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## Creator

Isamu Murata, Jun Kobayashi, [Shinsuke Ishihara](https://orcid.org/0000-0001-6854-6032), [Nobuo Iyi](https://orcid.org/0000-0001-5547-7031)

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[Inhaled nitric oxide as a rescue therapy in rat crush syndrome: translating bench research to field application](https://mdr.nims.go.jp/datasets/795969be-882c-4ae6-a868-2c710e1a4b87)

## Fulltext

Inhaled nitric oxide as a rescue therapy in rat crush syndrome: translating bench research to field application1  Inhaled nitric oxide as a rescue therapy in rat crush syndrome: translating bench 1 research to field application   2 Isamu Murata1(0000-0003-2237-4938)*, Jun Kobayashi2*(0000-0002-3490-2980), Shinsuke 3 Ishihara3(0000-0001-6854-6032), Nobuo Iyi3(0000-0001-5547-7031) 4    5 1 Laboratory of Pharmacotherapeutics and Neuropsychopharmacology, Faculty of Pharmacy 6 and Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, 7 Japan. E-mail: ismurata@josai.ac.jp   8 2 Laboratory of Pathophysiology, Department of Clinical Dietetics and Human Nutrition,   9 Faculty of Pharmaceutical Science, Josai University, 1-1 Keyakidai, Sakado, Saitama, 350-10 0295, Japan. E-mail: junkoba@josai.ac.jp   11 3 Research Center for Materials Nanoarchitectonics (MANA), National Institute for Materials 12 Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki 305 0044, Japan.  13 E-mail: ISHIHARA.Shinsuke@nims.go.jp (S.I.); IYI.Nobuo@nims.go.jp (N.I.) 14  15 Graphical abstract 16  17   18 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/2  Abstract   19 Crush syndrome (CS) is characterised by ischaemia/reperfusion-induced rhabdomyolysis, 20 leading to systemic inflammation and high mortality. Building on our previous findings that 21 intravenous nitric oxide (NO) donors improve survival in this condition, we investigated the 22 therapeutic efficacy of inhaled NO delivered via a portable, controlled-release device in an 23 experimental rat model of CS. Anaesthetised rats underwent bilateral hindlimb compression 24 using rubber tourniquets for 5 h, followed by reperfusion. Among the various inhalation 25 conditions tested, administration of NO (160 parts per million) for 2 h after reperfusion 26 significantly increased survival rate from 20 to 90%. Improvements in haemodynamic 27 parameters, biochemical markers, and histopathological findings correlated with enhanced 28 survival outcomes. These results suggest that on-site NO inhalation therapy may serve as an 29 effective first-line, emergency intervention for CS, particularly in disaster settings. 30  31   32 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/3  Crush syndrome (CS) is a potentially fatal condition that occurs following rescue from disasters, 33 such as earthquakes1, landslides2, vehicle accidents3, and combat scenarios4. The sudden relief 34 of prolonged and continuous compression exerted by heavy debris on lower limb skeletal 35 muscles induces rhabdomyolysis, subsequently leading to circulatory shock, metabolic 36 acidosis, acute respiratory distress syndrome (ARDS), and acute renal failure—complications 37 frequently result in death4,5.     38 The aetiology of rhabdomyolysis in CS has been well characterised5. Compressive injury to 39 skeletal muscle fibres imposes mechanical stress on the sarcolemma, activating non-selective 40 stretch-activated channels. This facilitates an influx of Na+ and Ca2+ into the myocyte cytosol, 41 triggering cellular swelling and calcium-dependent autolysis4. Simultaneously, local ischaemia 42 induces a metabolic shift from aerobic to anaerobic pathways, resulting in ATP depletion and 43 subsequent muscle cell damage6, culminating in rhabdomyolysis.    44 Importantly, rhabdomyolysis does not develop during ischaemia per se but rather upon 45 reperfusion of the injured muscle4. During reperfusion, oxidative stress leads to muscle cell 46 lysis and the release of intracellular constituents—such as potassium and myoglobin—into the 47 circulation, precipitating early and late fatal complications, including cardiac arrhythmias, 48 myoglobinuric renal failure, and systemic inflammation5.     49 Various animal models of CS have been established to simulate skeletal muscle 50 compression–decompression injury7,8, and the therapeutic effects of multiple pharmacological 51 agents have been investigated9–11. We have previously developed a simple rat model using 52 bilateral hindlimb compression with rubber tourniquets to study CS pathophysiology and 53 explore treatment strategies5. Using this model, we demonstrated the survival benefit of 54 intravenous nitrite—a nitric oxide (NO) donor— highlighting its anti-inflammatory properties 55 in CS treatment12.      56 In the present study, we evaluated the potential of NO inhalation as an alternative therapeutic 57 modality13. Following ischaemia–reperfusion (I/R) injury, lysates derived from damaged 58 skeletal muscle accumulate in the pulmonary vasculature, triggering inflammation and 59 respiratory failure5. The lungs—being the first capillary bed to receive systemic circulation—60 serve as a primary site of injury, often culminating in ARDS. Inhaled NO has been shown to 61 attenuate pulmonary endothelial inflammation and limit the downstream systemic propagation 62 of inflammatory mediators14. Moreover, recent studies indicate that inhaled NO confers 63 protection against myocardial I/R injury by forming stable S-nitrosothiols (RSNOs) in plasma 64 and erythrocytes during pulmonary circulation15–17. These RSNOs may exert NO-dependent 65 signalling effects in extrapulmonary organs, including the kidney and injured skeletal muscles. 66 Here, we employed a portable, controlled-release NO gas delivery device developed by 67 Ishihara and Iyi—co-authors of the present study. They have previously described its technical 68 specifications and proposed its clinical utility in emergency scenarios18. The findings of this 69 study highlight its substantial potential for use in first-aid and emergency interventions, 70 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/4  particularly in field settings.  71   72 Results  73 Effects of ischaemic duration, inhaled NO concentration, and timing of NO inhalation on 74 survival in a rat model of CS  75 In our previous study, we established that 5 h of bilateral hindlimb ischaemia induced by rubber 76 tourniquets produced the highest mortality in a rat model of CS; therefore, this duration was 77 selected for the present experiments5. To determine the optimal concentration of inhaled NO 78 for therapeutic intervention, we compared the effects of low (20 parts per million [ppm]) and 79 high (160 ppm) concentrations of inhaled NO on survival outcomes in the CS rat model. We 80 then evaluated the impact of the timing of 20 ppm NO inhalation using various regimens 81 relative to reperfusion: 1 h or 2 h before reperfusion, 1 h before and 1 h after reperfusion, and 82 1 h or 2 h after reperfusion. Untreated CS rats exhibited high mortality, with survival rates of 83 approximately 20% at both 24 and 48 h after reperfusion. Notably, rats receiving 2 h of inhaled 84 NO beginning after reperfusion demonstrated an improved survival rate of approximately 40% 85 at 48 h. In contrast, all other NO inhalation regimens yielded survival rates comparable to those 86 of the untreated group, remaining around 20% (Fig. 1a).   