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S. S. Atherton, M. Zhang; Virus-Free Supernatants From MCMV Infected Retinal Cultures Induce Apoptosis in Uninfected Cultured Retinas. Invest. Ophthalmol. Vis. Sci. 2007;48(13):555.
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MCMV infection of the cultured retina induces apoptosis of retinal neurons. The purpose of this study was to test the hypothesis that apoptosis is related to apoptosis inducer(s) secreted by activated microglia after MCMV infection but not to viral infection per se.
The retinas from C57BL/6 mice were cultured at 37°C and infected with MCMV (1 × 105 PFU/retina). At day 11 p.i., the cultures were harvested for RT-PCR (TNF-α, iNOS, MCP-1) or immunohistochemistry (F4/80, TNF-α, iNOS, MCMV EA) and TUNEL assay. The supernatants were collected and filtered through a Vivaspin ultrafiltration unit with a molecular weight cutoff of 300,000 (Sartorius) at 6,000g for 1 hour at 4°C. The filtrate, which was free of infectious virus (as demonstrated by plaque assay), was mixed with fresh retinal culture medium (1:1) and added to retinal cultures. The filtrate from supernatants of non-MCMV infected normal retinal cultures was used as control. The retinal cultures were harvested at day 4 post culturing with filtrate and prepared for TUNEL assay.
MCMV infected and apoptotic cells were observed in the retina at day 11 post MCMV infection and the majority of apoptotic cells were not virus infected. F4/80 positive activated microglia were also detected and some of the activated microglia were TNF-α or iNOS positive. RT-PCR results showed mRNA expression of TNF-α, iNOS, MCP-1 detected in the MCMV infected retina but not in the non-infected control retina. Only a few TUNEL positive cells were observed in the retina after 4 days of culturing with either fresh culture medium or the filtrate from supernatants of non-MCMV infected normal retinal cultures. In contrast, many apoptotic cells were noted in the retina after 4 days of culturing with the filtrate from supernatants of MCMV infected retinal cultures.
Apoptosis inducers, including TNF-α, and NO, were produced by activated microglia after MCMV infection of retinal cultures. The virus-free supernatants from MCMV infected retinal cultures induced apoptosis of retinal cells in uninfected retinal cultures.
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