Department of Neurology, Yale School of Medicine, New Haven, CT, USA.
Department of Biological Sciences, Dartmouth College, Hanover, NH, USA.
Nat Protoc. 2021 Jan;16(1):472-496. doi: 10.1038/s41596-020-00425-w. Epub 2020 Dec 9.
Mural cells (smooth muscle cells and pericytes) are integral components of brain blood vessels that play important roles in vascular formation, blood-brain barrier maintenance, and regulation of regional cerebral blood flow (rCBF). These cells are implicated in conditions ranging from developmental vascular disorders to age-related neurodegenerative diseases. Here we present complementary tools for cell labeling with transgenic mice and organic dyes that allow high-resolution intravital imaging of the different mural cell subtypes. We also provide detailed methodologies for imaging of spontaneous and neural activity-evoked calcium transients in mural cells. In addition, we describe strategies for single- and two-photon optogenetics that allow manipulation of the activity of individual and small clusters of mural cells. Together with measurements of diameter and flow in individual brain microvessels, calcium imaging and optogenetics allow the investigation of pericyte and smooth muscle cell physiology and their role in regulating rCBF. We also demonstrate the utility of these tools to investigate mural cells in the context of Alzheimer's disease and cerebral ischemia mouse models. Thus, these methods can be used to reveal the functional and structural heterogeneity of mural cells in vivo, and allow detailed cellular studies of the normal function and pathophysiology of mural cells in a variety of disease models. The implementation of this protocol can take from several hours to days depending on the intended applications.
壁细胞（平滑肌细胞和周细胞）是脑血管的固有组成部分，在血管形成、血脑屏障维持和调节局部脑血流（rCBF）方面发挥着重要作用。这些细胞与从发育性血管紊乱到与年龄相关的神经退行性疾病等各种病症有关。在这里，我们展示了使用转基因小鼠和有机染料进行细胞标记的互补工具，这些工具允许对不同壁细胞亚型进行高分辨率活体成像。我们还提供了详细的方法学，用于成像壁细胞中的自发性和神经活动诱发的钙瞬变。此外，我们描述了单光子和双光子光遗传学的策略，这些策略允许对单个和小簇壁细胞的活动进行操纵。与单个脑微血管直径和血流的测量相结合，钙成像和光遗传学允许研究周细胞和平滑肌细胞的生理学及其在调节 rCBF 中的作用。我们还展示了这些工具在阿尔茨海默病和脑缺血小鼠模型中研究壁细胞的实用性。因此，这些方法可用于揭示活体壁细胞的功能和结构异质性，并允许在各种疾病模型中对壁细胞的正常功能和病理生理学进行详细的细胞研究。根据预期应用，实施此方案可能需要数小时到数天的时间。
