N-methyl-D-aspartate (NMDA)-induced apoptosis in rat retina

PURPOSE. The involvement of apoptosis in N-methyl-D-aspartate (NMDA)-induce d excitotoxicity in adult rat retinas was examined. METHODS. Excitotoxic loss of inner retinal elements was induced by intravit real injections of various concentrations of neutralized NMDA in adult albi no Lewis rats. Tissue responses were quantified by measuring the inner reti nal thickness (IRT) in plastic sections of the retinas and cell counts in t he retinal ganglion cell layer in flatmount preparations of the whole retin as. Internucleosomal DNA fragmentation, a hallmark of apoptosis, was assaye d with agarose DNA gel electrophoresis. The in situ TdT-mediated biotin-dUT P nick end labeling (TUNEL) method was used to locate nicked DNA in paraffi n sections of the retinas. Ultrastructural changes of the degenerating cell s were examined by electron microscopy. The efficacy of Ac-Tyr-Val-Ala-Asp- CMK (YVAD-CMK), a peptidyl caspase inhibitor, and 3-aminobenzamide (ABA), a n inhibitor of poly(ADP-ribose) polymerase (PARP), in ameliorating the loss of inner retinal elements was evaluated using morphometry to examine the a poptotic pathways. RESULTS. Intravitreal injection of NMDA induced a dose-dependent loss of in ner retinal elements as evidenced by the measurements of IRT and RGCCs. The re were time- and dose-related appearances of internucleosomal fragmentatio n of retinal DNA and a time-related appearance of TUNEL-positive nuclei in the inner retinas after intravitreal NMDA injection. Ultrastructural featur es consistent with classic apoptotic changes were noted in degenerating cel ls in the retinal ganglion cell layer and the inner nuclear layer. Control retinas given vehicle, N-methyl-L-aspartate (the L-isomer of NMDA), or NMDA plus MK-801, a specific antagonist, did not show these changes. Simultaneo us administration of NMDA and YVAD-CMK or ABA abolished or attenuated the l oss of RGCCs in the posterior retinas. CONCLUSIONS. NMDA-induced excitotoxicity involved apoptosis and caspases an d PARP may play important roles in the pathways.