Diaphragmatic Activity Research Articles

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

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

Prodromal Physiologic Changes During Copper Sulfate‐Induced Nausea and Vomiting: Potential Use of Intestinal EMG Recordings as a Marker for Nausea

Although emetic responses are present during many disease states, there is no consensus regarding the physiologic changes that signal the onset of nausea. In this study, 10 adult felines (6 males and 4 females) were instrumented to chronically record, during the awake state, the electrocardiogram, diaphragm and abdominal muscle electromyographic (EMG) activity, and EMG activity from the stomach and duodenum before and after the gavage or intragastric infusion of saline or copper sulfate (CuSO4, doses ranging from 83 to 322 mg). Intragastric CuSO4 is a prototypical emetic stimulus that triggers emesis primarily by activating a gastrointestinal vagus nerve‐to‐brain pathway. CuSO4 infusion elicited a significant increase in heart rate, decrease in respiratory rate, and a disruption in gastric (baseline~6 CPM, cycles‐per‐minute) and intestinal (baseline~20 CPM) EMG activity several minutes prior to vomiting (see Fig. 1). The change in EMG activity was most consistent in the intestine. Administration of saline did not induce these physiologic changes. Increasing the dose of CuSO4 did not alter the physiologic changes induced by the treatment. In some animals, a second bout of vomiting occurred following the first episode, when prodromal changes of the intestinal EMG were less pronounced. It is postulated that the intestinal EMG activity was related to a retrograde movement of chyme from the intestine to the stomach. These findings suggest that monitoring of intestinal EMG activity may be the best indicator of the onset of nausea following treatments and in disease conditions associated with emesis.

Accessory Muscle Support of Breathing in Young Dystrophin‐deficient mdx Mice

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder that occurs due to the absence of the structural protein dystrophin and secondary consequences. DMD is characterized by severe and progressive skeletal muscle weakness, which extends to the respiratory muscles. In young dystrophin‐deficient mdx mice, peak inspiratory pressure‐generating capacity is preserved despite diaphragm muscle weakness and reduced electromyogram (EMG) activitya. Our overarching hypothesis is that accessory muscle compensation limits ventilatory deficit in early dystrophic disease. Four‐month‐old male wild‐type (n=39) and mdx (n=41) mice were studied. Breathing was assessed in conscious mice using whole body plethysmography during normoxia and chemochallenge (10% O2/6% CO2). Thoracic oesophageal pressure and obligatory (diaphragm and external intercostal) respiratory muscle EMG activities were recorded in urethane (1.7g/kg i.p.) anaesthetised spontaneously breathing mice during baseline and sustained tracheal occlusion (maximal muscle activation). Diaphragm and scalene (accessory respiratory muscle) force output were examined ex vivo. Data were statistically compared using unpaired Student’s t test or two‐way ANOVA with Bonferroni post hoc test. Peak diaphragm force ex vivo (P < 0.0001) and baseline diaphragm EMG activity (P = 0.01) were significantly lower in mdx compared with wild‐type. Minute ventilation was equivalent in age‐matched wild‐type and mdx mice during baseline and chemochallenge (P = 0.1). Peak diaphragm (P < 0.0001) and external intercostal (P = 0.01) EMG activities during obstruction were significantly lower in mdx mice compared to wild‐type, while peak inspiratory pressure was equivalent (P = 0.4). Interestingly, peak scalene muscle force ex vivo was preserved in 4‐month‐old mdx mice (P = 0.8). Despite a decline in diaphragm muscle performance (decreased EMG activity and mechanical weakness) at baseline, ventilatory capacity is preserved in mdx mice. Furthermore, despite lower peak electrical activation of the obligatory muscles of breathing and diaphragm muscle functional deficit, peak inspiratory pressure is preserved in 4‐month‐old mdx mice, confirming our previous findings in 2‐month‐old micea. Our finding of preserved scalene muscle force suggests that compensation may be provided by accessory muscles to support ventilatory and non‐ventilatory behaviours in early dystrophic disease. Current studies are examining the EMG activity of accessory respiratory muscles (e.g. scalene, sternomastoid, cleidomastoid and trapezius) in early and advanced dystrophic disease in mdx mice.References a Burns DP, Murphy KH, Lucking EF, O'Halloran KD. Inspiratory pressure‐generating capacity is preserved during ventilatory and non‐ventilatory behaviours in young dystrophic mdx mice despite profound diaphragm muscle weakness. J. Physiol. 2019;597(3):831‐848.

