【作者】 赵卫华；

【导师】 黄楹； 黄慧玲；

【作者基本信息】 天津医科大学 ， 外科学， 2007， 硕士

【摘要】 目的：改进枕大池2次注血法，建立蛛网膜下腔出血(SAH)导致迟发脑血管痉挛(DCVS)的大鼠模型；应用该模型动态监测SAH后脑血流(CBF)的变化，对基底动脉进行形态学测定和组织病理学检查，动态观察CVS的病理演变过程；探讨SAH导致DCVS的脑代谢变化规律，及其与皮质CBF变化的相关性；观察DCVS大鼠海马CA1区神经元的凋亡情况及演变过程，进一步探讨SAH后DCVS的发生机制及演变规律。方法：(1)模型建立及CBF监测：50只SD大鼠随机分为5组：对照组、SAH后1天、3天、5天、7天组，每组10只。应用鼠尾非抗凝自体动脉血，枕大池2次注血法制作动物模型。5只大鼠(n=5)用于大体观察：其中4只在2次注血后30分钟处死，观察血的大体分布；1只2次注入人工脑脊液后30分钟处死做为对照观察。激光多普勒(LDF)测定各组的皮层CBF；(2)各组在相应时段处死，取基底动脉应用光学显微镜对基底动脉进行形态学测定，应用透射电子显微镜观察基底动脉超微结构变化；(3)采用微透析(MD)仪监测各组脑细胞外液的葡萄糖(Glu)、乳酸(Lac)、丙酮酸(Pyr)、谷氨酸(Glut)及甘油(Gly)的浓度，并计算乳酸与丙酮酸比值(L／P)；(4)各组在相应时段处死取海马组织，应用原位细胞凋亡检测法(TUNEL)监测海马CA1区神经元的凋亡情况及演变过程。组间差异应用单因素方差分析，两两比较应用LSD法。结果：(1)模型建立情况：SAH组血液主要分布于基底池、脚间池及额叶底面。亚甲蓝与血液混合物主要沉积于颅底，同时在脑室及纵裂池亦可见血液分布。对照组大鼠注入人工脑脊液后未见发生颅内出血及血液沉积现象；LDF监测显示，SAH各组出现了严重的CVS，CBF明显低于对照组(P＜0.05)，其中第5天组降低最显著。表明模型建立成功；(2)形态学研究：与对照组相比，光镜下SAH组发生了明显的CVS，表现为血管直径减小、管壁增厚、管腔周长减少、内弹力膜皱褶，同时可见痉挛血管细胞增殖现象明显。尤以第5天组最显著(P＜0.05)。出血第1、3、5、7天组基底动脉直径分别减少了46.34％、33.95％、50.59％、17.21％；管壁厚度分别增加了98.55％、75.58％、159.27％、19.21％；管腔内周长分别减少了56.30％、45.97％、62.50％、25.77％。与对照组比较，电镜下SAH组内皮细胞出现类凋亡样变化，包括内皮细胞膜起泡，胞质凝聚，胞浆空泡变，核染色质凝聚、趋边。以第5天组最显著，伴有大量内皮细胞的剥离导致内弹力膜的裸露，平滑肌细胞的坏死。(3)脑代谢检测：SAH组大鼠脑ECF中Glu及Pyr含量明显降低(P＜0.05)；L／P明显升高(P＜0.05)，以第5天及第7天组升高最为明显(P＜0.05)；第5天组Lac含量亦明显升高(P＜0.05)；Glut及Gly浓度明显升高，以第5天组升高最明显(P＜0.05)。CBF的变化率与Lac、L／P比值、Glut、Gly呈明显正相关(r=0.477，0.721，0.804，0.718；均P＜0.05)，与Glu、Pyr呈明显负相关(r=-0.447，-0.579；均P＜0.05)；(4)凋亡研究：对照组未见凋亡细胞；SAH第1天组可见TUNEL阳性细胞，第3天组逐渐增多，第5天、7天组明显增多。结论：(1)大鼠SAH并发DCVS模型制备成功，该模型可以在一定程度上模拟人类SAH后CVS的双时相特点和病理演变过程；(2)DCVS的发生，与血管周围细胞的增殖以及基底动脉内皮细胞类凋亡样变化相一致，表明凋亡和血管细胞增殖可能是CVS发生机制中非常重要的因素；(3)微透析指标与CBF变化具有一致性，可作为SAH脑组织间液生化指标的动态监测工具，用于预测DCVS的发生。(4)SAH大鼠海马CA1区出现了明显的神经元的凋亡，并且随着DCVS的发生，神经元的凋亡现象更为严重，提示海马区的凋亡可能是SAH后伴发迟发性缺血性神经功能缺陷(DIND)的重要因素。更多还原