87 Compared with the survival rates observed in the 20 ppm NO inhalation groups, inhalation of 88 160 ppm NO—particularly when administered after reperfusion—markedly improved survival 89 outcomes. Specifically, survival rates were 60%, 70%, and 90% for NO inhalation administered 90 for 1 h after reperfusion, 1 h before plus 1 h after reperfusion, and 2 h after reperfusion, 91 respectively. These rates were significantly higher than those observed in the 160 ppm NO 92 groups administered 1 h or 2 h before reperfusion (30% and 40%, respectively) and in the CS 93 control group (20%). Among all experimental groups, 160 ppm NO inhalation for 2 h after 94 reperfusion yielded the highest survival rate (90%), second only to the sham group, which 95 showed 100% survival (Fig. 1b). These findings indicate that higher concentrations of inhaled 96 NO (160 ppm vs 20 ppm), longer durations of administration (2 h vs 1 h), and after reperfusion 97 delivery rather than before reperfusion are key determinants of improved survival in this CS 98 rat model. Accordingly, subsequent experiments were conducted using 160 ppm NO inhalation 99 to evaluate its therapeutic effects on haemodynamic parameters, biochemical markers, and 100 histopathological changes.    101  102 Effects of NO inhalation on electrocardiogram parameters in CS rats 103 Supplementary Figs. S1a–e show electrocardiogram (ECG) parameters from sham-operated 104 rats, CS controls, and NO-inhaled CS rats (2 h before, 1 h before /1 h after, and 2 h after 105 reperfusion) assessed at 3, 24, and 48 h after reperfusion. Compared with the sham group, the 106 QRS, PR, and QT intervals were significantly prolonged in CS control rats at all time points 107 (Supplementary Figs. S1a–c). In contrast, NO inhalation attenuated these interval 108 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/5  prolongations, with the most pronounced improvements observed in rats receiving NO for 2 h 109 after reperfusion (Supplementary Figs. S1a–c). In CS control rats, tall and peaked T wave 110 amplitudes—so-called tented T waves indicative of hyperkalaemia—were evident at 3, 24, and 111 48 h after reperfusion (Supplementary Fig. S1e). NO inhalation significantly reduced T-wave 112 amplitudes, again most prominently in the 2 h after reperfusion group. Additionally, although 113 P-wave amplitude was reduced in CS control rats compared with sham rats, it was restored by 114 NO inhalation, with the greatest recovery observed in the 2 h after reperfusion group 115 (Supplementary Fig. S1d). Collectively, these results indicate that NO inhalation mitigates I/R-116 induced electrophysiological abnormalities, including hyperkalaemia-associated T-wave 117 changes (Supplementary Fig. S1e), atrioventricular nodal conduction delays (Supplementary 118 Fig. S1b), and atrial myocardial dysfunction (Supplementary Figs. S1a, c, d). 119  120 Effects of NO inhalation on haemodynamic and biochemical parameters in CS rats 121 In CS rats, heart rate (HR) and blood pressure (BP) parameters—including systolic blood 122 pressure (SBP), diastolic blood pressure (DBP), and mean blood pressure (MBP)—were 123 significantly lower than those in the sham group. In contrast, NO inhalation significantly 124 improved BP in all treated groups (2 h before, 1 h before /1 h after, and 2 h after reperfusion). 125 Restoration of BP to near-sham levels was most pronounced in rats receiving NO for 2 h after 126 reperfusion (Table 1). 127 Table 2 summarises the time course of plasma biochemical parameters in sham-operated rats, 128 CS control rats, and NO-treated CS rats. Levels of creatine kinase (CK), myoglobin (Mb), 129 glucose (Glu), potassium (K+), blood urea nitrogen (BUN), and creatinine (Cre) were 130 significantly elevated in CS control rats following skeletal muscle I/R injury, consistent with 131 rhabdomyolysis, hyperkalaemia, and acute renal dysfunction. NO inhalation significantly 132 attenuated these elevations, with the greatest reductions observed in the 2 h after reperfusion 133 group. Collectively, these findings indicate that after reperfusion NO inhalation provides 134 superior protection against I/R-induced rhabdomyolysis, electrolyte imbalance, and renal 135 impairment. 136  137 Arterial blood gas and methaemoglobin analysis in CS rats 138 Arterial blood gas analysis revealed persistent acidosis in CS rats at 24 and 48 h after 139 reperfusion. NO inhalation corrected arterial pH from 7.28 in CS control rats to 7.39 in the 2 h 140 before and 1 h before /1 h after reperfusion groups and to 7.47 in the 2 h after reperfusion group 141 at 24 h after reperfusion. This improvement in arterial pH was sustained through 48 h (Table 142 3). Elevations in the anion gap and arterial lactate levels in CS rats indicated a primary 143 metabolic acidosis, further supported by reductions in base excess (BE) and bicarbonate 144 (HCO3-). However, the presence of hypercapnia exceeding the expected physiological 145 respiratory compensation at both 24 and 48 h after reperfusion suggests the coexistence of 146 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/6  mixed metabolic and respiratory acidosis.  147 Methaemoglobin (Met-Hb) levels were evaluated from 2 h before reperfusion through 48 h 148 after reperfusion in sham-operated rats, CS control rats, and NO-treated CS rats (160 ppm). 149 Met-Hb levels peaked at approximately 4% in the 2 h before and 1 h before /1 h after 150 reperfusion groups. In contrast, rats receiving NO for 2 h after reperfusion exhibited lower Met-151 Hb levels, remaining below 3% throughout the observation period (Supplementary Fig. S2). 152 These findings indicate that after reperfusion NO inhalation effectively improves acid–base 153 balance while maintaining a relatively lower risk of NO-induced methaemoglobinaemia. 154  155 Effects of NO inhalation on systemic cytokine profiles in CS rats  156 Plasma concentrations of the pro-inflammatory cytokines tumour necrosis factor-α (TNF-α), 157 interleukin (IL)-1β, and IL-6, as well as the anti-inflammatory cytokine IL-10, were measured 158 at 24 and 48 h after reperfusion in sham-operated rats, CS control rats, and NO-treated CS rats 159 (160 ppm for 2 h before , 1 h before /1 h after , and 2 h after reperfusion). Compared with sham 160 rats, CS rats exhibited significantly elevated plasma levels of TNF-α, IL-1β, and IL-6 at both 161 time points. NO inhalation significantly attenuated these elevations, with the most pronounced 162 reductions observed in the 2 h after reperfusion group (Fig. 2a). Although cytokine levels 163 remained elevated at 48 h after reperfusion, peak concentrations were generally lower than 164 those observed at 24 h, suggesting a gradual resolution of the inflammatory response. In 165 contrast to the pro-inflammatory cytokines, IL-10 levels were similarly elevated in CS control 166 and NO-treated groups at 24 h after reperfusion (Fig. 2a). This persistent elevation of IL-10 167 may reflect an endogenous anti-inflammatory response aimed at limiting I/R injury, potentially 168 through suppression of neutrophil recruitment and cytokine production, consistent with 169 previous reports on the regulatory role of IL-10 in myocardial and pulmonary I/Rinjury19–21. 170 At 48 h after reperfusion, IL-10 levels showed a declining trend in NO-treated rats, with a more 171 pronounced reduction in the 2 h after reperfusion group, potentially indicating progression 172 towards resolution of the inflammatory phase.  