氢气在兔蛛网膜下腔出血后早期的脑保护作用及机制研究

发布时间：2018-04-27 21:48

本文选题：蛛网膜下腔出血 + 氢气　；参考：《南京大学》2014年博士论文

【摘要】：研究背景：脑蛛网膜下腔出血(subarachnoid hemorrhage, SAH)是神经外科临床常见疾病,主要由颅内动脉瘤破裂引起。SAH常伴随高死亡率和高致残率,病理生理机制复杂,并发症较多,一直为神经外科研究热点。既往相关研究主要围绕SAH后脑血管痉挛(cerebral vasospasm, CVS)等机制展开,尤其多针对SAH后CVS引起的缺血性损伤及神经功能障碍。但几十年的相关基础和临床研究并无明确进展。而越来越多的研究表明SAH后早期(24-72小时)的脑损伤(early brain injury, EBI)在SAH的严重不良预后中起重要作用。并且,诸多证据显示SAH后的EBI与SAH后发生的脑氧化应激损伤和脑水肿密切相关。近年,大量研究证实氢气(H2)是一种能够选择性清除毒性自由基而不影响正常生理氧化反应的较温和的抗氧化剂,且H2在中枢神经内的保护作用已有较多报道。故本实验主要研究H2是否能对SAH后的早期脑损伤起保护作用,并研究相关机制。目的：1)建立稳定的兔枕大池二次自体动脉血注射的SAH模型；2)验证H2生理盐水溶液对兔SAH后EBI的保护效果及行为学效果；3)研究H2生理盐水溶液对SAH后EBI的可能保护机制。方法：使用兔枕大池二次注血法建立稳定的SAH模型。新西兰兔随机分为四组：对照组,SAH组,SAH+溶剂(生理盐水)对照组,SAH+ H2生理盐水溶液组。兔枕大池注血制作SAH模型后,使用H2生理盐水溶液或生理盐水于兔SAH后腹腔注射治疗；动物处死前进行行为学评分；于断头取脑后分光光度法检测双颞叶脑组织丙二醛含量；Western blot法检测对应脑组织内凋亡、抗凋亡相关蛋白；干湿重比法检测各组动物脑组织水含量；TUNEL染色法展示和定位神经元凋亡,并用尼氏染色法显示脑损伤程度；电泳迁移率改变实验(electrophoretic mobility shift assay, EMSA)和免疫荧光双染法分别定量和定位NF-κB在神经元内表达与激活情况；Real-time PCR法检测NF-κB下游相关基因的转录水平；同时ELISA法检测各组动物SAH后脑组织炎症因子(TNF-α、IL-β)的水平变化。结果：各实验组模型在SAH后72小时取脑时均被确定有效。检测结果表明较对照组,兔SAH后72小时脑组织丙二醛含量明显升高(P0.05),脑水肿明显(P0.05)；ELISA检测结果显示炎症因子(TNF-α、IL-β)水平明显增高；Western blot检测显示SAH后72小时凋亡相关蛋白caspase-12/3含量明显增多(P0.05)；同时,TUNEL染色、尼氏染色显示明显的神经元凋亡和脑损伤(P0.05)。溶剂对照组检测呈类似结果。然而,SAH后予以H2治疗可明显降低脑组织丙二醛含量,减少凋亡相关蛋白,减轻脑水肿,明显缓解早期脑损伤(P0.05)。另外,H2治疗SAH后,EMSA和免疫荧光显示NF-κB被明显激活,同时下游相关抗凋亡蛋白Bcl-xL基因转录及蛋白表达水平明显升高(P0.05)；但ELISA检测结果显示H2治疗并未明确降低炎症因子水平。此外,兔行为学评分结果显示各组动物神经功能评分未见明显差异。结论：我们发现兔SAH后EBI明确发生,且SAH后早期的氧化应激反应及脑水肿在SAH后EBI中起重要作用。H2治疗能够抑制SAH后氧化应激并减轻脑水肿。此外,我们在中枢神经系统首次发现SAH后H2可以激活NF-κB/Bcl-xL抗凋亡通路,但H2未能明显改变SAH后的NF-κB相关炎症反应。因此,H2可以通过抑制SAH后的氧化应激、减轻脑水肿、激活NF-κB/Bcl-xL通路而减轻SAH后的EBI。以上结果提示H2在SAH后的EBI治疗中有潜在治疗价值,有可能成为针对SAH的有效治疗剂。
[Abstract]:Background: subarachnoid hemorrhage (SAH) is a common clinical disease in the Department of neurosurgery. The rupture of intracranial aneurysms is mainly caused by the high mortality and high disability rate of.SAH, and the pathophysiological mechanism is complex and the complications are many. The previous related studies mainly focus on the blood of SAH after cerebral blood. Guan Jingluan (cerebral vasospasm, CVS) and other mechanisms, especially for ischemic injury and neurological dysfunction caused by CVS after SAH, have not been made clear for decades of related basic and clinical studies. More and more studies have shown that the early (24-72 hour) brain damage (early brain injury, EBI) after SAH is in SAH serious bad precondition. In addition, many evidence shows that EBI after SAH is closely related to brain oxidative stress and brain edema after SAH. In recent years, a large number of studies have confirmed that hydrogen (H2) is a mild antioxidant that can selectively remove toxic free radicals without affecting normal physiological oxidation, and the protection of H2 in the central nervous system More reports have been reported. Therefore, this experiment mainly studies whether H2 can protect the early brain injury after SAH and study the related mechanisms. Objective: 1) to establish a stable SAH model of two autologous arterial blood injection in the rabbit pillow big pool; 2) to verify the protective effect of H2 saline solution on rabbit SAH after SAH and the behavioral effect of EBI; and 3) to study H2 students. The possible protective mechanism of SAH EBI after SAH. Methods: a stable SAH model was established by using the rabbit pillow large pool blood injection method. New Zealand rabbits were randomly divided into four groups: control group, SAH group, SAH+ solvent (physiological