【作者】 刘珂舟；

【导师】 郑筱祥；

【作者基本信息】 浙江大学 ， 生物医学工程， 2009， 博士

【摘要】 缺血性脑损伤是一个复杂的病理生理过程，其主要病理变化是缺血性神经元损伤。以往对缺血性脑损伤的研究认为，脑血流中断后，脑细胞能量供应不足而导致脑细胞死亡。近年来研究发现脑血流中断和再灌注使脑组织细胞产生损伤级联反应，至少涉及4个不同的机制：能量障碍和兴奋性氨基酸毒性、梗死周围去极化、炎症及程序性细胞死亡。大量动物实验及临床研究表明，脑缺血及再灌注期间发生着复杂的病理生理变化。一方面，脑缺血再灌注既可以挽救濒临梗死的细胞，另一方面加重细胞损伤，导致细胞死亡。而在这个复杂的过程中，一氧化氮(Nitric Oxide，NO)起着十分重要的作用。其作为一种新型信使分子，同时具有神经介质和神经毒性作用。尤其在脑部的组织中的双重作用更是近年来研究的重点。一方面它能够增加皮层供血量，缩小梗死面积；另一方面它能够与缺血产生的氧自由基协同造成神经细胞损伤。本论文通过在体检测脑缺血及再灌注过程中大鼠脑海马内NO的释放情况，真实反应了该过程中NO的释放变化情况，为进一步研究NO的神经介质作用和神经毒性作用奠定基础。并以培养的大鼠海马神经细胞的缺氧缺糖(OGD)为离体脑缺血／再灌注模型(即对培养的海马神经细胞进行缺氧缺糖复氧复糖)，利用荧光标记和激光共聚焦实时扫描技术，对海马神经细胞内释放的NO变化进行了检测，从而完成了对脑缺血／再灌注过程中NO的动态变化过程的整体与细胞两个层面的研究。最新的关于脑缺血及再灌注损伤的治疗方法的研究是通过对再灌注过程的干预(post-conditioning)来减少脑部梗死面积，但该过程中NO的变化情况、具体的作用机理及究竟哪种干预方法更有效均未见报道。通过使用本论文中的在体检测NO方法，实时、连续地记录了该干预过程中NO的变化情况，从而阐明了NO确实参与了缺血后处理，提示NO通路有可能是该方法对脑缺血／再灌注损伤保护作用的一条途径。同时，利用TTC染色与流式细胞技术对比了不同后处理方法对于大脑的保护作用，为进一步优化该干预方法提供了一定的理论基础。所得结果如下：1．海马内NO释放减少，对血管的舒张作用降低，继而大鼠血压上升。这与理论“内皮依赖性舒张因子(endothelium-derived relaxing factor，EDRF)和一氧化氮同质”相符，进而验证了该在体检测NO技术的真实性和稳定性。同时得出结论，静脉注射一氧化氮合酶(nitric oxide synthase，NOS)抑制剂L-NAME 20μL-30μL后，NO的释放量减少了4．5 nM-6nM。2．大鼠脑缺血／再灌注初期中，海马内NO的变化经历了四个阶段：在脑缺血的最初10min，由于大脑供血的迅速减少，NO的释放量也迅速下降，并达到最低点；之后稳定维持在一个低水平；在再灌注初期，由于血液的恢复，NO释放量迅速升高，并在10．15min内达到最高点；之后维持在一个稳定的高水平。同时通过拟合定标曲线，计算得出：在脑缺血过程中海马内NO释放的减少浓度为0．806±0．221μM，在再灌注初期NO释放量增加浓度为0．768±0．029μM。3．在不同的时间点分别静脉注射内皮型NOS (eNOS)／神经元型NOS(nNOS)和诱生型NOS(iNOS)的抑制剂，结果表明在再灌注初期eNOS／nNOS起主要作用，与iNOS没有关系，在这个过程中海马内释放的NO对受损脑组织起到了保护和损伤双重作用。4．在离体培养的大鼠海马神经元细胞实验上，通过共聚焦显微镜得到了NO在OGD／复氧复糖模型中的变化过程。该过程实验结果与在体大鼠实验结果相比，NO释放的变化过程显得较为简单。在OGD／复氧复糖过程中，NO的表达均有明显上升，并在再灌注10-12 min后达到稳定的平台期。5．利用在体检测NO技术，实验发现缺血后处理(post-conditioning)能够使大鼠海马内NO的释放缓慢增加，从而进一步增加脑内血流量。我们认为，脑缺血后处理所引起的NO缓慢而大量的释放能够抑制NO的毒性作用而进一步加大NO对于脑血流的积极增加作用，从而减少脑缺血／再灌注损伤。不同后处理方法对于脑内NO释放的影响不同。6．对比6组不同的后处理方法(即改变缺血和再灌注的时间长短及交替的次数)，实验发现缺血后处理都可以一定程度上减少脑部梗死面积，提高大鼠海马内神经细胞的存活率。但其中3次30s／30s的再灌注／缺血循环能够最有效的减少大脑损伤。我们认为该方法可能能够最大程度的激发脑内NO的缓慢大量释放，说明NO很有可能是post-conditioning改善脑损伤的一条作用途径。本论文采用实时、连续、在体检测NO结合荧光标记与激光共聚焦实时扫描技术的方法，分别从整体与细胞两个层次上对脑缺血／再灌注过程中大鼠海马内NO浓度进行了检测，全面的阐述了该过程中NO的变化情况以及对脑组织的双重作用。另一方面，利用NO的在体检测技术，首次检测了在缺血后处理过程中NO的变化情况，揭示了NO可能是该干预方法作用的一个途径，并通过对比多种缺血后处理方法对于缺血／再灌注损伤的保护作用，进一步对该干预方法进行优化。NO在脑缺血／再灌注疾病中发挥着重要的作用，清楚了解NO在脑缺血／再灌注过程中的变化及其作用机制对于防治中风药物的开发与筛选以及临床上治疗中风引起的神经损伤都具有重要的指导意义。更多还原

