FOS Expression in Respiratory Motor Networks After Cervical Spinal Cord Stimulation and Injury

Low current stimulation of the dorsal surface of the spinal cord (epidural stimulation) has been shown to restore volitional control of various functions after spinal cord injury (SCI) but has not been clinically translated to breathing. Our lab uses closed-loop epidural stimulation to restore breathing after cervical SCI, using diaphragm EMG activity to trigger short trains of stimulation only during respiratory effort (e.g. closed-loop). We have previously demonstrated that this approach restores some ipsilesional diaphragm EMG activity in rats with C2 hemisection (C2HS) (Mickle et al., 2024); however, it is unknown what neural populations are activated by stimulation to achieve this motor output. This study investigates stimulation and injury-induced changes in neuronal activity within respiratory motor networks, assessed through FOS expression, to see if stimulation activates particular neurons in the spinal diaphragm and intercostal motor pools. Here, we investigate FOS expression at the spinal segments C4, the location of the stimulus electrodes and likely greatest effect of stimulation, and at T4, a distal respiratory motor pool isolated from direct stimulation. Male Sprague-Dawley rats (3–5 months old) were urethane anesthetized and mechanically ventilated. Stimulation electrodes were sutured to the dorsal surface of C4 and recording EMG electrodes sutured to the diaphragm. Rats were then either C2 hemisected or left intact before undergoing two bouts of contralesional EMG-triggered stimulation (in-phase and out-of-phase) separated by 20 minutes or a sham period. Rats were perfused 60 minutes after the end of stimulation and T4 and C4 spinal tissue sectioned at 15 µm and stained for FOS mRNA via RNAScope (sham intact C4: n = 4, T4: n =3; sham C2HS C4: n = 4, T4: n = 4; stim intact C4: n = 8, T4: n = 3; stim C2HS C4: n = 8, T4: n = 4). Cell nuclei were stained with DAPI and identified using StarDist. FOS positivity was detected using a channel intensity threshold classifier. At C4, there was no significant effect of injury or stimulation on the total percentage of all cells positive for FOS. However, stimulation resulted in more gray matter FOS positivity in both injured and intact rats (proportion of total FOS positivity in gray matter mean +/- SD: stim intact 64 +/- 13%, stim C2HS 68 +/- 16%, sham intact 38 +/- 11%, sham C2HS 35 +/- 14%; main effect of stimulation, p = 0.0001). Work is ongoing to colocalize FOS positive cells with various neural markers to identify a population of neurons necessary for stimulation-induced increases in diaphragm EMG. In contrast, at T4, C2HS resulted in elevated FOS positivity in gray matter compared to intact rats (% cells FOS+ in gray matter intact vs C2HS: 0.58 +/- 0.11% vs 0.95 +/- 0.10%, p = 0.025), suggesting injury increases neuronal firing at the intercostal motor pool, potentially as a compensatory mechanism, independent of stimulation. While stimulation did not alter gray matter FOS expression at T4, stimulation decreased white matter FOS positivity in hemisected rats while in intact rats white matter positivity relative to gray was increased with stimulation (proportion of total FOS positivity in white matter: stim intact 56.6 +/- 9.5%, sham intact 38.7 +/- 15.0%, stim C2HS 30.3 +/- 5.4%, sham C2HS 45.6 +/- 8.6%; interaction of stimulation and injury, p = 0.01). Thus, closed-loop epidural stimulation influences neuronal activity in respiratory motor networks after cervical SCI, with distinct effects at local (C4) and distal (T4) spinal segments. These findings contribute to the understanding of ES mechanisms and its potential application for restoring respiratory function after SCI. University Scholars Program (CB), R01HL153102 (ED), T32HL134621 (AM), Bryan Robinson Endowment Grant for the Neurosciences (AM), Grand Aerie Fraternal Order of Eagles (ED) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.