Cervical Repetitive Magnetic Stimulation Enhances Respiratory Recovery by Modulating Neuronal Plasticity After Cervical Spinal Cord Injury

10 Cervical spinal cord injury (SCI) frequently leads to life-threatening respiratory insufficiency by 11 disrupting descending phrenic pathways. There is growing interest in non-invasive 12 neuromodulatory approaches to enhance plasticity of spared respiratory circuits. We investigated 13 whether cervical repetitive magnetic stimulation (rMS) applied to the injured cervical spinal cord 14 promotes ventilatory recovery in a preclinical mouse model. Adult mice received a unilateral C3 15 hemicontusion followed by either rMS or sham stimulation. We found that rMS-treated mice 16 significantly improved recovery of tidal volume and minute ventilation at 21 days post injury(dpi) 17 compared to sham controls under various breathing conditions (isoflurane anesthesia, 18 poikilocapnic phase and hypercapnic challenge). Correspondingly, diaphragm EMG enhanced 19 ipsilateral hemidiaphragm activity in ventral and medial regions, and even contralateral 20 hemidiaphragm activity in its ventral part. This was associated with a marked attenuation of the 21 2 inflammatory response at the cervical spinal cord level. Indeed, rMS lowered astroglial, fibrotic 22 scarring, pro-inflammatory CD68-, Iba1- microglial/macrophage markers. Moreover, 23 perineuronal net expression (WFA positive staining) is globally reduced in the ventral spinal horn, 24 whereas at the lesion site it is markedly increased and tightly wrapped around motoneurons. 25 Together, these findings demonstrate that rMS promotes functional respiratory recovery after 26 cervical SCI through combined enhancement of diaphragmatic motor output and modulation of 27 the inflammatory and extracellular environment. Together, these functional and cellular findings 28 indicate that spinal rMS promotes a permissive, pro-regenerative environment supporting 29 respiratory circuit plasticity. We conclude that rMS significantly enhances ventilatory recovery 30 via reduced inflammatory response and improved intraspinal rewiring after high cervical SCI, 31 suggesting it is a promising non-invasive strategy. The ability of rMS to engage spared 32 respiratory networks and support neuroplasticity highlights its promise as a safe, non-invasive 33 therapeutic strategy with translational potential for rehabilitation of breathing function after SCI. 34 Key Word: Cervical spinal cord injury/Respiratory plasticity/Diaphragm electromyography/ 35 Neuroinflammation/Repetitive magnetic stimulation 36

37 Spinal cord injury (SCI) affects over 20.6 million people worldwide, with 900,000 new cases 38 reported each year (Ding et al, 2022; Qin et al, 2024; Safdarian et al, 2023). Among these, high 39 cervical SCI often results in chronic respiratory insufficiency and, in many cases, a lifelong 40 inability to breathe independently (Brown et al, 2006; Josefson et al, 2021; Zimmer et al, 2007). 41 Respiratory complications, including pneumonia and bronchitis, are the leading causes of death 42 in both the acute and chronic phases following SCI (Burns, 2007; Johansson et al, 2025). 43 Currently, mechanical ventilation remains the standard of care for patients with high-level 44 3 cervical injury with reduced ventilatory capacity (Korupolu et al, 2021; Schreiber et al, 2021). 45 While lifesaving, this approach introduces numerous complications, such as frequent suctioning 46 of respiratory secretions, impaired speech and smell, increased risk of infection, and significant 47 psychological and physical burden for both patients and caregivers (Galeiras Vazquez et al, 2013; 48 Hendershot & O'Phelan, 2022; Tollefsen & Fondenes, 2012). Moreover, prolonged mechanical 49 ventilation leads to diaphragm disuse and subsequent atrophy, further impeding the chance of 50 spontaneous respiratory recovery (Levine et al, 2008; Penuelas et al, 2019; Powers et al, 2009; 51 Smuder et al, 2016). 52 Diaphragm pacing, which stimulates the phrenic nerve to contract the diaphragm, offers a 53 potential alternative for selected patients. It can reduce ventilator dependence and improve 54 quality of life (Onders et al, 2022; Romero-Ganuza et al, 2011; Sharma et al, 2021). However, 55 this approach requires intact phrenic nerve conduction and a functional lower motor neuron pool. 56 Unfortunately, only about 5% of patients with cervical SCI meet these criteria (Skalsky et al, 57 2015; Vashisht & Chowdhury, 2025), severely limiting its applicability. Consequently, the 58 majority of individuals with high cervical injuries are left without practical options to regain 59 independent breathing. 60 One of the major barriers to recovery following SCI is the neuroinflammatory response 61 consecutive to the initial injury. The primary injury initiates a cascade of cellular and molecular 62 events that lead to long-lasting secondary damage. Resident microglia become rapidly activated 63 and release proinflammatory cytokines such as TNF-α , IL-1β , and IL-6, aiming at containing 64 tissue damage, promoting debris clearance, but at the same time contributing to demyelination, 65 oxidative stress, and neuronal apoptosis (Hellenbrand et al, 2021; Li et al, 2022a). Similarly, 66 monocyte-derived macrophages infiltrate the lesion site, sustaining chronic inflammation and 67 4 interfering with tissue repair. In addition, activated astrocytes undergo hypertrophy and form a 68 dense glial scar, secreting extracellular matrix molecules such as chondroitin sulfate 69 proteoglycans (CSPGs), which are highly repulsive to axonal growth and plasticity (Anderson et 70 al, 2016; Silver & Miller, 2004; Wanner et al, 2008; Yang et al, 2020). These CSPGs, along with 71 other extracellular matrix components, accumulate around neurons to form perineuronal nets 72 (PNNs), a structure that stabilizes synaptic connections but also severely limits synaptic 73 remodeling after injury (Bradbury & Burnside, 2019; Dyck et al, 2018; Dyck & Karimi-74 Abdolrezaee, 2015; Fawcett et al, 2022; Sorg et al, 2016). Additionally, PDGFRβ -positive 75 pericytes proliferate and contribute to fibrotic scarring, further reinforcing an environment that is 76 non-permissive to regeneration (Birbrair et al, 2014; Dias et al, 2021; Picoli et al, 2019; Yao et al, 77 2022). This chronic neuroinflammatory microenvironment is particularly detrimental to the 78 phrenic motor circuit, which spans the C3 to C5 segments. After cervical SCI, especially in the 79 region around C3/4, the surviving phrenic motor neurons are surrounded by reactive glia, 80 inflammatory mediators, and dense PNNs that limit their ability to reconnect or reorganize 81 (Lukacova et al, 2021; Sánchez-Ventura et al, 2023; Windelborn & Mitchell, 2012). These 82 cellular and molecular events act synergistically to prevent meaningful respiratory recovery. Thus, 83 strategies that can modulate inflammation and reduce extracellular matrix barriers are critical for 84 functional repair. 85 Currently, neurostimulation emerges as a promising therapeutic strategy for enhancing neural 86 plasticity after SCI (Dorrian et al, 2023; Inanici et al, 2018; Van Steenbergen et al, 2023). 87 Among available techniques, repetitive magnetic stimulation (rMS) is particularly attractive due 88 to its non-invasive nature and established safety in clinical settings (Mann & Malhi, 2025). 