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C.J. Lieven, L.A. Levin; Optic Nerve Crush Leads to a Rise in Superoxide Levels in Cultured Retinal Ganglion Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):154.
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Purpose: Acutely dissociated retinal ganglion cells (RGC) rapidly undergo apoptosis in the absence of growth factors (25–50% loss over 24 hours). Optic nerve crush leads to a slower loss of RGCs (50% loss over 1 week), and thus is a more representative model of optic neuropathy. Our previous studies demonstrated that intracellular superoxide levels in cultured RGCs rise after dissociation, and we have hypothesized that this rise signals apoptosis. We tested whether this was also true of axotomy by measuring superoxide levels in RGCs cultured from retinas at various time points after optic nerve crush. Methods: Primary rat mixed retinal cultures of DAPI–labeled axotomized RGCs were prepared at various time points after optic nerve crush. Cells from retinas undergoing the crush and uncrushed control eyes were prepared in parallel and treated with dihydroethidium (HEt) after one hour in culture. Oxidation of HEt to oxy–Et is indicative of superoxide production within the cell. The intensity of oxy–Et fluorescence in RGCs was measured and recorded in multiple cells after one hour in culture and compared between uncrushed and crushed conditions. Results: Superoxide levels rise in RGCs after optic nerve crush, but not synchronously. Superoxide levels rose by more than 50% in 9.5% of the RGCs cultured 4 hours after optic nerve crush, increasing to 13.2% at 8 hours, 16.7% at 16 hours, and 18.9% at 24 hours. RGCs from uncrushed retinas did not show these elevated levels of superoxide as prominently (5.3%, 2.4%, 7.3%, and 2.9% of RGC at the same time points). Looking at the normalized (to 1.0 in controls) mean superoxide levels of all RGCs at each time point, there was a significant increase in cells from crushed retinas at 8 hours compared to control retinas (1.17 ± 0.07 vs. 1.00 ± 0.04; p=0.032) and at 24 hours (1.20 ± 0.06 vs. 1.00 ± 0.07; p=0.038). This rise was about half that caused by acute dissociation (1.34 ± 0.08 of control at 24 hours). Conclusions: Optic nerve crush, a model of acute optic neuropathy, produces an early rise in superoxide levels in RGCs, similar but quantitatively less than that seen with acute dissociation. RGC death following axotomy may be mediated by superoxide anion, and these findings raise the possibility that scavenging superoxide or inhibiting its intracellular targets may be a useful approach for RGC neuroprotection.
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