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Tim T. Lam, Andrew S. Abler, Jacky M. K. Kwong, Mark O. M. Tso; N-Methyl-D-Aspartate (NMDA)–Induced Apoptosis in Rat Retina. Invest. Ophthalmol. Vis. Sci. 1999;40(10):2391-2397.
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purpose. The involvement of apoptosis in N-methyl-D-aspartate (NMDA)–induced
excitotoxicity in adult rat retinas was examined.
methods. Excitotoxic loss of inner retinal elements was induced by
intravitreal injections of various concentrations of neutralized NMDA
in adult albino Lewis rats. Tissue responses were quantified by
measuring the inner retinal thickness (IRT) in plastic sections of the
retinas and cell counts in the retinal ganglion cell layer in
flatmount preparations of the whole retinas. Internucleosomal
DNA fragmentation, a hallmark of apoptosis, was assayed with agarose
DNA gel electrophoresis. The in situ TdT-mediated biotin-dUTP nick end
labeling (TUNEL) method was used to locate nicked DNA in paraffin
sections of the retinas. Ultrastructural changes of the degenerating
cells were examined by electron microscopy. The efficacy of
Ac-Tyr-Val-Ala-Asp-CMK (YVAD–CMK), a peptidyl caspase inhibitor, and
3-aminobenzamide (ABA), an inhibitor of poly(ADP-ribose) polymerase
(PARP), in ameliorating the loss of inner retinal elements was
evaluated using morphometry to examine the apoptotic pathways.
results. Intravitreal injection of NMDA induced a dose-dependent loss of
inner retinal elements as evidenced by the measurements of IRT and
RGCCs. There were time- and dose-related appearances of
internucleosomal fragmentation of retinal DNA and a time-related
appearance of TUNEL-positive nuclei in the inner retinas after
intravitreal NMDA injection. Ultrastructural features consistent with
classic apoptotic changes were noted in degenerating cells 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.
Simultaneous administration of NMDA and YVAD–CMK or ABA abolished or
attenuated the loss of RGCCs in the posterior retinas.
conclusions. NMDA-induced excitotoxicity involved apoptosis and caspases and PARP
may play important roles in the pathways.
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