Optimization and validation of quantitative birefringence microscopy for assessment of myelin pathologies associated with cognitive impairments and motor deficits in young and old aging monkey brain

定量双折射显微镜的优化和验证，用于评估与年轻和年老猴脑认知障碍和运动缺陷相关的髓磷脂病理学

• 批准号: 10544518
• 负责人: IRVING J. BIGIO
• 金额: $ 55.91万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10544518
• 关键词: 3-Dimensional; Age-associated memory impairment; Aging; Alzheimer' s Disease; Animal Model; Animals; Anisotropy; Architecture; Archives; Autopsy; Axon; Behavioral; Biological Assay; Biological Markers; Birefringence; Boston; Brain; Brain imaging; Central Nervous System; Cephalic; Cognitive; Computers; Cryopreservation; Darkness; Dementia; Diffusion Magnetic Resonance Imaging; Disease; Electron Microscopy; Exhibits; Health; Human; Image; Imaging Device; Impaired cognition; Injury; Label; Lighting; Macaca mulatta; Measures; Medical; Mesenchymal Stem Cells; Methods; Microscope; Microscopic; Microscopy; Modeling; Monkeys; Motor; Motor Cortex; Movement; Multiple Sclerosis; Myelin; Myelin Sheath; Nerve Degeneration; Optics; Pathology; Peripheral Nerves; Phase; Polarization Microscopy; Positioning Attribute; Qualitative Methods; Quantitative Microscopy; Raman Spectrum Analysis; Recovery of Function; Refractive Indices; Reporting; Research Personnel; Resolution; Sampling; Stains; Stroke; Structure; Surveys; Sushi Domain; Testing; Therapeutic; Thick; Time; Tissue imaging; Tissues; Universities; Validation; Vesicle; Work; age related; age related neurodegeneration; arm; brain tissue; cognitive testing; computing resources; cost; density; extracellular vesicles; hand dysfunction; high resolution imaging; in vivo; in vivo imaging; light microscopy; microscopic imaging; motor deficit; motor recovery; myelin degeneration; nervous system disorder; neural; neurophysiology; neurotransmission; nonhuman primate; normal aging; parallel computer; pathology imaging; quantitative imaging; rapid technique; reflectance confocal microscopy; repaired; scale up; tool

Degeneration or breakdown of the myelin sheath that wraps and insulates axons of the central nervous system, as well as peripheral nerves, is a factor in a large array of neurological disorders. These include multiple sclerosis, stroke, age-related neurodegenerative diseases like Alzheimer’s, and in non-neurodegenerative age- related cognitive impairment. To date, myelin breakdown has been assessed indirectly with neurophysiological assays of conduction, post-mortem myelin staining, or in-vivo diffusion MRI. In the normal aging rhesus monkey, post-mortem electron microscopy (EM) has confirmed myelin breakdown at the ultrastructural level; however, EM studies cannot be scaled up to evaluate the entire 100-cc monkey brain or the larger human brain. Myelin stains suffer from various technical and practical limitations that impede quantification. Other methods, like label- free spectral confocal reflectance imaging (SCoRe), and coherent anti-Stokes Raman spectroscopy (CARS), are promising, but like EM, are expensive, slow and difficult to apply to large brain sections. While SCoRe and CARS microscopy work in reflectance, opening the possibility of in-vivo imaging through a cranial window in the small- animal brain, a complementary method is needed that is validated, quantitative and high-resolution, to survey the status of myelin in whole-brain sections of monkey or human brain. Here we propose to optimize and validate quantitative birefringence microscopy (qBRM) for high-resolution imaging of normal and abnormal myelin. The qBRM image provides, for every pixel, two quantitative measures: a) the relative phase retardance, which is linearly proportional to the density of the birefringent medium and the organization of its anisotropy; and b) the local orientation of the optic axis of the myelin, which corresponds to the direction of the structural anisotropy. Archived brain tissues from two rhesus monkey models of myelin damage are available for this project at the Boston University Medical Campus (BUMC). In Aim 1 we will automate the function of our birefringence microscope with computer-controlled, motorized polarizer components and stage-positioning, to facilitate fast, automated image acquisition (montaging) of large tissue sections (up to 40x40 mm). We will also employ massively-parallel computing resources at BU to rapidly render the multi-megapixel quantitative images. In Aim 2 we will validate our qBRM image measures by comparing them, in the same sections, with myelin identified by myelin stains, such as FluoroMyelin Red, and with label-free spectral confocal reflectance (SCoRe) microscopy. In Aim 3, We will demonstrate the untility of qBRM for quantifying myelin by imaging tissue sections from three monkey models of myelin pathology: a) behaviorally characterized young and old monkeys with cognitive assessment; b) behaviorally characterized old monkeys with impaired hand movement produced by motor-cortex injury and treated with mesenchymal stem-cell derived extracellular vesicles (EVs); c) behaviorally characterized old monkeys with demonstrated cognitive impairments treated with EVs. Together this will establish the validity and utility of qBRM for assessing myelin in the central nervous system in health and disease.

