线粒体途径介导的细胞凋亡在颅内动脉瘤生成中的作用机制研究
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摘要
第一部分：单纯血流动力学诱导的兔基底动脉尖部动脉瘤生成模型建立
目的：建立稳定的单纯血流动力学诱导的兔基底动脉尖部动脉瘤生成模型。方法：12只新西兰大白兔随机分为2组，假手术组（6只）仅暴露双侧颈动脉而未结扎，术后2天组（6只）则结扎双侧颈动脉。采用TCD、MRA血管重建及计算机流体动力学获得各组动物术前及术后2天的基底动脉平均血流速度、基底动脉直径及基底动脉分叉部各部分平均壁面切应力等数值。所有动物术后2天处死获取基底动脉分叉部组织，观察两组之间基底动脉尖部内弹力层损害、中层薄弱及血管腔外凸样改变等组织病理变化情况。
结果：在术后2天组内动物在术后2天时基底动脉尖部平均壁面切应力明显高于术前（P=.000）；基底动脉直径术后2天时较术前增加，但无统计学差别（P=.311）；基底动脉平均血流速度术后2天时较术前明显提高（P=.003）。而在假手术组内术后2天时与术前的上述指标相比均无统计学差异（P＞.05）。在两组各自组内动物术前及术后2天时其各自的基底动脉尖部平均壁面切应力均明显高于基底动脉干及左侧、右侧大脑后动脉（P＜.01）。两组所有动物基底动脉尖部平均壁面切应力与其对应的基底动脉平均血流速度呈正相关（R2=0.70, P <0.0001）。假手术组动物未发现基底动脉尖部的内弹力层损害、中层薄弱及血管腔外凸样改变；术后2天组所有动物基底动脉尖部即壁面切应力（WSS）最高区域均出现内弹力层损害，而部分动物（2/6）出现中层薄弱及血管腔外凸样改变。
结论：兔双侧颈总动脉结扎术后2天其最大壁面切应力位于基底动脉尖部，且基底动脉尖部平均壁面切应力较术前明显提高，与其对应的基底动脉平均血流速度呈正相关；所有动物结扎术后2天基底动脉尖部均有内弹力层损害，部分可见到中层薄弱和血管腔外凸等血管破坏性重塑形改变。稳定的单纯血流动力学诱导的兔基底动脉尖部动脉瘤生成模型成功建立。通过该模型，可以进一步研究单纯血流动力学诱导颅内动脉瘤生成的分子机制。
第二部分：线粒体途径介导的细胞凋亡在单纯血流动力学诱导的兔基底动脉尖部动脉瘤生成过程中的作用机制研究
目的：在采用双侧颈总动脉结扎的家兔基底动脉尖动脉瘤生成模型基础上，研究细胞凋亡参与颅内动脉瘤生成的分子机制。
方法：新西兰大白兔15只，分为3组，假手术组6只（其中3只用于实时荧光定量PCR分析），双侧颈动脉结扎术后2天组3只，双侧颈动脉结扎术后7天组6只(其中3只用于实时荧光定量PCR分析），获取动物基底动脉分叉部组织，观察其内弹力层损害、中层薄弱及血管腔外凸样改变等组织病理变化情况，TUNEL法检测凋亡细胞，免疫组化染色及定量分析炎症细胞分布及凋亡相关蛋白表达情况，实时荧光定量PCR方法检测凋亡相关蛋白mRNA表达情况。
结果：假手术组动物均未见内弹力层损害及凋亡细胞，而术后2天及7天组动物均发现凋亡细胞且主要位于基底动脉尖部即内弹力层损害部位，且该部位凋亡细胞数量均明显高于基底动脉干、左侧及右侧大脑后动脉部位，有统计学差异（p＜0.05）。所有动物血管壁内皮层基本保持完整，而炎症细胞在血管壁上分布少而散在，且位于外膜层，各组基底动脉尖部即内弹力层损害部位与其它部位相比，炎症细胞分布均无统计学差异（P＞0.05）。术后7天组动物基底动脉尖部caspase-9及caspase-3mRNA表达较假手术组均明显增高，有统计学差异（P＜0.01）；而假手术组与术后7天组动物基底动脉尖部caspase-8蛋白及mRNA表达比较均无统计学差异（P＞0.05）。