.i., 7.3% of apoptotic retinal cells were glial cells. Some CD45+ leukocytes in the retina were apoptotic. However, most apoptotic cells in the inner retina were not infiltrating leukocytes. The percentage of apoptotic leukocytes of the total apoptotic retinal cells was 4.6% at day 7 p.i. and 1.6% at day 10 p.i., respectively. The majority of the RPE cells remained in situ during the course of the infection. Conclusion: Taken together, results showing that most apoptotic cells in the inner retina were not infiltrating leukocytes, glial cells, or RPE cells suggest that the majority of apoptotic cells are neurons. Since uninfected neuronal cells become apoptotic, the amount of retinal damage is amplified beyond that caused by MCMV infection alone.

Keywords: 382 cytomegalovirus • 316 animal model • 568 retinitis