173  174 Effects of NO inhalation on tissue myeloperoxidase activity in CS rats  175 Myeloperoxidase (MPO) activity, a marker of leukocyte infiltration, was assessed in skeletal 176 muscle, lung, and kidney tissues at 24 and 48 h after reperfusion in sham-operated rats, CS 177 control rats, and NO-treated CS rats (160 ppm for 2 h before, 1 h before /1 h after, and 2 h after 178 reperfusion). MPO activity was significantly elevated in all examined organs of CS rats 179 compared with sham animals, reflecting enhanced leukocyte accumulation secondary to I/R 180 injury. Inhaled NO markedly suppressed MPO activity in each organ at both time points, with 181 the most pronounced reductions observed in the 2 h after reperfusion group (Fig. 2b). These 182 findings are consistent with the temporal profile of increased plasma pro-inflammatory 183 cytokines (Fig. 2a) and suggest that NO inhalation mitigates systemic and local inflammatory 184 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/7  responses by limiting leukocyte infiltration not only in tissues directly affected by I/R injury 185 but also in remote organs, including the lungs and kidneys. The mechanisms underlying this 186 multi-organ anti-inflammatory effect of inhaled NO are further addressed in the Discussion 187 section. 188  189 Effects of NO inhalation on histopathology of the lungs, skeletal muscles, and kidneys in CS 190 rats 191 In sham-operated rats, gastrocnemius muscles displayed normal histological architecture. In 192 contrast, CS rats exhibited marked interstitial oedema, leukocyte infiltration, muscle fibre 193 degeneration, and atrophy. These pathological changes were attenuated by NO inhalation, with 194 the most pronounced improvements observed in rats treated for 2 h after reperfusion at both 24 195 and 48 h after reperfusion (Fig. 3a and b, upper panels). 196 Similarly, histological examination of lung tissue revealed thin alveolar septa in sham rats, 197 whereas CS rats showed extensive intra-alveolar and interstitial oedema accompanied by 198 leukocyte infiltration. These inflammatory features were substantially reduced by NO 199 inhalation, particularly in the 2 h after reperfusion group (Fig. 3a and 3b, middle panels). 200 Renal histology in sham rats showed intact glomeruli and normal proximal and distal tubular 201 structures. In contrast, CS rats exhibited moderate dilation of distal tubules and epithelial 202 flattening, with these lesions more prominent at 48 than at 24 h after reperfusion. NO inhalation 203 ameliorated these pathological changes at both time points, again with greater efficacy in the 2 204 h after reperfusion group (Fig. 3a and b, lower panels, 3b). Consistent with these histological 205 findings, urinary levels of kidney injury molecule-1 (KIM-1), a transmembrane protein with 206 immunoglobulin and mucin domains and a recognised urinary marker of acute proximal tubular 207 injury22, were significantly elevated in CS rats. NO inhalation reduced KIM-1 excretion, with 208 a modest decrease at 24 h and a significant reduction at 48 h after reperfusion, particularly in 209 rats treated with NO for 2 h after reperfusion (Fig. 3c). Quantitative scoring of 210 histopathological variables in skeletal muscle, lung and kidney tissues corroborated these 211 observations, demonstrating significant improvements following NO inhalation. The 212 therapeutic effect was most pronounced in the 2 h after reperfusion group at both 24 and 48 h 213 across all organs examined (Fig. 3a and b).  214  215 Discussion   216 To the best of our knowledge, this is the first report to establish NO inhalation as a practical 217 and effective treatment strategy for CS that bypasses the need for intravenous drug 218 administration or fluid infusion. 219 CS remains a major cause of mortality at disaster sites, necessitating the urgent development 220 of effective, field-deployable therapeutic strategies. Among emerging interventions3, NO 221 donors have shown promise as protective agents against I/R injury, a central pathological 222 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/8  component of CS. Our group has previously demonstrated that intravenous administration of 223 nitrite, an NO donor, immediately before reperfusion significantly improves survival in a rat 224 model of CS, increasing survival rates from 24% in untreated CS controls to 36% and 64% 225 with 100 and 200–500 μmol/kg of NaNO₂, respectively12. However, systemic hypotension—a 226 well-recognised adverse effect of intravenous nitrite—raises safety concerns, particularly in 227 the setting of CS-associated hypovolaemic shock. In our earlier study, intravenous nitrite 228 administration did not restore mean arterial pressure to levels observed in sham-operated 229 animals12.  230 To address this limitation and evaluate the translational potential of NO-based therapy in real-231 world disaster settings, we examined the effects of inhaled NO in a rat model of CS. We utilised 232 a ready-to-use NO gas-releasing device, recently described by Ishihara and Iyi18, which offers 233 a practical and rapidly deployable method for delivering inhaled NO in austere environments.  234 In this study, we focused on optimising three key experimental variables—ischaemic duration, 235 inhaled NO concentration, and timing of NO administration (2 h before , 1 h before /1 h after , 236 and 2 h after reperfusion)—to establish conditions that closely mimic the mortality associated 237 with CS. Accurate determination of the ischaemic period is critical for the development of 238 effective therapeutic strategies. Belkin et al.23, using spectrophotometric analysis of skeletal 239 muscle injury in a rat hindlimb tourniquet model, demonstrated that significant tissue damage 240 begins at 3 h of ischaemia and progressively worsens with longer durations (4, 5, and 6 h). 241 Consistently, Murata et al.5 reported that tourniquet-induced 5 h of bilateral hindlimb ischaemia 242 in rats resulted in near-complete mortality within 24 h after reperfusion, whereas mortality 243 markedly declined to 0% and 10% with shorter (4 h) and longer (6 h) ischaemic durations, 244 respectively. These findings suggest a time-dependent ‘critical window’ of ischaemia that 245 maximally triggers systemic inflammatory responses and fatal crush injury. Blaisdell et al.24 246 further corroborated this observation, showing that systemic release of inflammatory mediators 247 from injured muscle was minimal when the crush period was ≤4 h (owing to limited tissue 248 necrosis) and ≥6 h (attributed to the no-reflow phenomenon caused by irreversible 249 microvascular occlusion). In the clinical setting, when limb ischaemia exceeds 6 h, survival 250 often necessitates amputation of the necrotic extremity. Although the precise ischaemic 251 threshold varies by organ, species, and experimental model, we selected a 5 h crush duration 252 in our rat model to reliably induce severe rhabdomyolysis, systemic inflammation, and lethal 253 CS, consistent with previous reports5.  254 In clinical practice, inhaled NO is commonly administered at concentrations ranging from 10 255 to 250 ppm for conditions such as idiopathic respiratory distress syndrome (IRDS) in neonates 256 and severe coronavirus disease 2019 in adults25–27. In the present study, we evaluated two 257 concentrations of inhaled NO—20 ppm (low) and 160 ppm (high)—to determine their 258 therapeutic efficacy in a rat model of CS. Notably, inhalation of 160 ppm NO significantly 259 improved survival compared with 20 ppm (Fig. 1). Furthermore, administration of 160 ppm 260 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/9  NO significantly improved blood pressure parameters (systolic, diastolic, and mean blood 261 pressure), as well as heart rate, particularly when administered after reperfusion, compared 262 with untreated CS control rats (Table 1). These findings suggest that NO-mediated pulmonary 263 vasodilation enhances ventilation–perfusion matching, augments pulmonary venous return, 264 and improves cardiac output and heart rate in the setting of CS-induced circulatory compromise. 