【作者】 李博；

【导师】 冯华；

【作者基本信息】 第三军医大学 ， 外科学， 2012， 博士

【摘要】 背景及目的：蛛网膜下腔出血（Subarachnoid hemorrhage，SAH）是最常见的出血性脑血管意外之一，主要为颅内动脉瘤破裂所致。而颅内动脉瘤本身是一种良性疾病，随着神经外科手术和影像技术的发展，颅内动脉瘤瘤体本身可通过外科手术或介入治疗获得治愈，但其伴发的SAH却导致超过40%的患者死残。因此，SAH继发性脑损伤是破裂动脉瘤治疗中的关键环节及研究热点。SAH继发性脑损伤机制的研究已持续了数十年，但迄今尚未被完全阐明。既往大部分研究着眼于SAH后颅内大动脉的痉挛上，但近些年研究显示大动脉痉挛并不是SAH后迟发性神经功能障碍及临床不良结果的主要原因。因此，国际上最近又提出了SAH后“早期脑损伤”理论，认为在SAH后早期（72h内）机体一系列病理过程即已启动。其中，以“皮质播散性去极化（CorticalSpreading Depolarization, CSD）”为代表的神经元兴奋性紊乱在SAH后继发性脑损伤中扮演着重要的角色。“超极化激活/环核苷酸依赖（HCN)通道”最早发现于上个世纪七十年代，三十多年来，其电生理功能的多样性一直吸引着众多科学家对其进行深入研究。近年来，HCN通道在中枢神经系统兴奋性维持中的重要作用备受关注。目前普遍观点认为HCN通道是神经环路兴奋性稳态调控的重要靶点，并将其比喻为神经系统中的“制动器（brake）”。尤其值得注意的是，在某些中枢神经系统疾病（例如癫痫、脑缺血等）中，HCN通道的功能状态变化将直接导致或者参与了相关神经环路兴奋性紊乱的发生。但迄今为止对于HCN通道在SAH后继发性脑损伤中的作用尚缺乏研究。我们推测在SAH后可能存在HCN通道的功能、表达变化，使神经元电活动发生了改变，进而诱导或者参与了SAH后CSD相关疾病现象的发生，并最终造成SAH后继发性神经功能障碍。本课题拟从HCN通道介导的神经兴奋性紊乱的新角度探讨SAH后神经功能损害发生的机制及防治途径，并在更现实的意义上，可能有助于为新的临床药物开发提供实验室依据，改变目前临床SAH后继发性脑损害治疗缺乏有效手段的现状。方法：1.建立线穿法大鼠蛛网膜下腔出血的动物模型，观察其死亡率及蛛网膜下腔出血情况，HE染色观察SAH后不同时间段海马附近脑室血凝块（blood clot）分布情况，常规免疫组化方法观察SAH后不同时间段血红蛋白（Hb）在脑室周围海马脑实质浸润情况；2.应用Western blot及RT-PCR技术检测SAH后不同时间段海马CA1区HCN1型通道的表达情况；3.应用含有氧合Hb的人工脑脊液体外模拟SAH出血环境，全细胞膜片钳技术观察记录脑组织片海马CA1区锥体神经元在血性脑脊液环境下HCN通道电流及神经兴奋性的变化情况。Biocytin标记对脑组织片海马CA1区记录细胞进行形态学鉴定。4.应用“NO试剂盒”检测海马CA1区组织片在血性脑脊液环境下NO释放浓度的变化，全细胞膜片钳技术观察记录脑组织片海马CA1区锥体神经元在分别给予NO释放剂NO/Sp和一氧化氮合酶（NOS）抑制剂L-NNA后HCN通道电流及神经元兴奋性的变化，然后进一步对比比较血性脑脊液环境下海马CA1区锥体神经元分别在上述两者干预后HCN通道电流和神经元兴奋性的变化情况。结果：1. SAH模型动物24h死亡率为38%，假手术组动物24h死亡率为0%。SAH模型动物24h后出血量评分为13±2\18。HE染色结果显示动物建模后24h及72h海马结构附近脑室有明显血凝块聚集。免疫组化结果显示SAH后早期溶血产物Hb侵入脑室周围海马脑实质，其中SAH后72h时Hb浸润程度明显重于SAH后24h时。2. Western blot结果显示SAH后24h及72h时海马CA1区HCN1型通道蛋白表达水平显著下降。进一步应用RT-PCR手段检测，结果显示SAH后24h及72h时海马CA1区HCN1mRNA水平与对照组相比分别下降了60.7%±5.4%和81.2%±4.1%。3.全细胞膜片钳记录显示海马CA1区锥体神经元具有HCN通道电流特性。给予含有Hb的血性人工脑脊液灌流后，海马CA1区锥体神经元膜电位波动明显，伴有动作电位密集发放。与此同时，记录细胞HCN通道电流幅值显著下降。应用CsCl阻断HCN通道后，CA1区锥体细胞在灌流含Hb人工脑脊液前后神经元兴奋性和HCN通道电流均无明显改变。Biocytin标记显示记录细胞具备海马CA1区锥体神经元特征性形态。4.给予含Hb的血性人工脑脊液灌流1h后，海马CA1区组织片NO释放浓度明显下降，灌流2h、3h、4h后，NO浓度继续缓慢下降，同时伴有CA1区HCN1通道蛋白的表达下降。全细胞膜片钳记录条件下，给予NO/Sp，海马CA1区锥体神经元兴奋性降低，表现为特定膜电位水平下的动作电位发放频率下降，与此同时，记录细胞HCN通道电流幅值较给药前有所增加。相反地，给予L-NNA后,海马CA1区锥体神经元兴奋性升高，同时伴有HCN通道电流幅值减小。更进一步地，Hb血性脑脊液灌流后给予NO/Sp可以部分逆转Hb对海马CA1区锥体神经元HCN通道电流的抑制作用，同时可以有效地下调Hb所致的神经元兴奋性增高。结论：1. SAH后早期血液及其溶血产物在“蛛网膜下腔-脑室”系统内迅速播散，并侵入脑实质；2. SAH后72h内，海马CA1区HCN1型通道蛋白和mRNA表达水平持续下降，此结果表明SAH后的病理环境影响海马CA1区HCN通道的正常表达分布；3. Hb血性脑脊液环境下，海马CA1区锥体神经元的兴奋性和HCN通道电流发生改变。Hb能够显著地抑制HCN通道电流，进而诱导或者参与了SAH后海马CA1区锥体神经元兴奋性紊乱的发生；4. Hb血性脑脊液环境下，脑组织NO含量急剧下降。而通过干预循环脑脊液中NO含量，能够明显地影响海马CA1区锥体神经元的兴奋性和HCN通道电流。此结果表明SAH后溶血产物Hb可能通过耗竭脑组织中的NO，进而使HCN通道功能发生改变，并最终导致了SAH后神经元兴奋性紊乱的发生。更多还原

