Apoptotic effects of metabolic challenge on retinal precursor cells in vitro

Purpose. Apoptosis is a common pathway for cell death occurring in normal retinal development, or as part of retinal degenerative disease. The study of retinal apoptotic mechanisms and interventions would be greatly simplified with the availability of a stable retinal cell culture model with differentiation potential. Methods. We utilized the R28 immortalized retinal precursor cell line (developed in this laboratory) which has been cultured over 100 passages, and expresses markers consistent with both glial and photoreceptor phenotypes. Cells were metabolically challenged with defined, depolarizing medium without serum. Loss of cell viability was measured first by a neutral red assay. Apoptosis was confirmed by DNA fragmentation analyses consisting of DNA gel electrophoresis, and a TUNEL-i'n situ method. Results. Death of R28 cells under defined, depolarizing conditions was documented by a fivefold decrease in neutral red viability over the course of three days, and the appearance of apoptotic bodies in culture. The apoptotic response was confirmed within 20 hours of treatment on a single cell basis by the TUNEL-in situ method, and within 48 hours by DNA gel electrophoresis. Defined, depolarizing conditions led to a consistent, density-dependent apoptotic cell death, with a plateau in the number of cells undergoing apoptosis from 44 hours through 60 hours of treatment. In addition. TUNEL-positive apoptotic cells appeared in groups or clusters, suggesting possible effects of cell-cell contact or microenvironment. Conclusions. R28 retinal precursor cells exhibit a consistent apoptotic response to metabolic challenge that would provide an excellent model system for further study of apoptotic mechanisms relevant to retinal degeneration as well as retinal cell differentiation.