Objective:To explore how trans-spinal intermittent theta burst stimulation (iTBS) might inhibit ferroptosis using a mouse model of acute spinal cord injury (SCI).Methods:Thirty C57BL/6J mice were randomly assigned to control, model, and iTBS groups, each of 10. SCI was induced at the T 9/T 10 vertebral level by laminectomy and contusion injury using an impactor. The control group underwent laminectomy only. On the 1st day post-injury (dpi), mice in the iTBS group began receiving intermittent theta burst stimulation of the spine daily. The resting motor threshold (RMT) was determined as 25% of the magnetic stimulator′s maximum output intensity, and the stimulation intensity was set at 80% of the average RMT. The treatment was administered twice daily, with each session consisting of 10 bursts at 50Hz, repeated 40 times at 5Hz intervals (3 pulses per burst). The treatment continued until 28dpi. Immunofluorescence was used to assess the expression of glutathione peroxidase 4 (GPX4) and acyl-CoA synthetase long-chain family member 4 (ACSL4). Western blotting quantified the levels of GPX4 and ACSL4 protein. Iron deposition in the spinal cord tissue was evaluated using Prussian blue staining. Iron concentration, glutathione (GSH), and malondialdehyde (MDA) levels in the spinal cord tissue were measured using commercial assay kits, while locomotor functioning was assessed using the Basso Mouse Scale (BMS) on 1st, 3rd, 7th, 14th and 28thdpi. Results:The model group exhibited significantly increased iron levels and prominent iron deposition in the spinal cord compared to the control group, while significantly reduced iron levels and iron deposition were observed in the iTBS group. The immunofluorescence and western blotting revealed that GPX4 expression was downregulated and ACSL4 expression was upregulated in the SCI model group compared to the control group. iTBS treatment significantly upregulated GPX4 and downregulated ACSL4 expression. In addition, the iTBS group showed significantly lower MDA levels and significantly higher GSH levels in their spinal cord tissue compared to the SCI model group. Locomotion, assessed in terms of BMS scores, was significantly improved in the iTBS group compared to the SCI model group on 7th, 14th, and 28thdpi.Conclusions:These findings suggest that iTBS delivered via the spinal cord effectively inhibits ferroptosis and improves locomotion after a SCI, potentially by restoring iron homeostasis, enhancing antioxidant capacity, and suppressing lipid peroxidation.

Objective:To explore how trans-spinal intermittent theta burst stimulation (iTBS) might inhibit ferroptosis using a mouse model of acute spinal cord injury (SCI).Methods:Thirty C57BL/6J mice were randomly assigned to control, model, and iTBS groups, each of 10. SCI was induced at the T 9/T 10 vertebral level by laminectomy and contusion injury using an impactor. The control group underwent laminectomy only. On the 1st day post-injury (dpi), mice in the iTBS group began receiving intermittent theta burst stimulation of the spine daily. The resting motor threshold (RMT) was determined as 25% of the magnetic stimulator′s maximum output intensity, and the stimulation intensity was set at 80% of the average RMT. The treatment was administered twice daily, with each session consisting of 10 bursts at 50Hz, repeated 40 times at 5Hz intervals (3 pulses per burst). The treatment continued until 28dpi. Immunofluorescence was used to assess the expression of glutathione peroxidase 4 (GPX4) and acyl-CoA synthetase long-chain family member 4 (ACSL4). Western blotting quantified the levels of GPX4 and ACSL4 protein. Iron deposition in the spinal cord tissue was evaluated using Prussian blue staining. Iron concentration, glutathione (GSH), and malondialdehyde (MDA) levels in the spinal cord tissue were measured using commercial assay kits, while locomotor functioning was assessed using the Basso Mouse Scale (BMS) on 1st, 3rd, 7th, 14th and 28thdpi. Results:The model group exhibited significantly increased iron levels and prominent iron deposition in the spinal cord compared to the control group, while significantly reduced iron levels and iron deposition were observed in the iTBS group. The immunofluorescence and western blotting revealed that GPX4 expression was downregulated and ACSL4 expression was upregulated in the SCI model group compared to the control group. iTBS treatment significantly upregulated GPX4 and downregulated ACSL4 expression. In addition, the iTBS group showed significantly lower MDA levels and significantly higher GSH levels in their spinal cord tissue compared to the SCI model group. Locomotion, assessed in terms of BMS scores, was significantly improved in the iTBS group compared to the SCI model group on 7th, 14th, and 28thdpi.Conclusions:These findings suggest that iTBS delivered via the spinal cord effectively inhibits ferroptosis and improves locomotion after a SCI, potentially by restoring iron homeostasis, enhancing antioxidant capacity, and suppressing lipid peroxidation.