Ruxolitinib: A new hope for ventilator-induced diaphragm dysfunction.

AIM: Mechanical ventilation (MV) results in diminished diaphragm size and strength, termed ventilator-induced diaphragm dysfunction (VIDD). VID increases dependence, prolongs weaning, and increases discharge mortality rates. The Janus kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway is implicated in VIDD, upregulated following MV. JAK/STAT inhibition alleviates chronic muscle wasting conditions. This study aimed to explore the therapeutic potential of Ruxolitinib, an FDA approved JAK1/2 inhibitor (JI) for the treatment of VIDD. METHODS: Rats were subjected to 5 days controlled MV (CMV) with and without daily Ruxolitinib gavage. Muscle fiber size and function were assessed. RNAseq, mitochondrial morphology, respirometry, and mass spectrometry were determined. RESULTS: CMV significantly reduced diaphragm size and specific force by 45% (p < 0.01), associated with a two-fold P-STAT3 upregulation (p < 0.001). CMV disrupted mitochondrial content and reduced the oxygen consumption rate (p < 0.01). Expression of the motor protein myosin was unaffected, however CMV alters myosin function via post-translational modifications (PTMs). Daily administration of JI increased animal survival (40% vs. 87%; p < 0.05), restricted P-STAT3 (p < 0.001), and preserved diaphragm size and specific force. JI was associated with preserved mitochondrial content and respiratory function (p < 0.01), and the reversal or augmentation of myosin deamidation PTMs of the rod and head region. CONCLUSION: JI preserved diaphragm function, leading to increased survival in an experimental model of VIDD. Functional enhancement was associated with maintenance of mitochondrial content and respiration and the reversal of ventilator-induced PTMs of myosin. These results demonstrate the potential of repurposing Ruxolitinib for treatment of VIDD.

Balanced modulation of neuromuscular synaptic transmission via M1 and M2 muscarinic receptors during inhibition of cholinesterases.

Organophosphorus (OP) compounds that inhibit acetylcholinesterase are a common cause of poisoning worldwide, resulting in several hundred thousand deaths each year. The pathways activated during OP compound poisoning via overstimulation of muscarinic acetylcholine receptors (mAChRs) play a decisive role in toxidrome. The antidotal therapy includes atropine, which is a nonspecific blocker of all mAChR subtypes. Atropine is efficient for mitigating depression in respiratory control centers but does not benefit patients with OP-induced skeletal muscle weakness. By using an ex vivo model of OP-induced muscle weakness, we studied the effects of the M1/M4 mAChR antagonist pirenzepine and the M2/M4 mAChR antagonist methoctramine on the force of mouse diaphragm muscle contraction. It was shown that weakness caused by the application of paraoxon can be significantly prevented by methoctramine (1 µM). However, neither pirenzepine (0.1 µM) nor atropine (1 µM) was able to prevent muscle weakness. Moreover, the application of pirenzepine significantly reduced the positive effect of methoctramine. Thus, balanced modulation of neuromuscular synaptic transmission via M1 and M2 mAChRs contributes to paraoxon-induced muscle weakness. It was shown that methoctramine (10 µmol/kg, i.p.) and atropine (50 µmol/kg, i.p.) were equieffective toward increasing the survival of mice poisoned with a 2xLD50 dose of paraoxon.

Spinally delivered ampakine CX717 increases phrenic motor output in adult rats.

Ampakines are synthetic molecules that allosterically modulate AMPA-type glutamate receptors. We tested the hypothesis that delivery of ampakines to the intrathecal space could stimulate neural drive to the diaphragm. Ampakine CX717 (20 mM, dissolved in 10 % HPCD) or an HPCD vehicle solution were delivered via a catheter placed in the intrathecal space at the fourth cervical segment in urethane-anesthetized, mechanically ventilated adult male Sprague-Dawley rats. The electrical activity of the phrenic nerve was recorded for 60-minutes following drug application. Intrathecal application of CX717 produced a gradual and sustained increase in phrenic inspiratory burst amplitude (n = 10). In contrast, application of HPCD (n = 10) caused no sustained change in phrenic motor output. Phrenic burst rate, heart rate, and mean arterial pressure were similar between CX717 and HPCD treated rats. We conclude that intrathecally delivered ampakines can modulate phrenic motor output. This approach may have value for targeted induction of spinal neuroplasticity in the context of neurorehabiliation.