265 A recognised clinical concern associated with high-dose NO inhalation is 266 methaemoglobinaemia, which typically warrants discontinuation when Met-Hb levels exceed 267 5%28. In our study, inhalation of 160 ppm NO resulted in peak Met-Hb levels of approximately 268 4% in the 2 h before and 1 h before /1 h after reperfusion groups and below 3% in the 2 h after 269 reperfusion group (Supplementary Fig. S2), remaining within clinically acceptable limits29. 270 The variation in Met-Hb levels appeared to depend on the timing of NO administration relative 271 to reperfusion. During the ischaemic phase, substantial tissue oedema in skeletal muscle likely 272 sequesters erythrocytes and plasma components within interstitial and intracellular spaces, 273 reducing their contribution to systemic circulation. Upon reperfusion, re-entry of these 274 components may dilute circulating Met-Hb concentration. Accordingly, in the before 275 reperfusion groups, Met-Hb levels peaked approximately 1 h after NO exposure and declined 276 rapidly after reperfusion, whereas in the after-reperfusion group, peak Met-Hb levels were 277 attenuated owing to immediate dilution by restored circulation. These observations underscore 278 the safety of 160 ppm NO inhalation with respect to methaemoglobinaemia in this CS model. 279 Among the NO intervention regimens, inhalation of 160 ppm NO initiated 2 h after 280 reperfusion conferred the greatest survival benefit in rats with CS (Fig. 1b). This timing was 281 associated with the most pronounced improvements in haemodynamics, biochemical 282 parameters, and renal function (Tables 1–3 and Fig. 3c). In CS, the lungs are the first organ 283 exposed to cellular debris and damage-associated molecular patterns released from ischaemic 284 skeletal muscle, triggering interactions with the pulmonary vascular endothelium, neutrophil 285 infiltration, induction of inducible NO synthase, and generation of reactive oxygen species. 286 These processes promote pulmonary inflammation and, in severe cases, ARDS30,31. Inhaled 287 NO mitigates this inflammatory cascade by suppressing the expression of adhesion molecules, 288 such as intercellular adhesion molecule-1 on the pulmonary vascular endothelium, thereby 289 limiting neutrophil adhesion and systemic propagation of inflammatory mediators14. 290 Furthermore, NO-induced pulmonary vasodilation improves ventilation–perfusion matching, 291 arterial oxygenation, and venous return, ultimately enhancing cardiac output and heart rate in 292 CS rats (Table 1).  293 We have previously reported that nitrite, an endocrine reservoir of NO under hypoxic 294 conditions, is depleted in ischaemic skeletal muscle of CS rats where it is reduced to NO within 295 mitochondria to support ATP generation under impaired oxidative phosphorylation12. This 296 depletion suggests that exogenous NO supplementation—via intravenous administration or 297 inhalation—may be required to protect organs from oxidative stress during reperfusion. In the 298 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/10  present study, we focused on inhaled NO as a strategy to deliver NO not only to the pulmonary 299 vasculature but also to distal tissues, including skeletal muscle. Notably, extrapulmonary 300 effects of inhaled NO have been demonstrated in the systemic circulation32. Fox-Robichaud et 301 al.33 showed that inhaled NO acts at the peripheral microvasculature, exerting anti-adhesive, 302 anti-vasoconstrictive, and anti-permeability effects in NO-depleted tissues. Additional clinical 303 and preclinical studies support systemic benefits of inhaled NO, including attenuation of 304 reperfusion-associated inflammation in ischaemic limbs32, reduction of hepatic injury 305 following liver transplantation34, and protection against myocardial I/R injury through 306 accumulation of NO metabolites, such as RSNOs in plasma and tissues17. 307 Although NO was previously thought to be rapidly inactivated in pulmonary circulation 308 through oxidation and scavenging by haemoglobin, forming biologically inert nitrate in plasma 309 and erythrocytes35,36, accumulating evidence indicates that inhaled NO (the NO· radical) forms 310 stable protein S-nitrosothiols (RSNOs; NO⁺ covalently bound to thiol groups of proteins or 311 low-molecular-weight thiols) in plasma and erythrocytes during pulmonary circulation37,14. 312 These RSNOs circulate systemically and mediate cGMP-independent NO signalling via 313 transnitrosylation, thereby conferring anti-inflammatory and cytoprotective effects in remote 314 organs, such as skeletal muscle and kidney15–17. The mechanisms underlying the 315 extrapulmonary effects of inhaled NO have been comprehensively reviewed elsewhere31.  316 An additional critical consideration is the timing of NO inhalation relative to reperfusion, 317 because the ability of inhaled NO-derived RSNOs to reach distant ischaemic organs depends 318 on when NO is administered. NO inhalation initiated 2 h before reperfusion failed to effectively 319 deliver RSNOs to erythrocytes and plasma proteins within ischaemic skeletal muscle tissues. 320 In contrast, NO inhalation initiated 2 h after reperfusion allowed RSNOs to be delivered to 321 tissues at the onset of reperfusion, resulting in more robust protective effects. This benefit 322 exceeded that observed with split timing (1 h before /1 h after reperfusion), indicating that NO 323 inhalation initiated immediately after reperfusion onset may represent the optimal strategy to 324 maximise RSNO delivery and improve survival outcomes in CS. The study focused on specific 325 pulmonary and systemic effects of inhaled NO, potentially overlooking other critical factors 326 influencing outcomes in CS, such as the variability in patient responses to treatment and 327 presence of co-morbid conditions. Furthermore, while the portable NO delivery device shows 328 promise for field deployment, its long-term efficacy, safety, and practicality in diverse disaster 329 scenarios have not been clarified. Finally, the integration of inhaled NO with other supportive 330 measures, while theoretically beneficial, necessitates further exploration to establish optimal 331 treatment protocols and timing to maximise clinical outcomes. 332  333 Conclusion 334 Inhaled NO exerted multifaceted pulmonary and systemic effects through both cGMP-335 dependent and -independent mechanisms, including reduced pulmonary vascular resistance, 336 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/11  decreased thrombotic risk, and suppression of leukocyte–endothelial interactions across 337 vascular beds, ultimately leading to improved survival in CS rats. Importantly, this study 338 highlights the practical advantages of inhaled NO—particularly its simplicity, portability, and 339 non-invasiveness—over conventional intravenous therapies, making it especially attractive for 340 use in disaster scenarios. Although NO inhalation alone was highly effective, further 341 improvements in clinical outcomes may be achievable by integrating this approach with 342 established supportive measures such as aggressive fluid resuscitation and targeted anti-343 inflammatory therapies.  