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Objective: To compare pulmonary function and gas exchange in ponies during maintenance of anaesthesia with isoflurane or by a total intravenous anaesthesia (TIVA) technique., Study Design: Experimental, cross-over study., Animals: Six healthy ponies weighing mean 286 (range 233-388) ± SD 61 kg, age 13 (9-16) ± 3 years., Methods: The ponies were anaesthetized twice, a minimum of two weeks apart. Following sedation with romifidine [80 μg kg(-1) intravenously (IV)], anaesthesia was induced IV with midazolam (0.06 mg kg(-1)) and ketamine (2.5 mg kg(-1), then maintained either with inhaled isoflurane (Fe'Iso = 1.1 vol%) (T-ISO) or an IV infusion of romifidine (120 μg kg(-1) hour(-1)), midazolam (0.09 mg kg(-1) hour(-1) IV) and ketamine (3.3 mg kg(-1) hour(-1)) (T-TIVA). Ponies were placed in lateral recumbency. Breathing was spontaneous and Fi'O(2) 60%. After an instrumentation/stabilisation period of 30 minutes, arterial and mixed venous blood samples were taken simultaneously every 10 minutes for 60 minutes and analysed immediately. Oxygen extraction ratio (O(2)ER) and venous admixture were calculated. Tidal volume (TV), minute volume (MV), respiratory rate (f(R)), packed cell volume (PCV), arterial blood pressure and heart rate (HR) were measured and recorded. Data were analysed with mixed model anova (α = 0.05). Treatments were compared overall and at two selected time points (T30 and T60) using Bonferroni correction., Results: Arterial and mixed venous partial pressures of O(2) and CO(2), and TV were significantly lower and MV and f(R) were higher in T-TIVA compared to T-ISO. Venous admixture did not differ between treatments. O(2) R was significantly higher in T-TIVA. Mean arterial pressure was higher and HR was lower in T-TIVA compared to T-ISO., Conclusions and Clinical Relevance: Whilst arterial CO(2) was within an acceptable range during both protocols, the impairment of oxygenation was more pronounced with the T-TIVA evidenced by lower arterial and venous oxygen partial pressures., (© 2014 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesia and Analgesia.)

Background: The aetiology for nonsteroidal anti-inflammatory drug (NSAID)-induced small intestinal injuries has not been well characterised., Aim: To determine the risk factors of symptomatic NSAID-induced small intestinal injuries, including diaphragm disease., Methods: Of the 1262 symptomatic patients who underwent videocapsule endoscopy and/or double-balloon enteroscopy, 156 consecutive patients were verified as having taken NSAIDs. Their CYP2C9*2, *3 and *13 single nucleotide polymorphisms (SNPs) were determined by allelic discrimination with Taqman 5'-nuclease assays., Results: Of the 156 NSAIDs users, 31 patients (20%) were diagnosed with NSAID-induced small intestinal injury. Multivariate analysis indicated that the presence of comorbidities and the use of oxicams (meloxicam, ampiroxicam and lornoxicam) or diclofenac were associated with an increased risk of NSAID-induced small intestinal injury (adjusted OR: 2.97, 95% CI: 1.05-8.41, P = 0.041 and adjusted OR: 7.05, 95% CI: 2.04-24.40, P = 0.002, respectively). The combination of aspirin and non-aspirin NSAID was more damaging than aspirin alone. Age, sex, concomitant use of proton pump inhibitors, indications for NSAIDs use, duration of NSAIDs use and CYP2C9*2, *3 and *13SNPs were unrelated. The use of meloxicam and CYP2C9*3SNPs were significantly associated with an increased risk for diaphragm disease (adjusted OR: 183.75, 95% CI: 21.34-1582.38; P < 0.0001 and adjusted OR: 12.94, 95% CI: 1.55-108.36, P = 0.018, respectively)., Conclusion: The use of specific NSAIDs and the factors interfering with NSAIDs metabolism might associate with small intestinal injury, especially with diaphragm disease., (© 2014 John Wiley & Sons Ltd.)

