Imaging signal transduction in the living mouse brain using two-photon microscopy

使用双光子显微镜对活体小鼠大脑中的信号转导进行成像

• 批准号: 7727062
• 负责人: Katerina Akassoglou
• 金额: $ 38.2万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7727062
• 关键词: Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Animal Model; Animals; Asthma; Autoimmune Diseases; Biosensor; Brain; Cells; Communities; Cyclic AMP-Dependent Protein Kinases; Diabetes Mellitus; Disease; Event; Goals; Health; Hypoxia; Image; Imaging Device; Immune; Infection; Inflammation; Inflammatory; Injury; Lead; Life; Malignant Neoplasms; Methods; Microglia; Microscopy; Molecular; Morphology; Multiple Sclerosis; Mus; Nervous system structure; Neuraxis; Neurobiology; Oxidation-Reduction; Pathway interactions; Peptide Signal Sequences; Process; Quantum Dots; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Slice; Stimulus; Testing; Time; Trauma; brain tissue; design; effective therapy; extracellular; in vivo; macrophage; nervous system disorder; novel; response; rho; spinal cord injury pain; tool; two-photon

In vivo imaging by two-photon microscopy has revolutionized our understanding of neurobiological and immunological processes and revealed unexpected information on the dynamic morphology of central nervous system structures and immune processes in health and disease. Currently, intracellular signaling cascades can only be studied in isolated cells or slices of brain tissue. The goal of this EUREKA proposal is to image signal transduction pathways in the living brain in mice, with the aim to study signaling events as they are regulated in real time by trauma, inflammation, and hypoxia. In this proposal we will (1) develop genetically modified biosensorexpressing mice to image the activation of five major signal transduction pathways-redox changes, NFB, Rho, PKA, and Ras- in microglia and macrophages in vivo; (2) generate novel genetically encoded biosensors for the PKA, Rho, and Ras pathways, optimized for in vivo imaging by two-photon microscopy; (3) develop the first pharmacologic tools (biosensor-conjugated Quantum Dots) to deliver biosensors to microglia and macrophages in the brain; and (4) demonstrate dynamic signaling events in microglia in living mouse brain in real time during hypoxia, inflammation, or traumatic injury. The tools we propose to develop will fundamentally change the methods we use to study the dynamic activation of intracellular signaling pathways. Results from this proposal will elucidate inflammatory signal transduction pathways in an unprecedented way-by correlating molecular mechanisms of microglial activation with dynamic morphologic alterations in response to extracellular stimuli in the brain of a living animal. This proposal will generate tools to make possible imaging of signal transduction in vivo. It will make available to the scientific community novel biosensors, delivery methods, and animal models to study the temporal and spatial activation of five major signal transduction pathways in the living animal. In vivo imaging of signal transduction pathways in microglia and macrophages can be applied to a wide range of diseases with an inflammatory component, including cancer, diabetes, asthma, infection, and autoimmune disorders, and nervous system disorders characterized by microglia activation, such as multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis, pain, and spinal cord injury.

两光子显微镜的体内成像已彻底改变了我们的 了解神经生物学和免疫学过程，并揭示了意外信息 关于中枢神经系统结构的动态形态和健康和免疫过程 疾病。当前，细胞内信号传导级联反应只能在孤立的细胞或切片中研究 脑组织。尤里卡提案的目的是图像生活中的信号转导途径 小鼠的大脑，目的是研究信号事件，因为它们是通过创伤实时调节的， 炎症和缺氧。在此提案中，我们将（1）开发经过转基因的BioSensorexpressing 小鼠成像五个主要信号转导途径的激活-REDOX变化NFB， 小胶质细胞和巨噬细胞中的Rho，PKA和Ras-在体内； （2）生成新的遗传编码 PKA，RHO和RAS途径的生物传感器，可通过两光片对体内成像进行优化 显微镜； （3）开发第一个药理工具（生物传感器偶联的量子点）以提供 大脑中小胶质细胞和巨噬细胞的生物传感器； （4）在 在缺氧，炎症或创伤性损伤期间，活小鼠大脑中的小胶质细胞实时实时。工具 我们建议开发从根本上改变我们用于研究动态激活的方法 细胞内信号通路。该提案的结果将阐明炎症信号 小胶质细胞的前所未有的相关分子机制中的转导途径 响应于A脑中细胞外刺激的动态形态改变的激活 活动物。该建议将生成工具以使体内信号转导的可能成像。 它将为科学社区的小说生物传感器，递送方法和动物提供服务 研究五个主要信号转导途径的时间和空间激活的模型 活动物。小胶质细胞和巨噬细胞中信号转导途径的体内成像可以是 应用于具有炎症成分的多种疾病，包括癌症，糖尿病， 哮喘，感染和自身免疫性疾病以及以小胶质细胞为特征的神经系统疾病 激活，例如多发性硬化症，阿尔茨海默氏病，肌萎缩性侧索硬化症，疼痛和 脊髓损伤。