3D visualization and quantification of microvessels in the whole ischemic mouse brain using solvent-based clearing and light sheet microscopy
3D visualization and quantification of microvessels in the whole ischemic mouse brain using solvent-based clearing and light sheet microscopy
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DOI：
10.1177/0271678x17698970
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发表时间：
2017-10-01
影响因子：
6.3
通讯作者：
Hermann, Dirk M.
中科院分区：
文献类型：
--
作者：
Lugo-Hernandez, Erlen;Squire, Anthony;Hermann, Dirk M.
脑微血管的可视化对于理解卒中后脑重塑至关重要。染料注射允许评价灌注血管，但具有与不完全微血管填充或渗漏相关的局限性。在传统的组织化学中，微血管的分析仅限于2D结构，在血管回路的解释方面具有明显的局限性。在此，我们开发了一种直接的技术来可视化整个缺血小鼠大脑中的微血管，将荧光标记的低粘度水凝胶缀合物的注射与3D溶剂清除结合，然后进行自动光片显微镜检查。我们在C57 BI/6 j小鼠中进行短暂的大脑中动脉闭塞，并从整个大脑中获取详细的3D血管图像。随后的图像处理、渲染和血管与细丝模型的拟合用于计算血管长度密度，健康组织中为0.922 +/- 0.176 m/mm（3），缺血组织中为0.329 +/- 0.131 m/mm（3）。该分析显示，直径10 μ m的毛细血管明显损失，直径> 10 μ m和20 μ m的微血管损失较中度，而直径> 20 μ m的血管不受局灶性脑缺血的影响。我们建议，该协议是非常适合研究微血管损伤和重塑中风后。
The visualization of cerebral microvessels is essential for understanding brain remodeling after stroke. Injection of dyes allows for the evaluation of perfused vessels, but has limitations related either to incomplete microvascular filling or leakage. In conventional histochemistry, the analysis of microvessels is limited to 2D structures, with apparent limitations regarding the interpretation of vascular circuits. Herein, we developed a straight-forward technique to visualize microvessels in the whole ischemic mouse brain, combining the injection of a fluorescent-labeled low viscosity hydrogel conjugate with 3D solvent clearing followed by automated light sheet microscopy. We performed transient middle cerebral artery occlusion in C57BI/6j mice and acquired detailed 3D vasculature images from whole brains. Subsequent image processing, rendering and fitting of blood vessels to a filament model was employed to calculate vessel length density, resulting in 0.922 +/- 0.176 m/mm(3) in healthy tissue and 0.329 +/- 0.131 m/mm(3) in ischemic tissue. This analysis showed a marked loss of capillaries with a diameter10 mu m and a more moderate loss of microvessels in the range > 10 and 20 mu m, whereas vessels > 20 mu m were unaffected by focal cerebral ischemia. We propose that this protocol is highly suitable for studying microvascular injury and remodeling post-stroke.