Roles of GABAergic and Glutamatergic Prebötzinger Complex Circuits in Opioid‐Induced Respiratory Depression

Introduction. Opioids are widely prescribed analgesic medication but are highly addictive and can be lethal with overdose. Respiratory depression is the primary cause of death by opioid overdose, and it limits the ability to use opioid analgesics safely and effectively. Respiratory depression by opioids is mediated by the inhibition of neural circuits in the brainstem, such as the preBötzinger Complex (preBötC), a small region of the medulla involved in respiratory rhythm generation. The generation of respiratory rhythm requires the activation of excitatory and inhibitory neurons in the preBötC. Glutamate is a main excitatory neurotransmitter, and preBötC glutamatergic neurons are involved in generating respiratory rhythm. γ‐aminobutyric acid (GABA) is a main inhibitory neurotransmitter, and GABAergic neurons are found in the preBötC. Interestingly, GABAergic neurons often co‐express µ‐opioid receptors, and disinhibition of GABAergic neurons by opioid drugs is involved in opioid addiction and euphoria. Here, we aim to determine the roles of excitatory glutamatergic and inhibitory GABAergic neurons in opioid‐induced respiratory depression.MethodsTo control glutamatergic and GABAergic neurons, we expressed opsin channels in cells expressing the vesicular glutamate transporter 2 (vGLUT2) and the vesicular GABA transporter (vGAT), respectively. Using a cre‐lox recombination approach, we injected into the preBötC cre‐dependent adeno‐associated viruses to locally express the inhibitory archaerhodopsin or the excitatory channelrhodopsin in vGLUT2 or vGAT‐expressing cells. Following a 4‐week incubation period, we applied 554 nm or 470 nm lights through optical fibres focused onto the preBötC while recording diaphragm and genioglossal muscle activities in anesthetized mice. Following systemic injection of fentanyl (5µg/kg), we inhibited or activated glutamatergic or GABAergic cells, respectively.ResultsOur preliminary data indicate that inhibition of vGLUT2 cells severely depressed respiratory rate, mimicking the respiratory depression observed with the opioid fentanyl. We are currently performing optical inhibition of vGAT preBötC cells in anesthetized mice and determining their role in respiratory depression by fentanyl.DiscussionWe will identify the role of GABAergic and glutamatergic preBötC cells in coordinating respiratory rhythm in preBötC cells and whether GABAergic cells contribute to respiratory depression by opioid drugs. Understanding the role of the populations of cells involved in respiratory depression by opioid drugs is critical to identify new molecular targets to reduce respiratory depression and provide safe and reliable pain killers without the risks associated with opioid pain killers.

Prolonged diaphragm activation using closed loop optogenetic stimulation

Diaphragm dysfunction in individuals with late stage neuromuscular disease and cervical spinal cord injury is a substantial problem, often resulting in respiratory insufficiencies and ultimately respiratory failure. Therefore, there is a need to develop new methods that can improve diaphragm function in these clinical populations. Here we used an optogenetic method of activating diaphragm myofibers to assess the feasibility of prolonged photostimulation of the diaphragm. Our hypothesis was that closed loop photostimulation can pace the diaphragm over a prolonged period (1 hour) without a decrease in evoked response magnitude. Wild type mice (C57/bl6, n= 5) received an injection of AAV9‐CAG‐ChR2‐mVenus (34 µl, titer: 1.8x1013 vg/ml) to the intrapleural space between the lung and chest wall to target the diaphragm. After an 8‐18 week incubation period, diaphragm EMG and respiratory airflow were measured under isoflurane anesthesia. A light stimulus was delivered to the inferior diaphragm surface using a high‐power, 472 nm, micro‐LED. Photostimulation was delivered in a closed loop fashion using the onset of the inspiratory EMG burst recorded in intact hemi‐diaphragm to trigger the stimulus. The photostimulation paradigm consisted of a 250 ms, ramping/decrementing train of light pulses (pulse duration = 0.1 ms, interpulse interval = 0.1 ms). After a 10‐minute baseline recording, a unilateral right phrenicotomy was performed to paralyze the right diaphragm. The photostimulation paradigm immediately restored EMG activity to the paralyzed hemi‐diaphragm. Further, the magnitude of the evoked diaphragm EMG bursts stayed consistent over the sixty‐minute period of closed loop photostimulation, and there was no evidence of a decrement in EMG activation. Post‐mortem histology confirmed mVenus expression in diaphragm myofibers. We conclude that stimulation of the diaphragm using optogenetics is effective for up to one hour. This is a first demonstration of prolonged photostimulation of the diaphragm and provides evidence to support the feasibility of this method for the treatment of chronic diaphragm paralysis.

