血流动力学诱导基底动脉动脉瘤样重构及机制研究

发布时间：2018-07-12 12:31

本文选题：血流动力学 + 颅内动脉瘤　；参考：《第二军医大学》2014年博士论文

【摘要】：第一部分血流动力学诱导兔基底动脉顶端动脉瘤样重构及机制目的：颅内动脉瘤是危害性极大的脑血管疾病，但具体生成机制仍不明确。本研究通过改变血流动力学，诱导兔基底动脉顶端动脉瘤样重构并探讨可能的机制。方法：新西兰大白兔36只，分3组，假手术组12只，术后1w组12只，术后2w组12只。术前即刻及术后1w、2w，分别行TOF法MRA检查及血流动力学分析。在术后1w、2w，处死兔子，动脉灌注，解剖出基底动脉顶端，纵向切片。病理切片分别行Masson，EVG染色，分别观察平滑肌及内弹力层及抗-RAM11（巨噬细胞），抗-eNOS、抗-iNOS（内皮细胞标志物），抗-NF-κB p65、抗-TNF-α（促炎因子）免疫组化染色和抗-α-SMA（平滑肌收缩蛋白）、抗-MMP-2、抗-MMP-9（细胞外基质降解酶）免疫荧光染色。基底动脉顶端组织分别采取ELISA法检测NF-κB、TNF-α蛋白相对含量，实时定量PCR法检测CD68、eNOS、iNOS、α-SMA、MMP-2、MMP-9mRNA含量。结果：双侧颈总动脉结扎后83.3%（10/12）基底动脉顶端壁面切应力升高，诱导顶端内弹力层变薄、断裂或缺失，平滑肌层变薄弱，并瘤壁向外突出，形成动脉瘤样改变。但两实验组各有一只动物（16.7%（2/12））基底动脉顶端壁面切应力较其他动物低，且基底动脉顶端未出现动脉瘤样改变。在重构的基底动脉中，三组之间内弹力层缺失长度、中膜变薄长度及中膜薄弱百分比有显著统计学差异(P 0.001，，P0.001，P 0.001)。基底动脉顶端动脉瘤样重构区域未见巨噬细胞浸润。血流动力学改变导致血管内皮细胞功能障碍，血管保护性因子eNOS分泌减少，而具有血管毒性作用的iNOS分泌增加，三组之间eNOS mRNA、iNOS mRNA表达有统计学差异（P=0.003，P=0.019）。血流动力学及细胞因子作用导致血管平滑肌细胞表型转化，收缩型细胞减少，促炎、促基质重构型细胞增多，三组α-SMA mRNA表达有统计学差异（P 0.001）。合成型平滑肌细胞通过分泌MMPs及使TNF-α表达增高，NF-κB激活，降解细胞外基质，损伤血管壁，导致管壁变薄、向外突出，三组之间有统计学差异（P 0.001，P 0.001，P 0.001）。结论：单纯基底动脉血流动力学改变，导致基底动脉顶端壁面切应力升高，出现动脉瘤样病理重构。壁面切应力较低的动物未诱导出动脉瘤样改变。这些动脉瘤样重构不是炎症细胞引起的炎症反应，而是血流动力学改变导致血管内皮细胞功能障碍，血管保护性因子eNOS分泌减少，而血管毒性作用的iNOS分泌增加、血管平滑肌细胞表型转化，大量促炎因子分泌增加及细胞外基质降解等，一系列生物力学反应过程。第二部分血流动力学诱导兔基底动脉干血管重构及其影响目的：血管重构在血管性疾病发挥重要作用。本研究通过新西兰大白兔双侧颈总动脉结扎，观察基底动脉干血管重构及其对基底动脉顶端的影响。方法：新西兰大白兔18只，假手术组6只，实验组术后1w6只，实验组术后2w6只。术前、术后1d、1w、2w，经颅多普勒测定基底动脉血流速度。术前即刻及术后1w、2w，分别行TOF法MRA检查并血管重建及计算血流动力学。所有兔子术后1w、2w分别行脑血管造影并分别测量分析血管直径、弯曲角度、弯曲指数。每组动物分别于术后1w、2w取出基底动脉，横断面切片，Masson、EVG染色及免疫组化染色。对壁面切应力较低动物基底动脉顶端行纵形切片及HE染色。结果：双侧颈总动脉结扎后，在每个时间点基底动脉血流速度较假手术组均明显加快，两组之间血流速度有显著统计学差异(P 0.001)。基底动脉壁面切应力自汇合部开始明显上升，至中部有所下降但仍高于假手术组；但术后1w、2w组各有一只动物壁面切应力低于同组其他动物。实验组术后基底动脉中部开始弯曲，术后2w基底动脉管腔向外扩张且弯曲成角更明显；但弯曲越明显，基底动脉顶端壁面切应力越低。血管造影显示基底动脉向外扩张并弯曲成角，假手术组、实验组术后1w、2w之间基底动脉直径、弯曲角度和弯曲指数有显著统计学差异(P 0.001，P=0.008，P 0.001)。且基底动脉直径越大，壁面切应力越低。基底动脉病理切片染色显示，实验组基底动脉直径较假手术组大；实验组内弹力层锯齿状皱褶拉伸变平，部分平滑肌层迁移入内膜；实验组术后1w平滑肌层较假手术组略有增生，术后2w有所减少。HE染色显示16.7%（2/12）基底动脉中段壁面切应力较低动物基底动脉尖未见动脉瘤样重构。免疫组化染色显示三组eNOS均表达，实验组术后1w、2w增多；假手术组iNOS不表达，实验组表达增多；术后1w组MMP-2、MMP-9表达增多，术后2w时表达减少。结论：双侧颈总动脉结扎后，基底动脉血流速度加快，83.3%（10/12）壁面切应力升高，导致兔基底动脉发生病理重构，管腔扩张并弯曲成角。16.7%基底动脉扩张及弯曲显著，导致壁面切应力降低，基底动脉顶端未出现动脉瘤样重构。病理染色示内弹力层完整，锯齿状拉伸，平滑肌层变薄弱并迁移入内膜。NO信号通路及MMP-2、MMP-9参与了基底动脉血管重构。
[Abstract]:Part one
Hemodynamics induced aneurysm like remodeling in the top of the basilar artery in rabbits and its mechanism
Objective: intracranial aneurysm is a very dangerous cerebrovascular disease, but the specific mechanism is still unclear. This study induced the remodeling of the aneurysm like the apical artery of the basilar artery in rabbits and explored the possible mechanism by changing the hemodynamics.
