Novel Optical Imaging Approach to Study Neurovascular Coupling System

了解神经活动与血脑屏障 (BBB) 通透性之间的关系 整合我们对神经血管耦合系统的知识非常重要。虽然付出了很多努力 专注于了解调节血流以响应的潜在机制 神经活动5，人们对神经活动如何调节组织灌注知之甚少。直接体内 脑血管细胞 Ca2 动力学测量和 BBB 定量 由于技术限制，渗透性一直很困难。研究人员一直在使用 用于 BBB 渗透性常规研究的大体积荧光标记溶质。然而体积庞大 荧光标记扰乱生物分子的结构和功能特性 遭受光漂白。由于这些原因，微小的拉曼标记分子与 SRS 成像是研究体内 BBB 通透性的理想选择。在这里，我们提出了一种新颖的光学 成像方法确定神经活动与血脑屏障通透性之间的关系 体内。该提案的主旨是使用生物正交探测刺激的组合 拉曼散射 (SRS)、多光子荧光和小鼠遗传学同时获得 Ca2 活性和 BBB 通透性的测量。血管内皮细胞将被标记 具有基因编码的钙指示剂，而具有特定生物正交标记的分子 振动模式将被注入脑脉管系统以测量 BBB 渗透性， 研究钙动力学与血脑屏障通透性之间的关系。 成功完成本提案中描述的目标将为以下领域提供一个新的框架： 血管动物模型体内血脑屏障通透性的未来机制研究 发育和脉管系统疾病。

Understanding the relationship between neural activity and Blood-brain barrier (BBB) permeability is important to integrate our knowledge of neurovascular coupling system. Although much effort is focused in understanding the underlying mechanisms that regulate blood flow in response to neural activity5, little is known about how neural activity regulates tissue perfusion. Direct in vivo measurements of Ca2+ dynamics in cerebral blood vessel cells and quantification of BBB permeability have been difficult largely due to technical limitation. Researchers have been using bulky fluorescence-labeled solutes for conventional studies on BBB permeability. However, bulky fluorescence labeling perturbs the structural and functional properties of the biomolecules and suffered from photo bleaching. For these reasons, tiny Raman tagged molecules combined with SRS imaging is ideal for studying BBB permeability in vivo. Here we propose a novel optical imaging approach to determine the relationship between neural activity and BBB permeability in vivo. The main thrust of this proposal is to use a combination of bioorthogonal probed stimulated Raman scattering (SRS), multi-photon fluorescence, and mouse genetics to obtain simultaneous measurements of Ca2+ activity and BBB permeability. Vascular endothelial cells will be labeled with a genetically encoded calcium indicator while bioorthogonal-tagged molecules with specific vibrational modes will be injected into the brain vasculature to measure the BBB permeability, and the relationship between calcium dynamics and permeability of the BBB will be studied. Successful completion of the aims described in this proposal will enable a novel framework for future mechanistic studies of in vivo BBB permeability in animal models for blood vessel development and vasculature diseases.