344 The portable, controlled NO delivery device employed in this study represents a clinically 345 feasible, field-deployable solution with strong potential to transform early-phase treatment of 346 CS and improve survival in mass-casualty events. 347  348 Methods   349 CS animal model 350 Male Wistar rats (250–300 g) were obtained from Japan SLC (Shizuoka, Japan) and housed 351 under controlled environmental conditions (23 ± 3°C, 55 ± 15% relative humidity) on a 12 h/12 352 h light–dark cycle, with ad libitum access to food and water. Anaesthesia was maintained with 353 inhaled isoflurane (2–5%), and body temperature was maintained using a heating pad 354 throughout the procedure. The CS model was established as previously described by Murata et 355 al.5. Briefly, a rubber tourniquet was applied bilaterally to the hind limbs of each rat, wrapped 356 five times around a 2.0-kg metal cylinder, and secured with adhesive tape (Supplementary Fig. 357 S3). After 5 h of compression, the tourniquet was released by cutting the band and removing 358 the device to initiate reperfusion. 359   360 Experimental design  361 This study consisted of four separate experiments using the CS rat model (Supplementary 362 Fig. S5). 363 Experiment 1. Survival rates of CS rats inhaling two concentrations of NO (20 and 160 ppm) 364 were assessed at 0, 1, 3, 6, 24, and 48 h after reperfusion. Based on superior survival outcomes 365 with 160 ppm NO, this concentration and three inhalation timing protocols (2 h before, 1 h 366 before /1 h after, and 2 h after reperfusion) were applied in subsequent experiments. 367 Experiment 2. CS rats inhaled 160 ppm NO for 2 h at different timings relative to reperfusion 368 (2 h before, 1 h before /1 h after, and 2 h after reperfusion). Vital signs, including blood pressure 369 and heart rate, were measured at 3, 24, and 48 h after reperfusion. 370 Experiment 3. Met-Hb levels were monitored over time following inhalation of 160 ppm NO 371 using the same three timing protocols described above. 372 Experiment 4. Biochemical parameters were assessed at 3, 24, and 48 h after reperfusion in CS 373 rats treated with 160 ppm NO under the same three timing conditions.  374 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/12   375 Device for the controlled release of NO gas 376 The NO generator used in this study was prepared as described in our previous reports18,38. 377 Briefly, two types of nitrite-type layered double hydroxides (NLDHs) were used to deliver low 378 (20 ppm) and high (160 ppm) concentrations of NO to CS rats. For the low-concentration 379 condition (20 ppm), NLDH synthesised via the reconstruction method (RC-NLDH, 400 mg)38 380 was mixed with iron(II) sulphate heptahydrate (FeSO₄·7H₂O; 400 mg) using a mortar and 381 pestle. One quarter of the solid mixture (200 mg total) was then loaded into a 6 mL plastic 382 syringe fitted with sponge caps to retain the powder mixture. The NO generator was dried under 383 vacuum overnight and subsequently sealed in a gas-barrier bag (Lamizip® AL-D, 384 Seisannipponsha, Ltd.) together with a zeolite-type strong desiccant (AZ10G-100, As One 385 Corp.). The packaged generator was stored under refrigerated conditions and opened 386 immediately before use. For the high-concentration condition (160 ppm), NLDH synthesised 387 via the anion-exchange method (AE-NLDH; 800 mg)18 was mixed with FeSO₄·7H₂O (800 mg) 388 using mortar and pestle. One quarter of this solid mixture (400 mg total) was similarly loaded 389 into a 6 mL plastic syringe with sponge caps, dried under vacuum, and packaged in the same 390 manner as described above. 391 NO generation was initiated by exposure of the NO generator to humid air, which triggered 392 an anion-exchange reaction between interlayer nitrite (NO₂-) and sulphate (SO₄²-), followed by 393 a redox reaction between NO₂⁻ and ferrous ions (Fe²⁺)18,38. The NO generator was connected 394 sequentially to a humidifier (a plastic column containing wet wipes; Wipers S-200, KIMWIPE), 395 an electric pump (GSP-400FT, GASTEC), a nitrogen dioxide (NO₂) remover (a plastic column 396 containing calcium hydroxide/lithium chloride pellets; Litholyme®, Allied Healthcare 397 Products, Inc.), a syringe filter (pore size, 0.45 μm), and an electrochemical NO sensor 398 (ToxiRAE Pro, RAE Systems), as illustrated in Supplementary Fig. S4. The flow rate of the 399 pump was set to 0.25 L min-¹ and adjusted as necessary to maintain the desired NO 400 concentration. Notably, the acidic impurity gas NO₂ is efficiently removed by basic adsorbents 401 such as calcium hydroxide (Ca(OH)₂), whereas neutral NO passes through unimpeded39. NO 402 generation was monitored at room temperature. 403  404 Analysis of haemodynamics, blood gas levels, biochemical parameters, coagulation, and 405 interleukin levels  406 The HR, SBP, MBP, DBP, and ECG parameters—including QRS interval, PR interval, QT 407 interval, P wave, and T wave—were recorded using a PowerLab data acquisition system (AD 408 Instruments, Nagoya, Japan). The carotid artery was cannulated with a polyethylene catheter 409 (PE-50 tubing) connected to a pressure transducer. Arterial blood samples were collected from 410 each rat via the carotid artery catheter at 3, 6, 24, and 48 h after reperfusion5.  411 Arterial levels of Glu, K+, BUN, haematocrit (Hct), pH, partial pressure of oxygen (pO₂), partial 412 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/13  pressure of carbon dioxide (pCO₂), BE, anion gap (AG), and lactate (Lac) were analysed using 413 an i-STAT 300F blood gas analyser with CG4+ and EC8+ cartridges (FUSO Pharmaceutical 414 Industries, Osaka, Japan). Creatine phosphokinase (CPK) levels were measured using a 415 creatine kinase assay kit (EnzyChrom, BioAssay Systems Co.). Plasma Mb concentrations 416 were determined using a solid-phase enzyme-linked immunosorbent assay (Rat Myoglobin 417 ELISA; Life Diagnostics, West Chester, PA, USA). Met-Hb levels in blood were assessed using 418 a manual spectrophotometric assay based on the disappearance of the Met-Hb absorption peak 419 at 635 nm (pH 6.6), following conversion of Met-Hb to cyan-Met-Hb by neutralised 420 cyanide40.  421 In each experimental group (3, 24, and 48 h after reperfusion; n=6), venous blood and 422 gastrocnemius muscle tissue samples were collected for the measurement of inflammatory 423 cytokines, tissue thiobarbituric acid-reactive substances (TBARS), and MPO activity41. Venous 424 blood samples were obtained at 3, 6, 24, and 48 h after reperfusion via a jugular vein catheter5. 425 Serum levels of IL-6, IL-10, IL-1β, and TNF-α were measured using Quantikine® ELISA kits 426 (R&D Systems, Inc., Minneapolis, MN, USA). KIM-1 levels were measured using a Rat TIM-427 1/KIM-1/HAVCR immunoassay (R&D Systems, Inc.), and plasma and urinary creatinine 428 levels were determined using a creatinine colourimetric assay kit (Cayman Chemical, Ann 429 Arbor, MI, USA). 430  431 Histological evaluation  432 For histological evaluation, tissue samples from skeletal muscle, lung, and kidney were fixed 433 in 10% neutral-buffered formalin, embedded in paraffin, sectioned, and stained with 434 haematoxylin and eosin5.  435 For lung and skeletal muscle tissues, histological variables scored included alveolar and 436 interstitial inflammation, alveolar and interstitial haemorrhage, oedema, atelectasis, and 437 necrosis. Each variable was graded on a 0–4 scale: 0, no injury; 1, injury involving 25% of 438 the field; 2, injury involving 50% of the field; 3, injury involving 75% of the field; and 4, 439 injury involving the entire field. The maximum possible composite score was 2842. 