Diaphragmatic Activity Research Articles

Inspiratory neural drive and dyspnea in interstitial lung disease: Effect of inhaled fentanyl

BackgroundExertional dyspnea in interstitial lung disease (ILD) remains difficult to manage despite advances in disease-targeted therapies. Pulmonary opioid receptors present a potential therapeutic target for nebulized fentanyl to provide dyspnea relief. MethodsILD patients were characterized with reference to healthy volunteers. A randomized, double-blind, placebo-controlled crossover comparison of 100 mcg nebulized fentanyl vs placebo on dyspnea intensity and inspiratory neural drive (IND) during constant work rate (CWR) cycle exercise was performed in 21 ILD patients. ResultsDyspnea intensity in ILD increased in association with an increase in IND (diaphragm activation) from a high resting value of 16.66 ± 6.52 %–60.04 ± 12.52 % of maximum (r = 0.798, p < 0.001). At isotime during CWR exercise, Borg dyspnea intensity ratings with fentanyl vs placebo were 4.1 ± 1.2 vs 3.8 ± 1.2, respectively (p = 0.174), and IND responses were also similar. ConclusionIND rose sharply during constant work rate exercise in association with dyspnea intensity in mild to moderate ILD but was not different after nebulized fentanyl compared with placebo.

Neuregulin-1β Protects the Rat Diaphragm during Sepsis against Oxidative Stress and Inflammation by Activating the PI3K/Akt Pathway.

Sepsis-induced diaphragm dysfunction (SIDD) which is mainly characterized by decrease in diaphragmatic contractility has been identified to cause great harms to patients. Therefore, there is an important and pressing need to find effective treatments for improving SIDD. In addition, acetylcholinesterase (AChE) activity is a vital property of the diaphragm, so we evaluated both diaphragmatic contractility and AChE activity. Though neuregulin-1β (NRG-1β) is known to exert organ-protective effects in some inflammatory diseases, little is known about the potential of NRG-1β therapy in the diaphragm during sepsis. Our study was aimed at exploring the effects of NRG-1β application on diaphragmatic contractility and AChE activity during sepsis. Proinflammatory cytokines, muscle injury biomarkers in serum, contractile force, AChE activity, proinflammatory cytokines, oxidative parameters, histological condition, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and expression of phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB/Akt) signaling proteins in the diaphragm were measured and compared between nonseptic and septic groups with or without NRG-1β treatment. In vitro, the effects of NRG-1β on reactive oxygen species (ROS) production in the lipopolysaccharide- (LPS-) stimulated L6 rat muscle skeletal cells with or without the Akt inhibitor MK-2206 were detected. NRG-1β inhibited proinflammatory cytokine release and muscle injury biomarkers soaring in serum and improved the sepsis-induced diaphragm dysfunction and AChE activity decrease significantly during sepsis. Meanwhile, the inflammatory response, oxidative stress, pathological impairment, and cell apoptosis in the diaphragm were mitigated by NRG-1β. And NRG-1β activated the PI3K/Akt signaling in the diaphragm of septic rats. Elevated ROS production in the LPS-stimulated L6 rat skeletal muscle cells was reduced after treatment with NRG-1β, while MK-2206 blocked these effects of NRG-1β. In conclusion, our findings underlined that NRG-1β could reduce circulating levels of proinflammatory cytokines in rats with sepsis, adjust diaphragmatic proinflammatory cytokine level, mitigate diaphragmatic oxidative injury, and lessen diaphragm cell apoptosis, thereby improving diaphragmatic function, and play a role in diaphragmatic protection by activating PI3K/Akt signaling.

Inhibition of central activation of the diaphragm: a mechanism of weaning failure.

