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Inflammation following ischemic stroke is known to contribute to injury. NADPH oxidase (NOX) is a major enzyme system originally studied in immune cells that leads to superoxide (O.*) generation. Apocynin is a NOX inhibitor that has been studied as a potential treatment in experimental stroke. Here we explored the effect of different doses of apocynin in a mouse model of 2 h transient middle cerebral artery occlusion (tMCAO) followed by 22 h reperfusion. Apocynin, given i.v. at a dose of 2.5 mg/kg 30 min before reperfusion, improved neurological function (P<0.01), reduced infarct volume (P<0.05), and reduced the incidence of cerebral hemorrhage (P<0.05), but not at higher doses of 3.75 and 5 mg/kg, where it actually increased brain hemorrhage. Apocynin also tended to reduce mortality at the lower dose, but not at higher doses. Using hydroethine fluorescence to delineate O.* in the brain, neurons and some microglia/macrophages, but not vascular endothelial cells were found to contain O.*. Apocynin at protective doses markedly prevented ischemia-induced increases in O.*. Our data suggested that apocynin can protect against experimental stroke, but with a narrow therapeutic window.

Metallothioneins (MTs) are a family of metal binding proteins that have been proposed to participate in a cellular defense against zinc toxicity and free radicals. In the present study, we investigated whether increased expression of MT in MT-1 isoform-overexpressing transgenic mice (MT-TG) affords protection against mild focal cerebral ischemia and reperfusion. Transient focal ischemia was induced in control (wild type) and MT-TG mice by occluding the right middle cerebral artery for 45 min. Upon reperfusion, cerebral edema slowly developed and peaked at 24 hr as shown by T2-weighted MRI. The volume of affected tissue was on the average 42% smaller in MT-TG mice compared with control mice at 6, 9, 24, and 72 hr and 14 days postreperfusion (P < 0.01). In addition, functional studies showed that 3 weeks after reperfusion MT-TG mice showed a significantly better motor performance compared with control mice (P = 0.011). Although cortical baseline levels of MT-1 mRNA were similar in control and MT-TG mice, there was an increase in MT-1 mRNA levels in the ischemic cortex of MT-TG mice to 7.5 times baseline levels compared with an increase to 2.3 times baseline levels in control mice 24 hr after reperfusion. In addition, MT-TG mice showed an increased MT immunoreactivity in astrocytes, vascular endothelial cells, and neurons 24 hr after reperfusion whereas in control mice MT immunoreactivity was restricted mainly to astrocytes and decreased in the infarcted tissue. These results provide evidence that increased expression of MT-1 protects against focal cerebral ischemia and reperfusion.

Gp91-phox is an integral component of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex that generates reactive oxygen species (ROS) in activated circulating phagocytes. The authors previously demonstrated that gp91-phox knockout (KO) mice show significant protection from neuronal injury after cerebral ischemia--reperfusion injury, suggesting a pivotal role for this enzyme. Moreover, results from chimeric mice suggested that elimination of gp91-phox from both circulating phagocytes and a putative central nervous system (CNS) source were required to confer neuroprotection. In the current study, the authors demonstrated gp91-phox-specific immunostaining of perivascular cells in the CNS of control rats. However, after transient cerebral ischemia, gp91-phox-positive phagocytes were observed within the core ischemic region and activated microglial cells were positive in the penumbra. Such activated microglial cells were also gp91-phox-positive in the CNS of a chimpanzee with mild meningitis. Finally, in humans, both normal adult CNS tissues and isolated fetal microglial cells expressed gp91-phox mRNA. These microglia also expressed mRNA for the five other known components that comprise the NADPH oxidase complex. These data strongly suggest that microglial cells may contain a functionally active NADPH oxidase capable of generating ROS during CNS inflammation.

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