89 Originally developed as a treatment for depression (Loo et al, 2008), rMS modulates cortical 90 5 excitability and synaptic plasticity, and has been approved for clinical use by regulatory agencies 91 worldwide (Cotovio et al, 2023; Lenz et al, 2016). In animal models, repetitive trans-spinal 92 magnetic stimulation (rTSMS) has shown the capacity to enhance functional recovery after SCI, 93 reduce glial reactivity, and stimulate axonal sprouting (Jiang et al, 2024; Liu et al, 2020; Robac 94 et al, 2021). Several studies have also reported that rMS can downregulate inflammatory 95 signaling and reduce microglial activation in models of traumatic brain injury and SCI (O'Leary 96 et al, 2025; Sasso et al, 2016; Sekar et al, 2019; Toledo et al, 2021; Zong et al, 2020). 97 Importantly, in cervical SCI models, rMS applied to the spinal cord has been shown to increase 98 diaphragm activity, likely by enhancing excitability of the phrenic circuit and possibly through 99 modulation of the local microenvironment (Lee & Vinit, 2024; Lv et al, 2023; Michel-Flutot et al, 100 2021). Despite these encouraging results, rMS protocols vary widely across studies, including 101 differences in stimulation frequency, intensity, duration, and anatomical targeting (Dufor et al, 102 2023; Kim et al, 2020). Such variability complicates comparisons between studies and limits 103 mechanistic interpretation, particularly in the context of respiratory recovery. Stimulation 104 frequency is a critical parameter, as it strongly influences the direction and magnitude of plastic 105 changes (Brihmat et al, 2022; Torii et al, 2012). High-frequency protocols, typically at or above 106 10 Hz, are generally associated with increase neuronal excitability and modulate the balance 107 between excitatory and inhibitory inputs, for example by enhancing phrenic motor neuron output 108 and attenuating GABAergic inhibition (Lenz et al., 2016; Michel-Flutot et al., 2021), whereas 109 lower frequencies tend to produce inhibitory outcomes. 110 In the present study, we therefore selected a 10 Hz rMS protocol, as this frequency has been 111 consistently shown to enhance excitability and promote plasticity in both cortical and spinal 112 6 circuits. Importantly, 10 Hz stimulation has also been used safely in clinical and preclinical 113 settings and has demonstrated efficacy in modulating motor and respiratory-related pathways. 114 We sought to determine whether 10 Hz rMS could reduce neuroinflammation and promote 115 respiratory recovery in a mouse model of C3/4HC. We hypothesized that chronic cervical rMS 116 would attenuate microglial and astrocyte activation, decrease CSPG deposition and/or PNN 117 formation, leading to a more preserved phrenic motor network. Furthermore, we aimed to 118 evaluate whether these cellular changes translate into improved diaphragm function. By targeting 119 both the inflammatory and functional components of injury, this study explores the potential of 120 rMS as a non-invasive therapeutic strategy to restore breathing in patients with high cervical SCI. 121

122 Physiological Effect and Histological Analysis in C3HC Mice Treated with rMS or Sham 123 Stimulation 124 A transient decrease in body weight was observed in both C3HC groups (sham rMS and rMS) 125 during the first week following surgery which then re-increased progressively similarly between 126 groups. By 21 days post-injury, animals had reached levels comparable to their preoperative 127 baseline. No significant difference in body weight was found between the sham rMS and rMS 128 groups at any of the three time points (D0, D7, or D21; Fig. 1C, Supplementary Table 1). 129 Histological quantification of the lesion extent revealed no significant difference between groups, 130 with comparable percentages of injured hemicord in the sham rMS and rMS-treated animals 131 (79.0/i3 ±/i3 6.5% vs. 74.8/i3 ±/i3 8.3%). 132 7 133 Fig. 1. Examples of extent of injury following a C3 hemicontusion treated with rMS or 134 sham stimulation. 135 (A) Representative examples of cresyl violet staining at the injured site for in sham rMS (top) and136 rMS treated groups (bottom). The lesion area is delineated by the dotted black outline; the 137 vertical dashed line indicates the spinal midline. Scale Bar: 500 μ m. (B) Quantification of injury 138 extent, calculated as the area of the lesion relative to the total area of the ipsilateral hemi-spinal 139 cord (set as 100%). Statistical analysis was performed using the unpaired Student’s t-test. (C) 140 Body weight measurements of animals across the four experimental groups including lami + 141 sham rMS, lami + rMS, C3HC + sham rMS, C3HC + rMS group at three time points: baseline 142 (D0), 7 days post-injury (D7), and 21 days post-treatment (D21). Data represent mean ± SD. 143 Statistical analysis was performed using the Two way repeated-measures (RM) ANOVA 144 followed by Tukey’s post hoc multiple comparisons test. 145 7 nd 8 Effects of rMS on Respiratory Function 146 Plethysmography measurements performed under light anesthesia (1% isoflurane) are presented 147 in Fig. 2 and table S1. In the whole group of mice, cervical SCI induced a significant reduction in 148 tidal volume (VT) in all animals when corrected for body weight. By day 7 (D7) post-injury, VT 149 had decreased by approximately 44% in the Sham group (5.81/i3 ±/i3 0.88 vs. 3.08/i3 ±/i3 0.88/i3 µL/g, 150 p<0.01), and by 32% in the rMS group (5.71/i3 ±/i3 0.61 to 3.88/i3 ±/i3 1.01µL/g, p<0.001), with no 151 significant difference between groups. However, recovery profiles differed by day 21 (D21). VT 152 was significantly higher in the rMS group (rMS vs sham rMS: 6.02/i3 ±/i3 1.15 vs. 3.87/i3 ±/i3 1.01 153 µL/g, p<0.01) compared to the sham group with limited spontaneous recovery. There was no 154 significant change observed for Breathing frequency (Bf) between groups or time points. As a 155 result, similar changes were observed for Minute ventilation (VE). Mice treated rMS group 156 recovered faster than those in the Sham group, and a significant difference was observed between 157 groups (rMS vs sham rMS: 36.53/i3 ±/i3 11.56 vs. 21.94/i3 ±/i3 6.48 mL/min, p<0.05). 158 9 159 160 Fig. 2. Respiratory parameters in C3HC mice treated with rMS or sham stimulation under 161 light anesthesia (1% isoflurane). 162 Histograms (Min to max, Box and Whiskers) present tidal volume (VT), breathing frequency (Bf163 and minute ventilation (VE) measured before surgery (D0), 7 days after injury (D7), and 21 days 164 after injury (D21) in C3HC mice treated with sham rMS or rMS. Statistical analysis was 165 9 r Bf) s 1 0 performed using the Two way repeated-measures (RM) ANOVA followed by Tukey’s post hoc 166 multiple comparisons test. *** p < 0.001, ** p < 0.01, * p < 0.05. 167 During hypercapnic challenge with 5% CO/i2 (Fig. 3), VT did not differ significantly between 168 baseline and 7 days post-injury in either the sham (8.05/i2 ±/i2 1.63 vs. 5.92/i2 ±/i2 2.29 µL/g, n.s) or 169 rMS groups (8.26/i2 ±/i2 2.19 vs. 6.12/i2 ±/i2 3.13/i2 µL/g, n.s). In the rMS group, VT significantly 170 increased by day 21 compared to day 7 (p < 0.01), while no significant recovery was observed in 171 the sham group (rMS vs sham rMS: 10.09/i2 ±/i2 3.71 vs. 7.36/i2 ±/i2 2.52/i2 µL/g, n.s). Although VT 172 tended to be higher in the rMS group at day 21, the between-group difference was not statistically173 significant. Bf remained unchanged across time points and treatment groups. VE followed a 174 similar pattern to VT, with a significant reduction at D7 in rMS groups (48.44/i2 ±/i2 13.20 vs. 175 31.05/i2 ±/i2 19.45 mL/min, p < 0.05) and with a similar trend in sham rMS group (49.01/i2 ±/i2 13.40176 vs. 31.52/i2 ±/i2 16.62 mL/min, p = 0.08). VE significantly increased in the rMS group at D21 177 compared to D7 (p < 0.001), while sham mice showed no recovery. No significant difference in 178 VE was found between groups at D21, although a trend toward higher values was observed in the 179 rMS group (rMS vs sham rMS: 54.58/i2 ±/i2 23.82 vs. 40.02/i2 ±/i2 14.72 mL/min, n.s). 