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We tested the hypothesis that the incidence and magnitude of diaphragm fatigue following high-intensity exercise would be lower in females with a high aerobic capacity (Hi-Fit) compared with healthy females with an average aerobic fitness (Avg-Fit). Participants were assigned to groups based on their peak O 2 uptake (V̇o 2peak ) obtained during cycle exercise: Hi-Fit n = 9, V̇o 2peak ≥ 56.1 ± 3.4 mL·kg -1 ·min -1 versus Avg-Fit n = 9, V̇o 2peak ≤ 35.7 ± 4.9 mL·kg -1 ·min -1 . On the second day, diaphragm fatigue was assessed before and after constant load exercise test to exhaustion. Magnetic stimulation of the phrenic nerve roots was used to nonvolitionally assess diaphragm fatigue by measurement of transdiaphragmatic twitch pressure (P di,tw ). Both groups exercised at >90% of V̇o 2peak for a similar duration (Hi-Fit: 546.1 ± 177.8 vs. Avg-Fit: 559.3 ± 175.0 s, P = 0.9). Diaphragm fatigue was defined as a ≥15% reduction in P di,tw , approximately two times greater than the coefficient of variation. The mean group average reduction in P di,tw following exercise in the Hi-Fit (17.5%) and Avg-Fit groups (12.2%) was not different between groups ( P = 0.2). The Hi-Fit group performed exercise at a higher absolute work rate that elicited significantly greater ventilatory work and inspiratory muscle force output. The Hi-Fit group did not experience greater fatigue compared with the Avg-Fit group, which we attribute to a greater reliance on accessory respiratory muscle recruitment, to training-induced increases in the aerobic capacity of the diaphragm, or a combination of the two. In summary, aerobic fitness is not predictive of exercise-induced diaphragm fatigue in healthy females. NEW & NOTEWORTHY We hypothesized that females with a high aerobic capacity would be resistant to exercise-induced diaphragm fatigue relative to healthy but untrained females. We assessed the pressure generated by the diaphragm following exhaustive exercise by stimulating the phrenic nerves. The magnitude and incidence of fatigue were similar between groups. Despite having higher ventilatory demands during exercise highly trained females were resistant to greater diaphragm fatigue. Aerobic fitness is not predictive of exercise-induced diaphragm fatigue in females.

Respiratory muscle paralysis due to trauma or neurodegenerative diseases can have devastating consequences. Only a few studies have investigated the reconstruction of motor function in denervated diaphragms caused by such conditions. Here, we studied the efficacy of transplanting E14 embryonic spinal motor neurons (SMNs) into peripheral nerve grafts for functionally reconstructing a denervated diaphragm in a rat model. The diaphragms of 8-week-old male Fischer 344 rats were first denervated by transecting the phrenic nerves. Subsequently, peripheral nerve grafts taken from the lower limb were used for neurotization of the denervated diaphragms. One week later, fetal E14 SMNs were transplanted into the peripheral nerve grafts. After 3 months, we observed functional contraction of the diaphragm following neuromuscular electrical stimulation (NMES) of the peripheral nerve graft. Additionally, we confirmed that SMN transplantation into the peripheral nerve graft had an inhibitory effect on diaphragm muscle atrophy. The SMNs transplanted into the peripheral nerve grafts formed a structure similar to the spinal cord, and the neuromuscular junction of the denervated diaphragm was reinnervated. These findings suggest the establishment of an ectopic motor neuron pool in the peripheral nerve graft. Free peripheral intra-nerve SMN transplantation in combination with NMES, which can be applied for diaphragmatic pacing, offers novel insights into the development of neuroregenerative therapies for treating life-threatening and intractable respiratory muscle paralysis caused by severe nerve damage and degenerative diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Hypoxia can trigger a sequence of breathing-related behaviors, from augmentation to apneusis to apnea and gasping. Gasping is an autoresuscitative behavior that, via large tidal volumes and altered intrathoracic pressure, can enhance coronary perfusion, carotid blood flow, and sympathetic activity, and thereby coordinate cardiac and respiratory functions. We tested the hypotheses that hypoxia-evoked gasps are amplified through a disinhibitory microcircuit within the inspiratory neuron chain and that this drive is distributed via an efference copy mechanism. This generates coordinated gasplike discharges concurrently in other circuits of the raphe-pontomedullary respiratory network. Data were obtained from six decerebrate, vagotomized, neuromuscularly blocked, and artificially ventilated adult cats. Arterial blood pressure, phrenic nerve activity, end-tidal CO 2 , and other parameters were monitored. Hypoxia was produced by ventilation with a gas mixture of 5% O 2 in nitrogen. Neuron spike trains were recorded at multiple pontomedullary sites simultaneously and evaluated for firing rate modulations and short-timescale correlations indicative of functional connectivity. Experimental perturbations evoked reconfiguration of raphe-pontomedullary circuits during initial augmentation, apneusis and augmented bursts, apnea, and gasping. Functional connectivity, altered firing rates, efference copy of gasp drive, and coordinated incremental blood pressure increases support a distributed brain stem network model for amplification and broadcasting of inspiratory drive during autoresuscitative gasping. Gasping begins with a reduction in inhibition by expiratory neurons and an initial loss of inspiratory drive during hypoxic apnea and culminates in autoresuscitative efforts. NEW & NOTEWORTHY Severe hypoxia evokes a sequence of breathing-related behaviors culminating in gasping. We report firing rate modulations and short-timescale correlations in spike trains recorded simultaneously in the raphe-pontomedullary respiratory network during hypoxia. Our findings support a disinhibitory microcircuit and a distributed efference copy mechanism for amplification of gasping. Coordinated increments in blood pressure lead to a model for autoresuscitative bootstrapping of peripheral chemoreceptor reflexes, breathing, and sympathetic activity, complementing and extending prior work.

Shape and size of the nasopharyngeal airway is controlled by muscles innervated facial, glossopharyngeal, vagal, and hypoglossal cranial nerves. Contrary to brainstem networks that drive facial, vagal and hypoglossal nerve activities (FNA, VNA, HNA) the discharge patterns and origins of glossopharyngeal nerve activity (GPNA) remain poorly investigated. Here, an in situ perfused brainstem preparation (n=19) was used for recordings of GPNA in relation to phrenic (PNA), FNA, VNA and HNA. Brainstem transections were performed (n=10/19) to explore the role of pontomedullary synaptic interactions in generating GPNA. GPNA generally mirrors FNA and HNA discharge patterns and displays pre-inspiratory activity relative to the PNA, followed by robust inspiratory discharge in coincidence with PNA. Postinspiratory (early expiratory) discharge was, contrary to VNA, generally absent in FNA, GPNA or HNA. As described previously FNA and HNA discharge was virtually eliminated after pontomedullary transection while an apneustic inspiratory motor discharge was maintained in PNA, VNA and GPNA. After brainstem transection GPNA displayed an increased tonic activity starting during mid-expiration and thus developed prolonged pre-inspiratory activity compared to control. In conclusion respiratory GPNA reflects FNA and HNA which implies similar function in controlling upper airway patency during breathing. That GPNA preserved its pre-inspiratory/inspiratory discharge pattern in relation PNA after pontomedullary transection suggest that GPNA premotor circuits may have a different anatomical distribution compared HNA and FNA and thus may therefore hold a unique role in preserving airway patency., (Copyright © 2024. Published by Elsevier B.V.)

Spinal cord injury poses considerable challenges, particularly in diaphragm paralysis. To address limitations in existing diaphragm pacing technologies, we report an implantable, self-driven diaphragm pacing system based on a microvibration triboelectric nanogenerator (MV-TENG). Leveraging the efficient MV-TENG, the system harvests micromechanical energy and converts this energy into pulses for phrenic nerve stimulation. In vitro tests confirm a stable MV-TENG output, while subcutaneous implantation of the device in rats results in a constant amplitude over 4 weeks with remarkable energy-harvesting efficacy. The system effectively induces diaphragmatic motor-evoked potentials, triggering contractions of the diaphragm. This proof-of-concept system has potential clinical applications in implantable phrenic nerve stimulation, presenting a novel strategy for advancing next-generation diaphragm pacing devices.

Background: In the United States in 2017, there were an estimated 903,745 hospitalizations involving mechanical ventilation (MV). Complications from ventilation can result in longer hospital stays, increased risk of disability, and increased healthcare costs. It has been hypothesized that electrically pacing the diaphragm by phrenic nerve stimulation during mechanical ventilation may minimize or reverse diaphragm dysfunction, resulting in faster weaning., Methods: The ReInvigorate Trial is a prospective, multicenter, randomized, controlled clinical trial evaluating the safety and efficacy of Stimdia's pdSTIM System for facilitating weaning from MV. The pdSTIM system employs percutaneously placed multipolar electrodes to stimulate the cervical phrenic nerves and activate contraction of the diaphragm bilaterally. Patients who were on mechanical ventilation for at least 96 h and who failed at least one weaning attempt were considered for enrollment in the study. The primary efficacy endpoint was the time to successful liberation from mechanical ventilation (treatment vs. control). Secondary endpoints will include the rapid shallow breathing index and other physiological and system characteristics. Safety will be summarized for both primary and additional analyses. All endpoints will be evaluated at 30 days or at the time of removal of mechanical ventilation, whichever is first., Discussion: This pivotal study is being conducted under an investigational device exception with the U.S. Food and Drug Administration. The technology being studied could provide a first-of-kind therapy for difficult-to-wean patients on mechanical ventilation in an intensive care unit setting., Trial Registration: Clinicaltrials.gov, NCT05998018 , registered August 2023., (© 2024. The Author(s).)

