MECHANISMS OF REDUCED STRIATAL NMDA EXCITOTOXICITY IN TYPE-I NITRIC-OXIDE SYNTHASE KNOCK-OUT MICE

We investigated the role of neuronal (type I) nitric oxide synthase (n NOS) in NMDA-mediated excitotoxicity in wild-type (SV129 and C57BL/6J) and type I NOS knack-out (nNOS (-/-)) mice and examined its relations hip to apoptosis. Excitotoxic lesions were produced by intrastriatal s tereotactic NMDA microinjections (10-20 nmol). Lesion size was dose-an d time-dependent, completely blocked by MK-801 pretreatment, and small er in nNOS knock-out mice compared with wild-type littermates (nNOS(+/ +), 11.7 +/- 1.7 mm(3); n = 8; nNOS(-/-), 6.4 +/- 1.8 mm(3); n = 7), T he density and distribution of striatal NMDA binding sites, determined by NMDA receptor autoradiography, did not differ between strains. Pha rmacological inhibition of nNOS by 7-nitroindazole (50 mg/kg, i.p.) de creased NMDA lesion size by 32% in wild-type mice (n = 7). Neurochemic al and immunohistochemical measurements of brain nitrotyrosine, a prod uct of peroxynitrite formation, were increased markedly in wild-type b ut not in the nNOS(-/-) mice. Moreover, elevations in 2,3- and 2,5-dih ydroxybenzoic acid levels were significantly reduced in the mutant str iatum, as a measure of hydroxyl radical production. The importance of apoptosis to NMDA receptor-mediated toxicity was evaluated by DNA ladd ering and by quantitative histochemistry [terminal deoxynucleotidyl tr ansferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) staining]., DNA laddering was first detected within lesioned t issue after 12-24 hr. TUNEL-positive cells were first observed at 12 h r, increased in number at 48 hr and 7 d, and were located predominantl y in proximity to the lesion border. The density was significantly low er in nNOS(-/-) mice. Hence, oligonucleosomal DNA breakdown suggesting apoptosis develops as a late consequence of NMDA microinjection and i s reduced in nNOS mutants. The mechanism of protection in nNOS(-/-) mi ce may relate to decreased oxygen free radical production and related NO reaction products and, in part, involves mechanisms of neuronal dea th associated with the delayed appearance of apoptosis.