Retinal ischemia-reperfusion (IR) injury remains a common cause of blindness and has a final pathway of retinal ganglion cell (RGC) death by apoptosis and necrosis. RGC apoptosis was intensively studied in IR injury, while RGC necrosis did not receive nearly enough consideration since it was viewed as an accidental and unregulated cellular event. However, there is evidence that necrosis, like apoptosis, can be implemented by a programmed mechanism. In this study, we tested the role of RGC programmed necrosis (necroptosis) in IR-induced retinal injury. We employed the mouse model of retinal IR injury for in vivo experiments. The oxygen and glucose deprivation (OGD) model was used as an IR model in vitro. Primary RGCs were isolated by an immunopanning technique. Necrostatin 1 (Nec1) was used to inhibit necroptosis in in vitro and in vivo experiments. The changes in gene expression were assessed by quantitative RT-PCR. The distribution of proteins in the retina and in RGC cultures was evaluated by immunohistochemistry and immunocytochemistry, respectively. Our data suggest that proteins (Ripk1 and Ripk3), which initiate necroptosis, were present in normal and ischemic RGCs. Treatment with Nec1 significantly reduced retinal damage after IR. Increased RGC survival and reduced RGC necrosis following OGD were observed in Nec1-treated cultures. We found significantly reduced expression of genes coding pro-inflammatory markers Il1b, Ccl5, Cxcl10, Nos2 and Cybb in Nec1-treated ischemic retinas. Thus, our findings suggest that RGC necroptosis contributes to retinal damage after IR through direct loss of cells and induction of associated inflammatory responses.
Keywords: Ripk1; Ripk3; ischemia–reperfusion; necroptosis; necrostatin 1; retinal damage; retinal ganglion cells.
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