Breath-by-breath comparison of a novel percutaneous phrenic nerve stimulation approach with mechanical ventilation in juvenile pigs: a pilot study.

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.

Postnatal survival of phrenic motor neurons is promoted by BDNF/TrkB.FL signaling.

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 TrkBF616A 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 TrkBF616A 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.

A physiological model of phrenic nerve excitation by electrical stimulation.

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 × 10-2and 1.64 × 10-2for 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.

Closed-loop parameter optimization for patient-specific phrenic nerve stimulation.

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.

The pre-Bötzinger complex is necessary for the expression of inspiratory and post-inspiratory motor discharge of the vagus.

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 GABAA 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.

Pattern sensitivity of ampakine-hypoxia interactions for evoking phrenic motor facilitation in anesthetized rat.

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 (Aprior) 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. Aprior) or after hypoxia (+23 ± 40% above BL, P = 0.005 vs. Aprior). 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.

Sonographic Phrenic Nerve Changes in Amyotrophic Lateral Sclerosis.

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.

Chloroquine impairs maximal transdiaphragmatic pressure generation in old mice.

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% O2)-hypercapnia (5% CO2) exposure, spontaneous deep breaths ("sighs"), and maximal activation elicited by bilateral phrenic nerve stimulation (Pdimax). 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 H2O), hypoxia-hypercapnia (∼10 cm H2O), or sigh (∼36 cm H2O), consistent with prior studies documenting a lack of aging effects on ventilatory behaviors. In vehicle and chloroquine-treated mice, average Pdimax was 61 and 46 cm H2O, respectively. Chloroquine decreased Pdimax by 24% compared to vehicle (P < 0.05). There were no sex or age effects on Pdi in older mice. The observed decrease in Pdimax 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.

Cervical spinal hemisection effects on spinal tissue oxygenation and long-term facilitation of phrenic, renal and splanchnic sympathetic nerve activity.

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 (PtO2). Splanchnic and renal sympathetic nerve activity was recorded in intact and complete C2Hx rats. RESULTS: Spinal PtO2 near phrenic motor neurons was decreased after C2Hx, an effect most prominent with complete vs. partial injuries; baseline PtO2 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.

Cannabinoid receptors involved in descending inhibition on spinal seizure-like activity in the phrenic output.

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.

Size-dependent differences in mitochondrial volume density in phrenic motor neurons.

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.

Safety and Feasibility of Noninvasive Electromagnetic Stimulation of the Phrenic Nerves.

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 H2O. 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.

Mild inflammation impairs acute intermittent hypoxia-induced phrenic long-term facilitation by a spinal adenosine-dependent mechanism.

Inflammation undermines neuroplasticity, including serotonin-dependent phrenic long-term facilitation (pLTF) following moderate acute intermittent hypoxia (mAIH: 3, 5-min episodes, arterial Po2: 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.

Novel regenerative drug, SPG302 promotes functional recovery of diaphragm muscle activity after cervical spinal cord injury.

Spinal cord hemisection at C2 (C2 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 C2 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 C2 SH on transdiaphragmatic pressure (Pdi ) 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 Pdi function 14 days post-C2 SH. In anaesthetised Sprague-Dawley rats, DIAm EMG and Pdi were assessed during eupnoea, hypoxia/hypercapnia and airway occlusion prior to surgery (C2 SH or sham), immediately post-surgery and at 14 days post-surgery. In C2 SH rats, 14 days of DMSO (vehicle) or SPG302 treatments (i.p. injection) occurred. At the terminal experiment, maximum Pdi was evoked by bilateral phrenic nerve stimulation. We show that significant EMG and Pdi deficits are apparent in C2 SH compared with sham rats immediately after surgery. In C2 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 Pdi deficits following C2 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 (C2 SH) on diaphragm muscle (DIAm) EMG activity, very little is understood about the impact of C2 SH on the gamut of ventilatory and non-ventilatory transdiaphragmatic pressures (Pdi ). Recovery of DIAm activity following C2 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 Pdi 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.

Diaphragm stimulation elicits phrenic afferent-induced neuromuscular plasticity.

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.

Altering magnetic field strength impacts the assessment of diaphragmatic function using cervical magnetic stimulation.

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 Pditw and M-waves during the incremental protocol (both p < 0.01). Similarly, 2-Stim resulted in greater potentiated Pditw (31 ± 8 vs. 41 ± 9 cmH2O; 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.

Voluntary activation of the diaphragm after inspiratory pressure threshold loading.

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 (PDI,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. PDI,TW and D-VA were measured using cervical magnetic stimulation of the phrenic nerves in combination with maximal inspiratory pressure maneuvers. At task failure, PDI,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 PDI,TW correlated with both the total cumulative diaphragm pressure generation (R2  = 0.43; p = 0.021) and the time to task failure (TTF, R2 = 0.40; p = 0.30) whereas the decrease in D-VA correlated only with TTF (R2  = 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.

BDNF-induced phrenic motor facilitation shifts from PKCθ to ERK dependence with mild systemic inflammation.

Moderate acute intermittent hypoxia (mAIH) elicits a form of phrenic motor plasticity known as phrenic long-term facilitation (pLTF), which requires spinal 5-HT2 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).