Diaphragm EMG activity during hyperbaric oxygen exposure in spinal injured rats

Cervical spinal cord injury can cause significant respiratory dysfunction and in severe cases necessitates mechanical ventilation. Accumulating evidence indicates that hyperbaric oxygen (HBO) therapy, initiated shortly after spinal cord injury (SCI), can mitigate spinal cord pathology. Further, a recent study from our group demonstrated that HBO therapy can attenuate diaphragm atrophy after SCI. The primary purpose of the present investigation was to evaluate diaphragm electromyogram (EMG) activity during HBO therapy in rats with cervical SCI. HBO therapy has not been widely adopted after SCI, and if HBO is to be translated to human use, we reasoned that it is important to determine how breathing is impacted during HBO exposure. Adult female Sprague‐Dawley rats were implanted with chronic indwelling EMG electrodes in the mid‐costal diaphragm two weeks prior to SCI. Following hemicontusion injury of the C4 spinal cord, one experimental group was treated with HBO (n=3), and another group was left untreated (n=3). The HBO treatment consisted of a one hour exposure to 100% O2 at 3 atmospheres (ATA). In all experiments, diaphragm EMG activity was recorded in freely moving rats, without anesthesia. Recordings were made for 10 minutes prior to treatment, throughout the HBO exposure, and for 10 minutes after. Diaphragm EMG recordings in the normobaric normoxia control group conditions were completed for the same duration of time as the treated group. Daily HBO treatment and EMG recordings were done until 10‐days post‐SCI. Peak diaphragm EMG burst amplitude was quantified before SCI, over the 10 days of treatment, and at 1 and 2 weeks post‐treatment. Prior to SCI, body weight was similar between groups (control: 227±4 g; HBO: 228±3 g). All rats with tolerated HBO treatment with no evidence of adverse events or impact on weight gain over the 10 day period (10 day body weight: control: 198 ± 14 g; HBO: 197 ± 13 g). The acute HBO exposure caused an increase in the area under the curve of the rectified and integrated inspiratory burst amplitude of the diaphragm recording made ipsilateral to the lesion. This increase averaged 126 ± 18% of baseline across the 10 HBO treatments. The ipsilateral EMG burst amplitude also remained elevated at 10 min post‐HBO exposure (115 ± 32%) compared to pre HBO exposure. At the conclusion of the 10 day treatment paradigm, ipsilateral diaphragm EMG output during quiet breathing was elevated in HBO (156 ± 50% of post‐injury baseline) as compared to the control group (111 ± 6% of baseline). However the ipsilateral diaphragm EMG burst amplitude of the two groups were similar when evaluated 2 weeks after the daily HBO sessions concluded. This ongoing study indicates that acute exposure to HBO therapy is associated with an increase in diaphragm EMG activity, and 10 days of HBO therapy accelerates the recovery of diaphragm activation following cervical contusion injury.

Locomotor Training Enhances Respiratory Plasticity in a Pre‐clinical Model of Cervical Spinal Cord Injury

Spinal cord injury (SCI) is a devastating and irreversible injury that impacts thousands of people each year. Most injuries occur at cervical levels, compromising the phrenic motor network, resulting in life‐threatening respiratory deficits. While there is mounting clinical and pre‐clinical evidence for spontaneous plasticity, it is limited, and life‐threatening deficits persist. One strategy for therapeutically enhancing plasticity is the use of activity‐based therapies, such as respiratory and locomotor training. Clinical studies using treadmill training have shown improved respiratory function for individuals with SCI. Therefore, the present work reverse translates these promising clinical findings back to animal models of mid‐cervical SCI to assess the effects of treadmill training on anatomical and functional respiratory plasticity after injury.Adult female Sprague‐Dawley rats (~200g) received a lateralized C3/4 contusive injury. One week post injury, the animals were blindly divided into two groups: untrained or treadmill trained (30 min, once a day, 5 days a week) for 4 weeks. Breathing behavior was measured each week using whole‐body plethysmography. After training completion, all animals were anatomically traced with retrograde and transsynaptic pseudorabies virus (PRV) to the left hemidiaphragm to label the phrenic motor network, ipsilateral to injury. Three days later, animals were anesthetized and diaphragm electromyography (dEMG) was performed to observe ipsilateral diaphragm deficits and respiratory recovery, and animals were perfused for histology. Immunohistological assessment was used to quantify interneuronal connectivity and density of descending serotonergic input to the phrenic motor network. Preliminary data demonstrated increased interneuronal connectivity with injured phrenic motor network, and increased serotonergic input to spinal networks caudal to injury. Electrophysiological assessment of diaphragm function revealed increased diaphragm activity ipsilateral to injury, after 4 weeks of training. Ongoing analyses will further elucidate the therapeutic effects of locomotor training for improving breathing post‐SCI.