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.

Orexin-mediated stimulation of orexin receptors 1/2 (OX[1/2]R) may stimulate the diaphragm and genioglossus muscle via activation of inspiratory neurons in the pre-Bötzinger complex, which are critical for the generation of inspiratory rhythm, and phrenic and hypoglossal motoneurons. Herein, we assessed the effects of OX2R-selective agonists TAK-925 (danavorexton) and OX-201 on respiratory function. In in vitro electrophysiologic analyses using rat medullary slices, danavorexton and OX-201 showed tendency and significant effect, respectively, in increasing the frequency of inspiratory synaptic currents of inspiratory neurons in the pre-Bötzinger complex. In rat medullary slices, both danavorexton and OX-201 significantly increased the frequency of inspiratory synaptic currents of hypoglossal motoneurons. Danavorexton and OX-201 also showed significant effect and tendency, respectively, in increasing the frequency of burst activity recorded from the cervical (C3-C5) ventral root, which contains axons of phrenic motoneurons, in in vitro electrophysiologic analyses from rat isolated brainstem-spinal cord preparations. Electromyogram recordings revealed that intravenous administration of OX-201 increased burst frequency of the diaphragm and burst amplitude of the genioglossus muscle in isoflurane- and urethane-anesthetized rats, respectively. In whole-body plethysmography analyses, oral administration of OX-201 increased respiratory activity in free-moving mice. Overall, these results suggest that OX2R-selective agonists enhance respiratory function via activation of the diaphragm and genioglossus muscle through stimulation of inspiratory neurons in the pre-Bötzinger complex, and phrenic and hypoglossal motoneurons. OX2R-selective agonists could be promising drugs for various conditions with respiratory dysfunction., Competing Interests: We have read the journal’s policy and the authors of this manuscript have the following competing interests: all authors are employees and stockholders of Takeda Pharmaceutical Company Limited., (Copyright: © 2024 Yamada et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Prolonged inhibition of respiratory neural activity elicits a long-lasting increase in phrenic nerve amplitude once respiratory neural activity is restored. Such long-lasting facilitation represents a form of respiratory motor plasticity known as inactivity-induced phrenic motor facilitation (iPMF). Although facilitation also occurs in inspiratory intercostal nerve activity after diminished respiratory neural activity (iIMF), it is of shorter duration. Atypical PKC activity in the cervical spinal cord is necessary for iPMF and iIMF, but the site and specific isoform of the relevant atypical PKC are unknown. Here, we used RNA interference to test the hypothesis that the zeta atypical PKC isoform (PKCζ) within phrenic motor neurons is necessary for iPMF but PKCζ within intercostal motor neurons is unnecessary for transient iIMF. Intrapleural injections of siRNAs targeting PKCζ (siPKCζ) to knock down PKCζ mRNA within phrenic and intercostal motor neurons were made in rats. Control rats received a nontargeting siRNA (NTsi) or an active siRNA pool targeting a novel PKC isoform, PKCθ (siPKCθ), which is required for other forms of respiratory motor plasticity. Phrenic nerve burst amplitude and external intercostal (T2) electromyographic (EMG) activity were measured in anesthetized and mechanically ventilated rats exposed to 30 min of respiratory neural inactivity (i.e., neural apnea) created by modest hypocapnia (20 min) or a similar recording duration without neural apnea (time control). Phrenic burst amplitude was increased in rats treated with NTsi (68 ± 10% baseline) and siPKCθ (57 ± 8% baseline) 60 min after neural apnea vs. time control rats (-3 ± 3% baseline), demonstrating iPMF. In contrast, intrapleural siPKCζ virtually abolished iPMF (5 ± 4% baseline). iIMF was transient in all groups exposed to neural apnea; however, intrapleural siPKCζ attenuated iIMF 5 min after neural apnea (50 ± 21% baseline) vs. NTsi (97 ± 22% baseline) and siPKCθ (103 ± 20% baseline). Neural inactivity elevated the phrenic, but not intercostal, responses to hypercapnia, an effect that was blocked by siPKCζ. We conclude that PKCζ within phrenic motor neurons is necessary for long-lasting iPMF, whereas intercostal motor neuron PKCζ contributes to, but is not necessary for, transient iIMF. NEW & NOTEWORTHY We report important new findings concerning the mechanisms regulating a form of spinal neuroplasticity elicited by prolonged inhibition of respiratory neural activity, inactivity-induced phrenic motor facilitation (iPMF). We demonstrate that the atypical PKC isoform PKCζ within phrenic motor neurons is necessary for long-lasting iPMF, whereas intercostal motor neuron PKCζ contributes to, but is not necessary for, transient inspiratory intercostal facilitation. Our findings are novel and advance our understanding of mechanisms contributing to phrenic motor plasticity.

About one in three critically ill patients requires mechanical ventilation (MV). Prolonged MV, however, results in diaphragmatic weakness, which itself is associated with delayed weaning and increased mortality. Inducing active diaphragmatic contraction via electrical phrenic nerve stimulation (PNS) not only provides the potential to reduce diaphragmatic muscular atrophy but also generates physiological-like ventilation and therefore offers a promising alternative to MV. Reasons why PNS is not yet used in critical care medicine are high procedural invasiveness, insufficient evidence, and lack of side-by-side comparison to MV. This study aims to establish a minimal-invasive percutaneous, bilateral electrode placement approach for sole PNS breathing and thereby enable, for the first time, a breath-by-breath comparison to MV. Six juvenile German Landrace pigs received general anesthesia and orotracheal intubation. Following the novel ultrasound-guided, landmark-based, 4-step approach, two echogenic needles per phrenic nerve were successfully placed. Stimulation effectiveness was evaluated measuring tidal volume, diaphragmatic thickening and tomographic electrical impedance in a breath-by-breath comparison to MV. Following sufficient bilateral phrenic nerve stimulation in all pigs, PNS breaths showed a 2.2-fold increase in diaphragmatic thickening. It induced tidal volumes in the lung-protective range by negative pressure inspiration and improved dorso-caudal regional ventilation in contrast to MV. Our study demonstrated the feasibility of a novel ultrasound-guided, percutaneous phrenic nerve stimulation approach, which generated sufficient tidal volumes and showed more resemblance to physiological breathing than MV in a breath-by-breath comparison., (© 2024. The Author(s).)

The number of motor neurons (MNs) declines precipitously during the final trimester before birth. Thereafter, the number of MNs remains relatively stable, with their connections to skeletal muscle dependent on neurotrophins, including brain-derived neurotrophic factor (BDNF) signaling through its high-affinity full-length tropomyosin-related kinase receptor subtype B (TrkB.FL) receptor. As a genetic knockout of BDNF leads to extensive MN loss and postnatal death within 1-2 days after birth, we tested the hypothesis that postnatal inhibition of BDNF/TrkB.FL signaling is important for postnatal phrenic MN (PhMN) survival. In the present study, we used a 1NMPP1-sensitive TrkB F616A mutant mouse to evaluate the effects of inhibition of TrkB kinase activity on phrenic MN (PhMN) numbers and diaphragm muscle (DIAm) fiber cross-sectional area (CSA). Pups were exposed to 1NMPP1 or vehicle (DMSO) from birth to 21 days old (weaning) via the mother's ingestion in the drinking water. Following weaning, the right phrenic nerve was exposed in the neck and the proximal end dipped in a rhodamine solution to retrogradely label PhMNs. After 24 h, the cervical spinal cord and DIAm were excised. Labeled PhMNs were imaged using confocal microscopy, whereas DIAm strips were frozen at ∼1.5× resting length, cryosectioned, and stained with hematoxylin and eosin to assess CSA. We observed an ∼34% reduction in PhMN numbers and increased primary dendrite numbers in 1NMPP1-treated TrkB F616A mice. The distribution of PhMN size (somal surface area) DIAm fiber cross-sectional areas did not differ. We conclude that survival of PhMNs during early postnatal development is sensitive to BDNF/TrkB.FL signaling. NEW & NOTEWORTHY During early postnatal development, BDNF/TrkB signaling promotes PhMN survival. Inhibition of BDNF/TrkB signaling in early postnatal development does not impact PhMN size. Inhibition of BDNF/TrkB signaling in early postnatal development does not impact the number or CSA of DIAm fibers.