180 181 182 0 in lly 0 he 11 Fig. 3. Respiratory parameters in C3HC mice treated with rMS or sham stimulation in the 183 5% CO/i1 phase. 184 Histograms (Min to max, Box and Whiskers) present tidal volume (VT), breathing frequency (Bf) 185 and minute ventilation (VE) measured before surgery (D0), 7 days after injury (D7), and 21 days 186 after injury (D21) in C3HC mice treated with sham rMS or rMS. Statistical analysis was 187 performed using the Two way repeated-measures (RM) ANOVA followed by Tukey’s post hoc 188 multiple comparisons test. *** p < 0.001, ** p < 0.01, * p < 0.05. 189 190 Under light anesthesia (1% isoflurane) after hypercapnic challenge (Fig. 4), VT tended to 191 decrease from baseline to day 7 in both sham (4.57/i3 ±/i3 1.08 vs. 3.03/i3 ±/i3 0.58 µL/g, p = 0.05) 192 and rMS groups (4.48/i3 ±/i3 1.23 vs. 3.27/i3 ±/i3 1.38 µL/g, n.s). By day 21, VT in the rMS group 193 showed a trend toward recovery, with higher values compared to the sham group, although the 194 difference was not statistically significant (rMS vs sham rMS: 4.31/i3 ±/i3 1.31 vs. 3.58/i3 ±/i3 1.12 195 µL/g, n.s). Bf remained stable across timepoints without intergroup differences. VE also declined 196 at D7 in both sham rMS (26.65/i3 ±/i3 7.01 vs. 14.91±/i3 5.69 mL/min, p = 0.08) and rMS 197 (25.60/i3 ±/i3 11.05 vs. 14.48±/i3 7.00 mL/min, p = 0.05) groups. At D21, VE showed partial 198 recovery in the both groups (rMS vs sham rMS: 18.51/i3 ±/i3 6.36 vs. 18.44/i3 ±/i3 5.15 mL/min, n.s). 199 Although the group difference was not significant, the upward trend in VE paralleled the 200 improvement observed in VT. 201 1 2 202 203 Fig. 4. Respiratory parameters in C3HC mice treated with rMS or sham stimulation in the 204 1% isoflurane (after hypercapnic challenge). 205 Histograms (Min to max, Box and Whiskers) present tidal volume (VT), breathing frequency (Bf206 and minute ventilation (VE) measured before surgery (D0), 7 days after injury (D7), and 21 days 207 after injury (D21) in C3HC mice treated with sham rMS or rMS. Statistical analysis was 208 performed using the Two way repeated-measures (RM) ANOVA followed by Tukey’s post hoc 209 multiple comparisons test. * p < 0.05. 210 211 During spontaneous breathing in ambient air (normal poikilocapnic breathing) (Fig. 5), 212 respiratory parameters remained relatively stable in the sham rMS group throughout the post-213 injury period. In contrast, animals treated with rMS showed significant recovery of VT and VE 214 by day 21 compared to baseline (D0 vs D21: p < 0.05), indicating a progressive improvement in 215 respiratory capacity. Although no significant difference was observed between rMS and sham 216 groups at each individual time point, the within-group recovery trend in the rMS group suggests a217 beneficial effect of rMS on restoring respiratory output under physiological breathing conditions. 218 2 Bf) s s a s. 1 3 Breathing frequency (Bf) significantly change after day 7 and day 21 in sham rMS group (D0 vs 219 D7 vs D21: 175/i2 ±/i2 29 vs. 210 ±/i2 17 vs. 222 ±/i2 30 breaths/min, p < 0.05), whereas a 220 nonsignificant upward trend in rMS group (D0 vs D7 vs D21: 203/i2 ±/i2 36 vs. 229 ±/i2 18 vs. 230 221 ±/i2 31 breaths/min, n.s). 222 223 224 Fig. 5. Respiratory parameters in C3HC mice treated with rMS or sham stimulation during 225 spontaneous (poikilocapnic) breathing. 226 Histograms (Min to max, Box and Whiskers) present tidal volume (VT), breathing frequency (Bf227 and minute ventilation (VE) measured before surgery (D0), 7 days after injury (D7), and 21 days 228 after injury (D21) in C3HC mice treated with sham rMS or rMS. Statistical analysis was 229 performed using the Two way repeated-measures (RM) ANOVA followed by Tukey’s post hoc 230 multiple comparisons test. * p < 0.05. 231 232 Effects of rMS on Diaphragm Activity 233 3 ng Bf) s 14 To investigate the mechanistic basis of improved respiratory function, we assessed dia-EMG 234 activity across three anatomical regions (ventral, medial, and dorsal) for both intact and injured 235 sides (Fig. 6). On the injured side, rMS significantly preserved dia-EMG amplitude close to 236 control values, while it tended to decrease in the sham-rMS group after C3HC injury. In the 237 ventral region of the diaphragm, a reduction in dia-EMG activity was observed in the C3HC + 238 sham rMS group compared to the lami + sham rMS group (0.40 ± 0.27 vs. 0.77 ± 0.38/i3μ V·s·s). 239 Remarkably, rMS treatment effectively preserved dia-EMG activity, restoring it to 0.77 ± 240 0.33/i3μ V·s·s, a level significantly higher than the C3HC + sham-rMS injury group (p < 0.05). In 241 the medial region, a similar pattern was observed, with reduced activity in the C3HC + sham rMS 242 group (0.47 ± 0.3/i3 vs. 0.68 ± 0.4 μ V·s·s) compared to the lami + sham rMS group and restored 243 following rMS treatment (0.86 ± 0.41/i3μ V·s·s; p < 0.05). No significant differences were 244 observed in the dorsal region among groups. 245 On the uninjured side, rMS also enhanced diaphragm activity in the ventral region. Dia-EMG 246 amplitude significantly increase in the C3HC + rMS group compared to the C3HC + sham rMS 247 group (1.1 ± 0.57/i3 vs. 0.59 ± 0.3/i3μ V·s·s), while the laminectomy group showed a baseline level 248 (0.85 ± 0.32 vs. 0.64 ± 0.26/i3μ V·s·s) and without significant difference. This increase in activity 249 may reflect a compensatory enhancement in the contralateral diaphragm. No significant 250 differences were observed among groups in the middle and dorsal regions on the uninjured side. 251 1 5 252 253 Fig. 6. Diaphragm activity in lami or C3HC mice treated with rMS or sham stimulation. 254 5 16 (A) Representative examples of diaphragmatic EMG in mice with sham rMS and rMS treated 255 groups from the ventral part of diaphragm on the injured side. (B) Quantification of peak 256 amplitude of double integrated diaphragm EMG (expressed in μ V·s·s) on both the injured and 257 uninjured sides. All values have been corrected for the amplifier gain factor to represent 258 biological source voltage. Data are presented as Histograms (Min to max, Box and Whiskers) in 259 four experimental groups: lami + sham rMS (black), lami + rMS (gray), C3HC + sham rMS (red), 260 and C3HC + rMS (blue). Each dot represents one animal. Comparisons are shown across the 261 ventral, medial, and dorsal regions of the diaphragm. Statistical significance between the C3HC + 262 sham rMS and C3HC + rMS groups was assessed using an unpaired student’s t test(#), # p < 0.05. 