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E. J. Knott, K. G. Sheets, D. R. Bergsma, W. C. Gordon, N. G. Bazan; Neuroprotectin D1 Attenuates Photoreceptor Apoptosis in Retinal Explants After Light Damage in vivo. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2995. doi: https://doi.org/.
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Bright light initiates damage that leads to photoreceptor apoptotic cell death and that, as stress continues, mDNA and nDNA are damaged (increased 8-oxoguanine [8-OG]), and repair mechanisms initiated (DNA polymerase β, 8-oxoguanine-DNA-glycosylase [OGG-1]) (Cortina et al. 2005). If repair is successful, photoreceptors survive, otherwise, cell death occurs. In retinal pigment epithelial cells (RPE), neuroprotectin D1 (NPD1), a derivative of docosahexaenoic acid (DHA), counteracts oxidative stress, caspase-3 activation, and apoptotic DNA damage. NPD1 up-regulates the anti-apoptotic proteins Bcl-2 and Bcl-xL, while decreasing pro-apoptotic Bax and Bad expression. Therefore, the purpose of this study was to determine if NPD1 attenuates or halts photoreceptor light-induced apoptosis.
Dark-adapted Rats were stimulated with bright light (5h); eyes were collected and retinas prepared for explants in DMEM/F12/FCS (1:1:10%) medium with and without 100nM NPD1. Quantification and localization of DNA fragmentation was monitored by counting TUNEL-positive cells in retinal sections and comparing them to dark controls.
Retina explants from rats exposed to light show an increase in TUNEL-positive photoreceptor labeling in superior retina as compared to inferior retina. This TUNEL labeling appears 6 h post light and increases 11-fold by 24 h. Intact retina has fewer TUNEL-positive cells compared to retina with detached RPE. NPD1- treated explants show an 85% decrease in the number of proximal TUNEL-positive photoreceptor cells in light-induced DNA damage. However, distal cells (photoreceptor nuclei just below inner segments) remained TUNEL-positive, even with NPD1 administration.
Appearance of TUNEL labeling at 6 to 24 hours indicates an increase in DNA fragmentation and apoptosis. Damage located in the superior retina is consistent with previous studies, which have shown this region to degenerate preferentially. DNA damage in distal regions of the retina suggests the absence of RPE survival mechanisms, or damage due to preparation of explants. Proximally located TUNEL-positive photoreceptor nuclei in retinal explants are protected upon addition of NPD1 to incubation medium. This suggests NPD1 is an inhibitor of the apoptotic pathway triggered by light-induced oxidative stress.
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