We have previously shown that melatonin reduces postischemic rises in the blood-brain barrier (BBB) permeability and improves neurovascular dysfunction and hemorrhagic transformation following ischemic stroke. It is known that activation of the matrix metalloproteinases (MMPs) plays a crucial role in the pathogenesis of brain edema and hemorrhagic transformation after ischemic stroke. We, herein, investigated whether melatonin would ameliorate MMP-2 and MMP-9 activation and expression in a rat model of transient focal cerebral ischemia. Adult male Sprague-Dawley rats were subjected to a 90-min middle cerebral artery (MCA) occlusion using an intraluminal filament. Melatonin (5 mg/kg) or vehicle was intravenously injected upon reperfusion. Brain infarction and hemorrhage within infarcts were measured, and neurological deficits were scored. The activity and expression of MMP-2 and MMP-9 were determined by zymography, in situ zymography and Western immunoblot analysis. Cerebral ischemia-reperfusion induced increased pro-MMP-9 and MMP-9 activity and expression 24 hr after reperfusion onset. Relative to controls, melatonin-treated animals, however, had significantly reduced levels in the MMP-9 activity and expression (P < 0.01), in addition to reduced brain infarct volume and hemorrhagic transformation as well as improved sensorimotor neurobehavioral outcomes. No significant change in MMP-2 activity was observed throughout the course experiments. Our results indicate that the melatonin-mediated reductions in ischemic brain damage and reperfusion-induced hemorrhage are partly attributed to its ability to reduce postischemic MMP-9 activation and increased expression, and further support the fact that melatonin is a suitable as an add-on to thrombolytic therapy for ischemic stroke patients.

We have shown that melatonin attenuated matrix metalloproteinase-9 (MMP-9) activation and decreased the risk of hemorrhagic transformation following cerebral ischemia-reperfusion. Herein, we investigate the possible involvement of the plasminogen/plasmin system and endogenous MMPs inhibitor underlying the melatonin-mediated MMP-9 inhibition. Mice were subjected to 1-hr ischemia and 48-hr reperfusion of the right middle cerebral artery. Melatonin (5 mg/kg) or vehicle was intravenously injected upon reperfusion. Brain infarction and hemorrhagic transformation were measured. Extracellular matrix damage was determined by Western immunoblot analysis for laminin protein. The activity and expression of MMP-2 and MMP-9 were determined by gelatin zymography, in situ zymography, and Western immunoblot analysis. In addition, the activities of tissue and urokinase plasminogen activators (tPA and uPA) were evaluated by plasminogen-dependent casein zymography. Endogenous plasminogen activator inhibitor (PAI) and tissue inhibitors of MMP (TIMP-1) were investigated using enzyme-linked immunosorbent assay (ELISA) and Western immunoblot analysis, respectively. Cerebral ischemia-reperfusion induced increased MMP-9 activity and expression at 12-48 hr after reperfusion onset. Relative to controls, melatonin-treated animals had significantly decreased MMP-9 activity and expression (P<0.05), in addition to reduced brain infarction and hemorrhagic transformation as well as improved laminin protein preservation. This melatonin-mediated MMP-9 inhibition was accompanied by reduced uPA activity (P<0.05), as well as increased TIMP-1 expression and PAI activity (P<0.05, respectively). These results demonstrate the melatonin's pluripotent mechanisms for attenuating postischemic MMP-9 activation and neurovascular damage, and further support it as an add-on to thrombolytic therapy for ischemic stroke patients.

Synapto-dendritic dysfunction and rearrangement takes place over time at the peri-infarct brain after stroke, and the event plays an important role in post-stroke functional recovery. Here, we evaluated whether melatonin would modulate the synapto-dendritic plasticity after stroke. Adult male Sprague-Dawley rats were treated with melatonin (5 mg/kg) or vehicle at reperfusion onset after transient occlusion of the right middle cerebral artery (tMCAO) for 90 min. Local cerebral blood perfusion, somatosensory electrophysiological recordings and neurobehavioral tests were serially measured. Animals were sacrificed at 7 days after tMCAO. The brain was processed for Nissl-stained histology, Golgi-Cox-impregnated sections, or Western blotting for presynaptic proteins, synaptosomal-associated protein of 25 kDa (SNAP-25) and synaptophysin (a calcium-binding protein found on presynaptic vesicle membranes). Relative to controls, melatonin-treated animals had significantly reduced infarction volumes (P < 0.05) and improved neurobehavioral outcomes, as accessed by sensorimotor and rota-rod motor performance tests (P < 0.05, respectively). Melatonin also significantly improved the SNAP-25, but not synaptophysin, protein expression in the ischemic brain (P < 0.05). Moreover, melatonin significantly improved the dendritic spine density and the somatosensory electrophysiological field potentials both in the ischemic brain and the contralateral homotopic intact brain (P < 0.05, respectively). Together, melatonin not only effectively attenuated the loss of presynaptic protein, SANP-25, and dendritic spine density in the ischemic territory, but also improved the reductions in the dendritic spine density in the contralateral intact brain. This synapto-dendritic plasticity may partly account for the melatonin-mediated improvements in functional and electrophysiological circuitry after stroke.

Nicotinamide effectively attenuated postischemic nuclear factor-kappa]B activation and exhibited robust anti-inflammatory actions against ischemic stroke.