PURPOSE Retinal light exposure is a source of oxidative stress, and retinal cells contain molecules that scavenge or inactivate reactive oxygen species (ROS). Yet, ROS also play a role in signal transduction, and some retinal cells (e.g., neurotrophin-dependent retinal ganglion cells, RGCs) may use ROS as part of the signaling process for cell death. RGCs might therefore have specialized mechanisms for regulating ROS levels. The hypothesis that RGCs might regulate ROS differently from other retinal cells was tested by studying their differential response to oxidative stress in vitro.
METHODS RGCs were retrogradely labeled by injecting the fluorescent tracer DiI into the superior colliculi of postnatal day 2 through 4 Long-Evans rats. At postnatal days 7 through 9 the retinas were dissociated with papain and cultured with and without specific ROS-generating systems and/or scavengers. RGCs were identified by their DiI positivity using rhodamine filters. Living cells, determined by metabolism of calcein-AM viewed with fluorescein filters, were counted in triplicate. Degenerate reverse transcription-polymerase chain reaction (RT-PCR) using primers specific to peroxidase homology regions was used to survey for novel peroxidases expressed within normal retinas.
RESULTS Compared with other retinal cells, RGCs were remarkably resistant to cell death induced by superoxide anion, hydrogen peroxide, or hydroxyl radical. Catalase counteracted the effect of each ROS-generating system on retinal cells, consistent with damage occurring via a hydrogen peroxide intermediate. Aminotriazole, L-buthionine sulfoximine, and sodium azide partly abrogated the RGC resistance to oxidative stress, suggesting that this resistance may be mediated by catalase and/or glutathione peroxidase. A limited expression survey within the retina using degenerate RT-PCR did not demonstrate novel peroxidases.
CONCLUSIONS These data suggest a role for one or more endogenous peroxidases within RGCs, which could possibly be protective under conditions of axonal damage. Exploration of the unique characteristics of RGC resistance and susceptibility to injury may help in better understanding the pathophysiology of diseases associated with primary axonal damage.