缺血性脑卒中微血管网络重塑的同步辐射三维成像实验研究

发布时间：2019-05-22 07:40

【摘要】：目的脑生理功能的维持依赖于微血管网络结构的完整。缺血性脑卒中后,机体启动瀑布式级联反应致微血管网络稳态失调,神经血管单元发生结构与功能重塑。脑缺血可引发自体血管新生,然而其发生机制、发展进程以及具体形式等目前尚不明确。传统的血管可视化研究手段如组织病理学方法等,对微血管空间结构的展示尚停留在二维平面层次,无法精确展示血管网络的时空分布。常规影像学手段如MRA、 CTA、DSA等,由于有限的时间空间分辨率,尚无法探测直径在50μm以下的微小血管。新兴的同步辐射技术突破传统光学成像方法的衍射极限,凭借亚微米至微米量级的极高分辨率能够清晰实现对物质显微结构的无损、三维成像。本研究应用上海光源同步辐射相位衬度成像进行正常和缺血性脑卒中模型的血管成像研究,明确脑微血管高分辨、三维成像的可行性和有效性,探索脑缺血后血管网络重塑的动态发展过程,力求以新的视角为缺血性卒中血管损伤与修复的研究提供丰富的3D影像学信息和形态计量学依据。方法调整成像参数,使用滤波反算法及成像软件多角度多平面重建正常大鼠全脑血管3D视图。在此基础上,对血管网络进行基于结构信息学的量化分析。同时体式显微镜、HE染色检测的脑组织样品进行比较。随后围绕大鼠缺血性脑卒中模型,利用同步辐射相位衬度成像观察缺血后不同时点自发性血管新生动态发展过程,探索定量分析血管新生的方法,同时结合免疫组织化学技术,从3D/2D可视化角度综合评估缺血性脑卒中的血管网络重塑进程。结果 1.利用同步辐射相位衬度成像建立了从投影图、二维slice到全脑血管网络3D图像的完整可视化体系。使用5.9μmCCD探测器,可分辨的最小血管直径约为9.4μm,接近毛细血管水平。二维平片与HE染色比较,无需造影剂和繁琐切片制备程序,即可清晰显示海马区域的血管走行。脑体表面的3D渲染图像和Microfil血管铸型剂灌注后体式显微镜原位摄片结果高度一致,软脑膜的多级血管吻合支均能清楚辨析。 2.进一步建立基于空间维度变换的全脑水平位、冠状位、矢状位虚拟切片和数字化3D脑血管断层视图,并对其功能区的血供配布进行解析,从2D、3D视角构建出一系列数字化的脑血管3D解剖图集。 3.血管网络骨骼提取结果显示,大鼠全脑血管网络主要是在20μm以下的微小血管骨架基础上构建而成的。基于3D网络的形态计量学分析,进一步明确了正常大鼠脑血管容积、血管密度、血管分支数、节点数、相邻血管间距、直径分布频数等各项生理参考指标。 4.此后围绕大鼠缺血性脑卒中模型的一系列同步辐射光源2D、3D成像研究结果显示：缺血后2小时、4小时、6小时微血管数目持续增高,密集分布在缺血区周缘,由缺血半暗带向梗死区延伸包绕。然而随着缺血时间的延长,缺血1天、3天后新生血管的分布范围和密度均有所减小,微血管形态扭曲,形成不规则血管襻。缺血7天梗死灶液化成蜂窝状囊腔,胶质瘢痕已逐渐形成,缺血18天病灶侧脑体积明显减小,脑室受到挤压,皮质和纹状体梗死灶发生机化,形成高密度的瘢痕组织和无实质组织的“中风囊腔”。 5.定量分析研究显示：随着缺血时间的持续增加,病灶侧脑体积自3天后逐渐减小,其中缺血后18天病灶侧脑体积较对照组显著缩小(P0.05)。缺血后2小时、4小时、6小时血管密度均较对照组升高,差异有统计学意义(P0.05),而缺血后7天、18天血管密度逐渐降低(P0.05)。缺血后2小时、4小时、6小时血管分支数、节点数目较对照组显著升高(P0.05),随着时间的推移,缺血后3天、7天、18天血管分支、节点数目逐渐降低(P0.05)。缺血后2小时、4小时、6小时10-20μm区段的微小血管和对照组相比,频数明显增多,差异具有统计学意义(P0.05)。随着时间的推移,频数逐渐下降,缺血7天、18天频数显著减少(P0.05)。缺血后2小时、4小时、6小时、1天、3天的血管迂曲度明显增高(P0.05)。 6.CD31免疫组化结果缺血后2小时、4小时、6小时纹状体CD31阳性染色微血管持续增多,密集排布缺血边缘区域,血管呈点状、弯曲短状或圆管状,形态不一。随着时间推移,缺血1天、3天、7天,新生微血管数目逐渐减少。以上微血管的变化趋势与同步辐射成像结果一致。结论 1.基于同步辐射的脑微血管可视化体系的建立,可多平面多角度解析全脑和局部靶区域的3D血供配布情况,并实现血管网络空间结构参数的量化分析。 2.缺血性损伤导致微血管网络发生结构与功能重塑,血管新生在缺血后极早期即可启动,并迅速达到高峰,新生血管迂曲度增加,分支增多,形成繁复的血管网,对局部血流有重要的代偿意义。 3.同步辐射相衬成像可同时从3D和2D角度,提供血管新生动态发展的影像学证据。
[Abstract]:Purpose The maintenance of the physiological function of the brain depends on the completion of the microvessel network In the whole, after the ischemic stroke, the body starts the waterfall type cascade reaction to cause the steady state disorder of the microvessel network, and the structure and function of the neurovascular unit Plasmoplastic. Cerebral ischemia can trigger the neogenesis of the autograft. However, the mechanism, the development process, the specific form and so on are not yet known. It is true that the traditional methods of vascular visualization, such as the method of histopathology and the like, can not accurately show the time and space of the vascular network in the two-dimensional plane of the display of the microvessel space structure. The conventional imaging means, such as MRA, CTA, DSA, etc., can not detect the minute blood of diameter below 50 & mu; m due to the limited time-space resolution the new synchronous radiation technology breaks through the diffraction limit of the traditional optical imaging method, and can clearly realize the non-destructive and three-dimensional formation of the material microstructure by virtue of the extremely high resolution of the sub-micron to the micron order, Objective: To study the feasibility and effectiveness of brain microvessel high resolution and three-dimensional imaging, and to explore the dynamic development of vascular network remodeling after cerebral ischemia by using the phase contrast imaging of the synchrotron radiation phase contrast imaging of Shanghai light source to study the blood vessel imaging of the normal and ischemic stroke model. In order to provide a rich 3D imaging and morphometric method for the study of vascular injury and repair of ischemic stroke with a new angle of view. It was reported. Methods: The imaging parameters were adjusted, and the whole brain blood of the normal rats was reconstructed from multi-angle and multi-plane by using the filtering inverse algorithm and the imaging software. The 3D view of the tube is based on the structural information of the vascular network. Quantitative analysis of the brain tissue of the body microscope and HE staining The model of the ischemic stroke of the rat was then used to observe the dynamic development of the spontaneous blood vessel at the same time after the ischemia, and the method of quantitative analysis of the new blood vessel was explored, and the immune group was combined. Comprehensive evaluation of vascular network of ischemic stroke from 3D/ 2D visual angle winding weight Results 1. Using the synchrotron radiation phase contrast imaging, the three-dimensional