The aim of this study was to investigate the influence of chronic dexamethasone (Dex) administration on rat diaphragm sensitivity to non-depolarizing muscle relaxants (NDMRs) and muscular nicotinic acetylcholine receptor (nAChR) expression, which may help direct future administration of NDMRs. Adult male Sprague-Dawley rats were randomized to receive a daily intraperitoneal injection of Dex (600 μg/kg body mass) or an equivalent volume of saline (N = 20 in each group) for 14 days. We evaluated isometric twitch tensions of nerve-hemidiaphragm preparations elicited by indirect supramaximal stimulation at 0.1 Hz. Real-time quantitative PCR was performed to determine the mRNA expression of two nAChR subunits (ε-subunit and γ-subunit) in the diaphragm. Dex administration markedly (P < 0.01) increased the 50% twitch depression (IC50) of the three NDMRs. The IC50 ratio, which standardized the magnitudes of the resistance, was the largest for atracurium, with the second largest for vecuronium and the smallest for rocuronium (P < 0.01). The ε- and γ-subunit mRNAs were both upregulated with an increased γ/ε ratio in rats exposed to Dex. The results indicated that chronic Dex administration induces hyposensitivity to NDMRs, the degree of which depends on the kind of neuromuscular blocker, and is associated with increased nAChR expression.

Haemorrhagic shock (HS) and fluid resuscitation can lead to increased reactive oxygen species (ROS), contributing to ischaemia-reperfusion injury and organ damage. Ubiquinol is a potent antioxidant that decreases ROS. This study examined the effects of ubiquinol administered with fluid resuscitation following controlled HS. Adult male Sprague-Dawley rats were randomly assigned to treatment [ubiquinol, 1 mg (100 g body weight)(-1)] or control groups. Rats were subjected to 60 min of HS by removing 40% of the total blood volume to a mean arterial pressure ∼45-55 mmHg. The animals were resuscitated with blood and lactated Ringer solution, with or without ubiquinol, and monitored for 120 min. At the end of the experiments, the rats were killed and the lungs, diaphragm, heart and kidneys harvested. Leucocytes were analysed for mitochondrial superoxide at baseline, end of shock and 120 min following fluid resuscitation using MitoSOX Red. Diaphragms were examined for hydrogen peroxide using dihydrofluorescein diacetate and confocal microscopy. The apoptosis in lungs, diaphragm, heart and kidneys was measured using fluorescence microscopy with acridine orange and ethidium bromide. Leucocyte mitochondrial superoxide levels were significantly lower in rats that received ubiquinol than in the control animals. Production of hydrogen peroxide and apoptosis were significantly reduced in the organs of rats treated with ubiquinol. These findings suggest that ubiquinol, administered with fluid resuscitation after HS, attenuates ROS production and apoptosis. Thus, ubiquinol is a potent antioxidant that may be used as a potential treatment to reduce organ injury following haemorrhagic events., (© 2014 The Authors. Experimental Physiology © 2014 The Physiological Society.)