Ampakines Stimulate Diaphragm Activity after Spinal Cord Injury.

Respiratory compromise after cervical spinal cord injury (SCI) is a leading cause of mortality and morbidity. Most SCIs are incomplete, and spinal respiratory motoneurons as well as proprio- and bulbospinal synaptic pathways provide a neurological substrate to enhance respiratory output. Ampakines are allosteric modulators of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are prevalent on respiratory neurons. We hypothesized that low dose ampakine treatment could safely and effectively increase diaphragm electromyography (EMG) activity that has been impaired as a result of acute- or sub-acute cervical SCI. Diaphragm EMG was recorded using chronic indwelling electrodes in unanesthetized, freely moving rats. A spinal hemi-lesion was induced at C2 (C2Hx), and rats were studied at 4 and 14 days post-injury during room air breathing and acute respiratory challenge accomplished by inspiring a 10% O2, 7% CO2 gas mixture. Once a stable baseline recording was established, one of two different ampakines (CX717 or CX1739, 5 mg/kg, intravenous) or a vehicle (2-hydroxypropyl-beta-cyclodextrin [HPCD]) was delivered. At 4 days post-injury, both ampakines increased diaphragm EMG output ipsilateral to C2Hx during both baseline breathing and acute respiratory challenge. Only CX1739 treatment also led to a sustained (15 min) increase in ipsilateral EMG output. At 14 days post-injury, both ampakines produced sustained increases in ipsilateral diaphragm EMG output and enabled increased output during the respiratory challenge. We conclude that low dose ampakine treatment can increase diaphragm EMG activity after cervical SCI, and therefore may provide a pharmacological strategy that could be useful in the context of respiratory rehabilitation.

Rationale and design of a mechanistic clinical trial of JAK inhibition to prevent ventilator-induced diaphragm dysfunction.

INTRODUCTION: Ventilator-induced diaphragm dysfunction (VIDD) is an important phenomenon that has been repeatedly demonstrated in experimental and clinical models of mechanical ventilation. Even a few hours of MV initiates signaling cascades that result in, first, reduced specific force, and later, atrophy of diaphragm muscle fibers. This severe, progressive weakness of the critical ventilatory muscle results in increased duration of MV and thus increased MV-associated complications/deaths. A drug that could prevent VIDD would likely have a major positive impact on intensive care unit outcomes. We identified the JAK/STAT pathway as important in VIDD and then demonstrated that JAK inhibition prevents VIDD in rats. We subsequently developed a clinical model of VIDD demonstrating reduced contractile force of isolated diaphragm fibers harvested after ∼7 vs ∼1 h of MV during a thoracic surgical procedure. MATERIALS AND METHODS: The NIH-funded clinical trial that has been initiated is a prospective, placebo controlled trial: subjects undergoing esophagectomy are randomized to receive 6 preoperative doses of the FDA-approved JAK inhibitor Tofacitinib (commonly used for rheumatoid arthritis) vs. placebo. The primary outcome variable will be the difference in the reduction that occurs in force generation of diaphragm single muscle fibers (normalized to their cross-sectional area), in the Tofacitinib vs. placebo subjects, over 6 h of MV. DISCUSSION: This trial represents a first-in-human, mechanistic clinical trial of a drug to prevent VIDD. It will provide proof-of-concept in human subjects whether JAK inhibition prevents clinical VIDD, and if successful, will support an ICU-based clinical trial that would determine whether JAK inhibition impacts clinical outcome variables such as duration of MV and mortality.

A randomized controlled trial to determine whether beta-hydroxy-beta-methylbutyrate and/or eicosapentaenoic acid improves diaphragm and quadriceps strength in critically Ill mechanically ventilated patients.

