C2 Hemisection Research Articles

Enhanced phrenic motor neuron BDNF expression elicited by daily acute intermittent hypoxia is undermined in rats with chronic cervical spinal cord injury

Acute intermittent hypoxia (AIH) elicits spinal neuroplasticity and is emerging as a potential therapeutic modality to improve respiratory and non-respiratory motor function in people with chronic incomplete spinal cord injury (SCI). Brain-derived neurotrophic factor (BDNF) is necessary and sufficient for moderate AIH-induced phrenic long-term facilitation, a well-studied form of respiratory motor plasticity. Repetitive daily AIH (dAIH) enhances BDNF expression within the phrenic motor neurons of normal rats, but its effects on BDNF after chronic cervical spinal cord injury (cSCI) are unknown. In contrast to AIH, chronic intermittent hypoxia (CIH), simulating that experienced during sleep apnea, elicits neuropathology and undermines plasticity. Here, we tested the hypothesis that daily AIH vs CIH differentially regulate phrenic motor neuron BDNF expression in spinally intact and injured rats. Rats with and without C2 hemisection (C2Hx; 8 weeks post-injury) were exposed to 28 days of: 1) sham normoxia (Nx, 21% O2); 2) daily AIH (dAIH: 10, 5min of 10.5% O2 per day; 5min normoxic intervals); 3) mild CIH (CIH5/5: 5min of 10.5% O2, 5min intervals, 8 hrs/day); or 4) moderate CIH (CIH2/2: 2min of 10.5% O2, 2min intervals, 8 hrs/day). After 28 days of daily exposure (i.e., 12 weeks post-injury), BDNF immunoreactivity was assessed within phrenic motor neurons identified via retrograde cholera toxin B fragment labeling. In intact rats, daily AIH increased BDNF protein levels in phrenic motor neurons (~31%) but not in rats with C2Hx. CIH had no effects on phrenic motor neuron BDNF levels in intact rats, although there was a trend towards increased phrenic motor neuron BDNF after C2Hx, suggesting the need for further study. Since dAIH effects on phrenic motor neuron BDNF are not observed in rats with chronic cervical SCI, the potential of dAIH to enhance BDNF-dependent phrenic motor plasticity may be suppressed by conditions prevailing with chronic cSCI.

Sex differences in spontaneous respiratory recovery following chronic C2 hemisection.

Respiratory deficits after C2 hemisection (C2Hx) have been well documented through single-sex investigations. Although ovarian sex hormones enable enhanced respiratory recovery observed in females 2 wk post-C2Hx, it remains unknown if sex impacts spontaneous respiratory recovery at chronic time points. We conducted a longitudinal study to provide a comprehensive sex-based characterization of respiratory neuromuscular recovery for 8 wk after C2Hx. We recorded ventilation and chronic diaphragm electromyography (EMG) output in awake, behaving animals, phrenic motor output in anesthetized animals, and performed diaphragm muscle histology in chronically injured male and female rodents. Our results show that females expressed a greater recovery of tidal volume and minute ventilation compared with males during subacute and chronic time points. Eupneic diaphragm EMG amplitude during wakefulness and phrenic motor amplitude are similar between sexes at all time points after injury. Our data also suggest that females have a greater reduction in ipsilateral diaphragm EMG amplitude during spontaneous deep breaths (e.g., sighs) compared with males. Finally, we show evidence for atrophy and remodeling of the fast, fatigable fibers ipsilateral to injury in females, but not in males. To our knowledge, the data presented here represent the first study to report sex-dependent differences in spontaneous respiratory recovery and diaphragm muscle morphology following chronic C2Hx. These data highlight the need to study both sexes to inform evidence-based therapeutic interventions in respiratory recovery after spinal cord injury (SCI).NEW & NOTEWORTHY In response to chronic C2 hemisection, female rodents display increased tidal volume during eupneic breathing compared with males. Females show a greater reduction in diaphragm electromyography (EMG) amplitude during spontaneous deep breaths (e.g., sighs) and atrophy and remodeling of fast, fatigable diaphragm fibers. Given that most rehabilitative interventions occur in the subacute to chronic stages of injury, these results highlight the importance of considering sex when developing and evaluating therapeutics after spinal cord injury.

Assessing Functional Recovery of Eupneic Diaphragm Activity Following Unilateral Cervical Spinal Cord Injury in Rats.

Following cSCI, activation of the DIAm can be impacted depending on the extent of the injury. The present manuscript describes a unilateral C2 hemisection (C2SH) model of cSCI that disrupts eupneic ipsilateral diaphragm (iDIAm) electromyographic (EMG) activity during breathing in rats. To evaluate recovery of DIAm motor control, the extent of deficit due to C2SH must first be clearly established. By verifying a complete initial loss of iDIAm EMG during breathing, subsequent recovery can be classified as either absent or present, and the extent of recovery can be estimated using the EMG amplitude. Additionally, by measuring the continued absence of iDIAm EMG activity during breathing after the acute spinal shock period following C2SH, the success of the initial C2SH may be validated. Measuring contralateral diaphragm (cDIAm) EMG activity can provide information about the compensatory effects of C2SH, which also reflects neuroplasticity. Moreover, DIAm EMG recordings from awake animals can provide vital physiological information about the motor control of the DIAm after C2SH. This article describes a method for a rigorous, reproducible, and reliable C2SH model of cSCI in rats, which is an excellent platform for studying respiratory neuroplasticity, compensatory cDIAm activity, and therapeutic strategies and pharmaceuticals.

