Calpain and N–Methyl–D–Aspartate (NMDA)–Induced Excitotoxicity in Rat Retina

Abstract

Abstract: : Purpose: To examine the involvement of µ– and m–calpain in N–methyl–D–aspartate (NMDA)–induced excitotoxicity in the adult rat retina. Methods: Forty–five to fifty–five day–old albino Sprague Dawley rats were given intravitreal injections of 8 nmoles NMDA. Animals were euthanized at 1, 3, 6, 9, 12, 18 or 24 hours after injection. Immunohistochemistry and Western blotting of µ– and m–calpain were performed on retinas. Colocalization studies of TdT–mediated biotin–dUTP nick end labeling (TUNEL) and µ– or m–caplain immunoreacticity were conducted on retinal sections from animals 12 hours after NMDA injection. The effect of a calpain inhibitor on NMDA–induced excitotoxicity was evaluated by counting retinal ganglion cell (RGC)–like cells 7 days after injection and counting TUNEL positive cells 18 hours after injection. Results: Increased immunoreactivity of µ–calpain was noted in cells at the RGC layer and inner nuclear layer with a maximal expression at 12 hours after NMDA injection. This was confirmed by Western blotting. TUNEL positive cells in the inner retina colocalized with moderate or intense µ–calpain immunoreactivity. In contrast, there was no remarkable change in m–calpain immunoreactivity at all time points after NMDA injection. Simultaneous injection of 2 nmoles of a calpain inhibitor (calpain inhibitor II) significantly reduced the number of TUNEL positive cells in the inner retina (P=0.01, 98% in RGC layer; P=0.04, 97% in inner nuclear layer) 18 hours after NMDA injection, and preserved RGC–like cells counted in central retina (P=0.01, 89%) and peripheral retina (P=0.02, 100%) 7 days after injection. Conclusions: µ–Calpain may be involved in mediating NMDA–induced excitotoxicity in retinas and calpain inhibitors may play a therapeutic role in NMDA related diseases.

Keywords: neuroprotection • ganglion cells • apoptosis/cell death