C. Ayata et al., MECHANISMS OF REDUCED STRIATAL NMDA EXCITOTOXICITY IN TYPE-I NITRIC-OXIDE SYNTHASE KNOCK-OUT MICE, The Journal of neuroscience, 17(18), 1997, pp. 6908-6917
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.