在术后2天及7天组各种凋亡相关蛋白（caspase-3、caspase-9、Bcl-2、phospho-Bad、Bax、Bid）在基底动脉尖部表达均明显高于基底动脉干部、左侧及右侧大脑后动脉，有统计学差异（P＜0.05）。在基底动脉尖部的caspase-3、caspase-9、Bcl-2、Bax、Bid的表达均在术后7天组最高，术后2天组次之，假手术组最低，有统计学差异（P＜0.05）；而Phospho-Bad的表达在术后2天组最高，术后7天组次之，假手术组最低，有统计学差异（P＜0.05）。
结论：单纯血流动力学诱导的基底动脉尖部动脉瘤早期生成过程中，血管壁的破坏性重塑形改变并不是由炎症细胞浸润导致，而是来源于管壁本身固有细胞的作用。细胞凋亡参与了单纯血流动力学诱导的基底动脉尖部动脉瘤的早期生成，其分子发生机制是通过caspase-9激活的由Bcl-2介导的线粒体途径。
第三部分：Ac-DEVD-CHO对兔基底动脉尖部动脉瘤生成的影响
目的：选择caspase-3抑制剂Ac-DEVD-CHO作用于单纯血流动力学诱导的基底动脉尖部动脉瘤生成模型，验证细胞凋亡在动脉瘤发生中的作用，确认细胞凋亡是否是颅内动脉瘤发生主要机制。
方法：新西兰大白兔30只，随机分为假手术组、术后2天组、术后DMSO2天组、术后抑制剂2天组、术后7天组、术后DMSO7天组、术后抑制剂7天组、术后1月组、术后DMSO1月组、术后抑制剂1月组，每组3只。观察双侧颈动脉结扎术后半小时应用caspase-3抑制剂Ac-DEVD-CHO能否抑制兔基底动脉尖部caspase-3蛋白表达及细胞凋亡，能否阻断或延缓术后动物基底动脉尖部的组织病理变化进程（采用内弹力层损害长度、中层薄弱长度及程度、血管腔外凸改变长度及动脉瘤发展评分进行评估)。
结果：术后2天组及术后DMSO2天组均发现凋亡细胞主要位于基底动脉尖部，而术后抑制2天组基底动脉尖部均未见凋亡细胞，且术后抑制2天组基底动脉尖部caspase-3蛋白表达较术后2天组及术后DMSO2天组明显降低（P＜0.05）。应用caspase-3抑制剂、DMSO与未应用caspase-3抑制剂时，双侧颈动脉结扎术后动物基底动脉尖部组织病理变化有同样的随时间变化趋势；且在相同时间点应用caspase-3抑制剂、DMSO与未应用caspase-3抑制剂三种情况相比基底动脉尖部组织病理变化均无统计学差异（P＞0.05）。双侧颈动脉结扎术后1月内随着时间延长动脉瘤发展评分（ADS）逐步升高，有统计学差异（P＜0.05）。
结论：Caspase-3抑制剂Ac-DEVD-CHO后虽然能抑制双侧颈动脉结扎术后基底动脉尖部的细胞凋亡，但其破坏性重塑形改变进程及动脉瘤生成并未被抑制。在颅内动脉瘤生成早期细胞凋亡可能仅仅是对血流动力学改变的一个反应结果，它不是引起基底动脉尖部血管壁的早期破坏性重塑形进程的主要因素，因此细胞凋亡可能并不是单纯血流动力学诱导的颅内动脉瘤生成的主要机制。动脉瘤发展评分（ADS）是实验性颅内动脉瘤发展程度评估的有效方法。
Part one：Establishment of a stable aneurysm formation model caused solely byhemodynamics at the basilar terminus of rabbit.
Objective: To establish a stable aneurysm formation model caused solely byhemodynamics at the basilar terminus of rabbit.
