Transcriptomic and Functional Studies Reveal Undermined Chemotactic and Angiostimulatory Properties of Aged Microglia During Stroke Recovery

Age-dependent alterations in microglia behavior have been implicated in neurodegeneration and CNS injuries. Here, we compared the transcriptional profiles of young versus aged microglia during stroke recovery. CD45intermediateCD11b+ microglia were FACS-isolated from the brains of young (10-week-old) and aged (18-month-old) male mice 14 days after distal middle cerebral artery occlusion (dMCAO) or sham operation and subjected to RNA-sequencing analysis. Functional groups enriched in young microglia are indicative of upregulation in cell movement, cell interactions, inflammatory responses and angiogenesis, while aged microglia exhibited a reduction or no change in these features. We confirmed reduced chemoattractive capacities of aged microglia toward ischemic brain tissue in organotypic slide co-cultures, and delayed accumulation of aged microglia around dead neurons injected into the striatum in vivo. In addition, aging is associated with an overall failure to increase the expression of microglial genes involved in cell-cell interactions, such as CXCL10. Finally, impaired upregulation of pro-angiogenic genes in aged microglia was associated with a decline in neovascularization in aged mice compared to young mice after dMCAO. This study provides a new resource to understand the mechanisms underlying microglial alterations in the aged brain milieu and sheds light on new strategies to improve microglial functions in aged stroke victims. Overall design: CD45intermediateCD11b+ microglia were FACS-isolated from the brains of young (10-week-old) and aged (18-month-old) male mice 14 days after distal middle cerebral artery occlusion (dMCAO) or sham operation and subjected to RNA-sequencing analysis.

小胶质细胞（microglia）的年龄依赖性行为改变已被证实与神经退行性疾病及中枢神经系统（Central Nervous System, CNS）损伤相关。本研究对比了脑卒中恢复期间年轻与衰老小胶质细胞的转录组谱。我们从大脑中动脉远端闭塞（distal middle cerebral artery occlusion, dMCAO）或假手术后14天的年轻（10周龄）与衰老（18月龄）雄性小鼠脑中，通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）分离得到CD45中间型CD11b阳性小胶质细胞，并开展RNA测序（RNA-sequencing）分析。
富集于年轻小胶质细胞的功能群提示，细胞运动、细胞互作、炎症应答及血管生成通路均出现上调；而衰老小胶质细胞在上述特征上则表现为表达下调或无显著变化。我们通过器官型玻片共培养实验证实，衰老小胶质细胞对缺血脑组织的趋化能力减弱；同时在体内实验中，注射至纹状体（striatum）的死亡神经元周围，衰老小胶质细胞的聚集出现延迟。
此外，衰老与小胶质细胞参与细胞间互作的基因（如CXCL10）的整体表达上调失败密切相关。最后，与年轻小鼠相比，大脑中动脉远端闭塞术后的衰老小鼠，其小胶质细胞中促血管生成基因的上调受损，这与新生血管形成（neovascularization）能力下降相关。
本研究为理解衰老脑微环境中小胶质细胞改变的潜在机制提供了全新的研究资源，并为改善衰老脑卒中患者的小胶质细胞功能提供了新的治疗策略方向。
整体实验设计：我们从dMCAO或假手术后14天的年轻（10周龄）与衰老（18月龄）雄性小鼠脑中，通过FACS分离CD45中间型CD11b阳性小胶质细胞，并进行RNA测序分析。