263 264 rMS Modulates Scar-associated and Inflammatory Cellular Responses After C3HC 265 rMS Reduces Fibrotic Cells and Astrocytic Scar Formation 266 Immunohistofluorescence analysis of PDGFRβ and GFAP at the lesion level is shown in Fig. 7, 267 and around lesion level are presented in fig. S1. PDGFRβ and GFAP were co-expressed within 268 the fibrotic and astroglial components of the glial scar. A significantly higher percentage area of 269 PDGFRβ -positive staining was observed in the C3HC + sham rMS group compared to the 270 laminectomy group (p < 0.01). rMS treatment markedly reduced the PDGFRβ -positive area, with 271 a significant difference between the sham and rMS-treated C3HC groups (20.98/i3 ±/i3 2.98 % vs. 272 12.89/i3 ±/i3 2.58 %, p < 0.001). Similarly, PDGFRβ -positive cells counts were elevated in the 273 C3HC + sham rMS group compared to the laminectomy group and significantly reduced 274 following rMS treatment (278/i3 ±/i3 56 vs. 216/i3 ±/i3 47 cells, p < 0.05). Furthermore, GFAP-275 positive staining was significantly increased in the C3HC injury group compared to the 276 1 7 laminectomy group (p < 0.05). The GFAP-positive area was higher in the C3HC + sham rMS 277 group and tended to be decrease by rMS treatment (51.57/i2 ±/i2 14.78 % vs. 40.70/i2 ±/i2 9.80 %, p =278 0.08). Consistently, the number of GFAP-positive cells was significantly higher at the lesion site 279 in the sham group and was reduced by rMS (584/i2 ±/i2 80 vs. 469/i2 ±/i2 90 cells, p < 0.05). 280 Concordant reductions around the lesion level are shown in fig. S1. 281 282 283 Fig. 7. PDGFRβ and GFAP immunoreactivity at 21 days post-injury in the cervical spinal 284 cord lesion level. 285 7 = te 18 (A) Representative immunofluorescence images showing PDGFRβ (red), GFAP (yellow), and 286 merged channels in the lesion area of Laminectomy + sham rMS, Laminectomy + rMS, C3HC + 287 Sham rMS, and C3HC + rMS-treated animals at 21 days post-injury. (Scale bar: 500μ m) (B) 288 Quantification of PDGFRβ + percentage area, GFAP+ percentage area, and cell number in the 289 lesion site at the ventral part of spinal cord. Each dot represents one animal. Histograms (Min to 290 max, Box and Whiskers) show data distribution across the four groups. Statistical significance 291 across the four groups was determined using a Kruskal-Wallis test followed by Dunn’s post-hoc 292 multiple comparisons test (*) *p<0.05, **p<0.01, ***p<0.001, while direct comparisons between 293 the C3HC + sham rMS and C3HC + rMS-treated groups were specifically performed using a 294 Mann-Whitney test (#) #p<0.05, ##p<0.01, ###p<0.001. 295 296 rMS Attenuates Microglial/Macrophage Activation 297 CD68 and Iba1, both associated with activated pro-inflammatory microglia/macrophages, were 298 used to assess neuroinflammation (Fig. 8). At the lesion level, the CD68-positive area was 299 significantly increased in the C3HC + sham rMS group compared to the laminectomy group (p < 300 0.01). rMS treatment significantly reduced the percentage area of CD68-positive labeling in the 301 C3HC model (21.22/i3 ±/i3 5.04 % vs. 14.94/i3 ±/i3 3.92%, p < 0.05). The number of CD68-positive 302 cells decreased following rMS, (423/i3 ±/i3 117 vs. 304/i3 ±/i3 55 cells, p = 0.05). The percentage area 303 of Iba1-positive staining was significantly higher in the C3HC + sham rMS group compared to 304 the rMS-treated group (34.09/i3 ±/i3 9.15 % vs. 24.70/i3 ±/i3 6.53 %, p < 0.01). The number of Iba1-305 positive cells was also likewise reduced by rMS treatment (417/i3 ±/i3 88 vs. 329/i3 ±/i3 74 cells, p = 306 0.05). Similar trends were observed around lesion level (Supplementary Figure S2). However, for 307 CD206, a marker of anti-inflammatory microglia/macrophages, the percentage area and the 308 1 9 number of CD206-positive cells did not differ between groups (1.21/i2 ±/i2 1.64 % vs. 309 3.51/i2 ±/i2 6.26 %; 65 ±/i2 77 vs. 66/i2 ±/i2 76 cells). A reduction in fibrotic and glial scarring, as well 310 as pro-inflammatory microglial activation, was also observed around the lesion site following 311 rMS (Supplementary Figure S4). 312 313 314 Fig. 8. CD68 and Iba1 immunoreactivity at 21 days post-injury in the cervical spinal cord 315 lesion level. 316 9 ell 20 (A) Representative immunofluorescence images showing CD68 (purple), Iba1 (cyan), and 317 merged channels in the lesion area of Laminectomy + sham rMS, Laminectomy + rMS, C3HC + 318 Sham rMS, and C3HC + rMS-treated animals at 21 days post-injury. Scale bar: 500 µm. (B) 319 Quantification of CD68+ percentage area, Iba1+ percentage area, and cell number in the lesion 320 site at the ventral part of spinal cord. Each dot represents one animal. Histograms (Min to max, 321 Box and Whiskers) show data distribution across the four groups. Statistical significance across 322 the four experimental groups was determined using a Kruskal-Wallis test followed by Dunn’s 323 post-hoc multiple comparisons test (*) *p<0.05, **p<0.01, ***p<0.001, while direct comparisons 324 between the C3HC + sham rMS and C3HC + rMS-treated groups were specifically performed 325 using a Mann-Whitney test (#) #p<0.05, ##p<0.01, ###p<0.001. 326 rMS Modulates WFA labeled Perineuronal Nets and Neuronal Preservation 327 WFA staining showed a trend toward increased CSPG-rich perineuronal net (PNN) signal in the 328 C3HC + sham rMS group compared to the lami + sham rMS (5.86/i3 ±/i3 3.39 % vs. 1.71 ± 0.91%), 329 with a trend toward reduction following rMS treatment (5.86/i3 ±/i3 3.39 % vs. 3.10/i3 ±/i3 2.09 %, p 330 =0.1). In parallel, C3HC injury led to a clear reduction in neuronal density (280/i3 ±/i3 19 vs. 119 331 ±/i3 30 cells, p < 0.05) compared to the Lami + sham rMS group, and neurons in the ventral horn 332 tended to be higher at the lesion site in the rMS group compared to sham stimulation group 333 (119/i3 ±/i3 30 vs. 146/i3 ±/i3 27 cells, p = 0.08). Morphologically, WFA-labeled CSPGs appeared 334 forming dense, cage-like structures condensed around the somata of large ventral horn neurons in 335 sham-stimulated injury animals. In contrast, rMS treatment showed a trend toward reducing this 336 peri-neuronal condensation. Similar trends were observed around lesion level without 337 significance (fig. S3). 338 2 1 339 340 Fig. 9. WFA staining and ventral neuron survival at 21 days post-injury in the cervical 341 spinal cord lesion level. 342 1 22 (A) Representative immunofluorescence images showing WFA (green), NeuroTrace™ 435/455 343 (neuronal marker, red), and merged channels in the lesion area of Laminectomy + sham rMS, 344 Laminectomy + rMS, C3HC + sham rMS, and C3HC + rMS-treated animals at 21 days post-345 injury. NeuroTrace™ 435/455, which possesses intrinsic blue fluorescence, has been 346 pseudocolored red using image processing software to enhance visualization and contrast within 347 the merged channels. Scale bar: 500 µm. (B) Quantification of WFA-positive percentage area, 348 number of ventral neurons at the lesion site. Each dot represents one animal. Histograms (Min to 349 max, Box and Whiskers) show data distribution across the four groups. Statistical significance 350 across the four experimental groups was determined using a Kruskal-Wallis test followed by 351 Dunn’s post-hoc multiple comparisons test (*) *p<0.05, **p<0.01. 352 353