Methods: Twelve New Zealand white rabbits were randomly divided into2groups: shamoperation (6animals) and operation group (6animals). The rabbits’ bilateral carotidarteries were exposed but not ligated in the sham operation group, while exposed andligated in the operation group. The data before and two days after operation, such as meanblood flow velocity, diameter and mean wall shear stress, were got from the animals byTCD, MRA vascular reconstruction and computational fluid dynamics. Basilar terminiwere harvested2days after the operation. The histopathological changes, such as internalelastic lamina (IEL) loss, media thinning and bulging of the vessel wall at the basilarterminus, were observed between the two groups.
Results: In the operation group, the mean wall shear stress of the basilar terminus2daysafter operation was significantly increased (P=.000) than that before the operation, whilethe basal artery diameter increased without statistical differences (P=.311) when comparedto that before the operation. Also a significant increase (P=.003) in mean blood flowvelocity of the basal artery was observed. In the sham operation group, there was nostatistical difference (P>.05) in mean wall shear stress of the basilar terminus, diameter andmean blood flow velocity of the basal artery while compared with those before theoperation. All animals’ mean wall shear stress of the basilar terminus was significantlyhigher (P<.01) than that of Basilar artery stem, left or right posterior cerebral artery in eachgroup, while the mean wall shear stress of the basilar terminus and mean blood flowvelocity of the basal artery has a positive correlation (R2=0.70, P <0.0001) in all animalsincluding the operation group and the sham operation group. In addition, the animalshasn’t internal elastic lamina damage or media thinning and bulging of the vessel wall inthe sham operation group, while in in the operation group the basilar terminus of allanimals, which was the region that experiences the highest WSS, appeared internal elasticlamina damage, and that of some animals (2/6) had media thinning and bulging of thevessel wall.
Conclusion: The mean wall shear stress of the basilar terminus, which appeared positivecorrelation with mean blood flow velocity of the basal artery, increased significantly twodays after ligation of bilateral carotid artery when compared with that before operation inthe rabbits. Also, the basilar termini of all animals appeared internal elastic lamina damageafter ligation, and that of some animals had media thinning and bulging of the vessel wall.We have succeeded in establishing a stable aneurysm formation model at the basilarterminus of rabbit which caused solely by hemodynamics. Based on the model we couldstudy the molecular mechanisms of intracranial aneurysms formation caused solely byhemodynamics.
Part two：Mitochondrial Pathway-Mediated Apoptosis during Intracranial AneurysmInitiation caused solely by hemodynamics at the basilar terminus of rabbit.
Objective: To study the mechanism of apoptosis during intracranial aneurysm initiation inrabbits, whose bilateral carotid artery were ligated.
Methods: Fifteen New Zealand white rabbits were divided into3groups: sham operationgroup (6animals,3animals for real time PCR),2days after operation group (3animals)and7days after operation group (6animals,3animals for real time PCR). Basilar arterialbifurcation tissues were harvested to analyze the histopathological changes, such asinternal elastic lamina damage, media thinning and bulging of the vessel wall. Apoptoticcells were detected by TUNEL staining. The distribution of inflammatory cells andexpression of apoptosis-related protein were analyzed by immunohistochemical staining,whlie the real-time quantitative PCR was used to detect the expression of apoptosis-relatedprotein mRNA.