Halofuginone has been shown to prevent fibrosis via the transforming growth factor-β/Smad3 pathway in muscular dystrophies. We hypothesized that halofuginone would reduce apoptosis--the presumed cause of satellite-cell depletion during muscle degradation-in the mdx mouse model of Duchenne muscular dystrophy. Six-week-old mdx mouse diaphragm exhibited fourfold higher numbers of apoptotic nuclei compared with wild-type mice as determined by a TUNEL assay. Apoptotic nuclei were found in macrophages and in Pax7-expressing cells; some were located in centrally-nucleated regenerating myofibers. Halofuginone treatment of mdx mice reduced the apoptotic nuclei number in the diaphragm, together with reduction in Bax and induction in Bcl2 levels in myofibers isolated from these mice. A similar effect was observed when halofuginone was added to cultured myofibers. No apparent effect of halofuginone was observed in wild-type mice. Inhibition of apoptosis or staurosporine-induced apoptosis by halofuginone in mdx primary myoblasts and C2 myogenic cell line, respectively, was reflected by less pyknotic/apoptotic cells and reduced Bax expression. This reduction was reversed by a phosphinositide-3-kinase and mitogen-activated protein kinase/extracellular signal-regulated protein kinase inhibitors, suggesting involvement of these pathways in mediating halofuginone's effects on apoptosis. Halofuginone increased apoptosis in α smooth muscle actin- and prolyl 4-hydroxylase β-expressing cells in mdx diaphragm and in myofibroblasts, the major source of extracellular matrix. The data suggest an additional mechanism by which halofuginone improves muscle pathology and function in muscular dystrophies., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Duchenne muscular dystrophy (DMD) is a degenerative skeletal muscle disease caused by mutations in the gene encoding dystrophin (DYS). Tumor necrosis factor (TNF) has been implicated in the pathogenesis since short-term treatment of mdx mice with TNF blocking drugs proved beneficial; however, it is not clear whether long-term treatment will also improve long-term outcomes of fibrosis and cardiac health. In this investigation, short and long-term dosing studies were carried out using the TNF blocking drug Remicade and a variety of outcome measures were assessed. Here we show no demonstrable benefit to muscle strength or morphology with 10mg/kg or 20mg/kg Remicade; however, 3mg/kg produced positive strength benefits. Remicade treatment correlated with reductions in myostatin mRNA in the heart, and concomitant reductions in cardiac and skeletal fibrosis. Surprisingly, although Remicade treated mdx hearts were less fibrotic, reductions in LV mass and ejection fraction were also observed, and these changes coincided with reductions in AKT phosphorylation on threonine 308. Thus, TNF blockade benefits mdx skeletal muscle strength and fibrosis, but negatively impacts AKT activation, leading to deleterious changes to dystrophic heart function. These studies uncover a previously unknown relationship between TNF blockade and alteration of muscle growth signaling pathways., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Organ dysfunction consequent to infection ('severe sepsis') is the leading cause of admission to an intensive care unit (ICU). In both animal models and early clinical studies the calcium channel sensitizer levosimendan has been demonstrated to have potentially beneficial effects on organ function. The aims of the Levosimendan for the Prevention of Acute oRgan Dysfunction in Sepsis (LeoPARDS) trial are to identify whether a 24-hour infusion of levosimendan will improve organ dysfunction in adults who have septic shock and to establish the safety profile of levosimendan in this group of patients., Methods/design: This is a multicenter, randomized, double-blind, parallel group, placebo-controlled trial. Adults fulfilling the criteria for systemic inflammatory response syndrome due to infection, and requiring vasopressor therapy, will be eligible for inclusion in the trial. Within 24 hours of meeting these inclusion criteria, patients will be randomized in a 1:1 ratio stratified by the ICU to receive either levosimendan (0.05 to 0.2 μg.kg⁻¹.min⁻¹ or placebo for 24 hours in addition to standard care. The primary outcome measure is the mean Sequential Organ Failure Assessment (SOFA) score while in the ICU. Secondary outcomes include: central venous oxygen saturations and cardiac output; incidence and severity of renal failure using the Acute Kidney Injury Network criteria; duration of renal replacement therapy; serum bilirubin; time to liberation from mechanical ventilation; 28-day, hospital, 3 and 6 month survival; ICU and hospital length-of-stay; and days free from catecholamine therapy. Blood and urine samples will be collected on the day of inclusion, at 24 hours, and on days 4 and 6 post-inclusion for investigation of the mechanisms by which levosimendan might improve organ function. Eighty patients will have additional blood samples taken to measure levels of levosimendan and its active metabolites OR-1896 and OR-1855. A total of 516 patients will be recruited from approximately 25 ICUs in the United Kingdom., Discussion: This trial will test the efficacy of levosimendan to reduce acute organ dysfunction in adult patients who have septic shock and evaluate its biological mechanisms of action., Trial Registration: Current controlled trials ISRCTN12776039 (19 September 2013).

