Screening the optimal rTSMS frequency to orchestrate immune-fibrotic remodeling for adult spinal cord repair

Introduction: The clinical translation of magnetic stimulation for central nervous system trauma is severely hindered by "parameter ambiguity"-the lack of evidence-based screen of stimulation protocol. Repetitive trans-spinal magnetic stimulation (rTSMS) holds therapeutic promise, yet its frequency-dependent effects on the complex spinal microenvironment remain poorly understood. To address this gap, this study aimed to decode the frequency-response relationship of rTSMS and establish an optimal, evidence-based frequency standard to orchestrate immune-fibrotic remodeling and promote functional recovery following spinal cord injury (SCI). Methods: label-free second-harmonic generation (SHG) imaging to visualize the real-time microglia activation, we performed a high-fidelity close-loop screen of various rTSMS frequencies (10 Hz, 40 Hz, and 80 Hz). In an adult mice SCI model, we integrated transcriptomic profiling, morphological analysis, electrophysiological recordings, and behavioral assessments to comprehensively evaluate the neuroregenerative potential. Results: We identified 40 Hz as a privileged therapeutic frequency that specifically modulates microglia and the extracellular matrix. Unlike 10 Hz or 80 Hz regimens, 40 Hz rTSMS uniquely reprogrammed the transcriptomic landscape, driving microglia toward a restorative M2 phenotype, and most importantly, suppressing collagen fibrillogenesis. This targeted modulation effectively attenuated fibrotic scarring and preserved mitochondrial dynamics and axonal integrity. Consequently, the 40 Hz protocol significantly enhanced corticospinal tract conduction and drove robust, long term sensorimotor recovery. Conclusion: Our findings define 40 Hz as the critical therapeutic standard for coupling immune modulation with fibrotic remodeling in SCI. By overcoming the barrier of inconsistent parameters, this study provides a precise, clinically translatable framework for the application of rTSMS in neurorestorative medicine.