Neuroprotective Mechanisms Of Hypothermia Against Ischemia/reperfusion Induced Cerebral Injury

Cerebral ischemia/reperfusion injury is a very complex,involving a variety of pathological physiological processes.Conventional neuroprotective drugs are usually used for one node to the damage network.Combined with previous work,we use therapeutic hypothermia and antioxidant drug to protect cerebral reperfusion injury.We establish an optimal condition of local brain hypothermia in mouse with middle cerebral artery occlusion(MCAo).Recanalization will be achieved by retract the thread after one hour occlusion.After reperfusion,ice cap with temperature of(15,10 or 4 C)was applied to ischemia cerebral and sustain 60 minutes.With the above optimal condition on structure and function of NYU in the setting of ischemia reperfusion injury,explore the underlying mechanisms of therapeutic hypothermia protecting the microvascular permeability barrier by evaluating following parameters:neurological function,volume size of infarction,structure of NVU,permeability of blood brain barrier,and the expression of structural protein(ZO-1,occludin,claudin5),primary matrix proteinase for basal mambrane degradation(MMP-2,MMP-9),and the association between these proteins and cold-inducible RNA binding protein(CIRBP).The results of our project may provide an important theoretical foundation and experimental basis for this combined therapeutic strategy in treatment of acute cerebral infarction.The results as following:1.(Sal B)against cerebral I/R injury and investigate the underlying mechanism.Sal B significantly reduced cerebral infarct size,and accompanied with improved neurobehavioral functions as indicated by the modified Bederson score and Longa five-point scale.Sal B decreased the production of reactive oxygen species(p<0.05).The data of Western blotting and reverse transcription quantitative real time polymerase chain reaction(qRT-PCR)analyses showed that the expression of GFAP,Ibal,IL-1?,IL-6,TNF-a and Cleaved-caspase 3 was significantly reduced by Sal B in I/R injured brain tissues as compared to corresponding controls(p<0.05).Over activation of astrocytes and microglia were inhibited by Sal B as shown by immunostaining of GFAP and Iba 1.These data suggest that Sal B has neural protective effects against I/R-induced cerebral injury and could be an effective candidate for further development of clinical therapy.2.The ice cap is applied to the infarction side,Set up different therapeutic hypothermia(4?,10? and 15?)and different time point(hypothermia on cerebral ischemia,hypothermia after ischemia/reperfusion,hypothermia 1h after ischemia/reperfusion,hypothermia 2 h after ischemia/reperfusion).Data showed that therapeutic hypothermia(4? on cerebral ischemia,4? after ischemia/reperfusion and 10? on cerebral ischemia)reduce cerebral infarct size,and accompanied with improved neurobehavioral functions.Expression of Claudin-5,occludin and ZO-1 mRNA and protein has significantly increased compared with model group(I/R),the results have significant difference(p<0.05).Expression of mRNA and protein of MMP-2 and MMP-9 are increasing in the model group compare with 4? on cerebral ischemia group,4?after ischemia/reperfusion group and 10? on cerebral ischemia group.CIRBP is found in brain cortex and hippocampus after ischemia,and increase more obvious in the ischemic brain exposed to low temperature environment.CIRBP mRNA and protein increase significantly in the group of 4? on cerebral ischemia,4? after ischemia/reperfusion and 10? on cerebral ischemia.The results have significant difference(p<0.01).3.We generate CIRBP gene knockout mice by CRISPR/Cas9 system.The sgRNA targeting sites were designed for CIRBP targeting.The sgRNA with the best gene editing efficiency was chosen.Then,the sgRNA was mixed with Cas9 mRNA and microinjected into fertilized eggs of C57BL/6 mice to generate mice with targeted mutation.Eight mice containing the CIRBP gene mutations were generated.The CIRBP gene mutations were found to differ among these mice.4.According the clinical treatment to the onset of cerebral ischemia,we choose 4?to intervene cerebral ischemia after reperfusion.Wild type mice and CIRBP-/-mice are used for ischemia reperfusion(I/R)injury and 4? intervention after reperfusion therapy.CIRBP knockout mice protected by 4? after I/R,cerebral infarction volume is not reducing compare with mice without 4? protection after I/R(p>0.05).CIRBP knockout activation of matrix metalloproteinases,lead to a capillary barrier related structural protein and microvascular basement membrane degradation,caused the destruction of the blood brain barrier.5.Brain microvascular endothelial cell model of hypoxia for 2 h,then reoxygenation and give normal culture,the cell have to apoptosis,AKT phosphorylation reduced.4 ?protection after OGD/R can reduce injury caused by lack of oxygen and glucose,strengthen AKT phosphorylation.CIRBP cause of AKT phosphorylation,CIRBP has a specific mode of action through the PI3K/AKT signaling pathway.CIRBP could activate PI3K/AKT signal pathway and protect the blood brain barrier.Reactive oxygen free radicals producted after cerebral ischemia hypoxia,causes astrocytes and microglia activation.The infiltration of lymphocytes,product inflammatory cytokines,inflammatory reaction and cause secondary damage.