Infections induce severe respiratory muscle weakness. Currently there are no treatments for this important clinical problem. We tested the hypothesis that β-hydroxy-β-methylbutyrate (HMB) would prevent sepsis-induced diaphragm weakness. Four groups of adult male mice were studied: controls (saline-injected), sepsis (intraperitoneal lipopolysaccharide), sepsis+HMB (injected intravenously), and HMB. Diaphragm force generation and indices of caspase 3, calpain, 20S proteasomal subunit, and double-stranded RNA-dependent protein kinase (PKR) activation were assessed after 24h. Sepsis elicited large reductions in diaphragm specific force generation at all stimulation frequencies. Endotoxin also activated caspase 3, calpain, the 20S proteasomal subunit and PKR in the diaphragm. HMB blocked sepsis-induced caspase 3, 20S proteasomal and PKR activation, but did not prevent calpain activation. Most importantly, HMB administration significantly attenuated sepsis-induced diaphragm weakness, preserving muscle force generation at all stimulation frequencies (p<0.01). We speculate that HMB may prove to be an important therapy in infected patients, with the potential to increase diaphragm strength, to reduce the duration of mechanical ventilation and to decrease mortality in this patient population., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Capnography is a standard monitoring tool during general anaesthesia. Diaphragmatic movement with the weaning of muscle relaxant effect produces the characteristic "curare cleft" on capnography. Various artefacts can mimick this trace intraoperatively. Cautious interpretation and identification of these is essential to avoid any undue overdosing of the patients with muscle relaxants. We report "curare cleft" like artefact during ventilation with a single lumen tube in a patient with unilateral capnothorax undergoing minimally invasive esophagectomy.

The early research found that the spiderlings of black widow spider (Latrodectus tredecimguttatus) exhibited obvious toxicity to animals. The present work performed a systematical analysis of the aqueous extract of newborn black widow spiderlings. The extract was shown to contain 69.42% of proteins varying in molecular weights and isoelectric points. Abdominal injection of the extract into mice and cockroaches caused obvious poisoning symptoms as well as death, with LD50 being 5.30 mg/kg in mice and 16.74 µg/g in Periplaneta americana. Electrophysiological experiments indicated that the extract at a concentration of 10 µg/mL could completely block the neuromuscular transmission in isolated mouse nerve-hemidiaphragm preparations within 21 ± 1.5 min, and 100 µg/mL extract could inhibit a certain percentage of voltage-activated Na⁺, K⁺, and Ca²⁺ channel currents in rat dorsal root ganglion neurons. These results demonstrate that the spiderlings are rich in neurotoxic components, which play important roles in the spiderling toxicity., (© 2014 Wiley Periodicals, Inc.)

Context: Traditional medicine is used by over 60% of the world's population for health care. Mukia maderaspatana (L.) M. Roem. (Cucurbitaceae) (Mukia) is extensively used in folklore medicine as an antidiabetic plant. It is rich in phenolics that contribute to its medicinal properties., Objective: Mukia extract and phenolics such as quercetin and phloroglucinol are investigated for their in vitro antidiabetic activity., Materials and Methods: Quercetin, phloroglucinol, and methanol extract of the dried whole plant (0.25 and 0.5 mg/ml) were studied for the inhibition of gluconeogenesis in rat liver slices and glucose uptake in isolated rat hemi-diaphragm (50 and 100 µg/ml). Phenolics of Mukia were analyzed by HPLC., Results and Discussion: Glucose (1.2 mg/g/h) was synthesized from pyruvate and the synthesis was completely inhibited by insulin (1 U/ml). Quercetin at 0.25 and 0.5 mg/ml caused 65% and 89% inhibition (0.42 mg/g/h and 0.13 mg/g/h glucose). Addition of insulin did not increase inhibition. Phloroglucinol inhibited 100% glucose production with or without insulin. Mukia (0.25 mg/ml) inhibited gluconeogenesis (0.65 mg/g/h) by 45%, and with insulin, inhibition increased to 50% (0.59 mg/g/h). At 0.5 mg/ml, glucose production was stimulated by1.2-fold, but with insulin it was inhibited by 89% (0.13 mg/g/h glucose). Mukia had no effect on glucose uptake, but potentiated the action of insulin mediated glucose uptake (152.82 ± 13.30 mg/dl/g/30 min) compared with insulin control (112.41 ± 9.14 mg/dl/g/30 min) (p < 0.05). HPLC analysis revealed the presence of phenolics., Conclusion: Results provide scientific rationale for the use of Mukia in folk medicine as an antidiabetic nutraceutical.