NIV-NAVA versus NCPAP immediately after birth in premature infants: A randomized controlled trial

ObjectiveTo evaluate whether noninvasive-neurally adjusted ventilatory assist (NIV-NAVA) decrease respiratory efforts compared to nasal continuous positive airway pressure (NCPAP) during the first hours of life. MethodsTwenty infants born between 28+0 and 31+6 weeks were randomized to NIV-NAVA or NCPAP. Positive end-expiratory pressure was constantly kept at 6 cmH2O for both groups and the NAVA level was 1.0 cmH2O/µV for NIV-NAVA group. The electrical activity of diaphragm (Edi) were recorded for the first two hours. ResultsPeak and minimum Edi decreased similarly in both groups (P = 0.98 and P = 0.59, respectively). Leakages were higher in the NIV-NAVA group than in the NCPAP group (P < 0.001). The neural apnea defined as a flat Edi for ≥ 5 s were less frequent in NIV-NAVA group than in NCPAP group (P = 0.046). ConclusionsImmediately applied NIV-NAVA in premature infants did not reduce breathing effort, measured as peak Edi. However, NIV-NAVA decreased neural apneic episodes compared to NCPAP.

Optogenetic activation of the diaphragm

Impaired diaphragm activation is common in many neuromuscular diseases. We hypothesized that expressing photoreceptors in diaphragm myofibers would enable light stimulation to evoke functional diaphragm activity, similar to endogenous bursts. In a mouse model, adeno-associated virus (AAV) encoding channelrhodopsin-2 (AAV9-CAG-ChR2-mVenus, 6.12 × 1011 vg dose) was delivered to the diaphragm using a minimally invasive method of microinjection to the intrapleural space. At 8–18 weeks following AAV injection, mice were anesthetized and studied during spontaneous breathing. We first showed that diaphragm electromyographic (EMG) potentials could be evoked with brief presentations of light, using a 473 nm high intensity LED. Evoked potential amplitude increased with intensity or duration of the light pulse. We next showed that in a paralyzed diaphragm, trains of light pulses evoked diaphragm EMG activity which resembled endogenous bursting, and this was sufficient to generate respiratory airflow. Light-evoked diaphragm EMG bursts showed no diminution after up to one hour of stimulation. Histological evaluation confirmed transgene expression in diaphragm myofibers. We conclude that intrapleural delivery of AAV9 can drive expression of ChR2 in the diaphragm and subsequent photostimulation can evoke graded compound diaphragm EMG activity similar to endogenous inspiratory bursting.

Indirect Lung Absorbed Dose Verification by 90Y PET/CT and Complete Lung Protection by Hepatic Vein Balloon Occlusion: Proof of Concept.

Postradioembolization lung absorbed dose verification was historically problematic and impractical in clinical practice. We devised an indirect method using 90Y PET/CT. Methods: Conceptually, true lung activity is simply the difference between the total prepared activity minus all activity below the diaphragm and residual activity within delivery apparatus. Patient-specific lung mass is measured by CT densitovolumetry. True lung mean absorbed dose is calculated by MIRD macrodosimetry. Results: Proof of concept is shown in a hepatocellular carcinoma patient with a high lung shunt fraction of 26%, where evidence of technically successful hepatic vein balloon occlusion for radioembolization lung protection was required. Indirect lung activity quantification showed the postradioembolization lung shunt fraction to be reduced to approximately 1% with a true lung mean absorbed dose of approximately 1 Gy, suggesting complete lung protection by hepatic vein balloon occlusion. Conclusion: We discuss possible clinical applications such as lung absorbed dose verification, refining the limits of lung tolerance, and the concept of massive activity radioembolization.

Diaphragmatic Activity and Respiratory Function Following C3 or C6 Unilateral Spinal Cord Contusion in Mice.

