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

包裹和隔离中枢神经系统轴突的髓鞘变性或破裂， 以及周围神经，是一系列神经疾病的一个因素。其中包括多个 硬化症、中风、阿尔茨海默氏症等与年龄相关的神经退行性疾病，以及非神经退行性疾病- 相关的认知障碍。到目前为止，髓鞘的破坏是通过神经生理学间接评估的。 传导分析、死后髓鞘染色或体内扩散磁共振成像。在正常衰老的恒河猴身上， 死后电子显微镜(EM)证实髓鞘在超微结构水平上破裂；然而， EM研究不能扩大到评估整个100毫升的猴脑或更大的人脑。髓鞘 染色受到各种技术和实践上的限制，阻碍了定量。其他方法，比如贴标签- 自由光谱共焦反射成像(SCORE)和相干反斯托克斯拉曼光谱(CARS) 前景看好，但和EM一样，价格昂贵、速度慢，很难应用于大型脑部。而分数和汽车 显微镜的工作原理是反射率，这为通过小脑窗进行体内成像提供了可能。 动物大脑，需要一种经过验证的、定量的、高分辨率的辅助方法来进行调查 髓鞘在猴脑和人脑全脑切片中的地位。在这里，我们建议优化和 验证定量双折射显微镜(QBRM)对正常和 髓鞘异常。QBRM图像为每个像素提供两个定量测量：a)相对相位 延迟性，它与双折射介质的密度及其组织成线性正比 各向异性；以及b)髓鞘视轴的局部方向，它对应于 结构各向异性。两个恒河猴髓鞘损伤模型的存档脑组织可用于 该项目在波士顿大学医学院(BUMC)进行。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部件和工作台定位，以 便于快速、自动获取(蒙太奇)大型组织切片(高达40x40 mm)的图像。我们还将 在BU使用大规模并行计算资源来快速渲染数百万像素的定量图像。 在目标2中，我们将验证我们的qBRM图像测量方法，在相同的部分中，将它们与髓鞘进行比较 通过髓鞘染色，如荧光髓鞘红和无标记光谱共聚焦反射(SCORE)进行鉴定 显微镜。在目标3中，我们将通过成像组织切片来证明qBRM在定量髓鞘方面的有效性 来自三种髓鞘病理学的猴子模型：a)幼猴和老年猴的行为特征 认知评估；b)由以下原因引起的手部运动障碍的老年猴子的行为特征 运动皮质损伤与间充质干细胞来源的细胞外小泡(EVS)治疗；c)行为学 描述了患有电动车治疗的认知障碍的老年猴子的特征。合在一起，这将是 建立qBRM在健康和疾病中评估中枢神经系统髓鞘的有效性和实用性。

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.