Single-cell analyses reveal the dynamic functions of Itgb2+ microglia subclusters at different stages of cerebral ischemia-reperfusion injury in transient middle cerebral occlusion mice model

The underlying pathophysiological mechanisms of cerebral ischemia-reperfusion injury (CIRI) is intricate, and current studies suggest that neuron, astrocyte, microglia, endothelial cell, and pericyte all have different phenotypic changes of specific cell types after ischemic stroke. And microglia account for the largest proportion after CIRI. Previous transcriptomic studies of ischemic stroke have typically focused on the 24 hours after CIRI, obscuring the dynamics of cellular subclusters throughout the disease process. Therefore, traditional methods for identifying cell types and their subclusters may not be sufficient to fully unveil the complexity of single-cell transcriptional profile dynamics caused by an ischemic stroke. In this study, to explore the dynamic transcriptional profile of single cells after CIRI, we used scSTAR, a new bioinformatics method, to analyze the single cell transcriptional profile of day 1, 3 and 7 of transient middle cerebral artery occlusion (tMCAO) mice. Combining our bulk RNA sequence and proteomics data, we found that the Itgb2+ subclusters of microglia exhibited specific functions at three different time points after the tMCAO. Our further analysis revealed that the Itgb2+ microglia subcluster was mainly involved in energy metabolism, cell cycle, angiogenesis, neuronal myelin formation and repair at 1, 3 and 7 days after tMCAO, respectively. Our results suggested that Itgb2+ microglia act as a time-specific multifunctional immunomodulatory subcluster during CIRI, and the underlying mechanisms remain to be further investigated. Single-cell sequencing of the ipsilateral cerebral hemispheres of mice at 1, 3, 7 days after tMCAO and at day 7 of sham group.

脑缺血再灌注损伤（cerebral ischemia-reperfusion injury, CIRI）的潜在病理生理机制极为复杂，现有研究表明，缺血性脑卒中发生后，神经元、星形胶质细胞、小胶质细胞、内皮细胞以及周细胞均会出现各自特异性的表型改变。其中，小胶质细胞在CIRI后占比最高。既往缺血性脑卒中转录组学研究多聚焦于CIRI发生后的24小时内，这掩盖了疾病全程中细胞亚群的动态变化过程。因此，传统的细胞类型及其亚群鉴定方法，或不足以全面揭示缺血性脑卒中所引发的单细胞转录组动态复杂性。本研究为探究CIRI后单细胞的动态转录组谱特征，采用新型生物信息学方法scSTAR，对暂时性大脑中动脉闭塞（transient middle cerebral artery occlusion, tMCAO）模型小鼠在造模后第1、3、7天的单细胞转录组数据进行分析。结合本研究的批量RNA测序（bulk RNA sequence）与蛋白质组学数据，我们发现小胶质细胞的Itgb2+亚群在tMCAO后的三个不同时间点呈现出特异性功能。进一步分析显示，Itgb2+小胶质细胞亚群分别在tMCAO后第1、3、7天主要参与能量代谢、细胞周期、血管生成、神经元髓鞘形成与修复过程。本研究结果表明，Itgb2+小胶质细胞在CIRI进程中发挥时序特异性的多功能免疫调节亚群作用，其潜在调控机制仍有待进一步探索。本研究的测序样本包括tMCAO模型小鼠造模后第1、3、7天的缺血侧大脑半球，以及假手术组小鼠第7天的同侧大脑半球。