Methods: 36 New Zealand white rabbits were divided into 3 groups, 12 sham operation groups, 12 1W after operation and 12 in group 2W after operation. MRA examination and hemodynamic analysis were performed immediately before operation and 1W, 2W after operation. After operation, 1W, 2W, rabbits were executed and arterial perfusion, and the apical and longitudinal sections of the basilar artery were dissected. Masson, EVG staining, respectively, were observed, respectively. The pathological sections were stained respectively to view the pathological sections respectively. The pathological sections were stained respectively, respectively, to observe the pathological sections, respectively. Inspection of smooth muscle and internal elastic layer and anti -RAM11 (macrophage), anti -eNOS, anti -iNOS (endothelial cell marker), anti -NF- kappa B p65, anti -TNF- alpha (pro-inflammatory factor) immuno histochemical staining and anti a -SMA (smooth muscle contractile protein), anti -MMP-2, anti -MMP-9 (extracellular matrix degrading enzyme) immunofluorescence staining. The apical tissue of basilar artery took ELISA method. The relative content of NF- kappa B and TNF- alpha protein was detected. CD68, eNOS, iNOS, alpha -SMA, MMP-2 and MMP-9mRNA contents were detected by real-time quantitative PCR.
Results: 83.3% (10/12) basilar artery apical wall shear stress increased after bilateral common carotid artery ligation. The elastic layer in the top of the basilar artery was thinner, broken or missing, the smooth muscle layer became weak, and the wall of the tumor protruded out to form an aneurysm like change. But the two experimental group had each animal (16.7% (2/12)) of the basilar artery wall shear stress compared with the other animals. There was no aneurysmal change at the top of the basilar artery. In the reconstructed basilar artery, the length of the inner elastic layer, the thinning length of the middle membrane and the weak percentage of the middle membrane were statistically significant differences (P 0.001, P0.001, P 0.001). Vascular endothelial cell dysfunction, vascular protective factor eNOS secretion decreased, and vascular toxic effect of iNOS secretion increased, between the three groups of eNOS mRNA, iNOS mRNA expression is statistically significant (P=0.003, P=0.019). Hemodynamic and cytokine effects lead to the vascular smooth muscle cell phenotype transformation, contractile type cell reduction, proinflammatory The expression of matrix remodeling type cells increased and the expression of alpha -SMA mRNA in the three group was statistically different (P 0.001). The synthetic smooth muscle cells were activated by MMPs and TNF- alpha expression, NF- kappa B was activated, the extracellular matrix was degraded and the vascular wall was damaged. The wall of the tube became thinner and outbound, and there were statistical differences between the three groups (P 0.001, P 0.001, P 0.001).
