Apoptosis in the retina during MCMV retinitis in immunosuppressed BALB/c mice

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Abstract

Background: Cytomegalovirus (CMV) retinitis is the most common opportunistic ocular infection observed in immunosuppressed (IS) adult and pediatric patients. Due to the species restriction of the cytomegaloviruses, mice infected with murine CMV (MCMV) have been used to study the pathogenesis of CMV retinitis. Objectives: The objectives of this study were to determine if retinal glial cells are the targets of MCMV infection and to determine which cells in the retina become apoptotic following inoculation of MCMV via the supraciliary route. Study design: Adult female BALB/c mice were IS with methylprednisolone; one half of the mice were injected with MCMV and one half of the mice were injected with an equivalent volume of tissue culture medium via the supraciliary route. Animals were sacrificed and frozen sections of eyes were stained for MCMV early antigen, RPE65, CD45 or TUNEL; additional slides were double stained with combinations of the above reagents. Results and conclusions: The results indicate that most apoptotic cells in the retina were not virus infected, most apoptotic cells were not infiltrating CD45 positive leukocytes, and retinal glial cells were infected with MCMV but only late in infection. Together, these results suggest that retinal cells that undergo apoptosis during MCMV infection are neurons and that apoptosis of uninfected bystander cells is an important component of the pathogenesis of CMV retinitis.

Introduction

Cytomegalovirus (CMV) retinitis is the most common sight-threatening opportunistic infection observed in adult and pediatric patients who are immunosuppressed (IS) as a result of chemotherapy, malignancy, or the acquired immunodeficiency syndrome (AIDS) (Culbertson, 1989, Drew, 1992, Cohen, 1995, Jabs, 1996). CMV chorioretinitis is also the most frequent ocular abnormality seen in congenital CMV infection involving about 15% of infants with symptomatic infection (Weller et al., 1962, McCracken et al., 1969, Stagno et al., 1977, Boppana et al., 1994, Buggage et al., 2000).

Our laboratory has established a mouse model of CMV retinitis with features resembling those observed in human patients with CMV retinitis (Atherton et al., 1992, Atherton et al., 1991). In this model, inoculation of 5×102–5×103 PFU of MCMV into IS BALB/c mice via the supraciliary route results in progressive focal necrotizing retinitis. In contrast, supraciliary injection of the same dose of MCMV into immunocompetent mice results in minimal retinal involvement. Our previous results showed that the retinas of immunocompetent mice contained many apoptotic cells while MCMV infected cells were observed only in the choroid and RPE, observations which together suggested that MCMV infection causes apoptosis of uninfected retinal cells (Bigger et al., 2000). It remains to be determined which retinal cells are the targets of MCMV infection and whether neuronal or non-neuronal cells undergo apoptosis.

Glial cells are the major non-neuronal cells of the nervous system. They perform many important functions in the brain and eye such as providing physical support, responding to injury, participating in the blood–brain and blood–retinal barriers, and forming the myelin insulation of nervous pathways. Glial cells also participate in the immune response to viral infection of neuronal tissue (Weinstein et al., 1990, Card et al., 1993, Persidsky et al., 2000). The aims of this study were to determine if glial cells are the targets of MCMV ocular infection and also to determine which cells in the retina become apoptotic.

Section snippets

Virus and virus titration

The original stock of MCMV (k181 strain) was a generous gift of Dr Edward S. Mocarski (Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA). Virus was prepared from the salivary glands of MCMV-infected BALB/c mice as described previously (Atherton et al., 1992). Virus stocks were titered by plaque assay on monolayers of Swiss Brown mouse embryo fibroblast (MEF) cells (Bale et al., 1990). Virus stocks were stored at −70 °C. The average titer of the

MCMV infection of the retina after supraciliary inoculation

As expected, MCMV-infected retinal cells and retinitis were observed in the injected eye of IS mice inoculated with MCMV via the supraciliary route (Atherton et al., 1992, Atherton et al., 1991, Duan et al., 1994).

At day 3 p.i., virus infected cells were seen in the anterior segment and in the choroid. At this time, although a few RPE cells were virus positive, no virus positive cells were observed in the retina. At day 5 p.i., many RPE cells were viral antigen positive and MCMV antigen

Discussion

Apoptosis or programmed cell death is an actively controlled process of cell suicide characterized by distinctive morphological and biochemical changes that occur in response to a wide variety of stimuli (White, 1996). The process of apoptosis is important in eliminating cells whose survival might otherwise prove harmful to the organism as a whole, thereby providing a defense against viral infection and the development of cancer. Although, virus-infected cells can often be recognized and

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