Endogenous Neuroprotective Mechanism of Retinal Ganglion Cells following Transection of the Optic Nerve

Abstract

Optic nerve transection has been shown to induce retinal ganglion cell (RGC) death mediated by caspase-3 and -9 activation. In this study, we examined the possible role of cytochrome c release in axotomized RGC death. Axotomized RGCs are proposed to die via apoptosis. Caspase-3 activation after axotomy has been shown, and caspase-3 inhibitors or Bcl-2 over-expression has been observed to enhance RGC survival. These studies suggest that caspase-3 may participate in the loss of axotomized RGCs. One of the upstream initiators of the apoptotic pathway is cytochrome c release from the intermembrane space of mitochondria. Released cytochrome c binds to Apaf-1 and activates caspase-9. Activated caspase-9 in turn activates caspase-3. To examine the involvement of the cytochrome c pathway in the apoptosis of axotomized RGCs, the release of cytochrome c post-axotomy was analyzed using immunohistochemistry. At 1, 2, 3, 5, 7, 10 and 14 days post-axotomy (dpa), animals were sacrificed and their eyeballs sectioned. Ten-micrometer retinal sections where the RGCs were prelabeled with retrograde transport of fluoro-Gold were used. The sections were counter-stained with DAPI to reveal nuclear morphology. We found that by 1 dpa, there was a significant increase in cytochrome c immunoreactivity compared to normal, undamaged retinas. The increase peaked at 3 dpa, and declined thereafter. By 10 dpa, cytochrome c immunoreactivity has returned to control level. Based on the findings from other apoptosis studies, this upregulation of cytochrome c immunoreactivity may contribute to axotomized RGC death. Interestingly, all the RGCs that were stained positive for cytochrome c exhibited normal nuclear morphology. Immunohistochemical staining of activated caspase-3 revealed that no caspase-3 activation was observed until 3 dpa. This suggests that cytochrome c release may act upstream of caspase-3 activation and precede any visible nuclear changes. Double immunohistochemical analysis for cytochrome c and activated caspase-3 showed that the two staining exhibit no co-localization. The lack of co-localization and the lag between the onset of cytochrome c and activated caspase- 3 staining suggests that there is a temporal gap between the two events. To examine the mechanism underlying the apparent temporal gap between cytochrome c release and caspase activation, the effect of axotomy on Akt activation was investigated. We found that axotomy induced a rapid increase in Akt phosphorylation at 3 h postaxotomy. Phospho-Akt content returned to control level at 3 dpa, coinciding with the onset of caspase activation and RGC loss. Our data suggest that this transient increase may contribute to the delayed activation of caspase-3/9 in axotomized retinas. Attenuating the increase in Akt phosphorylation following axotomy by intravitreal injections of wortmannin, a PI3K inhibitor, resulted in the presence of activated caspase-3 and -9-positive apoptotic cells in the ganglion cell layer. The activation of PI3K/Akt pathway following axotomy may serve as an endogenous protective machinery to counteract the death signals initiated by axotomy.