Simple SummaryTetraplegia is one of the most devastating conditions that an individual can sustain and affects more than 2.5 million people worldwide. Tetraplegia not only affects mobility but also impacts spontaneous breathing such that survivors can be rendered ventilator dependent and have increased mortality. Although no treatment can restore respiratory function after tetraplegia, there is a need for more exploratory studies defining therapeutic approaches to ameliorate respiratory decline in tetraplegia. Here, we studied two models of tetraplegia mimicking human forms of injury, using spinal cord contusion in mice, either above the third cervical metameric segment (C3 model) or below the sixth cervical metameric segment (C6 model) innervation of the phrenic nerves. These nerves are responsible for contraction of the diaphragm, the main inspiratory muscle. Using measurements of spontaneous breathing and muscle activity of the diaphragm, we found reduced diaphragmatic activity in both models, but only the C3 model led to reduced spontaneous breathing similar to what is seen in humans with tetraplegia. Moreover, we found a decline in basal contractility of the diaphragm in the C3 model only. We conclude that the C3 model is an appropriate model to explore interventions aimed at restoring breathing following tetraplegia.The majority of spinal cord injuries (SCIs) are cervical (cSCI), leading to a marked reduction in respiratory capacity. We aimed to investigate the effect of hemicontusion models of cSCI on both diaphragm activity and respiratory function to serve as preclinical models of cervical SCI. Since phrenic motoneuron pools are located at the C3–C5 spinal level, we investigated two models of preclinical cSCI mimicking human forms of injury, namely, one above (C3 hemicontusion—C3HC) and one below phrenic motoneuron pools (C6HC) in wild-type swiss OF-1 mice, and we compared their effects on respiratory function using whole-body plethysmography and on diaphragm activity using electromyography (EMG). At 7 days post-surgery, both C3HC and C6HC damaged spinal cord integrity above the lesion level, suggesting that C6HC potentially alters C5 motoneurons. Although both models led to decreased diaphragmatic EMG activity in the injured hemidiaphragm compared to the intact one (−46% and −26% in C3HC and C6HC, respectively, both p = 0.02), only C3HC led to a significant reduction in tidal volume and minute ventilation compared to sham surgery (−25% and −20% vs. baseline). Moreover, changes in EMG amplitude between respiratory bursts were observed post-C3HC, reflecting a change in phrenic motoneuronal excitability. Hence, C3HC and C6HC models induced alteration in respiratory function proportionally to injury level, and the C3HC model is a more appropriate model for interventional studies aiming to restore respiratory function in cSCI.

Comparison of ultrasound assessment for diaphragmatic workload during spontaneous breathing trial between automatic tube compensation and pressure support ventilation: Study protocol

Background: The process of weaning from mechanical ventilation is crucial. Less demanding spontaneous breathing trials (SBT) can be done by either automatic tube compensation (ATC) or pressure support ventilation (PSV) to decrease inspiratory effort by endotracheal tube resistance compensation. This study aimed to assess the patient’s effort, by diaphragm ultrasonography with ATC compared to PSV during SBT. Methods: Patients who have been on mechanical ventilation for more than 48 hours and meet the weaning requirements are given 30 minutes for ATC and 30 minutes for PSV in this randomized control experiment. The diaphragm workload difference, as assessed by diaphragm thickness fraction, was the primary outcome. The sensitivity and specificity of ultrasound-measured diaphragmatic muscle activity measures in predicting ventilator weaning and effective extubation were secondary outcomes. Hypothesis: Intubated patients should be (1) weaning with lower effort SBT mode (2) predicted weaning success with more accurately parameters Ethics and dissemination: Ramathibodi Human Research Ethics Committee has approved the trial. The findings plan to summitted in peer-reviewed publications and conferences in critical care medicine or anesthesiology. Trial registration number: TCTR20210317004

Role of Glucocorticoid Signaling and HDAC4 Activation in Diaphragm and Gastrocnemius Proteolytic Activity in Septic Rats.

Sepsis increases glucocorticoid and decreases IGF-1, leading to skeletal muscle wasting and cachexia. Muscle atrophy mainly takes place in locomotor muscles rather than in respiratory ones. Our study aimed to elucidate the mechanism responsible for this difference in muscle proteolysis, focusing on local inflammation and IGF-1 as well as on their glucocorticoid response and HDAC4-myogenin activation. Sepsis was induced in adult male rats by lipopolysaccharide (LPS) injection (10 mg/kg), and 24 h afterwards, rats were euthanized. LPS increased TNFα and IL-10 expression in both muscles studied, the diaphragm and gastrocnemius, whereas IL-6 and SOCS3 mRNA increased only in diaphragm. In comparison with gastrocnemius, diaphragm showed a lower increase in proteolytic marker expression (atrogin-1 and LC3b) and in LC3b protein lipidation after LPS administration. LPS increased the expression of glucocorticoid induced factors, KLF15 and REDD1, and decreased that of IGF-1 in gastrocnemius but not in the diaphragm. In addition, an increase in HDAC4 and myogenin expression was induced by LPS in gastrocnemius, but not in the diaphragm. In conclusion, the lower activation of both glucocorticoid signaling and HDAC4-myogenin pathways by sepsis can be one of the causes of lower sepsis-induced proteolysis in the diaphragm compared to gastrocnemius.