During a T-tube trial following disconnection of mechanical ventilation, patients failing the trial do not develop contractile diaphragmatic fatigue despite increases in inspiratory pressure output. Studies in volunteers, patients, and animals raise the possibility of spinal and supraspinal reflex mechanisms that inhibit central-neural output under loaded conditions. We hypothesized that diaphragmatic recruitment is submaximal at the end of a failed weaning trial despite concurrent respiratory distress. Tidal transdiaphragmatic pressure (ΔPdi) and electrical activity (ΔEAdi) were recorded with esophago-gastric catheters during a T-tube trial in 20 critically ill patients. During the T-tube trial, ∆EAdi was greater in weaning failure patients than in weaning success patients (P = 0.049). Despite increases in ΔPdi, from 18.1 ± 2.5 to 25.9 ± 3.7 cm H2O (P < 0.001), rate of transdiaphragmatic pressure development (from 22.6 ± 3.1 to 37.8 ± 6.7 cm H2O/s; P < 0.0004), and concurrent respiratory distress, ∆EAdi at the end of a failed T-tube trial was half of maximum, signifying inhibition of central neural output to the diaphragm. The increase in ΔPdi in the weaning failure group, while ∆EAdi remained constant, indicates unexpected improvement in diaphragmatic neuromuscular coupling (from 46.7 ± 6.5 to 57.8 ± 8.4 cm H2O/%; P = 0.006). Redistribution of neural output to the respiratory muscles characterized by a progressive increase in rib cage and accessory muscle contribution to tidal breathing and expiratory muscle recruitment contributed to enhanced coupling. In conclusion, diaphragmatic recruitment is submaximal at the end of a failed weaning trial despite concurrent respiratory distress. This finding signifies that reflex inhibition of central neural output to the diaphragm contributes to weaning failure.NEW & NOTEWORTHY Research into pathophysiology of failure to wean from mechanical ventilation has excluded several factors, including contractile fatigue, but the precise mechanism remains unknown. We recorded transdiaphragmatic pressure and diaphragmatic electrical activity in patients undergoing a T-tube trial. Diaphragmatic recruitment was submaximal at the end of a failed trial despite concurrent respiratory distress, signifying that inhibition of central neural output to the diaphragm is an important mechanism of weaning failure.

PERSONALIZED INTENSIVE CARE OF CARDIOVASCULAR DISORDERS IN CHILDREN WITH RESPIRATORY FAILURE

PERSONALIZED INTENSIVE CARE OF CARDIOVASCULAR DISORDERS IN CHILDREN WITH RESPIRATORY FAILURE

Feasibility of neurally synchronized and proportional negative pressure ventilation in a small animal model.

RationaleSynchronized positive pressure ventilation is possible using diaphragm electrical activity (EAdi) to control the ventilator. It is unknown whether EAdi can be used to control negative pressure ventilation.AimTo evaluate the feasibility of using EAdi to control negative pressure ventilation.MethodsFourteen anesthetized rats were studied (380–590 g) during control, resistive breathing, acute lung injury or CO2 rebreathing. Positive pressure continuous neurally adjusted ventilatory assist (cNAVAP+) was applied via intubation. Negative pressure cNAVA (cNAVAP−) was applied with the animal placed in a sealed box. In part 1, automatic stepwise increments in cNAVA level by 0.2 cmH2O/µV every 30 s was applied for cNAVAP+, cNAVAP−, and a 50/50 combination of the two (cNAVAP±). In part 2: During 5‐min ventilation with cNAVAP+ or cNAVAP− we measured circuit, box, and esophageal (Pes) pressure, EAdi, blood pressure, and arterial blood gases.ResultsPart 1: During cNAVAP+, pressure in the circuit increased with increasing cNAVA levels, reaching a plateau, and similarly for cNAVAP−, albeit reversed in sign. This was associated with downregulation of the EAdi. Pes swings became less negative with cNAVAP+ but, in contrast, Pes swings were more negative during increasing cNAVAP− levels. Increasing the cNAVA level during cNAVAP± resulted in an intermediate response. Part 2: no significant differences were observed for box/circuit pressures, EAdi, blood pressure, or arterial blood gases. Pes swings during cNAVAP− were significantly more negative than during cNAVAP+.ConclusionNegative pressure ventilation synchronized and proportional to the diaphragm activity is feasible in small animals.

Sex-specific vagal and spinal modulation of breathing with chest compression.

Lung volume is modulated by sensory afferent feedback via vagal and spinal pathways. The purpose of this study was to systematically alter afferent feedback with and without a mechanical challenge (chest compression). We hypothesized that manipulation of afferent feedback by nebulization of lidocaine, extra-thoracic vagotomy, or lidocaine administration to the pleural space would produce differential effects on the motor pattern of breathing during chest compression in sodium pentobarbital anesthetized rats (N = 43). Our results suggest that: 1) pulmonary stretch receptors are not the sole contributor to breathing feedback in adult male and female rats; 2) of our manipulations, chest compression had the largest effect on early expiratory diaphragm activity (“yield”); 3) reduction of spinally-mediated afferent feedback modulates breathing patterns most likely via inhibition; and 4) breathing parameters demonstrate large sex differences. Compared to males, female animals had lower respiratory rates (RR), which were further depressed by vagotomy, while chest compression increased RR in males, and decreased yield in females without changing RR. Collectively, our results suggest that balance between tonic vagal inhibition and spinal afferent feedback maintains breathing characteristics, and that it is important to specifically evaluate sex differences when studying control of breathing.

