Abstract: Objective To study the distribution characteristics of cerebral blood vessels in mice and the spatial relationship with adjacent nerve cells using fast three-dimensional microscopic imaging technology.Methods The adult Tie2-Cre::Ai9 hybrid transgenicmice(C57BL/6) with fluorescent protein labeled vascular endothelial cells were perfused with 4% paraformaldehyde in the heart to obtain complete brain tissue,clearing tissue,andstained the nerve cells of the whole brain.The rapid three-dimensional imaging system (VISoR) was used to obtain the microscopic images of the whole brain vessels and nerve cells in mice with 1×1×3.5 μm³ voxel resolution,and the two-dimensional image sequence was reconstructed into a three-dimensional whole brain image.Part of cortex,hippocampus and thalamus were selected to segment and extract blood vessels and analyze their morphological characteristics;Automatically identify nerve cells and quantitatively analyze the distribution characteristics of the distance between nerve cells and the nearest blood vessels.Results The three-dimensional images of the distribution of blood vessels and nerve cells in the whole brain of mice were successfully collected.The whole brain view of C57BL/6 mouse's complex vascular network was provided through high-resolution brain atlas.The analysis showed that the density of bifurcation points and the volume fraction of blood vessels in hippocampus were smaller than those in cortex and thalamus,and the difference was statistically significant.The staining of neurons with Nissl stain showed that there were differences in the distribution of blood vessels and nerve cells in different brain regions.In the regions of interest of different brain regions,the distance between neurons and blood vessels was counted,and the results were statistically different.Conclusion The whole brain vessels and neurons of mice can be imaged by fast 3D microscopic imaging technology.Through the quantitative analysis of regional blood vessels and nerves,it was found that there were regional distribution characteristics between blood vessels and nerves in mice.

Key words: Fast 3D imaging technology, Tissue clearing, Whole brain vascular imaging, Nessler stain, Neurocellular imaging

摘要： 目的 应用快速三维显微成像技术研究小鼠脑血管分布特征及与临近神经细胞的空间关系。方法 采用荧光蛋白标记血管内皮细胞的成年Tie2-Cre::Ai9杂交转基因小鼠(C57BL/6),经心脏灌注4%多聚甲醛获取完整脑组织,组织透明化处理,并对全脑神经细胞进行染色。应用快速三维成像系统(VISoR)以1×1×3.5 μm³体素分辨率获取小鼠全脑血管和神经细胞的显微图像,将二维图像序列重建为三维全脑图像。选取皮质、海马及丘脑中部分区域进行分割、提取血管,分析其形态特征;自动化识别神经细胞,量化分析神经细胞与最邻近血管距离的分布特征。结果 成功采集了小鼠全脑血管与神经细胞分布的三维图像。通过高分辨率脑图谱提供了C57BL/6小鼠复杂的血管网络全脑视图。分析显示在海马中,血管的分叉点密度以及血管的体积分数均小于皮层和丘脑,差异具有统计学意义。尼氏染色剂对神经细胞的染色显示了在不同脑区中,血管和神经细胞的分布存在差异。在不同脑区的感兴趣区域中,统计了神经细胞到血管之间的距离,结果有统计学差异。结论 采用快速三维显微成像技术可以对小鼠全脑血管与神经细胞进行三维成像。通过对区域血管神经进行量化分析,发现了小鼠血管神经之间具有区域分布特点。

关键词: 快速三维成像技术, 组织透明化, 全脑血管成像, 尼氏染色剂, 神经细胞成像