Comprehensive Quantitative Profiling of Cellular Alterations Caused by Injury

摘要 目前，使用低复杂性研究与病理条件相关的细胞改变， 免疫组织化学（IHC）测定，通常利用2-5种抗体，仅揭示了一个微小的子集， 正在发生的变化，缺乏全面的细胞背景，并且不能提供定量的读数， 整个组织的细胞变化。例如，损伤或疾病可以引发复杂的 病理改变跨细胞类型，并在多个尺度，从单个细胞到多细胞 单位和分层的大脑细胞结构。然而，技术限制阻碍了更多的 全面了解这些病理变化。这种缺乏了解的情况阻碍了 我们智能设计有效治疗方案的能力，可能导致了 针对单一细胞类型或特定蛋白质的临床试验。为了弥合我们理解上的这一差距，我们 我建议开发一个全面的脑细胞变化分析工具包（CBAT），一个仔细的 经过验证且广泛适用的图像分析工具包，具有加速调查的前所未有的潜力 和开发下一代脑疾病治疗方法。CBAT，与灵活和 用于高度多重IHC的模块化方案，将能够同时分析所有主要脑细胞类型， 它们的功能/病理状态（例如，静息、反应性、凋亡）。它将提供 从所有类型的单个细胞到多个细胞， 细胞单位（例如小生境）、脑细胞层和脑区域。全面的细胞分析和 使用CBAT产生的测量将使人们能够更深入地了解病理细胞变化， 将加速治疗干预的设计、测试和优化。此外，它将减少 通过用单一的综合性分析代替大量信息量较少的分析， 比色法从长远来看，这将提高我们进行系统级调查的能力， 需要充分理解，并成功地治疗，多种脑部病变。为了实现这些目标， 我们提出了以下目标：目标1：开发和验证一个灵活的、可伸缩的、可扩展的和可复制的 立体定向排列的大鼠中所有主要脑细胞类型的全面整体载玻片成像方法 目标2：开发和验证一个完整的软件系统，分析细胞的识别和状态 从单个细胞到多细胞单位、脑细胞层和脑解剖结构的多个尺度 目标3：测试CBAT系统在全面分析震荡生物学方面的效用， 评估药物组合减少新发现的病理的有效性。经过发展 和验证，CBAT将免费传播给研究界，用于其特定的 研究项目。

ABSTRACT Currently, cellular alterations associated with pathological conditions are studied using low complexity immunohistochemical (IHC) assays, typically utilizing 2-5 antibodies, that only reveal a tiny subset of the alterations that are occurring, lack comprehensive cellular context, and do not provide quantitative readouts of cellular changes throughout the tissue. For example, a injury or disease can initiate a complex web of pathological alterations across cell types, and at multiple scales ranging from individual cells to multi-cellular units and the layered brain cytoarchitecture. However, technological limitations are hindering a more comprehensive global understanding of these pathological changes. This lack of understanding is hampering our ability to intelligently design effective treatment regimens, and may have contributed to the failures of clinical trials that targeted a single cell type or specific protein. To bridge this gap in our understanding, we propose to develop a Comprehensive Brain Cellular Alteration Profiling Toolkit (CBAT), a carefully validated and broadly applicable image analysis toolkit with unprecedented potential to accelerate investigation & development of next-generation treatments for brain diseases. CBAT, in association with a flexible and modular protocol for highly multiplexed IHC, will enable simultaneous profiling of all major brain cell types and their functional/pathological status (e.g., resting, reactive, apoptotic) across whole brain sections. It will provide quantitative readouts of cellular alterations at multiple scales ranging from individual cells of all types to multi- cellular units (e.g. niches), brain cell layers, and brain regions. Comprehensive cellular profiling and measurements generated using CBAT will enable a deeper understanding of pathological cellular changes that will enable accelerated design, testing, and optimization of therapeutic interventions. Further, it will reduce overall experimental costs by replacing a large number of less-informative assays with a single comprehensive assay. In the longer term, it will enhance our ability to conduct the systems-level investigations that will be required for fully understanding, and successfully treating, multiple brain pathologies. To achieve these goals, we propose the following aims: Aim 1: Develop and validate a flexible, scalable, extensible, and reproducible method for comprehensive whole slide imaging of all the major brain cell types in stereotactically aligned rat whole brain sections; Aim 2: Develop and validate a turnkey software system profiling cell identify and status at multiple scales ranging from individual cells to multi-cellular units, brain cell layers, and brain anatomic regions; and Aim 3: Test the utility of the CBAT system to comprehensively profile concussion biology, and assess the effectiveness of a drug combination to reduce newly identified pathologies. After its development and validation, CBAT will be disseminated to the research community at no cost for use in their specific research projects.