saline) control group, SAH+ H2 physiological saline solution group. After the rabbit pillow big pool was injected with blood to make SAH model, a solution of H2 physiological saline solution or birth was used. The saline was treated with intraperitoneal injection after SAH in rabbits; the behavior score was evaluated before the death of the animals; the content of malondialdehyde in the brain tissue of the double temporal lobe was detected by the spectrophotometric method after taking the head off the brain; the Western blot method was used to detect the apoptosis and anti apoptosis related protein in the brain tissue, and the water content of the brain tissue in each group was detected by the dry wet weight ratio method; the TUNEL staining method was used. The neuron apoptosis was shown and located, and the degree of brain injury was detected by Nissl staining. The expression and activation of NF- kappa B in the neurons were quantified and identified by electrophoretic mobility shift assay (EMSA) and immunofluorescence double staining, and Real-time PCR method was used to detect the transcriptional level of the downstream related genes of NF- kappa B; At the same time, ELISA method was used to detect the level changes of inflammatory factors (TNF- alpha, IL- beta) in the brain tissue of each group of animals. Results: all experimental groups were determined to be effective when taking brain 72 hours after SAH. The results showed that the content of malondialdehyde in brain tissue increased significantly (P0.05) and brain edema (P0.05) at 72 hours after SAH in the control group (P0.05), and the results of ELISA detection were obvious. The levels of inflammatory factors (TNF- alpha, IL- beta) were significantly higher; Western blot detection showed that the apoptosis related protein caspase-12/3 content increased significantly (P0.05) 72 hours after SAH; meanwhile, TUNEL staining, Nissl staining showed obvious neuronal apoptosis and brain damage (P0.05). Decrease the content of malondialdehyde in brain tissue, reduce apoptosis related protein, reduce brain edema and alleviate early brain injury (P0.05). In addition, after the treatment of SAH, EMSA and immunofluorescence showed that NF- kappa B was obviously activated, while the downstream related anti apoptotic protein Bcl-xL gene transcriptional and protein expression level increased significantly (P0.05), but ELISA detection results showed that the results showed that the level of NF- kappa B was significantly increased (P0.05). H2 treatment did not clearly reduce the level of inflammatory factors. Furthermore, the results of rabbit behavior score showed no significant difference in the neurological function score of each group. Conclusion: we found that the EBI after SAH was clearly found in rabbits, and the early oxidative stress reaction after SAH and the important role of brain edema after SAH EBI could inhibit the oxidative stress after SAH and In addition, H2 can activate the anti apoptotic pathway of NF- kappa B/Bcl-xL after the first discovery of SAH in the central nervous system, but H2 does not significantly alter the NF- kappa B related inflammatory response after SAH. Therefore, H2 can be reduced by inhibiting oxidative stress after SAH and reducing brain edema. 2 there is potential therapeutic value in EBI therapy after SAH, and it may become an effective therapeutic agent for SAH.

【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743.35

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