440 Renal injury was scored by calculating the percentage of tubules exhibiting tubular dilation, 441 cast formation, and tubular necrosis, according to a previously described method43. For each 442 kidney, 12 cortical tubules from at least four different regions (three tubules per region) were 443 evaluated, with care taken to avoid repeated scoring of different convolutions of the same 444 tubule. Higher scores reflected more severe injury (maximum score per tubule, 7). Points 445 were assigned for tubular epithelial cell flattening (1 point), brush-border loss (1 point), cell 446 membrane bleb formation (1 point), interstitial oedema (1 point), cytoplasmic vacuolisation 447 (1 point), cell necrosis (1 point), and tubular lumen obstruction (1 point). 448  449 Statistical analyses    450 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/14  Data are presented as mean ± standard error of the mean (s.e.m.). Survival rates were 451 analysed using the log-rank test. 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Intensive Care 7, 90 (2017).  553 42.  Su, X., Wang, L., Song, Y. & Bai, C. Inhibition of inflammatory responses by 554 ambroxol, a mucolytic agent, in a murine model of acute lung injury induced by 555 lipopolysaccharide. Intensive Care Med. 30, 133-40 (2004). 556 43.  Paller, M. S., Hoidal, J. R. & Ferris, T. F. Oxygen free radicals in ischemic acute renal 557 failure in the rat. J. Clin. Invest. 74: 1156‑1164 (1984). 558  559  560 Declarations 561 Ethical approval 562 All animal experiments were conducted in accordance with institutional guidelines for animal 563 use and were approved by the Life Science Research Centre of Josai University (approval no. 564 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/17  JU18030). 565  566 Acknowledgements   567 This work was supported by JSPS KAKENHI Grant Number JP22K09148 (to I.M., J.K., and 568 S.I.), a Grant-in-Aid for Scientific Research Presidential Research Grant of Josai University 569 (to I.M. and J.K., 2023–2024), and the World Premier International Research Center Initiative 570 (WPI), MEXT, Japan (to S.I. and N.I.). 571  572 Authors and affiliations 573 Faculty of Pharmacy and Pharmaceutical Sciences, Josai University, Saitama, Japan 574 Isamu Murata and Jun Kobayashi 575 *Correspondence to: Isamu Murata (ismurata@josai.ac.jp) and Jun Kobayashi 576 (junkoba@josai.ac.jp)  577  578 Author contributions 579 I.M. developed the experimental system for the rat crush syndrome model, performed most of 580 the animal experiments, and generated key data. J.K. proposed the concept of nitric oxide 581 inhalation for crush syndrome and contributed to data interpretation and manuscript preparation 582 together with I.M. S.I. and N.I. developed the nitric oxide gas delivery system and provided it 583 for the experiments. All authors reviewed the manuscript and approved its submission to this 584 journal. 585  586 Competing interests  587 The authors declare no competing financial interests. 588  589   590 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/18  Figures and Table Legends 591  592  593  594 Fig. 1| Survival rates with nitric oxide inhalation 595 a) 20 ppm nitric oxide inhalation. 596 Survival rates of sham-operated rats, CS rats, and CS rats inhaling 20 ppm NO (20 1 h pre, 20 597 2 h pre, 20 1 h post, 20 2 h post, and 20 1 h pre/1 h post) were assessed at 0, 1, 3, 6, 24, and 48 598 h after reperfusion. Survival curves are generated using the Kaplan–Meier method (n=15 per 599 group). *p<0.05 vs sham group, #p<0.05 vs CS group (log-rank test). 600 b) 160 ppm nitric oxide inhalation. 601 Survival rates of sham-operated rats, CS rats, and CS rats inhaling 160 ppm NO (160 1 h pre, 602 160 2 h pre, 160 1 h post, 160 2 h post, and 160 1 h pre/1 h post) at 0, 1, 3, 6, 24, and 48 h after 603 reperfusion. Abbreviations: NO, nitric oxide; CS, crush syndrome; ppm, parts per million; 20 604 1 h pre, 20 ppm NO inhalation 1 h before reperfusion; 20 2 h pre, 20 ppm NO inhalation 2 h 605 before reperfusion; 20 1 h post, 20 ppm NO inhalation 1 h after reperfusion; 20 2 h post, 20 606 ppm NO inhalation 2 h after reperfusion; 20 1 h pre/1 h post, 20 ppm NO inhalation 1 h before 607 and 1 h after reperfusion. 160 1 h pre, 160 ppm NO inhalation 1 h before reperfusion; 160 2 h 608 pre, 160 ppm NO inhalation 2 h before reperfusion; 160 1 h post, 160 ppm NO inhalation 1 h 609 after reperfusion; 160 2 h post, 160 ppm NO inhalation 2 h ter reperfusion; 160 1 h pre/1 h post, 610 160 ppm NO inhalation 1 h before and 1 h after reperfusion. 611  612  613  614 0204060801000 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48survival rate (%)reperfused (h)#*0204060801000 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48survival rate (%)reperfused (h)0204060801000 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48survival rate (%)reperfused (h)160 ppm NO inhalationshamCS160 1h pre160 2h pre160 1h pre/1hr post160 1h post160 2h post* *#** *#,†#,†,♭#,†#,†#,†#,†,♭*#*#a) b)0204060801000 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48survival rate (%)reperfused (h)20 ppm NO inhalationshamCS20 1h pre20 2h pre20 1h pre/1hr post20 1h post20 2h post0100200300400500600serum IL-1β (pg/mL)**### ##0100200300400500600700serum IL-6(pg/mL)*####*050100150200250300serum IL-10 (pg/mL)*#*#020406080100120140160180200serum TNF-α(pg/mL)*###*24           48after reperfusion (h)012345678910muscle MPO activity (unit /g tissue)**###0510152025303540lung MPO activity (unit/ g tissue)**## #00.511.522.5kidney MPO activity (unit/ g tissue)**# ##,†# ##shamCS160 2h pre160 1h pre/1h post160 2h post#24           48after reperfusion (h)24           48after reperfusion (h)24           48after reperfusion (h)a) b).CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/19   615 Fig. 2 | Plasma levels of inflammatory, anti-inflammatory cytokines and tissue 616 myeloperoxidase activity levels 617 a) Plasma levels of inflammatory, and anti-inflammatory cytokines. Plasma levels of the pro-618 inflammatory cytokines, TNF-α, IL-1β, and IL-6, and the anti-inflammatory cytokine (IL-10), 619 are measured at 24 and 48 h after reperfusion in sham-operated rats, CS control rats, and CS 620 rats treated with inhaled NO (160 ppm). 621 *p<0.05, vs sham group, #p<0.05, vs CS group (Tukey–Kramer test). Values are presented as 622 the mean ± s.e.m. (n=6 per group). 623 b) Tissue myeloperoxidase activity levels. MPO activity in skeletal muscle, lung, and kidney 624 tissues is measured at 24 and 48 h after reperfusion in sham-operated rats, CS control rats, and 625 CS rats treated with inhaled NO (160 ppm). 626 *p<0.05, vs sham group; #p<0.05, vs CS group; †p<0.05, vs 160 ppm before reperfusion group 627 (Tukey–Kramer test). Values are presented as mean ± s.e.m. (n=6 per group). 628  629 Abbreviations: NO, nitric oxide; CS, crush syndrome; ppm, parts per million; TNF-α, tumour 630 necrosis factor-α; IL-1β, interleukin-1β; IL-6, interleukin-6; IL-10, interleukin-10; s.e.m., 631 standard error of mean. Pre and post indicate timing relative to reperfusion 632  633  634  635 Fig. 3 | Effects of NO inhalation on the histopathology of skeletal muscle, lung, and kidney 636 in CS rats 637 a) Representative histological sections of gastrocnemius muscle, lung, and kidney tissues 638 obtained at 24 and 48 h after reperfusion from sham-operated rats, CS rats, and CS rats treated 639 with inhaled NO (160 ppm) for 2 h before, 1 h pre/1 h post, or 2 h after reperfusion.  