Sex hormone supplementation improves breathing and restores respiratory neuroplasticity following C2 hemisection in rats.

In addition to loss of sensory and motor function below the level of the lesion, traumatic spinal cord injury (SCI) may reduce circulating steroid hormones that are necessary for maintaining normal physiological function for extended time periods. For men, who comprise nearly 80% of new SCI cases each year, testosterone is the most abundant circulating sex steroid. SCI often results in significantly reduced testosterone production and may result in chronic low testosterone levels. Testosterone plays a role in respiratory function and the expression of respiratory neuroplasticity. When testosterone levels are low, young adult male rats are unable to express phrenic long-term facilitation (pLTF), an inducible form of respiratory neuroplasticity invoked by acute, intermittent hypoxia (AIH). However, testosterone replacement can restore this respiratory neuroplasticity. Complicating the interpretation of this finding is that testosterone may exert its influence in three possible ways: 1) directly through androgen receptor (AR) activation, 2) through conversion to dihydrotestosterone (DHT) by way of the enzyme 5α-reductase, or 3) through conversion to 17β-estradiol (E2) by way of the enzyme aromatase. DHT signals via AR activation similar to testosterone, but with higher affinity, while E2 activates local estrogen receptors. Evidence to date supports the idea that exogenous testosterone supplementation exerts its influence through estrogen receptor signaling under conditions of low circulating testosterone. Here we explored both recovery of breathing function (measured with whole body barometric plethysmography) and the expression of AIH-induced pLTF in male rats following C2-hemisection SCI. One week post injury, rats were supplemented with either E2 or DHT for 7days. We hypothesized that E2 would enhance ventilation and reveal pLTF following AIH in SCI rats. To our surprise, though E2 did beneficially impact overall breathing recovery following C2-hemisection, both E2 supplementation and DHT restored the expression of AIH-induced pLTF 2weeks post-SCI.

Closed-loop Epidural Stimulation Elicits Respiratory Plasticity in a Rat Model of Acute Spinal Cord Injury

Epidural stimulation restores motor control in animal models and humans after spinal cord injury (SCI) with the most notable example being locomotion; however, it has not yet been clinically translated to breathing. Further, neuroplasticity elicited by epidural stimulation is largely unexplored. We showed in a rat C2 hemisection (C2HS) model of SCI that one bout of closed-loop epidural stimulation (CLES) restores some ipsilesional (ipsi) diaphragm EMG acutely post paralysis, and that in some rats, activity briefly lasts post CLES (<15 seconds) (Mickle et al., 2023). Here we explore if this brief plasticity increases in magnitude and duration via multiple bouts of CLES, respiratory challenge, or altered sensory input. Adult Sprague Dawley (n=5) rats were urethane anesthetized, implanted with diaphragm EMG recording electrodes and dorsal C4 stimulating electrodes and given a C2HS plus >15 minute recovery period to stabilize and confirm hemidiaphragm paralysis. Rats were delivered two <5 minute periods of CLES during which current amplitude increased by 25 uA steps every 30 breaths until breathing instability. Afferent input was removed via bilateral cervical dorsal rhizotomy (CDR) prior to CLES (n = 2) or between the first two bouts (n = 3). During the first two bouts of CLES, rats were administered 50% O2 via a cycle triggered ventilator which delivers breaths in-phase with contralesional (contra) EMG. For the 3rd bout, rats were challenged with room air and removal of ventilatory support. CLES increased contra EMG output in 5/5 and restored ipsi EMG in 4/5 rats during all bouts. There were no differences in PCO2 prior to each bout (mmHg +/- SEM: 44 +/- 6, 43 +/- 4, 41 +/- 4) though PO2 was much lower under room air (mmHg +/- SEM 172 +/- 24. 165 +/- 31, 73 +/- 5). Contra peak amplitude was elevated in the 30 seconds post-CLES vs baseline for the 1st bout of CLES (77 vs 93 mV, SE of diff. 3, p = 0.009) with no difference in rats with or without CDR (20 vs 23% increase from baseline, SE of diff. 10), and no difference in the 1st and 2nd bouts (93 vs 90 mV, SE of diff. 5). While contra peak EMG did not change between bouts, there was a trend towards an increase in length of residual ipsi activity post bout 2 vs 1 (223 vs 54 sec, SE of diff. 170). Removing the ventilator increased contra peak (77 vs 132 mV, SE of diff. 16, p = 0.025) and unveiled ipsi activity in 3/5 rats. CLES during challenge trended towards increased ipsi response during CLES (15 vs 61 V cumulative sum, SE of diff. 8) and length of residual ispi activity (223 vs 1819 sec, SE of diff. 1031). In 3/5 rats, the residual ipsi activity post challenge CLES bout continued until the end of the experimental period, in one case lasting >20 min. Finally, across all CLES bouts, the magnitude of ipsi activity in the 30 seconds post CLES was correlated with the magnitude of response during CLES (r2 0.34, p = 0.05) indicating conditions that increase response may be best for neuroplasticity. Work is ongoing to clarify the roles of multiple bouts, cervical afferents, challenge, and time post injury in eliciting the enduring activity seen in some rats, but this work represents the first evidence that CLES can elicit respiratory neuroplasticity on a timescale and magnitude which may be relevant for functional benefit. T32HL134621 (AM), R01HL153102 (ED), SPARC OT2OD023854 (ED), Craig H. Neilsen Pilot Grant (ED). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Strategies for protecting lung health after spinal cord injury and gastric reflux

