【作者】 谢晓娜；

【导师】 吴江；

【作者基本信息】 吉林大学 ， 神经病学， 2012， 博士

【摘要】 背景：中风是由于脑血流中断导致的以急性神经功能受损为特征的一组综合征，具有发病率高，致残率高及死亡率高的特点。治疗中风最有效的方法是血管再通，通过溶栓及介入治疗实现早期再灌注，尽早实现缺血组织的血液供应。但是，再灌注本身产生大量的活性氧簇或自由基，导致发生再灌注损伤。脑缺血后适应是指脑组织缺血一段时间后，在进行再灌注时实施几次短暂的、重复的缺血与再灌注，能够对缺血的脑组织提供保护作用，减轻脑缺血再灌注损伤。Zhao等研究证实：在大鼠永久性大脑中动脉阻塞联合15min、30min及60min颈总动脉阻塞的局灶性脑缺血模型，缺血后适应能够减少脑梗死面积分别达80%、51%及17%；Pignataro等采用大脑中动脉梗塞100min的脑缺血模型大鼠，同样证实缺血后适应能够减少脑梗死体积最高达70%。上述缺血后适应的保护作用均是在血糖正常的动物模型上获得的。但是在临床上，缺血性脑卒中合并糖尿病的患者非常多见。糖尿病是目前公认的脑卒中的重要危险因素之一。探讨糖尿病状态下，缺血后适应是否仍具有抑制缺血再灌注损伤的作用，对于缺血后适应的临床应用具有重要意义。目的：探讨缺血后适应在糖尿病状态下是否仍然具有脑保护作用，并进一步探讨高血糖对缺血后适应作用机制的影响。方法：本研究采用高脂喂养4周加链脲佐菌素注射的方法制备糖尿病大鼠，采用线栓法制备大脑中动脉梗死模型。缺血再灌注组：缺血时间为1h，然后进行再灌注；后适应组为再灌注开始时立即进行再灌注10s/缺血10s，共5次循环，在缺血/再灌注24h留取标本。我们比较了缺血后适应对血糖正常大鼠和糖尿病大鼠局灶性脑缺血再灌注损伤是否都有保护作用。我们通过TTC染色法测定脑梗死体积，大鼠的神经功能评分及光镜下观察脑组织病理改变，判断缺血后适应对脑缺血的保护作用，并进一步探讨了缺血后适应对血糖正常大鼠及糖尿病大鼠的脑组织氧化应激反应及细胞凋亡、凋亡相关蛋白Bcl-2、Bax及凋亡执行蛋白caspase-3表达的影响，同时我们还探讨了缺血后适应对PI3K-Akt通路中的关键蛋白Akt、p-Akt表达的影响。结果：对于血糖正常的大鼠，缺血后适应能够明显降低局灶性脑缺血再灌注24h的脑梗死体积百分比，明显改善大鼠的神经功能评分，减少脑缺血周围区域的细胞凋亡数量。而对于糖尿病大鼠，缺血后适应的上述脑保护作用不明显。对于血糖正常的大鼠，缺血后适应能够明显降低脑组织脂质过氧化产物丙二醛（MDA）的水平，升高脑组织超氧化物歧化酶（SOD）和谷胱甘肽还原酶（GSH-pX）的活性，提高抗凋亡蛋白Bcl-2的表达，降低促凋亡蛋白Bax及凋亡执行蛋白caspase-3的表达，同时能够使再灌注损伤抢救激酶途径中的p-Akt蛋白表达明显增加；而对于糖尿病大鼠，缺血后适应对脑组织MDA水平及SOD、GSH-pX活性及上述凋亡相关蛋白及p-Akt的蛋白表达无明显的改变。结论：缺血后适应对血糖正常的大鼠局灶性脑缺血再灌注损伤有明显的保护作用，其主要机制为抑制氧化应激及细胞凋亡；而慢性高血糖抵消了缺血后适应的这种保护作用，其机制可能与高血糖诱发氧化应激、细胞凋亡及Akt的磷酸化障碍有关。更多还原

【Abstract】 Backgrounds：Stroke is a group of syndromes due to the interruption ofcerebral blood flow. It is characterized by a high incidence and high morbidityand high mortality. Advances in intravascular techniques and thrombolyticagents have reduced functional deficits within an optimal time window instroke patients. However, reperfusion itself generates an overproduction ofreactive oxygen species (ROS) or free radicals, leading to reperfusion injury.Ischemic postconditioning is defined in the field of cerebral ischemia researchas a series of brief occlusions and reperfusions applied at the onset ofestablishing reflow, which has proved to be effective against cerebral ischemia.Zhao et al found that postconditioning reduced the infarct size by80%,51%,and17%, respectively, in15,30, or60-min common carotid artery occlusioncombined with permanent distal middle cerebral artery occlusion. Pignataro etal had also shown a very strong protection with postconditioning in anotherfocal ischemic model, in which the middle cerebral artery was occluded for100min. Clinical ischemic stroke in diabetic patients is very common. Diabetesmellitus (DM) is widely recognized as important risk factor for stroke. It is ofgreat significance for ischemic postconditioning in the clinical application toinvestigate whether ischemic postconditioning still has the role of inhibition ofischemia-reperfusion injury in diabetic state.Objective: To investigate whether ischemic postconditioning in thediabetic state still has a protective effect on focal cerebral ischemia and discussthe probable mechanism of ischemic postconditioning in normal blood glucosestate and diabetic state.Methods: We fed the male Sprague-Dawley rats with high fat diet for4 weeks and then injected streptozotocin (STZ) in order to establish diabetic ratmodels. Normal SD rats and diabetic SD rats were treated with ischemicpostconditioning after60minutes of middle cerebral artery occlusion at theonset of reperfusion. Neurologic scores, infarct volumes with TTC staining andhistopathology of cerebral ischemia were assessed at24hours. Oxidative stresswas evaluated by detecting the activity of superoxidase dismutase (SOD),glutathione peroxidase (GSH-pX) and malondialdehyde (MDA) assay.Apoptosis-related molecules such as Bcl-2, Bax and caspase-3were studied byimmunohistochemical staining. Protein kinase B (PKB/Akt) was also tested byWestern blotting and immunohistochemical staining. We compared the effect ofischemic postconditioning in normal rats and diabetic rats.Results: Our results showed that compared with ischemia/reperfusiongroup, ischemic postconditioning significantly reduced the size of ischemicinfarction and improved the neurologic scores at24hours of ischemic/reperfusion injury in normal blood glucose state. Ischemic postconditioningcould also reduce the number of TUNEL positive cells in the ischemicpenumbra in normal rats. But the protective effect of ischemic postconditioningwas not seen in the diabetic state. Postconditioning failed to decrease infarctvolume or improve the neurologic scores in diabetic rats. Postconditioningincreased the activity of SOD and GSH-pX in the brain tissue in the normalstate but not in the diabetic rats when measured at ischemia-reperfusion24hours; Postconditioning decreased the level of MDA in the brain tissue innormal rats but not in the diabetic rats; Postconditioning treatment upregulatedBcl-2and p-Akt protein expression and downregulated Bax and caspase-3activity in normal rats but not in the diabetic rats.Conclusions: Ischemic postconditioning has protective effect for focalischemia-reperfusion injury in normal rats. Chronic hyperglycemia canabrogate the protection of postconditioning at least in part via oxidative stress, cell apoptosis and Akt phosphorylation dysfunction.更多还原