Background: Ventilator-induced diaphragm dysfunction occurs rapidly following the onset of mechanical ventilation and has significant clinical consequences. Phrenic nerve stimulation has shown promise in maintaining diaphragm function by inducing diaphragm contractions. Non-invasive stimulation is an attractive option as it minimizes the procedural risks associated with invasive approaches. However, this method is limited by sensitivity to electrode position and inter-individual variability in stimulation thresholds. This makes clinical application challenging due to potentially time-consuming calibration processes to achieve reliable stimulation., Methods: We applied non-invasive electrical stimulation to the phrenic nerve in the neck in healthy volunteers. A closed-loop system recorded the respiratory flow produced by stimulation and automatically adjusted the electrode position and stimulation amplitude based on the respiratory response. By iterating over electrodes, the optimal electrode was selected. A binary search method over stimulation amplitudes was then employed to determine an individualized stimulation threshold. Pulse trains above this threshold were delivered to produce diaphragm contraction., Results: Nine healthy volunteers were recruited. Mean threshold stimulation amplitude was 36.17 ± 14.34 mA (range 19.38-59.06 mA). The threshold amplitude for reliable nerve capture was moderately correlated with BMI (Pearson's r = 0.66, p = 0.049). Repeating threshold measurements within subjects demonstrated low intra-subject variability of 2.15 ± 1.61 mA between maximum and minimum thresholds on repeated trials. Bilateral stimulation with individually optimized parameters generated reliable diaphragm contraction, resulting in significant inhaled volumes following stimulation., Conclusion: We demonstrate the feasibility of a system for automatic optimization of electrode position and stimulation parameters using a closed-loop system. This opens the possibility of easily deployable individualized stimulation in the intensive care setting to reduce ventilator-induced diaphragm dysfunction., (© 2023 The Authors. Artificial Organs published by International Center for Artificial Organ and Transplantation (ICAOT) and Wiley Periodicals LLC.)

The mammalian three-phase respiratory motor pattern of inspiration, post-inspiration and expiration is expressed in spinal and cranial motor nerve discharge and is generated by a distributed ponto-medullary respiratory pattern generating network. Respiratory motor pattern generation depends on a rhythmogenic kernel located within the pre-Bötzinger complex (pre-BötC). In the present study, we tested the effect of unilateral and bilateral inactivation of the pre-BötC after local microinjection of the GABA A receptor agonist isoguvacine (10 mM, 50 nl) on phrenic (PNA), hypoglossal (HNA) and vagal nerve (VNA) respiratory motor activities in an in situ perfused brainstem preparation of rats. Bilateral inactivation of the pre-BötC triggered cessation of phrenic (PNA), hypoglossal (HNA) and vagal (VNA) nerve activities for 15-20 min. Ipsilateral isoguvacine injections into the pre-BötC triggered transient (6-8 min) cessation of inspiratory and post-inspiratory VNA (p < 0.001) and suppressed inspiratory HNA by - 70 ± 15% (p < 0.01), while inspiratory PNA burst frequency increased by 46 ± 30% (p < 0.01). Taken together, these observations confirm the role of the pre-BötC as the rhythmogenic kernel of the mammalian respiratory network in situ and highlight a significant role for the pre-BötC in the transmission of vagal inspiratory and post-inspiratory pre-motor drive to the nucleus ambiguus., (Copyright © 2023. Published by Elsevier B.V.)

Repeated hypoxic episodes can produce a sustained (>60 min) increase in neural drive to the diaphragm. The requirement of repeated hypoxic episodes (vs. a single episode) to produce phrenic motor facilitation (pMF) can be removed by allosteric modulation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors using ampakines. We hypothesized that the ampakine-hypoxia interaction resulting in pMF requires that ampakine dosing precedes the onset of hypoxia. Phrenic nerve recordings were made from urethane-anesthetized, mechanically ventilated, and vagotomized adult male Sprague-Dawley rats during isocapnic conditions. Ampakine CX717 (15 mg/kg iv) was given immediately before ( n = 8), during ( n = 8), or immediately after ( n = 8) a 5-min hypoxic episode (arterial oxygen partial pressure 40-45 mmHg). Ampakine before hypoxia (A prior ) resulted in a sustained increase in inspiratory phrenic burst amplitude (i.e., pMF) reaching +70 ± 21% above baseline (BL) after 60 min. This was considerably greater than corresponding values in the groups receiving ampakine during hypoxia (+28 ± 47% above BL, P = 0.005 vs. A prior ) or after hypoxia (+23 ± 40% above BL, P = 0.005 vs. A prior ). Phrenic inspiratory burst rate, heart rate, and systolic, diastolic, and mean arterial pressure (mmHg) were similar across the three treatment groups (all P > 0.3, treatment effect). We conclude that the presentation order of ampakine and hypoxia impacts the magnitude of pMF, with ampakine pretreatment evoking the strongest response. Ampakine pretreatment may have value in the context of hypoxia-based neurorehabilitation strategies. NEW & NOTEWORTHY Phrenic motor facilitation (pMF) is evoked after repeated episodes of brief hypoxia. pMF can also be induced when an allosteric modulator of AMPA receptors (ampakine) is intravenously delivered immediately before a single brief hypoxic episode. Here we show that ampakine delivery before hypoxia (vs. during or after hypoxia) evokes the largest pMF with minimal impact on arterial blood pressure and heart rate. Ampakine pretreatment may have value in the context of hypoxia-based neurorehabilitation strategies.

Mechanical ventilation is essential in intensive care treatment but leads to diaphragmatic atrophy, which in turn contributes to prolonged weaning and increased mortality. One approach to prevent diaphragmatic atrophy while achieving pulmonary ventilation is electrical stimulation of the phrenic nerve. To automize phrenic nerve stimulation resulting in lung protective tidal volumes with lowest possible currents, mathematical models are required. Nerve stimulation models are often complex, so many parameters have to be identified prior to implementation. This paper presents a novel, simplified approach to model phrenic nerve excitation to obtain an individualized patient model using a few data points. The latter is based on the idea that nerve fibers are excited when the electric field exceeds a threshold. The effect of the geometry parameter on the model output was analyzed, and the model was validated with measurement data from a pig trial (RMSE in between 0.44 × 10 -2 and 1.64 × 10 -2 for parameterized models). The modeled phrenic nerve excitation behaved similarly to the measured tidal volumes, and thus could be used to develop automated phrenic nerve stimulation systems for lung protective ventilation., (© 2024 IOP Publishing Ltd.)

Aging results in increased neuromuscular transmission failure and denervation of the diaphragm muscle, as well as decreased force generation across a range of motor behaviors. Increased risk for respiratory complications in old age is a major health problem. Aging impairs autophagy, a tightly regulated multistep process responsible for clearing misfolded or aggregated proteins and damaged organelles. In motor neurons, aging-related autophagy impairment may contribute to deficits in neurotransmission, subsequent muscle atrophy, and loss of muscle force. Chloroquine is commonly used to inhibit autophagy. We hypothesized that chloroquine decreases transdiaphragmatic pressure (Pdi) in mice. Old mice (16-28 mo old; n = 26) were randomly allocated to receive intraperitoneal chloroquine (50 mg/kg) or vehicle 4 h before measuring Pdi during eupnea, hypoxia (10% O 2 )-hypercapnia (5% CO 2 ) exposure, spontaneous deep breaths ("sighs"), and maximal activation elicited by bilateral phrenic nerve stimulation (Pdi max ). Pdi amplitude and ventilatory parameters across experimental groups and behaviors were evaluated using a mixed linear model. There were no differences in Pdi amplitude across treatments during eupnea (∼8 cm H 2 O), hypoxia-hypercapnia (∼10 cm H 2 O), or sigh (∼36 cm H 2 O), consistent with prior studies documenting a lack of aging effects on ventilatory behaviors. In vehicle and chloroquine-treated mice, average Pdi max was 61 and 46 cm H 2 O, respectively. Chloroquine decreased Pdi max by 24% compared to vehicle ( P < 0.05). There were no sex or age effects on Pdi in older mice. The observed decrease in Pdi max suggests aging-related susceptibility to impairments in autophagy, consistent with the effects of chloroquine on this important homeostatic process. NEW & NOTEWORTHY Recent findings suggest that autophagy plays a role in the development of aging-related neuromuscular dysfunction; however, the contribution of autophagy impairment to the maintenance of diaphragm force generation in old age is unknown. This study shows that in old mice, chloroquine administration decreases maximal transdiaphragmatic pressure generation. These chloroquine effects suggest a susceptibility to impairments in autophagy in old age.