Spinal cord injuries have a significant and far-reaching impact, affecting more than 300,000 people in the United States alone. Among these injuries, cervical-level injuries are the most common, accounting for approximately 60% of all spinal cord injuries. Pulmonary complications, specifically aspiration-related pneumonia, rank as the primary cause of death in individuals with these injuries. Notably, those with cervical spinal cord injuries have an elevated risk of experiencing acute lung injury and acute respiratory distress syndrome. Moreover, spinal cord injuries, regardless of their location, often lead to gastric dysmotility, a condition frequently linked with gastroesophageal reflux disease. This research aims to untangle the intricate relationship between these interconnected health issues and their collective impact on pulmonary function. Here we tested the hypotheses 1) that cervical spinal cord injury increases the susceptibility for developing acute lung injury associated with gastric juice aspiration and 2) that TRPV4 inhibitors will mitigate gastric fluid/SCI-induced lung injury. These studies employed a rat model of spinal cord injury, C2 hemisection (C2Hx). This model mimics the reduced inspiratory tidal volume observed in individuals with cervical spinal cord injuries and has more recently been shown to recapitulate certain aspects of lung injury associated with spinal cord injuries. Additionally, to produce the effects of gastric reflux, gastric juices were introduced into the trachea in a subset of animals following spinal cord injury. Furthermore, TRPV4 inhibitors were administered to assess their potential to mitigate lung damage after spinal injury, gastric juice instillation or at both times. Preliminary data suggest that C2Hx and low levels of gastric juice increase inflammatory markers in the lung and circulation above even high levels of gastric instillation. Further, preliminary data supports the hypothesis that TRVP4 inhibition may protect the lung. The outcomes of these investigations will provide valuable insights into how spinal cord injuries might predispose individuals to lung injury. Additionally, they will evaluate the effcacy of a promising therapeutic approach using TRPV4 inhibitors to restore lung barrier function, potentially reversing this susceptibility. R21NS121966-01. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Exploring the Impact of Fibroblast Growth Factor 17 on Functional Recovery and Neuroinflammation in Spinal Cord Injury

Spinal cord injury (SCI) affects more than 300,000 people in the United States, making it a significant and widespread problem. This includes injuries at the cervical level, which pose greater challenges due to respiratory in addition to mobility issues. Though there is a large societal impact there is still standard of care following spinal cord injury. While previous preclinical research has shown benefits from FGF1 and FGF2, there is weak data to suggest their clinical effcacy. A related compound, Fibroblast Growth Factor 17 (FGF17), has recently been described as a component within the CSF of young animals that when delivered to aged animals improved cognitive function. These improvements were associated with oligodendrogenesis. Spinal cord injury results in secondary demyelination, which may be mitigated by increasing oligodendrogenesis; this led us to examine how FGF17 may be used as a therapeutic agent following SCI. We used a rat C2 hemisection injury model to evaluate the effects of FGF17 at 4 hours, 2 and 6 weeks after injury. FGF17 was administered via intrathecal infusion at 25 μg/μl for 14 days at 0.5 μL/hour or a single application of 20 μL (4-hour timepoint). Following treatment, whole body plethysmography was performed weekly. Tissues were collected at endpoint to assess brainstem neuroinflammation via immunohistochemistry and molecular signaling pathways via western blotting. As anticipated, animals that were not treated with FGF17 had reduced tidal volume following spinal cord injury. Treatment with FGF17 partially preserved tidal volume as early as 3 days post injury. 6 weeks following spinal cord injury we observed a reduction in GFAP+ and IBA1+ staining in the brainstem near regions associated with respiratory control in animals treated with FGF17. Further, when we quantified the protein content in the spinal cord near the lesion epicenter we saw an increase in p-ERK 1/2 in all injured animals. However, in FGF17 treated animals we saw an increase in downstream transcription factors, STAT6 and Serum Response Factor. Serum Response Factor was implicated in oligodendrogenesis, ongoing studies will directly address this. These data suggest that FGF17 may be a viable alternative to the traditional fibroblast growth factor family members when treating spinal cord injury. R21NS121966, R01NS116068. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

