Marked Induction of Calcium-Independent Nitric Oxide Synthase Activity after Focal Cerebral Ischemia

Abstract

We studied the effect of focal cerebral ischemia on inducible (iNOS) and constitutive (cNOS) nitric oxide synthase enzymatic activities in the affected brain. The middle cerebral artery (MCA) was occluded in spontaneously hypertensive rats. Animals were killed 1, 2, 4, and 7 days later. cNOS and iNOS enzymatic activities were determined in the infarcted cortex using the assay of Bredt and Snyder. cNOS was assayed in the presence of calcium, whereas iNOS was assayed in the absence of calcium and in the presence of tetrahydrobiopterin. The validity of the iNOS assay was verified in rats treated with bacterial lipopolysaccharide. In these animals, the magnitude of the induction of iNOS enzymatic activity in lung, spleen, and brain paralleled the expression of iNOS mRNA, assessed by reverse-transcription polymerase chain reaction. After MCA occlusion, calcium-dependent (cNOS) activity was markedly reduced only in lesioned cerebral cortex at days 1–7 ( p < 0.001; analysis of variance and Tukey's test). In contrast to cNOS, calcium-independent (iNOS) activity was induced substantially in the infarct ( p < 0.005) but not in the contralateral intact cortex ( p > 0.05). iNOS activity peaked at day 2 and was not different from baseline at day 7 ( p > 0.05). No NADPH diaphorase-positive neurons were observed in the area of the lesion at days 1–7. Macrophages appeared at day 2 and invaded the infarcted tissue by day 7. At this time, numerous glial fibrillary acidic protein-positive astrocytes were observed within the lesion. The results suggest that the decline in calcium-dependent (cNOS) activity reflects loss of NOS neurons within the lesion. The induction of calcium-independent activity is likely to reflect induction of iNOS in nonneuronal cells. Sustained nitric oxide production by iNOS may contribute to the late phase of tissue damage in focal cerebral ischemia.