【作者】 梁成；

【导师】 王纪佐； 李新；

【作者基本信息】 天津医科大学 ， 神经病学， 2009， 博士

【摘要】 目的：再建血运(再灌注)如溶栓治疗是被公认和各国FDA批准的的急性缺血性卒中的唯一治疗手段，但3小时治疗时间窗的限制使多数多病人不能获益。亚低温(28—35℃)是最早在临床实践中被证实有确切疗效的的神经保护手段，而且缺血性脑损伤实验也证实亚低温的神经保护效果是最好的。为此，我们探讨了再灌注期亚低温能否延长再建血运(再灌注)治疗时间窗及其机制，并探讨再灌注期亚低温的脑保护机制。方法：1将120只SD雄性大鼠随机分为假手术组、大脑中动脉完全闭塞24小时组(MCAO24h组)、常温组：按再灌注时间点不同分为以下亚组：大脑中动脉闭塞2小时、3小时、4小时、5小时、6小时／灌注24小时组(MCAO2h再灌注组、MCAO3h再灌注组、MCAO4h再灌注组、MCAO5h再灌注组、MCAO6h再灌注组)；亚低温组：按再灌注时间点不同分成与常温组对应的亚组。2实施亚低温时将大鼠放入冰屋内，调整冰屋内冰袋数量，使大鼠肛温维持在31士1℃，5h时后，将大鼠移至室内环境复温。亚低温在大脑中动脉再通后即刻实施并持续5小时。线拴法制备大鼠大脑中动脉缺血／再灌注模型。3采用免疫组化法检测大鼠缺血／再灌注模型的AIF、Cyt C、HSP70和MMP-9、LN的表达，并进行TTC染色观察梗死体积，用干-湿重法测定脑含水量，对大鼠模型进行Longa评分。结果：一再灌注期亚低温与再建血运(再灌注)治疗时间窗的关系及其脑保护的研究结果1、与脑梗死的近期结局MCAO24h组的梗死体积、Longa评分比较：常温组MCAO2—4h再灌注各组有显著性差异(P＜0．01)，MCAO5—6h再灌注各组没有显著性差异(P＞0．05)；亚低温组MCAO2—5h再灌注各组有显著性差异(P＜0．01)；MCAO6h再灌注各组没有显著性差异(P＞0．05)2亚低温各组的梗死体积、Longa评分和含水量均显著低于相应常温组(MCAO2-5h各组P＜0．01，MCAO6h组P＜0．05)。二、再灌注期亚低温延长再建血运(再灌注)治疗时间窗及其脑保护机制的研究结果1细胞凋亡角度研究的结果1．1缺血／再灌注各组(常温组、亚低温组)的AIF、Cyt C、HSP70均显著高于假手术组(P＜0．01)。1．2亚低温各组的AIF、Cyt C均显著低于相应常温组，而HSP70各组阳性细胞计数均显著高于相应常温组(MCAO2-5h各组P＜0．01，MCAO6h组P＜0．05)。1．3与脑梗死近期结局MCAO24h组的AIF、Cyt C、HSP70：常温组MCAO2—4h再灌注组有显著性差异(P＜0．01)，MCAO5-6h再灌注组无显著性差异(P＞0．05)；亚低温组MCAO2-5h再灌注各组有显著性差异(P＜0．01)；MCAO6h再灌注组无显著性差异(P＞0．05)。2．血管通透性角度研究的结果2．1缺血／再灌注各组(常温组、亚低温组)的MMP-9的表达与层粘连蛋白表达呈显著负相关(r=-0．817，P＜0．01)。2．2再灌注各组(常温组、亚低温组)的MMP-9显著高于假手术组(P＜0．01)；层粘连蛋白显著低于假手术组(P＜0．01)。2．3亚低温各组的MMP-9均显著低于相应常温组，而层粘连蛋白MCAo2-6h各组阳性细胞计数均显著高于相应常温组(MCAO2-5h各组P＜0．01，MCAO6h组P＜0．05)。2．4与脑梗死近期结局MCAO24h的MMP-9、层粘连蛋比较：常温组MCAO2-4h再灌注组有显著性差异(P＜0．01)，MCAO5-6h再灌注组无显著性差异(P＞0．05)；亚低温组MCAO2-5h再灌注组有显著性差异(P＜0．01)；MCAO6h再灌注组无显著性差异(P＞0．05)。结论：1．再灌注期亚低温可延长再建血运(再灌注)治疗的时间窗，通过发挥再灌注期亚低温的脑保护作用(通过同时抑制非caspase和caspase两种凋亡途径，提高HSP70的表达，以全面抑制半暗带细胞的凋亡；对MMP-9的有效抑制使脑微循环破坏减轻，微循环承受再灌注能力上升。)，实现延长治疗时间窗的目的。2．再灌注期亚低温是一项理想的延长再建血运(再灌注)治疗时间窗的手段：它从抑制半暗带细胞的凋亡和减轻脑血管损伤提高微循环承受再灌注能力等多个角度阻断或减缓缺血／再灌注后的恶性病理生理进程，全面保护缺血／再灌注的脑组织，延长时间窗的效果明显。而其它延长时间窗的治疗手段，作用机制单一，针对性较差(一般不直接针对缺血／再灌注的病理生理)，效果较弱；特别是临床实际上，病人就诊时不可能提前给予亚低温治疗，错过治疗时间窗的病人较多等因素，应用再灌注期亚低温以延长时间窗的临床实用性较大，并且亚低温基础研究和临床使用的手段已经比较成熟，推广起来比较容易。同时由于再灌注期亚低温具有全面的脑保护作用且实用方便，它与其它神经保护剂及各种血管再通手段的联合应用以治疗急性期脑缺血必将有着良好的研究应用前景。更多还原