BACKGROUND: Intensive care unit acquired weakness is a serious problem, contributing to respiratory failure and reductions in ambulation. Currently, there is no pharmacological therapy for this condition. Studies indicate, however, that both beta-hydroxy-beta-methylbutyrate (HMB) and eicosapentaenoic acid (EPA) increase muscle function in patients with cancer and in older adults. The purpose of this study was to determine whether HMB and/or EPA administration would increase diaphragm and quadriceps strength in mechanically ventilated patients. METHODS: Studies were performed on 83 mechanically ventilated patients who were recruited from the Medical Intensive Care Units at the University of Kentucky. Diaphragm strength was assessed as the trans-diaphragmatic pressure generated by supramaximal magnetic phrenic nerve stimulation (PdiTw). Quadriceps strength was assessed as leg force generated by supramaximal magnetic femoral nerve stimulation (QuadTw). Diaphragm and quadriceps thickness were assessed by ultrasound. Baseline measurements of muscle strength and size were performed, and patients were then randomized to one of four treatment groups (placebo, HMB 3 gm/day, EPA 2 gm/day and HMB plus EPA). Strength and size measurements were repeated 11 days after study entry. ANCOVA statistical testing was used to compare variables across the four experimental groups. RESULTS: Treatments failed to increase the strength and thickness of either the diaphragm or quadriceps when compared to placebo. In addition, treatments also failed to decrease the duration of mechanical ventilation after study entry. CONCLUSIONS: These results indicate that a 10-day course of HMB and/or EPA does not improve skeletal muscle strength in critically ill mechanically ventilated patients. These findings also confirm previous reports that diaphragm and leg strength in these patients are profoundly low. Additional studies will be needed to examine the effects of other anabolic agents and innovative forms of physical therapy. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01270516. Registered 5 January 2011, https://clinicaltrials.gov/ct2/show/NCT01270516?term=Supinski&draw=2&rank=4 .

Effects of sevoflurane and adenosine receptor antagonist on the sugammadex-induced recovery from rocuronium-induced neuromuscular blockade in rodent phrenic nerve-hemidiaphragm tissue specimens.

Sevoflurane affects on the A1 receptor in the central nervous system and potentiates the action of neuromuscular blocking agents. In the present study, we investigated whether sevoflurane (SEVO) has the ability to potentiate the neuromuscular blocking effect of rocuronium and if the specific antagonist of adenosine receptor (SLV320) can reverse this effect. In this study, phrenic nerve-hemidiaphragm tissue specimens were obtained from 40 Sprague-Dawley (SD) rats. The specimens were immersed in an organ bath filled with Krebs buffer and stimulated by a train-of-four (TOF) pattern using indirect supramaximal stimulation at 20 s intervals. The specimens were randomly allocated to control, 2-chloroadenosine (CADO), SEVO, or SLV320 + SEVO groups. In the CADO and SLV320 + SEVO groups, CADO and SLV320 were added to the organ bath from the start to a concentration of 10 µM and 10 nM, respectively. We then proceeded with rocuronium-induced blockade of >95% depression of the first twitch tension of TOF (T1) and TOF ratio (TOFR). In the SEVO and SLV320 + SEVO groups, SEVO was added to the Krebs buffer solution to concentration of 400-500 µM for 10 min. Sugammadex-induced T1 and TOFR recovery was monitored for 30 min until >95% of T1 and >0.9 of TOFR were confirmed, and the recovery pattern was compared by plotting these data. T1 recovery in the SEVO and CADO groups was significantly delayed compared with the control and SLV320 + SEVO groups (p < .05). In conclusion, sevoflurane affects on the A1 receptor at the neuromuscular junction and delays sugammadex-induced recovery from neuromuscular blockade.

Overdoses due to fentanyl and its analogues (F/FAs) push naloxone to the limit.

WHAT IS KNOWN AND OBJECTIVE: Food and Drug Administration (FDA) risk evaluation and mitigation strategies (REMs) encourage emergency responders, paramedics, law enforcement agents, and even laypeople to be trained in the administration of naloxone with the intent of rescuing individuals from a known or suspected opioid overdose. COMMENT: Although naloxone is generally safe and effective at reversing respiratory depression caused by a conventional opioid such as morphine or heroin by competing with the opioid and displacing it from the µ-opioid receptor, questions increasingly are arising as to whether naloxone can adequately reverse opioid overdoses that may involve the potent opioids fentanyl and its analogues (F/FAs). In other words, as more and more opioid overdoses involve F/FAs, can naloxone keep up? WHAT IS NEW AND CONCLUSION: As a competitive antagonist at µ-opioid receptors, naloxone is often a life-saving agent in cases of overdose caused by conventional opioids, but it may not be versatile or powerful enough to combat the rising tide of overdoses due to fentanyl and its illicit analogues, or in cases of overdose involving combinations of opioids and non-opioids.

