rTMS-induced motor cortex activation drives neural network tissueoid mediated spinal motor neural pathway reconstruction

<b>Rationale:</b> The integration of biological and physical interventions represents a promising therapeutic strategy for spinal cord injury (SCI), offering a novel approach to restore disrupted motor pathways. This study investigates whether repetitive transcranial magnetic stimulation (rTMS) can prevent cerebral neuroapoptosis and promote the regeneration and integration of brain-derived nerve fibers with neural network tissueoids (NNToids) following SCI. <b>Methods:</b> Neural stem cell-derived NNToids were transplanted into rats with complete SCI and simultaneously treated with 10 Hz rTMS. Neuroinflammatory responses, neuroapoptosis, neuronal activation, and axonal regeneration were systematically evaluated using transcriptomic sequencing, histological validation, Western blotting, and neural tract tracing. The responsiveness of NNToids to 10 Hz rTMS in facilitating motor neural pathway reconstruction was also assessed. <b>Results:</b> 10 Hz rTMS significantly enhanced cFOS expression in layer V pyramidal neurons of the sensorimotor cortex (SMC), markedly reduced microglial activation and neuroapoptosis, and upregulated the expression of mitochondrial-related protein TOM20, axonal regeneration marker p-S6, and synaptic plasticity-associated protein Arc in SMC neurons. NNToids facilitated the ingrowth of corticospinal tract (CST) and 5-hydroxytryptamine (5-HT) - positive nerve fibers into the transplantation site. Retrograde PRV tracing demonstrated that 10 Hz rTMS enhanced the capacity of NNToid neurons to relay CST and 5-HT signals to hindlimb motor neurons. Functional assessments and cortical motor evoked potentials confirmed that the rTMS-NNToid combination improved the transmission of motor-related neural signals to the hindlimbs. Histological analysis further demonstrated that activated NNToid neurons exhibited increased expression of N-methyl-D-aspartate receptors (NMDAR) and formed more synaptic connections with vGluT-positive axon terminals. <b>Conclusion:</b> These findings demonstrate that rTMS mitigates motor cortex inflammation, promotes the regeneration and integration of brain-derived nerve fibers with NNToid neurons, thereby establishing a foundation for motor function recovery. Moreover, the study identifies the mechanism through which NNToid neurons mediate motor neural pathway reconstruction under rTMS modulation. Although based on a rat model, this work provides a promising framework for future biophysical therapies that combine patient-derived autologous iPSC-based NNToids with non-invasive brain stimulation.