Results: In the sham operation group there wasn’t internal elastic lamina damage orapoptotic cells, while in the other groups apoptotic cells were mainly detected in the basilarterminus, where the internal elastic lamina damage appeared. There were more apoptoticcells at the basilar terminus than basilar artery stem, left or right posterior cerebral artery,and there was statistical difference (P<0.05). All animals’ vessel wall intima basicallyremained intact, and the distribution of inflammatory cells in the adventitia of vessel wallwas little and scattered. The distribution of inflammatory cells at basilar terminus hadn’tsignificantly statistical difference (P>0.05) compared with other parts. In the7days afteroperation group mRNA expression of caspase-3and caspase-9at the basilar terminus wassignificantly higher than that in the sham operation group (P<0.01). The expression ofcaspase-8protein and its mRNA in the seven days after operation group were notstatistically different (P>0.05) compared with the sham operation group. In2days and7days after operation groups various apoptosis-related proteins (caspase-3, caspase-9, Bcl-2,phospho-Bad, Bax, Bid) expression at the basilar terminus were significantly higher thanthe basilar artery stem, the left or right posterior cerebral artery (P <0.05). At the basilarterminus the expression of caspase-3, caspase-9, Bcl-2, Bax, Bid was the highest in sevendays after operation group,2days group followed and the sham operation group was thelowest. There was a statistical difference (P <0.05) among the three groups. Phospho-Badexpression in two days group was the highest,7days group followed, the sham operation group was the lowest, there was statistical difference (P <0.05) among the three groups.
Conclusion: During the early process of basilar terminus aneurysm formation caused solel-y by hemodynamics, the destructive remodeling of the vascular wall arises from intrinsicmural cells, rather than through inflammatory cell infiltration. Apoptosis was involved inthe early process of basilar terminus aneurysm formation caused solely by hemodynamics,activation of which is mediated predominantly by the Bcl-2-mediated intrinsic pathwaythrough the activation of caspase-9.
Part three：Effects of Ac-DEVD-CHO to basilar terminus aneurysm formation at inrabbit
Objective: Caspase-3inhibitor Ac-DEVD-CHO was chose to role in the basilar terminusaneurysm formation model caused solely by hemodynamic, which was to verify the role ofapoptosis in the initiation of intracranial aneurysm and confirm whether apoptosis is themain mechanism during the intracranial aneurysms formation.
Methods: Thirty New Zealand white rabbits were randomly divided into10groups: shamoperation group,2days after operation group, DMSO group2days after operation,Ac-DEVD-CHO group2days after operation,7days after operation group, DMSO group7days after operation, Ac-DEVD-CHO group7days after operation,30days afteroperation group, DMSO group30days after operation, Ac-DEVD-CHO group30daysafter operation, and there were3animals in each group. With application ofAc-DEVD-CHO half hour after operation we studied whether it could inhibit caspase-3protein expression and apoptosis and whether Ac-DEVD-CHO could block or delay theprocess of histopathological changes at the basilar terminus which were assessed bycomputing the length of internal elastic lamina loss, media thinning and bulging of thevessel wall and aneurysm development score.
Results: In the2days after operation group and DMSO group2days after operation,apoptotic cells were mainly detected at the basilar terminus. While in the Ac-DEVD-CHOgroup2days after operation apoptotic cells were not detected at the basilar terminus, andthe expression of caspase-3was significantly lower than that in the2days after operationgroup and DMSO group2days after operation (P<0.05). With application ofAc-DEVD-CHO, DMSO and without application of Ac-DEVD-CHO, pathologicalchanges of basilar terminus after bilateral carotid artery ligation had the same trend overtime and had not statistical difference (P>0.05) at the same time among the threeconditions. The aneurysm development score increased gradually with time during30daysafter bilateral carotid artery ligation, and there was a statistically difference (P <0.05).
Conclusion: Caspase-3inhibitor Ac-DEVD-CHO could inhibit apoptosis at the basilarterminus after bilateral carotid artery ligation, but process of its destructive remodeling andaneurysm formation had not been suppressed. Apoptosis might be just a reaction tohemodynamic changes during the early stage of intracranial aneurysm formation, andcould not cause the early process of vessel wall destructive remodeling at the basilar terminus, so it might be not the main mechanism in intracranial aneurysms formationcaused solely by hemodynamic. Aneurysm development score is an effective way to assessthe development degree of experimental intracranial aneurysms.
