Therapeutic mechanism of transcranial iTBS on nerve regeneration and functional recovery in rats with complete spinal cord transection
Background: After spinal cord transection injury, the inflammatory microenvironment formed in the injury site and the cascade of secondary injury results in limited regeneration of injured axons and the apoptosis of neurons in the sensorimotor cortex (SMC). It is crucial to reverse these adverse processes for the recovery of voluntary movement. In this study, rats which had 2-mm spinal cord resection at the T10 level were exposed to transcranial intermittent theta-burst stimulation (iTBS) for the treatment of injury. The mechanism of transcranial iTBS as a new non-invasive neural regulation paradigm in promoting axonal regeneration and motor function repair was explored.  Methods: Rats from the iTBS group were treated with transcranial iTBS 72h after spinal cord injury (SCI). Once a day, 5 days a week, a total of 8 weeks of treatment. Each rat was received behavioral testing every week. Inflammation, neuronal apoptosis, neuroprotective effect, regeneration and synaptic plasticity were measured by immunofluorescence staining, western blotting and mRNA sequencing two or four weeks after SCI. Each rat was received anterograde tracings in the SMC or the long descending propriospinal neurons and tested for motor evoked potentials. Regeneration of corticospinal tract (CST) and 5-hydroxytryptamine (5-HT) nerve fibers were detected eight weeks after SCI. Results: The iTBS group showed reduced inflammatory responses and neuronal apoptosis in the SMC two weeks after treatment when compared to the control group. After four weeks, the neuroimmune microenvironment at the injury site was improved, and neuroprotective effects were seen to promote axonal regeneration and synaptic plasticity. After eight weeks of iTBS treatment, the regeneration of CST in the region rostral to the injured site, 5-HT nerve fibers in the center of the injured area and long descending propriospinal tract (LDPT) fibers in the region caudal to the injured site was significantly increased. Moreover, motor evoked potentials and hindlimb motor function were significantly improved at eight weeks.  Conclusions: The results of neural tracing and neuronal activation further verified that iTBS has the potential to provide neuroprotective effects at early injury stages and pro-regeneration effects related to the 1) CST–5-HT; 2) CST–LDPT; and 3) CST–5-HT–LDPT descending motor pathways and revealed the relationships among neural pathway activation, neuroimmune regulation, neuroprotection, and axonal regeneration, as well as the interaction network of key genes. The proposed non-invasive transcranial iTBS treatment is expected to provide a serviceable practical and theoretical support for spinal cord injury.