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

项目总结 中风是美国第五大死亡原因，也是导致长寿的第一大原因。 成年人的长期残疾。急性卒中有效药物治疗的研究进展 一直局限于神经元的保存，尽管广泛而有力的数据表明 包括小胶质细胞在内的胶质细胞在缺血性损伤的病理生理学中的作用。缺血预适应(IPC) 是一段短暂的缺血期，对随后的缺血事件提供强大的神经保护。 阐明IPC的细胞和分子介质被认为是卒中研究中的一个关键挑战 具有很好的治疗应用前景。我们团队和其他人的研究表明，先天 免疫信号通路对IPC介导的神经保护至关重要，我们有 特别确定了小胶质细胞对这些先天免疫途径的需求。研究来自 其他组织也提出了渗透外周免疫细胞在支持IPC中的可能作用。 中枢神经系统(CNS)的中介保护，但个体免疫细胞的作用 都没有被很好地理解。此外，越来越多的数据表明，对伤害的反应不同。 在白质和灰质小胶质细胞之间。在这项研究中，我们将收集单细胞RNA测序 动物暴露于IPC后大脑皮层髓系细胞的(scRNA-seq)数据 刺激，延长脑缺血(中风)，或两者兼而有之。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群 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 后者被先前的IPC刺激调节到神经保护环境。此外，我们还将表演 这些实验在脑白质缺血/再灌注损伤模型中阐明区别 长时间脑缺血后脑白质和灰质损伤的反应。采集 由于受影响的脑白质体积很大，因此脑白质占优势的区域的数据很重要 在人类中风患者与活体小鼠模型相比，这是灰质占主导地位。这些 研究将帮助我们更好地了解免疫细胞在缺血/再灌注损伤中的作用 并确定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.