Tissue‐plasminogen activator‐induced ischemic brain injury is reversed by melatonin: role of iNOS and Akt

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Abstract
In vivo studies showed that tissue‐plasminogen activator (t‐PA) may aggravate neuronal injury after focal cerebral ischemia. We hypothesized that t‐PA impairs survival‐promoting cell signaling in the ischemic brain, which may be reversed by a neuroprotectant, i.e. melatonin. We examined the effects of t‐PA (10 mg/kg, i.v.), administered alone or in combination with melatonin (4 mg/kg, i.p.), on ischemic injury, inducible nitric oxide synthase (iNOS) expression as well as Akt, Bcl‐XL and caspase‐3 signaling following 90 min of intraluminal middle cerebral artery (MCA) occlusion in mice. t‐PA, delivered immediately after reperfusion onset, increased infarct volume at 24 hr after MCA occlusion, in accordance with previous findings. Melatonin reduced infarct size when administered alone and reversed the t‐PA‐induced brain injury. Immunohistochemical studies showed that t‐PA treatment was associated with an accumulation of iNOS positive cells in ischemic brain areas, which was abolished after co‐delivery of melatonin. Western blots revealed that t‐PA decreased phosphorylated Akt levels, but did not influence Bcl‐XL expression and caspase‐3 activity in ischemic brain lysates. Co‐treatment with melatonin restored phosphorylated Akt levels, increased Bcl‐XL expression and reduced caspase‐3 activity. We provide evidence that t‐PA‐induced brain injury is accompanied by an activation of iNOS and inhibition of phosphatidylinositol‐3 kinase/Akt. That melatonin reversed these signaling changes and the t‐PA‐induced brain injury makes this indole attractive as an add‐on treatment with thrombolytics.