钙离子信号在弥漫性轴索损伤发生发展中的初步研究

发布时间：2018-03-09 03:18

本文选题：法医病理学　切入点：DAI　出处：《华中科技大学》2013年硕士论文　论文类型：学位论文

【摘要】：【研究背景、目的】弥漫性轴索损伤（diffuse axonal injury， DAI），也称“弥漫性轴突损伤”，是外力引起的广泛性脑白质轴索损伤。脑外伤死亡病人中，DAI占29％～43％。DAI以头部外伤后意识障碍为其典型表现，无脑挫裂伤等常见的局灶性损伤症状和体征，CT、MRI等常见影像学检查不能直接显示轴索损伤，目前DAI尚无统一的诊断标准。虽然近20年来对颅脑损伤研究有了很大进展，诊断技术及治疗措施也在不断完善，但颅脑损伤的病死率及致残率仍未见明显下降。大量的研究显示DAI与Ca~(2+)超载、轴浆运输障碍、线粒体损伤、轴索的细胞骨架破坏、自由基、炎症因子等有关。其中Ca~(2+)超载被认为是导致轴索损伤瀑布反应过程中的中枢环节。在治疗上，DAI迄今尚无特异方法能够阻断DAI进展，因此预后较差。DAI临床及基础研究的进展缓慢也限制了其在法医鉴定中的应用，实际检案中DAI应用较少，目前仍然是法医学鉴定的难点及争议的焦点。因此，本研本实验拟通过在动物模型中使用Ca~(2+)拮抗剂尼莫地平（Nimodepine,ND）阻断来观察其对轴索和血管损伤的影响来探讨Ca~(2+)在DAI发病中确切作用及机制，以期为DAI临床诊治及法医学应用提供研究基础。【材料和方法】将28只雄性SD实验大鼠随机分为对照组、实验组及干预组。参照Marmarou实验动物模型，建立大鼠DAI模型，实验组及干预组打击后分别在12h、24h及72h处死，对照组为假手术组（大鼠未进行打击），实验组及干预组为直线加速度打击负荷组，实验组打击后不作处理，干预组打击并加用尼莫地平（Nimodipine，ND）进行干预。每组每个时间点4只。用过量10%水合氯醛腹腔麻醉处死，，开颅取脑，打开颅腔取出大脑、小脑及脑干，沿正中矢状面将大脑、脑干及小脑分为左、右两半，其中一半脑用于脑湿干比例测定，另外一半脑用于组织形态学及免疫组化观察：（1）取一部分脑组织称重后放入烤箱内（85℃）烘烤24小时，在烘干后再次进行脑称重，进行脑湿干比例计算；（2）另一半脑组织用4%多聚甲醛固定24h，然后进行脱水、透明、包埋等制成石蜡切片，用苏木素-伊红（HE）染色、β-APP及vWF免疫组织化学染色进行组织学及免疫组化观察。【结果】实验组及干预组脑组织湿干比例呈先升高后降低的趋势，脑组织含水量最高值出现在打击后24h,此后脑组织含水量呈下降趋势；给予Nimodepine（ND）干预后，脑水肿成下降趋势，但仍然高于对照组：实验组与干预组HE染色见轴索变粗、扭曲、肿胀、断裂且随时间呈逐渐加重趋势，干预组给予ND干预后轴索损伤程度有所减轻，但仍高于对照组；实验组与干预组各时间点β-APP免疫组织化学染色见β-APP表达随时间延长表达增强，干预组给予ND干预后β-APP表达有所减少，但仍高于对照组；vWF表达部位位于蛛网膜下腔、脑实质血管内皮细胞及脑干部位轴索，打击后vWF表达明显增强，24h尤其明显，脑实质血管内皮细胞阳性表达较强的区域与脑水肿严重区域较一致，尼莫地平干预减轻vWF表达，特别是脑实质血管的表达显著减轻。【结论】本研究模拟Marmarou动物模型构建DAI模型，观察打击后出现脑水肿、轴索损伤并见点灶状蛛网膜下腔出血；运用钙离子拮抗剂ND干预后可以减轻脑水肿和轴索损伤并血管损伤局限化，提示钙离子信号在弥漫性轴索损伤中有重要作用；脑水肿在打击后24h达到高峰，推测创伤后24h可能是治疗弥漫性轴索损伤的较佳时间窗。
[Abstract]:[research background, purpose]
Diffuse axonal injury (diffuse axonal, injury, DAI), also known as "diffuse axonal injury", is the external cause of extensive cerebral white matter axonal injury. The patient died of cerebral trauma, DAI accounted for 29% ~ 43%.DAI in head injury of disturbance of consciousness is the typical performance, no brain contusion and other common office focal injury symptoms and signs, CT, MRI and other common imaging cannot directly display axonal injury, at present there is no uniform diagnostic criteria of DAI. Although nearly 20 years study of brain injury has made great progress, diagnostic techniques and treatment measures are continuously improved, but the mortality rate of brain injury and the disability rate is there was no obvious decline. A large number of studies show that DAI and Ca~ (2+) overload, damage to mitochondria impaired axonal transport, cytoskeleton, cable shaft damage, free radicals, inflammatory factors and so on. The Ca~ (2+) overload is considered to be the cause of axonal injury cascade The central link in the process. In the treatment of DAI, so far there is no specific method to block the progression of DAI, so.DAI has a poor prognosis in clinical and basic research of the slow also limits its application in forensic identification, the DAI application is less practical cases, is still the focus of forensic identification of the difficulties and disputes.
Therefore, the research in this study through the use of Ca~ in animal models (2+) antagonist nimodipine (Nimodepine, ND) to observe the effect of blocking axonal and vascular injury of Ca~ (2+) and the exact mechanism in the pathogenesis of DAI, in order to provide the basis for the study of DAI clinical diagnosis and forensic medicine the application of science.
[materials and methods]