Hydrogen sulfide (H2S) is a gasotransmitter synthesized from cysteine (Cys) by pyridoxal-5'-phosphate-dependent enzymes. We investigated the potential roles of H2S in the regulation of central rhythmic respiration in adult rats in vivo. Sodium hydrosulfide (NaHS: 2.5 mM, 10 mM, and 5 mM) as a source of exogenous H2S, Cys (2.5 mM, 10 mM and 5 mM) as a source of endogenous H2S, 2.5 mM Cys+10 mM hydroxylamine (NH2OH), and 10 mM NH2OH, respectively, were intracerebroventricularly injected into rats. The rhythmic discharge of the diaphragm, including burst duration (BD), burst interval (BI), burst frequency (BF), and integrated amplitude (IA), and arterial blood pressure (BP) were measured at different time points. The results were analyzed by analysis of variance. A total of 2.5 mM NaHS did not significantly affect changes in BD, BI, BF, IA, or BP (P>0.05), whereas 2.5 mM Cys significantly altered BD, BI, and BF (P<0.05); however, there was no change in IA and BP (P>0.05). A concentration of 5 mM Cys had effects similar to those of 5 mM NaHS; both induced biphasic respiratory responses and changed the BF (P<0.05). A concentration of 10 mM NH2OH irreversibly inhibited rhythmic discharge of the diaphragm except for IA. No change was seen in BI, BF, IA, or BP (P>0.05) except for BD was temporarily decreased (P<0.05) in the 2.5 mM Cys+10 mM NH2OH group. These results suggest that exogenous and endogenous H2S may participate in the regulation of respiratory activity in adult rats.

Respiratory muscle dysfunction documented in sleep apnoea patients is perhaps due to oxidative stress secondary to chronic intermittent hypoxia (CIH). We sought to explore the effects of different CIH protocols on respiratory muscle form and function in a rodent model. Adult male Wistar rats were exposed to CIH (n = 32) consisting of 90 s normoxia-90 s hypoxia (either 10 or 5% oxygen at the nadir; arterial O2 saturation ∼ 90 or 80%, respectively] for 8 h per day or to sham treatment (air-air, n = 32) for 1 or 2 weeks. Three additional groups of CIH-treated rats (5% O2 for 2 weeks) had free access to water containing N-acetyl cysteine (1% NAC, n = 8), tempol (1 mM, n = 8) or apocynin (2 mM, n = 8). Functional properties of the diaphragm muscle were examined ex vivo at 35 °C. The myosin heavy chain and sarco(endo)plasmic reticulum Ca(2+)-ATPase isoform distribution, succinate dehydrogenase and glyercol phosphate dehydrogenase enzyme activities, Na(+)-K(+)-ATPase pump content, concentration of thiobarbituric acid reactive substances, DNA oxidation and antioxidant capacity were determined. Chronic intermittent hypoxia (5% oxygen at the nadir; 2 weeks) decreased diaphragm muscle force and endurance. All three drugs reversed the deleterious effects of CIH on diaphragm endurance, but only NAC prevented CIH-induced diaphragm weakness. Chronic intermittent hypoxia increased diaphragm muscle myosin heavy chain 2B areal density and oxidized glutathione/reduced glutathione (GSSG/GSH) ratio. We conclude that CIH-induced diaphragm dysfunction is reactive oxygen species dependent. N-Acetyl cysteine was most effective in reversing CIH-induced effects on diaphragm. Our results suggest that respiratory muscle dysfunction in sleep apnoea may be the result of oxidative stress and, as such, antioxidant treatment could prove a useful adjunctive therapy for the disorder.

