Spatiotemporal R-loop accumulation orchestrates microenvironmental remodeling after spinal cord injury.

Spinal cord injury (SCI) induces severe neurological dysfunction through direct mass cell damage and secondary inflammatory molecular cascades. These cascades-initiated by damage-recruit immune cells and amplify cytokine release, exacerbating neuronal death and tissue destruction. We initially report that R-loop accumulation (three-stranded RNA-DNA hybrids with displaced ssDNA) in neural injury contexts drives neurodegeneration via neuroinflammation. Utilizing annotated R-loop-associated genes (loci harboring RNA-DNA hybrids, resolvases, or functionally impacted by R-loops), we characterized fresh spinal cord tissues (50-100 mg) harvested from the cavity of injury (COI) (including gray/white matter) at 0, 1, 3, 7, and 14 days post-injury(dpi) in clip contusion models. Our research characterized dynamic post-injury expression patterns, including stage-specific upregulation of Cdk1, Top2a, Tp53, Tln1, and Flna. Spatial transcriptomics and immunofluorescence revealed significant R-loop accumulation in COI, correlating with tissue damage progression (loss of normal cellular architecture and expanding tissue cavitation quantified by HE/LFB staining). Applying cellular deconvolution-a computational approach inferring the proportional composition of distinct cell types by analyzing bulk-level molecular data-to SCI models, we observed dynamic microenvironment remodeling in COI. This process featured depletion of oligodendrocytes and neurons within post-injury, along with recruitment of diverse immune subsets including peripheral macrophages, bordering leukocytes, and activated microglia. Single-cell analysis and immunofluorescence staining ultimately identified Cdk1 as potentially involved in R-loop-associated microglial (Cd68+) inflammatory infiltration in COI at 7 dpi. These results provide the first evidence of R-loop's potential role in SCI progression, offering new insights for developing therapies aimed at preserving neurological function and promoting repair.