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S. Mareninov, L.K. Gordon; Survival–Death of Retinal Ganglion Cells: Necrotic Aspect of Adult RGCs Loss in Short Term Culture . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1290.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Acquired retinal ganglion cell (RGC) loss, associated with glaucoma or other optic neuropathies, is a major cause of visual morbidity in adults. RGC primary culture studies are used to clarify mechanisms of cell death and potential applications for neuroprotective therapy, however many in vitro studies use cells derived from early postnatal rats. We previously observed different survival characteristics of RGCs isolated either from early postnatal or from adult rats. The purpose of this study was to further characterize mechanisms of cell death and factors that alter survival of cultured RGCs from adult animals. Methods: RGCs were isolated from 8–10–day (P10) and 60–day (P60) postnatal Long– Evans female rats. A modified immunomagnetic bead protocol for RGC isolation was used to purify RGCs to greater than 92% as determined using Thy–1.1 labeling. Cells were plated on poly–D–lysine and laminin coated cover slips in serum–free medium under various conditions, and analyzed for cell survival and death mode. Cell survival was detected by calcein AM and ethidium homodimer–1 staining, performed at multiple time points. The mode of RGC death was analyzed using propidium iodide to determine necrosis and YO–PRO–1 to detect apoptosis. Results: In agreement with previous studies, RGCs from P10 postnatal rats, exhibit a stable decline to about 12% in a 7 day culture, with death from apoptosis. In contrast, RGCs from adult rats exhibit an abrupt loss of about 65% of the cells within the first 24 hours of cell culture and this early cell death is via a necrotic pathway. About 26% of the P60 RGCs and 11% of the P10 RGCs survive at day 7 in culture. Cellular debris from either P10 or P60 RGCs, obtained through cycles of freezing and thawing, enhance RGC survival in culture. Conditioned media (CM) from the P60 RGCs, but not from P10 RGCs, exhibited at least a 2 fold increase in RGC survival at 7 days(p<0.001). This survival factor was present in the high molecular weight fraction of the CM. Conclusions: RGCs from P10 and P60 rats show different survival rates and employ different cell death pathways during a short term cell culture model. In contrast to RGCs from P10 rats, necrosis was the primary mode of early cell death in P60 RGCs. Furthermore, necrotic cell death with its associated cellular debris produced an RGC survival factor that prolonged survival of the remaining cells. Apparently, early necrosis, a previously underestimated RGC cell death pathway, may release survival factors and enhance survival of adjacent at–risk ganglion cells in adult animals.
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