【作者】 陈笛；

【导师】 王莎莉；

【作者基本信息】 重庆医科大学 ， 组织工程与细胞工程， 2011， 博士

【摘要】 研究背景蛛网膜下腔出血是一种严重的神经外科急症。虽然SAH发病率仅占全部脑卒中发病率的5%～10%,但总体死亡率却占整个卒中患者群死亡率的25%。近年来,对SAH的发病机制和病理生理过程的研究日趋深入,各种预防和治疗措施在临床上的广泛开展,使其预后有了一定改观,但其病死率和致残率依旧居高不下。许多患者遗留永久性神经功能缺损和/或认知障碍,临床疗效并不尽如人意。研究证实:SAH后继发性脑损伤是造成患者预后不良的主要原因,因此,有效防治继发性脑损伤可能是改善SAH总体预后的关键环节。当归是我国传统中医使用频率最高的药用植物之一,具有上千年的使用历史,藁苯内酯是其最主要的活性成分之一。LIG药理作用广泛,具有良好的临床应用潜力和开发价值。近年来的研究证明,LIG对短暂性前脑缺血、永久性局灶性脑缺血和永久性前脑缺血等模型动物具有神经保护作用。还可以通过抑制钙内流或钙库释放而对大鼠主动脉或肠系膜动脉产生舒血管效应,并能抑制血管平滑肌的增殖和改善微循环。但LIG对蛛网膜下腔出血后继发性脑损伤是否具有保护作用却未见报道。本课题旨在证实LIG对SAH后脑损伤是否具有积极的治疗作用,然后从多层次多角度深入探讨其作用机理,找寻其作用靶点。希望能为进一步开发和利用我国丰富的当归资源,为临床治疗SAH提供新的理论与实验依据。第一部分藁苯内酯对大鼠蛛网膜下腔出血的神经保护作用目的建立有明显神经损伤和迟发性脑血管痉挛的蛛网膜下腔出血模型,探讨藁苯内酯对SAH大鼠的神经保护作用。方法1将雄性Sprague-Dawley(SD)大鼠随机分为:(1)假手术组:Sham-1d、3d、5d、10d、14d组;(2)SAH组:SAH-1d、3d、5d、10d、14d组,采用改良的枕大池二次注血法建立SAH模型。通过检测大鼠的生存率、神经功能学评分、脑组织含水量;光镜观察脑组织和基底动脉组织病理学改变;测量基底动脉血管壁厚度、管腔面积与直径,对模型进行综合评价。2将雄性SD大鼠随机分为:(1)假手术组、(2)SAH+vehicle组、(3)SAH+LIG5(LIG 5mg/kg)组、(4)SAH+LIG20(LIG 20mg/kg)组。通过观察实验大鼠的一般生存情况和生存率、检测神经功能学评分、脑组织含水量、血脑屏障通透性,以及脑组织的HE和尼氏染色,综合评价蒿苯内酯对SAH大鼠的保护作用。结果1与假手术组相比,采用改良的枕大池二次注血法建立SAH大鼠模型的:(1)术后生存时间明显缩短,神经功能学评分降低。(2)大脑组织含水量在二次注血后1 d和3d明显增加。(3)脑组织HE染色显示皮层和海马等处出现缺血性脑损伤的表现,水肿变性或凋亡,神经元数量减少。(4)基底动脉管腔面积和直径明显减小,血管壁增厚;出现一定程度的病理组织学改变,内弹力膜皱褶,内皮细胞皱缩,平滑肌细胞收缩等;上述变化于建模后的第5天达高峰。2蒿苯内酯(20mg/kg)治疗SAH模型大鼠后,可以:(1)改善神经功能学评分,但对死亡率无明显影响。(2)减少大脑组织含水量;降低血脑屏障通透性。(3)改善皮层和海马等处神经元的病理性损伤。结论1应用改良的枕大池二次注血法能够成功建立大鼠SAH模型;该模型脑组织损伤明显,脑血管痉挛发生的时间窗典型,是研究SAH所致脑损伤和脑血管痉挛的发病机制和疗效观察的较为理想的动物模型。2 LIG对SAH模型大鼠有确切的神经保护作用,可改善神经功能,降低脑水肿和BBB通透性,减轻神经元凋亡和坏死。第二部分藁苯内酯对大鼠蛛网膜下腔出血的神经保护作用的机制研究目的探讨LIG对大鼠蛛网膜下腔出血的神经保护作用的可能机制。方法1实验分组:将雄性SD大鼠随机分为:(1)假手术组、(2)SAH+vehicle组、(3)SAH+LIG5(LIG 5mg/kg)组、(4)SAH+LIG20(LIG 20mg/kg)组。2 LIG对SAH后脑血管痉挛的治疗作用及其机制:(1)光镜和电镜观察基底动脉的组织病理学改变。(2)光镜下测量基底动脉管腔面积及管壁厚度。(3)TUNEL法检测基底动脉内皮细胞的凋亡。(4)免疫组织化学法和蛋白印迹法检测基底动脉的p53、Bax、Bcl -2及caspase -3等凋亡通路相关蛋白的表达。3 LIG对SAH后血液流变学的影响:全自动血液粘度动态分析仪测定血液流变学的改变。4 LIG对SAH后大脑皮层自由基清除和神经元凋亡的影响:(1)黄嘌呤氧化酶法检测大脑皮层脑组织的超氧化物岐化酶活力。(2)硫化巴比妥酸法检测大脑皮层脑组织中丙二醛含量。(3)TUNEL法检测大脑皮层神经元的凋亡。(4)免疫组织化学法和蛋白印迹法检测大脑皮层神经元的p53、Bax、Bcl -2及caspase -3等凋亡通路相关蛋白的表达。结果LIG治疗可以:1明显改善SAH大鼠基底动脉光镜和电镜下的组织病理学损害。2增加基底动脉的管腔面积,降低管壁厚度。3减少基底动脉内皮细胞和大脑皮层神经元中的TUNEL阳性细胞;4下调基底动脉内皮细胞和大脑皮层神经元中促凋亡蛋白p53和cleaved caspase-3的表达,上调抗凋亡蛋白Bcl-2的表达,但对促凋亡蛋白Bax的表达无明显影响。5使SAH大鼠的全血粘度、全血还原粘度、全血高切和低切相对粘度、红细胞聚集指数、红细胞刚性指数和红细胞变形指数等血液流变学指标有所改善,但其差异并无统计学意义。6使脑组织SOD活性增加,MDA含量降低。结论LIG可能主要通过抗内皮细胞凋亡缓解SAH后迟发性CSV、抗自由基损伤和抗神经元凋亡等途径减轻SAH所致的继发性脑缺血损伤。更多还原

【Abstract】 BackgroundSubarachnoid hemorrhage is a severe neurosurgical emergency. Although SAH comprises about 5%–10% of all strokes, it is responsible for 25% of all stroke deaths. Numerous intensive researches on mechanisms and pathophysiology of SAH were performed recently. Although various preventative and therapeutic interventions have been applied in clinic practice to improve the outcome of SAH, its mortality and disability still remain at a relatively high level. A considerable percentage of patients still experience permanent neurologic and/or cognitive impairments. The overall prognosis of patients harboring SAH remains poor. Previous studies have demonstrated that secondary brain damage as a result of SAH is one of the leading causes of poor outcome. Thus, prevention and management of secondary brain damage is critical for improving the overall outcome of patients with SAH.Radix Angelica sinensis is one of most popular medicinal