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A.E. Khalyfa, I. Charles, B. Srinivasan, R. Roque, N. Cooper, N. Agarwal; Serum Deprivation Induced Apoptosis of Retinal Ganglion Cells Involves Both the Intrinsic and Extrinsic Signal Transduction Pathways . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4395.
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Purpose: To determine the mechanisms of apoptosis in rat retinal ganglion cells deprived of growth factors and to establish an in vitro model of glaucoma. Methods: An established line of transformed rat retinal ganglion cells, RGC-5, was subjected to serum deprivation for 3-6 days. Control cells were cultured in growth medium containing 10% fetal calf serum. Cell viability was measured by neutral red assay. Secreted neurotrophins were measured by ELISA assays and several apoptosis associated genes were studied by RT-PCR and/or immunoblot analysis. Results: Serum deprivation of RGC-5 cells for three days resulted in a 50% cell loss due to apoptosis as established by DNA laddering and propidium iodide staining. Serum deprivation also resulted in increased oxidative stress as revealed by lowering of reduced glutathione (GSH) and increased levels of malonydialdehyde (MDA). Reduced levels of NF-kB binding activity and Bcl-2 and increased levels of Bax mRNA and protein were observed under serum deprived conditions. In addition, expression of caspases 3, 8, and 9 and death receptors (Dr3 and Dr4) proteins were elevated. Serum deprivation was also associated with a loss of mitochondrial function as revealed by cytochrome-c release and rhodamine 123 staining. The RGC-5 cell death correlated with reduced levels of various neurotrophins and cell death was further augmented by K252a, a Trk- receptor inhibitor. Conclusions: These results indicate that blockade of retrograde transport of neurotrophins may result in retinal ganglion cell death by means of both the intrinsic as well as extrinsic apoptotic pathways involving oxidative stress, and trk receptors. Supported by AHAF-National Glaucoma Program (NA) and NIH: P20 RR16481 (NC).
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