目的：建立稳定的单纯血流动力学诱导的兔基底动脉尖部动脉瘤生成模型。方法：12只新西兰大白兔随机分为2组，假手术组（6只）仅暴露双侧颈动脉而未结扎，术后2天组（6只）则结扎双侧颈动脉。采用TCD、MRA血管重建及计算机流体动力学获得各组动物术前及术后2天的基底动脉平均血流速度、基底动脉直径及基底动脉分叉部各部分平均壁面切应力等数值。所有动物术后2天处死获取基底动脉分叉部组织，观察两组之间基底动脉尖部内弹力层损害、中层薄弱及血管腔外凸样改变等组织病理变化情况。
结果：在术后2天组内动物在术后2天时基底动脉尖部平均壁面切应力明显高于术前（P=.000）；基底动脉直径术后2天时较术前增加，但无统计学差别（P=.311）；基底动脉平均血流速度术后2天时较术前明显提高（P=.003）。而在假手术组内术后2天时与术前的上述指标相比均无统计学差异（P＞.05）。在两组各自组内动物术前及术后2天时其各自的基底动脉尖部平均壁面切应力均明显高于基底动脉干及左侧、右侧大脑后动脉（P＜.01）。两组所有动物基底动脉尖部平均壁面切应力与其对应的基底动脉平均血流速度呈正相关（R2=0.70, P <0.0001）。假手术组动物未发现基底动脉尖部的内弹力层损害、中层薄弱及血管腔外凸样改变；术后2天组所有动物基底动脉尖部即壁面切应力（WSS）最高区域均出现内弹力层损害，而部分动物（2/6）出现中层薄弱及血管腔外凸样改变。
结论：兔双侧颈总动脉结扎术后2天其最大壁面切应力位于基底动脉尖部，且基底动脉尖部平均壁面切应力较术前明显提高，与其对应的基底动脉平均血流速度呈正相关；所有动物结扎术后2天基底动脉尖部均有内弹力层损害，部分可见到中层薄弱和血管腔外凸等血管破坏性重塑形改变。稳定的单纯血流动力学诱导的兔基底动脉尖部动脉瘤生成模型成功建立。通过该模型，可以进一步研究单纯血流动力学诱导颅内动脉瘤生成的分子机制。
第二部分：线粒体途径介导的细胞凋亡在单纯血流动力学诱导的兔基底动脉尖部动脉瘤生成过程中的作用机制研究
目的：在采用双侧颈总动脉结扎的家兔基底动脉尖动脉瘤生成模型基础上，研究细胞凋亡参与颅内动脉瘤生成的分子机制。
方法：新西兰大白兔15只，分为3组，假手术组6只（其中3只用于实时荧光定量PCR分析），双侧颈动脉结扎术后2天组3只，双侧颈动脉结扎术后7天组6只(其中3只用于实时荧光定量PCR分析），获取动物基底动脉分叉部组织，观察其内弹力层损害、中层薄弱及血管腔外凸样改变等组织病理变化情况，TUNEL法检测凋亡细胞，免疫组化染色及定量分析炎症细胞分布及凋亡相关蛋白表达情况，实时荧光定量PCR方法检测凋亡相关蛋白mRNA表达情况。
结果：假手术组动物均未见内弹力层损害及凋亡细胞，而术后2天及7天组动物均发现凋亡细胞且主要位于基底动脉尖部即内弹力层损害部位，且该部位凋亡细胞数量均明显高于基底动脉干、左侧及右侧大脑后动脉部位，有统计学差异（p＜0.05）。所有动物血管壁内皮层基本保持完整，而炎症细胞在血管壁上分布少而散在，且位于外膜层，各组基底动脉尖部即内弹力层损害部位与其它部位相比，炎症细胞分布均无统计学差异（P＞0.05）。术后7天组动物基底动脉尖部caspase-9及caspase-3mRNA表达较假手术组均明显增高，有统计学差异（P＜0.01）；而假手术组与术后7天组动物基底动脉尖部caspase-8蛋白及mRNA表达比较均无统计学差异（P＞0.05）。在术后2天及7天组各种凋亡相关蛋白（caspase-3、caspase-9、Bcl-2、phospho-Bad、Bax、Bid）在基底动脉尖部表达均明显高于基底动脉干部、左侧及右侧大脑后动脉，有统计学差异（P＜0.05）。在基底动脉尖部的caspase-3、caspase-9、Bcl-2、Bax、Bid的表达均在术后7天组最高，术后2天组次之，假手术组最低，有统计学差异（P＜0.05）；而Phospho-Bad的表达在术后2天组最高，术后7天组次之，假手术组最低，有统计学差异（P＜0.05）。
结论：单纯血流动力学诱导的基底动脉尖部动脉瘤早期生成过程中，血管壁的破坏性重塑形改变并不是由炎症细胞浸润导致，而是来源于管壁本身固有细胞的作用。细胞凋亡参与了单纯血流动力学诱导的基底动脉尖部动脉瘤的早期生成，其分子发生机制是通过caspase-9激活的由Bcl-2介导的线粒体途径。
第三部分：Ac-DEVD-CHO对兔基底动脉尖部动脉瘤生成的影响