High frequency repetitive Transcranial Magnetic Stimulation promotes long lasting phrenic motoneuron excitability via GABAergic networks.

Repetitive transcranial magnetic stimulation (rTMS) is a promising, innovative, and non-invasive therapy used clinically. Efficacy of rTMS has been demonstrated to ameliorate psychiatric disorders and neuropathic pain through neuromodulation of affected neural circuits. However, little is known about the mechanisms and the specific neural circuits via which rTMS facilitates these functional effects. The aim of this study was to begin revealing the mechanisms by which rTMS may tap into existing neural circuits, by using a well characterized spinal motor circuit - the phrenic circuit. Here we hypothesized that rTMS can be used to enhance phrenic motoneuron excitability in anesthetized Sprague Dawley rats. Multiple acute rTMS protocols were used revealing 10 Hz rTMS protocol induced a robust, long-lasting increase in phrenic motoneuron excitability, functionally evaluated by diaphragm motor evoked potentials (59.1 ± 21.1 % of increase compared to baseline 60 min after 10 Hz protocol against 6.0 ± 5.8 % (p = 0.007) for Time Control, -5.8 ± 7.4 % (p < 0.001) for 3 Hz, and 5.2 ± 12.5 % (p = 0.008) for 30 Hz protocols). A deeper analyze allowed to discriminate "responder" and "non-responder" subgroups among 10 Hz rTMS treated animals. Intravenous injections of GABAA and GABAB receptor agonists prior to 10 Hz rTMS treatment, abolished the enhanced phrenic motoneuron excitability, suggesting GABAergic input plays a mechanistic role in rTMS-induced phrenic excitability. These data demonstrate that a single high frequency rTMS protocol at 10 Hz increases phrenic motoneuron excitability, mediated by a local GABAergic "disinhibition". By understanding how rTMS can be used to affect neural circuits non-invasively we can begin to harness the therapeutic potential of this neuromodulatory strategy to promote recovery after disease or injury to the central nervous system.

Acute intrathecal BDNF enhances functional recovery after cervical spinal cord injury in rats.

Unilateral C2 hemisection (C2SH) disrupts descending inspiratory-related drive to phrenic motor neurons and thus, silences rhythmic diaphragm muscle (DIAm) activity. There is gradual recovery of rhythmic DIAm EMG activity over time post-C2SH, consistent with neuroplasticity, which is enhanced by chronic (2 wk) intrathecal BDNF treatment. In the present study, we hypothesized that acute (30 min) intrathecal BDNF treatment also enhances recovery of DIAm EMG activity after C2SH. Rats were implanted with bilateral DIAm EMG electrodes to verify the absence of ipsilateral eupneic DIAm EMG activity at the time of C2SH and at 3 days post-C2SH. In those animals displaying no recovery of DIAm EMG activity after 28 days (n = 7), BDNF was administered intrathecally (450 mcg) at C4. DIAm EMG activity was measured continuously both before and for 30 min after BDNF treatment, during eupnea, hypoxia-hypercapnia, and spontaneous sighs. Acute BDNF treatment restored eupneic DIAm EMG activity in all treated animals to an amplitude that was 78% ± 9% of pre-C2SH root mean square (RMS) (P < 0.001). In addition, acute BDNF treatment increased DIAm RMS EMG amplitude during hypoxia-hypercapnia (P = 0.023) but had no effect on RMS EMG amplitude during sighs. These results support an acute modulatory role of BDNF signaling on excitatory synaptic transmission at phrenic motor neurons after cervical spinal cord injury.NEW & NOTEWORTHY Brain-derived neurotrophic factor (BDNF) plays an important role in promoting neuroplasticity following unilateral C2 spinal hemisection (C2SH). BDNF was administered intrathecally in rats displaying lack of ipsilateral inspiratory-related diaphragm (DIAm) EMG activity after C2SH. Acute BDNF treatment (30 min) restored eupneic DIAm EMG activity in all treated animals to 78% ± 9% of pre-C2SH level. In addition, acute BDNF treatment increased DIAm EMG amplitude during hypoxia-hypercapnia but had no effect on EMG amplitude during sighs.

