Retinal ganglion cell axotomy induces an increase in intracellular superoxide anion.
OBJECTIVE
Retinal ganglion cells (RGCs) undergo apoptosis after axonal injury. The time course of cell death is variable and depends in part on the degree of injury sustained. Decreasing reactive oxygen species (ROS) levels or shifting the redox state to reduction promotes the survival of RGCs in tissue culture after axotomy. It was hypothesized that a specific ROS, superoxide anion, acts as an intracellular signaling molecule for RGC death after axotomy.


METHODS
Intracellular superoxide levels were measured after dissociation in retrograde-labeled rat RGCs with use of the superoxide-sensitive fluorophores hydroethidium and MitoSOX Red. Having found a significant increase, the effect of axotomy was determined on superoxide levels independent of dissociation with an optic nerve crush model.


RESULTS
Optic nerve crush caused RGCs to undergo a superoxide burst. The burst was asynchronous and was manifested in only a fraction of cells at any given time. Neurotrophin deprivation was not responsible for the superoxide burst because it was not prevented by incubation with the neurotrophic factors brain-derived neurotrophic factor, ciliary neurotrophic factor, forskolin, or insulin. Several inhibitors of intracellular superoxide generation were studied, but only antimycin A, which inhibits complex III of the mitochondrial electron transport chain, blocked the increase in superoxide.


CONCLUSIONS
These findings suggest that superoxide generated in the mitochondrial electron transport chain could be a parallel system to neurotrophic deprivation for signaling cell death after axonal injury.
Department of Ophthalmology and Visual Sciences, University of Wisconsin Medical School, Madison, Wisconsin 53792, USA.
Christopher J
CJ
Lieven
Mark J
MJ
Hoegger
Christopher R
CR
Schlieve
Leonard A
LA
Levin
D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D001369:Axons / Q000502:physiology*; D019771:Axotomy; D018929:Cell Culture Techniques; D002470:Cell Survival; D014450:Electron Transport Complex III / Q000037:antagonists & inhibitors; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009409:Nerve Crush; D009414:Nerve Growth Factors / Q000494:pharmacology; D009900:Optic Nerve / Q000502:physiology*; D010617:Phenanthridines / Q000032:analysis*; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats; D020318:Rats, Long-Evans; D012165:Retinal Ganglion Cells / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction; D013482:Superoxide Dismutase / Q000494:pharmacology; D013481:Superoxides / Q000037:antagonists & inhibitors / Q000378:metabolism*
C521281:5-(6'-triphenylphosphoniumhexyl)-5,6-dihydro-6-phenyl-3,8-phenanthridinediammine; D009414:Nerve Growth Factors; D010617:Phenanthridines; D013481:Superoxides; D011092:Polyethylene Glycols; D000968:Antimycin A; D013482:Superoxide Dismutase; C063471:polyethylene glycol-superoxide dismutase; D014450:Electron Transport Complex III