Hypotheses: Moderate acute intermittent hypoxia (mAIH) elicits plasticity in both respiratory (phrenic long-term facilitation; pLTF) and sympathetic nerve activity (sympLTF) in rats. Although mAIH produces pLTF in normal rats, inconsistent results are reported after cervical spinal cord injury (cSCI), possibly due to greater spinal tissue hypoxia below the injury site. There are no reports concerning cSCI effects on sympLTF. Since mAIH is being explored as a therapeutic modality to restore respiratory and non-respiratory movements in humans with chronic SCI, both effects are important. To understand cSCI effects on mAIH-induced pLTF and sympLTF, partial or complete C2 spinal hemisections (C2Hx) were performed and, 2 weeks later, we assessed: 1) ipsilateral cervical spinal tissue oxygen tension; 2) ipsilateral & contralateral pLTF; and 3) ipsilateral sympLTF in splanchnic and renal sympathetic nerves., Methods: Male Sprague-Dawley rats were studied intact, or after partial (single slice) or complete C2Hx (slice with ∼1 mm aspiration). Two weeks post-C2Hx, rats were anesthetized and prepared for recordings of bilateral phrenic nerve activity and spinal tissue oxygen pressure (PtO 2 ). Splanchnic and renal sympathetic nerve activity was recorded in intact and complete C2Hx rats., Results: Spinal PtO 2 near phrenic motor neurons was decreased after C2Hx, an effect most prominent with complete vs. partial injuries; baseline PtO 2 was positively correlated with mean arterial pressure. Complete C2Hx impaired ipsilateral but not contralateral pLTF; with partial C2Hx, ipsilateral pLTF was unaffected. In intact rats, mAIH elicited splanchnic and renal sympLTF. Complete C2Hx had minimal impact on baseline ipsilateral splanchnic or renal sympathetic nerve activity and renal, but not splanchnic, sympLTF remained intact., Conclusion: Greater tissue hypoxia likely impairs pLTF and splanchnic sympLTF post-C2Hx, although renal sympLTF remains intact. Increased sympathetic nerve activity post-mAIH may have therapeutic benefits in individuals living with chronic SCI since anticipated elevations in systemic blood pressure may mitigate hypotension characteristic of people living with SCI., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects both the upper and lower motor neurons in the nervous system, causing muscle weakness and severe disability. The progressive course of the disease reduces the functional capacity of the affected patients, limits daily activities, and leads to complete dependence on caregivers, ultimately resulting in a fatal outcome. Respiratory dysfunction mostly occurs later in the disease and is associated with a worse prognosis. Forty-six participants were included in our study, with 23 patients in the ALS group and 23 individuals in the control group. The ultrasound examination of the phrenic nerve (PN) was performed by two authors using a high-resolution "Philips EPIQ 7" ultrasound machine with a linear 4-18 MHz transducer. Our study revealed that the phrenic nerve is significantly smaller on both sides in ALS patients compared to the control group ( p < 0.001). Only one significant study on PN ultrasound in ALS, conducted in Japan, also showed significant results ( p < 0.00001). These small studies are particularly promising, as they suggest that ultrasound findings could serve as an additional diagnostic tool for ALS.

Aims: Sleep disorder breathing is an important non-cardiovascular comorbidity in patients with heart failure (HF). However, central sleep apnoea (CSA) remains poorly diagnosed and treated. This post hoc analysis examined symptoms and quality of life in patients with CSA and HF following 12 months of transvenous phrenic nerve stimulation (TPNS) therapy., Methods and Results: Patients enrolled in the remedē System Pivotal trial were invited to complete self-reported questionnaires. Symptoms and responses to three validated questionnaires were examined. Percentage of patients noting an impairment was calculated at baseline. At 12 months, % of patients experiencing improvement, no change, or worsening was calculated. Shifts from symptom presence at baseline to absence at 12 months were assessed for those symptoms experienced by ≥50% of patients at baseline. Seventy-five patients were included. Most frequently reported symptoms were fatigue and daytime sleepiness. Following 12 months of TPNS, a variety of subjective improvements were observed; 45% of patients indicating cessation of daytime sleepiness, 44% cessation of fatigue/weakness, and 52% no longer having difficulty falling/staying asleep. Specific questions related to tiredness/fatigue, motivation, and chance of dozing provided an insight into potential areas of improvement. Furthermore, at least 60% of patients reported resolution of insomnia/fragmented sleep and snoring on therapy., Conclusion: Adult patients with CSA and HF experience distressing symptoms and limitations. Transvenous phrenic nerve stimulation was found to improve many of these. Awareness of key symptoms or limitations patients experience can be used to inform the development of a CSA-specific patient questionnaire to identify CSA sooner and aid treatment decisions., Competing Interests: Conflict of interest: L.H. and M.L.: no conflict of interest; T.M. and S.M.: employee of ZOLL Respicardia; Q.A.A.: consultant for ZOLL Respicardia, and practising physician caring for patients with the technology receiving no compensation., (© The Author(s) 2022. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Spinal cord hemisection at C 2 (C 2 SH), sparing the dorsal column is widely used to investigate the effects of reduced phrenic motor neuron (PhMN) activation on diaphragm muscle (DIAm) function, with reduced DIAm activity on the injured side during eupnoea. Following C 2 SH, recovery of DIAm EMG activity may occur spontaneously over subsequent days/weeks. Various strategies have been effective at improving the incidence and magnitude of DIAm recovery during eupnoea, but little is known about the effects of C 2 SH on transdiaphragmatic pressure (P di ) during other ventilatory and non-ventilatory behaviours. We employ SPG302, a novel type of pegylated benzothiazole derivative, to assess whether enhancing synaptogenesis (i.e., enhancing spared local connections) will improve the incidence and the magnitude of recovery of DIAm EMG activity and P di function 14 days post-C 2 SH. In anaesthetised Sprague-Dawley rats, DIAm EMG and P di were assessed during eupnoea, hypoxia/hypercapnia and airway occlusion prior to surgery (C 2 SH or sham), immediately post-surgery and at 14 days post-surgery. In C 2 SH rats, 14 days of DMSO (vehicle) or SPG302 treatments (i.p. injection) occurred. At the terminal experiment, maximum P di was evoked by bilateral phrenic nerve stimulation. We show that significant EMG and P di deficits are apparent in C 2 SH compared with sham rats immediately after surgery. In C 2 SH rats treated with SPG302, recovery of eupneic, hypoxia/hypercapnia and occlusion DIAm EMG was enhanced compared with vehicle rats after 14 days. Treatment with SPG302 also ameliorated P di deficits following C 2 SH. In summary, SPG302 is an exciting new therapy to explore for use in spinal cord injuries. KEY POINTS: Despite advances in our understanding of the effects of cervical hemisection (C 2 SH) on diaphragm muscle (DIAm) EMG activity, very little is understood about the impact of C 2 SH on the gamut of ventilatory and non-ventilatory transdiaphragmatic pressures (P di ). Recovery of DIAm activity following C 2 SH is improved using a variety of approaches, but very few pharmaceuticals have been shown to be effective. One way of improving DIAm recovery is to enhance the amount of latent local spared connections onto phrenic motor neurons. A novel pegylated benzothiazole derivative enhances synaptogenesis in a variety of neurodegenerative conditions. Here, using a novel therapeutic SPG302, we show that 14 days of treatment with SPG302 ameliorated DIAm EMG and P di deficits compared with vehicle controls. Our results show that SPG302 is a compound with very promising potential for use in improving functional outcomes post-spinal cord injury., (© 2023 The Authors. The Journal of Physiology © 2023 The Physiological Society.)

Neuromotor control of diaphragm muscle (DIAm) motor units is dependent on an orderly size-dependent recruitment of phrenic motor neurons (PhMNs). Slow (type S) and fast, fatigue resistant (type FR) DIAm motor units, which are frequently recruited to sustain ventilation, comprise smaller PhMNs that innervate type I and IIa DIAm fibers. More fatigable fast (type FF) motor units, which are infrequently recruited for higher force, expulsive behaviors, comprise larger PhMNs that innervate more type IIx/IIb DIAm fibers. We hypothesize that due to the more frequent activation and thus higher energy demand of type S and FR motor units, the mitochondrial volume density (MVD) of smaller PhMNs is greater compared with larger PhMNs. In eight adult (6 mo old) Fischer 344 rats, PhMNs were identified via intrapleural injection of Alexa488-conjugated cholera toxin B (CTB). Following retrograde CTB labeling, mitochondria in PhMNs were labeled by transdural infusion of MitoTracker Red. PhMNs and mitochondria were imaged using multichannel confocal microscopy using a ×60 oil objective. Following optical sectioning and three-dimensional (3-D) rendering, PhMNs and mitochondria were analyzed volumetrically using Nikon Elements software. Analysis of MVD in somal and dendritic compartments was stratified by PhMN somal surface area. Smaller PhMNs (likely S and FR units) had greater somal MVDs compared with larger PhMNs (likely FF units). By contrast, proximal dendrites or larger PhMNs had higher MVD compared with dendrites of smaller PhMNs. We conclude that more active smaller PhMNs have a higher mitochondrial volume density to support their higher energy demand in sustaining ventilation. NEW & NOTEWORTHY Type S and FR motor units, comprising smaller phrenic motor neurons (PhMNs) are regularly activated to perform indefatigable ventilatory requirements. By contrast, type FF motor units, comprising larger PhMNs, are infrequently activated to perform expulsive straining and airway defense maneuvers. This difference in activation history is mirrored in the mitochondrial volume density (MVD), with smaller PhMNs having higher MVD than larger PhMNs. In proximal dendrites, this trend was reversed, with larger PhMNs having higher MVD than smaller PhMNs, likely due to the maintenance requirements for the larger dendritic arbor of FF PhMNs.

