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）：神经胶质细胞的数量远远超过大脑中的神经元，并且在发育、神经传递调节、健康和疾病中发挥积极作用。然而，与神经元相比，人们对神经胶质细胞的了解相对较少。关于神经胶质细胞的了解主要来自对分离细胞或活脑切片的研究。然而，这些方法尚未阐明活体大脑中星形胶质细胞和神经元之间微妙动态等现象。多光子荧光显微镜可以促进活的、完整的小鼠大脑（体内）的研究，但前提是细胞距离大脑表面小于约 0.5 毫米。因此，大多数神经胶质细胞无法在体内观察到。细光纤已被植入小鼠大脑中，该光纤可以到达很深的地方以可视化荧光标记的细胞体，而不会破坏太多的脑组织。然而，当神经胶质细胞与神经元相互作用时，它们无法解析神经胶质细胞的细膜过程。这对于研究正常发育和衰老，或吸毒、神经退行性变或损伤的影响是有用的信息。最近，内窥镜已小型化，用于小鼠大脑的体内研究。高分辨率版本被植入玻璃鞘中，与多光子显微镜一起使用时可以解析精细的细胞过程。然而，它的 1800 微米直径明显比光纤更宽，光纤的总直径约为 300 微米。因此，它取代的脑组织比纤维多 25 倍以上，并且需要在植入前去除脑组织。因此，它们的应用有限，不应该被植入大脑很深的地方。该项目将开发一种可植入的直径 350 微米的透镜，与多光子荧光显微镜一起使用，该透镜像光纤一样薄，具有高分辨率，可以观察体内精细的细胞过程。它将具有光纤和微型内窥镜的最佳属性，而没有它们的缺点。为了证明其实用性，长版本的晶状体将被植入足够深的位置，以便在三个月的时间内观察体内成体出生的神经胶质细胞。这将是对当前使用脑切片进行短期研究的方法的重大改进。切片研究观察结果高度依赖于技术，并且对迁移率的估计产生了相互矛盾的结果。在对其能力的另一项测试中，用于注射钙敏感染料的小端口将与植入物结合在一起。将在稍后的时间点注射染料以观察 神经胶质细胞内钙的释放。钙释放是神经胶质功能的一种测量方法，可能为神经元功能的调节提供重要线索。由于荧光标记工具的扩展，该工具预计将有许多其他用途，包括在小鼠中启动子定向表达荧光蛋白，可以标记神经胶质细胞亚群，以及在数小时、数天或数月内对同一大脑区域进行成像的能力。 公共健康相关性：该项目将为研究人员创建一个工具，以发现大脑中的神经胶质细胞如何发挥作用以及它们如何参与衰老和疾病，例如阿尔茨海默病和帕金森病。直径如针的微小玻璃晶状体将被植入实验室小鼠的大脑中，这些小鼠的神经胶质细胞中含有荧光染料（或蛋白质）。使用显微镜观察晶状体，研究人员将能够通过照射荧光染料来记录细胞的数量和形状，并确定衰老或某些疾病是否存在重大变化，以及潜在的治疗是否可以使它们恢复正常状态。