Dynamic fibroblast-immune interactions shape recovery after brain injury [snRNASeq_Timecourse]

Fibroblasts and immune cells coordinate tissue regeneration and necessary scarring after injury. In the brain, fibroblasts are border-enriched cells whose dynamic molecular states and immune interactions after injury remain unclear. We used single nuclear RNA sequencing at multiple timepoints to profile stromal and immune responses to sterile (photothrombotic, PT) injury. We find that early pro-fibrotic myofibroblasts transition into several states, including meningeal and lymphocyte-interactive fibroblasts. Bulk nuclei (DAPI+) were isolated and sort-purified from WT mice at rest (dural meninges and cortex), 2 days post injury (dpi; PT lesion and perilesional cortex), 7dpi, and 21dpi. 1 male and 1 female mouse were harvested per timepoint and were processed together. Microanatomical regions from each timepoint were processed separately and barcoded using 10X CellPlex barcodes; microanatomical regions from a given timepoint were combined prior to sorting and sequenced together. Resting dural meninges were enriched for fibroblasts using Col1a2(creER); R26(Sun1GFP) nuclear signal. Mice with impaired Tgfb signaling were harvested at 7 and 21dpi but were discarded after initial clustering and not analyzed separately due to low yield.