Systemic Administration of Tempol, a Superoxide Dismutase Mimetic, Augments Upper Airway Muscle Activity in Obese Zucker Rats.

Obstructive sleep apnea (OSA) is characterized by repetitive partial/complete collapse of the pharynx during sleep, which results in apnea/hypopnea leading to arterial oxygen desaturations and arousals. Repetitive apnea/hypopnea-arousal episodes cause hypoxia/reoxygenation cycles, which increase free radical generation and oxidative stress that cause motor/sensory nerve impairments and muscle damage. We hypothesize that antioxidants may protect and/or reverse from oxidative stress-induced damage in OSA patients. To understand the acute protective effects of antioxidants on respiratory muscles, we studied the systemic effects of a membrane permeable superoxide dismutase mimetic, Tempol, on genioglossus (EMGGG) and diaphragmatic (EMGDIA) electro-myographic activities, hypoglossal motoneuron (HMN) nerve activity and cardiorespiratory parameters (mean arterial blood pressure, heart rate) in adult isoflurane-anesthetized obese Zucker rats (OZR) and age-matched lean Zucker rats (LZR). Tempol dose-dependently (1–100 mg/kg) increased EMGGG without changing EMGDIA in OZR and LZR. Tempol increased respiratory rate and tidal volume in OZR and LZR. Tempol (1–25 mg/kg) dose-dependently increased HMN nerve activity in healthy Sprague Dawley rats. Tempol (100 mg/kg) increased EMGGG output by 189% in OZR and 163% in LZR. With respect to mechanisms of effect, Tempol (100 mg/kg) did not augment EMGGG after bilateral HMN transection in Sprague Dawley rats. Although future studies are warranted, available data suggest that in addition to its antioxidant and antihypertensive properties, Tempol can selectively augment EMGGG through modulating HMN and this effect may prevent collapsibility and/or improve stability of the upper airway pharyngeal dilator muscles during episodes of partial and/or complete collapse of the upper airway in OSA human subjects.

Rostral-Caudal Effect of Cervical Magnetic Stimulation on the Diaphragm Motor Evoked Potential after Cervical Spinal Cord Contusion in the Rat.

The present study was designed to investigate the rostral-caudal effect of spinal magnetic stimulation on diaphragmatic motor-evoked potentials after cervical spinal cord injury. The diaphragm electromyogram was recorded in rats that received a laminectomy or a left midcervical contusion at the acute (1 day), subchronic (2 weeks), or chronic (8 weeks) injury stages. The center of a figure-eight coil was placed at 30 mm lateral to bregma on the left side, and the effect of magnetic stimulation was evaluated by stimulating the rostral, middle, and caudal cervical regions in spontaneously breathing rats. The results demonstrated that cervical magnetic stimulation induced intensity-dependent motor-evoked potentials in the bilateral diaphragm in both uninjured and contused rats; however, the left diaphragm exhibited a higher amplitude and earlier onset than the right diaphragm. Moreover, the intensity-response curve was shifted upward in the rostral-to-caudal direction of magnetic stimulation, suggesting that caudal cervical magnetic stimulation produced more robust diaphragmatic motor-evoked potentials compared with rostral cervical magnetic stimulation. Interestingly, the diaphragmatic motor-evoked potentials were similar between uninjured and contused rats during cervical magnetic stimulation despite weaker inspiratory diaphragmatic activity in contused rats. In addition, in contused animals but not uninjured animals, diaphragmatic motor-evoked potential amplitudes were greater at the chronic stage than during earlier injury stages. These results demonstrated that cervical magnetic stimulation can excite the residual phrenic motor circuit to activate the diaphragm in the presence of a significant lesion in the cervical spinal cord. These findings indicate that this non-invasive approach is effective for modulating diaphragmatic excitability after cervical spinal cord injury.

