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M.J. Hoegger, L.A. Levin; Sources for Superoxide Generation in Axotomized Retinal Ganglion Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):190.
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Purpose: Retinal ganglion cells (RGC) undergo apoptosis after axotomy. Our prior studies demonstrated that reactive oxygen species (ROS) such as superoxide play a role in signaling apoptosis after axotomy. Understanding the cellular processes generating the rise in superoxide would help determine the upstream mechanism by which axotomy induces this signal. Methods: Postnatal Longs Evans rats aged two to four days old were retrogradely labeled with the fluorescent dye 4 6–diamidino–2–phenylindole (DAPI) to distinguish RGCs from other retinal cells. Dissociated retinas were plated and superoxide levels measured by treating with dihydroethidium (HEt), a nonfluorescent molecule which becomes fluorescent when oxidized by superoxide and bound to DNA. Fluorescence was measured at 2 and 24 hours after dissociation, in the presence of the following specific inhibitors of superoxide production: rotenone 0.1 µM (inhibits mitochondrial complex I); antimycin A 1 µM (inhibits mitochondrial complex III); indomethacin 100 µM (inhibits cyclooxygenase); allopurinol 100 µM (inhibits xanthine oxidase); diphenyleneiodinium 10 µM (inhibits flavoproteins); or control medium alone. Results: At 2 hours after dissociation the superoxide level was similar for all treatments (p = 0.84 by ANOVA). There was a rise in RGC superoxide at 24 hours, reflecting the effect of axotomy (29.9±1.5 vs. 42.1±2.3 fluorescence units; p = 0.00007). Treatment with antimycin A alone eliminated the rise in fluorescence (20.8±2.5 at 2 hr vs. 21.2±2.5 at 24 hr; p = 0.89). Treatment with other blockers of superoxide generation did not inhibit superoxide production, and in the case of diphenyleneiodinium there was a significant increase in superoxide compared to control (62.4±4.7 vs. 42.1±2.3; p < 0.01 by Tukey HSD post hoc test). Conclusions: Complex III of the mitochondrial electron transport chain may be a source of superoxide generation in RGCs after axotomy, and may represent a target for preventing signaling of apoptosis.
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