Intra-arterial Hypothermia May Reduce The Ischaemia-reperfusion Injury By Upregulating CIRP

Part Ⅰ Exploration of optimal perfusion conditions for intra-arterial local brain hypothermiaBackground and Objective—Ischemia/reperfusion injury（IRI）is an important factor affecting clinical outcome after revascularization of acute ischaemic stroke（AIS）.None of the existing neuroprotectants have yet achieved a satisfied clinical transformation.Therapeutic hypothermia is a promising neuroprotective measure,which could comprehensively inhibit of cerebral ischemia-reperfusion injury（CIR）network by simultaneously influencing multiple steps of the cascade of pathophysiological process,including inflammation,oxidative stress,excitoxity and ect.While systemic hypothermia is difficult to implement due to its side effects and complications.In recent years,with the extensive development of endovascular thrombectomy,it has been possible to treat AIS by intra-arterial hypothermia.This part explores the feasibility and optimal intervention conditions of Intra-arterial hypothermia（IAH）on reduring intracerebral ischemia-reperfusion injury in the middle cerebral artery occlusion（MCAO）model rats.Methods—The adult male Sprague-Dawley（SD）rat（240-265 g）were subjected to right middle cerebral artery occlusion（MCAO）for 2 h by using micro-filament.The local saline perfusion was carried by the interventional microcatheter inserted into the internal carotid artery（ICA）through the external carotid artery（ECA）.The hypothermia group were randomly divided into H₁-H₉ subgroups using orthogonal experimental design by the conditions of local saline perfusion based on three factors and three levels,including perfusion temperature（4,10,15°C）,perfusion rate(1/3,1/2,2/3 of ICA blood flow velocity,that is 1/3 V_ICA,1/2 V_ICA,2/3 V_ICA))and continuous perfusion time（10,20,30 min）.Vital signs,blood routines,biochemical parameters,and hypothermia side effects and related complications were monitored during perfusion to assess tolerance of model animals and safety of IAH;and the temperatures of perfusate（T₀）,brain tissue（T_b）and Jugular venous blood（Tjvb）,as well as systemic body core temperature（Tcore）were monitored to analyze the relationship between the temperature of each other.After 24 hours of ischemia-reperfusion,the degree of neurological deficit was assessed by animal behavior（modified Neurological Severity Score,mNSS）.Tissue infarct volume was measured by TTC staining,and brain water content was calculated to evaluate brain edema degree.The optimal intervention conditions for local hypothermia were determined according to the tolerance of the model rats,the degree of neurological deficit,the infarct volume and cerebral edema after hypothermia intervention.Results—The vital signs of rats were stable during the intra-arterial cold saline perfusion,and there were no significant changes in heart rate,pulse rate and respiratory rate.The oxygen saturation was reduced by 0.5-5.4%,but were still above 90%.The blood routine and biochemical indexes of rats had different degrees of changes before and after perfusion,but there were no obvious changes.The side effects and associated complications of the local brain cooling was lower compared with the systematic cooling,including chills（0-14.8%）,polyuria（3.7-25.9%）,pulmonary edema/infection（0-11.1%）,and delayed wound healing（0-18.5%）.The survival rates ranged from 70.4 to 85.2%.Each subgroup the ischemic T_b could be reduced to a therapeutic hypothermia level below 35°C within 10 minutes,and be maintained for 10-90 minutes with little effect on systemic core body temperature;while the Tcore was reduced no more than 1°C.The lower the temperature of the perfusate and faster the perfusion rate,the faster T_b can be lowered to the therapeutic mild hypothermic level.After the end of perfusion,T_b and T_jvbvb slowly and steadily restored to the level before perfusion,and the declined level of T_jvb was lower than T_b.There was a significant correlation between them（Rho>0.98,P<0.01）.Orthogonal experimental design analysis of variance for mNSS scores,cerebral infarction volume,and brain tissue water content in each H subgroup showed that lowest mNSS score,the smallest cerebral infarction volume,the lowest brain water content,and the least cerebral edema was observed when perfusion conditions were 2/3 V_ICA perfusion with 4°C saline for 20min.Conclusion—It is safe and feasible to treat the MCAO/R model rats with intra-arterial cold saline infusion.It can effectively reduce the local brain tissue temperature of MCAO/R model rats in a short time with little effect on the body core temperature.Monitoring the temperature of the ipsilateral jugular venous return blood can help to predict the cooling of the local brain tissue and guide the implementation of local hypothermia in the arterial brain.Our research has shown that 4°C saline perfusion at 2/3V_ICA perfusion rate for 20 min is the optimal intervention conditions for local brain hypothermia which could obviously improve neurological deficits,reduce brain volume and cerebral edema.Part Ⅱ The protective effect of intra-arterial local hypothermia on neurovascular unit during ischemia-reperfusionObjective—To study the neuroprotective effect of intra-arterial intracerebral hypothermia（IAH）on cerebral ischemia-reperfusion injury in MCAO/R model rats under optimal perfusion conditions,and observe its protective effect on preserving microvascular barrier of neurovascular