Introduction
Nitric oxide (NO) plays an important role in the pathogenesis of neuronal injury during cerebral ischemia. 1 It was reported that infarct volumes in nNOS knockout mice decreased significantly 24 hours after middle cerebral artery occlusion and was suggested that nNOS exacerbated acute ischemic injury. Therefore, we investigated the dynamics of cerebral NO production in nNOS knockout mice during cerebral ischemia and reperfusion.
Methods
Male nNOS knockout mice (n = 6) and littermate mice (control group) (n = 7) were anesthetized by halothane. NO production was continuously monitored by in vivo microdialysis. Microdialysis probes were inserted into the left striatum and perfused with Ringer's solution at a constant rate 2 μl/min. Laser Doppler probes were also inserted into the right striatum. After 2 hours equilibrium period, fractions were collected every 10 minutes. Forebrain cerebral ischemia was produced by occlusion of both common carotid arteries for 10 minutes. Levels of nitric oxide metabolites, nitrite (NO2-) and nitrate (NO3-), in the dialysate were determined using the Griess reaction. Brain sections were immunostained with an anti-nNOS antibody (polyclonal, 1:400, Zymed). To determine the fractional area density of nNOS-immunoreactive pixels to total pixels of the whole field, the captured images were analyzed. Mann-Whitney U test was used for group comparison analysis.
The level of NO3−
Results
Blood Pressure: There were no significant difference between nNOS knockout mice (78. 9 ± 6.9 mmHg; mean ± SD) and control group (66.5 ± 8.0). Only in 120 minutes after reperfusion, nNOS knockout mice (77.5 ± 15.4) showed significantly higher blood pressure than those of control group (57.9 ± 14.7) in 120 minutes after repurfusion. Cerebral Blood Flow (CBF): During cerebral ischemia, no significant differences in CBF were observed between the two groups. CBF in nNOS knockout mice (61.1 ± 19.5 % of the baseline level) was significantly higher than that of control group (42.1 ± 9.5 %) in 70, 90∼120 minutes after the start of reperfusion. Nitric oxide:
NO2-: No significant differences were seen in the two groups. NO3-: The levels of NO3- in nNOS knockout mice were significantly lower than those of control group before and during ischemia, and during reperfusion (Fig.1). nNOS activity: The percentage of nNOS-immunoreactive pixels to whole area in nNOS knockout mice (0.06.±0.12%) was significantly lower than that of the control group (0.62 ± 0.57 %).
Conclusion
The above data suggest that nNOS leads to neurotoxicity by producing NO during cerebral ischemia and reperfusion.