Expression of nitric oxide synthases and nitrotyrosine during blood-brain barrier breakdown and repair after cold injury

This study was undertaken to determine whether the blood-brain barrier (BBB ) breakdown and cerebral edema occurring post-trauma are associated with ov erexpression of the endothelial (e) and inducible (i) nitric oxide synthase s (NOS), enzymes responsible for nitric oxide (NO) biosynthesis. These enzy mes were determined quantitatively at the mRNA level and qualitatively at t he protein level in the rat cerebral cortical cold injury model, during a p eriod up to 6 days post-injury. In addition, peroxynitrite generation at th e lesion site was detected by immunolocalization of nitrotyrosine as a mark er of NO-superoxide interactions. These studies were correlated with the pe rmeability status of the BBB by immunohistochemical detection of endogenous fibronectin extravasation in the same brains. BBB breakdown was immediate in lesion vessels, it was present as early as 10 minutes post-lesion and de layed in perilesional vessels that showed maximal BBB breakdown between 2-4 days. The BBB was restored to normal at 6 days post-lesion. An increase in both eNOS and iNOS mRNA was observed at the lesion site as compared with t he contralateral hemisphere at 12 hours, 2 days, and 4 days. The mRNA retur ned to resting levels by 6 days. Increased eNOS protein was observed in the endothelium of permeable perilesional vessels and neovessels and in the en dothelium of the hyperplastic pial vessels overlying the lesion site. iNOS protein was observed initially in polymorphonuclear leukocytes at the lesio n site and later in macrophages, endothelial cells, and the smooth muscle c ells of the overlying pial vessels. Furthermore, nitrotyrosine was demonstr ated at the lesion site up to 5 days. Up-regulation of the NO synthases at both the mRNA and protein level accompanied by presence of nitrotyrosine! d uring BBB breakdown and angiogenesis suggests that NO has a role in the pat hogenesis of these processes.