Single cell RNA-seq characterization of CNS myeloid cells after ischemic preconditioning

项目摘要 中风是美国的第五大死亡原因，也是长期死亡的第一大原因。 成年人的长期残疾。急性脑卒中有效药物治疗进展 尽管有大量可靠的数据表明， 神经胶质细胞，包括小胶质细胞，在缺血性损伤的病理生理学。缺血预处理（IPC） 是短暂的局部缺血，其赋予针对随后的局部缺血事件的强有力的神经保护。 阐明IPC的细胞和分子介质被认为是卒中研究的关键挑战 具有很好的治疗应用前景。我们小组和其他人的研究表明， 免疫信号通路对于IPC介导的神经保护至关重要，我们已经 特别确定了小胶质细胞中这些先天免疫途径的要求。的研究 其他研究小组提出了浸润性外周免疫细胞在支持IPC中的可能作用， 在中枢神经系统（CNS）介导的保护，然而，个体免疫细胞的贡献 并没有得到很好的理解。此外，越来越多的数据表明， 在白色和灰质小胶质细胞之间。在本研究中，我们将收集单细胞RNA测序， 在动物暴露于IPC后，来自皮质的骨髓细胞的scRNA-seq数据 刺激、长期脑缺血（中风）或两者。我们将使用此数据集来识别差异 基于这些不同实验范例中的基因表达谱的细胞簇， 确定神经免疫和外周免疫细胞对IPC和脑缺血的反应，以及如何 后者通过先前的IPC刺激被调节到神经保护环境。此外，我们将执行 这些实验在缺血/再灌注损伤的白色物质模型中进行， 长时间脑缺血后白色物质和灰质损伤之间的反应。收集 由于受影响的白色物质体积较大，因此白色物质占优势的束的数据很重要 与以灰质为主的体内小鼠模型相比，这些 研究将帮助我们更好地了解免疫细胞对缺血/再灌注损伤的作用 并鉴定用于中风患者治疗干预的潜在细胞和分子靶点。

PROJECT SUMMARY Stroke is the 5th leading cause of death in the United States and the number one leading cause of long- term disability in adults. Progress in the development of effective pharmacotherapies for acute stroke has been limited and centered on neuronal preservation despite extensive and robust data implicating glial cells, including microglia, in the pathophysiology of ischemic injury. Ischemic preconditioning (IPC) is a brief period of ischemia that confers robust neuroprotection against subsequent ischemic events. Elucidating cellular and molecular mediators of IPC is considered a critical challenge in stroke research with promising therapeutic applications. Research by our group and others has implicated innate immune signaling pathways as being critical for IPC-mediated neuroprotection, and we have specifically identified a requirement for these innate immune pathways in microglia. Research from other groups have suggested possible roles for infiltrating peripheral immune cells in supporting IPC- mediated protection in the central nervous system (CNS), however individual immune cell contributions are not well understood. Furthermore, a growing body of data suggest differential responses to injury between white and grey matter microglia. In this study we will collect single cell RNA sequencing (scRNA-seq) data from the myeloid cells of the cortex after animals have been exposed to an IPC stimulus, prolonged cerebral ischemia (stroke), or both. We will use this dataset to identify differential clusters of cells based on gene expression profiles in these different experimental paradigms to determine neuroimmune and peripheral immune cell responses to IPC and cerebral ischemia, and how the latter is modulated to a neuroprotective milieu by a prior IPC stimulus. Additionally, we will perform these experiments in a white matter model of ischemia/reperfusion injury to elucidate differential responses between white matter and grey matter injury after prolonged cerebral ischemia. Gathering data on white matter predominant tracts is important due to the large volume of white matter impacted in human stroke patients compared to in vivo mouse models, which are grey matter predominant. These studies will help us better understand the contribution of immune cells to ischemia/reperfusion injury and identify potential cellular and molecular targets for therapeutic intervention in stroke patients.