Background: Mechanical ventilation is widely used in ICU patients as a lifesaving intervention. Diaphragmatic atrophy and thinning occur from lack of contractions of the diaphragm during mechanical ventilation. It may prolong weaning and increase the risk of respiratory complications. Noninvasive electromagnetic stimulation of the phrenic nerves may ameliorate the atrophy seen with ventilation. The objective of this study was to show that noninvasive repetitive electromagnetic stimulation is safe, feasible, and effective to stimulate the phrenic nerves in both awake individuals and anesthetized patients., Methods: A single-center study with 10 subjects overall, 5 awake volunteers and 5 anesthetized subjects. We used a prototype electromagnetic, noninvasive, simultaneous bilateral phrenic nerve stimulation device in both groups. In the awake volunteers, we assessed time-to-first capture of the phrenic nerves and safety measures, such as pain, discomfort, dental paresthesia, and skin irritation. In the anesthetized subjects, time-to-first capture as well as tidal volumes and airway pressures at 20%, 30%, and 40% stimulation intensity were assessed., Results: Diaphragmatic capture was achieved in all the subjects within a median (range) of 1 min (1 min to 9 min 21 s) for the awake subjects and 30 s (20 s to 1 min 15 s) for the anesthetized subjects. There were no adverse or severe adverse events in either group, nor any dental paresthesia, skin irritation, or subjective pain in the stimulated area. Tidal volumes increased in all the subjects in response to simultaneous bilateral phrenic nerve stimulation and increased gradually with increasing stimulation intensity. Airway pressures corresponded to spontaneous breathing of ∼2 cm H 2 O., Conclusions: Noninvasive phrenic nerve stimulation can be safely performed in awake and anesthetized individuals. It was feasible and effective in stimulating the diaphragm by induction of physiologic and scalable tidal volumes with minimum positive airway pressures., Competing Interests: The other authors have disclosed no conflicts of interest., (Copyright © 2023 by Daedalus Enterprises.)

Inflammation undermines neuroplasticity, including serotonin-dependent phrenic long-term facilitation (pLTF) following moderate acute intermittent hypoxia (mAIH: 3, 5-min episodes, arterial Po 2 : 40-50 mmHg; 5-min intervals). Mild inflammation elicited by a low dose of the TLR-4 receptor agonist, lipopolysaccharide (LPS; 100 µg/kg, ip), abolishes mAIH-induced pLTF by unknown mechanisms. In the central nervous system, neuroinflammation primes glia, triggering ATP release and extracellular adenosine accumulation. As spinal adenosine 2 A (A2A) receptor activation impairs mAIH-induced pLTF, we hypothesized that spinal adenosine accumulation and A2A receptor activation are necessary in the mechanism whereby LPS impairs pLTF. We report that 24 h after LPS injection in adult male Sprague Dawley rats: 1 ) adenosine levels increase in ventral spinal segments containing the phrenic motor nucleus (C3-C5; P = 0.010; n = 7/group) and 2 ) cervical spinal A2A receptor inhibition (MSX-3, 10 µM, 12 µL intrathecal) rescues mAIH-induced pLTF. In LPS vehicle-treated rats (saline, ip), MSX-3 enhanced pLTF versus controls (LPS: 110 ± 16% baseline; controls: 53 ± 6%; P = 0.002; n = 6/group). In LPS-treated rats, pLTF was abolished as expected (4 ± 6% baseline; n = 6), but intrathecal MSX-3 restored pLTF to levels equivalent to MSX-3-treated control rats (120 ± 14% baseline; P < 0.001; n = 6; vs. LPS controls with MSX-3: P = 0.539). Thus, inflammation abolishes mAIH-induced pLTF by a mechanism that requires increased spinal adenosine levels and A2A receptor activation. As repetitive mAIH is emerging as a treatment to improve breathing and nonrespiratory movements in people with spinal cord injury or ALS, A2A inhibition may offset undermining effects of neuroinflammation associated with these neuromuscular disorders. NEW & NOTEWORTHY Mild inflammation undermines motor plasticity elicited by mAIH. In a model of mAIH-induced respiratory motor plasticity (phrenic long-term facilitation; pLTF), we report that inflammation induced by low-dose lipopolysaccharide undermines mAIH-induced pLTF by a mechanism requiring increased cervical spinal adenosine and adenosine 2 A receptor activation. This finding advances the understanding of mechanisms impairing neuroplasticity, potentially undermining the ability to compensate for the onset of lung/neural injury or to harness mAIH as a therapeutic modality.

We hypothesized that activation of phrenic afferents induces diaphragm motor plasticity. In anesthetized and spontaneously breathing rats we delivered 40 Hz, low threshold (twitch and 1.5X twitch threshold), inspiratory-triggered stimulation to the left hemidiaphragm for 30 min to activate ipsilateral phrenic afferents. Diaphragm amplitude ipsilateral and contralateral to stimulation were increased for 60 min following both currents compared to time controls not receiving stimulation. Diaphragm stimulation was repeated in laminectomy controls or following a unilateral C3-C6 dorsal rhizotomy to eliminate phrenic afferent volleys. Laminectomy controls expressed neuromuscular plasticity post-stimulation. In contrast, ipsilateral and contralateral diaphragm amplitude following dorsal rhizotomy was lower than laminectomy controls and no different than time controls, suggesting diaphragm motor plasticity was not induced post-rhizotomy. Our results indicate that diaphragm stimulation induces a novel form of plasticity in the phrenic motor system which requires phrenic afferent activation. Respiratory motor plasticity elicited by diaphragm stimulation may have value as a therapeutic strategy to improve diaphragm output in neuromuscular conditions., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Quantifying diaphragm neuromuscular function using cervical magnetic stimulation (CMS) typically uses only a single stimulator (1-Stim) which may be inadequate to maximally stimulate the phrenic nerves. We questioned if using two stimulators (2-Stim) together alters diaphragm neuromuscular function at baseline and following inspiratory pressure threshold loading. Six (n = 3 female) healthy young participants were instrumented with esophageal and gastric balloon tipped catheters and electrodes over the 7-8th intercostal space. With either 1-Stim or 2-Stim an incremental protocol, where the stimulator intensity was progressively increased was completed prior to a series of potentiated twitches. The inspiratory threshold loading test consisted of loaded breathing to failure. Compared to 1-Stim, 2-Stim resulted in significantly greater unpotentiated Pdi tw and M-waves during the incremental protocol (both p < 0.01). Similarly, 2-Stim resulted in greater potentiated Pdi tw (31 ± 8 vs. 41 ± 9 cmH 2 O; p = 0.02) and M-waves (6.4 ± 2.9 vs. 8.6 ± 2.4 V; p = 0.02). Our findings suggest that CMS using 1-Stim is unlikely to generate a sufficient magnetic field to maximally stimulate the phrenic nerves and may underestimate diaphragm function., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Moderate acute intermittent hypoxia (mAIH) elicits a form of phrenic motor plasticity known as phrenic long-term facilitation (pLTF), which requires spinal 5-HT 2 receptor activation, ERK/MAP kinase signaling, and new brain-derived neurotrophic factor (BDNF) synthesis. New BDNF protein activates TrkB receptors that normally signal through PKCθ to elicit pLTF. Phrenic motor plasticity elicited by spinal drug administration (e.g., BDNF) is referred to by a more general term: phrenic motor facilitation (pMF). Although mild systemic inflammation elicited by a low lipopolysaccharide (LPS) dose (100 µg/kg; 24 h prior) undermines mAIH-induced pLTF upstream from BDNF protein synthesis, it augments pMF induced by spinal BDNF administration through unknown mechanisms. Here, we tested the hypothesis that mild inflammation shifts BDNF/TrkB signaling from PKCθ to alternative pathways that enhance pMF. We examined the role of three known signaling pathways associated with TrkB (MEK/ERK MAP kinase, PI3 kinase/Akt, and PKCθ) in BDNF-induced pMF in anesthetized, paralyzed, and ventilated Sprague Dawley rats 24 h post-LPS. Spinal PKCθ inhibitor (TIP) attenuated early BDNF-induced pMF (≤30 min), with minimal effect 60-90 min post-BDNF injection. In contrast, MEK inhibition (U0126) abolished BDNF-induced pMF at 60 and 90 min. PI3K/Akt inhibition (PI-828) had no effect on BDNF-induced pMF at any time. Thus, whereas BDNF-induced pMF is exclusively PKCθ-dependent in normal rats, MEK/ERK is recruited by neuroinflammation to sustain, and even augment downstream plasticity. Because AIH is being developed as a therapeutic modality to restore breathing in people living with multiple neurological disorders, it is important to understand how inflammation, a common comorbidity in many traumatic or degenerative central nervous system disorders, impacts phrenic motor plasticity. NEW & NOTEWORTHY We demonstrate that even mild systemic inflammation shifts signaling mechanisms giving rise to BDNF-induced phrenic motor plasticity. This finding has important experimental, biological, and translational implications, particularly since BDNF-dependent spinal plasticity is being translated to restore breathing and nonrespiratory movements in diverse clinical disorders, such as spinal cord injury (SCI) and amyotrophic lateral sclerosis (ALS).

