Targeting Dnmt3a/m5C/RelA Axis Attenuates Microglia Inflammatory Response and Improves Postoperative Recovery in Chronic Compressive Cervical Spinal Cord Injury

Chronic compressive cervical spinal cord injury (cCSCI) is a major cause of adult spinal cord dysfunction. Surgical decompression is the primary treatment; however, microglia-driven neuroinflammation often hampers postoperative recovery. This study was conducted to investigate the role of 5-methylcytosine (m5C) in sustaining pro-inflammatory microglial states following decompression. Using a mouse cCSCI model, we performed single-nucleus RNA sequencing (snRNA-seq) to map microglial states, calculated an m5C-regulator activity score, and validated m5C changes via dot blot. We examined the Dnmt3a-RelA pathway in primary microglia through siRNA knockdown, m5C-RIP, and mRNA stability assays. Therapeutic potential was assessed with intrathecal AAV-shDnmt3a. SnRNA-seq revealed a dominant pro-inflammatory microglial subpopulation (C1) post-decompression, enriched for NF-κB signaling. The C1 subset exhibited an elevated m5C score and increased Dnmt3a expression. Dnmt3a knockdown reduced m5C enrichment on RelA transcripts, destabilized RelA mRNA, and suppressed NF-κB activation. In vitro, Dnmt3a silencing reduced pro-inflammatory marker expression and cytokine release, and microglia-targeted AAV-shDnmt3a improved neurological recovery in cCSCI mice. These findings underscore the potential of targeting the microglia Dnmt3a/m5C/RelA axis to enhance postoperative recovery.