640 Upper panels: Sham rats show normal gastrocnemius muscle architecture at both time points. 641 CS rats exhibit marked oedema, muscle fibre degeneration, and atrophy. These pathological 642 changes are attenuated by NO inhalation, most prominently in the 2 h after reperfusion group.  643 Middle panels: Sham rats display normal alveolar architecture with thin septa. CS rats show 644 pronounced intra-alveolar and interstitial oedema with inflammatory cell infiltration, which is 645 reduced by NO inhalation at both 24 and 48 h after reperfusion.  646 Lower panels: Sham kidneys exhibit preserved cortical architecture. CS rats show dilation of 647 muscle24h48hlung24h48hkidney24h48hsham CS100µmNO inhalation160 2h pre160 1h pre/1 h post160 2h postshamCS160 2h pre160 1h pre/1h post160 2h post012345score of muscle injury012345score of lung injury012345score of kidney injury＊＊＊＊＊＃＃＃＃ ＃＃＃＃＃＃＊24                        48after reperfusion (h)00.511.522.533.5urine KIM-1 / Cre ratio24                        48after reperfusion (h)###＊＊a) b) c).CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/20  distal tubules with epithelial flattening, particularly at 48 h after reperfusion. NO inhalation 648 mitigates these renal pathological changes at both time points. Haematoxylin and eosin 649 staining; original magnification, ×200.  650 b) Quantitative analysis of the variables scored in skeletal muscles, lungs and kidneys before 651 and after NO inhalation. Quantitative analysis of histopathological variables in skeletal muscle, 652 lung, and kidney tissues at 24 and 48 h after reperfusion in sham-operated rats, CS rats, and CS 653 rats treated with inhaled NO (160 ppm). NO inhalation significantly improves 654 histopathological scores across all organs, with the most pronounced therapeutic effects 655 observed in the 2 h after reperfusion group at both time points. 656 c) Urinary excretion of kidney injury molecule-1. Urinary levels of KIM-1 measured at 24 657 and 48 h after reperfusion in sham-operated rats, CS rats, and CS rats treated with inhaled NO 658 (160 ppm). CS rats show significantly increased urinary KIM-1 excretion compared with 659 sham rats. NO inhalation reduces KIM-1 levels, with a decreasing trend at 24 h and a 660 significant reduction at 48 h after reperfusion, particularly in the 2 h after reperfusion group.  661 *p<0.05, vs sham group; #p<0.05, vs CS group (Tukey–Kramer test). Values are presented as 662 the mean ± s.e.m. (n=6 per group). 663 Abbreviations: NO, nitric oxide; CS, crush syndrome; KIM-1, kidney injury molecule-1; ppm, 664 parts per million. Pre and post indicate timing relative to reperfusion. 665  666 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/21  Tables 667 Table 1 | Effects of 160 ppm NO inhalation on heart rate and blood pressure in a rat model of crush syndrome   668  669     after reperfused (h) 3 24 48 HR  (bpm) sham 394.3 ± 13.7   388.5 ± 19.6   388.2 ± 15.6   CS 271.0 ± 19.7 * 306.8 ± 11.2   274.5 ± 34.6 * 160 2h pre 312.5 ± 26.8   282.5 ± 26.0   284.3 ± 30.3   160 1h pre/1h post 364.5 ± 16.1 # 342.0 ± 33.4   304.3 ± 33.7   160 2h post 391.2 ± 9.4 # 375.7 ± 22.4   351.0 ± 26.2   SBP  (mmHg) sham 127.0 ± 3.4   127.8 ± 5.3   120.3 ± 6.8   CS 68.3 ± 4.2 * 67.0 ± 7.6 * 63.8 ± 4.6 * 160 2h pre 85.2 ± 8.8   95.3 ± 6.3   77.5 ± 2.1   160 1h pre/1h post 124.8 ± 5.2 # 108.5 ± 6.6 # 77.8 ± 4.9   160 2h post 149.7 ± 4.3 # 123.0 ± 2.6 # 121.5 ± 7.7 # MBP  (mmHg) sham 123.2 ± 3.1   117.8 ± 5.3   118.5 ± 3.8   CS 59.7 ± 3.9 * 60.7 ± 6.0 * 60.7 ± 5.5 * 160 2h pre 72.8 ± 8.0   85.3 ± 6.3   69.2 ± 4.4   160 1h pre/1h post 104.3 ± 6.1 # 85.8 ± 3.6   67.8 ± 4.9   160 2h post 138.7 ± 4.8 # 104.2 ± 2.2 # 111.5 ± 7.7 # DBP  (mmHg) sham 111.5 ± 4.4   102.7 ± 3.4   109.7 ± 3.1   CS 62.3 ± 2.8 * 57.7 ± 6.8 * 50.2 ± 3.6 * 160 2h pre 71.3 ± 5.6   79.2 ± 6.9   61.5 ± 2.6   160 1h pre/1h post 91.2 ± 7.2 # 81.3 ± 3.8 # 63.5 ± 5.7   .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/22  160 2h post 110.0 ± 3.8 # 99.8 ± 2.0 # 102.8 ± 7.8 # Statistics: *p<0.05, vs sham group; #p<0.05, vs CS group (Tukey–Kramer test). Values are presented as mean ± s.e.m. (n=6 per group). 670 Abbreviations: HR, heart rate; SBP, systolic blood pressure; MBP, mean blood pressure; DBP, diastolic blood pressure; bpm, beats per minute; 671 NO, nitric oxide; CS, crush syndrome. 672 Pre and post indicate timing relative to reperfusion.  673  674  675 676 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/23  Table 2 | Effects of 160 ppm NO inhalation on biochemical parameters in a rat model of crush syndrome 677   678     after reperfused (h) 3 24 48 CPK  (×104 IU/L) sham 0.2 ± 0.0   0.2 ± 0.0   0.2 ± 0.0   CS 6.8 ± 1.4 * 20.6 ± 3.2 * 22.2 ± 1.8 * 160 2h pre 4.4 ± 0.9   18.2 ± 0.8   16.9 ± 2.6   160 1h pre/1h post 3.4 ± 0.7 # 12.0 ± 1.6 # 12.1 ± 1.4 # 160 2h post 3.0 ± 0.3 # 8.0 ± 0.6 # 6.6 ± 0.6 # Myoglobin  (mg/mL) sham 10.8 ± 2.2   4.8 ± 0.2   4.7 ± 0.2   CS 246.5 ± 56.6 * 512.2 ± 58.1 * 566.0 ± 82.4 * 160 2h pre 238.3 ± 53.5   446.0 ± 22.8   504.0 ± 38.2   160 1h pre/1h post 227.7 ± 34.2   318.3 ± 36.7   332.3 ± 14.4   160 2h post 150.2 ± 15.0 # 302.5 ± 25.1 # 306.0 ± 39.1 # Glu  (mg/dL) sham 155.5 ± 6.9   170.2 ± 9.1   181.0 ± 6.1   CS 224.7 ± 21.8 * 257.3 ± 22.4 * 260.2 ± 24.1 * 160 2h pre 195.2 ± 21.2   211.2 ± 7.8   233.8 ± 13.7   160 1h pre/1h post 183.2 ± 10.1   204.2 ± 29.3   214.0 ± 29.3   160 2h post 176.7 ± 18.2   190.3 ± 26.2   242.8 ± 36.5   pottasium   (mEq/L) sham 3.93 ± 0.13   4.47 ± 0.08   4.03 ± 0.14   CS 6.07 ± 0.17 * 8.10 ± 0.32 * 6.63 ± 0.31 * 160 2h pre 5.97 ± 0.15   5.93 ± 0.31   5.93 ± 0.20   160 1h pre/1h post 5.17 ± 0.23   5.78 ± 0.31 # 4.82 ± 0.16 # 160 2h post 4.77 ± 0.16 # 5.00 ± 0.12 # 3.95 ± 0.11 # .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/24  BUN  (mg/dL) sham 20.2 ± 0.8   23.3 ± 1.7   22.5 ± 1.5   CS 41.2 ± 3.3 * 113.8 ± 6.4 * 113.5 ± 6.4 * 160 2h pre 38.5 ± 2.6   59.0 ± 1.9   59.2 ± 4.4   160 1h pre/1h post 24.7 ± 1.5 # 41.2 ± 3.0 # 46.5 ± 5.5 # 160 2h post 19.0 ± 1.1 # 45.8 ± 8.4 # 30.2 ± 1.2 # Cre  (mg/dL) sham 0.21 ± 0.02   0.21 ± 0.02   0.22 ± 0.01   CS 1.59 ± 0.16 * 1.22 ± 0.26 * 1.15 ± 0.18 * 160 2h pre 1.07 ± 0.21   0.95 ± 0.14   1.19 ± 0.09   160 1h pre/1h post 0.98 ± 0.15   0.64 ± 0.20   1.10 ± 0.10   160 2h post 1.13 ± 0.22   0.84 ± 0.15   0.47 ± 0.04 # Statistics: *p<0.05, vs sham group; #p<0.05, vs CS group (Tukey–Kramer test). Values are presented as the mean ± s.e.m. (n=6 per group). 679 Abbreviations: CPK, creatine phosphokinase; Glu, glucose; BUN, blood urea nitrogen; Cre, creatinine; NO, nitric oxide; CS, crush syndrome; 680 ppm, parts per million; s.e.m., standard error of mean; Pre and post indicate timing relative to reperfusion. 681  682 683 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/25  Table 3 | Effects of 160 ppm NO inhalation on arterial blood gas parameters in a rat model of crush syndrome 684  685     after reperfused (h) 3 24 48 Hct  (%) sham 45.0 ± 0.9   46.7 ± 0.8   45.8 ± 1.2   CS 52.3 ± 1.3 * 59.2 ± 1.0 * 59.3 ± 0.8 * 160 2h pre 50.8 ± 1.5   50.3 ± 3.2   52.0 ± 1.2   160 1h pre/1h post 48.0 ± 0.6   45.0 ± 3.4   45.7 ± 0.6   160 2h post 47.3 ± 0.9   45.2 ± 1.8   44.8 ± 0.9   pH sham 7.45 ± 0.01   7.44 ± 0.01   7.43 ± 0.02   CS 7.41 ± 0.01 * 7.28 ± 0.03 * 7.30 ± 0.00 * 160 2h pre 7.44 ± 0.02   7.39 ± 0.01   7.36 ± 0.02   160 1h pre/1h post 7.43 ± 0.01   7.39 ± 0.02   7.38 ± 0.02   160 2h post 7.46 ± 0.02   7.47 ± 0.02 # 7.42 ± 0.02 # pO2  (mmHg) sham 84.2 ± 2.1   83.8 ± 1.1   79.5 ± 0.7   CS 123.0 ± 2.4 * 114.2 ± 4.9 * 105.3 ± 5.0 * 160 2h pre 114.8 ± 4.0   93.2 ± 10.2   90.7 ± 10.4   160 1h pre/1h post 110.3 ± 3.2   77.2 ± 6.1   76.8 ± 6.0 # 160 2h post 103.5 ± 6.0 # 76.7 ± 5.6 # 79.3 ± 1.8 # pCO2  (mmHg) sham 40.3 ± 0.7   39.4 ± 0.9   44.0 ± 1.5   CS 34.3 ± 2.3 * 62.0 ± 4.6 * 56.7 ± 2.0 * 160 2h pre 30.7 ± 0.7   44.3 ± 6.2   53.1 ± 5.4   160 1h pre/1h post 35.3 ± 2.2   45.3 ± 2.3   49.7 ± 4.6   .