Mechanisms of reduced striatal NMDA excitotoxicity in type I nitric oxide synthase knock-out mice

Abstract

We investigated the role of neuronal (type I) nitric oxide synthase (nNOS) in NMDA-mediated excitotoxicity in wild-type (SV129 and C57BL/6J) and type I NOS knock-out (nNOS-/-) mice and examined its relationship to apoptosis. Excitotoxic lesions were produced by intrastriatal stereotactic NMDA microinjections (10-20 nmol). Lesion size was dose- and time-dependent, completely blocked by MK-801 pretreatment, and smaller in nNOS knock-out mice compared with wild-type littermates (nNOS+/+, 11.7 +/- 1.7 mm3; n = 8; nNOS-/-, 6. 4 +/- 1.8 mm3; n = 7). The density and distribution of striatal NMDA binding sites, determined by NMDA receptor autoradiography, did not differ between strains. Pharmacological inhibition of nNOS by 7-nitroindazole (50 mg/kg, i.p.) decreased NMDA lesion size by 32% in wild-type mice (n = 7). Neurochemical and immunohistochemical measurements of brain nitrotyrosine, a product of peroxynitrite formation, were increased markedly in wild-type but not in the nNOS-/- mice. Moreover, elevations in 2,3- and 2,5-dihydroxybenzoic acid levels were significantly reduced in the mutant striatum, as a measure of hydroxyl radical production. The importance of apoptosis to NMDA receptor-mediated toxicity was evaluated by DNA laddering and by quantitative histochemistry [terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) staining]. DNA laddering was first detected within lesioned tissue after 12-24 hr. TUNEL-positive cells were first observed at 12 hr, increased in number at 48 hr and 7 d, and were located predominantly in proximity to the lesion border. The density was significantly lower in nNOS-/- mice. Hence, oligonucleosomal DNA breakdown suggesting apoptosis develops as a late consequence of NMDA microinjection and is reduced in nNOS mutants. The mechanism of protection in nNOS-/- mice may relate to decreased oxygen free radical production and related NO reaction products and, in part, involves mechanisms of neuronal death associated with the delayed appearance of apoptosis.

Fig. 3.

Autoradiograms of agarose gels showing DNA breakdown after intrastriatal NMDA injection (20 nmol). Brains were removed rapidly, and 2-mm-thick coronal slices were cut. DNA was isolated from the second slice containing both cortex and striatum, and a [³²P]dideoxy-ATP end-labeling method with terminal transferase was performed. A, DNA breakdown both in the form of nonspecific fragmentation (smearing) and oligonucleosomal breakdown (laddering) appears at 12–24 hr and intensifies 48 hr after NMDA in SV129 mice. Each lanecontains tissue obtained from different animals killed at 12, 24, and 48 hr after NMDA injection. B, Both nonspecific DNA fragmentation and oligonucleosomal breakdown are observed to a lesser extent in nNOS^−/− compared with nNOS^+/+ littermates 48 hr after NMDA. The gel is representative of three independent experiments. M, Molecular weight marker.

Fig. 1.

Production of 3-nitrotyrosine and 2,3- and 2,5-DHBA 1 hr after intrastriatal injection of NMDA in wild-type (SV129, n = 11; C57/B6, n = 5) and nNOS^−/− (n = 9) mice.A, NMDA (20 nmol) significantly increased striatal 3-nitrotyrosine levels in both wild-type strains but not in nNOS^−/− mice (open bars) 1 hr after injection, compared with the contralateral unlesioned (filled bars) striatum. 3-Nitrotyrosine was measured electrochemically. B, NMDA (20 nmol) increased striatal 2,3- and 2,5-DHBA production by ∼100% and 50% in SV129 and C57BL/6 mice, respectively (open bars), 1 hr after injection, compared with the contralateral unlesioned (filled bars) striatum. Levels in nNOS^−/− mice showed no change. Using the salicylate OH^⋅-trapping method, 2,3- and 2,5-DHBA were measured by HPLC. *p < 0.05 versus contralateral hemisphere. Error bars indicate ±SEM.

Fig. 2.

Histopathology and 3-nitrotyrosine immunohistochemistry of striatal NMDA lesions in wild-type (A, C, E) and nNOS^−/− (B, D,F) mice 12 hr after injection. Nissl staining of NMDA-lesioned brains identifies the extent of the damage (staining pallor) in both the wild-type (A) and nNOS^−/− mice (B). In adjacent cut sections, expression of 3-nitrotyrosine immunoreactivity is markedly increased in the wild-type mouse (C,E), with no immunolabel observed within the nNOS^−/− mouse (D,F). 3-Nitrotyrosine immunoreactivity is found in both neuron soma and neuropil in the wild-type animals. Scale bars:A, 2 mm, same for B–D; F, 200 μm, same for E.

Fig. 4.

Density and distribution of TUNEL-positive cells in the striatum 48 hr after intrastriatal NMDA injection.A, Diagram illustrating the method used for counting TUNEL-positive cells. TUNEL-positive cells located within a 100-μm-wide horizontal band (extending from the medial to lateral edge of the lesion, ∼2.5 mm) were counted in a single tissue section containing the largest diameter of NMDA lesion and expressed as the number of TUNEL-positive cells per square millimeter. The distribution of TUNEL-positive cells along the horizontal band is shown by dividing the diameter of the lesion into five equal segments,a–e, with a the most medial ande the most lateral, to normalize for variations in lesion size. B, The distribution of TUNEL-positive cells shifted from the center of the lesion to the periphery from 48 hr to 7 d after NMDA injection. There were more TUNEL-positive cells in the medial striatum compared with the lateral striatum. There were no TUNEL-positive cells in the lesion at 3 and 6 hr. C, The density of TUNEL-positive cells was less in nNOS^−/− mice than in nNOS^+/+mice 48 hr after NMDA injection. The difference was more marked in the lateral striatum. There was no difference in the morphology of apoptotic cells between nNOS^−/− and nNOS^+/+ mice. The area of the TUNEL-positive region was 5.94 ± 0.64 and 5.22 ± 0.31 mm²in nNOS^+/+ and nNOS^−/−groups, respectively (p > 0.05). The distances from the center of the TUNEL-positive area to the midline and to the dorsal cortical surface were similar in both nNOS^+/+ and nNOS^−/− groups (data not shown). Error bars indicate ±SEM. D, Photomicrograph of TUNEL-stained section, taken from the periphery of the lesion, 48 hr after NMDA injection in an SV129 mouse, illustrating densely stained pyknotic nuclei with occasional apoptotic bodies.E, Photomicrograph of H&E-stained section, taken from a region similar to D, 48 hr after NMDA injection in an SV129 mouse. A wide variety of cellular morphology is observed, including necrotic as well as intact cells. Cells with pyknotic nuclei and occasional apoptotic bodies are observed along with necrotic cells. Cells with a normal morphology are also seen.