plants in traditional Chinese Medicine, which has been used in clinic for thousands of years. Z-Ligustilide, one of the main active ingredients of Radix Angelica sinensis, has various pharmacological effects. Recent researches have reported that LIG provided significant neuroprotective effects on transient forebrain ischemia in mice, permanent forebrain ischemia in rats and focal cerebral ischemia in rats. Ligustilide induces vasodilatation in murine aorta and superior mesenteric artery via inhibiting Ca²⁺ influx and release. It also inhibits proliferation of smooth muscle cells and improves microcirculation. However, the protective effects of LIG on the secondary brain injury following SAH have been not yet reported.The aim of this study was to evaluate the potential therapeutic use of LIG as a treatment option following a SAH-induced brain injury and explore the mechanisms and targets of the neuroprotection of LIG. The results of the present study may provide new theoretical and experimental data for the management of SAH and development and utilization of Angelica sinensis.PART ONE THE NEUROPROTECTIVE EFFECTS OF Z-LIGUSTILIDE AGAINST SUBARACHNOID HEMORRHAGE IN RATSObjective1 To establish a subarachnoid hemorrhage model with secondary neurological injury and delay cerebral vasospasm.2 To assess neuroprotective effects of Z-ligustilide on subarachnoid hemorrhage in rats.Methods1 The male Sprague-Dawley rats were randomly assigned to ten groups as follows: （1） Sham groups: Sham-1d, 3d, 5d, 10d, 14d groups; （2） SAH groups: SAH-1d, 3d, 5d, 10d, 14d groups.SAH model was established by a modified rat double hemorrhage model. Survival analysis, neurological evaluation, brain water content, histological examination of brains and basilar arteries, and diameter, wall thickness and cross-section area of basilar arteries were investigated to assess this model.2 The male SD rats were randomly assigned to the following four groups: （1） Sham group, （2） SAH+vehicle group, （3） SAH+LIG5 group （5mg/kg LIG treatment）, and （4） SAH+LIG20 group （20mg/kg LIG treatment）. General observations, survival analysis, neurological evaluation, brain water content, and blood brain barrier permeability were measured, and morphologic changes in the brains were observed by Hematoxylin-Eosin staining and Nissl staining to evaluate protective effects of LIG on SAH model rats.Results1 Compared with the Sham group, the survival time was significantly shortened and neurological scores were reduced in SAH group. The brain water content was increased on 1d and 3d after SAH. The ischemic brain injury was observed in the cortex and hippocampus of SAH group. Edema and typical neural apoptosis were observed and the number of neurons was reduced in the above-mentioned regions. HE staining of basilar artery showed a decrease in diameter and cross-section area, and an increase in wall thickness in SAH group. Some histopathological changes of BA were observed in SAH group, including a corrugated internal elastic lamina, a shrunken endothelium, and contracted smooth muscle cells. The spasm of BA reached its peak on 5d after SAH.2 The administration of LIG （20 mg/kg） improved neurological scores, reduced brain edema and BBB permeability. LIG treatment also alleviated histopathological changes in the cortex and hippocampus. LIG