PURPOSE: The aim of this study was to determine whether NOX2, one of the homologs of NADPH oxidase, plays a role in neuronal cell death during retinal ischemia. METHODS: Ischemia reperfusion (I/R) injury was generated in C57/BL6 and NOX2(-/-) mice by increasing the intraocular pressure (IOP) to 110 mm Hg for 40 minutes followed by reperfusion. Quantitative PCR and Western blot analysis were performed to measure NOX2 expression. Reactive oxygen species (ROS) formation was assessed by dihydroethidium imaging of superoxide formation and Western blot analysis for tyrosine nitration. TUNEL assay was performed to determine cell death at 3 days after I/R. Survival of neurons within the ganglion cell layer (GCL) was assessed at 7 days after I/R by confocal morphometric imaging of retinal wholemounts immunostained with NeuN antibody. Activation of mitogen-activated protein kinases and nuclear factor κB (NF-κΒ) was measured by Western blot analysis. RESULTS: NOX2 mRNA and protein and ROS were significantly increased in wild-type I/R retinas. This effect was associated with a 60% decrease in the number of GCL neurons and a 10-fold increase in TUNEL-positive cells compared with the fellow sham control eyes. Phosphorylation of ERK and NF-κB was significantly increased in wild-type I/R retinas. Each of these effects was markedly attenuated in the NOX2(-/-) retina (P < 0.01). CONCLUSIONS: These data demonstrate that the deletion of NOX2 can reduce I/R-induced cell death and preserve retinal GCL neurons after I/R injury. The neuronal cell injury caused by I/R is associated with the activation of ERK and NF-κB signaling mechanisms.
PURPOSE: The aim of this study was to determine whether NOX2, one of the homologs of NADPH oxidase, plays a role in neuronal cell death during retinal ischemia. METHODS:Ischemia reperfusion (I/R) injury was generated in C57/BL6 and NOX2(-/-) mice by increasing the intraocular pressure (IOP) to 110 mm Hg for 40 minutes followed by reperfusion. Quantitative PCR and Western blot analysis were performed to measure NOX2 expression. Reactive oxygen species (ROS) formation was assessed by dihydroethidium imaging of superoxide formation and Western blot analysis for tyrosine nitration. TUNEL assay was performed to determine cell death at 3 days after I/R. Survival of neurons within the ganglion cell layer (GCL) was assessed at 7 days after I/R by confocal morphometric imaging of retinal wholemounts immunostained with NeuN antibody. Activation of mitogen-activated protein kinases and nuclear factor κB (NF-κΒ) was measured by Western blot analysis. RESULTS:NOX2 mRNA and protein and ROS were significantly increased in wild-type I/R retinas. This effect was associated with a 60% decrease in the number of GCL neurons and a 10-fold increase in TUNEL-positive cells compared with the fellow sham control eyes. Phosphorylation of ERK and NF-κB was significantly increased in wild-type I/R retinas. Each of these effects was markedly attenuated in the NOX2(-/-) retina (P < 0.01). CONCLUSIONS: These data demonstrate that the deletion of NOX2 can reduce I/R-induced cell death and preserve retinal GCL neurons after I/R injury. The neuronal cell injury caused by I/R is associated with the activation of ERK and NF-κB signaling mechanisms.
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