The carotid bodies exercise control over active expiration but not peak performance during high-intensity treadmill running.

The carotid bodies exercise control over active expiration but not peak performance during high-intensity treadmill running.

Effect of different mechanical ventilation modes on patient-ventilator synchrony and diaphragm function in rabbit model of acute respiratory distress syndrome

Objective: To observe the effect of different modes of mechanical ventilation on patient-ventilator synchrony and diaphragm function in rabbits with acute respiratory distress syndrome(ARDS). Methods: Eighteen New Zealand rabbit models of ARDS were induced by intratracheal infusion hydrochloric acid until the oxygenation index (PaO(2)/FiO(2)) was less than 200 mmHg, and then divided into three groups with random number: assisted-controlled mechanical ventilation (A/C) group, pressure support ventilation (PSV) group and neurally adjusted ventilatory assist (NAVA) group. All of them were ventilated for four hours with the targeted tidal volume (V(T)) (6 ml/kg) and the positive end-expiratory pressure (PEEP) titrated with the maximum oxygenation method. Gas exchange, pulmonary mechanics and patient-ventilator synchrony were determined during 4 h of ventilation and the concentrations of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) in diaphragm were measured after 4 h of ventilation. The q test was used for the multiple comparison of the sample mean. Results: There were no significant differences in PaO(2)/FiO(2) between three groups during ventilation 1-4 h (F=1.029, P>0.05). The V(T) in NAVA group was obviously lower than that in PSV group and the respiratory rate (RR) and the electrical activity of diaphragm(EAdi) were higher than those in A/C group(all P<0.05).The trigger delay and off cycle delay the in NAVA group were markedly lower than those in A/C and PSV group during ventilation 1-4 h(F=14.312, 9.342, both P<0.05). Asynchrony index in NAVA group (3.1%±1.0%) was obviously lower than those in A/C group (22.3%±5.2%) and PSV group(8.4%±2.3%) (F=7.192, P<0.05). In NAVA group, peak EAdi (EAdi(peak)) and peak airway pressure (Ppeak) were markedly correlated (r=0.97±0.16, P<0.05), while Ppeak delivery in A/C and PSV group was not correlated to EAdi(peak) (r=0.38±0.13,0.46±0.15, both P>0.05).Compared with A/C group, the concentration of MDA in the diaphragm in NAVA group was obviously lower(P<0.05). SOD and GSH level inthe diaphragm in NAVA group were both obviously higher than those in A/C group (both P<0.05). Conclusions: It is helpful to avoid eccentric contraction of diaphragm, lessen oxidative stress and alleviate ventilator-related diaphragm dysfunction by keeping spontaneous breathing as far as possible and subject-ventilator synchrony when ventilation in ARDS with NAVA.

Neurally Adjusted Ventilatory Assist in Difficult Weaning: Promising Findings on a Prickly Issue.

Neurally Adjusted Ventilatory Assist in Difficult Weaning: Promising Findings on a Prickly Issue.

Recruitment pattern of the diaphragm and extradiaphragmatic inspiratory muscles in response to different levels of pressure support

BackgroundInappropriate ventilator assist plays an important role in the development of diaphragm dysfunction. Ventilator under-assist may lead to muscle injury, while over-assist may result in muscle atrophy. This provides a good rationale to monitor respiratory drive in ventilated patients. Respiratory drive can be monitored by a nasogastric catheter, either with esophageal balloon to determine muscular pressure (gold standard) or with electrodes to measure electrical activity of the diaphragm. A disadvantage is that both techniques are invasive. Therefore, it is interesting to investigate the role of surrogate markers for respiratory dive, such as extradiaphragmatic inspiratory muscle activity. The aim of the current study was to investigate the effect of different inspiratory support levels on the recruitment pattern of extradiaphragmatic inspiratory muscles with respect to the diaphragm and to evaluate agreement between activity of extradiaphragmatic inspiratory muscles and the diaphragm.MethodsActivity from the alae nasi, genioglossus, scalene, sternocleidomastoid and parasternal intercostals was recorded using surface electrodes. Electrical activity of the diaphragm was measured using a multi-electrode nasogastric catheter. Pressure support (PS) levels were reduced from 15 to 3 cmH2O every 5 min with steps of 3 cmH2O. The magnitude and timing of respiratory muscle activity were assessed.ResultsWe included 17 ventilated patients. Diaphragm and extradiaphragmatic inspiratory muscle activity increased in response to lower PS levels (36 ± 6% increase for the diaphragm, 30 ± 6% parasternal intercostals, 41 ± 6% scalene, 40 ± 8% sternocleidomastoid, 43 ± 6% alae nasi and 30 ± 6% genioglossus). Changes in diaphragm activity correlated best with changes in alae nasi activity (r2 = 0.49; P < 0.001), while there was no correlation between diaphragm and sternocleidomastoid activity. The agreement between diaphragm and extradiaphragmatic inspiratory muscle activity was low due to a high individual variability. Onset of alae nasi activity preceded the onset of all other muscles.ConclusionsExtradiaphragmatic inspiratory muscle activity increases in response to lower inspiratory support levels. However, there is a poor correlation and agreement with the change in diaphragm activity, limiting the use of surface electromyography (EMG) recordings of extradiaphragmatic inspiratory muscles as a surrogate for electrical activity of the diaphragm.