treatment had no significant effect on the survival time of SAH rats.Conclusion1 The rat SAH model established by a modified rat double hemorrhage model could induce obvious secondary brain injury and CSV with a typical time course. It is a reliable model for studying mechanisms of both brain injury and CSV induced by SAH2 LIG treatment provided exact neuroprotective effects for SAH model rats via ameliorate neurological deficits, reduce brain edema and BBB permeability, and decrease the neuronal apoptosis and necrosis. PART TWO THE MECHANISMS OF NEUROPROTECTION OF Z-LIGUSTILIDE AGAINST SUBARACHNOID HEMORRHAGE IN RATSObjectiveTo investigate the possible mechanisms of neuroprotection of LIG against SAH.Methods1 Grouping: The male SD rats were randomly assigned to the following four groups: （1） Sham group, （2） SAH+vehicle group, （3） SAH+LIG5 group （5mg/kg LIG treatment）, and （4） SAH+LIG20 group （20mg/kg LIG treatment）.2 The effect of LIG treatment on CSV following SAH and the possible mechanism was investigated.（1） Morphologic changes in the basilar arteries were observed using transmission electron microscopy and light microscopy.（2） Morphometric analysis was performed to detect wall thickness and cross-section area of basilar arteries.（3） TUNEL staining was used for assessing apoptosis of the endothelial cells of BA.（4） The levels of specific apoptosis-associated protein in the BA, including p53, Bax, Bcl-2, and caspase-3, were evaluated using immunohistochemistry and Western blot analysis.3 The effect of LIG treatment on hemorrheology in the SAH model rats was evaluated.The variables of hemorrheology were measured using an automatic blood viscometer dynamic analysis apparatus.4 The effects of LIG treatment on free radical clearance and neural apoptosis in the cortex after SAH was evaluated.（1） Superoxide dismutase activity in the cerebral cortex was determined.（2） Malondialdehyde content in the cerebral cortex was determined.（3） TUNEL staining was used for assessing apoptosis of the neurons in the cortex.（4） The levels of specific apoptosis-associated protein in the cortical neurons, including p53,Bax, Bcl-2, and caspase-3, were evaluated using immunohistochemistry and Western blot analysis.Results1 LIG treatment significantly ameliorated pathological changes of BAs.2 LIG enlarged cross-section area, and decreased wall thickness of BAs after SAH.3 It also reduced TUNEL-positive endothelial cells of BAs and cerebral neurons. 4 The levels of pro-apoptotic proteins p53 and cleaved caspase-3 were down-regulated and anti-apoptotic protein Bcl-2 was up-regulated in endothelial cells of BAs and cerebral neurons by LIG treatment, whereas it had no effect on pro-apoptotic protein Bax expression.5 Administration of LIG slightly improved whole blood viscosity, whole blood reduced viscosity, erythrocyte aggregation index, e rythrocyte rigidity index, and erythrocyte deformability index, although these findings were not statistically significant.6 The activity of SOD was enhanced and the level of MDA was reduced by LIG treatment.ConclusionLIG could protect the secondary ischemic brain injury induced by SAH. The underlying mechanisms may be partly related to relieving the delay CSV following SAH by anti-endothelial apoptosis, reducing free radical in the brains, and anti-neural apoptosis.更多还原