Diaphragm Pacing Enhances Breathing and Activates Spinal Neurons After Cervical Spinal Cord Injury

Cervical spinal cord injury (cSCI) disrupts descending respiratory motor drive and can lead to profound breathing impairments. In a rodent model of cSCI, we previously demonstrated that four days of intermittent diaphragm pacing improves breathing for at least 24 hours after pacing ends. We also showed that phrenic afferent activation during pacing is necessary to elicit these improvements, suggesting plasticity is mediated by central neural circuits. Here we tested the hypothesis that intermittent pacing activates a greater number of spinal neurons in laminae associated with proprioceptive feedback. To test this hypothesis male animals received a left C2 hemisection (C2Hx) and 1-week later were assigned to one of the following groups: control (n=7), diaphragm pacing (n=6), and diaphragm pacing + C4-C6 dorsal rhizotomy (n=6). Animals received 1hr of intermittent diaphragm pacing (30Hz, 0.8mA for 5 minutes on, 5 minutes off) ipsilateral to injury for 4 consecutive days. We performed immunohistochemistry for the immediate early gene cFos to label activated cells and NeuN to label neurons in spinal sections (4-6 per animal) from C3-C5. We quantified cFos positive neurons in quadrants above and below the central canal, ipsilateral and contralateral to diaphragm pacing. We found that animals receiving pacing showed a significant increase in activated neurons compared to controls in the ipsilateral dorsal (62 ± 9 vs 24 ± 8; p = 0.0308; Tukey’s post-hoc), contralateral dorsal (58 ± 7 vs 21 ± 7; p = 0.0023; Tukey’s post-hoc) and contralateral ventral (48 ± 9 vs 16 ± 8; p = 0.0308; Tukey’s post-hoc) regions of the spinal cord. In the contralateral dorsal region this increase after pacing is also increased compared to rhizotomy treated animals (58 ± 7 vs 27 ± 5; p = 0.0138; Tukey’s post-hoc), suggesting neuronal activation in the contralateral dorsal quadrant may be important for pacing-induced improvements in breathing. To further identify the specific location of activated neurons, we examined cFos expression within spinal lamina based on Paxinos and Watson, Brain Atlas 2007 for one representative section per animal. On the contralateral side we found a significant lamina × group interaction (F(16,128) = 2.309, p = 0.0051; Two-way RM ANOVA). Pacing treated animals showed a significant increase in activated neurons compared to controls in lamina 2 (5 ± 2 vs 1 ± 0.5; p=0.0381; Tukey’s post-hoc) and lamina 3 (7 ± 2 vs 2 ± 0.4; p=0.0070; Tukey’s post-hoc). Within lamina 3 we found that this increase is different than rhizotomy treated animals (Lamina 3: 7 ± 2 vs 3 ± 1; p=0.0070; Tukey’s post-hoc). Our data suggest that intermittent diaphragm pacing improves breathing after spinal cord injury by activating the spinal neural network within the contralateral dorsal region, particularly lamina 3 neurons that process proprioceptive input. Identifying the network activated in response to diaphragm pacing is critical to design targeted interventions to improve breathing after spinal cord injury. Funding: This work was supported by funding from the National Institutes of Health, grant numbers: R00HL143207 (Streeter) and R01HL179209 (Streeter) and the National Center for Advancing Translational Sciences Grant Number 2TL1 TR001437 (Holmes). This abstract was presented at the American Physiology Summit 2026 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.