Assessing Diaphragmatic Function.

The diaphragm is vulnerable to injury during mechanical ventilation, and diaphragm dysfunction is both a marker of severity of illness and a predictor of poor patient outcome in the ICU. A combination of factors can result in diaphragm weakness. Both insufficient and excessive diaphragmatic contractile effort can cause atrophy or injury, and recent evidence suggests that targeting an appropriate amount of diaphragm activity during mechanical ventilation has the potential to mitigate diaphragm dysfunction. Several monitoring tools can be used to assess diaphragm activity and function during mechanical ventilation, including pressure-derived parameters, electromyography, and ultrasound. This review details these techniques and presents the rationale for a diaphragm-protective ventilation strategy.

Surface respiratory electromyography and dyspnea in acute heart failure patients.

Dyspnea is the most common symptom among hospitalized patients with heart failure (HF) but besides dyspnea questionnaires (which reflect the subjective patient sensation and are not fully validated in HF) there are no measurable physiological variables providing objective assessment of dyspnea in a setting of acute HF patients. Studies performed in respiratory patients suggest that the measurement of electromyographic (EMG) activity of the respiratory muscles with surface electrodes correlates well with dyspnea. Our aim was to test the hypothesis that respiratory muscles EMG activity is a potential marker of dyspnea severity in acute HF patients. Prospective and descriptive pilot study carried out in 25 adult patients admitted for acute HF. Measurements were carried out with a cardio-respiratory portable polygraph including EMG surface electrodes for measuring the activity of main (diaphragm) and accessory (scalene and pectoralis minor) respiratory muscles. Dyspnea sensation was assessed by means of the Likert 5 questionnaire. Data were recorded during 3 min of spontaneous breathing and after breathing at maximum effort for several cycles for normalizing data. An index to quantify the activity of each respiratory muscle was computed. This assessment was carried out within the first 24 h of admission, and at day 2 and 5. Dyspnea score decreased along the three measured days. Diaphragm and scalene EMG index showed a positive and significant direct relationship with dyspnea score (p<0.001 and p = 0.003 respectively) whereas pectoralis minor muscle did not. In our pilot study, diaphragm and scalene EMG activity was associated with increasing severity of dyspnea. Surface respiratory EMG could be a useful objective tool to improve assessment of dyspnea in acute HF patients.

Swallow Motor Pattern Is Modulated by Fixed or Stochastic Alterations in Afferent Feedback.

Afferent feedback can appreciably alter the pharyngeal phase of swallow. In order to measure the stability of the swallow motor pattern during several types of alterations in afferent feedback, we assessed swallow during a conventional water challenge in four anesthetized cats, and compared that to swallows induced by fixed (20 Hz) and stochastic (1-20Hz) electrical stimulation applied to the superior laryngeal nerve. The swallow motor patterns were evaluated by electromyographic activity (EMG) of eight muscles, based on their functional significance: laryngeal elevators (mylohyoid, geniohyoid, and thyrohyoid); laryngeal adductor (thyroarytenoid); inferior pharyngeal constrictor (thyropharyngeus); upper esophageal sphincter (cricopharyngeus); and inspiratory activity (parasternal and costal diaphragm). Both the fixed and stochastic electrical stimulation paradigms increased activity of the laryngeal elevators, produced short-term facilitation evidenced by increasing swallow durations over the stimulus period, and conversely inhibited swallow-related diaphragm activity. Both the fixed and stochastic stimulus conditions also increased specific EMG amplitudes, which never occurred with the water challenges. Stochastic stimulation increased swallow excitability, as measured by an increase in the number of swallows produced. Consistent with our previous results, changes in the swallow motor pattern for pairs of muscles were only sometimes correlated with each other. We conclude that alterations in afferent feedback produced particular variations of the swallow motor pattern. We hypothesize that specific SLN feedback might modulate the swallow central pattern generator during aberrant feeding conditions (food/liquid entering the airway), which may protect the airway and serve as potentially important clinical diagnostic indicators.