Background: Every year, more than 2.5 million critically ill patients in the ICU are dependent on mechanical ventilation. The positive pressure in the lungs generated by the ventilator keeps the diaphragm passive, which can lead to a loss of myofibers within a short time. To prevent ventilator-induced diaphragmatic dysfunction (VIDD), phrenic nerve stimulation may be used., Objective: The goal of this study is to show the feasibility of transesophageal phrenic nerve stimulation (TEPNS). We hypothesize that selective phrenic nerve stimulation can efficiently activate the diaphragm with reduced co-stimulations., Methods: An in vitro study in saline solution combined with anatomical findings was performed to investigate relevant stimulation parameters such as inter-electrode spacing, range to target site, or omnidirectional vs. sectioned electrodes. Subsequently, dedicated esophageal electrodes were inserted into a pig and single stimulation pulses were delivered simultaneously with mechanical ventilation. Various stimulation sites and response parameters such as transdiaphragmatic pressure or airway flow were analyzed to establish an appropriate stimulation setting., Results: Phrenic nerve stimulation with esophageal electrodes has been demonstrated. With a current amplitude of 40 mA, similar response figures of the diaphragm activation as compared to conventional stimulation with needle electrodes at 10mA were observed. Directed electrodes best aligned with the phrenic nerve resulted in up to 16.9 % higher amplitude at the target site in vitro and up to 6 cmH20 higher transdiaphragmatic pressure in vivo as compared to omnidirectional electrodes. The activation efficiency was more sensitive to the stimulation level inside the esophagus than to the inter-electrode spacing. Most effective and selective stimulation was achieved at the level of rib 1 using sectioned electrodes 40 mm apart., Conclusion: Directed transesophageal phrenic nerve stimulation with single stimuli enabled diaphragm activation. In the future, this method might keep the diaphragm active during, and even support, artificial ventilation. Meanwhile, dedicated sectioned electrodes could be integrated into gastric feeding tubes., (© 2023. The Author(s).)

Seizure-like burst activities are induced by blockade of GABAA and/or glycine receptors in various spinal ventral roots of brainstem-spinal cord preparation from neonatal rodents. We found that this is not applicable to the phrenic nerve and that a new inhibitory descending pathway may suppress seizure-like activity in the phrenic nerve. Experiments were performed in brainstem-spinal cord preparation from newborn rats (age: 0-1 day). Left phrenic nerve and right C4 activities were recorded simultaneously. When GABAA and glycine receptors were blocked by 10 μM bicuculline and 10 μM strychnine (Bic+Str), seizure-like burst activities appeared in the fourth cervical ventral root (C4) but not the phrenic nerve. After making a transverse section at C1, the inspiratory burst activity disappeared from both C4 and the phrenic nerve, whereas seizure-like activity appeared in both nerves. We hypothesized that inhibitory descending pathways other than those via GABAA and/or glycine receptors (from the medulla to the spinal cord) work to avoid disturbance of regular respiratory-related diaphragm contraction by seizure-like activity. We found that cannabinoid receptor antagonist, AM251 was effective for the induction of seizure-like activity by Bic+Str in the phrenic nerve in brainstem-spinal cord preparation. Cannabinoid receptors may be involved in this descending inhibitory system.

After a bout of isolated inspiratory work, such as inspiratory pressure threshold loading (IPTL), the human diaphragm can exhibit a reversible loss in contractile function, as evidenced by a decrease in transdiaphragmatic twitch pressure (P DI,TW ). Whether or not diaphragm fatigability after IPTL is affected by neural mechanisms, measured through voluntary activation of the diaphragm (D-VA) in addition to contractile mechanisms, is unknown. It is also unknown if changes in D-VA are similar between sexes given observed differences in diaphragm fatigability between males and females. We sought to determine whether D-VA decreases after IPTL and whether this was different between sexes. Healthy females (n = 11) and males (n = 10) completed an IPTL task with an inspired duty cycle of 0.7 and targeting an intensity of 60% maximal transdiaphragmatic pressure until task failure. P DI,TW and D-VA were measured using cervical magnetic stimulation of the phrenic nerves in combination with maximal inspiratory pressure maneuvers. At task failure, P DI,TW decreased to a lesser degree in females vs. males (87 ± 15 vs. 73 ± 12% baseline, respectively, p = 0.016). D-VA decreased after IPTL but was not different between females and males (91 ± 8 vs. 88 ± 10% baseline, respectively, p = 0.432). When all participants were pooled together, the decrease in P DI,TW correlated with both the total cumulative diaphragm pressure generation (R 2  = 0.43; p = 0.021) and the time to task failure (TTF, R 2 = 0.40; p = 0.30) whereas the decrease in D-VA correlated only with TTF (R 2  = 0.24; p = 0.041). Our results suggest that neural mechanisms can contribute to diaphragm fatigability, and this contribution is similar between females and males following IPTL., (© 2023 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Phrenic motoneurons (PhrMNs) innervate diaphragm myofibers. Located in the ventral gray matter (lamina IX), PhrMNs form a column extending from approximately the third to sixth cervical spinal segment. Phrenic motor output and diaphragm activation are impaired in many neuromuscular diseases, and targeted delivery of drugs and/or genetic material to PhrMNs may have therapeutic application. Studies of phrenic motor control and/or neuroplasticity mechanisms also typically require targeting of PhrMNs with drugs, viral vectors, or tracers. The location of the phrenic motoneuron pool, however, poses a challenge. Selective PhrMN targeting is possible with molecules that move retrogradely upon uptake into phrenic axons subsequent to diaphragm or phrenic nerve delivery. However, nonspecific approaches that use intrathecal or intravenous delivery have considerably advanced the understanding of PhrMN control. New opportunities for targeted PhrMN gene expression may be possible with intersectional genetic methods. This article provides an overview of methods for targeting the phrenic motoneuron pool for studies of PhrMNs in health and disease.

Background: Rapid magnetic stimulation (RMS) of the phrenic nerves may serve to attenuate diaphragm atrophy during mechanical ventilation. With different coil shapes and stimulation location, inspiratory responses and side-effects may differ. This study aimed to compare the inspiratory and sensory responses of three different RMS-coils either used bilaterally on the neck or on the chest, and to determine if ventilation over 10 min can be achieved without muscle fatigue and coils overheating., Methods: Healthy participants underwent bilateral anterior 1-s RMS on the neck (RMS BAMPS ) (N = 14) with three different pairs of magnetic coils (parabolic, D-shape, butterfly) at 15, 20, 25 and 30 Hz stimulator-frequency and 20% stimulator-output with + 10% increments. The D-shape coil with individual optimal stimulation settings was then used to ventilate participants (N = 11) for up to 10 min. Anterior RMS on the chest (RMS aMS ) (N = 8) was conducted on an optional visit. Airflow was assessed via pneumotach and transdiaphragmatic pressure via oesophageal and gastric balloon catheters. Perception of air hunger, pain, discomfort and paresthesia were measured with a numerical scale., Results: Inspiration was induced via RMS BAMPS in 86% of participants with all coils and via RMS aMS in only one participant with the parabolic coil. All coils produced similar inspiratory and sensory responses during RMS BAMPS with the butterfly coil needing higher stimulator-output, which resulted in significantly larger discomfort ratings at maximal inspiratory responses. Ten of 11 participants achieved 10 min of ventilation without decreases in minute ventilation (15.7 ± 4.6 L/min)., Conclusions: RMS BAMPS was more effective than RMS aMS, and could temporarily ventilate humans seemingly without development of muscular fatigue. Trial registration This study was registered on clinicaltrials.gov (NCT04176744)., (© 2022. The Author(s).)