目的：选择caspase-3抑制剂Ac-DEVD-CHO作用于单纯血流动力学诱导的基底动脉尖部动脉瘤生成模型，验证细胞凋亡在动脉瘤发生中的作用，确认细胞凋亡是否是颅内动脉瘤发生主要机制。
方法：新西兰大白兔30只，随机分为假手术组、术后2天组、术后DMSO2天组、术后抑制剂2天组、术后7天组、术后DMSO7天组、术后抑制剂7天组、术后1月组、术后DMSO1月组、术后抑制剂1月组，每组3只。观察双侧颈动脉结扎术后半小时应用caspase-3抑制剂Ac-DEVD-CHO能否抑制兔基底动脉尖部caspase-3蛋白表达及细胞凋亡，能否阻断或延缓术后动物基底动脉尖部的组织病理变化进程（采用内弹力层损害长度、中层薄弱长度及程度、血管腔外凸改变长度及动脉瘤发展评分进行评估)。
结果：术后2天组及术后DMSO2天组均发现凋亡细胞主要位于基底动脉尖部，而术后抑制2天组基底动脉尖部均未见凋亡细胞，且术后抑制2天组基底动脉尖部caspase-3蛋白表达较术后2天组及术后DMSO2天组明显降低（P＜0.05）。应用caspase-3抑制剂、DMSO与未应用caspase-3抑制剂时，双侧颈动脉结扎术后动物基底动脉尖部组织病理变化有同样的随时间变化趋势；且在相同时间点应用caspase-3抑制剂、DMSO与未应用caspase-3抑制剂三种情况相比基底动脉尖部组织病理变化均无统计学差异（P＞0.05）。双侧颈动脉结扎术后1月内随着时间延长动脉瘤发展评分（ADS）逐步升高，有统计学差异（P＜0.05）。
结论：Caspase-3抑制剂Ac-DEVD-CHO后虽然能抑制双侧颈动脉结扎术后基底动脉尖部的细胞凋亡，但其破坏性重塑形改变进程及动脉瘤生成并未被抑制。在颅内动脉瘤生成早期细胞凋亡可能仅仅是对血流动力学改变的一个反应结果，它不是引起基底动脉尖部血管壁的早期破坏性重塑形进程的主要因素，因此细胞凋亡可能并不是单纯血流动力学诱导的颅内动脉瘤生成的主要机制。动脉瘤发展评分（ADS）是实验性颅内动脉瘤发展程度评估的有效方法。
Part one：Establishment of a stable aneurysm formation model caused solely byhemodynamics at the basilar terminus of rabbit.
Objective: To establish a stable aneurysm formation model caused solely byhemodynamics at the basilar terminus of rabbit.
Methods: Twelve New Zealand white rabbits were randomly divided into2groups: shamoperation (6animals) and operation group (6animals). The rabbits’ bilateral carotidarteries were exposed but not ligated in the sham operation group, while exposed andligated in the operation group. The data before and two days after operation, such as meanblood flow velocity, diameter and mean wall shear stress, were got from the animals byTCD, MRA vascular reconstruction and computational fluid dynamics. Basilar terminiwere harvested2days after the operation. The histopathological changes, such as internalelastic lamina (IEL) loss, media thinning and bulging of the vessel wall at the basilarterminus, were observed between the two groups.