APOE and sex impacts functional recovery after cervical spinal cord injury

Genetic factors are key to understanding recovery following spinal cord injury (SCI). Prior work conducted in our laboratory demonstrated that APOE genotype impacts respiratory plasticity after cervical SCI. Specifically, female mice expressing the APOE4 allele had impairments in their ability to generate and maintain robust diaphragmatic activity following cervical SCI and intermittent hypoxia treatment relative to APOE3 counterparts. Subsequently we showed that different APOE alleles leads to altered neural respiratory control even without a spinal cord injury and that the effect is sex dependent. To extend these findings, we are now investigating the impact of APOE genotype and sex on locomotor and respiratory motor recovery in a humanized APOE knock-in mouse model homozygous for either the APOE3 and E4 alleles. Further, we examined how genotype and sex effected plasticity and serotonin levels below the lesion. We hypothesized that animals with the APOE4 allele will demonstrate impaired recovery following SCI across all measures. We performed a left C2 hemisection injury on male and female mice of both APOE3 and APOE4 genotype (n = 8/group). We recorded whole body plethysmography and CatWalk gait analysis weekly beginning at pre-injury through three weeks post injury. Consistent with our previous findings male APOE3 mice responded better than APOE4 counterparts during respiratory challenge even following SCI. Specifically APOE3 male mice showed greater improvements in respiratory rate during both normoxia and hypoxia challenge at early timepoints following injury. Interestingly during catwalk gait analysis APOE4 male mice recovered function faster than APOE3 counterparts in the weeks following SCI. This work is critical because it lays the foundation for understanding how diverse genotypes in the clinical SCI population may indicate the need to personalize treatment for individuals following injury. AG065220 AG060056 Nielsen Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Diaphragm Pacing Elicits Ventilatory Plasticity in a Rodent Model of Spinal Cord Injury

Cervical spinal cord injury (cSCI) often results in respiratory impairments requiring the use of chronic mechanical ventilation (MV). Diaphragm pacing (DP) is an emerging clinical intervention designed to combat the deleterious effects of chronic MV by electrically stimulating the diaphragm muscle to maintain ventilation. Anecdotal clinical evidence suggests a rehabilitative benefit of DP; however, current clinical practice is limited by a lack of controlled investigations. Thus, the purpose of this ongoing study was to determine if daily DP could be used as a rehabilitative tool following cSCI. We hypothesized that following cSCI, DP would 1) induce a long-lasting increase in tidal volume 60 minutes after stimulation, and 2) result in sustained improvements in tidal volume after four days of daily DP in awake, behaving rats. To test these hypotheses, we implanted custom electromyography electrodes in the mid-costal region of the left and right hemidiaphragms of male Sprague-Dawley rats. One week later, all animals received a left C2 hemisection (C2Hx; n=14) and a subset also received a left C4-6 dorsal rhizotomy (n=3). One week after injury, rats received stimulation on the paralyzed hemidiaphragm (biphasic 30Hz, 0.8mA, 80 μs pulse width) in phase with breathing (Pacing n=5, Rhizotomy n=3), or served as controls (n=6). We delivered DP to animals within plethysmography chambers in an intermittent pattern (5 minutes on/5 minutes off) for 1 hour/day over 4 consecutive days. Using whole body plethysmography, we recorded ventilation before, during, and for 60 minutes after to examine the acute effects of DP. Additionally, we measured ventilation during normoxia (21% FiO2) and respiratory challenge (10.5% FiO2, 7% FiCO2) before and after C2Hx, and 24 hours after four days of DP to assess the rehabilitative potential of pacing. DP increased tidal volume (% change from pre-pacing) compared to control animals during stimulation (P =0.021). Animals receiving DP displayed an elevated tidal volume compared to controls for at least 60 minutes after DP (P=0.015). When compared to control animals 24 hours after the completion of the pacing protocol, DP animals displayed an increased tidal volume (mL/breath/100g) during both normoxia (P =0.006) and respiratory challenge (P =0.019). In contrast, animals pre-treated with rhizotomy showed a similar tidal volume (mL/breath/100g) compared to controls (normoxia: P=0.597; respiratory challenge: P=0.191), suggesting that DP-induced ventilatory plasticity requires phrenic afferents. Taken together, our data suggest that diaphragm pacing has rehabilitative potential and elicits functional respiratory improvements in rodents following C2Hx. Acknowledgements: This work was supported by funding from the National Institute of Health, grant numbers: R00 HL143207-01 (KAS), UL1TR001436 (TCH) and the APTA Acute Care Physical Therapy Seed Grant (TCH). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

V2a neurons restore diaphragm function in mice following spinal cord injury

The specific roles that different types of neurons play in recovery from injury is poorly understood. Here, we show that increasing the excitability of ipsilaterally projecting, excitatory V2a neurons using designer receptors exclusively activated by designer drugs (DREADDs) restores rhythmic bursting activity to a previously paralyzed diaphragm within hours, days, or weeks following a C2 hemisection injury. Further, decreasing the excitability of V2a neurons impairs tonic diaphragm activity after injury as well as activation of inspiratory activity by chemosensory stimulation, but does not impact breathing at rest in healthy animals. By examining the patterns of muscle activity produced by modulating the excitability of V2a neurons, we provide evidence that V2a neurons supply tonic drive to phrenic circuits rather than increase rhythmic inspiratory drive at the level of the brainstem. Our results demonstrate that the V2a class of neurons contribute to recovery of respiratory function following injury. We propose that altering V2a excitability is a potential strategy to prevent respiratory motor failure and promote recovery of breathing following spinal cord injury.

Contrasting Experimental Rodent Aftercare With Human Clinical Treatment for Cervical Spinal Cord Injury: Bridging the Translational "Valley of Death".

