Genetic lineage tracing and single cell RNAseq analysis identifies unique feature and cell interactions in resident microglia and infiltrating monocyte derived macrophage at different stages of ischemic stroke

Ischemic stroke elicits a strong neuroinflammatory response, characterized by the activation of microglia and infiltrating immune cells from the very acute stages onward. Although microglia participate in many critical aspects of stroke pathology, their exact role at different stages of ischemic injury remains unclear and debated. Similarly, infiltrating peripheral monocytes/macrophages (iMM) have also been implicated in lesion formation and expansion. Microglia and iMM have been speculated to have differential transcriptomic profiles and functional roles in CNS injury. However, because the gene expression profiles of microglia and iMM overlap in the injured brain, distinguishing these two cell populations has posed a challenge for the field. Using a recently developed and characterized microglia-specific TMEM119CreER-Ai14 tdTomato reporter mouse model, we prelabeled microglia in the CNS prior to stroke, enabling detailed characterization of the transcriptomic and spatial distributions of microglia vs iMMs across different stages of stroke. Here, we report that microglia and iMMs are enriched at distinct locations and exhibit differential temporal dynamics in the stroke brain, around the ischemic core and peri-infarct area. We also observed that iMM markedly increased in the infarct core on D3 after stroke, peaked at D7, and was no longer detected at D30, suggesting that iMM in the stroke brain is not long-living. Additionally, Single-cell RNAseq further revealed distinct transcriptomic states of microglia and iMM at D7 and D14, which also show overlapping and distinct cell-cell interactions with other immune cells in the stroke brain. Genetic tracing also allowed us to identify novel markers for activated microglia vs iMM in stroke brain, such as Gm21188, which is enriched specifically in iMM after stroke. Finally, our data also showed that Igf1 was up-regulated in both microglia and iMM after stroke. In contrast to the recently reported protective role of microglial IGF-1 in the hyperacute stage of stroke, our data show that Cx3cr1ERT2 Igf1fl/fl mediated Igf1 gene KO at 5-9 Days post-stroke results in expedited sensorimotor function recovery but worsened anxiety-like behavior after stroke. This is accompanied by decreased T-cell infiltration in the stroke brain in the Igf1 iKO stroke mice. In summary, this study provides valuable insights for future investigations aimed at modulating microglia, iMM, and their intercellular communication to promote stroke resolution and functional recovery in vivo.