GABAergic neurons of the medullary raphe regulate active expiration during hypercapnia.

The parafacial respiratory group (pFRG), located in the lateral aspect of the rostroventral lateral medulla, has been described as a conditional expiratory oscillator that emerges mainly in conditions of high metabolic challenges to increase breathing. The convergence of inhibitory and excitatory inputs to pFRG and the generation of active expiration may be more complex than previously thought. We hypothesized that the medullary raphe, a region that has long been described to be involved in breathing activity, is also responsible for the expiratory activity under hypercapnic condition. To test this hypothesis, we performed anatomical and physiological experiments in urethane-anesthetized adult male Wistar rats. Our data showed anatomical projections from serotonergic (5-HT-ergic) and GABAergic neurons of raphe magnus (RMg) and obscurus (ROb) to the pFRG region. Pharmacological inhibition of RMg or ROb with muscimol (60 pmol/30 nL) did not change the frequency or amplitude of diaphragm activity and did not generate active expiration. However, under hypercapnia (9-10% CO2), the inhibition of RMg or ROb increased the amplitude of abdominal activity, without changing the increased amplitude of diaphragm activity. Depletion of serotonergic neurons with saporin anti-SERT injections into ROb and RMg did not increase the amplitude of abdominal activity during hypercapnia. These results show that the presumably GABAergic neurons within the RMg and ROb may be the inhibitory source to modulate the activity of pFRG during hypercapnia condition.NEW & NOTEWORTHY Medullary raphe has been involved in the inspiratory response to central chemoreflex; however, these reports have never addressed the role of raphe neurons on active expiration induced by hypercapnia. Here, we showed that a subset of GABA cells within the medullary raphe directly project to the parafacial respiratory region, modulating active expiration under high levels of CO2.

Phrenic motor neuron activation using temporal interference stimulation

Based on deep brain stimulation data, we are using a method called temporal interference (T‐I) stimulation to activate phrenic motor neurons in adult rats. The premise is to target the deep ventral motor pools of the spinal cord with two high frequency but low amplitude electrical waveforms. The waveforms are delivered at kilohertz frequencies that are well above values that directly stimulate neurons, and amplitudes insufficient to activate neurons until the electric field combine in the tissue. The frequency of the two waveforms is offset by a small amount (e.g. 1–5 Hz), and where the two electrical fields combine to form a difference‐frequency time‐modulated signal in the tissue, neuronal populations are recruited. This work tested the hypotheses that 1) T‐I stimulation using epidural electrodes can activate phrenic motoneurons via direct depolarization, and 2) T‐I stimulation using percutaneously electrodes in the neck can activate the diaphragm and sustain breathing after opioid overdose. For epidural stimulation, electrodes were placed at C3 and experiments were conducted in ventilated, urethane anesthetized adult rats. Initial experiments showed that a pair of electrodes, one medial and one lateral, over the left hemicord was sufficient to enable diaphragm activation via T‐I. Intraspinal delivery of CNQX and AP5 was used to block excitatory synaptic inputs to phrenic motoneurons, confirmed by a complete cessation of endgoenous diaphragm activity. Under these conditions, epidural T‐I (5000Hz and 5001Hz 360–1800μA) could activate the diaphragm, but the response was attenuated by 50–75% as compared to pre‐drug conditions. Blocking inhibitory synaptic inputs using strychnine and bicuculline increased the impact of epidural T‐I by 100–170%. Opioid overdose (fentanyl, 30mcg/kg) in spontaneously breathing rats produced severe hypoventilation and mortality in 4 of 4 cases. However, when T‐I stimulation was given via intramuscular wires placed in the neck (5000Hz and 5001Hz 8–10mA), 4 of 4 rats survived the fentanyl overdose and were able to eventually resume spontaneous breathing. Ongoing efforts by our group are aimed at understanding the flow of electrical current in the spinal cord during epidural and percutaneous delivery to T‐I. To this end, in silico models of spinal cord stimulation are being developed using the Sim4Life software (image‐based electromagnetic and neural simulations in a rat anatomical model with detailed spinal cord structures). The modeling data are intended to help refine the electrode design, placement, and stimulation parameters, to minimize off‐target muscle activation. At this time, we conclude that 1) epidural T‐I of the cervical spinal cord can activate phrenic motoneurons by a combination of pre‐ and post‐synaptic inputs, and 2) percutaneous T‐I is capable of producing airflow which can sustain life through opioid overdose.Support or Funding InformationF31 HL145831‐01 (MDS), R21‐NS109571 (DDF)