28 male SD rats were randomly divided into control group, experimental group and intervention group. According to the Marmarou experimental animal model DAI rat model, the experimental group and the intervention group after the attack at 12h, 24h and 72h were control group (sham operation group rats were not hit), experiment group and intervention group for linear acceleration combat load group, the experimental group against no treatment, the intervention group combat and added nimodipine (Nimodipine, ND) to intervene. In each group 4 rats at each time point. With an excess of 10% chloral hydrate intraperitoneal anesthesia were craniotomy for brain, open the cranial cavity out of the brain, cerebellum and brainstem the brain, along the sagittal plane, brainstem and cerebellum is divided into left and right halves, one half of the brain to brain wet to dry ratio determination, the other half of the brain for histological and immunohistochemical observation: (1) take part in brain tissue after weighing into a baking box (85 DEG C) baking 24 hours in baking Dry again after brain weight, brain wet dry ratio; (2) the other half of the brain tissue was fixed with 4% paraformaldehyde 24h, then dehydrated, transparent, etc. embedded into paraffin sections, with hematoxylin eosin (HE) staining, beta -APP and vWF immunohistochemical staining of tissue pathological and immunohistochemical observation.
[results] the experimental group and the intervention group of brain tissue wet to dry ratio first increased and then decreased, the water content of brain tissue had the highest value in the fight against 24h, then the brain tissue water content decreased; Nimodepine (ND) intervention, brain edema and a downward trend, but still higher than the control group: the experiment group and intervention group HE staining showed axonal diameter, distortion, swelling, fracture and showed a gradually increasing trend in the intervention group were given ND intervention to axonal damage has been reduced, but still higher than the control group; the experimental group and the intervention group at different time points P -APP immunohistochemical staining showed the expression of -APP beta with time prolonged expression, the intervention group was given ND after the intervention of beta -APP expression decreased, but still higher than the control group; the expression of vWF located in the subarachnoid space, cerebral vascular endothelial cells and brain stem axons, the expression of vWF was significantly enhanced after the attack, especially 24h Obviously, the areas with strong positive expression of cerebral parenchymal vascular endothelial cells were more consistent with severe areas of cerebral edema. Nimodipine intervention alleviated the expression of vWF, especially the expression of cerebral parenchymal vessels.
[Conclusion] model to construct the DAI model this study simulated Marmarou animal brain edema were observed after the attack, axonal injury and focal subarachnoid hemorrhage; use of calcium antagonists after ND intervention can alleviate brain edema and axonal injury and localized vascular injury, suggesting that calcium signaling in diffuse axonal there is an important role in the fight against injury; brain edema after 24h and reached the peak at 24h after trauma that may be a better treatment of diffuse axonal injury time window.

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：D919.4

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