Objective: HF-SCS is a novel technique of inspiratory muscle activation which results in coincident activation of the diaphragm and inspiratory intercostal muscles via spinal cord pathways and has the potential to provide respiratory support in ventilator dependent persons with spinal cord injury. The purpose of the present study was to examine the phrenic-to-intercostal reflex during HF-SCS., Methods: In 5 anesthetized and C2 spinalized dogs, electrical stimulation was applied via a stimulating electrode located on the ventral surface of the upper thoracic spinal cord at the T2 level. Fine wire recording electrodes were used to assess single motor unit (SMU) activity of the left and right external intercostal muscles (EI) in the 3rd interspace before and after sequential left and right phrenicotomy., Results: Mean control peak firing frequency of the right EI and left EI was 11.4 ± 0.3 Hz and 10.6 ± 0.3 Hz respectively. Following unilateral right phrenic nerve section, mean SMU peak firing frequency of right EI (ipsilateral to the section) was significantly greater when compared to control (15.9 ± 0.5 Hz vs 11.4 ± 0.3 Hz; p = 0.01). Mean SMU peak firing frequency of the contralateral left EI remained unchanged (10.2 ± 0.3 Hz vs 10.6 ± 0.3 Hz, p = 0.40). Subsequent, section of the left phrenic nerve resulted in significantly higher mean SMU peak firing frequency of the left EI (16.2 ± 0.5 Hz vs 10.2 ± 0.3 Hz) when compared to before section p = 0.01). Contralateral, right EI peak firing frequency was not different if compared to before left phrenic nerve section (16.9 ± 0.4 Hz vs. 15.9 ± 0.5 Hz; p = 0.14)., Conclusion: This study demonstrates that during HF-SCS: 1) unilateral diaphragmatic afferents reflexly inhibit motor activity to the ipsilateral EI muscles, 2) the neural circuitry mediating the phrenic-to-intercostal reflex is preserved at a spinal level and does not require supraspinal input and 3) unilateral compensatory increases were observed in EI muscle activation following ipsilateral diaphragm paralysis., Competing Interests: Conflict of interest A. DiMarco is a founder of and has a significant financial interest in Synapse BioMedical, a manufacturer of diaphragm pacing systems and holds patents for spinal cord stimulation to restore cough and respiration (nos. 5,678,535; 5,911,218; 5,999,855; and 8,751,004). A. DiMarco and K. Kowalski hold the U.S. patents for technology related to the content of this manuscript, Respiratory Muscle Activation by Spinal Cord Stimulation (no. 8,352,036)., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Diaphragm muscle atrophy during mechanical ventilation begins within 24 h and progresses rapidly with significant clinical consequences. Electrical stimulation of the phrenic nerves using invasive electrodes has shown promise in maintaining diaphragm condition by inducing intermittent diaphragm muscle contraction. However, the widespread application of these methods may be limited by their risks as well as the technical and environmental requirements of placement and care. Non-invasive stimulation would offer a valuable alternative method to maintain diaphragm health while overcoming these limitations., Methods: We applied non-invasive electrical stimulation to the phrenic nerve in the neck in healthy volunteers. Respiratory pressure and flow, diaphragm electromyography and mechanomyography, and ultrasound visualization were used to assess the diaphragmatic response to stimulation. The electrode positions and stimulation parameters were systematically varied in order to investigate the influence of these parameters on the ability to induce diaphragm contraction with non-invasive stimulation., Results: We demonstrate that non-invasive capture of the phrenic nerve is feasible using surface electrodes without the application of pressure, and characterize the stimulation parameters required to achieve therapeutic diaphragm contractions in healthy volunteers. We show that an optimal electrode position for phrenic nerve capture can be identified and that this position does not vary as head orientation is changed. The stimulation parameters required to produce a diaphragm response at this site are characterized and we show that burst stimulation above the activation threshold reliably produces diaphragm contractions sufficient to drive an inspired volume of over 600 ml, indicating the ability to produce significant diaphragmatic work using non-invasive stimulation., Conclusion: This opens the possibility of non-invasive systems, requiring minimal specialist skills to set up, for maintaining diaphragm function in the intensive care setting., (© 2022 The Authors. Artificial Organs published by International Center for Artificial Organ and Transplantation (ICAOT) and Wiley Periodicals LLC.)

RespiCardia remedē System (Minnetonka, MN 2017), a transvenous phrenic nerve stimulator, is indicated to treat central sleep apnea (CSA) by stimulating the phrenic nerve to cause diaphragmatic contraction to restore normal breathing during sleep. CSA is associated with decreased patient quality of life and worsens cardiovascular outcomes. Systematic review was conducted according to the Preferred Reporting of Systematic Reviews and Meta-Analysis guidelines. PubMed/MEDLINE, Cochrane, EBSCO CINAHL, and Ovid databases were queried by 2 independent reviewers for English-language studies published between 2000 and 2021. The initial search screened for all occurrences of "remedē" then was further refined to include studies evaluating use of the RespiCardia remedē System as a treatment for CSA in multiple patients. A total of 124 articles were identified from the initial search results. A total of 37 articles were identified through screening of title and abstracts from initial results. Full-text review of all the articles was then completed. Of the 13 articles included, a total of 232 patients underwent device implantation. We sought to summarize the available evidence regarding patient selection for implantation, immediate and delayed complications, adherence to therapy, and polysomnographic evidence of efficacy. All 13 articles detailed significant decreases in central apnea index and many patients reported significant mild to marked improvement in quality of life. In conclusion, the remedē System has been demonstrated to improve sleep and respiratory parameters, with few complications. This device demonstrates safe and effective treatment of moderate to severe CSA in adult patients, including those with heart failure., Competing Interests: Disclosures The authors have no conflicts of interest to declare., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Purpose: To evaluate the phrenic nerve compound muscle action potential (CMAP) in rats after diabetes mellitus (DM) induction., Methods: Twenty DM animals (intravenous streptozotocin, 45 mg.kg -1 ) and 25 controls underwent CMAP analysis before and 30, 60 and 90 days after DM induction., Results: Amplitude (mV) progressively declined in DM group after 30 (Mean difference (MD): -0.915, 95 % Confidence interval (CI) -1.580 to -0.250, p < 0.01), 60 (MD: -1.122, 95 % CI -1.664 to -0.581, p < 0.001) and 90 days (MD: -2.226, 95 % CI -3.059 to -1.393, p < 0.001); as well as the area (mV.ms) after 30 (MD: -3.19, 95 % CI -5.94 to -0.44, p < 0.05), 60 (MD: -3.94, 95 % CI -6.24 to -1.64, p < 0.001) and 90 days (MD: -8.64, 95 % CI -12.08 to -5.21, p < 0.001). Transient differences were observed in latency and duration at 60 days., Conclusions: The progressive changes in phrenic nerve CMAP observed during DM suggest a decrement in axonal function rather than substantial demyelination., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Objective: To report a new patient friendly and convenient technique for phrenic nerve conduction with alternative sites of stimulation and recording., Methods: Phrenic nerve conduction was performed in forty volunteers and ten patients of peripheral neuropathy. Active recording electrode was placed in tenth intercostal space 2.5 cm away from para-spinal muscles (mid-scapular line), reference electrode in eighth intercostal space just medial to subcostal margin with ground between stimulating and recording electrode. Stimulation was done at the level of crico-thyroid space near or under the posterior margin of sternocleidomastoid muscle. This new method was compared with existing ones., Analysis: Data was analysed using SPSS 23 version. Correlation between height, weight, body mass index, age, and chest expansion was done using bi-variate correlation. Mean latency and amplitude of the study method were compared with other methods using MANNOVA test., Results: Total of forty subjects were studied. Thirty-seven were male subjects. Mean age was 28.03 ± 9.63 years, height 168.0 ± 9.60 cm and chest expansion 3.53 ± 0.64 cm. Right sided phrenic nerve mean latency was 5.99 ± 0.629 ms and amplitude 1.088 ± 0.178 mV. Left sided phrenic nerve conductions showed mean latency of 6.02 ± 1.82 ms, amplitude of 1.092 ± 0.2912 mV. These standard deviations were smaller than what were observed with other methods suggesting increased consistency of our results. There was no correlation between phrenic nerve conduction with age, height, gender or chest expansion., Conclusion: This study method gave a better as well as consistent morphology, higher amplitude and required lower amount of current strength. It was superior to previously reported methods in consistency of normative data., Competing Interests: None

This study characterised the hemidiaphragm elevation on 3-month interval chest X-rays (CXRs) of patients post COVID-19 pneumonia. 467 CXRs were screened; 19 (4.1%) had an elevated hemidiaphragm. There were 15 (3.2%) patients of interest with new hemidiaphragm elevation, persisting on average 7 months post COVID-19 diagnosis. Symptomatic patients underwent diaphragm ultrasound (n=12), pulmonary function test (n=10), muscle function test (n=6) and neurophysiology (n=5), investigating phrenic nerve function. Ultrasound demonstrated reduced/paradoxical diaphragmatic movements in eight; four of eight had reduced thickening fraction. Neurophysiology peripheral limb studies did not support the differential diagnoses of critical illness neuropathy/myopathy. We propose that, in selected patients, COVID-19 may cause phrenic nerve mononeuritis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Spasticity is a common symptom in many developmental motor disorders, including spastic cerebral palsy (sCP). In sCP, respiratory dysfunction is a major contributor to morbidity and mortality, yet it is unknown how spasticity influences respiratory physiology or diaphragm muscle (DIAm) function. To investigate the influence of spasticity on DIAm function, we assessed in vivo transdiaphragmatic pressure (Pdi - measured using intraesophageal and intragastric pressure catheters under conditions of eupnea, hypoxia/hypercapnia and occlusion) including maximum Pdi (Pdi max via bilateral phrenic nerve stimulation), ex vivo DIAm-specific force and fatigue (using muscle strips stimulated with platinum plate electrodes), and type-specific characteristics of DIAm fiber cross sections (using immunoreactivity against myosin heavy chain slow and 2A) in spa and wildtype mice. Spa mice show reduced Pdi max , reduced DIAm specific force, and altered fatigability and atrophy of type IIx/IIb fibers. These findings suggest marked DIAm dysfunction may underlie the respiratory phenotype of sCP. NEW & NOTEWORTHY Developmental motor control dysfunctions, including spastic cerebral palsy (sCP) often have respiratory components. Spa mutant mice exhibit a spastic phenotype closely resembling sCP symptoms. Using the spa mouse model of spastic cerebral palsy (sCP), we quantified transdiaphragmatic pressure deficits, diaphragm muscle weakness, and fiber type-specific atrophy, improving our understanding of respiratory dysfunctions in sCP.