Conclusion: the changes in the basilar artery hemodynamics lead to the increase of the wall shear stress at the top of the basilar artery and the pathological remodeling of the aneurysm. The animals with lower wall shear stress do not induce the changes of the aneurysm like changes. These aneurysmal remodeling is not an inflammatory reaction caused by inflammatory cells, but the hemodynamic changes lead to the vascular endothelium. Cell dysfunction, vascular protective factor eNOS secretion decreased, and vascular toxic effect of iNOS secretion increased, vascular smooth muscle cell phenotype transformation, a large number of proinflammatory cytokines secretion increase and extracellular matrix degradation, a series of biomechanical reactions.
The second part
Hemodynamic remodeling of basilar artery in rabbits and its influence
Objective: vascular remodeling plays an important role in vascular diseases. This study was conducted by ligating bilateral common carotid artery in New Zealand white rabbits to observe the revasculature of the basilar artery and its effect on the top of the basilar artery.
Methods: 18 New Zealand white rabbits and 6 sham operation group, 1w6 only in experimental group, 2w6 only after operation in experimental group. Before operation, 1D, 1W, 2W, and transcranial Doppler were used to determine the velocity of basilar artery blood flow. Preoperative and postoperative 1W, 2W, TOF method MRA examination, vascular reconstruction and hemodynamics were performed respectively. All rabbits were performed cerebrovascular angiography and 2W after operation, respectively. The diameter of blood vessel, bending angle and bending index were measured and analyzed respectively. The basilar artery was taken out of 1W, 2W after operation, cross section section, Masson, EVG staining and immunohistochemical staining. The longitudinal section and HE staining were performed on the top of the basilar artery at the lower wall shear stress.
Results: after the ligation of bilateral common carotid artery, the velocity of basilar artery blood flow at each time point was significantly faster than that in the sham operation group, and the velocity of blood flow was significantly different between the two groups (P 0.001). The wall shear stress of the basilar artery began to rise obviously from the confluence part, to the middle of the middle, but still higher than that in the sham operation group; but after the operation, the group of 1W and 2W had each group. The wall shear stress of one animal was lower than that of the other animals. The central basilar artery in the experimental group began to bend after the operation, and the basilar artery cavity expanded outward and the curved angle was more obvious after the operation, but the more obvious the bending, the lower the wall shear stress of the basilar artery was lower. The angiography showed that the basal artery of the basilar artery was dilated and curved into the angle, the sham operation group, the experiment showed the 2W. The basilar artery diameter, bending angle and bending index between 1W and 2W after operation were significantly different (P 0.001, P=0.008, P 0.001). The larger the diameter of the basilar artery, the lower the wall shear stress. The basilar artery diameter in the basilar artery showed that the diameter of the basilar artery in the experimental group was larger than that in the artificial hand, and the stretch of the serrated fold in the elastic layer in the experimental group was leveled. In the experimental group, the 1W smooth muscle layer was slightly proliferated in the experimental group than the sham operation group. After the operation, the 2W decreased by.HE staining and showed 16.7% (2/12) the middle basilar artery wall shear stress in the middle basilar artery was lower than that of the basilar artery. The immunohistochemical staining showed that all the three groups of eNOS were expressed, and the 1W and 2W increased after operation in the experimental group. In the sham operation group, iNOS was not expressed, and the expression of the experimental group increased. After operation, the expression of MMP-2 and MMP-9 in group 1W increased, and the expression of 2W decreased after operation.
Conclusion: after the ligation of bilateral common carotid artery, the velocity of the basilar artery is accelerated and the wall shear stress of 83.3% (10/12) increases, causing pathological remodeling of the basilar artery in the rabbit, dilation of the basilar artery and the dilatation and bending of the.16.7% basilar artery, which leads to the decrease of the wall shear stress and no aneurysm like reconstruction at the top of the basilar artery. Pathological staining shows that the artery of basilar artery is not reconstructed. The inner elastic layer is complete, zigzag stretch, smooth muscle layer becomes weak and migration into the intimal.NO signaling pathway and MMP-2, MMP-9 participates in basilar artery remodeling.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743

【共引文献】