Results: In the operation group, the mean wall shear stress of the basilar terminus2daysafter operation was significantly increased (P=.000) than that before the operation, whilethe basal artery diameter increased without statistical differences (P=.311) when comparedto that before the operation. Also a significant increase (P=.003) in mean blood flowvelocity of the basal artery was observed. In the sham operation group, there was nostatistical difference (P>.05) in mean wall shear stress of the basilar terminus, diameter andmean blood flow velocity of the basal artery while compared with those before theoperation. All animals’ mean wall shear stress of the basilar terminus was significantlyhigher (P<.01) than that of Basilar artery stem, left or right posterior cerebral artery in eachgroup, while the mean wall shear stress of the basilar terminus and mean blood flowvelocity of the basal artery has a positive correlation (R2=0.70, P <0.0001) in all animalsincluding the operation group and the sham operation group. In addition, the animalshasn’t internal elastic lamina damage or media thinning and bulging of the vessel wall inthe sham operation group, while in in the operation group the basilar terminus of allanimals, which was the region that experiences the highest WSS, appeared internal elasticlamina damage, and that of some animals (2/6) had media thinning and bulging of thevessel wall.
Conclusion: The mean wall shear stress of the basilar terminus, which appeared positivecorrelation with mean blood flow velocity of the basal artery, increased significantly twodays after ligation of bilateral carotid artery when compared with that before operation inthe rabbits. Also, the basilar termini of all animals appeared internal elastic lamina damageafter ligation, and that of some animals had media thinning and bulging of the vessel wall.We have succeeded in establishing a stable aneurysm formation model at the basilarterminus of rabbit which caused solely by hemodynamics. Based on the model we couldstudy the molecular mechanisms of intracranial aneurysms formation caused solely byhemodynamics.
Part two：Mitochondrial Pathway-Mediated Apoptosis during Intracranial AneurysmInitiation caused solely by hemodynamics at the basilar terminus of rabbit.
Objective: To study the mechanism of apoptosis during intracranial aneurysm initiation inrabbits, whose bilateral carotid artery were ligated.
Methods: Fifteen New Zealand white rabbits were divided into3groups: sham operationgroup (6animals,3animals for real time PCR),2days after operation group (3animals)and7days after operation group (6animals,3animals for real time PCR). Basilar arterialbifurcation tissues were harvested to analyze the histopathological changes, such asinternal elastic lamina damage, media thinning and bulging of the vessel wall. Apoptoticcells were detected by TUNEL staining. The distribution of inflammatory cells andexpression of apoptosis-related protein were analyzed by immunohistochemical staining,whlie the real-time quantitative PCR was used to detect the expression of apoptosis-relatedprotein mRNA.
Results: In the sham operation group there wasn’t internal elastic lamina damage orapoptotic cells, while in the other groups apoptotic cells were mainly detected in the basilarterminus, where the internal elastic lamina damage appeared. There were more apoptoticcells at the basilar terminus than basilar artery stem, left or right posterior cerebral artery,and there was statistical difference (P<0.05). All animals’ vessel wall intima basicallyremained intact, and the distribution of inflammatory cells in the adventitia of vessel wallwas little and scattered. The distribution of inflammatory cells at basilar terminus hadn’tsignificantly statistical difference (P>0.05) compared with other parts. In the7days afteroperation group mRNA expression of caspase-3and caspase-9at the basilar terminus wassignificantly higher than that in the sham operation group (P<0.01). The expression ofcaspase-8protein and its mRNA in the seven days after operation group were notstatistically different (P>0.05) compared with the sham operation group. In2days and7days after operation groups various apoptosis-related proteins (caspase-3, caspase-9, Bcl-2,phospho-Bad, Bax, Bid) expression at the basilar terminus were significantly higher thanthe basilar artery stem, the left or right posterior cerebral artery (P <0.05). At the basilarterminus the expression of caspase-3, caspase-9, Bcl-2, Bax, Bid was the highest in sevendays after operation group,2days group followed and the sham operation group was thelowest. There was a statistical difference (P <0.05) among the three groups. Phospho-Badexpression in two days group was the highest,7days group followed, the sham operation group was the lowest, there was statistical difference (P <0.05) among the three groups.