Acute and Subacute Toxicity and Cytotoxicity of Opuntia Dillenii (Ker-Gawl) Haw. Seed Oil and Its Impact on the Isolated Rat Diaphragm Glucose Absorption.

This study aims to assess the safety of the Opuntia dillenii (Ker-Gawl) haw. seed oil (ODSO) and its effect on the glucose absorption activity of the isolated rat hemidiaphragm. This oil's safety was studied by exploring its acute (doses 1, 3, 5, and 7 mL/kg) and subacute (doses 1 and 2 mL/kg) toxicities in albino mice and Wistar rats, respectively. The safety of the ODSO was also assessed by studying its effect on the HepG2 cell viability in vitro. The effect of ODSO, or combined with the insulin, on the glucose absorption activity of isolated rat hemidiaphragm was evaluated at the dose 1 g/L in vitro. The results demonstrated the safety of ODSO. Indeed, this study showed that this oil does not produce any mortality or signs of toxicity after the single-dose administration in mice. Additionally, the daily intake of the ODSO during four weeks does not induce a significant variation in the biochemical parameters and body weight of rats compared with the control group. Besides, the cell viability of HepG2 did not change in the presence of ODSO. On the other hand, the ODSO increased the glucose absorption activity of the isolated rat hemidiaphragm, and this activity was significantly enhanced when combined with insulin. This study confirms, on one side, the safety of this oil and its efficacy and, on the other side, encourages its potential use as a complement to treat diabetes.

Angiotensin 1-7 protects against ventilator-induced diaphragm dysfunction.

Mechanical ventilation (MV) is a life-saving instrument used to provide ventilatory support for critically ill patients and patients undergoing surgery. Unfortunately, an unintended consequence of prolonged MV is the development of inspiratory weakness due to both diaphragmatic atrophy and contractile dysfunction; this syndrome is labeled ventilator-induced diaphragm dysfunction (VIDD). VIDD is clinically important because diaphragmatic weakness is an important contributor to problems in weaning patients from MV. Investigations into the pathogenesis of VIDD reveal that oxidative stress is essential for the rapid development of VIDD as redox disturbances in diaphragm fibers promote accelerated proteolysis. Currently, no standard treatment exists to prevent VIDD and, therefore, developing a strategy to avert VIDD is vital. Guided by evidence indicating that activation of the classical axis of the renin-angiotensin system (RAS) in diaphragm fibers promotes oxidative stress and VIDD, we hypothesized that activation of the nonclassical RAS signaling pathway via angiotensin 1-7 (Ang1-7) will protect against VIDD. Using an established animal model of prolonged MV, our results disclose that infusion of Ang1-7 protects the diaphragm against MV-induced contractile dysfunction and fiber atrophy in both fast and slow muscle fibers. Further, Ang1-7 shielded diaphragm fibers against MV-induced mitochondrial damage, oxidative stress, and protease activation. Collectively, these results reveal that treatment with Ang1-7 protects against VIDD, in part, due to diminishing oxidative stress and protease activation. These important findings provide robust evidence that Ang1-7 has the therapeutic potential to protect against VIDD by preventing MV-induced contractile dysfunction and atrophy of both slow and fast muscle fibers.

TrkB signaling contributes to transdiaphragmatic pressure generation in aged mice.

