Reduced redox state allows prolonged survival of axotomized neonatal retinal ganglion cells
Section snippets
Animals
All experiments were performed in accordance with institutional, federal, and state guidelines regarding animal research. All efforts were made to minimize the number of animals used and their suffering.
Materials
Cell culture reagents were obtained from Gibco (Grand Island, NY, USA). The retrograde fluorescent tracers 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiIC18; DiI) and 4′-6-diamidino-2-2 phenylindole, and the fluorescent viability agent calcein-acetoxymethyl ester (calcein-AM) were
ROS scavenging protects against RGC death in vitro
We hypothesized that scavenging of ROS would increase the viability of axotomized RGCs, similar to what is seen with neurotrophin-deprived sympathetic neurons (Greenlund et al., 1995). Mixed retinal cultures containing RGCs retrogradely labeled with the fluorescent dye DiI were incubated with the peroxide scavenger catalase (500 U/ml), the superoxide dismutase mimetic Mn(III)-tetrakis-(1-methyl-4-pyridyl)-porphyrin pentachloride (MnTMPyP; 50 μM), or the peroxynitrite scavenger (as well as
Discussion
These data demonstrate that specific ROS scavengers and hypoxia reduce the death of neonatal RGCs after acute dissociation, and that RGC survival is dependent on redox state, with greatest survival under mildly reduced conditions, consistent with findings of Castagne et al. in chick retina (Castagne and Clarke, 1996, Castagne et al., 1999). Furthermore, maintenance of an axotomized neonatal RGC in a reduced state prevents death to an extent greater than that seen with either ROS scavenging or
Acknowledgements
Supported by the Retina Research Foundation, the Glaucoma Foundation, NIH EY12492, and an unrestricted departmental grant from Research to Prevent Blindness. L.A.L. is a Research to Prevent Blindness Dolly Green scholar.
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