354 Our study shows for the first time that rMS can improve tidal volume recovery after cervical SCI. 355 Indeed, spontaneous recovery remained limited without treatment, whereas rMS accelerated and 356 amplified functional restoration. These functional improvements occurred alongside reduced 357 glial and fibrotic scarring as well as suppression of chronic M1-like activation, suggesting that 358 rMS mitigates inhibitory and cytotoxic cues that normally consolidate lesion pathology. 359 Moreover, rMS showed a trend toward reducing perineuronal net density and was associated with 360 a trend toward neuronal survival, which suggests that rMS may stabilize spared phrenic 361 motoneurons and may facilitate the re-establishment of functional connections within the phrenic 362 motor pool. These effects, likely arising from the modulation of the neuroinflammatory 363 environment, provide a plausible mechanism by which rMS supports the restoration of 364 respiratory capacity. 365 23 Neuroprotection via reduced glial/fibrotic scarring 366 At 21 days post-injury, the spinal inflammatory response is transitioning from the subacute to the 367 early chronic phase, a stage where the glial and fibrotic scar consolidates into a persistent barrier 368 to repair (Hellenbrand et al., 2021; Orr & Gensel, 2018). This scar, formed by proliferating 369 PDGFRβ + fibroblasts and GFAP+ astrocytes, is known to constrain axonal sprouting and limit 370 the reconnection of spared neural circuits. (Bradbury & Burnside, 2019; Li et al, 2022b; Orr & 371 Gensel, 2018; Pang et al, 2021). In this study, rMS appeared to shift this balance toward a less 372 restrictive milieu by reducing both PDGFRβ + fibroblasts and GFAP+ labeled cells. Rather than 373 simply being an anatomical structure, the scar functions as a dynamic cellular and extracellular 374 interface that either fosters or obstructs regeneration depending on its composition. Intriguingly, 375 other studies using trans-spinal magnetic stimulation have reported inhibition of fibrotic scar by 376 increasing in turn astrogliosis reactivity (Chalfouh et al, 2020; Robac et al., 2021). However, 377 astrocytes are highly heterogeneous in their responses after SCI (Qian et al, 2025). On one hand, 378 reactive astrocytes rapidly proliferate and upregulate GFAP to form the glial scar, which 379 physically and chemically restricts axonal growth (Perez-Gianmarco & Kukley, 2023). On the 380 other hand, a subset of astrocytes can adopt a protective role, limiting lesion expansion, isolating 381 inflammatory infiltrates, and providing trophic support (Faulkner et al, 2004). Thus, an apparent 382 increase in astrogliosis in some contexts may actually reflect the predominance of this reparative, 383 neuroprotective phenotype (Wang et al, 2022). In contrast, the reduced GFAP signal observed in 384 our study may indicate that rMS specifically dampened the maladaptive component of astrocytic 385 activation that contributes to deposition of inhibitory extracellular matrix molecules such as 386 chondroitin sulfate proteoglycans (CSPGs), which are known to inhibit axonal growth and 387 24 synaptic remodeling since we also observed a reduction in CSPG (Francos-Quijorna et al, 2022; 388 Yang et al, 2024). 389 Suppression of pro-inflammatory microglia/macrophage activation 390 Pro-inflammatory Microglia and Macrophages are known to remain persistently activated after 391 SCI within the lesion environment, contributing to the maintenance of glial and fibrotic scarring 392 (Bradbury & Burnside, 2019; Pang et al., 2021). In the present study, rMS significantly 393 suppressed this chronic inflammatory activation, as reflected by lower CD68+ and Iba1+ labeling, 394 suggesting a shift away from the deleterious M1-like phenotype. These findings are consistent 395 with prior reports that repetitive transcranial magnetic stimulation (rTMS) downregulates 396 microglial activity and suppresses key pro-inflammatory cytokines such as IL-1β , TNF-α , and IL-397 6 (Guo et al, 2023), while promoting anti-inflammatory mediators including IL-10 and 398 neurotrophic factors like BDNF (Bai et al, 2023; Luo et al, 2022). An intriguing aspect of our 399

is that this reduction in M1-associated markers was not accompanied by a corresponding 400 increase in CD206, a commonly used marker of M2-like macrophages. One possible explanation 401 lies in the role of extracellular matrix components such as CSPGs, which have been shown to 402 constrain Microglia/Macrophage phenotypic conversion. In rodent SCI models, CSPGs abundant 403 in the post-SCI scar, exert only modest effects on M1 cells but strongly prevent M2-like 404 macrophages from maintaining a reparative state (Dyck et al., 2018; Francos-Quijorna et al., 405 2022). This blockade occurs through TLR4 signaling and is already evident by 7 days post-injury, 406 the stage when inflammation would normally begin to resolve (Francos-Quijorna et al., 2022). 407 Consequently, persistent CSPG accumulation interferes with the resolution phase and locks 408 macrophages into a pro-inflammatory profile. Our data suggest that rMS effectively alleviates 409 chronic M1-like neuroinflammation, which occurred alongside a trend toward reduced CSPG 410 25 deposition. However, the absence of increased CD206 expression at 21 day post injury likely 411 reflects the fact that the resolution window had already been disrupted earlier, such that M2-like 412 populations could not expand despite reduced CSPG levels. Alternatively, it is possible that the 413 magnitude of CSPG attenuation achieved in this study was insufficient to independently trigger a 414 significant phenotypic shift toward an anti-inflammatory M2-like state at this chronic stage. 415 Additionally, CD206 alone may not capture the full spectrum of reparative phenotypes, and 416 future work including complementary markers may clarify these dynamics. 417 Promotion of respiratory function through improved diaphragm activity 418 High cervical spinal cord injury (SCI) often results in significant disruption of descending 419 respiratory pathways, leading to impaired neuromuscular control of the diaphragm and reduced 420 respiratory efficiency (National Spinal Cord Injury Statistical, 2005; Warren et al, 2014; 421 Winslow & Rozovsky, 2003). As expected, a significant reduction in VT (~40%) was observed 422 across all animals within the first 7 days post-injury, reflecting diminished neuromuscular drive 423 stemming from phrenic pathway interruption, the present results suggest partial spontaneous 424 recovery (25%) after 21 days post injury, although this increase was not sufficient to restore 425 normal ventilation. By contrast, rMS-treated mice exhibited a significantly greater restoration of 426 VT at 21 dpi, which reached almost complete recovery, indicating that rMS accelerated and 427 amplified the limited spontaneous recovery. 428 During hypercapnia, which strongly activates chemoreceptor-driven respiratory drive and tests 429 the ability to recruit ventilatory reserve, rMS animals exhibited stronger within-group recovery at 430 D7 vs. D21, whereas sham-injured mice showed deficits in VT (and therefore VE). This suggests 431 that rMS facilitates the recruitment of spared bulbospinal and interneuronal pathways under 432 conditions of increased demand, thereby enhancing respiratory reserve capacity. These findings 433 26 are consistent with previous reports showing that mid-cervical SCI impairs the ventilation during 434 hypercapnic challenge, reflecting a loss of reserve capacity (Choi, Liao et al. 2005, Golder, Fuller 435 et al. 2011). During normal poikilocapnic breathing, rMS produced a within-group recovery of 436 VT and VE by day 21, whereas sham animals failed to restore VT and instead compensated by 437 increasing breathing frequency. These divergent patterns suggest two distinct mechanisms that 438 rMS enhanced inspiratory strength through greater phrenic motoneuron excitability, more 439 effective motor-unit recruitment, and improved neuromuscular transmission, while sham animals 440 relied on a less efficient “rapid shallow breathing” strategy to maintain ventilation. The absence 441 of significant between-group differences likely reflects high variability in spontaneous breathing, 442 yet the within-group recovery (D0 vs. D21) in rMS indicates a potential treatment effect. 