Yu Yokoyama1, Kazuichi Maruyama1, Kotaro Yamamoto1, Kazuko Omodaka1, Masayuki Yasuda1, Noriko Himori1, Morin Ryu1, Koji M Nishiguchi1, Toru Nakazawa2. 1. Department of Ophthalmology, Tohoku University Graduate School of Medicine, Miyagi, Japan. 2. Department of Ophthalmology, Tohoku University Graduate School of Medicine, Miyagi, Japan; Department of Retinal Disease Control, Tohoku University Graduate School of Medicine, Miyagi, Japan; Department of Advanced Ophthalmic Medicine, Tohoku University Graduate School of Medicine, Miyagi, Japan. Electronic address: ntoru@oph.med.tohoku.ac.jp.
Abstract
PURPOSE: In this study, we set out to establish an in vivo animal model of oxidative stress in the retinal ganglion cells (RGCs) and determine whether there is a link between oxidative stress in the RGCs and the activation of calpain, a major part of the apoptotic pathway. MATERIALS AND METHODS: Oxidative stress was induced in the RGCs of C57BL/6 mice by the intravitreal administration of 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH, 30mM, 2μl). Control eyes were injected with 2μl of vehicle. Surviving Fluorogold (FG)-labeled RGCs were then counted in retinal flat mounts. Double staining with CellROX and Annexin V was performed to investigate the co-localization of free radical generation and apoptosis. An immunoblot assay was used both to indirectly evaluate calpain activation in the AAPH-treated eyes by confirming α-fodrin cleavage, and also to evaluate the effect of SNJ-1945 (a specific calpain inhibitor: 4% w/v, 100mg/kg, intraperitoneal administration) in these eyes. RESULTS: Intravitreal administration of AAPH led to a significant decrease in FG-labeled RGCs 7days after treatment (control: 3806.7±575.2RGCs/mm(2), AAPH: 3156.1±371.2RGCs/mm(2), P<0.01). CellROX and Annexin V signals were co-localized in the FG-labeled RGCs 24h after AAPH injection. An immunoblot assay revealed a cleaved α-fodrin band that increased significantly 24h after AAPH administration. Intraperitoneally administered SNJ-1945 prevented the cleavage of α-fodrin and had a neuroprotective effect against AAPH-induced RGC death (AAPH: 3354.0±226.9RGCs/mm(2), AAPH+SNJ-1945: 3717.1±614.6RGCs/mm(2), P<0.01). CONCLUSION: AAPH administration was an effective model of oxidative stress in the RGCs, showing that oxidative stress directly activated the calpain pathway and induced RGC death. Furthermore, inhibition of the calpain pathway protected the RGCs after AAPH administration.
PURPOSE: In this study, we set out to establish an in vivo animal model of oxidative stress in the retinal ganglion cells (RGCs) and determine whether there is a link between oxidative stress in the RGCs and the activation of calpain, a major part of the apoptotic pathway. MATERIALS AND METHODS: Oxidative stress was induced in the RGCs of C57BL/6 mice by the intravitreal administration of 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH, 30mM, 2μl). Control eyes were injected with 2μl of vehicle. Surviving Fluorogold (FG)-labeled RGCs were then counted in retinal flat mounts. Double staining with CellROX and Annexin V was performed to investigate the co-localization of free radical generation and apoptosis. An immunoblot assay was used both to indirectly evaluate calpain activation in the AAPH-treated eyes by confirming α-fodrin cleavage, and also to evaluate the effect of SNJ-1945 (a specific calpain inhibitor: 4% w/v, 100mg/kg, intraperitoneal administration) in these eyes. RESULTS: Intravitreal administration of AAPH led to a significant decrease in FG-labeled RGCs 7days after treatment (control: 3806.7±575.2RGCs/mm(2), AAPH: 3156.1±371.2RGCs/mm(2), P<0.01). CellROX and Annexin V signals were co-localized in the FG-labeled RGCs 24h after AAPH injection. An immunoblot assay revealed a cleaved α-fodrin band that increased significantly 24h after AAPH administration. Intraperitoneally administered SNJ-1945 prevented the cleavage of α-fodrin and had a neuroprotective effect against AAPH-induced RGC death (AAPH: 3354.0±226.9RGCs/mm(2), AAPH+SNJ-1945: 3717.1±614.6RGCs/mm(2), P<0.01). CONCLUSION:AAPH administration was an effective model of oxidative stress in the RGCs, showing that oxidative stress directly activated the calpain pathway and induced RGC death. Furthermore, inhibition of the calpain pathway protected the RGCs after AAPH administration.
Authors: Maria D Pinazo-Duran; Kian Shoaie-Nia; Vicente Zanon-Moreno; Silva M Sanz-Gonzalez; Javier Benitez Del Castillo; Jose J Garcia-Medina Journal: Curr Neuropharmacol Date: 2018 Impact factor: 7.363