【Abstract】 Background:It has been estimated that1–6%of the world population may harbor anintracranial aneurysm and that each year～10/100,000people suffer from ananeurysmal subarachnoid hemorrhage (SAH). Despite major advances in surgicaltechniques, radiology, and anesthesiology, the mortality and morbidity rates afterspontaneous SAH have not changed in recent years. Early brain injury includingelectrophysiological disorders, rather than cerebral vasospasm, may contribute tothe high mortality and morbidity rate of SAH. Electrophysiological disorders suchas “cortical spreading depolarization,” first appeared at the early stage after SAH.This kind of neuronal hyperexcitability originates from a temporary disruption oflocal ionic homeostasis and ultimately contributes to delayed ischemic neurologicaldeficit in patients with SAH.The hyperpolarization-activated/cyclic nucleotide (HCN)-gated channels are amixed-cation conductance, encoded by4genes (HCN1–4), and widely distributedin peripheral and central neurons.An abundance of evidence has demonstrated thatHCN1channels are critical for the regulation of neuronal excitability inhippocampus CA1region and neocortex. In these brain areas, HCN1subunits aredensely distributed and expressed on the dendritic spines of pyramidal neurons,and are involved in the integration of excitatory synaptic input and therebyinfluences the excitability of neural network. Interestingly, cortical spreadingdepolarization is more readily provoked in hippocampus CA1sector and neocortex,and its generation as well as propagation is also greatly dependent on the apicaldendrites of pyramidal neurons. Therefore, we speculate that HCN1channels arepotential regulative targets which contribute to the formation of neuronalhyperexcitability after SAH. Methods:1. The endovascular perforation model of SAH was established in vivo. Firstly,the mortality rat and extent of SAH were observed. Then, HE staining was appliedto observe the blood clots distributed within the cerebral ventricles nearhippocampus tissue at24and72h after SAH. Furthermore, the extent of Hbpenetrated into hippocampus tissue around cerebral ventricles was evaluated bythe method of immuno-histochemistry.2. Western Blot and RT-PCR were applied to detect the changes of expression ofHCN1in hippocampus CA1region at24and72h after SAH.3. For patch-clamp studies, Sprague–Dawley rat pups (p21-24) were used forslice preparation. Whole-cell recordings of HCN channels and neural activity wereconducted in hippocampus CA1pyramidal neurons in the presence and absence ofhemoglobin (Hb)-containing artificial cerebrospinal fluid (CSF). Additionally, somecells were intracellularly labeled with biocytin (0.5%) to confirm morphologicalidentification.4. At1,2,3and4h after perfusion of hippocampus slices with Hb, the NOlevels of hippocampus CA1tissue were assayed according to the instructions in theNO detection kit. For patch-clamp studies, NO/Sp or L-NNA was applied duringthe perfusion of Hb on CA1pyramidal neurons before the excitatory effect of Hbevaluated. Simultaneouly, the influence of Hb on HCN channel was observedagain in the presence of NO/Sp or L-NNA.Results:1. The