Sympathetic Preganglionic Neurons (SPNs): a Promising Target to Regain Cardiorespiratory Control in Spinal Cord Injured (SCI) Patients

BackgroundThe consequences of spinal cord injuries (SCI) are massive and become more severe with higher levels of injury. Lesions located on cervical portions, specifically between C1‐C3 cause an interruption of the descending bulbospinal pathways. These pathways include a key component of the neural control of respiration, linking the brainstem breathing rhythm generator with phrenic motor neurons that activate the diaphragm. Disruption to these pathways result in respiratory muscle paresis and/or paralysis, requiring mechanical ventilation. Respiratory complications, such as pneumonia and atelectasis complicate the process of weaning patients from ventilators, contributing to patient mortality. Previous data collected in the Wilson lab demonstrated that sympathetic preganglionic neurons (SPNs) located at the thoracic spinal cord have remarkable oxygen and pressure‐sensitive capabilities. Here, we hypothesized that SPNs can stimulate phrenic motor neurons independent of the brainstem.AimTo study the novel direct influence of SPNs on the phrenic motor nucleus.MethodsAn in situ artificially‐perfused cervico‐thoracic rat spinal cord preparation was used to record phrenic and splanchnic nerve activity while having the cervical and thoracic spinal cord separately perfused. This approach allowed us to selectively expose the thoracic segment to acute hypoxia (30 Torr; 10 min) in order to observe if cardiorespiratory responses elicited by the SPN’s could stimulate phrenic motor neurons located at the cervical spinal cord. Additionally, we recorded the electromyography activity from the diaphragm during hypoxia and ischemia (0 mmHg; 10 min).ResultsThe results demonstrated that stimulation of the SPNs with hypoxia evoked phrenic nerve and diaphragm activity. This indicates that even without inputs from the brainstem, SPNs are responsive to hypoxia and ischemia and might have capability to stimulate phrenic motor neurons.ConclusionThe implications of these findings point to the thoracic SPNs as a unique and promising target to regain cardiorespiratory control in SCI patients. Further pharmacological investigations may reveal effective treatment strategies to improve life quality and reduce mortality of SCI patients.Support or Funding InformationAlberta Innovates Health Solutions (LL), Eyes High (NB), NSERC (HY), Canadian Institute of Health Research (RW).

Development of a Phrenic Nerve Injury Model in Rats to Study Axonal Regeneration and Compensatory Respiratory Neuroplasticity

Phrenic nerve injury (PNI) is an unintended consequence of some clinical procedures, including heart and lung transplants. Injury can occur in multiple ways, including an accidental stretching, heating via an electrocautery device or (less often) crushing the phrenic nerve. These types of phrenic nerve damage can severely impair function of the ipsilateral diaphragm, causing respiratory impairments. Currently, the strategy to resolve phrenic nerve injuries is to wait for spontaneous recovery, leaving patients compromised for prolonged periods. Thus, there is a need to study PNI and mechanisms of recovery to guide new treatments that would accelerate this process. When studying regrowth after peripheral nerve injuries, it is essential to report complete and sustained loss of axons and, therefore, function. Although nerve crush injury models have been described, there is high variability in the completeness of injury between published methods. Thus, the purpose of this study was to: 1) develop a ventral approach to perform reproducible PNI; 2) identify a surgical tool that produces a robust and persistent loss of phrenic nerve and ipsilateral diaphragm muscle function; and 3) monitor acute compensatory responses in diaphragm and intercostal muscle activities. We monitored the functional extent of phrenic nerve crush injury via ipsilateral diaphragm EMG recordings in anesthetized, spontaneously breathing rats (n=6). Since we are also interested in mechanisms of compensation immediately following injury, we monitored compensatory responses in contralateral diaphragm EMGs (n=6) and bilateral intercostal muscle EMGs (n=1). We performed the nerve crush on the right phrenic nerve since: 1) the right phrenic nerve has ~20% more axons than the left (Song et al., 1999); and 2) during human lung transplantation, the right phrenic nerve is injured more often (Sheridan et al., 1995). PNI induced with a serrated, micro‐mosquito hemostat crush (n=1) resulted in spared nerve activity, as shown by an initial decrease in phrenic activity followed by immediate recovery of 50% compared to baseline. PNI induced with Dumont 3C forceps (n=5) induced a sustained decrease in phrenic activity (&gt;1 hour). A major goal of this project is to study variability in compensation from spared nerve activity/function. Since it is well documented that the contralateral diaphragm compensates for loss of ipsilateral diaphragm function, we were interested in mechanisms of compensation during a PNI. We observed an average increase of 15% in activity in the contralateral diaphragm immediately after phrenic nerve crush (n=3). Additionally, we observed a 30% increase in ipsilateral intercostal activity (n=1). Arterial blood samples taken 2, 30, and 60 minutes post‐crush (n=3) indicated increasing PaCO2 over time post‐PNI (~55 mmHg immediately post‐crush to ~70 mmHg one hour post‐crush). Increasing PaCO2 may recruit silent but spared neural pathways in the affected nerve and/or recruit activities in the contralateral diaphragm and accessory muscles. This model of PNI will enable studies concerning cellular mechanisms promoting phrenic nerve regeneration post‐injury, and new ways to accelerate that growth.Support or Funding InformationNIH R01 HL148030, T32 HL134621 (MNK), and the UF McKnight Brain Institute.