Ultrasound-Derived Diaphragm Contractile Reserve as a Marker of Clinical Status in Patients With Cystic Fibrosis.

Introduction: In patients with cystic fibrosis (CF), the monitoring of respiratory muscle activity using electromyography can provide information on the demand-to-capacity ratio of the respiratory system and act as a clinical marker of disease activity, but this technique is not adapted to routine clinical care. Ultrasonography of the diaphragm could provide an alternative, simpler and more widely available alternative allowing the real-time assessment of the diaphragm contractile reserve (DCR), but its relationship with recognized markers of disease severity and clinical outcomes are currently unknown.Methods: Stable patients with CF were prospectively recruited. Diaphragm ultrasound was performed and compared to forced expiratory volume in 1 s (FEV1), residual volume (RV), handgrip strength, fat-free mass index (FFMI), serum vitamin levels, dyspnea levels and rate of acute exacerbation (AE). Diaphragm activity was reported as DCR (the ratio of tidal-to-maximal thickening fractions, representing the remaining diaphragm contractility available after tidal inspiration) and TFmax (representing maximal diaphragm contractile strength). Inter-observer reliability of the measurement of DCR was evaluated using intra-class correlation analysis.Results: 110 patients were included [61 males, median (interquartile range), age 31 (27–38) years, FEV1 66 (46–82)% predicted]. DCR was significantly correlated to FEV1 (rho = 0.46, p < 0.001), RV (rho = −0.46, p < 0.001), FFMI (rho = 0.41, p < 0.001), and handgrip strength (rho = 0.22, p = 0.02), but TFmax was not. In a multiple linear regression analysis, both RV and FFMI were independent predictors of DCR. DCR, but not TFmax, was statistically lower in patients with > 2 exacerbations/year (56 ± 25 vs. 71 ± 17%, p = 0.001) and significantly lower with higher dyspnea levels. A ROC analysis showed that DCR performed better than FEV1 (mean difference in AUROC 0.09, p = 0.04), RV (mean difference in AUROC 0.11, p = 0.03), and TFmax at identifying patients with an mMRC score > 2. Inter-observer reliability of DCR was high (ICC = 0.89, 95% CI 0.84–0.92, p < 0.001).Conclusion: In patients with CF, DCR is a reliable and non-invasive marker of disease severity that is related to respiratory and extra-pulmonary manifestations of the disease and to clinical outcomes. Future studies investigating the use of DCR as a longitudinal marker of disease progression, response to interventions or target for therapy would further validate its translation into clinical practice.

Effects of Dexmedetomidine on Diaphragm Activity Measured by Ultrasonography in Spontaneously Breathing Patients.

Background: Diaphragm movement is well correlated with inspired volume of the lung. Dexmedetomidine (DEX) has less effect on respiratory functions than other sedatives. The objective of this study was to investigate diaphragmatic movement using ultrasound (US) during DEX infusion for sedation in spontaneously breathing patients undergoing unilateral upper limb surgery.Methods: A total of 33 consecutive patients were enrolled in this study. Patients were sedated using DEX with ipsilateral axillary brachial nerve plexus block. Diaphragmatic activity was evaluated using diaphragmatic thickening at end-inspiration (TEI), diaphragmatic thickening at end-expiration (TEE), and diaphragmatic thickening fraction (DTF) measured by diaphragmatic US at three time-points; T0, baseline; T1, after DEX sedation; and T2, after DEX recovery. Supplementary oxygen was applied with a simple mask at 5 L/min. Peripheral oxygen saturation (SpO2), end tidal CO2 (EtCO2), and respiratory rate (RR) were recorded.Results: TEI and TEE showed no significant changes during the study period (P = 0.394 and P = 0.205, respectively). DTF was maintained at both T0 and T1 (P = 1.000). At recovery after DEX infusion discontinued, DTF was increased by 3.85%, although such increase was not statistically significant (T0 vs. T2, P = 0.525). SpO2 remained above 99% and EtCO2 remained below 36 mmHg. Desaturation episodes were not observed during the study period.Conclusions: Results of this study showed that DEX sedation did not affect the diaphragmatic movement in situation of decreased RR induced by DEX. This finding implies that DEX-induced sedation does not result in clinically significant respiratory depression.

