Reduced redox state allows prolonged survival of axotomized neonatal retinal ganglion cells

doi:10.1016/S0306-4522(01)00493-6

Neuroscience

Volume 109, Issue 3, 14 February 2002, Pages 635-642

https://doi.org/10.1016/S0306-4522(01)00493-6 Get rights and content

Abstract

Axonal injury to CNS neurons results in apoptotic cell death. The processes by which axotomy signals apoptosis are diverse, and may include deprivation of target-derived factors, induction of injury factors, bursts of reactive oxygen species (ROS), and other mechanisms. Our previous studies demonstrated that death of a dissociated retinal ganglion cell, an identified CNS neuron, is ROS-dependent. To better define the mechanisms by which ROS induce retinal ganglion cell death after axotomy, we studied their effects in dissociated neonatal rat retinal cultures. Postnatal day 2–4 Long–Evans rat retinal ganglion cells were retrogradely labeled with the fluorescent tracer 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI). Postnatal day 7–9 retinas were dissociated and cultured in the presence of specific ROS generating systems, scavengers, or redox modulators. Retinal ganglion cells were identified by DiI positivity and viability determined by metabolism of calcein-acetoxymethyl ester.

We found that ROS scavengers protected against retinal ganglion cell death after acute dissociation, and the effects of ROS appeared to be due to shifts in the redox potential, as retinal ganglion cell survival was critically dependent on redox state, with greatest survival under mildly reducing conditions. Culture of retinal ganglion cell with the non-thiol-containing reducing agent tris(carboxyethyl)phosphine resulted in long-term survival equivalent to or better than with neurotrophic factors.

Our data suggest that axotomy-associated neuronal death induced by acute dissociation may be partly dependent on ROS production, acting to shift the redox state and oxidize one or more key thiols. Understanding the mechanisms by which ROS signal neuronal death could result in strategies for increasing their long-term survival after axonal injury.

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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Reduced redox state allows prolonged survival of axotomized neonatal retinal ganglion cells

Abstract

Section snippets

Animals

Materials

ROS scavenging protects against RGC death in vitro

Discussion

Acknowledgements

Neuron

Neuroscience

Biochim. Biophys. Acta.

Toxicol. Appl. Pharmacol.

Neuron

J. Biol. Chem.

Cell

J. Biol. Chem.

J. Biol. Chem.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

Neurosci. Lett.

Neuron

Effects of neurotrophins on the survival and regrowth of injured retinal neurons

Ciba Found. Symp.

Axotomy results in delayed death and apoptosis of retinal ganglion cells in adult rats

J. Neurosci.

A critical role of the N-methyl-D-aspartate (NMDA) receptor subunit (NR) 2A in the expression of redox sensitivity of NR1/NR2A recombinant NMDA receptors

J. Pharmacol. Exp. Ther.

Axotomy-induced retinal ganglion cell death in development: its time-course and its diminution by antioxidants

Proc. R. Soc. Lond. Biol. Sci.

Regulation of neuronal survival by the serine-threonine protein kinase Akt

Science