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Eric J. Knott, William C. Gordon, Nicolas G. Bazan; Docosahexaenoic Acid Pretreatment Protects Arpe-19 Cells From Subsequent Oxidative Stress. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4432.
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Docosahexaenoic acid (DHA) enrichment protects Neuro2A cells from serum starvation or staurosporine treatment via Akt signaling pathways. Highest concentrations of DHA in the body are found in the rod photoreceptor outer segment (ROS), and when ROS are phagocytized by RPE cells, neuroprotectin D1 (NPD1) is synthesized. We have shown that DHA and NPD1 protect RPE cells when applied during oxidative stress. The purpose of this study was to determine if DHA pretreatment enables RPE protection from subsequent oxidative stress.
ARPE-19 cells where cultured for 72 h to achieve 90% confluencey. Cells were serum starved for 18 hours in 0.5% FCS, DMEM/F12 1:1 media. After which 100nM DHA or ETOH (equal volume) was added to the media. After 6 hours of incubation in 100nM DHA or ETOH, media was replaced with fresh 0.5% FCS, DMEM/F12 1:1 media for 24h. Cells were then challenged with 400 or 600 µM H2O2 for 16 hours. Cells were fixed with neutral buffered formalin and stained with Hoestch 33258 in 1% triton X100. Cells were counted using automated unbiased image analysis of nuclear morphology. Cell death was determined by nuclear size (pixels) via nuclear condensation.
Control cells display a survival 95 ± 3%. Cells treated with 400 and 600 µM H2O2 for 16 hours result in nuclear condensation and a cell survival of 76 ± 4 % and 43 ± 9%. Cells pretreated with 100 nM DHA 24 hours prior to stress exhibit protection with a survival of 86 ± 6% and 79% ± 7%.
Our results demonstrate that DHA pretreatment elicits protection of human-ARPE-19 cells from oxidative stress-induced cell death. This data suggests protection via DHA derived NPD1 synthesis which is achieved via NPD1 mediated-PI3K/Akt signaling. This implies that application of DHA/NPD1 could be used as a potential target for the prevention or attenuation of the initiation or early progression of retinal degenerations.
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