443 Respiratory frequency (Bf) remained stable across time points and groups, indicating preserved 444 central rhythm genic activity and suggesting that changes in VE were primarily driven by 445 modulation of VT under 1% isoflurane anesthesia. Given the anesthetic suppression of accessory 446 respiratory muscles, these findings suggest that the functional recovery mediated by rMS 447 specifically arises from enhanced neural drive and mechanical output of the diaphragm. This 448 finding is consistent with the idea that rMS may have triggered a long-term facilitation (LTF)-449 like effect that preferentially enhanced the amplitude of respiratory motor outputs rather than the 450 central rhythm. Given its train-based, intermittent structure, our rMS protocol (9 trains with inter-451 train pauses) plausibly fulfils these temporal requirements, and previous work has likewise 452 shown that rMS can activate BDNF/ERK signaling cascades (Wang, Crupi et al. 2011, Peng, 453 Zhou et al. 2018). In support of this interpretation, spinal iTBS has been reported to increase 454 diaphragm EMG burst amplitude for periods extending beyond the stimulation session, indicating 455 a sustained enhancement of inspiratory drive at the motoneuron level (Lee and Vinit 2024). Thus, 456 27 the absence of frequency modulation in our rMS data likely reflects a mechanism in which spinal 457 plasticity strengthens inspiratory force generation without altering the central rhythm generator. 458 The Diaphragm activity recovery found in our study is in line with previous evidences that 459 magnetic stimulation can restore inspiratory drive, increase diaphragm excitability, and induce 460 neuroplastic changes in descending respiratory circuits (Lee & Vinit, 2024; Michel-Flutot et al, 461 2022; Randelman et al, 2021). At the same time, the observed trend toward reduced CSPG 462 levelswas associated with a trend toward an increased number of labeled neurons at the level of 463 the lesion, where phrenic motoneuron are located. Thus, one hypothesis is that the combination 464 of rMS-induced trends in CSPG attenuation and enhanced neuronal survival may have 465 strengthened spared motoneuron and connections at the level of the PMN pool and around. 466 Beyond reducing inhibitory CSPGs and enhancing neuronal survival, additional evidence 467 indicates that rMS/rTMS promotes regenerative plasticity in spared phrenic motoneurons and 468 their descending inputs. In C2 hemisection rats, high-frequency rTMS increased GAP-43 469 expression in ventrolateral cervical tracts, consistent with axonal sprouting within corticospinal-470 phrenic pathways (Michel-Flutot et al., 2022). Similar upregulation of GAP-43 has been reported 471 after iTBS in incomplete compression SCI models (Marufa et al, 2021) and trans-spinal iTBS has 472 been shown to augment inspiratory drive may via enhanced synaptic activity within the phrenic 473 nucleus and likely modulation of excitatory and inhibitory inputs to phrenic motoneurons in a C3 474 contusion model (Lee & Vinit, 2024). Together, these mechanisms together are likely to 475 contribute to the improved functional outcomes seen in rMS-treated animals. Similarly, previous 476 work showed that rTSMS minimizes cavity formation and preserves axons in both contusion and 477 transection models, resulting in better locomotor recovery (Bai et al., 2023). Such mechanistic 478 insights will be essential to refine stimulation protocols and enhance their translational relevance. 479 28 Clinical Relevance 480 These findings have clear translational relevance. Non-invasive rMS is already FDA-approved 481 for the treatment of depression and has demonstrated safety and efficacy in managing spasticity 482 and neuropathic pain in SCI patients (Benavides et al, 2025; Cotovio et al., 2023). Our data 483 suggest that rMS could be repurposed to directly target the spinal cord injury site. By reducing 484 glial scarring, mitigating neuroinflammation, and enhancing neuroprotection and plasticity, rMS 485 may synergize with rehabilitative therapies to accelerate functional recovery. Importantly, as a 486 non-invasive and repeatable modality, rMS presents minimal clinical risk and strong therapeutic 487 potential. These results warrant further clinical exploration and may pave the way toward 488 incorporating rMS as a standard adjunct in SCI recovery protocols. 489

and Strengths 490 One limitation of the present study is the use of a single model of cervical spinal cord injury, C3 491 hemi-contusion, which may not fully reflect the heterogeneity and complexity of human cervical 492 SCI. Different injury types, severities, and segmental levels may engage distinct respiratory and 493 inflammatory responses, potentially affecting the generalizability of our findings. However, the 494 C3HC model is relatively recent and less widely used but clinically relevant paradigm that 495 closely mimics many key features of incomplete cervical SCI in humans, including partial 496 preservation of descending motor pathways and compromise in ventilatory capacity.. Its 497 reproducibility and translational fidelity make it a suitable starting point for evaluating the 498 therapeutic potential of rMS in modulating respiratory recovery. Another limitation lies in the 499 temporal resolution of the respiratory assessments, which were conducted at three discrete time 500 points, baseline, 7 days post-injury, and 21 days post-injury. While these time points were 501 strategically selected to represent the pre-injury state, the acute phase of injury, and the early 502 29 subacute recovery phase, they may miss transient or delayed neuroplastic changes occurring 503 outside this window. Nonetheless, this focused time-course design enabled clear comparisons 504 across critical stages of injury and recovery while minimizing the impact of biological variability. 505 Importantly, the observed effects of rMS on both ventilatory parameters and diaphragm activity 506 were robust despite the limited temporal sampling (Michel-Flutot et al., 2022). A critical area for 507 future exploration lies in the optimization of rMS parameters, including frequency, burst pattern, 508 and stimulation intensity. While our standardized 10 Hz protocol elicited functional gains, the 509 field lacks a comprehensive "dose-response" map for respiratory recovery. Different frequencies 510 may engage distinct molecular pathways. For instance, intermittent theta burst stimulation (iTBS) 511 is often associated with more potent long-term potentiation (LTP)-like effects via rapid BDNF-512 TrkB signaling, whereas continuous low-frequency stimulation might more effectively modulate 513 homeostatic plasticity or suppress hyperexcitability. Finally, regarding treatment duration, our 514 short-term rMS regimen elicited significant improvements in tidal volume, respiratory reserve, 515 and diaphragm electromyographic output by Day 21 suggests a strong capacity for promoting 516 early-phase neuroplasticity. This aligns with recent evidence f that extended stimulation protocols 517 do not necessarily yield superior outcomes (Lee & Vinit, 2024). Similarly, comparative studies 518 demonstrating that a two-week intervention was sufficient to reach a therapeutic plateau in 519 respiratory recovery, with a four-week regimen offering no additional functional gain (Michel-520 Flutot et al., 2022). These findings support the notion that even limited, subacute stimulation 521 protocols can yield meaningful functional gains and may serve as an efficient entry point for 522 optimizing stimulation parameters in future translational studies. 523