Ventilatory deficits are common in old age and may result from neuromuscular dysfunction. Signaling via the tropomyosin-related kinase receptor B (TrkB) regulates neuromuscular transmission and, in young mice, is important for the generation of transdiaphragmatic pressure (Pdi). Loss of TrkB signaling worsened neuromuscular transmission failure and reduced maximal Pdi, and these effects are similar to those observed in old age. Administration of TrkB agonists such as 7,8-dihydroxyflavone (7,8-DHF) improves neuromuscular transmission in young and old mice (18 mo; 75% survival). We hypothesized that TrkB signaling contributes to Pdi generation in old mice, particularly during maximal force behaviors. Old male and female TrkBF616A mice, with a mutation that induces 1NMPP1-mediated TrkB kinase inhibition, were randomly assigned to systemic treatment with vehicle, 7,8-DHF, or 1NMPP1 1 h before experiments. Pdi was measured during eupneic breathing (room air), hypoxia-hypercapnia (10% O2/5% CO2), tracheal occlusion, spontaneous deep breaths ("sighs"), and bilateral phrenic nerve stimulation (Pdimax). There were no differences in the Pdi amplitude across treatments during ventilatory behaviors (eupnea, hypoxia-hypercapnia, occlusion, or sigh). As expected, Pdi increased from eupnea and hypoxia-hypercapnia (∼7 cm H2O) to occlusion and sighs (∼25 cm H2O), with no differences across treatments. Pdimax was ∼50 cm H2O in the vehicle and 7,8-DHF groups and ∼40 cm H2O in the 1NMPP1 group (F8,74 = 2; P = 0.02). Our results indicate that TrkB signaling is necessary for generating maximal forces by the diaphragm muscle in old mice and are consistent with aging effects of TrkB signaling on neuromuscular transmission.NEW & NOTEWORTHY TrkB signaling is necessary for generating maximal forces by the diaphragm muscle. In 19- to 21-mo-old TrkBF616A mice susceptible to 1NMPP1-induced inhibition of TrkB kinase activity, maximal Pdi generated by bilateral phrenic nerve stimulation was ∼20% lower after 1NMPP1 compared with vehicle-treated mice. Treatment with the TrkB agonist 7,8-dihydroxyflavone did not affect Pdi generation when compared with age-matched mice. Inhibition of TrkB kinase activity did not affect the forces generated during lower force behaviors in old age.

Suppression of Hypoxia-Inducible Factor 1α by Low-Molecular-Weight Heparin Mitigates Ventilation-Induced Diaphragm Dysfunction in a Murine Endotoxemia Model.

Mechanical ventilation (MV) is required to maintain life for patients with sepsis-related acute lung injury but can cause diaphragmatic myotrauma with muscle damage and weakness, known as ventilator-induced diaphragm dysfunction (VIDD). Hypoxia-inducible factor 1α (HIF-1α) plays a crucial role in inducing inflammation and apoptosis. Low-molecular-weight heparin (LMWH) was proven to have anti-inflammatory properties. However, HIF-1α and LMWH affect sepsis-related diaphragm injury has not been investigated. We hypothesized that LMWH would reduce endotoxin-augmented VIDD through HIF-1α. C57BL/6 mice, either wild-type or HIF-1α-deficient, were exposed to MV with or without endotoxemia for 8 h. Enoxaparin (4 mg/kg) was administered subcutaneously 30 min before MV. MV with endotoxemia aggravated VIDD, as demonstrated by increased interleukin-6 and macrophage inflammatory protein-2 levels, oxidative loads, and the expression of HIF-1α, calpain, caspase-3, atrogin-1, muscle ring finger-1, and microtubule-associated protein light chain 3-II. Disorganized myofibrils, disrupted mitochondria, increased numbers of autophagic and apoptotic mediators, substantial apoptosis of diaphragm muscle fibers, and decreased diaphragm function were also observed (p < 0.05). Endotoxin-exacerbated VIDD and myonuclear apoptosis were attenuated by pharmacologic inhibition by LMWH and in HIF-1α-deficient mice (p < 0.05). Our data indicate that enoxaparin reduces endotoxin-augmented MV-induced diaphragmatic injury, partially through HIF-1α pathway inhibition.

Comparative Efficacy of Angiotensin II Type 1 Receptor Blockers Against Ventilator-Induced Diaphragm Dysfunction in Rats.

