PURPOSE: To analyze the influence of oxidative stress on retinal ganglion cells (RGCs) by using a selective culture system of rat RGCs. METHODS: Rat RGCs were purified by a two-step immunopanning procedure and cultured either with or without antioxidant (AO) compounds. Reactive oxygen species (ROS) in RGCs were analyzed using dihydroethidium. Expression of angiotensin II, cleaved caspase 3, and netrin-1 was analyzed by immunocytochemistry. Live RGCs were detected by use of calcein-acetoxymethyl ester. The roles of angiotensin II type 1 receptor (AT1R) signaling and netrin-1 were analyzed by use of an AT1R blocker (telmisartan) and an anti-netrin-1 neutralizing antibody, respectively. RESULTS: ROS and angiotensin II were induced in RGCs cultured without AO compounds (AO-). In these cultures, the number of live RGCs decreased and expression of cleaved and activated caspase 3 increased, but these changes were attenuated by addition of the AT1R blocker. Reduction in netrin-1 expression under the AO- condition was also prevented by the AT1R blocker. The AT1R blocker's effects on RGC survival and reduction in cleaved caspase 3-positive cells were cancelled by the anti-netrin-1 neutralizing antibody. CONCLUSIONS: Oxidative stress induced cell death through AT1R signaling and netrin-1 reduction in cultured RGCs.
PURPOSE: To analyze the influence of oxidative stress on retinal ganglion cells (RGCs) by using a selective culture system of rat RGCs. METHODS:Rat RGCs were purified by a two-step immunopanning procedure and cultured either with or without antioxidant (AO) compounds. Reactive oxygen species (ROS) in RGCs were analyzed using dihydroethidium. Expression of angiotensin II, cleaved caspase 3, and netrin-1 was analyzed by immunocytochemistry. Live RGCs were detected by use of calcein-acetoxymethyl ester. The roles of angiotensin II type 1 receptor (AT1R) signaling and netrin-1 were analyzed by use of an AT1R blocker (telmisartan) and an anti-netrin-1 neutralizing antibody, respectively. RESULTS:ROS and angiotensin II were induced in RGCs cultured without AO compounds (AO-). In these cultures, the number of live RGCs decreased and expression of cleaved and activated caspase 3 increased, but these changes were attenuated by addition of the AT1R blocker. Reduction in netrin-1 expression under the AO- condition was also prevented by the AT1R blocker. The AT1R blocker's effects on RGC survival and reduction in cleaved caspase 3-positive cells were cancelled by the anti-netrin-1 neutralizing antibody. CONCLUSIONS: Oxidative stress induced cell death through AT1R signaling and netrin-1 reduction in cultured RGCs.
Authors: Brent D Wilson; Masaaki Ii; Kye Won Park; Arminda Suli; Lise K Sorensen; Fréderic Larrieu-Lahargue; Lisa D Urness; Wonhee Suh; Jun Asai; Gerhardus A H Kock; Tina Thorne; Marcy Silver; Kirk R Thomas; Chi-Bin Chien; Douglas W Losordo; Dean Y Li Journal: Science Date: 2006-06-29 Impact factor: 47.728