Brain-wide quantitative mapping of microglia activation

摘要 最近的技术发展，如组织清除和光片成像，允许三个- 以细胞分辨率对整个大脑进行三维可视化。半透明生物系统公司推动了这一进程 通过开发a)专有的中尺度成像系统来进一步技术，该系统允许可视化整个 老鼠大脑比以前的技术快得多，b)基于机器学习的算法，识别个人 整个完整小鼠大脑中的细胞和c)快速确定1,200个脑中细胞密度的常规程序 由带注释的艾伦脑图谱定义的区域。 在目前的提案中，我们将通过可视化小胶质细胞密度来展示我们的技术的力量 整个小鼠大脑作为神经炎症的生物标志物。小胶质细胞是脑内的常驻免疫细胞 大脑。虽然它们在健康的大脑功能中发挥着重要作用，但它们也介导了神经炎症过程。 对多种神经退行性疾病有显著影响，如阿尔茨海默氏症、帕金森氏症和 多发性硬化症以及在几种神经发育和神经疾病中发挥可能的作用(例如， 精神分裂症、自闭症谱系障碍、脑化疗)。一个用于三维成像的工具 整个大脑的神经炎症模式将有助于我们对神经炎性疾病的理解 在神经系统疾病的过程中，并协助评估治疗方法。 为了可视化小胶质细胞，我们将评估多种策略，包括CX3CR1-GFP小鼠系，这 在小胶质细胞中表达GFP，IBA1免疫标记和其他激活的小胶质细胞的抗体标记。该实用程序 我们监测神经炎症的方法将在CX3CR1-GFP小鼠身上通过治疗它们来演示 用炎症剂脂多糖(LPS)。小鼠将被注射三种不同剂量的内毒素，以及 然后，小胶质细胞将被可视化，并自动对1200个大脑区域进行计数。我们将确认 通过测定炎症蛋白标记物对内毒素引起的神经炎性反应。 我们的技术对神经退行性疾病的适用性将通过研究一只小鼠来证明。 LINE被用作阿尔茨海默病的模型，表现出明显的神经炎症模式。这些 实验证明，我们的方法能够提供可靠和详细的信息描述 我们的结果在速度、分辨率和信息的丰富性方面都好于 任何其他可用的技术。 一旦第一阶段的目标实现，我们将把我们的小胶质细胞激活试验发展成 精确和完整的神经炎症组织学检测的新黄金标准。在第二阶段，我们 计划1)开发机器学习工具，以建立区分休息和休息的形态标准 激活的小胶质细胞，2)表征小胶质细胞在各种已知或未知疾病小鼠模型中的特征 疑似神经炎症成分，以及3)验证抗炎的小胶质细胞靶向 治疗候选人。

SUMMARY Recent technological developments such as tissue clearing and light sheet imaging have allowed for the three- dimensional visualization of the entire brain at cellular resolution. Translucence Biosystems has advanced this technology further by developing a) a proprietary Mesoscale Imaging System that allows visualization of an entire mouse brain much faster than prior techniques, b) machine learning-based algorithms that identify individual cells across the entire intact mouse brain and c) routines that rapidly determine cell densities in >1,200 brain regions defined by the annotated Allen Brain Atlas. In the present proposal we will show the power of our technique by visualizing microglia density across the entire mouse brain as a biological marker for neuroinflammation. Microglia are the resident immune cells in the brain. While they play important roles in healthy brain function, they also mediate neuroinflammatory processes that have a significant impact in multiple neurodegenerative diseases such as Alzheimer's, Parkinson's, and multiple sclerosis as well as play possible roles in several neurodevelopmental and neurological disorders (e.g., schizophrenia, autism spectrum disorder, chemo brain). A tool for three-dimensional imaging of neuroinflammation patterns across the whole brain will help advance our understanding of neuroinflammatory processes in neurological diseases and aid in the evaluation of therapeutic approaches. To visualize microglia, we will evaluate multiple strategies, including the CX3CR1-GFP mouse line, which expresses GFP in microglia, Iba1 immunolabeling and other antibody markers of activated microglia. The utility of our approach for monitoring neuroinflammation will be demonstrated in CX3CR1-GFP mice by treating them with the inflammatory agent lipopolysaccharide (LPS). Mice will be injected with three different LPS doses, and then microglia will be visualized and automatically counted across >1,200 brain regions. We will confirm the neuroinflammatory response to LPS by measuring protein markers of inflammation. The applicability of our technique to neurodegenerative disease will be demonstrated by studying a mouse line used as a model of Alzheimer's disease that presents pronounced patterns of neuroinflammation. These experiments are designed to prove that our approach can provide reliable and detailed information describing neuroinflammation and that our results are better in terms of speed, resolution, and richness of information than any other technique available. Once the goals for Phase I are met, we will be positioned to develop our microglia activation assay into a new gold standard for precise and complete histological detection of neuroinflammation. During phase II, we plan to 1) develop machine learning tools to establish morphological criteria that differentiate resting from activated microglia, 2) characterize microglial signatures in various mouse models of diseases with known or suspected neuroinflammation components, and 3) validate the microglial targeting of anti-inflammatory therapeutic candidates.