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 是整合我们对神经血管耦合系统认识的重要环节。尽管我们付出了很多努力 重点是了解调节血液流动的潜在机制， 神经活动5，很少有人知道神经活动如何调节组织灌注。直接体内 脑血管细胞中Ca2+动力学的测量和BBB的定量 很大程度上由于技术限制而难以渗透。研究人员一直在使用 大体积荧光标记的溶质用于BBB渗透性的常规研究。然而， 荧光标记扰乱了生物分子的结构和功能特性， 遭受了照片漂白由于这些原因，微小的拉曼标记分子与 SRS成像是研究体内BBB通透性的理想方法。在这里，我们提出了一种新的光学 成像方法来确定神经活动和BBB通透性之间的关系， vivo.这项建议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，以获得同时 测量Ca2+活性和BBB通透性。将标记血管内皮细胞 与基因编码的钙指示剂，而生物正交标记的分子与特定的 将振动模式注入脑脉管系统以测量BBB渗透性， 并研究钙动力学与血脑屏障通透性的关系。 成功完成本提案中所述的各项目标将有助于建立一个新的框架， 在血管动物模型中的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.