Melatonin protects against ischemic stroke by modulating microglia/macrophage polarization toward anti-inflammatory phenotype through STAT3 pathway.

AIMS: Microglia and infiltrated macrophages play important roles in inflammatory processes after ischemic stroke. Modulating microglia/macrophage polarization from pro-inflammatory phenotype to anti-inflammatory state has been suggested as a potential therapeutic approach in the treatment of ischemic stroke. Melatonin has been shown to be neuroprotective in experimental stroke models. However, the effect of melatonin on microglia polarization after stroke and underlying mechanisms remain unknown. METHODS: In vivo, cerebral ischemia was induced by distal middle cerebral artery occlusion (dMCAO) in C57BL/6J mice. Melatonin was injected intraperitoneally (20 mg/kg) at 0 and 24 hours after ischemia. In vitro, the microglial cell line BV2 was stimulated to the pro-inflammatory state with conditioned media (CM) collected from oxygen-glucose deprivation (OGD) challenged neuronal cell line Neuro-2a (N2a). Real-time PCR was utilized to detect the mRNA expression of microglia phenotype markers. Activation of signal transducer and activator of transcription 3 (STAT3) pathway was determined by Western blot of phosphorylated STAT3 (pSTAT3). A neuron-microglia co-culture system was used to determine whether melatonin can inhibit the neurotoxic effect of pro-inflammatory microglia to post-OGD neurons. RESULTS: Melatonin treatment reduced brain infarct and improved neurological functions 3 days after dMCAO, which was accompanied by decreased expression of pro-inflammatory markers and increased expression of anti-inflammatory markers in the ischemic brain. In vitro studies confirmed that melatonin directly inhibited the pro-inflammatory responses in BV2 cells upon exposure to OGD neuron CM. The microglia possessing pro-inflammatory phenotype exacerbated post-OGD N2a cells death, whereas melatonin reduced such neurotoxic effect. Further, melatonin enhanced the otherwise inhibited pSTAT3 expression in BV2 cells treated with OGD neuron CM. STAT3 blockade significantly reduced the effect of melatonin on microglial phenotype shift. CONCLUSION: Melatonin treatment ameliorates brain damage at least partially through shifting microglia phenotype from pro-inflammatory to anti-inflammatory polarity in a STAT3-dependent manner.

Remote Ischemic Preconditioning-Mediated Neuroprotection against Stroke is Associated with Significant Alterations in Peripheral Immune Responses.

AIMS: Remote ischemic preconditioning (RIPC) of a limb is a clinically feasible strategy to protect against ischemia-reperfusion injury after stroke. However, the mechanism underlying RIPC remains elusive. METHODS: We generated a rat model of noninvasive RIPC by four repeated cycles of brief blood flow constriction (5 min) in the hindlimbs using a tourniquet. Blood was collected 1 h after preconditioning and 3 days after brain reperfusion. The impact of RIPC on immune cell and cytokine profiles prior to and after transient middle cerebral artery occlusion (MCAO) was assessed. RESULTS: Remote ischemic preconditioning protects against focal ischemia and preserves neurological functions 3 days after stroke. Flow cytometry analysis demonstrated that RIPC ameliorates the post-MCAO reduction of CD3(+)CD8(+) T cells and abolishes the reduction of CD3(+)/CD161a(+) NKT cells in the blood. In addition, RIPC robustly elevates the percentage of B cells in peripheral blood, thereby reversing the reduction in the B-cell population after stroke. RIPC also markedly elevates the percentage of CD43(+)/CD172a(+) noninflammatory resident monocytes, without any impact on the percentage of CD43(-)/CD172a(+) inflammatory monocytes. Finally, RIPC induces IL-6 expression and enhances the elevation of TNF-α after stroke. CONCLUSION: Our results reveal dramatic immune changes during RIPC-afforded neuroprotection against cerebral ischemia.