Modulation Of Neurovascular Inflammation In Ischemic Stroke

Background:Neurovascular unit(NVU)is comprised of serveral cell types including vascular cells,neuroglia and neurons.Brain ischemia activates the immune system.The recruitment of immune cells into the ischemic brain compromises NVU integrity and accelerate brain infarction.In this study,we investigated two approaches of immune modulation to preserve NVU function and reduce ischemic brain injury.Sphingosine-1-phosphate receptor(S1PR)modulation provides protection in preclinical and clinical studies of ischemic stroke,but the underlying mechanisms remain poorly understood.To explore the hypothesis that the protective effect of S1PR modulation is mediated in part by beneficial vascular mechanisms,we investigated the impact of a selective S1PR1 modulation on microvascular hemodynamics in a mouse model of laser-induced microvascular thrombosis.Translocator protein 18 kDa(TSPO)mainly localized to the mitochondrial outer membrane that is predominantly expressed in microglia within the central nervous system during inflammatory conditions.We investigated the effect of a TSPO agonist,etifoxine,on neuroinflammation and brain injury after ischemia/reperfusion.Methods:(1)The flow velocity of cortical arterioles in mice were measured in vivo under two-photon laser-scanning microscopy.Microthrombosis was induced in cortical arterioles by laser irritation.At 30 min after laser-induced thrombosis,mice were treated with either RP101075 or vehicle.The blood flow velocity after thrombosis and the change in thrombus volume was measured were observed using a two-photon microscope.The effect of RP101075 on heart rate and blood pressure in mice were monitored using a small animal electrocardiogram and a small animal non-invasive sphygmomanometer.Using immunofluorescence staining to investigate the effect of RP101075 on thrombus composition in mice after thrombosis.(2)After induction of a mouse model of cerebral ischemia in the middle of the brain for 60minutes,the expression of TSPO in the brain was detected by flow cytometry and pathological staining.After the model of the middle cerebral artery occlusion model was induced for 60 minutes After induction of cerebral ischemia model of middle cerebral artery occlusion at 30 minutes and 90 minutes,administration of 50mg/kg/day of Etifoxine after surgery,control group Mice were given the same dose of vehicle solution,and the infarct volume and behavioral scores of the two groups of mice were observed to explore the drug effects.Flow cytometry and immunofluorescence staining were used to detect the effect of drugs on the regulation of microglia.Finally,the small molecule inhibitor PLX3397 was used to specifically delete microglia in the brain,and the drug was used again to interfere with cerebral infarction.Prognosis to evaluate the role of microglia in drug actionResults:(1)RP101075 did not alter the flow velocity of cortical arterioles under physiological conditions.Laser-induced thrombosis led to a pronounced reduction(~80%)of flow velocity in cortical arterioles that persisted for at least 90 min.The reduction of flow velocity in cortical arterioles following thrombosis was significantly attenuated following RP101075 treatment.RP101075 did not significantly affect coagulation time,bleeding time,heart rate and blood pressure.In addition,RP101075 treatment reduced thrombus volume that is accompanied by a reduction of leukocyte content in thrombus.(2)TSPO was upregulated in Iba1~+or CD11b~+CD45int cells from mice subjected to MCAO and reperfusion.Etifoxine significantly attenuated neurodeficits and infarct volume after MCAO and reperfusion.The attenuation was pronounced in mice subjected to 30,60,or 90 min MCAO.Etifoxine reduced production of pro-inflammatory factors in the ischemic brain.In addition,etifoxine treatment led to decreased expression of interleukin-1?,interleukin-6,tumor necrosis factor-?,and inducible nitric oxide synthase by microglia.Notably,the benefit of etifoxine against brain infarction was ablated in mice depleted of microglia using a colony-stimulating factor 1 receptor inhibitor.Conclusions:Our findings demonstrate that the selective S1PR1 modulations improves microvascular circulation after thrombosis,implying a component of improved vascular hemodynamics to the benefit of S1PR modulation in cerebral ischemia.TSPO acgtivation reduces neuroinflammation and ischemic brain injury.The therapeutic potential of targeting TSPO requires further investigations in ischemic stroke.