髓鞘变性或破坏，髓鞘包裹和隔离中枢神经系统的轴突， 以及周围神经，是一系列神经系统疾病的一个因素。其中包括多个 硬化症，中风，老年性神经退行性疾病，如阿尔茨海默氏症，以及非神经退行性疾病， 相关的认知障碍。到目前为止，髓磷脂的破坏已经通过神经生理学方法间接评估。 传导测定、死后髓鞘染色或体内扩散MRI。在正常衰老的恒河猴中， 死后电子显微镜（EM）已经证实髓磷脂在超微结构水平上的分解;然而， EM研究不能扩大到评估整个100 cc的猴脑或更大的人脑。髓鞘 染色剂受到阻碍量化的各种技术和实际限制。其他方法，如标签- 自由光谱共焦反射成像（SCoRe）和相干反斯托克斯拉曼光谱（汽车）， 但是像EM一样，昂贵，缓慢并且难以应用于大的大脑切片。当SCore和汽车 显微镜在反射下工作，打开了通过小的颅骨窗口进行体内成像的可能性， 动物大脑，需要一种有效的、定量的和高分辨率的补充方法来调查 髓鞘在猴或人脑全脑切片中的状态。在这里，我们建议优化和 验证定量双折射显微镜（qBRM）用于正常和 异常髓鞘qBRM图像为每个像素提供两个定量测量： 延迟，其与双折射介质的密度及其结构成线性比例， 各向异性;和B）髓磷脂的光轴的局部取向，其对应于各向异性的方向。 结构各向异性来自两个髓磷脂损伤的恒河猴模型的存档脑组织可用于 这个项目在波士顿大学医学院（BUMC）。在目标1中，我们将自动化我们的 双折射显微镜，具有计算机控制的电动偏振器组件和载物台定位， 便于快速、自动采集大组织切片（最大40 x40 mm）的图像（剪辑）。我们还将 利用BU的并行计算资源快速绘制多兆像素的定量图像。 在目标2中，我们将通过在相同的切片中将它们与髓鞘进行比较来验证我们的qBRM图像测量 通过髓磷脂染色剂（如FluoroMyelin Red）和无标记光谱共聚焦反射（SCoRe）鉴定 显微镜在目标3中，我们将通过成像组织切片来证明qBRM用于定量髓鞘的有效性 来自髓鞘病理学的三种猴子模型：a）行为特征为年轻和年老的猴子， 认知评估; B）行为特征的老年猴，其手部运动受损， 运动皮层损伤并用间充质干细胞衍生的细胞外囊泡（EV）治疗; c）行为学上 这些研究描述了用EV治疗的表现出认知障碍的老年猴子的特征。这将共同 建立qBRM评估健康和疾病中中枢神经系统髓鞘的有效性和实用性。