Brain-wide quantitative mapping of microglia activation

总结 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在这方面取得了进展， a）专有的中尺度成像系统，可以可视化整个 B）基于机器学习的算法， c）快速测定> 1，200个脑中细胞密度的程序 由注释的艾伦脑图谱定义的区域。 在本提案中，我们将通过可视化小胶质细胞密度来展示我们技术的力量。 整个小鼠大脑作为神经炎症的生物标志物。小胶质细胞是免疫系统中的常驻免疫细胞。 个脑袋虽然它们在健康的大脑功能中发挥重要作用，但它们也介导神经炎症过程 对多种神经退行性疾病如阿尔茨海默氏症、帕金森氏症和 多发性硬化症以及在几种神经发育和神经障碍中发挥可能的作用（例如， 精神分裂症、自闭症谱系障碍、化疗脑）。一种三维成像工具， 整个大脑的神经炎症模式将有助于推进我们对神经炎症的理解 神经系统疾病的过程，并在治疗方法的评估援助。 为了可视化小胶质细胞，我们将评估多种策略，包括CX 3CR 1-GFP小鼠系， 在小胶质细胞中表达GFP，Iba 1免疫标记和活化的小胶质细胞的其他抗体标记。本实用 我们监测神经炎症的方法将在CX 3CR 1-GFP小鼠中得到证实， 与炎症因子脂多糖（LPS）。小鼠将注射三种不同剂量的LPS， 然后小胶质细胞将被可视化并在> 1，200个大脑区域中自动计数。我们将确认 通过测量炎症的蛋白质标志物来检测LPS对神经炎症反应的影响。 我们的技术对神经退行性疾病的适用性将通过研究小鼠来证明 线用作阿尔茨海默病的模型，呈现明显的神经炎症模式。这些 实验证明，我们的方法可以提供可靠和详细的信息描述， 我们的结果在速度、分辨率和信息丰富性方面优于 任何其他可用的技术。 一旦达到第一阶段的目标，我们将定位于将我们的小胶质细胞活化试验发展成一种新的方法。 精确、完整的神经炎症组织学检测的新金标准。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 活化的小胶质细胞，2）表征具有已知或 可疑的神经炎症成分，和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.