Respiratory Muscle Activity during Maximal Efforts in ALS Patients and Healthy Controls

Amyotrophic lateral sclerosis (ALS) is a type of neurodegenerative disease involving upper and lower motor neuron loss. ALS patients usually die of respiratory failure due to breathing muscle weakness from motor neuron death. It is important to better understand how breathing changes in ALS to design effective treatments to target breathing insufficiency. Breathing capacity is preserved until late stages of disease in the SOD1G93A rodent model of ALS. Possible mechanisms preserving breathing in ALS rats include: 1) spinal synaptic plasticity; 2) neuromuscular junction plasticity; and 3) transition in balance from more affected muscles (eg. diaphragm) to less affected accessory respiratory muscles. If these mechanisms also exist in humans with ALS, we predict that characteristic patterns of altered respiratory muscle activity would precede ventilatory failure, assessed via traditional pulmonary function tests. Thus, using surface EMGs (sEMG) in ALS patients, we tested the hypothesis that ALS patients exhibit differential recruitment patterns of inspiratory muscle activity during a maximal inspiratory pressure (MIP) test versus healthy age‐ and sex‐ matched controls. EMG electrodes (Bagnoli surface electrode, Delsys, Massachusetts, USA) were bilaterally placed on the diaphragm, 2nd parasternal, scalene and sternocleidomastoid muscles. Subjects (8 patients and 8 controls) were instructed to perform the MIP test. The test was repeated until 3 measurements were obtained within 10% variability. RMS EMG amplitude of MIP testing was calculated using a 100ms sliding window with MATLAB v.9.2., and relationships between MIP and EMG activity were evaluated using a linear regression. A moderate relationship (r=0.65) was seen between parasternal muscle activity and MIP in ALS patients, while the controls showed moderate correlations between neck muscle activity (sternocleidomastoid and scalene) and MIP (rSCM= 0.56 and rscalene=0.69). Moreover, MIP‐generated parasternal (r=0.76) and diaphragm (r=0.7) activity corresponded strongly with disease severity of patients, as measured by the ALS‐FRS scale. Interestingly, we did not see a clear relationship between diaphragm surface EMG activity and MIP in either patients or controls. Several factors could account for this, including the use of surface electrodes and the mass‐effort nature of the MIP test. Further analyses are underway to assess respiratory muscle activity during other inspiratory challenges and compare the amount of sEMG output distribution between controls and ALS patients. These preliminary results support the idea that ALS patients use a different pattern of muscle activity during maximal breathing effort from controls.Support or Funding InformationSupported by: UF Clinical and Translational Science Institute (supported byNIH UL1TR001427), UF “Moonshot” grant initiative, and UF McKnight Brain Institute

Selective optogenetic stimulation of RTN neurons drives ventilation and active expiration in unanesthetized rats