【Abstract】 Brain ischemic injury is a complex pathophysiologic progress and it causes theneurons ischemic injuries. It was considered that these injuries were a kind of celldeath because of the energy insufficiency in the cerebral cells after the cerebral bloodflow was interrupted. Recently many researches found that cerebral ischemia andreperfusion caused cell injuries through a series of at least 4 reactions, includingenergy barriers and excitatory amino acid toxicity, depolarization of infarct area,inflammation and program cell death. Abundant animal experiments and clinicalresearches approved that there was a complicated pathophysiologic progress incerebral ischemia and reperfusion. The reperfusion could save the dying cells. On theother hand it could aggravate the cell injury and cause cell death.Nitric oxide (NO) plays an important role in this process. As an importantmolecular messenger, NO works as a nerve mediator as well as a detrimental factor innervous system. In central and peripheral nervous systems NO participates in manyforms of synaptic transmission and pathological progress. Recently its dual effectbecomes a hot spot in experimental and clinical researches, especially under the statusof cerebral ischemia and reperfusion (I/R).In the present study, we measured NO levels in rat hippocampus using acarbon-fiber NO sensor during the process of global cerebral ischemia and the initialstage of reperfusion. Meanwhile, we separately observed the effects of two differentNOS inhibitors to the production of NO in this process. Based on the real-timemeasurement of NO concentration in rat hippocampus, the dynamic variance of NOconcentration in global cerebral ischemia and reperfusion has been revealed. Besidesthe animal experiments, we cultured hippocampal neurons in 20 min OGD andreperfusion to detect the changes of NO in OGD/reperfusion neurons. So we revealedthe dynamic changes of NO during ischemia and reperfusion, both in animal and celllevels.Although extensive research for I/R injury treatment has been performed in thepast several decades, few neuroprotectants and methods have been successfully translated from basic research into a clinical application. Recently ischemicpost-conditioning is a relatively novel concept. Unlike pre-conditioning,post-conditioning is composed of several repeated cycles of brief reperfusion andreocculusion of the artery applied at the onset of full reperfusion. Thie method hasbeen proved had powerful protective effects on ischemia, and may eventually lead tomore extensive clinical application. However, the underlying protective mechanism ofpost-conditioning remains illusive and in our experiment, we detected the dynamicchange of NO during the ischemic postconditioning against global cerebral I/R in vivo,and compared the effects of different numbers of cycles and periods forreperfusion/occlusion, to investigate the role of NO in ischemic postconditioning.Combining the cerebral blood flow (CBF) and the death ratio of neuron cells in rathippocampus, we could explore the mechanisms underlying the interruptingreperfusion. Furthermore, our results discover the best parameters that generate thestrongest protection and may provide important clues for cerebral I/R treatment.The results as follows:1. Alternation of NO levels in hippocampus resulted in a change of arterial bloodpressure and the electrochemical signal of NO could be recorded in real time.Intravenous administration of L-NAME (50mg/mL, 20μL-30μL) induced adecrease in NO concentration (4.5nM-6nM).2. In global cerebral ischemia and the initial stage of reperfusion, the whole NOconcentration profile could be divided into four stages as follows: the earlyischemia stage, during which there was a rapid and significant decrease of NOconcentration.; after the NO declined to the minimum, there was a platform withNO concentration keeping roughly unchanged; upon the onset of reperfusion, therewas an acute elevation stage lasted for 15 minutes; another steady stage appearedafter NO concentration reached the peak of reperfusion.3. We also observed the effects of two inhibitors of NOS on NO concentration.The two inhibitors (7-NI and 1400W) were respectively administratedintravenously at the onset of reperfusion and 1 h later. NO biosynthesis during the initial stage of reperfusion was mainly nNOS-dependent.4. We observed the dynamic changes of NO in hippocampal neurons inOGD/reperfusion. NO concentration was increased slowly and reached a platformafter 10-12 min.5. The speed of NO concentration increase was slowed down, but the extent onincrease was remarkably enlarged after ischemic post-conditioning, and CBF wasalso increased. We thought that post-conditioning induced a gentle and increasedNO synthesis, which inhibited the NO toxicity and strengthened the effect of NO toCBF, and finally decreased I/R lesion.6. Different numbers of cycles as well as different durations of reperfusion andocclusion had different effect to I/R injury. We found that 3 cycles of 30 sreperfusion and 30 s occlusion had the best protective effect.In conclusion, we detected the NO concentration in rat hippocampus in globalcerebral ischemia and the initial stage of reperfusion in vivo, and calculated thechange concentration of NO during this process. Meanwhile, we observed thechanges of NO in hippocampal neurons in 20 min OGD and reperfusion. So werevealed the dynamic changes of NO during ischemia and reperfusion, both inanimal and cell levels. It is very useful to reveal the behavior of NO concentrationin stroke and reperfusion. For post-conditioning, we detected the dynamic changeof NO during the ischemic postconditioning, and compared the effects of differentnumbers of cycles and periods for reperfusion/occlusion. We believed that NO alsoworked as an essential infactor in post-conditioning pathway.Our dynamic measurements of NO during this process would offer a timetherapeutic window for I/R treatment, which is very helpful for clinical therapies inthe future.更多还原