Conclusion: During the early process of basilar terminus aneurysm formation caused solel-y by hemodynamics, the destructive remodeling of the vascular wall arises from intrinsicmural cells, rather than through inflammatory cell infiltration. Apoptosis was involved inthe early process of basilar terminus aneurysm formation caused solely by hemodynamics,activation of which is mediated predominantly by the Bcl-2-mediated intrinsic pathwaythrough the activation of caspase-9.
Part three：Effects of Ac-DEVD-CHO to basilar terminus aneurysm formation at inrabbit
Objective: Caspase-3inhibitor Ac-DEVD-CHO was chose to role in the basilar terminusaneurysm formation model caused solely by hemodynamic, which was to verify the role ofapoptosis in the initiation of intracranial aneurysm and confirm whether apoptosis is themain mechanism during the intracranial aneurysms formation.
Methods: Thirty New Zealand white rabbits were randomly divided into10groups: shamoperation group,2days after operation group, DMSO group2days after operation,Ac-DEVD-CHO group2days after operation,7days after operation group, DMSO group7days after operation, Ac-DEVD-CHO group7days after operation,30days afteroperation group, DMSO group30days after operation, Ac-DEVD-CHO group30daysafter operation, and there were3animals in each group. With application ofAc-DEVD-CHO half hour after operation we studied whether it could inhibit caspase-3protein expression and apoptosis and whether Ac-DEVD-CHO could block or delay theprocess of histopathological changes at the basilar terminus which were assessed bycomputing the length of internal elastic lamina loss, media thinning and bulging of thevessel wall and aneurysm development score.
Results: In the2days after operation group and DMSO group2days after operation,apoptotic cells were mainly detected at the basilar terminus. While in the Ac-DEVD-CHOgroup2days after operation apoptotic cells were not detected at the basilar terminus, andthe expression of caspase-3was significantly lower than that in the2days after operationgroup and DMSO group2days after operation (P<0.05). With application ofAc-DEVD-CHO, DMSO and without application of Ac-DEVD-CHO, pathologicalchanges of basilar terminus after bilateral carotid artery ligation had the same trend overtime and had not statistical difference (P>0.05) at the same time among the threeconditions. The aneurysm development score increased gradually with time during30daysafter bilateral carotid artery ligation, and there was a statistically difference (P <0.05).
Conclusion: Caspase-3inhibitor Ac-DEVD-CHO could inhibit apoptosis at the basilarterminus after bilateral carotid artery ligation, but process of its destructive remodeling andaneurysm formation had not been suppressed. Apoptosis might be just a reaction tohemodynamic changes during the early stage of intracranial aneurysm formation, andcould not cause the early process of vessel wall destructive remodeling at the basilar terminus, so it might be not the main mechanism in intracranial aneurysms formationcaused solely by hemodynamic. Aneurysm development score is an effective way to assessthe development degree of experimental intracranial aneurysms.
引文
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[5]. Kondo S, Hashimoto N, Kikuchi H, et al. Apoptosis of medial smooth muscle cells in thedevelopment of saccular cerebral aneurysms in rats. Stroke,1998,29:18l-189.
[6]. Tada Y, Kitazato KT, Yagi K, Shimada K, Matsushita N, Kinouchi T, et al. Statins promote thegrowth of experimentally induced cerebral aneurysms in estrogen-deficient rats. Stroke,2011,42:2286-2293.
[7]. Wong LC, Langille BL. Developmental remodeling of the internal elastic lamina of rabbitarteries: effect of blood flow. Circ Res,1996,78:799–805.
[8]. Hazama F, Kataoka H, Yamada E, Kayembe K, Hashimoto N, Kojima M, et al. Early changes ofexperimentally induced cerebral aneurysms in rats.Light-microscopic study.Am J Pathol,1986,124:399–404.
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