Coefficient of kinetic friction (μ) of rabbit pleural mesothelium increased after short treatment of specimens with phospholipase C. This increase was removed by addition of a solution with hyaluronan or sialomucin, as previously shown in post-blotting Ringer or after short pronase treatment. After phospholipase μ decreased with increase in sliding velocity, but at highest velocity it was still greater than control; this difference was removed by addition of hyaluronan or sialomucin, as in post-blotting Ringer or after short pronase treatment. Hyaluronan placed on specimen before phospholipase treatment reduced increase in μ by protecting phospholipids from enzyme, as shown by others for alveolar and synovial phospholipids. Samples of parietal pleura stained with silver nitrate showed that mesothelial cells were not disrupted by short phospholipase treatment. Instead, they were disrupted if this treatment was preceded by a short pronase treatment; but even after this disruption addition of hyaluronan or sialomucin brought μ back to control., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Mechanical ventilation is crucial for patients with respiratory failure. The mechanical takeover of diaphragm function leads to diaphragm dysfunction and atrophy (ventilator-induced diaphragmatic dysfunction), with an increase in oxidative stress as a major contributor. In most patients, a sedative regimen has to be initiated to allow tube tolerance and ventilator synchrony. Clinical data imply a correlation between cumulative propofol dosage and diaphragm dysfunction, whereas laboratory investigations have revealed that propofol has some antioxidant properties. The authors hypothesized that propofol reduces markers of oxidative stress, atrophy, and contractile dysfunction in the diaphragm., Methods: Male Wistar rats (n = 8 per group) were subjected to either 24 h of mechanical ventilation or were undergone breathing spontaneously for 24 h under propofol sedation to test for drug effects. Another acutely sacrificed group served as controls. After sacrifice, diaphragm tissue was removed, and contractile properties, cross-sectional areas, oxidative stress, and proteolysis were examined. The gastrocnemius served as internal control., Results: Propofol did not protect against diaphragm atrophy, oxidative stress, and protease activation. The decrease in tetanic force compared with controls was similar in the spontaneous breathing group (31%) and in the ventilated group (34%), and both groups showed the same amount of muscle atrophy. The gastrocnemius muscle fibers did not show atrophy., Conclusions: Propofol does not protect against ventilator-induced diaphragmatic dysfunction or oxidative injury. Notably, spontaneous breathing under propofol sedation resulted in the same amount of diaphragm atrophy and dysfunction although diaphragm activation per se protects against ventilator-induced diaphragmatic dysfunction. This makes a drug effect of propofol likely.

Background: Snakebite is a significant public health issue in tropical countries. In Brazil, some of the most common snake envenomations are from Bothrops. Bothrops bites trigger local and systemic effects including edema, pain, erythema, cyanosis, infections, and necrosis. Vellozia flavicans is a plant from the Brazilian "cerrado" (savanna) that is popularly used as an anti-inflammatory medicine. Since inflammation develops quickly after Bothrops bites, which can lead to infection, the aim of the present study was to observe possible anti-snake venom and antimicrobial activities of V. flavicans (Vf)., Methods: The chromatographic profile of the main constituents from the Vf leaf hydroalcoholic extract was obtained by thin-layer chromatography (TLC). The anti-snake venom activity was measured by Vf's ability to neutralize the in vitro neuromuscular blockade caused by Bothrops jararacussu venom (Bjssu) in a mouse phrenic nerve-diaphragm model (PND). After a 20 min incubation, preparations of PND were added to Tyrode's solution (control); Vf (0.2, 0.5, 1, and 2 mg/mL); 40 μg/mL Bjssu; pre-incubation for 30 min with Bjssu and 1 mg/mL Vf; and a Bjssu pretreated preparation (for 10 min) followed by 1 mg/mL Vf. Myographic recording was performed, and the contractile responses were recorded. The antimicrobial activity (minimum inhibitory concentration [MIC] and minimum bactericidal concentration [MBC]) was obtained for Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecalis, using gentamicin and vancomycin as positive controls., Results: TLC analysis yielded several compounds from Vf, such as flavonoids (quercetin) and phenolic acids (chlorogenic acid). Bjssu completely blocked the contractile responses of PND preparations, while Vf preserved 97% (±10%) of the contractile responses when incubated with Bjssu. In the PND pretreated with Bjssu, Vf was able to inhibit the neuromuscular blockade progress. MIC and MBC of Vf ranged from 2.5 to 5.0 mg/mL for P. aeruginosa and S. aureus strains, while no antimicrobial activity was observed for E. coli and E. faecalis., Conclusions: The hydroalcoholic extract from Vf leaves was able to neutralize and decrease the in vitro neuromuscular blockade caused by Bjssu. However, it did not show significant antimicrobial activity against the tested bacteria.