Mechanical ventilation (MV) is a life-saving intervention for many critically ill patients. Unfortunately, prolonged MV results in the rapid development of inspiratory muscle weakness due to diaphragmatic atrophy and contractile dysfunction (termed ventilator-induced diaphragm dysfunction (VIDD)). Although VIDD is a major risk factor for problems in weaning patients from MV, a standard therapy to prevent VIDD does not exist. However, emerging evidence suggests that pharmacological blockade of angiotensin II type 1 receptors (AT1Rs) protects against VIDD. Nonetheless, the essential characteristics of AT1R blockers (ARBs) required to protect against VIDD remain unclear. To determine the traits of ARBs that are vital for protection against VIDD, we compared the efficacy of two clinically relevant ARBs, irbesartan and olmesartan; these ARBs differ in molecular structure and effects on AT1Rs. Specifically, olmesartan blocks both angiotensin II (AngII) binding and mechanical activation of AT1Rs, whereas irbesartan prevents only AngII binding to AT1Rs. Using a well-established preclinical model of prolonged MV, we tested the hypothesis that compared with irbesartan, olmesartan provides greater protection against VIDD. Our results reveal that irbesartan does not protect against VIDD whereas olmesartan defends against both MV-induced diaphragmatic atrophy and contractile dysfunction. These findings support the hypothesis that olmesartan is superior to irbesartan in protecting against VIDD and are consistent with the concept that blockade of mechanical activation of AT1Rs is a required property of ARBs to shield against VIDD. These important findings provide a foundation for future clinical trials to evaluate ARBs as a therapy to protect against VIDD.

LAR inhibitory peptide promotes recovery of diaphragm function and multiple forms of respiratory neural circuit plasticity after cervical spinal cord injury.

Chondroitin sulfate proteoglycans (CSPGs), up-regulated in and around the lesion after traumatic spinal cord injury (SCI), are key extracellular matrix inhibitory molecules that limit axon growth and consequent recovery of function. CSPG-mediated inhibition occurs via interactions with axonal receptors, including leukocyte common antigen- related (LAR) phosphatase. We tested the effects of a novel LAR inhibitory peptide in rats after hemisection at cervical level 2, a SCI model in which bulbospinal inspiratory neural circuitry originating in the medullary rostral ventral respiratory group (rVRG) becomes disconnected from phrenic motor neuron (PhMN) targets in cervical spinal cord, resulting in persistent partial-to-complete diaphragm paralysis. LAR peptide was delivered by a soaked gelfoam, which was placed directly over the injury site immediately after C2 hemisection and replaced at 1 week post-injury. Axotomized rVRG axons originating in ipsilateral medulla or spared rVRG fibers originating in contralateral medulla were separately assessed by anterograde tracing via AAV2-mCherry injection into rVRG. At 8 weeks post-hemisection, LAR peptide significantly improved ipsilateral hemidiaphragm function, as assessed in vivo with electromyography recordings. LAR peptide promoted robust regeneration of ipsilateral-originating rVRG axons into and through the lesion site and into intact caudal spinal cord to reach PhMNs located at C3-C5 levels. Furthermore, regenerating rVRG axons re-established putative monosynaptic connections with their PhMNs targets. In addition, LAR peptide stimulated robust sprouting of both modulatory serotonergic axons and contralateral-originating rVRG fibers within the PhMN pool ipsilateral/caudal to the hemisection. Our study demonstrates that targeting LAR-based axon growth inhibition promotes multiple forms of respiratory neural circuit plasticity and provides a new peptide-based therapeutic strategy to ameliorate the devastating respiratory consequences of SCI.

Tempol improves redox status in mdx dystrophic diaphragm muscle.

Oxidative stress is a critical element in relationship to the pathophysiology of Duchenne muscular dystrophy (DMD). In the mice the diaphragm (DIA) is most resembles the dystrophic human pathology. In this study we have evaluated the consequences of a synthetic antioxidant (tempol) on oxidative stress parameters in the DIA muscle of mdx mice. The mdx mice were separated into two groups: mdx, the control group receiving intraperitoneal (i.p.) injections of saline solution (100 µL), and mdxT, the treated group receiving i.p. injections of tempol (100 mg/kg). The tempol-treated group showed reduced oxidative stress markers, decreasing the dihydroethidium reaction (DHE) area; autofluorescent lipofuscin granules; and 4-hydroxynonenal (4-HNE)-protein adduct levels. DIA muscle of mdx mice. At the same time, the manganese-superoxide dismutase 2 (SOD2) levels were increased in the tempol-treated group. In addition, the tempol-treated group showed reduced levels of glutathione-disulphide reductase (GSR), glutathione peroxidase 1 (GPx1) and catalase (CAT) in immunoblots. The tempol-treated group has also shown lower relative gene expression of SOD1, CAT and GPx than the non-treated group. Our data demonstrated that tempol treatment reduced oxidant parameters and increased anti-oxidant SOD2 levels in the DIA muscle of mdx mice, which may contribute to the normalization of the redox homeostasis of dystrophic muscles.

