Hakim Md A, Behringer Erik J
Department of Basic Sciences, Loma Linda University.
Department of Basic Sciences, Loma Linda University;
J Vis Exp. 2019 Jan 20(143). doi: 10.3791/58832.
Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca [Ca]i increases and subsequent activation of small- and intermediate conductance Ca-activated K (SKCa/IKCa) channels. Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 °C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial "tubes" are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.
脑动脉及其各自的微循环通过血流调节为大脑输送氧气和营养物质。内皮细胞排列在血管腔内，并根据需要控制血管直径的变化，以满足神经元的代谢需求。膜电位（Vm）超极化和一氧化氮的主要内皮依赖性信号通路通常并行运作，以介导血管舒张，从而增加血流量。尽管内皮源性超极化（EDH）的成分对于协调数毫米血管长度上的血管舒张至关重要，但从历史上看，这些成分一直难以测量。EDH的这些成分需要细胞内钙[Ca]i增加，随后激活小电导和中电导钙激活钾（SKCa/IKCa）通道。在这里，我们展示了一个简化的流程，包括从小鼠脑动脉中分离新鲜内皮；分别使用Fura-2光度法和细胞内尖锐电极同时测量内皮[Ca]i和Vm；以及在生理条件（pH 7.4，37°C）下连续灌注盐溶液和药物制剂。从 Willis 环分离出大脑后动脉，使其不与后交通动脉和基底动脉相连。对清理后的大脑后动脉段进行酶消化，随后研磨，有助于去除外膜、血管周围神经和平滑肌细胞。然后将得到的大脑后动脉内皮“管”固定在显微镜下，在连续灌注的同时，使用相机、光电倍增管和一到两个静电计进行检查。总的来说，这种方法除了可以测量EDH通过缝隙连接在完整内皮上传播至毫米距离外，还能同时测量离散细胞位置的内皮[Ca]i和Vm的变化。预计这种方法将对正常和患病大脑中血流调节机制背后的脑内皮功能进行高通量分析。