BackgroundThe retrotrapezoid nucleus (RTN) consists of neurons that are chemosensitive (i.e., CO2/pH sensitive), glutamatergic, Phox2b+ and Neuromedin B+ (Nmb+). Lentiviral vectors currently used to transduce RTN neurons with various opto‐ or pharmacogenetic actuators contain a Phox2b‐activated promoter, PRSx8. When injected into the RTN, these vectors also transduce substantial numbers of nearby C1 and catecholaminergic neurons. The purpose of the present study was to determine the effects on breathing elicited by selectively activating RTN neurons.MethodsTo selectively transduce RTN neurons we microinjected a mixture of Lenti‐PRSx8‐ChR2‐mCherry and AAV5‐FLEX‐taCasP3‐TEVp into the RTN of adult TH‐Cre rats. One month after the surgery, RTN neurons were stimulated with blue light (473 nm, 10 ms pulse, 20 Hz, for 20 s). All the measurements were conducted inside a plethysmography chamber. Diaphragm (DIA) and abdominal (ABD) muscle activity were recorded in conscious conditions. Results (mean ± SD), were analyzed using one‐ or two‐way ANOVA as required and differences were considered significant at P&lt;0.05.ResultsRTN neurons (Nmb+, TH−) were selectively transduced (96.8 % ± 4) with the PRSx8 vector, the FLEX‐caspase AAV5 having caused the apoptotic death of C1 neurons (TH+) in the area where neurons were transduced with ChR2. Selective stimulation of RTN (NMB+ neurons; n=6) increased respiratory rate during wake and NREM sleep but not during REM sleep (Δ 30 ± 7 vs. 22.7 ± 6 vs. 4 ± 4 bpm from baseline), increased tidal volume also during quiet waking and non‐REM but not REM sleep (Δ 0.15 ± 0.07 vs. 0.13 ± 0.04 vs. 0.02 ± 0.007 mL/100g) and VE also during wake and non‐REM sleep but not REM (27.8 ± 11 vs. 21.4 ± 8 vs. 3.1 ±1 mL/100g/min). Selective RTN stimulation in rats breathing room air elicited active expiration (from 8.4 ± 7 to 15.9 ± 8 % of normalized abdominal EMG recorded at 9% FiCO2, n=3). RTN stimulation produced greater active expiration in rats exposed to 6% FiCO2 (from 56.3 ± 32 to 125.3 ± 104 % of normalized abdominal EMG).ConclusionsSelective RTN neuron stimulation drives breathing amplitude and frequency during wake, NREM but not in REM sleep, and elicits active expiration, especially at elevated levels of FiCO2.Support or Funding InformationSupported by grants from the National Institutes of Health (HL28785, 18 HL074011) and the Congenital Central Hypoventilation Syndrome Foundation to PGG.

Neural control of respiratory musculature in the mdx mouse model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an inherited fatal neuromuscular disease. Despite deficits in respiratory muscle performance being well recognised, there is a paucity of knowledge in respect of the neural control of breathing in dystrophinopathies. We sought to examine neural control of the diaphragm and upper airway musculature in dystrophin deficient mdx mice.Wild‐type (WT) and mdx mice were studied to examine diaphragm and genioglossus (GG) EMG activity using fine concentric needle electrodes in urethane (1.7mg/kg) anaesthetized spontaneously breathing mice. Basal EMG and responsiveness to chemostimulation were determined. In separate studies, phrenic and hypoglossal (XII) motor output were assessed in anaesthetized, vagotomized, paralyzed and mechanically ventilated WT and mdx mice.Diaphragm and upper airway (sternohyoid) muscle form and function were assessed. Monoamine concentrations, density of glial cells, and pro‐inflammatory gene expression were determined in the spinal cord (C3–C5) and brainstem. Diaphragm and GG EMG activities were depressed in mdx compared to WT during baseline and hypoxic‐hypercapnic challenge. Phrenic and XII nerve recordings revealed enhanced neural drive to diaphragm and GG in mdx mice during hypoxic‐hypercapnic challenge. Excitatory monoamine concentrations were elevated in mdx spinal cord (noradrenaline and serotonin) and brainstem (serotonin). Putative phrenic and hypoglossal motor nuclei in mdx mice had unaltered expression of microglia and astrocytes compared to WT. NFkB and GFAP mRNA expression were increased in mdx brainstem compared to WT, while GFAP mRNA was reduced in mdx spinal cord.Our study revealed a potentiated neural drive in phrenic and XII motor pathways in mdx mice during chemoactivation, suggesting plasticity in motor pathways facilitating breathing. Depressed EMG activity despite potentiated neural outputs, suggests compromised neuromuscular transmission, which we have also observed in respiratory EMG recordings during protracted airway obstruction (Burns et al., 2019, J Physiol). Studies of the fundamental control of breathing in models of DMD should provide a comprehensive vista of the disease, with the potential to identify novel therapeutic targets.