Ampakines stimulate phrenic motor output after cervical spinal cord injury.

Activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors increases phrenic motor output. Ampakines are a class of drugs that are positive allosteric modulators of AMPA receptors. We hypothesized that 1) ampakines can stimulate phrenic activity after incomplete cervical spinal cord injury (SCI), and 2) pairing ampakines with brief hypoxia could enable sustained facilitation of phrenic bursting. Phrenic activity was recorded ipsilateral (IL) and contralateral (CL) to C2 spinal cord hemisection (C2Hx) in anesthetized adult rats. Two weeks after C2Hx, ampakine CX717 (15 mg/kg, i.v.) increased IL (61 ± 46% baseline, BL) and CL burst amplitude (47 ± 26%BL) in 8 of 8 rats. After 90 min, IL and CL bursting remained above baseline (BL) in 7 of 8 rats. Pairing ampakine with a single bout of acute hypoxia (5-min, arterial partial pressure of O2 ~ 50 mmHg) had a variable impact on phrenic bursting, with some rats showing a large facilitation that exceeded the response of the ampakine alone group. At 8 weeks post-C2Hx, 7 of 8 rats increased IL (115 ± 117%BL) and CL burst amplitude (45 ± 27%BL) after ampakine. The IL burst amplitude remained above BL for 90-min in 7 of 8 rats; CL bursting remained elevated in 6 of 8 rats. The sustained impact of ampakine at 8 weeks was not enhanced by hypoxia exposure. Intravenous vehicle (10% 2-Hydroxypropyl-ß-cyclodextrin) did not increase phrenic bursting at either time point. We conclude that ampakines effectively stimulate neural drive to the diaphragm after cervical SCI. Pairing ampakines with a single hypoxic exposure did not consistently enhance phrenic motor facilitation.

Neuregulin-1ß 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-1ß (NRG-1ß) is known to exert organ-protective effects in some inflammatory diseases, little is known about the potential of NRG-1ß therapy in the diaphragm during sepsis. Our study was aimed at exploring the effects of NRG-1ß 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-1ß treatment. In vitro, the effects of NRG-1ß 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-1ß 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-1ß. And NRG-1ß 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-1ß, while MK-2206 blocked these effects of NRG-1ß. In conclusion, our findings underlined that NRG-1ß 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.

Effects of hyperthermia on the effective concentration of rocuronium and sugammadex-mediated reversal in isolated phrenic nerve hemidiaphragm preparations of rats.

BACKGROUND: Hyperthermia is relatively rare during general anesthesia; however, a few studies have been conducted on hyperthermia and the neuromuscular blockade (NMB) induced by rocuronium, and the reversal of NMB by sugammadex. We investigated the effect of hyperthermia status on the NMB induced by rocuronium, and its reversal by sugammadex, in isolated phrenic nerve hemidiaphragm (PNHD) preparations of the rat. METHODS: Thirty-three male Sprague-Dawley rat PNHD preparations were randomly assigned to three groups at different temperatures (36 °C, 38 °C, and 40 °C; each group, n = 11, in Krebs solution). The train-of-four (TOF) and twitch height responses were checked mechanomyographically. The PNHD were treated with progressively increasing doses of rocuronium and three effective concentrations (ECs), EC50, EC90, and EC95, of rocuronium were analyzed in each group via nonlinear regression analysis. Then, sugammadex was administered in doses equimolar to rocuronium. Thereafter, the T1 height (%), TOFR (%) and the duration index were measured. RESULTS: The EC of rocuronium (EC50, EC90, and EC95) decreased significantly in accordance with increasing temperature. The groups at 36 °C and 40 °C showed clear differences in all areas (all P < 0.001). Moreover, the T1 height (%) and the duration index upon sugammadex administration showed faster recovery results in the36 °C than the 38 °C and 40 °C groups. CONCLUSION: A rise of temperature from 38 °C to 40 °C in rat PNHD preparations proportionally enhanced the NMB induced by rocuronium. In addition, equimolar doses of sugammadex to the administered rocuronium showed a slower recovery time as the temperature rises.