【Abstract】 Objective:The most effective treatment for acute ischemic stroke is the early re--storation of blood flow.(reperfusion therapy)to salvage the ischemic penumbrae,thefunc--tionally impaired yet still viable brain tissue,thereby improving clinical outco--me.,but many patient can not benefit from it due to the narrow treatmemt windowtime of 3 h--ours.Hypothermia has been suggested to be the only potent cerebrop--rotection appro--ach in clinic and also been proved to be the best cerebroprotectionapproach.thus,we study whether mild hypothermia (28-35℃)applied during rep--erfusion can prolong the time window ofrestorationofblood (reperfusion)andmechanisms of that and study it’s neuroprotection mechanism.Methods:Methods:1.The adult rats were randomly divided into sham-operationgroup,MCAO24h,NT and HT respectively further divided into 6 subgroups(subjected ischemia for2,3,4,5and6h respectively and then all reperfused for 24h);2.the rats of hypothermia therapy is immediately loaded into ice-room.changingthe number of ice bags to keep natal temperatures by 31℃±10C.After 5hours,make them rewarm in the room Brain mild hypothermiawas achieved 5hoursimmediately after reperfusion period.The cerebral ischemia/reperfusion model of ratswere achieved by middle cerebral artery occlusion.3.The expressions ofAIF CytC HSP70and MMP-9 LN were detected byimmunohistochemistry.The infarct volumes were evaluated by TTC staining.Theneurological deficit score were measure by Longa.The water content of rats braintissue were measured by using dry-wet weight method.Result:The relationship between mild hypothermia applied during reperfusion withthe treatment time window of restoration of blood (reperfusion)and it’s protectionduring ischemia/reperfusion.1.Compared with MCAO24h group in term of the infarction volume and score ofLonga:MCAO2-4h reperfusion groups of NT have significant difference (P＜0.01),MCAO5-6h groups reperfusion of NT have not significant difference (P＞0.05);MCAO2-5h reperfusion groups of HT have significant difference (P＜0.01),MCAO6h reperfusiongroups of NT have not significant difference (P＞0.05). 2.HT were lower significantly than NT in term of the infarction volume and score ofLonga and the water content of infarcted cerebra(MCAO2-5h reperfusion groups P＜0.01,MCAO6h reperfusion groups P＜0.05).The mechanisms of prolonging treatment time window of restoration of bloodflow.(reperfusion)by mild hypothermia applied during reperfusion and cerebralprotection of it.1.The study in the view of apoptosis1.1HT and NT were higher significantly than sham-operation in term of AIF Cyt CandHSP70 (P＜0.01).1.2HT were lower significantly than NT in term of AIF and Cyt C,HT were highersignificantly than NT in term of HSP70 (MCAO2-5h reperfusion groups P＜0.01,MCAO6h reperfusion groups P＜0.05).1.3Compared with MCAO24h group in term ofAIF CytC and HSP70:MCAO2-4hreperfusion groups of NT have significant difference (P＜0.01),MCAO5-6h groupsreperfusion of NT have not significant difference (P＞0.05);MCAO2-5h reperfusiongroups of HT have significant difference (P＜0.01),MCAO6h reperfusiongroupsof NT have not significant difference (P＞0.05).2.The study in the view of cerebral vasopermeability2.1There was negative relationship between MMP-9and LN r=-0.817,P＜0.01).2.2HT and NT were higher significantly than sham-operation in term of MMP-9 (P＜0.01).HT and NT were lower significantly than sham-operation in term of LN (P＜0.01)2.3HT were lower significantly than NT in term of MMP-9,HT were highersignificantly than NT in term ofLN (MCAO2-5h reperfusion groups P＜0.01,MCAO6h reperfusion groups P＜0.05).2.4Compared with MCAO24h group in term of MMP-9 and LN:MCAO2-4hreperfusion groups of NT have significant difference (P＜0.01),MCAO5-6h groupsreperfusion of NT have not significant difference (P＞0.05);MCAO2-5h reperfusiongroups of HT have significant difference (P＜0.01),MCAO6h reperfusiongroupsof NT have not significant difference (P＞0.05). Conclusion:1.The study demonstrated that the mild hypothermia applied duringreperfusion can prolong the treatment time window of restoration of blood flow.(reperfusion therapy).The reason for that is its potential capability of cerebral protection,via inhibiting capas-e-dependent and caspase-independent pathways of apoptosis andpromoting the expr--ession of Hsp70 protein.In one word,mild hypothermia canthoroughly inhibit apoptosis;inhibiting the expression of MMP-9 protein so allevia--ting the injury of micro--circulation,promoting endurance of microcirculation suffe--red ischemia/reperfusion.2.The study indicated that the mild hypothermia applied during reperfusion is thebest approach to extend the time window of restoration blood flow.(reperfusion).therapy,it can inhibit apoptosis in ischemic penumbra and alleviate the injury ofmicrocirculation and promote endurance of microcirculation suffered ischemia/reperfusion,thus in multiple way to interruput pathophysiology processing.It cansingnificently prolongthe time window.In conclusion,mild hypothermia is superior to other treatment approach aimed atprolonging time window,whoses mechanism were single,pertinence were poor (notdirectly to pathophysiology),effection were less power;In particular,it accuratelyreplicated the acute ischemic stroke in human being.since it is impossible thatpatients with stroke would be able to be treated with mild hypothermia before onsetof symptoms of stroke.,so that the mild hypothermia applied during reperfusion stagehave better clinical practicability.,the way of hypothermia was maturate,it’s easy tobe popularized;because the mild hypothermia applied during reperfusion haveall-around cerebral protection,,it must have the good perspective that combine itwith the means of restoration of blood flow such as thrombolysis and/or brainprotection agents as well.更多还原