Amplification of a Reactive Oxygen Species Signal in Axotomized Retinal Ganglion Cells

Abstract

Abstract: : Purpose: Axotomy of retinal ganglion cells (RGC) induces mitochondion-mediated apoptosis. However, the exact events that trigger the mitochondrial signaling for apoptosis remain unclear. Results from our laboratory suggest that reactive oxygen species (ROS) may play a role in this signal transduction, as specific ROS scavengers and hypoxia have been shown to reduce the death of cultured RGCs after axotomy. To study the mechanism by which ROS could induce apoptosis signaling in axotomized RGCs, we exposed them to oxidative stress using H₂O₂ and measured the amount of superoxide produced. Methods: RGCs were retrogradely labeled by injecting the fluorescent tracer DAPI in the superior colliculi of postnatal day 2-4 Long Evans rats. At postnatal days 7-9, the retinas were dissociated with papain and cultured without specific ROS-generating systems or scavengers. RGCs were identified by their DAPI positivity using the appropriate filters under epifluorescence. The cells were then treated with Sytox Green and dihydroethidium (HEt). The oxidation of HEt to Et serves as a measure of superoxide production, while Sytox Green negativity identifies living cells. The intensity of Et fluorescence in living RGCs was measured over time using digital imaging and Metafluor software. After baseline acquisition, cells were exposed to 10 mM H₂O₂ or vehicle and Et intensities acquired for 30 minutes. Results: Compared with baseline, there was a significant increase in the intensity of Et fluorescence after H₂O₂ was added to the axotomized RGCs. However, this increase only occurred when imaging 24 hours after culturing the cells; there was no increase when imaging 2 hours after culturing. This apparent secondary oxidative burst is blocked by TCEP, suggesting that it is dependent on the oxidation of some intracellular target(s). The rise in superoxide is also blocked by 24 hour treatment with cycloheximide, implying that it requires protein synthesis. Conclusions: These results demonstrate that induction of an oxidative stress in cultured retinal ganglion cells induces a secondary burst of superoxide anion, resulting in amplification of the original oxidative signal. There may be intracellular targets, probably proteins, which have sulfhydryls oxidized by H₂O₂, resulting in the induction of an apoptosis program, an early event of which is a superoxide burst.

Keywords: apoptosis/cell death • ganglion cells • imaging/image analysis: non-clinical