Mitochondrial Pathway-mediated Apoptosis During Intracranial Aneurysm Initiation

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 inrabbitObjective: 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.