524 30 This study provides converging evidence that targeted repetitive magnetic stimulation (rMS) 525 applied to the injured cervical spinal cord markedly enhances respiratory recovery after high 526 cervical SCI. rMS-treated animals recovered larger tidal volumes and minute ventilation across 527 awake and challenge conditions, and exhibited stronger diaphragm activity on EMG in both 528 hemidiaphragms. These functional gains were paralleled by dramatic changes in the injury 529 microenvironment. rMS reduced indicators of reactive astrogliosis (GFAP) and fibrotic scarring 530 (PDGFRβ ), and lowered pro-inflammatory microglial activation (Iba1, CD68), without affecting 531 CD206+ cell levels. Neuronal survival was improved (more NeuN+ cells), and inhibitory 532 extracellular matrix (PNNs) tends to diminish. Thus, rMS appears to relieve multiple barriers to 533 plasticity, creating a pro-regenerative milieu. These findings are consistent with prior reports that 534 high-frequency magnetic stimulation can suppress astrocyte and microglial reactivity and 535 inflammation, reduce glial scar formation, and support axonal preservation. Importantly, our 536

highlight the translational promise of spinal rMS as a non-invasive neuromodulatory 537 therapy for cervical SCI. By attenuating scar and inflammation while strengthening the spared 538 MN and connections at the level of the PMN pool and around , rMS effectively accelerated and 539 amplified the limited spontaneous recovery. In summary, targeted rMS significantly promotes 540 ventilatory function and neural plasticity after cervical SCI, underscoring its potential as a 541 therapeutic strategy. Future studies should optimize stimulation protocols a nd explore 542 combination with rehabilitation to maximize respiratory recovery, with the ultimate goal of 543 improving breathing outcomes for individuals with cervical SCI. 544

545 Animal Care and Use Statement 546 3 1 The animal protocol was designed to minimize pain or discomfort to the animals. All 547 experimental procedures were in accordance with the European Community guiding principles on548 the care and use of animals (EU Directive 2010/63/EU), and approved by the Ethics Committee 549 Charles Darwin CEEACD/N 5 (Project authorization APAFIS No. 201901301500576). 550 Experimental Design 551 A total of 29 adult male Swiss mice (5 weeks old) were used to investigate the effects of 552 repetitive trans-spinal magnetic stimulation (rMS) on respiratory function after cervical spinal 553 cord injury. Mice were randomly assigned to four experimental groups: (1) laminectomy + sham 554 rMS (n = 4); (2) laminectomy + rMS (n = 4); (3) C3 hemi-contusion (C3HC) + sham rMS (n 555 =10); and (4) C3HC + rMS (n =11). Respiratory function was assessed using whole-body 556 plethysmography at baseline, post-injury, and after the rMS intervention, with tidal volume (Vt) 557 and respiratory frequency (Bf) recorded. Diaphragm activity was further evaluated through in-558 situ electromyography (EMG) under terminal anesthesia. Spinal cord tissues were harvested for 559 immunofluorescence to assess neuroinflammatory and structural markers. rMS or sham 560 stimulation was applied once daily for two weeks starting 7 days post-surgery. 561 562 563 1 on m 32 Fig. 10. Experimental timeline. 564 Schematic representation of the experimental design to evaluate the effects of rMS on respiratory 565 function after cervical spinal cord injury. Baseline respiratory function was performed before 566 surgery (Day 0), followed by C3/4 hemi-contusion or laminectomy (Day 1). Respiratory function 567 was reassessed on Day 7. rMS or sham stimulation was applied daily from Day 8 to Day 21. 568 Final assessments were performed, including respiratory function, diaphragm activity, and tissue 569 collection for immunofluorescence. 570 Surgical procedures 571 Animals were placed in a closed chamber for anesthesia induction with isoflurane (4%) and 572 maintained throughout the procedure with a facial mask (1.5-2.5% isoflurane) in 100% O2. The 573 dorsal skin and underlying muscles above the second cervical up to the first thoracic vertebrae 574 was retracted. A dorsal laminectomy were performed at C3/C4 to expose the spinal cord. A 575 precision Impactor Device (RWD life science; 68,099 II) with a 1.5 mm tip impactor will be used 576 to perform the C3/4 hemi-contusion. The impactor parameters are set-up as follows: a mean 577 depth of 2.6 ± 0.41 mm, a speed of 1.0 ± 0.1 m/s and a dwell time of 0.60 ±0.01 s (mean ± SEM). 578 After contusion, the wounds and skin were closed. The isoflurane vaporizer was turned off, and 579 the mice received subcutaneous injections of sulfadoxine (Borgal, 0.2 mL per mouse; 0.1 mL 580 diluted in 1 mL NaCl) and buprenorphine (Buprécare, 0.5 mL per mouse; 0.05 mL diluted in 1 581 mL NaCl). After surgery, the animals were placed on a heated pad to recover. The animals were 582 then placed in a cage containing water and a recovery diet gel, both in a small Petri dish, and then 583 solid food were provided in the cage. Food and water were also provided on the top grid of the 584 cage. Animals that showed a reduction of more than 20% tidal volume (Vt) from baseline were 585 divided into two groups: (1) C3HC + sham rMS (n = 7); (2) C3HC + rMS (n = 10). 586 3 3 Repetitive Magnetic Stimulation (rMS) Protocol 587 rMS protocol was performed using the magnetic stimulator MAGPRO R30 (Magventure, Farum, 588 Denmark) connected to a figure-of-eight coil (Cool-B65), delivering a unique biphasic pulse with 589 the intensity of the stimulus expressed as a percentage of a maximum output of the stimulator (% 590 MO). The protocol (10 Hz, 9 trains of 100 biphasic pulses, separated by 30 s intervals between 591 trains delivered at 80% MO, 900 stimulations per protocol) was applied in awake restrained 592 animals. This protocol induced a long-lasting increase in phrenic excitability in anesthetized, 593 intact rats(Michel-Flutot et al., 2022). Control animals received a Sham rMS protocol (e.g., no 594 stimulation but the same time spent in the custom-designed restraining device, Figure 2). This 595 rMS protocol was applied 7 days postinjury for 2 weeks (once a day, 5 days per week) (Figure 2).596 597 598 Figure 11. rMS experimental setup and stimulation protocol. (A) Schematic of the phrenic 599 motor circuit and rMS coil placement targeting the rVRG and cervical phrenic motoneurons. The 600 diaphragm is shown as the functional output. (B) HF-rMS protocol parameters (top) and 601 3 , ith % . he 34 experimental configuration for sham and active 10 Hz stimulation in awake, restrained mice 602 (bottom). 603 Breathing Recording Using Plethysmography 604 Respiratory function was assessed via whole-body plethysmography (Emka, France) at baseline, 605 7 days post-surgery, and following rMS treatment (Day 21). The mice were placed in a 606 hermetically sealed chamber (comprising a 250 mL animal compartment and a 150 mL reference 607 chamber) under a continuous 0.5 L/min bias flow. Subsequently mice were exposed to the 608 following test gases in chronological order: 5 min 1% Isoflurane, 5 min 5% CO2, 5 min 1% 609 Isoflurane, 10 min ambient air. This phase limit stress and resulted in calm animals, thereby 610 allowing successful recordings. Plethysmography signals were digitized and analyzed using 611 LabChart 8 Pro software (ADInstruments, Dunedin, New Zealand). Quantified ventilatory 612 parameters included tidal volume (VT), breathing frequency (Bf), minute ventilation (VE), and 613 inspiratory/expiratory times (Ti and Te). 