Long Term 3D Imaging of Mouse Brain In Vivo to Study Glial Cells and Gliogenesis

小鼠大脑体内长期 3D 成像研究神经胶质细胞和神经胶质生成

• 批准号: 8450379
• 负责人: Teresa Ann Murray
• 金额: $ 20.87万
• 依托单位: LOUISIANA TECH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-10 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8450379
• 关键词: Acids; Adult; Aging; Alzheimer' s Disease; Astrocytes; Behavioral; Biological Markers; Blood - brain barrier anatomy; Blood specimen; Brain; Brain region; Calcium; Calcium Signaling; Caliber; Cell Shape; Cell physiology; Cells; Communities; Conflict (Psychology); Dendritic Spines; Development; Devices; Disease; Disease Progression; Drug Addiction; Drug usage; Dyes; Electrophysiology (science); Endoscopes; Epigenetic Process; Equilibrium; Excision; Fiber; Fiber Optics; Fluorescent Dyes; Glass; Glial Fibrillary Acidic Protein; Goals; Health; Hippocampus (Brain); Hour; Image; Implant; In Vitro; Injection of therapeutic agent; Injury; Label; Laboratories; Laboratory mice; Life; Longitudinal Studies; Measures; Membrane; Methods; Microscope; Morphology; Multiphoton Fluorescence Microscopy; Mus; Needles; Nerve Degeneration; Neuroglia; Neurons; Onset of illness; Operative Surgical Procedures; Parkinson Disease; Pharmaceutical Preparations; Physiological; Process; Proteins; Protocols documentation; Research; Research Personnel; Resolution; Role; Shapes; Slice; Surface; System; Techniques; Testing; Therapeutic; Therapeutic Intervention; Three-Dimensional Imaging; Time; Tissues; Viral Vector; Work; adult stem cell; base; brain tissue; comparative; drug of abuse; expression vector; fluorescence microscope; gliogenesis; implantation; in vivo; indexing; innovation; lens; migration; miniaturize; neuronal cell body; neurotransmission; new technology; optical fiber; precursor cell; promoter; prototype; research study; stem cell therapy; subventricular zone; tool; ultra high resolution

DESCRIPTION (provided by applicant): Glial cells greatly outnumber neurons in the brain and have active roles in development, modulation of neurotransmission, health and disease. Yet, relatively little is known about glial cells compared to neurons. What is known about glial cells i derived largely from studies of dissociated cells or live brain slices. Yet these methods have not elucidated phenomena such as the nuanced dynamics between astrocytes and neurons in the living brain. Multiphoton fluorescence microscopy can facilitate studies in the living, intact mouse brain (in vivo), but only when the cells are less than ~0.5-mm below the brain surface. Thus, most glial cells cannot be observed in vivo. Thin optical fibers have been implanted in the mouse brain that can reach great depths to visualize fluorescently labeled cell bodies without disrupting much brain tissue. However, they cannot resolve fine membrane processes of glial cells as they interact with neurons. This would be useful information to study normal development and aging, or the effects of drug use, neurodegeneration, or injury. More recently, endoscopes have been miniaturized for in vivo studies in mouse brain. The high-resolution version is implanted in a glass sheath and can resolve fine cellular processes when used with a multiphoton microscope. Yet, it's 1800-um diameter is markedly wider than fiber optics, which are on the order of 300-um in overall diameter. Therefore, it displaces over 25 times more brain tissue than fibers and requires brain tissue removal prior to implantation. Thus, these have limited applications and should not be implanted very deep into the brain. This project will develop an implantable, 350-um diameter lens to use with multiphoton fluorescence microscopy that is thin, like an optical fiber, and has high resolution to observe fine cellular processes in vivo. It will have the best attributes of optical fibers and miniaturized endoscopes without their drawbacks. To demonstrate its utility, a long version of the lens will be implanted deeply enough to observe adult-born glial cells in vivo over a period of three months. This will offer a major improvement over the current method of using brain slices for short-term studies. The slice study observations are highly dependent on technique and have produced conflicting estimates of migration rates. In another test of its ability, a small port for injection of a calcium-sensitie dye will be incorporated with the implant. The dye will be injected at later time points to observe the release of calcium inside glial cells. Calcium release is one measure of glial function and may provide important clues to their modulation of neuron function. This tool is expected to have numerous other uses because of the expansion of fluorescent labeling tools, including promoter-directed expression of fluorescent proteins in mice that could label subpopulations of glial cells, and the ability to image the same brain region over hours, days or months. PUBLIC HEALTH RELEVANCE: This project will create a tool for researchers to discover how glial cells in the brain function and how they are involved in aging and disorders, such as Alzheimer's and Parkinson's diseases. A tiny glass lens with needle-like diameter will be implanted in the brain of laboratory mice that have a fluorescent dye (or protein) in their glial cells. Using a microscope to look into the lens, researchers will be able to record the numbers and shapes of the cells by illuminating the fluorescent dye and determine if there are major changes in aging or certain diseases, and if potential treatments return them to a normal state.

