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S. Mareninov, L.K. Gordon; Age–Dependent Survival of Retinal Ganglion Cells In Vitro . Invest. Ophthalmol. Vis. Sci. 2004;45(13):5377.
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Purpose: Short term retinal ganglion cell (RGC) cultures are used to study cell death pathways, survival factors, and neuroprotective agents. Most blindness from RGC loss occurs in adults, therefore the purpose of this study was to determine if RGCs from young or adult animals differed in their survival in vitro. Methods: RGCs were isolated from 12–day and 60–day old rats. Attachment to CD 11 b/c coated beads was used to deplete macrophages and the RGCs were enriched by positive selection through attachment to Thy–1.1 coated magnetic beads. The resultant cells were 90% pure for RGC. Cells were plated on poly–D–lysine and laminin coated cover slips in serum–free medium. Survival ratio was evaluated at multiple time points using calcein AM and ethidium homodimer–1 staining. Cells were quantified using confocal microscopy to determine the numbers of live and dead cells. Changes in both the culture medium and scheduled medium changes were tested in these studies. Results: Distinct and reproducible differences were observed in survival of RGC from young (P12) and adult (P60) animals. The number of surviving P12 RGCs decreases at a stable rate (11.8%/day) for the first 7 days, followed by slow loss of 1.4% per day. An abrupt loss of RGCs (64%) from the adult (P60) animals was observed in the first 24 hours of culture. The surviving cells were then stable for 7 days followed by a slow, progressive decline (2.9%/day). Surprisingly, the viability of the P60 cells at 7 days improved by 250% if the medium was not changed during that time period. Conclusions: Survival of Thy–1.1 positive retinal cells, presumably RGCs, in short term culture is different in young (P12) and adult (P60) animals. It is likely that a factor or factors secreted by P60 RGCs within first 7 days may enhance survival in cultures. Additional studies are required to determine the critical factors producing the observed differences between the P12 and P60 RGCs,however these result may provide important implication for other studies of RGC survival.
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