Melatonin, the secretory product of the pineal gland, is known to be neuroprotective in cerebral ischemia, which is so far mostly attributed to its antioxidant properties. Here we show that melatonin directly inhibits the mitochondrial permeability transition pore (mtPTP). mtPTP contributes to the pathology of ischemia by releasing calcium and cytochrome c (cyt c) from mitochondria. Consistently, NMDA-induced calcium rises were diminished by melatonin in cultured mouse striatal neurons, similar to the pattern seen with cyclosporine A (CsA). When the mouse striatal neurons were subjected to oxygen-glucose deprivation (OGD), melatonin strongly prevented the OGD-induced loss of the mitochondrial membrane potential. To assess the direct effect of melatonin on the mtPTP activity at the single channel level, recordings from the inner mitochondrial membrane were obtained by a patch-clamp approach using rat liver mitoplasts. Melatonin strongly inhibited mtPTP currents in a dose-dependent manner with an IC50 of 0.8 microM. If melatonin is an inhibitor of the mtPTP, it should prevent mitochondrial cyt c release as seen in stroke models. Rats underwent middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion. Melatonin (10 mg/kg ip) or vehicle was given at the time of occlusion and at the time of reperfusion. Indeed, infarct area in the brain sections of melatonin-treated animals displayed a considerably decreased cyt c release along with less activation of caspase-3 and apoptotic DNA fragmentation. Melatonin treatment diminished the loss of neurons and decreased the infarct volume as compared with untreated MCAO rats. Our findings suggest that the direct inhibition of the mtPTP by melatonin may essentially contribute to its anti-apoptotic effects in transient brain ischemia.

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Blood-brain barrier (BBB) breakdown after stroke is linked to the up-regulation of metalloproteinases (MMPs) and inflammation. This study examines the effects of progesterone (PROG) and its neuroactive metabolite allopregnanolone (ALLO) on BBB integrity following permanent middle cerebral artery occlusion (pMCAO). Rats underwent pMCAO by electro-coagulation and received intraperitoneal injections of PROG (8 mg/kg), ALLO (8 mg/kg) or vehicle at 1 h post-occlusion and then subcutaneous injections (8 mg/kg) at 6, 24, and 48 h. MMP activation and expression were analyzed by Western blot, immunohistochemistry and gelatin zymography 72 h post-pMCAO. Occludin1, claudin5, tumor necrosis factor-alpha (TNF-α) and Interleukin-6 (IL-6) were analyzed at 72 h post-pMCAO with Western blots. BBB permeability was measured by Evans blue extravasation and infarct size was evaluated by cresyl violet at 72 h after pMCAO. Ischemic injury significantly (p<0.05) increased the expression of MMP-9, MMP-2, TNF-α and IL-6, and reduced the level of occludin1 and claudin5. These changes were followed by increased infarct size (% contralateral hemisphere) and Evans blue extravasation into the brain indicating compromise of the BBB. PROG and ALLO attenuated BBB disruption and infarct size following pMCAO by reducing MMPs and the inflammatory response and by preventing the degradation of occludin1 and claudin5. We conclude that PROG and ALLO can help to protect BBB disruption following pMCAO.