Chapter 14 - Phrenic nerve paralysis and phrenic nerve reconstruction surgery

Phrenic nerve injury results in paralysis of the diaphragm muscle, the primary generator of an inspiratory effort, as well as a stabilizing muscle involved in postural control and spinal alignment. Unilateral deficits often result in exertional dyspnea, orthopnea, and sleep-disordered breathing, whereas oxygen or ventilator dependency can occur with bilateral paralysis. Common etiologies of phrenic injuries include cervical trauma, iatrogenic injury in the neck or chest, and neuralgic amyotrophy. Many patients have no identifiable etiology and are considered to have idiopathic paralysis. Diagnostic evaluation requires radiographic and pulmonary function testing, as well as electrodiagnostic assessment to quantitate the nerve deficit and determine the extent of denervation atrophy. Treatment for symptomatic diaphragm paralysis has traditionally been limited. Medical therapies and nocturnal positive airway pressure may provide some benefit. Surgical repair of the nerve injury to restore functional diaphragmatic activity, termed phrenic nerve reconstruction, is a safe and effective alternative to static repositioning of the diaphragm (diaphragm plication), in properly selected patients. Phrenic nerve reconstruction has increasingly become a standard surgical treatment for diaphragm paralysis due to phrenic nerve injury. A multidisciplinary approach at specialty referral centers combining diagnostic evaluation, surgical treatment, and rehabilitation is required to achieve optimal long-term outcomes.

Influence of mild pulmonary congestion on diaphragmatic mobility and activities of daily living in chronic kidney disease: An experimental and clinical study

BackgroundPulmonary congestion is a strong predictor of mortality and cardiovascular events in chronic kidney disease (CKD); however, the effects of the mild form on functionality have not yet been investigated. The objective of this study was to assess the influence of mild pulmonary congestion on diaphragmatic mobility (DM) and activities of daily living (ADL) in hemodialysis (HD) subjects, as well as compare ADL behavior on dialysis and non-dialysis days. In parallel, experimentally induce CKD in mice and analyze the resulting pulmonary and functional repercussions. MethodsThirty subjects in HD underwent thoracic and abdominal ultrasonography, anthropometric assessment, lung and kidney function, respiratory muscle strength assessment and symptoms analysis. To measure ADL a triaxial accelerometer was used over seven consecutive days. Twenty male mice were randomized in Control and CKD group. Thoracic ultrasonography, TNF-α analysis in kidney and lung tissue, exploratory behavior and functionality assessments were performed. ResultsMild pulmonary congestion caused a 26.1% decline in DM (R2=.261; P=.004) and 20% reduction in walking time (R2=.200; P=.01), indicating decreases of 2.23mm and 1.54min, respectively, for every unit increase in lung comet-tails. Regarding ADL, subjects exhibited statistically significant differences for standing (P=.002), walking (P=.034) and active time (P=.002), and number of steps taken (P=.01) on days with and without HD. In the experimental model, CKD resulted in increased levels of TNF-α on kidneys (P=.037) and lungs (P=.02), attenuation of exploratory behavior (P=.01) and significant decrease in traveled distance (P=.034). Thoracic ultrasonography of CKD mice showed presence of B-lines. ConclusionThe mild pulmonary congestion reduced DM and walking time in subjects undergoing HD. Individuals were less active on dialysis days. Furthermore, the experimental model implies that the presence of pulmonary congestion and inflammation may play a decisive role in the low physical and exploratory performance of CKD mice.

The influence of gravity on respiratory kinematics during phonation measured by dynamic magnetic resonance imaging

Respiratory kinematics are important for the regulation of voice production. Dynamic MRI is an excellent tool to study respiratory motion providing high-resolution cross-sectional images. Unfortunately, in clinical MRI systems images can only be acquired in a horizontal subject position, which does not take into account gravitational effects on the respiratory apparatus. To study the effect of body posture on respiratory kinematics during phonation, 8 singers were examined both in an open-configuration MRI with a rotatable gantry and a conventional horizontal MRI system. During dynamic MRI the subjects sang sustained tones at different pitches in both supine and upright body positions. Sagittal images of the respiratory system were obtained at 1–3 images per second, from which 6 anatomically defined distances were extracted to characterize its movements in the anterior, medium and posterior section of the diaphragm as well as the rip cage (diameter at the height of the 3rd and 5th rip) and the anterior–posterior position of the diaphragm cupola. Regardless of body position, singers maintained their general principles of respiratory kinematics with combined diaphragm and thorax muscle activation for breath support. This was achieved by expanding their chest an additional 20% during inspiration when singing in the supine position but not for sole breathing. The diaphragm was cranially displaced in supine position for both singing and breathing and its motion range increased. These results facilitate a more realistic extrapolation of research data obtained in a supine position.