More than half of all spinal cord injuries (SCIs) occur at the cervical level and often lead to life-threatening breathing motor dysfunction. The C2 hemisection (C2Hx) and high cervical contusion mouse and rat models of SCI are widely utilized both to understand the pathological effects of SCI and to develop potential therapies. Despite rigorous research effort, pre-clinical therapeutics studied in those animal models of SCI sometimes fail when evaluated in the clinical setting. Differences between standard-of-care treatment for acute SCI administered to clinical populations and experimental animal models of SCI could influence the heterogeneity of outcome between pre-clinical and clinical studies. In this review, we have summarized both the standard clinical interventions used to treat patients with cervical SCI and the various veterinary aftercare protocols used to care for rats and mice after experimentally induced C2Hx and high cervical contusion models of SCI. Through this analysis, we have identified areas of marked dissimilarity between clinical and veterinary protocols and suggest the modification of pre-clinical animal care particularly with respect to analgesia, anticoagulative measures, and stress ulcer prophylaxis. In our discussion, we intend to inspire consideration of potential changes to aftercare for animal subjects of experimental SCI that may help to bridge the translational "Valley of Death" and ultimately contribute more effectively to finding treatments capable of restoring independent breathing function to persons with cervical SCI.

Closed-loop cervical epidural stimulation partially restores ipsilesional diaphragm EMG after acute C2 hemisection

Cervical spinal cord injury creates lasting respiratory deficits which can require mechanical ventilation long-term. We have shown that closed-loop epidural stimulation (CL-ES) elicits respiratory plasticity in the form of increased phrenic network excitability (Malone et. al., E Neuro, Vol 9, 0426–21.2021, 2022); however, the ability of this treatment to create functional benefits for breathing function per se after injury has not been demonstrated. Here, we demonstrate in C2 hemisected anesthetized rats, a 20-minute bout of CL-ES administered at current amplitudes below the motor threshold restores paralyzed hemidiaphragm activity in-phase with breathing while potentiating contralesional activity. While this acute bout of stimulation did not elicit the increased network excitability seen in our chronic model, a subset of stimulated animals continued spontaneous ipsilesional diaphragm activity for several seconds after stopping stimulation. These results support the use of CL-ES as a therapeutic to rescue breathing after high cervical spinal cord injury, with the potential to lead to lasting recovery and device independence.

Sex differences in spontaneous respiratory recovery following chronic c2 hemisection

Respiratory deficits after cervical spinal cord injury (cSCI) have been well documented through single sex investigations. To our knowledge, only one report has evaluated sex in respiratory recovery after cSCI. This study showed that females have an enhanced tidal volume and phrenic burst amplitude 2 weeks post-injury. Despite this documented sex difference, it remains unknown if sex impacts spontaneous respiratory recovery at chronic time points. Thus, we conducted a longitudinal study to provide a comprehensive sex-based characterization of respiratory deficits and recovery after cSCI. We hypothesized that female rats would have a greater respiratory motor recovery compared to male rats at chronic time points post-injury. To test this hypothesis, we implanted custom electromyography (EMG) electrodes in the mid-costal region of the left and right hemidiaphragms and wires were connected to a headcap to enable chronic recordings. One-week after implantation, animals received a left C2 hemisection (C2Hx). We recorded whole-body plethysmography and bilateral diaphragm EMG activity before, 1 day, and weekly after C2Hx in awake, behaving male (n=5) and female (n=6) Sprague-Dawley rats for 8 weeks. At 9 weeks post-injury, we performed terminal phrenic nerve recordings in anesthetized and ventilated male (n=7) and female (n=6) rats. Consistent with published data, all rats showed a decrease in tidal volume (mL/breath/100g) that persisted for 8 weeks (male: P=0.0013; females: P=0.0036 vs. pre-injury), and a decrease in ipsilateral diaphragm EMG amplitude (% change pre-injury) at 1-week post-injury (males: P=0.0119; females: P=0.0004 vs. pre-injury). Female rats showed a greater improvement in tidal volume (mL/breath/100g) at 3 weeks post-cSCI which remained elevated for 8 weeks (all P≤0.0467 vs. male rats). Interestingly, males displayed an increased rate of weight gain (% baseline) compared to females, beginning 2 weeks post-cSCI (all P≤0.0418), which may have masked improvements in tidal volume in males. No sex differences in ipsilateral diaphragm EMG amplitude were observed at any time point post-injury (P≥0.8601). Similarly, no sex-based differences were observed in ipsilateral phrenic nerve amplitude during respiratory challenges (% change from baseline; all P≥0.2221). Despite using apneic threshold to set baseline CO2, arterial CO2 was higher in females (P=0.0003 vs. males), indicating a differential CO2 ventilatory threshold post-cSCI. Our data illustrate sex differences in tidal volume and CO2 ventilatory threshold at chronic time points after cSCI. These results highlight the need for sex to be included as a biological variable in the development of respiratory therapeutic strategies following cSCI. This work was supported by funding from the National Institute of Health, grant numbers: K99/R00 HL143207-01 (KAS). The authors declare no competing financial interests. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Extracellular traps formation following cervical spinal cord injury.