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/26  160 2h post 42.7 ± 1.8 # 40.1 ± 2.6 # 39.8 ± 2.3 # BE  (mmol/L) sham 2.5 ± 0.5   2.5 ± 0.5   3.0 ± 0.7   CS -2.7 ± 0.8 * -5.0 ± 0.8 * -3.3 ± 0.5 * 160 2h pre 0.3 ± 0.7   -3.3 ± 0.6   -1.5 ± 0.8   160 1h pre/1h post 2.0 ± 0.6 # -0.2 ± 0.7   3.2 ± 0.7 # 160 2h post 2.8 ± 0.3 # 1.7 ± 0.6 # 3.2 ± 0.4 # HCO3-  (mmol/L) sham 28.6 ± 1.0   27.8 ± 0.4   25.5 ± 0.8   CS 22.8 ± 0.8 * 18.4 ± 0.8 * 18.1 ± 0.7 * 160 2h pre 22.7 ± 1.0   22.6 ± 1.1   23.0 ± 1.1   160 1h pre/1h post 24.6 ± 1.8   25.2 ± 1.6 # 24.6 ± 0.5 # 160 2h post 27.8 ± 1.1 # 29.0 ± 0.7 # 26.9 ± 0.8 # AG  (mmol/L) sham 12.3 ± 0.6   15.2 ± 0.7   13.2 ± 0.5   CS 16.5 ± 1.7   20.0 ± 1.6 * 23.2 ± 0.8 * 160 2h pre 15.7 ± 0.6   18.7 ± 1.0   20.7 ± 1.0   160 1h pre/1h post 16.0 ± 0.6   17.8 ± 0.5   17.5 ± 1.6   160 2h post 15.5 ± 0.8   14.2 ± 0.5 # 14.3 ± 0.5 # Lac  (mmol/L) sham 1.16 ± 0.07   1.21 ± 0.03   1.34 ± 0.10   CS 1.99 ± 0.22   3.02 ± 0.28 * 3.04 ± 0.32 * 160 2h pre 1.92 ± 0.26   2.74 ± 0.25   2.36 ± 0.29   160 1h pre/1h post 1.59 ± 0.23   2.89 ± 0.29   1.98 ± 0.41   160 2h post 1.37 ± 0.25   1.41 ± 0.41 # 1.46 ± 0.29 # Statistics: *p<0.05, vs sham group; #p<0.05, vs CS group (Tukey–Kramer test). Values are presented as the mean ± s.e.m. (n=6 per group). 686 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/27  Abbreviations: Hct, haematocrit; pO2, partial pressure of oxygen; pCO2, partial pressure of carbon dioxide; BE, base excess; HCO3-, bicarbonate; 687 AG, anion gap; Lac, lactate; NO, nitric oxide; CS, crush syndrome; ppm, parts per million. Pre and post indicate timing relative to reperfusion.  688   689 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/28  Supplementary Information 690  691 Supplementary figures 692  693  694  695 Supplementary Fig. S1 | Electrocardiogram parameters of sham, CS control, and NO-696 inhaled CS rats 697 ECG parameters measured at 3, 24, and 48 h after reperfusion in sham-operated rats, CS control 698 rats, and CS rats NO for 2 h pre-, 1 h pre-/1 h post-, or 2 h post-reperfusion. The QRS, PR, and 699 QT intervals (Supplementary Figs. S1a–c), as well as T-wave amplitude (Supplementary Fig. 700 S1e), are increased in CS control rats and show a decreasing trend with NO inhalation, 701 particularly in the 2 h post-reperfusion group across all time points. P-wave amplitude is 702 reduced in CS control rats compared with sham rats but increased by NO inhalation, most 703 notably in the 2 h post-reperfusion group (Supplementary Fig. S1d).  704 *p < 0.05 vs sham group, #p < 0.05 vs CS group, †p < 0.05 vs 160 ppm pre-reperfusion group, 705 ♭p < 0.05 vs 160 ppm 1 h 160 ppm 1 h pre-/1 h post-reperfusion group (Tukey–Kramer test). 706 Values are presented as the mean ± s.e.m. (n = 6 per group). 707 Abbreviations: NO, nitric oxide; ECG, electrocardiogram; CS, crush syndrome; ppl, parts per 708 million; s.e.m., standard error of mean; 160 2 h pre, 160 ppm NO inhalation 2 h before 709 reperfusion; 160 2 h post, 160 ppm NO inhalation 2 h after reperfusion; 160 1 h pre/1 h post, 710 160 ppm NO inhalation 1 h before and 1 h after reperfusion.  711  712  713  714   715 010203040506070QRS interval (msec)***# ###†♭01020304050607080PR interval (msec)**##020406080100120QT interval (msec)***# # ####05101520P wave amplitude (mV)**####05101520253035T wave amplitude (mV)***#####3              24             48after reperfusion (h)3              24             48after reperfusion (h)3              24             48after reperfusion (h)a)                                                b)                                              c)d)                                                e)shamCS160 2h pre160 1h pre/1h post160 2h post.CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/29   716  717 Supplementary Fig. S2 Met-Hb levels of sham, CS controls, and NO inhaled CS rats  718 The acute phase of Met-Hb levels from 2 h pre-reperfusion to 48 h post-reperfusion show that 719 the inhaled NO at 2 h pre- and 1 h pre-/1 h post-reperfusion reaches approximately 4% of the 720 Met-Hb level. Inhaled NO at 2 h post-reperfusion shows less than 3% Met-Hb from 2 h pre-721 reperfusion to 48 h post-reperfusion.  722 Abbreviations: NO, nitric oxide; CS, crush syndrome; Met-Hb, methaemoglobin; 60 2 h pre, 723 160 ppm NO inhalation 2 h before reperfusion;160 2 h post, 160 ppm NO inhalation 2 h after 724 reperfusion; 160 1 h pre/1 h post, 160 ppm NO inhalation 1 h before and 1 h after reperfusion. 725  726   727 012345678-5 -4 -3 -2 -1 0 1 2 3 4 5 6 7Met-Hb(%)period (h)shamCS160 2h pre160 1h pre/1h post160 2h postNOgasNOgasNOgas# ###compression period24           48.CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/30   728  729  730 Supplementary Fig. S3 | Establishment of the crush syndrome rat model.  731 a) Rubber tourniquet applicator used for hind limb compression. 732 b) Application of tourniquets by sliding in the direction indicated by the arrow. 733 c) Representative positioning of rubber tourniquets on both hind limbs. 734 d) Experimental image showing CS rats receiving inhaled nitric oxide. 735 Abbreviations: CS, crush syndrome; NO, nitric oxide. 736  737  738  739  740  741   742 a)                                     b)                                                  d)c).CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/31   743  744  745 Supplementary Fig. S4 | Generation of nitric oxide gas 746 Schematic representation of the NO generation system. An NO generator (a plastic column 747 containing nitrite-type layered double hydroxides (NLDH)/iron(II) sulphate heptahydrate 748 (FeSO₄·7H₂O)) is connected sequentially to a syringe filter (pore size, 0.45 μm), a humidifier 749 (a plastic column containing wet wipes; Wipers S-200, KIMWIPE), an electric pump (GSP-750 400FT, GASTEC), an NO₂ remover (a plastic column containing calcium hydroxide 751 (Ca(OH)₂)), and an NO sensor (ToxiRAE Pro, RAE Systems). The pump flow rate is typically 752 set to 0.25 L/min, and NO generation is monitored at room temperature (20 ± 2 °C). 753 Abbreviations: NO, nitric oxide; NO2, nitrogen dioxide; NLDH, nitrite-type layered double 754 hydroxide; NO2−, nitrite ion; FeSO4∙7H2O, iron(II) sulphate heptahydrate; Ca(OH)2, calcium 755 hydroxide. 756   757 AirPumpNOsensorAirNO generator columnN-LDH/FeⅡSO4 7H2O mixtureHumidifier(Wet paper)Syringe filterinhalationNO2 remover columnCa(OH)2Humid air2[NO2−]LDH + SO42−           [SO42−]LDH + 2NO2− (eq. 1)NO2−  + Fe2+ + H2O          NO + Fe3+ + 2OH− (eq. 2) .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/32   758  759  760 Supplementary Fig. S5 | Experimental design of the crush syndrome rat model. 761 Schematic overview of the experimental protocol used in this study, comprising four separate 762 experiments (Experiments 1–4) performed in the CS rat model.  763 Abbreviations: CS, crush syndrome; NO, nitric oxide; Met-Hb, methaemoglobin. 764  765  766 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 12, 2026. ; https://doi.org/10.64898/2026.03.09.710439doi: bioRxiv preprint https://doi.org/10.64898/2026.03.09.710439http://creativecommons.org/licenses/by-nc-nd/4.0/