614 Electrophysiological Recording of the Diaphragm 615 After 21 days post-rMS treatment, terminal diaphragmatic electromyography (dia-EMG) were 616 performed to evaluate respiratory motor output. Mice were placed in a supine position on a 617 regulated heating pad to maintain a constant core body temperature (37.5 ± 1 °C). Following a 618 midline laparotomy to expose the diaphragm, dia-EMG activity was recorded from the ventral, 619 medial, and dorsal regions of both the ipsilateral and contralateral hemi-diaphragms during 620 spontaneous breathing. Signals were obtained using custom-made bipolar silver surface 621 electrodes positioned on the muscle fibers. The dia-EMG signals were amplified (Model 1800; 622 gain: 100; A-M Systems, Everett, WA, USA) and bandpass-filtered (100 Hz – 10 kHz). Data 623 35 were digitized using a PowerLab acquisition system (Acquisition rate: 4 k/s; ADInstruments, 624 Dunedin, New Zealand). For quantitative analysis, raw signals were rectified and double-625 integrated (50 ms decay constant) using LabChart 8 Pro software (ADInstruments). The 626 amplitude of at least 10 double-integrated diaphragmatic EMG inspiratory bursts during 627 normoxia was calculated for each animal from the injured and the intact sides. After the 628 experiment, the animals will be euthanized by exsanguination, followed by intracardiac perfusion 629 of 4% paraformaldehyde (4°C) for tissue fixation and subsequent harvesting. 630 Tissue Processing 631 After fixation, the C1-C8 segment of the spinal cord will be dissected and immediately placed in 632 cold 4% paraformaldehyde (P6148 Sigma-Aldrich, Darmstadt, Germany) for 24 h and then 633 cryoprotect in 30% sucrose (in 0.9% NaCl, S9888, Sigma-Aldrich, Darmstadt, Germany) for 48 h 634 and store at -80°C. Frozen transversal (C1- C8 spinal cord) free-floating sections (30 μ m) will be 635 obtained using a Thermo Fisher CryoStar NX70 cryostat. Spinal cord sections will be stored in a 636 cryoprotectant solution (sucrose 30% (pharma grade, 141621, AppliChem, Darmstadt, Germany), 637 ethylene glycol 30% (BP230-4, Fisher Scientific, Illkirch, France) and polyvinylpyrrolidone 40 638 (PVP40-100G; Sigma-Aldrich 1%) in phosphate-buffered saline (PBS) 1X (BP665-1; Fisher 639 Scientific, Illkirch, France) at -22°C. Every fifth section from C1- C8 will be used for lesion 640 evaluation to examine the extent of injury using cresyl violet histochemistry: 30 min in cresyl 641 violet solution (0.001% cresyl violet acetate (C5042-10G, Sigma-Aldrich, Darmstadt, Germany) 642 and 0.125% glacial acetic acid (A/0400/PB15, Fisher Scientific, Illkirch, France) in distilled 643 water), 30 s in 70% ethanol, 30 s in 95% ethanol, 2 × 10 s in 100% ethanol (E/0600DF/17, Fisher 644 Scientific, Illkirch, France) and 2 min in xylene (X/0100/PB17, Fischer Scientific, Illkirch, 645 France). Then, they will be coverslipped using Eukitt® mounting medium, and slide 646 36 microphotographs will be taken with a slide scanner (Aperio AT2, Leica, France). Each injury 647 was then digitized and analyzed with ImageJ 1.54k software (National Institutes of Health, 648 Bethesda, MD, USA; Schneider et al., 2012). The extent of the injury on the injured side was 649 calculated using a reference to a complete hemicontusion (which is 100% of the hemicord). 650 Immunohistochemistry 651 Several markers of interest were used on spinal cord samples from C3HC for qualitative 652 assessments. Free-floating transverse sections of the C1-C4 and C5-C8 spinal cord stored in 653 cryoprotectant solution have been washed 3 times in PBS 1X and placed in blocking solution 654 (normal donkey serum (NDS) 5% and 0.2% Triton 100X in PBS 1X) for 30 min at room 655 temperature. For primary labeling, sections were incubated overnight on an orbital shaker at 4 °C. 656 The following primary antibodies and/or biotinylated lectins were used: PDGFRβ (Abcam 657 ab32570, 1/1000, rabbit polyclonal), GFAP (PA5-18598, 1/1000, goat polyclonal), Iba1 (Abcam 658 ab5076, 1/400, goat polyclonal), CD68 (ThermoFisher, 14-0681-82, 1/100, rat polyclonal), 659 CD206 (ThermoFisher, MA5-16869, 1/100, rat polyclonal), Wisteria Floribunda Lectin (WFA, 660 Vector laboratories, Les Ulis, 1/2000) was used to labeled chondroitin sulfate proteoglycans 661 (CSPGs). After 3 times PBS 1X washes, sections were incubated for 2 h at room temperature 662 with the appropriate secondary reagents. Antibodies were visualized using Alexa Fluor 647 663 Donkey anti-rabbit (ThermoFisher, A-31573, 1/1000), Alexa Fluor® 594 donkey anti-goat 664 (ThermoFisher, A-11058, 1/1000), Alexa Fluor 647 Donkey anti-rat (ThermoFisher, A78947, 665 1/1000), biotinylated WFA binding was detected using Alexa Fluor 488 Avidin (Molecular 666 Probes, Illkirch, 1/1000) and they were washed again 3 times with PBS 1X. Sections were 667 incubated with NeuroTrace™435/455 (ThermoFisher, N21479, 1/1000), a neuronal marker 668 (Nissl stain), for 10 min and then wash again 3 times with PBS 1X. Images of the different 669 37 sections will be captured with a Hamamatsu ORCA-R2 camera mounted on an Olympus IX83 670 P2ZF scanning microscope. 671 Data Processing and Statistical Analyses 672 All data are presented as mean/i3 ±/i3 standard deviation (SD). Statistical analyses were performed 673 using GraphPad Prism (version 10.1.2, GraphPad Software, USA). The normality of data 674 distributions was assessed using the Shapiro-Wilk test. A significance threshold of p < 0.05 was 675 applied throughout. Comparisons between two groups (e.g., sham rMS vs. rMS) for continuous 676 variables such as body weight, lesion extent, diaphragmatic EMG activity, respiratory parameters, 677 and immune marker quantifications were analyzed using unpaired t-test for normally distributed 678 data, or the Mann-Whitney test for non-parametric data. For comparisons among more than two 679 independent groups (e.g., laminectomy + sham rMS, laminectomy + rMS, C3HC + sham rMS, 680 C3HC + rMS), one-way ANOVA followed by Tukey’s multiple comparisons test was used for 681 parametric data. For non-parametric data, the Kruskal-Wallis test followed by Dunn’s post hoc 682 test was applied. Repeated measures two-way ANOVA was used to assess the effects of 683 treatment (sham rMS vs. rMS) over time (D0, D7, D21) for longitudinal variables such as 684 respiratory measurements. Multiple comparisons were corrected using the Šidák or Tukey 685 method. Sample sizes and statistical test details are reported in each figure legend. 686 687 Acknowledgments 688 Funding: 689 This work was supported by Projet-ANR-24-CE19-4519 (IV); SATT Lutech Paris (IV), CNRS 690 (IV), Inserm (IV) and Sorbonne University (IV); Chancellerie des Universités de Paris (Legs 691 38 Poix) (SV), the Fondation de France (SV), the Fondation Médisite (SV), Projet ANR-RESPIRe-692 cSCI (SV) and Université de Versailles Saint-Quentin-en- Yvelines (SV), and the support of the 693 China Scholarship council program [Project ID: 202208330023](WC). 694 Author contributions: 695 Conceptualization: WC, SV, IV 696 Methodology: WC, SV 697 Investigation: WC, SV, IV 698 Visualization: WC 699 Funding acquisition: WC, SV 700 Project administration: WC, SV, IV 701 Resources: SV, IV 702 Supervision: SV, IV 703 Writing – original draft: WC 704 Writing – review & editing: WC, SV, IV 705 Competing interests: The authors declare that they have no competing interests. 706 Data and materials availability: All data needed to evaluate the conclusions in the paper are 707 present in the paper and/or the Supplementary Materials. 708

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