mortality rate in the SAH group was38%. None of the sham operatedcontrol animals died during experiment. At24h post hemorrhage, the SAH scorewas13±2out of a possible18in the SAH groups. HE staining showed dense redblood clots distributed within the cerebral ventricles near hippocampus tissue at24and72h after SAH. Furthermore, the immune-positive results of Hb demonstratedHb penetrated into hippocampus tissue around cerebral ventricles after SAH.Especially, the distribution of Hb in hippocampus tissue at72h is more extensivethan that at24h after SAH. 2. HCN1protein expression in SAH group was significantly reducedcompared with control group, and the decrease at the72h post-SAH point wasmore pronounced. Similar to the results of Western blot, HCN1mRNA expressionreduced obviously in SAH group. Concretely, HCN1mRNA expression reduced by60.7%±5.4%at24h post-SAH point and81.2%±4.1%at72h post-SAH point,respectively.3. Hippocampus CA1pyramidal neurons display electrophysiological featureof HCN currents. Hb induced a moderate fluctuation of membrane potential,accompanied with a rapid firing of action potentials (APs). Interestingly, Hbsynchronously produced a significant decrease in the amplitude of HCN currents.CsCl, a HCN blocker, was pretreated before Hb administration. The resultsindicated that there was no significant increase of spike firing between applicationof CsCl alone and CsCl/Hb combined. Additionally, all the biocytin-labeled cellshad morphological features of CA1pyramidal neurons as described previously.4. The level of NO released from hippocampus slices had a dramatic drop at1h after Hb perfusion, and then slowly decreased at2,3, and4h after Hb perfusion.Simultaneously, the protein expression of HCN1in CA1region decreasedmoderately at1and2h after Hb perfusion, and then had a significant decrease at3and4h after Hb perfusion. NO/Spermine, a controlled releaser of nitric oxide,attenuated neuronal excitability and enhanced HCN currents in CA1pyramidalneurons. L-NNA, an inhibitor of nitric oxide synthase (NOS) reduced the HCNcurrents. The inhibitory action of Hb on HCN currents was reversed by applicationof NO/Sp, which also reduced neuronal hyper-excitability.Conclusion:These observations demonstrated a reduction of HCN channels expressionafter SAH and Hb reduced HCN currents in hippocampus CA1pyramidal neurons.These results revealed a functional interaction between Hb and HCN channels afterSAH. Hb, released from blood clot after SAH, exhausted the NO signaling, thusinhibited HCN channels, and consequently induced or facilitated the formation ofneuronal hyperexcitability in hippocampus CA1region after SAH. In a word, thepresent results implied that the change of HCN channels may be a novel process involved in the formation of neuronal hyperexcitability after SAH, and mayprovide new therapeutic clues in patients with SAH.更多还原