Purpose: Axonal injury to central nervous system (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 (RGC) is ROS-dependent. To better define the mechanisms by which ROS induce RGC death after axotomy, we studied their effects in dissociated neonatal rat retinal cultures.
Methods:
P2-4 Long–Evans rat RGC’s were retrogradely labeled with the fluorescent tracer
DiI. P7-9 retinas were dissociated and cultured in the presence of specific ROS
generating systems, scavengers, or redox modulators. RGC’s were identified by
DiI positivity and viability
determined by metabolism of calcein–AM.
Results: We found that ROS scavengers protected against RGC death after acute dissociation, and the effects of ROS appeared to be due to shifts in the redox potential, as RGC survival was critically dependent on redox state, with greatest survival under mildly reducing conditions. Culture of RGC with the non-thiol-containing reducing agent tris-(2-carboxyethyl) phosphine (TCEP) resulted in survival equivalent to or better than with neurotrophic factors.
Conclusions:
These 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
survival after axonal injury.