unit（NVU）.Methods—SD rats were randomized to shame group（group S）and ischemic group subjected to right middle cerebral artery occlusion（MCAO）for 2 h by using micro-filament.The ischemic group was randomly divided into:Sham group,permanent cerebral ischemia group（I group）,i.e.permanent middle cerebral artery occlusion（pMCAO）group,ischaemia/reperfusion group（I/R group）,i.e.middle cerebral artery occlusion/recanalization（MCAO/R）group,and I/R+intra-arterial hypothermia group（H group）.The perfusion conditions of intracarotid saline in H group were treated with 4°C saline,2/3 V_ICACA for 20 min.After 24 hours of ischemia-reperfusion,the neurological deficit was assessed by mNSS,TTC staining and magnetic resonance imaging were used to detect the volume of cerebral infarction,brain water content were calculated to assess the degree of brain edema;Evans blue（EB）tracer staining to observe the integrity of blood-brain barrier;Western blotting（WB）and immunofluorescence staining（IF）were used to detect the expression and distribution of major structural proteins（ZO-1,Occludin,Claudin5 and AQP-4）to assess the damage of blood-brain barrier（BBB）,the protein expression levels of matrix metalloproteinases（MMPs）and cold-inducible RNA-binding proteins（CIRP）were also assessed.Results—Compared with the I/R group,the mNSS scores in H group was significantly reduced（P=0.007,n=20）.TTC staining,MRI images and water content of brain tissue were also showed a significant decrease（p<0.0001,p<0.0001,n=7）;BBB permeability assessed by EB content of brain tissues,which decreased as well（P<0.0001,n=7）.The expression of ZO-1,Occludin and Claudin5 were increased（p<0.01,p<0.01,p<0.01,n=7）,and the distribution of ZO-1 protein were dense and continuous（p<0.01,n=7）;while the level of expression of AQP-4 protein decreased（p=0.012,n=7）.Furthermore,the level of expression of MMP-2 and MMP-9 protein also decreased（p=0.025,p=0.015,n=7）;interestingly the expression of CIRP was elevated（p=0.02,n=7）.Conclusion—Intra-arterial local brain hypothermia can down-regulate the expression of MMPs,maintain the integrity of BBB,reduce cerebral infarction,edema and improve neurological function.The expression of CIRP in brain tissue was increased under local brain cooling,which may related with the hypothermic neuroprotection;yet,the specific mechanism needs further investigation.Part Ⅲ Local hypothermia in the arterial brain may reduce ischemia-reperfusion injury of neurovascular unit by up-regulating CIRP Objective—The main aim of this part was to investigate whether CIRP could enhance the neuroprotective effects of local brain hypothermia by alleviating ischemia-reperfusion injury in MCAO/R model rats,especially to clear the molecular mechanism of CIRP in the regulation of matrix metalloproteinases and protecting the structural integrity of microvascular barrier of neurovascular unit.Methods—The genetically modified SD rats（100-120 g）were constructed by injecting CIRP-knockdown and overexpression of lentivirus（synthesis by Shanghai Genechem Co.,Ltd.）into the rats’lateral ventricle by using a brain stereotaxic instrument for rats;and Western blotting were used to verify modification of CIRP in rats after 2 weeks.The experiment was randomly divided into negative control(CIRP^NC),CIRP gene knockdown(CIRP^sh)and overexpression(CIRP^over)groups,each group was further divided into sham operation（S）,ischemia/reperfusion（I/R）and local brain hypothermia（H）Subgroup.Immediately after recanalization,interventional microcatheter was sent to ICA of ischemic side in MCAO/R rats to perfuse cold saline for local hypothermia(4°C,2/3 V_ICA,20 min)in H group.After 24 hours of reperfusion,the expressed levels of major structural proteins of BBB（ZO-1,Occludin and AQP4and MMPs）were detected by Western blotting.Results—SD rats with CIRP knockdown(CIRP^sh)and over-expression(CIRP^over)were successfully obtained by the injection of lentivirus into lateral ventricle（p<0.01,p<0.01,n=7）.In the I/R subgroup,the CIRP^over group had a lower mNSS score（p=0.016,n=9）;the expression of ZO-1 and Occludin were upregulated（p=0.007,p=0.015,n=7）and AQP4（p=0.042,n=7）were increased compared with the CIRP^NC group;also the expression of MMP-2 and MMP-9 was significantly down-regulated（p=0.015,p=0.02,n=7）.In the CIRP^sh group,the mNSS score was increased（p=0.03,n=9）;the expression of ZO-1 and Occludin were down-regulated（p=0.001,p=0.025,n=7）and AQP4（p=0.012,n=7）were increased compared with the CIRP^NC group;the expression of MMP-2 and MMP-9 was significantly down-regulated（p=0.004,p=0.019,n=7）.In the CIRP^NC and CIRP^shh groups,the mNSS scores were lower in each H subgroup than that in I/R subgroup（p=0.033,p=0.022,p=0.018,n=9）,and the degree of neurological impairment was reduced;the expression of ZO-1（p=0.004,p=0.005,n=7）and Occludin（p=0.006,p=0.034,n=7）was increased,while the expression of AQP4 was decreased（p=0.015,p=0.011,n=7）;the expression of CIRP protein was further increased（p=0.001,p<0.001,n=7）,while The expression levels of MMP-2（p=0.022,n=7）and MMP-9（p=0.044,p=0.018,n=7）were all decreased.Conclusion—Local brain cooling may reduce the expression of matrix metalloproteinases（MMPs）to maintain the integrity of the BBB by up-regulating CIRP,which meanwhile could improve the outcome of neurological function.There may exist relationship between CIRP and neuroprotection in MCAO rats with local brain cooling and this may be expected to be a potential therapeutic target for ischemic stroke for neuroprotection.