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W.C. Gordon, K.G. Sheets, N.G. Bazan; In Normal Light–Treated and Rhodopsin S334ter–4 Mutant Rats, DNA Repair Enzymes in Distal Photoreceptor Nuclei May Provide Enhanced Resistance to Oxidative Stress . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4825.
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© ARVO (1962-2015); The Authors (2016-present)
To determine if the rate of oxidative stress–induced apoptotic cell death varies among photoreceptors within the superior–central retina.
Eyes from rat pups containing the human rhodopsin mutation S334ter–4 were collected from postnatal days 9–45 and retinas prepared for fluorescence microscopy. TUNEL labeling (indicating fragmented DNA) and immunolocalization for activated caspase–3 (indicating activation of the nDNA fragmentation process), 8–oxoguanine (caused by oxidative damage), and DNA polymerase beta (a nDNA repair enzyme) were performed on cryosections. Sprague–Dawley rats, 150–175 g, were treated for 5 h with fluorescent light, 18 kLx, and retinas collected until +24 h. These were similarly prepared for fluorescence microscopy. Only photoreceptors within the light–sensitive superior–central region of the retina were studied.
The product of guanine oxidation, 8–oxoguanine, was found distributed throughout the outer nuclear later (ONL) in S334ter–4 and light–treated animals, indicating that oxidative stress had occurred in all photoreceptors. Activated caspase–3 was also evenly distributed throughout the ONL, indicating that mitochondria had been compromised and photoreceptor nDNA fragmentation had commenced. DNA polymerase beta was localized to the distal ONL, while, initially, in both S334ter–4 mutants and light–treated rats, only nuclei within the deepest half of the ONL became TUNEL–positive. As photoreceptor loss occurred and the ONL thinned, only proximal nuclei showed DNA fragmentation. This TUNEL–positive pattern continued until 50% cell loss had occurred.
Once up–regulated, nDNA repair enzymes must move from their location of synthesis on inner segment endoplasmic reticula to the site of nDNA damage. Nuclei within the distal portion of the ONL receive these repair molecules before those located farther away from inner segments, within the proximal ONL. This results in a wave of photoreceptor apoptosis that commences deep in the ONL and sweeps in a distal direction. Cone nuclei occur within the distal–most third of the rodent ONL, and it has been demonstrated that cone photoreceptors endure apoptosis–inducing events longer than rods. Thus, prolonged cone and "distal" rod survival may be due, in part, to the shorter distance between their inner segments and nuclei, which results in a faster response to nDNA damage.
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