slice from the projection, the two-dimensional slice to the full-vascular network is established. The complete visualization system of the image. A 5.9. m u.M CCD detector is used, with a resolution of a minimum vessel diameter of about 9.4. m And is close to the capillary level. The two-dimensional plain film is compared with the HE staining, and the preparation procedures of the contrast agent and the complex slice are not needed, so that the blood capillary level can be clearly displayed. The blood vessels in the hippocampus of the hippocampus. The 3-D rendered image on the surface of the brain and the microfil blood vessel cast-in-place microscope after-perfusion of the body-type microscope in situ were highly consistent, and the multi-stage blood vessels of the dura mater 2. The whole-brain horizontal position, the coronal, the sagittal and the digital 3D cerebral angiography were further established, and the blood supply and distribution of the functional areas were analyzed, and a series of numbers were constructed from the 2D and 3D perspectives. 3. The results of the blood vessel network skeleton extraction show that the whole blood vessel network of the rat is mainly below 20. m Based on the morphometric analysis of the 3D network, the cerebral vascular volume, the vessel density, the number of vessel branches, the number of nodes, the adjacent blood vessel spacing, the diameter, 4. The results of 2D and 3D imaging of a series of synchronous radiation sources around the ischemic stroke model in rats showed that the number of microvessels increased continuously at 2 hours,4 hours and 6 hours after the ischemia, and the number of microvessels in the ischemic area was densely distributed at the periphery of the ischemic area. However, with the prolongation of the time of the ischemia, the distribution and the density of the new blood vessels decreased after 3 days. The shape of the vessel was distorted and the irregular blood vessel was formed. The ischemic 7-day infarction was liquefied into a honeycomb-like capsule, and the glial scar was gradually formed. The volume of the lateral brain of the focal side of the lesion was significantly reduced in the 18-day-after-ischemia. The cerebral ventricle was compressed, and the cortical and striatal infarction foci were developed to form a high-density scar tissue and a non-parenchymal tissue-free> "apoplex. Quantitative analysis showed that with the increase of the time of the ischemia, the volume of the lateral brain of the lesion was gradually decreased after 3 days, of which the focal side of the lesion was 18 days after the ischemia. The volume of blood vessel was significantly reduced in the control group (P0.05). The blood vessel density of 2 hours,4 hours and 6 hours after the ischemia was higher than that in the control group (P0.05). The blood vessel density was gradually decreased in 8 days (P0.05). The number of blood vessels in 2 hours,4 hours and 6 hours after the ischemia was significantly higher than that in the control group (P0.05). The blood vessel score was 3 days,7 days and 18 days after the ischemia. The number of the branches and the number of the nodes decreased gradually (P0.05). The frequency of the small blood vessels and the control group of 2 hours,4 hours,6 hours and 10-20. m The difference was statistically significant (P0.05). The frequency gradually decreased with the lapse of time and the ischemia was 7 days. The frequency of 18 days was significantly decreased (P0.05). After the ischemia,2 hours,4 hours,6 hours,1 day and 3 days The blood vessel tortuosity of CD31 was significantly higher (P0.05). The vessel is in the form of a punctiform, curved, short or round tube with varying forms. Over time, ischemia 1 The number of newly-born microvessels was gradually reduced on day,3 and 7 days. microblood Conclusion 1. The development trend of the tube is consistent with the results of the synchronous radiation imaging. Conclusion 1. The 3D blood supply of the whole brain and the local target area can be resolved by multi-plane multi-angle based on the establishment of the visualization system of the brain microvessel based on the synchronous radiation. 2. The structure and function remodeling of the microvessel network are caused by the ischemic injury, and the angiogenesis can be started at the early stage of the ischemia, and the peak is rapidly reached, the tortuosity of the new blood vessel is increased, and the branch and the synchronous radiation phase contrast imaging can
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743.3

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