Spinal cord injuries involve a primary injury that can lead to permanent loss of function and a secondary injury associated with pathologic and inflammatory processes. Extracellular traps are extracellular structures expressed by immune cells that are primarily composed of chromatin, granular enzymes and histones. Extracellular traps are known to induce tissue damage when overexpressed and could be associated in the occurrence of secondary damage. In the present study, we used flow cytometry to demonstrate that at 1day following a C2 spinal cord lateral hemisection in male Swiss mice, resident microglia form vital microglia extracellular traps, and infiltrating neutrophils form vital neutrophil extracellular traps. We also used immunolabelling to show that microglia near the lesion area are most likely to form these microglia extracellular traps. As expected, infiltrating neutrophils are located at the site of injury, though only some of them engage in post-injury extracellular trap formation. We also observed the formation of microglia and neutrophil extracellular traps in our sham animal models of durotomy, but formation was less frequent than following the C2 hemisection. Our results demonstrate for the first time that microglia form extracellular traps in the spinal cord following injury and durotomy. It remains however to determine the exact mechanisms and kinetics of neutrophil and microglia extracellular traps formation following spinal cord injury. This information would allow to better mitigate this inflammatory process that may contribute to secondary injury and to effectively target extracellular traps to improve functional outcomes following spinal cord injury.

Effects of C2 hemisection on respiratory and cardiovascular functions in rats.

High cervical spinal cord injuries induce permanent neuromotor and autonomic deficits. These injuries impact both central respiratory and cardiovascular functions through modulation of the sympathetic nervous system. So far, cardiovascular studies have focused on models of complete contusion or transection at the lower cervical and thoracic levels and diaphragm activity evaluations using invasive methods. The present study aimed to evaluate the impact of C2 hemisection on different parameters representing vital functions (i.e., respiratory function, cardiovascular, and renal filtration parameters) at the moment of injury and 7 days post-injury in rats. No ventilatory parameters evaluated by plethysmography were impacted during quiet breathing after 7 days post-injury, whereas permanent diaphragm hemiplegia was observed by ultrasound and confirmed by diaphragmatic electromyography in anesthetized rats. Interestingly, the mean arterial pressure was reduced immediately after C2 hemisection, with complete compensation at 7 days post-injury. Renal filtration was unaffected at 7 days post-injury; however, remnant systolic dysfunction characterized by a reduced left ventricular ejection fraction persisted at 7 days post-injury. Taken together, these results demonstrated that following C2 hemisection, diaphragm activity and systolic function are impacted up to 7 days post-injury, whereas the respiratory and cardiovascular systems display vast adaptation to maintain ventilatory parameters and blood pressure homeostasis, with the latter likely sustained by the remaining descending sympathetic inputs spared by the initial injury. A better broad characterization of the physiopathology of high cervical spinal cord injuries covering a longer time period post-injury could be beneficial for understanding evaluations of putative therapeutics to further increase cardiorespiratory recovery.

Phrenic Motor Neuron Fractalkine Expression After Intermittent Hypoxia and Spinal Cord Injury

Acute intermittent hypoxia (AIH) elicits spinal respiratory motor plasticity, and has emerged as a promising therapeutic approach to restore lost respiratory (and non‐respiratory) motor function following cervical spinal cord injury (cSCI). Conversely, most individuals with cSCI experience sleep disordered breathing, generating chronic intermittent hypoxia (CIH) at intensities sufficient to trigger pathology. Since glia play a critical role in regulating neuroplasticity, they may play a key role in differentiating responses to therapeutic (AIH) versus pathogenic (CIH) intermittent hypoxia. Motor neuron‐microglial interactions regulate AIH‐induced phrenic motor plasticity. Specifically, phrenic motor neuron fractalkine, a chemokine unique to neurons released during hypoxia activates its high affinity receptor, CX3CR1—which is unique to microglia in the CNS. In response, microglia release ATP/adenosine, inhibiting AIH‐induced phrenic motor plasticity. Thus, if intermittent hypoxia or cSCI impact phrenic motor neuron fractalkine expression, they may directly impact plasticity and the potential of therapeutic AIH. In this study, we tested the hypothesis that phrenic motor neuron fractalkine expression is attenuated by daily AIH (but not CIH) in spinally intact rats, but not in rats with cSCI. Fractalkine was assessed in phrenic motor neurons of male rats with/without C2 hemisection (12 weeks post‐injury) exposed to 28 days of: 1) normoxia; 2) daily AIH (10, 5‐min episodes per day, 10.5% O2; 5‐min intervals); and 3) moderate CIH (2‐min 10.5% O2 episodes; 2‐min normoxic intervals; 8 hr/day). Fourteen days before hemisection, intrapleural Cholera toxin B injections were made to retrogradely label phrenic motor neurons. Fractalkine expression was assessed in 40 mm sections using a custom MATLAB algorithm to assess immunofluorescence optical density within labeled phrenic motor neurons. Spinally intact and injured rats have differential responses to daily AIH vs CIH. Whereas injured rats had no change in phrenic motor neuron fractalkine expression after daily AIH or CIH, fractalkine expression was marginally reduced in intact rats following daily AIH (p = 0.07; cohen’s d= 132%), but not CIH (p = 0.88; cohen’s d= 23%). Thus, daily (therapeutic) AIH may decrease motor neuron fractalkine and microglial adenosine release, thereby enhancing moderate AIH‐induced phrenic motor plasticity.