While mechanical ventilation (MV) can lead to ventilator-induced diaphragmatic atrophy due to diaphragm inactivity, electrical phrenic nerve stimulation (PNS) can keep the diaphragm active and therefore prevent diaphragmatic weakness. To quantify the effectivity of PNS, an identification experiment during PNS is presented, and its data is used in Gaussian process regression (GPR) of the tidal volume based on the constant voltage amplitude of the stimulation pulses. The measurements were split into training data of variable size and test data for cross validation. For variable training sizes and different PNS settings, the GPR had a root mean square deviation (RMSD) between 0.39 and 0.91 mL/kg. An identification experiment as short as one and a half minutes was able to characteristically capture the relationship between tidal volume and voltage amplitude. The proposed method needs to be validated in further experiments.

Patients with central apnoea may use electro ventilation, provided their phrenic nerves and diaphragm muscles are normal. A tendency towards better survival has been found, and both an improved quality of life and facilitated nursing have been claimed with electro ventilation compared to mechanical ventilation. The high investment for the device may form a hurdle for fund providers like our hospital administration board. We, therefore, from our first patient onwards, collected clinically meaningful data in a special register of all patients using electro ventilation and their controls on mechanical ventilation. Since 1988 172 patients left our institution dependent on a respiratory device. Of these, all 48 patients with preserved phrenic nerves chose phrenic nerve stimulation. A patient on mechanical ventilation who agreed to participate was chosen as a control (n = 44). All patients were seen at least once a year. 90 patients suffered high tetraplegia, and 2 suffered central apnoea for other reasons. There is a tendency towards better survival, and there is a lower frequency of decubital ulcers (0.02) and respiratory tract infections (p0.000) with electro than with mechanical ventilation. The frequency of respiratory infections turned out to be a better measure of the quality of respiratory care than survival. The resulting decrease in the need for airway nursing, and the reduced incidence of respiratory infections repaid the high investment in electro ventilation within one year in our setting. Informed patients prefer electro to mechanical ventilation; fund providers might also agree with this preference., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Introduction: The female diaphragm develops less fatigue after high-intensity exercise compared with males. Diaphragm fatigability is typically defined as a decrease in transdiaphragmatic twitch pressure (Pdi,TW) and represents the contractile function of the muscle. However, it is unclear whether this sex difference persists when examining changes in voluntary activation, which represents a neural mechanism contributing to fatigability., Purpose: This study aimed to determine if high-intensity cycling results in a decrease in diaphragm voluntary activation (D-VA) and to explore if the decrease in D-VA is different between sexes., Methods: Twenty-five participants (15 females) completed a single bout of high-intensity constant load cycling. D-VA and Pdi,TW were measured before and after exercise using cervical magnetic stimulation of the phrenic nerves to assess diaphragm fatigability., Results: Participants were of similar aerobic fitness when expressed relative to predicted values (females: 114% ± 25% predicted, males: 111% ± 11% predicted; P = 0.769). Pdi,TW decreased relative to baseline to 85.2% ± 16.7% and 70.3% ± 12.4% baseline (P = 0.012) in females and males, respectively, immediately after exercise. D-VA also decreased in both females and males immediately after exercise. The decrease in D-VA was less in females compared with males (95.4% ± 4.9% baseline vs 87.4% ± 10.8% baseline, respectively; P = 0.018)., Conclusions: D-VA decreases after whole-body exercise in both females and males, although the magnitude of the decrease is not as large in females compared with males. The findings of this study suggest that the female diaphragm is more resistant to both contractile and neural mechanisms of fatigability after whole-body exercise., (Copyright © 2022 by the American College of Sports Medicine.)

Upper cervical spinal cord injuries (SCI) disrupt descending inputs to phrenic motor neurons (PhMNs), impairing respiratory function. Unilateral spinal hemisection at C2 (C2SH) results in loss of ipsilateral rhythmic diaphragm muscle (DIAm) EMG activity associated with lower force behaviors accomplished by recruitment of smaller PhMNs in rats. Activity during higher force, non-ventilatory behaviors that recruit larger PhMNs is minimally impaired following C2SH. We previously showed neuroplasticity in glutamatergic receptor expression in PhMN post-C2SH with changes in NMDA receptor expression reflecting functional recovery over time. We hypothesize that C2SH-induced changes in glutamatergic receptor (AMPA and NMDA) mRNA expression in PhMNs vary with motor neuron size, with more pronounced changes in smaller PhMNs. Retrogradely-labelled PhMNs were classified in tertiles according to somal surface area and mRNA expression was measured using single-cell, multiplex fluorescence in situ hybridization. Ipsilateral to C2SH, a pronounced reduction in NMDA mRNA expression in PhMNs was evident at 3 days post-injury with similar impact on PhMNs in the lower size tertile (~68% reduction) and upper tertile (~60%); by 21 days, there was near complete restoration of NMDA receptor mRNA expression across all PhMNs. There were no changes in NMDA mRNA expression contralateral to C2SH. There were no changes in AMPA mRNA expression at PhMNs on either side of the spinal cord or at any time-point post-C2SH. In summary, following C2SH there is ipsilateral reduction in PhMN NMDA mRNA expression at 3 days that is not limited to smaller PhMN recruited in the generation of lower force ventilatory behaviors. The recovery of NMDA mRNA expression by 21 days post-C2SH is consistent with evidence of spontaneous recovery of ipsilateral DIAm activity at this timepoint. These findings suggest a possible role for NMDA receptor mediated glutamatergic signaling in mechanisms supporting postsynaptic neuroplasticity at the PhMN pool and recovery of DIAm activity after cervical SCI., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Introduction: Patients with high cervical Spinal Cord Injury (SCI) usually require mechanical ventilation support. Phrenic Nerve Stimulation (PNS) both direct and indirect is the main alternative for these patients to wean off ventilator although PNS has several limitations and phrenic nerve could be also damaged after cervical spinal cord injury., Objective: In this study, we assessed if the spinal cord Epidural Electrical Stimulation (EES) at the segments T2-T5, related to intercostal muscles, can facilitate respiratory function and particularly inspired tidal volume during mechanic ventilation., Methods: Two patients with a high cervical injury were selected for this study with ethical committee permission and under review board supervision. A phrenic nerve conduction study with diaphragm electromyography (DEMG) was performed before and after trial of EES., Results: Results demonstrate that EES at T2-T5 substantially increase the inspired volume. The results of this study also demonstrate that EES at spinal segments T2-T5 can bring patients dependent from mechanical ventilation to pressure support (on CPAP), preventing Baro-trauma and other complications related to mechanical ventilation., Conclusion: These findings suggest that tested approach applied alone or in combination with the phrenic nerve stimulation could help to reduce time on mechanical ventilation and related complications., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Acute intermittent hypoxia (AIH) elicits long-term facilitation (LTF) of respiration. Although LTF is observed when CO 2 is elevated during AIH in awake humans, the influence of CO 2 on corticospinal respiratory motor plasticity is unknown. Thus, we tested the hypotheses that acute intermittent hypercapnic-hypoxia (AIHH): (1) enhances cortico-phrenic neurotransmission (reflecting volitional respiratory control); and (2) elicits ventilatory LTF (reflecting automatic respiratory control). Eighteen healthy adults completed four study visits. Day 1 consisted of anthropometry and pulmonary function testing. On Days 2, 3 and 4, in a balanced alternating sequence, participants received: AIHH, poikilocapnic AIH, and normocapnic-normoxia (Sham). Protocols consisted of 15, 60 s exposures with 90 s normoxic intervals. Transcranial (TMS) and cervical (CMS) magnetic stimulation were used to induce diaphragmatic motor-evoked potentials and compound muscle action potentials, respectively. Respiratory drive was assessed via mouth occlusion pressure (P 0.1 ), and minute ventilation measured at rest. Dependent variables were assessed at baseline and 30-60 min after exposures. Increases in TMS-evoked diaphragm potential amplitudes were observed following AIHH vs. Sham (+28 ± 41%, P = 0.003), but not after AIH. No changes were observed in CMS-evoked diaphragm potential amplitudes. Mouth occlusion pressure also increased after AIHH (+21 ± 34%, P = 0.033), but not after AIH. Ventilatory LTF was not observed after any treatment. We demonstrate that AIHH elicits central neural mechanisms of respiratory motor plasticity and increases resting respiratory drive in awake humans. These findings may have important implications for neurorehabilitation after spinal cord injury and other neuromuscular disorders compromising breathing. KEY POINTS: The occurrence of respiratory long-term facilitation following acute exposure to intermittent hypoxia is believed to be dependent upon CO 2 regulation - mechanisms governing the critical role of CO 2 have seldom been explored. We tested the hypothesis that acute intermittent hypercapnic-hypoxia (AIHH) enhances cortico-phrenic neurotransmission in awake healthy humans. The amplitude of diaphragmatic motor-evoked potentials induced by transcranial magnetic stimulation was increased after AIHH, but not the amplitude of compound muscle action potentials evoked by cervical magnetic stimulation. Mouth occlusion pressure (P 0.1 , an indicator of neural respiratory drive) was also increased after AIHH, but not tidal volume or minute ventilation. Thus, moderate AIHH elicits central neural mechanisms of respiratory motor plasticity, without measurable ventilatory long-term facilitation in awake humans., (© 2022 The Authors. The Journal of Physiology © 2022 The Physiological Society.)