【Abstract】 Objective: To establish the animal model of subarachnoid hemorrhage(SAH) -induced delayed cerebral vasospasm(DCVS) by modifying the two injections of nonheparinized autologous arterial blood into the cisterna magna. Using the animal model, we dynamically monitor the changes of cerebral blood fluid(CBF) and chose basilar artefies(BA) for morphometric Analysis and histopathological examination; To investigate the changes of the cerebral energy metabolism and the neuronal apoptosis in hippocampal CA1 region following SAil-induced DCVS, and explore the correlation with CBF changes; To further explore the pathogenesis of DCVS.Methods: (1) Establishment of animal model: Male Sprague-Dawley rats(n=50) were divided into five groups: control group and 1d、3d、5d、7d group following SAH. The animal model was induced by two injections of nonheparinized autologous arterial blood into the cistema magna. Five rats(n=5) were used for the general observation: Four rats were euthanized at 30 min after dobble-hemorrhage for blood distribution; One rat was injected twice into the cisterna magna with artificial cerebrospinal fluid for control.; The cortical CBF was measured by Laser Doppler Flowmetry(LDF); (2) Each group was sacrificed respectively, We chose basilar artedes(BA) for morphometdc Analysis with light microscope and for observation the changes of ultrastructure with transmission electron microscopy; (3) Microdialysis(MD) was applied to monitor the concentration of Glu、Lac、Pyr、Glut、Gly in the cerebral extracellular fluid(ECF)in each group, and calculated the I/P; (4) we chose hippocamp for the study on the neuronal apoptosis with TUNEL and observe the time course of apoptosiso Results:(1) The distribution of blood clot was seen in the basal cisterns, interpeduncular cisterns, on the base of the frontal lobe. Methylthioninium Chloride with blood was mainly observed in the subarachnoid space on the base of the brain, blood was seen in the ventricle and diastematia cistern as well. However, in control group none of the rats developed blood and no blood distribution was seen. the LDF measurements show severe vasospasm and there was an obviously decrease in CBF compared with baseline in each group following SAH(P＜0.05), especially in 5d group the CBF decreased significantly(P＜0.05); (2) Morphometric Analysis and histopathological examination: The basal artery revealed luminal narrowing, reduction in diameter, increased wall thickness, corrugation of the internal elastic lamina(IEL), and cellular proliferation were observed in the spastic BA as well, especially severe in 5d group(P＜0.05). In 1d、3d、5d、7d group, the mean diameter of BA was reduced from control levels by46.34%, 33.95%, 50.59%, 17.21%; the increases in wall thickness of BA were 98.55%, 75.58%, 159.27%, 19.21%; the mean luminal perimeter reduced by 56.30%, 45.97%, 62.50%, 25.77%. Apoptotic-like changes were noted in endothelial cells in each group following SAH and consisted of blebbing of the cell membrane, condensation of the cytoplasm, condensation of peripheral nuclear chromatin, and cytoplasmic vacuolization. The morphological changes in endothelial were most severe and wide spread in 5d group with some endothelial cells and smooth muscle cells showed evidence of necrotic .changes. (3) MDanalysis: [Glu]、[Pyr] was lower ineach group following SAH, whereas L/P、[Glut] and [Gly] were significantly higher than that in control group(P＜0.05), at same time, L/P in 5d、7d group was higher than that in other groups(P＜0.05), [Lac]、[Glut] and [Gly] were significantly elevated in 5d group(P＜0.05); Changes in CBF has significantly positive correlation with L/P、Lac、Glut and Gly (r=0.721, 0.477, 0.814, 0.718 P＜0.05), whereas, it has strongly negative correlation with Glu and Pyr (r=-0.447, -0.579; P＜0.05); (4) Neuron apoptosis in the hippocampal CA1 region was no found in control . the number of TUNEL—positive cells showed a significant increase in each SAIl group, especially in the 5d、7d group (P＜0.05).Conclusion: (1) The model of SAH was induced successfully and it can accurately simulate the time course of vasospasm in humans following SAH; (2) The course of CVS following SAH coincided with the progress of apoptosis changes in ehdothelial cells and proliferation of cells in the vascular wall. Endothelial apoptosis and cellular proliferation might play an important role in the pathogenesis of vasospasm; (3) MD markers are in good accordance with changes in CBF, MD is an ideal tool for dynamically monitoring neurochemical substances in the ECF following SAH and can indicate early the onset of delayed cerebral ischemia; (4) Neuron apoptosis in the hippocamp may play an important role in the pathogenesis of DIND.更多还原