Central nervous system 5‐HT plays a role in ventilatory and arousal responses to hypoxia and hypercapnia in spinal cord injured mice

PurposeThe present investigation was designed to explore the impact of serotonin (5HT) on the arousal and ventilatory response to hypercapnia and hypoxia following spinal cord injury (SCI) in mice.MethodsTelemetry transmitters were surgically implanted in wild type mice (Tph2+/+) (n =7 for SCI, n=7 for Sham) and tryptophan hydroxylase 2 knockout mice (Tph2‐/‐) (n=6 for SCI, n=6 for Sham) to measure electroencephalography, electromyography (EMG) of the genioglossus (GG) muscle, core body temperature and gross motor activity. Following recovery, the Tph2+/+ and Tph2‐/‐ mice were placed in whole body plethysmographs and measures of the ventilatory and arousal response to hypoxia and hypercapnia during non‐rapid eye movement sleep were obtained. Following these measures, a C2 hemi‐section of the spinal cord was completed and the physiological measurements were repeated 4 and 21 days following SCI. The ventilatory data is presented as a fraction of baseline.ResultsThe breathing frequency, tidal volume, and minute ventilation response to hypercapnia was greater in Tph2+/+ compared to Tph2‐/‐ mice at 21 days post SCI (Breathing frequency: 1.37 ± 0.12 vs. 1.02 ± 0.10, p = 0.036, Tidal volume: 1.70 ± 0.21 vs. 0.91 ± 0.09, p = 0.002, Minute Ventilation: 2.87 ± 0.48 vs. 0.93 ± 0.12, p = 0.001). In addition, the tidal volume and minute ventilation response to hypoxia was greater in Tph2+/+ compared to Tph2‐/‐ mice at 21 days post SCI (Tidal Volume: 1.27 ± 0.09 vs. 0.94 ± 0.08, p = 0.009, Minute Ventilation: 1.45 ± 0.14 vs. 0.98 ± 0.09, p = 0.012). The arousal latency to hypercapnia was reduced in Tph2+/+ compared to Tph2‐/‐ mice at 4 days post SCI (16.22 ± 4.15 vs. 73.83 ± 15.78 s, p = 0.013). The number of arousals during exposure to hypercapnia was greater in Tph2+/+ compared to Tph2‐/‐ mice at 21 days post SCI (5.31 ± 0.50 vs. 3.40 ± 0.51, p = 0.021). The magnitude of the GG response to hypercapnia and hypoxia was greater in Tph2+/+ compared to Tph2‐/‐ mice at 21 days post SCI (Hypercapnia: 47.42 ± 10.17 vs. 0.73 ± 0.18, p = 0.012, Hypoxia: 43.82 ± 11.25 vs. 3.30 ± 1.65, p = 0.019). The fraction of episodes in which there was no EMG response to hypercapnia and hypoxia was lower in Tph2+/+ compared to Tph2‐/‐mice at 4 days post SCI (Hypercapnia: 0.43 ± 0.10 vs. 0.92 ± 0.08, p = 0.001 Hypoxia: 0.19 ± 0.12 vs. 0.85 ± 0.15, p = 0.008). The fraction of episodes in which there was no EMG response to hypercapnia was lower in Tph2+/+ compared to Tph2‐/‐ mice at 21 days post SCI (0.19 ± 0.06 vs. 0.79 ± 0.10, p = 0.001).ConclusionThe ventilatory, arousal and GG response to hypercapnia and hypoxia is greater in Tph2+/+ compared to Tph2‐/‐ mice following SCI. Our results suggest that deficiencies in serotonin have a role in regulating respiratory and arousal responses to hypercapnia and hypoxia following SCI.

Ampakine CX717 Stimulates Diaphragm Activity following Mid‐cervical Spinal Contusion Injury

Cervical spinal cord injuries result in significant respiratory compromise and there is a need to develop therapies to counter respiratory insufficiency in this patient population. Ampakines are positive allosteric modulators of AMPA receptors, and can stimulate breathing during states of hypoventilation, such as following opioid overdose or in neuromuscular disorders. Our laboratory has previously shown that acute treatment with ampakine CX717 can stimulate phrenic activity in spinally injured animals under urethane anesthesia. Recently, we extended these findings by showing that a low dose of CX717 was sufficient to enhance diaphragm electromyography (EMG) activity and tidal volume in unanaesthetized animals with a C2 hemisection injury. The next step in the translational pathway, and the goal of the current study, was to test the efficacy of ampakines in a model of contusion injury that is more similar to the injuries typically experienced by humans. The C4 contusion injury used in the current study results in direct phrenic motor neuron loss and significant damage of the spinal pathways innervating respiratory motor pools. This model is also associated with strong spontaneous recovery of diaphragm function, such that pronounced deficits are likely to be detected only in the initial week following injury. Animals were implanted with in‐dwelling bilateral diaphragm EMG electrodes and ventilation was measured using whole body plethysmography. At 4 and 14 days following a 150 kDy C4 unilateral contusion injury (C4Ct), male and female rats were given a single intravenous bolus of CX717 (5 mg/kg, n=4) or vehicle (2‐hydroxypropyl‐beta‐cyclodextrin; HPCD; n=4). At the 4‐day time point, infusion of CX717 increased peak diaphragm EMG (recorded ipsilateral to C4Ct) by 40‐50% when compared to vehicle group. Respiratory rate was also elevated in the CX717 group by 80% post‐infusion, indicating a likely supraspinal mechanism of the impact of ampakines. In the CX717 group, tidal volume was increased by 25±13% during infusion, 20±16% at 5 min post infusion and returned to baseline levels by 15 mins post CX717 infusion. There was no change in tidal volume in the vehicle group. During an acute respiratory challenge with hypoxia (10% O2), CX717 treated animals were able to increase diaphragm EMG (100±15%) to a greater extent as compared to vehicle (50 ± 23%, P=0.004). Similarly, during a combined hypoxia‐hypercapnia challenge (10% O2, 7% CO2), CX717 treated animals displayed an increased diaphragm EMG response (200±44%) compared to vehicle (100 ± 55%, P=0.0144). In contrast to the 4‐day data, there was no discernable impact of ampakines on baseline or challenged breathing at 14 days post C4Ct. This is perhaps not surprising because prior work establishes that in this injury model, robust compensation has occurred after 2 weeks, and deficits in diaphragm activation are difficult to detect. In summary, our data indicate that low dose, low impact ampakine treatment can increase diaphragm muscle output at an acute timepoint following mid‐cervical contusion type injuries. These studies pave the path for future experiments to test if ampakine treatment can be combined with rehabilitation techniques to further enhance the therapeutic effect of ampakines in this injury model.

