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Ming Zhang, Brendan Marshall, Juan Mo, Sylvia B Smith, Zheng Dong; Photoreceptor cell death following choroidal murine cytomegalovirus (MCMV) infections of deeply immunosuppressed mice. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3627.
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© ARVO (1962-2015); The Authors (2016-present)
Our previous studies indicated that systemic MCMV spread to the choroid of deeply immunosuppressed mice, and subsequent infection of the choroid was associated with death of photoreceptors in the overlying retina. The purpose of this study was to explore the mechanism of photoreceptor cell death.
Rip3-/- and wild type mice were deeply immunosuppressed with methylprednisolone and monoclonal antibodies to CD4 and CD8. At several time points post MCMV intravenous inoculation, eyes were removed and were examined by immunohistochemical staining or by immunogold EM (electron microscopy) for MCMV early antigen (EA), AIF and Iba-1. Eye-cups (consisting of RPE, choroid, and sclera) and neural retinas were separated from eyes of MCMV-infected and uninfected control mice under a microscope. Western blots were performed to detect cleaved caspase 1, active NFkB, RIP1, RIP3, iNOS, PARP1, cleaved caspase3, and caspase 12.
Gold labeled MCMV EA was observed in the nuclei of many vascular endothelia in the choroid of MCMV infected mice. Although Bruch’s membrane appeared intact and MCMV EA was rarely observed in the RPE cells, apoptotic photoreceptors, characterized by nuclear shrinkage and strong chromatin condensation under EM were observed in the eyes of MCMV infected wild type mice. IBA1+ microglia were also noted in the subretinal space. Active AIF was found in the nuclei of photoreceptor cells, indicating that AIF was released from mitochondria and translocated into the nucleus. Western blots showed that higher levels of cleaved caspase 1, activated NFκB, RIP3 and iNOS were observed in the eye cups of MCMV infected wild type mice compared to eye cups of uninfected mice. In addition, higher levels of cleaved caspase 12 and cleaved PARP1 were also observed in neural retinas of MCMV infected wild type mice compared to retinas of uninfected mice. No cleaved caspase 3 was detected in neural retinas of MCMV infected mice or control mice. Although MCMV was observed in the choroid of deeply immunosuppressed RIP3-/-mice, TUNEL-positive cells were noted only in the outernuclear layer of wild type mice.
Our results suggest that most photoreceptor cell death induced by choroidal MCMV injection is due to caspase 3-independent PARP1 and AIF mediated apoptosis/necroptosis, and that RIP3 plays a significant role in choroidal inflammation and photoreceptor death.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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