Inducible nitric oxide synthase (iNOS), an enzyme that produces toxic amounts of nitric oxide, is expressed in a number of brain pathologies, including cerebral ischemia. We used mice with a null mutation of the iNOS gene to study the role of iNOS in ischemic brain damage. Focal cerebral ischemia was produced by occlusion of the middle cerebral artery (MCA). In wild-type mice, iNOS mRNA expression in the post-ischemic brain begun between 24 and 48 hr peaked at 96 hr and subsided 7 d after MCA occlusion. iNOS mRNA induction was associated with expression of iNOS protein and enzymatic activity. In contrast, mice lacking the iNOS gene did not express iNOS message or protein after MCA occlusion. The infarct and the motor deficits produced by MCA occlusion were smaller in iNOS knockouts than in wild-type mice (p < 0.05). Such reduction in ischemic damage and neurological deficits was observed 96 hr after ischemia but not at 24 hr, when iNOS is not yet expressed in wild-type mice. The decreased susceptibility to cerebral ischemia in iNOS knockouts could not be attributed to differences in the degree of ischemia or vascular reactivity between wild-type and knockout mice. These findings indicate that iNOS expression is one of the factors contributing to the expansion of the brain damage that occurs in the post-ischemic period. iNOS inhibition may provide a novel therapeutic strategy targeted specifically at the secondary progression of ischemic brain injury.

Cyclooxygenase-2 (COX-2), a prostanoid-synthesizing enzyme that contributes to the toxicity associated with inflammation, has recently emerged as a promising therapeutic target for several illnesses, ranging from osteoarthritis to Alzheimer's disease. Although COX-2 has also been linked to ischemic stroke, its role in the mechanisms of ischemic brain injury remains controversial. We demonstrate that COX-2-deficient mice have a significant reduction in the brain injury produced by occlusion of the middle cerebral artery. The protection can be attributed to attenuation of glutamate neurotoxicity, a critical factor in the initiation of ischemic brain injury, and to abrogation of the deleterious effects of postischemic inflammation, a process contributing to the secondary progression of the damage. Thus, COX-2 is involved in pathogenic events occurring in both the early and late stages of cerebral ischemia and may be a valuable therapeutic target for treatment of human stroke.middle cerebral artery occlusion ͉ prostanoids ͉ cerebral blood flow ͉ NS398 ͉ stroke

Focal cerebral ischemia is associated with expression of both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), enzymes whose reaction products contribute to the evolution of ischemic brain injury. We tested the hypothesis that, after cerebral ischemia, nitric oxide (NO) produced by iNOS enhances COX-2 activity, thereby increasing the toxic potential of this enzyme. Cerebral ischemia was produced by middle cerebral artery occlusion in rats or mice. Twenty-four hours after ischemia in rats, iNOS-immunoreactive neutrophils were observed in close proximity (<20 m) to COX-2-positive cells at the periphery of the infarct. In the olfactory bulb, only COX-2 positive cells were observed. Cerebral ischemia increased the concentration of the COX-2 reaction product prostaglandin E 2 (PGE 2 ) in the ischemic area and in the ipsilateral olfactory bulb. The iNOS inhibitor aminoguanidine reduced PGE 2 concentration in the infarct, where both iNOS and COX-2 were expressed, but not in the olfactory bulb, where only COX-2 was expressed. Postischemic PGE 2 accumulation was reduced significantly in iNOS null mice compared with wild-type controls (C57BL͞6 or SV129). The data provide evidence that NO produced by iNOS inf luences COX-2 activity after focal cerebral ischemia. Proinf lammatory prostanoids and reactive oxygen species produced by COX-2 may be a previously unrecognized factor by which NO contributes to ischemic brain injury. The pathogenic effect of the interaction between NO, or a derived specie, and COX-2 is likely to play a role also in other brain diseases associated with inf lammation.Focal cerebral ischemia is associated with a local inflammatory response that participates in the progression of ischemic brain injury (e.g., refs. 1, 2, 3, and 4; for a review, see ref. 5). Recent evidence indicates that nitric oxide (NO), produced by the ''immunologic'' isoform of NO synthase (iNOS), is one of the mechanisms by which postischemic inflammation contributes to cerebral ischemic damage (see ref. 6 for a review). Occlusion of the rat middle cerebral artery (MCA) leads to iNOS induction in the ischemic brain (7). iNOS expression peaks 24-48 hr after ischemia and occurs in neutrophils infiltrating the ischemic brain and in cerebral vascular cells (7). Delayed administration of the relatively selective iNOS inhibitor aminoguanidine (AG) reduces the size of the infarct produced by MCA occlusion (8). Furthermore, iNOS null mice have smaller infarcts and a better neurological outcome after focal ischemia (9). These observations suggest that large amounts of NO produced by iNOS are toxic to the injured brain and contribute to the late stages of cerebral ischemia.Cerebral ischemia also enhances the expression of the prostaglandin-synthesizing enzyme cyclooxygenase-2 (COX-2) (10,11,12,13). Although the time course of the up-regulation is similar to that of iNOS, COX-2 is expressed predominantly in neurons located at the border between normal and ischemic brain (10). The COX-2 inhibitor NS-398 reduces the s...

Flow-sensitive alternating inversion recovery (FAIR) is a recently introduced MRI technique for assessment of perfusion that uses blood water as an endogenous contrast agent. To characterize the FAIR signal dependency on spin tagging time (inversion time (TI)) and to validate FAIR for cerebral blood flow (CBF) quantification, studies were conducted on the rat brain at 9.4 T using a conventional gradient-recalled echo sequence. The T1 of cerebral cortex and blood was found to be 1.9 and 2.2 s, respectively, and was used for CBF calculations. At short TIs (<0.8 s), the FAIR signal originates largely from vascular components with fast flows, resulting in an overestimation of CBF. For TI > 1.5 s, the CBF calculated from FAIR is independent of the spin tagging time, suggesting that the observed FAIR signal originates predominantly from tissue/capillary components. CBF values measured by FAIR with TI of 2.0 s were found to be in good agreement with those measured by the iodoantipyrine technique with autoradiography in rats under the same conditions of anesthesia and arterial pCO2. The measured pCO2 index on the parietal cortex using the FAIR technique was 6.07 ml/100 g/min per mmHg, which compares well with the pCO2 index measured by other techniques. The FAIR technique was also able to detect the regional reduction in CBF produced by middle cerebral artery occlusion in rats.