Respiratory Response‐Based Intermittent Hypoxia Treatment Leads to Diaphragmatic Long Term Facilitation in Adult Rats

Spinal cord injury (SCI) most commonly occurs at the cervical level and can lead to severe respiratory dysfunction such as paresis of the diaphragm, the primary mammalian inspiratory muscle. Intermittent hypoxia (IH) treatment (tx) can be applied to induce a prolonged increase in breathing motor output, known as long term facilitation (LTF), which is achievable following SCI in humans. However, this does not potentiate following multiple days of tx, indicating a need for optimization of existing IH protocols. Many seminal experiments demonstrating LTF in uninjured animals employ phrenic nerve recordings to measure respiratory response to moderate IH protocols consisting of 3, 5‐minute‐long periods of hypoxia (~11% O2) interspersed with periods of normoxia (~20.9% O2). This tx increases respiratory motor drive, stimulating serotonin (5‐HT) and growth factor‐dependent plasticity within the cervical spinal cord. Importantly, many studies have shown that the pattern and severity of IH dramatically affect LTF expression. Indeed, fundamental literature indicates that the details of 5‐HT‐based or other stimulation protocol are crucial for induction of plasticity across multiple phenomena, species, and preparations. In these studies, real‐time verification of the stimulus’ effect is key. Since LTF is a neuroplastic paradigm which depends on sequelae of effective stimulation of respiratory drive, we constructed an IH protocol which adapts to the animal’s real‐time respiratory response during treatment. We hypothesized that novel, respiratory response‐based (RRB) IH tx would induce a greater degree of diaphragmatic (dia) LTF than would standard fixed duration (FD) tx in uninjured adult rats. To develop RRB treatment, we first derived an initial setpoint from previous EMG/IH experiments in rats which reflected the magnitude of diaEMG amplitude increase expressed during hypoxic exposure. We then utilized this setpoint during each of three IH cycles such that the animal would qualify for progression from hypoxic to normoxic exposure by expressing a sufficiently robust diaEMG amplitude increase with respect to output measured in previous normoxic conditions. To evaluate our hypothesis, we conducted diaphragm EMG recordings in isoflurane‐anesthetized Sprague‐Dawley retired breeder rats before, during, and after three cycles of either moderate RRB or FD tx administered by nose cone. Preliminary data demonstrated that RRB treatment (n=2) resulted in LTF reaching 113.2% and 130.8% of baseline diaphragm amplitude for the two subjects at least 1‐hr. post‐IH. Further elucidation of appropriate hypoxia‐induced diaphragm amplitude setpoints is a necessary next step, followed by completion of greater numbers of both RRB and FD treatments to achieve adequate power for statistical comparison before applying RRB to the rat C2 hemisection injury model to explore the treatment’s utility in restoring breathing motor function once lost.

Closed-Loop, Cervical, Epidural Stimulation Elicits Respiratory Neuroplasticity after Spinal Cord Injury in Freely Behaving Rats.

Over half of all spinal cord injuries (SCIs) are cervical, which can lead to paralysis and respiratory compromise, causing significant morbidity and mortality. Effective treatments to restore breathing after severe upper cervical injury are lacking; thus, it is imperative to develop therapies to address this. Epidural stimulation has successfully restored motor function after SCI for stepping, standing, reaching, grasping, and postural control. We hypothesized that closed-loop stimulation triggered via healthy hemidiaphragm EMG activity has the potential to elicit functional neuroplasticity in spinal respiratory pathways after cervical SCI (cSCI). To test this, we delivered closed-loop, electrical, epidural stimulation (CLES) at the level of the phrenic motor nucleus (C4) for 3 d after C2 hemisection (C2HS) in freely behaving rats. A 2 × 2 Latin Square experimental design incorporated two treatments, C2HS injury and CLES therapy resulting in four groups of adult, female Sprague Dawley rats: C2HS + CLES (n = 8), C2HS (n = 6), intact + CLES (n = 6), intact (n = 6). In stimulated groups, CLES was delivered for 12–20 h/d for 3 d. After C2HS, 3 d of CLES robustly facilitated the slope of stimulus-response curves of ipsilesional spinal motor evoked potentials (sMEPs) versus nonstimulated controls. To our knowledge, this is the first demonstration of CLES eliciting respiratory neuroplasticity after C2HS in freely behaving animals. These findings suggest CLES as a promising future therapy to address respiratory deficiency associated with cSCI.