Department of Basic Sciences, Loma Linda University.
Departments of Physiology, Surgery and Neurosurgery and Sarver Heart Center, University of Arizona College of Medicine Tucson.
J Vis Exp. 2022 Mar 11(181). doi: 10.3791/63463.
Cerebral blood flow is conveyed by vascular resistance arteries and downstream parenchymal arterioles. Steady-state vascular resistance to blood flow increases with decreasing diameter from arteries to arterioles that ultimately feed into capillaries. Due to their smaller size and location in the parenchyma, arterioles have been relatively understudied and with less reproducibility in findings than surface pial arteries. Regardless, arteriolar endothelial cell structure and function-integral to the physiology and etiology of chronic degenerative diseases-requires extensive investigation. In particular, emerging evidence demonstrates that compromised endothelial function precedes and exacerbates cognitive impairment and dementia. In the parenchymal microcirculation, endothelial K channel function is the most robust stimulus to finely control the spread of vasodilation to promote increases in blood flow to areas of neuronal activity. This paper illustrates a refined method for freshly isolating intact and electrically coupled endothelial "tubes" (diameter, ~25 µm) from mouse brain parenchymal arterioles. Arteriolar endothelial tubes are secured during physiological conditions (37 °C, pH 7.4) to resolve experimental variables that encompass K channel function and their regulation, including intracellular Ca dynamics, changes in membrane potential, and membrane lipid regulation. A distinct technical advantage versus arterial endothelium is the enhanced morphological resolution of cell and organelle (e.g., mitochondria) dimensions, which expands the usefulness of this technique. Healthy cerebral perfusion throughout life entails robust endothelial function in parenchymal arterioles, directly linking blood flow to the fueling of neuronal and glial activity throughout precise anatomical regions of the brain. Thus, it is expected that this method will significantly advance the general knowledge of vascular physiology and neuroscience concerning the healthy and diseased brain.
脑血流由血管阻力动脉和下游实质小动脉输送。从动脉到最终流入毛细血管的小动脉，血流的稳态血管阻力随着直径的减小而增加。由于其较小的尺寸和在实质中的位置，小动脉的研究相对较少，并且与脑表面软脑膜动脉相比，其发现的可重复性也较低。然而，小动脉内皮细胞的结构和功能——对慢性退行性疾病的生理学和病因学至关重要——需要广泛的研究。特别是，新出现的证据表明，内皮功能障碍先于并加剧认知障碍和痴呆。在实质微循环中，内皮 K 通道功能是精细控制血管扩张传播的最有力刺激因素，可促进神经元活动区域的血流增加。本文展示了一种从鼠脑实质小动脉中新鲜分离完整且电偶联的内皮“管”（直径约 25 µm）的改良方法。在生理条件下（37°C，pH 7.4）固定小动脉内皮管，以解决包括 K 通道功能及其调节在内的实验变量，包括细胞内 Ca 动力学、膜电位变化和膜脂质调节。与动脉内皮相比，一个明显的技术优势是细胞和细胞器（例如线粒体）尺寸的形态分辨率增强，这扩展了该技术的用途。终身健康的脑灌注需要实质小动脉中强大的内皮功能，直接将血流与大脑精确解剖区域的神经元和神经胶质活动的供能联系起来。因此，预计该方法将大大提高关于健康和患病大脑的血管生理学和神经科学的一般知识。