描述（由申请人提供）：神经胶质细胞在大脑中的人数大大超过了神经元，并且在发育，神经传递，健康和疾病的调节中具有积极作用。然而，与神经元相比，对神经胶质细胞的了解很少。关于神经胶质细胞I的知识主要来自解离细胞或活脑切片的研究。然而，这些方法尚未阐明现象，例如活体大脑中星形胶质细胞和神经元之间的细微动态。多光子荧光显微镜可以促进生活完整的小鼠脑（体内）的研究，但仅当细胞低于脑表面低于0.5 mm时。因此，大多数神经胶质细胞在体内无法观察到。稀薄的光纤已植入了小鼠大脑中，可以达到深度，以使荧光标记的细胞体可视化而不破坏大脑组织。但是，它们无法在与神经元相互作用时解析神经胶质细胞的细膜过程。这将是研究正常发育和衰老的有用信息，或药物使用，神经退行性或损伤的影响。最近，内窥镜已被小型化，用于小鼠脑中的体内研究。高分辨率版本被植入玻璃鞘中，并在与多光子显微镜一起使用时可以解决细胞过程。然而，它的直径比光纤宽了，光纤的总直径为300-UM。因此，它置换了脑组织的25倍以上，并且需要在植入前去除脑组织。因此，这些应用有限，不应将其植入大脑。该项目将开发一个可植入的350-UM直径透镜，用于与薄的多光子荧光显微镜一起使用，如光纤，并且具有高分辨率，可以观察体内细胞过程。它将具有光纤和微型内窥镜的最佳属性，而无需它们的缺点。为了证明其效用，将深入植入长期的镜头，以在三个月的时间内观察体内成人神经胶质细胞。这将为当前使用大脑切片进行短期研究的方法提供了重大改进。切片研究的观察高度依赖于技术，并产生了迁移率的估计相互矛盾。在另一个测试其能力的测试中，将与植入物一起掺入一个用于钙敏感染料的小端口。染料将在以后的时间点注入以观察 胶质细胞内钙的释放。钙释放是神经胶质功能的一种度量，可以为其神经元功能的调节提供重要的线索。由于荧光标记工具的扩展，包括荧光蛋白在小鼠中的启动子指导的表达，可以在小鼠中标记荧光蛋白的启动子指导的表达，并且可以在数小时，几天或几个月内对同一大脑区域成像相同的大脑区域。 公共卫生相关性：该项目将为研究人员创建一种工具，以发现大脑功能中的神经胶质细胞如何以及它们如何参与衰老和疾病，例如阿尔茨海默氏症和帕金森氏病。带有针直径的微小玻璃透镜将植入在其神经胶质细胞中含有荧光染料（或蛋白质）的实验室小鼠的大脑中。使用显微镜来研究镜头，研究人员将能够通过照明荧光染料并确定衰老或某些疾病是否有重大变化，以及潜在的治疗方法是否会使它们恢复正常状态，从而记录细胞的数量和形状。