Unraveling the mechanism of transcutaneous spinal cord stimulation in mitigating autonomic dysreflexia in experimental spinal cord injury

Background: Autonomic dysreflexia (AD) is a life-threatening hypertensive condition that occurs after cervical or thoracic spinal cord injury (SCI) due to uncontrolled sympathetic reflexes. Sympathetic preganglionic neurons (SPNs), primarily localized within the intermediolateral cell column (IML) undergo maladaptive plasticity post-SCI and are strongly activated by visceral stimuli such as colorectal distension (CRD), and induce AD. Non-invasive transcutaneous spinal cord stimulation (tSCS) has shown promise in reducing AD. However, its underlying neuronal mechanisms remain unknown. Hence, this study investigated whether tSCS prevents AD by recruiting inhibitory spinal interneurons (INs). Methods: Forty-six adults male Wistar rats were assigned to five groups: sham-injured controls: T3 Laminectomy without SCI; SCI+CRD: SCI with CRD; SCI+CRD+tSCS: SCI with CRD and tSCS; SCI+tSCS: SCI with tSCS only. A T3 contusion injury was performed in all SCI groups. At six weeks post-injury, CRD was delivered (60-second inflation–deflation cycles, using 2 mL air filling in balloon tipped catheter). We delivered concurrent tSCS at 30 Hz, biphasic 1 ms, 5 mA via a surface electrode at the T7 spinal segment. Fluoro-Gold (FG) was injected (i.p.) at five-weeks post-SCI, immunofluorescence staining was performed with markers; c-Fos, NeuN (Neuronal nuclei) choline acetyltransferase (ChAT), Glutamic acid decarboxylase-67 (GAD67), and Glycine receptor alpha-1(GlyR-α1). Results: Compared to sham group, expression of c-Fos was significantly elevated across the sampled neuraxis in the SCI+CRD, SCI+tSCS, and SCI+CRD+tSCS groups (p<0.01). However, the combined application of CRD and tSCS markedly reduced this CRD-evoked c-fos activation compared to CRD only group (p<0.01). Similarly, c-Fos⁺ChAT⁺ SPNs were significantly increased in SCI+CRD animals relative to sham, whereas their activation was substantially diminished in SCI+CRD+tSCS animals. In contrast, inhibitory INs showed the opposite pattern. SCI+CRD+tSCS animals exhibited a robust increase in activated GAD67⁺ (GABAergic) neurons, and GlyR-α1⁺ (glycinergic input receiving neurons) compared to their activation in SCI+CRD animals. Conclusion: tSCS prevents CRD-induced spinal neurons and SPNs activation in parallel to recruiting inhibitory INs in the spinal cord. These findings provide mechanistic insight into how tSCS modulates spinal autonomic circuits involved in AD mechanism.