The role of calpain in an in vivo model of oxidative stress-induced retinal ganglion cell damage

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, 30 mM, 2 mu l). Control eyes were injected with 2 mu l 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 a-fodrin cleavage, and also to evaluate the effect of SNJ-1945 (a specific calpain inhibitor: 4% w/v, 100 mg/kg, intraperitoneal administration) in these eyes. Results: Intravitreal administration of AAPH led to a significant decrease in FG-labeled RGCs 7 days after treatment (control: 3806.7 +/- 575.2 RGCs/mm(2), AAPH: 3156.1 +/- 371.2 RGCs/mm(2), P < 0.01). CellROX and Annexin V signals were co-localized in the FG-labeled RGCs 24 h after AAPH injection. An immunoblot assay revealed a cleaved alpha-fodrin band that increased significantly 24 h after AAPH administration. Intraperitoneally administered SNJ-1945 prevented the cleavage of a-fodrin and had a neuroprotective effect against AAPH-induced RGC death (AAPH: 3354.0 +/- 226.9 RGCs/mm2, AAPH+SNJ-1945: 3717.1 +/- 614.6 RGCs/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. (C) 2014 Elsevier Inc. All rights reserved.