N-methyl-D-aspartate (NMDA)--induced apoptosis in rat retina.
OBJECTIVE
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.
Lam
T T
TT
Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, USA. timlam@cuhk.edu.hk
Abler
A S
AS
Kwong
J M
JM
Tso
M O
MO
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001549:Benzamides / Q000494:pharmacology; D061945:Caspase Inhibitors; D020169:Caspases / Q000502:physiology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004247:DNA / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D004587:Electrophoresis, Agar Gel; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology*; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D007267:Injections; D008297:Male; D016202:N-Methylaspartate / Q000494:pharmacology*; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000502:physiology; D051381:Rats; D011917:Rats, Inbred Lew; D012160:Retina / Q000187:drug effects* / Q000473:pathology; D013997:Time Factors; D014822:Vitreous Body / Q000187:drug effects
D000590:Amino Acid Chloromethyl Ketones; D001549:Benzamides; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D004338:Drug Combinations; D018690:Excitatory Amino Acid Agonists; D018691:Excitatory Amino Acid Antagonists; C098738:N-acetyl-tyrosyl-valyl-alanyl-aspartyl chloromethyl ketone; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D016202:N-Methylaspartate; C025160:3-aminobenzamide; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases