【作者】 袁江伟；

【导师】 陈铎；

【作者基本信息】 中国医科大学 ， 外科学， 2010， 硕士

【摘要】 前言动脉瘤性蛛网膜下腔出血(subarachnoid hemorrhage S AH)发生率在西方国家约10人／10万人,蛛网膜下腔出血后的脑血管痉挛和早期脑损伤是致死和致残的主要原因,约12.4%患者在入院前已发生猝死,出血48小时内的死亡率高达40%-60%,约35%在SAH后24小时内死亡。尽管现在在血管内技术、诊断方法、手术及围手术期的管理方面取得了很大的进步,蛛网膜下腔出血患者的预后仍然不佳。蛛网膜下腔出血幸存者中,大多数存在认知缺陷并影响功能状态和生活质量.而迟发性血管痉挛发生于出血后4-5天。这表明,早期死亡的病人并不是因为SAH后迟发性血管痉挛,John H Zhang等学者最近提出“早期脑损伤”概念,它指的是蛛网膜下腔出血后形成脑血管痉挛之前的72小时内的直接损伤,包括继发性损伤。早期脑损伤的特点是颅内压(ICP)急性增高,随后出现脑灌注压(CPP)及脑血流量(CBF)逐渐减少,导致全脑缺血。这又引起继发损伤,如脑水肿的形成,急性血管收缩；在分子水平上表现为细胞厌氧呼吸、能量耗竭、兴奋性毒性和自由基的形成。蛛网膜下腔出血(SAH)早期脑损伤病理生理学包括血脑屏障(BBB)的破坏、脑水肿及脑缺血,但机制未明.研究表明,紧密连接(tight juctions, TJs)是保持血脑屏障(BBB)完整性的重要因素.目前认为,TJs由一组分子蛋白元件所构成,如,Claudins、occludin等,他们是调节物质通过细胞间连接的关键成分,其中Claudin-5是脑微血管内皮细胞中的特异性蛋白。有研究表明,Claudin-5是形成紧密连接的充要条件。ZO-1是第一个被确定的TJs相关蛋白,越来越多的证据显示ZO-1的缺失会导致TJs结构的破环。本实验采用标准大鼠血管内线穿刺法建立SAH动物模型,运用透射电镜观察BBB超微结构的变化,Western blot检测BBB紧密连接中ZO-1、Claudin-5的表达情况,进一步探讨ZO-1、Claudin-5在SAH后早期脑损伤中的作用。材料与方法按文献所述,建立标准大鼠血管内线穿刺法SAH动物模型。健康成年SD大鼠120只(体重300-350mg,雄性),随机分为6组：对照组(n=20),假手术组(n= 20), SAH组(n= 20), SAH+DMSO组(n= 20), SAH+SP600125(10mg/kg) (n= 20)和SAH+SP600125(30mg/kg) (n= 20)。SP600125 (JNK抑制剂)溶解于DMSO溶液中,分别于制备蛛网膜下腔出血动物模型前1小时和蛛网膜下腔出血后6小时腹腔注射.同样剂量的DMSO(不含抑制剂)腹腔注射SAH组作为SAH+DMSO组。结果1.SAH 24小时,透射电镜观察各组脑微血管血内皮细胞紧密连接(TJs)均显示有不同程度的开放；紧密连接相关蛋白ZO-1、Claudin-5表达水平：Sham组同Control组比较无统计学意义(ANOVA, P>0.05); SAH组和SAH+DMSO组同Sham组比较有显著统计学差异(ANOVA, P<0.05); SAH组同SAH+DMSO组比较无统计学意义(ANOVA, P>0.05)。2.JNK抑制剂SP600125干预后,透射电镜观察脑微血管血内皮细胞紧密连接(TJs)显示开放同程度减小；紧密连接相关蛋白ZO-1、Claudin-5表达水平：SAH+SP600125(10mg/kg)组同SAH组和SAH+DMSO组比较无统计学意义(ANOVA,P>0.05); SAH+SP600125(30mg/kg)组同SAH组和SAH+DMSO组比较有显著统计学差异(ANOVA, P<0.05); SAH+SP600125(30mg/kg)组同S AH+SP600125(1 Omg/kg)组比较有显著统计学差异(ANOVA, P<0.05)。3.JNK抑制剂SP600125可改善蛛网膜下腔出血存活大鼠24小时后神经行为功能障碍；Sham组无死亡,SAH组、SAH+DMSO组、SAH+SP600125(10mg/kg)、SAH+SP600125(30mg/kg)死亡率分别为30%、35%、25%、15%,组间比较无统计学意义(χ2 test, P>0.05)。结论1.本实验证明血脑屏障破坏是蛛网膜下腔出血后早期脑损伤的表现之一,紧密连接相关蛋白ZO-1、Claudin-5起重要作用。2.JNK抑制剂(SP600125)可改善蛛网膜下腔出血后血脑屏障破坏程度,从而起到对早期脑损伤保护作用。更多还原

【Abstract】 ObjectiveAneurysmal subarachnoid hemorrhage (SAH) affects 10 per 100000 population in the Western world. For survivors of the initial hemorrhage, cerebral vasospasm and early brain injury are major causes of subsequent morbidity and mortality,12.4% sudden death before receiving medical attention, and up to 40%to 60%of patients will die within the first 48 hours because of the initial bleeding and about 35%within 24 hours after SAH. Although considerable advances have been made in endovascular techniques, diagnostic methods, and surgical and perioperative management paradigms, outcome for patients with SAH still remains poor. Many survivors of SAH experience persistent cognitive deficits, with an effect on functional status and quality of life. The vasospasm is delayed in onset, usually 4-5days after SAH. This shows that patients with early death were not due to delayed vasospasm after SAH. Recently, the term "early brain injury" has been generated referring to the immediate injury within 72 h and includes secondary events to SAH before the development of cerebral vasospasm. Early brain injury is characterized by an acute increase in intracranial pressure (ICP) and subsequent reductions of cerebral perfusion pressure (CPP) and cerebral blood flow (CBF) resulting in global cerebral ischemia. These primary mechanisms trigger secondary phenomena such as brain edema formation, acute vasoconstriction, and, on the molecular level, anaerobic cellular respiration, energy depletion, excitotoxicity, and the formation of free radicals. Early brain injury comprising blood-brain barrier (BBB)disruption, brain edema, and global ischemia is important in the pathophysiology of subarachnoid hemorrhage (SAH); however, the mechanisms are not clearly understood. The study demonstrated tight junction(TJ) is the inportant factor to maintain blood-brain barrier(BBB) integrity. TJs constituted the barrier of ion and molecule passing membrance transport through the paracellular pathway. TJ was constituted by a group of molecular protein elements, such as claudins, occludin. Claudin-5 is the brain microvascular endothelial cell-specific protein in the TJ. Study found that claudin-5 was the necessary and suficient condition in the constitution of TJ. ZO-1 is the first protein identified in TJs, and more and more evidence shows that the lack of ZO-1 may lead to TJs broken.In present study, we established endovascular perforation model of SAH in rats as described previously and observed blood-brain barrier ultrastructual changes by transmission electron microscopy (TEM), meanwhile examined the tight junction proteins ZO-1 and Claudin-5 expression in cerebral hemisphere by Western blot, and to further explore the roles of which in early brain injury after subarachnoid hemorrhage in rats.Materials and methodsThe endovascular perforation SAH model was used as previously described in adult male Sprague Dawley rats. One hundred and twenty animals weighing 300mg to 350mg were were randomly divided into 6 groups:control(n=20), sham(n=20), SAH(n=20), SAH+DMSO(n=20), SAH+SP600125 (10mg/kg) (n=20)and SAH+ SP600125 (30mg/kg) (n=20). SP600125(a inhibitor of JNK) was intraperitoneally injected 1 hour before and 6 hour after the induction of SAH which was dissolved in DMSO. The same volume of DMSO without the inhibitor was injected in rats as the SAH+DMSO group.Results1. The tight junctions of brain microvessel endothelial cells were open to different degrees 24 h after SAH, tight junction associated protein ZO-1 and Claudin-5 expression levels:Sham group compared with the Control group was not significant (ANOVA, P> 0.05); SAH group and SAH+DMSO group compared with the Sham group had significant statistical difference (ANOVA, P<0.05); SAH group compared with the SAH+DMSO group was not significant (ANOVA, P> 0.05).2. The blood brain microvascular endothelial cell tight junctions (TJs) open lower, and SAH+10mg/kg(SP600125) group with SAH group and SAH+DMSO group was not statistically significant (ANOVA, P> 0.05); SAH+30mg/kg(SP600125) group with SAH group and SAH+DMSO group was statistically significant differences (ANOVA, P<0.05); SAH+30mg/kg(SP600125) group with SAH+ 10mg/kg(SP600125) group was statistically significant differences (ANOVA, P<0.05).3. JNK inhibitor SP600125 group improved the neurological deficits 24 h after subarachnoid hemorrhage; the mortality at 24 h after SAH was 30%in SAH group, 35%in SAH+DMSO group,25%in SAH+SP600125(10mg/kg)group and 15%in SAH+SP600125(30mg/kg)group respectively,and sham group had zero mortality,no significant differences were observed between the groups (x2 test, P>0.05).Conclusions1. Blood-brain barrier damage after subarachnoid hemorrhage is one of manifestations in early brain injury, in which the tight junctions associated protein ZO-1 and Claudin-5 play an important role.2. JNK inhibitor SP600125 can amend the damage of blood-brain barrier in early brain injury after subarachnoid hemorrhage.更多还原