Guillain-Barré syndrome, which is a variant of acute inflammatory neuropathy, is associated with anti-GM1 antibodies and causes ataxia. We investigated the effects of IgG anti-GM1 monoclonal antibody (IgG anti-GM1 mAb) on spontaneous muscle action potentials in a rat spinal cord-muscle co-culture system and the localization of IgG anti-GM1 mAb binding in the rat hemi-diaphragm. The frequency of spontaneous muscle action potentials in innervated muscle cells was acutely inhibited by IgG anti-GM1 mAb. When cultures were pretreated with GM2 synthase antisense oligodeoxynucleotide, IgG anti-GM1 mAb failed to inhibit spontaneous muscle action potentials, demonstrating the importance of the GM1 epitope in the action of IgG anti-GM1 mAb. Immunohistochemistry of rat hemi-diaphragm showed that IgG anti-GM1 mAb binding overlapped with neurofilament 200 (NF200) antibodies staining, but not α-bungarotoxin (α-BuTx) staining, demonstrating that IgG anti-GM1 mAb was localized at the presynaptic nerve terminal. IgG anti-GM1 mAb binding overlapped with syntaxin antibody and S-100 antibody in the nerve terminal. After collagenase treatment, IgG anti-GM1 mAb and NF200 antibodies did not show staining, but α-BuTx selectively stained the hemi-diaphragm. IgG anti-GM1 mAb binds to the presynaptic nerve terminal of neuromuscular junctions. Therefore, we suggest that the inhibitory effect of IgG anti-GM1 mAb on spontaneous muscle action potentials is related to the GM1 epitope in presynaptic motor nerve terminals at the NMJs.

The aim of the current study was to investigate the effect of chronic dexamethasone (Dex) administration on rat diaphragm function and sensitivity to rocuronium and muscle‑fiber transformation. Adult male Sprague‑Dawley rats were randomized to receive a daily intraperitoneal injection of Dex to evaluate whether alterations in diaphragm function and susceptibility to rocuronium would be induced. In addition, diaphragm contractile properties, histopathology and isometric twitch tensions of nerve‑hemidiaphragm preparations were evaluated. Dex administration led to impaired diaphragm force generation, increased fatigue resistance and a prolonged half‑relaxation time, as well as time‑to‑peak tension. Dex treatment led to desensitization of the rat diaphragm to rocuronium, as demonstrated by a shift of the rocuronium concentration‑twitch tension curves to the right. Histochemical analysis of adenosine triphosphatase revealed that the distribution and cross‑sectional area of type II fibers were decreased in rats exposed to Dex. The present study indicates that chronic Dex treatment induced alterations in muscle function and that susceptibility to rocuronium is associated with muscle fiber‑type transformation, which may aid in directing future administration of muscle relaxants.

Neuromuscular transmission is clinically monitored using the train-of-four ratio (TOFratio), which is the quotient between twitch tension produced by the fourth (T4) and by the first (T1) stimulus within a train-of-four stimulation at 2Hz. Neostigmine causes a paradoxical depression of the TOFratio (TOFfade) that is amplified by intra-arterial atropine in cats. This led us to question the usefulness of the TOFratio as a sole testing element to control neostigmine-induced reversal of neuromuscular transmission block caused by non-depolarizing agents. We hypothesized that the inhibition of cholinesterase activity might increase acetylcholine bioavailability and consequently cholinoceptor activation, leading to concomitant adenosine release from nerve endings and skeletal muscle fibers. The involvement of presynaptic muscarinic and adenosine receptors in neostigmine-induced TOFfade in the rat phrenic nerve diaphragm was investigated. Blockade of adenosine A2A receptors with ZM241385 and of muscarinic M2 receptors with methoctramine or atropine amplified neostigmine-induced TOFfade. Notwithstanding TOFfade amplification, the blockade of M2 or A2A receptors increased both T1 and T4 twitch tensions above control during the first 3min of neostigmine application. Beyond that period, the T1 twitch tension returned to baseline, whereas T4 decreased further until the control value with neostigmine alone. Blockade of M1 receptors by pirenzepine did not change neostigmine-induced TOFfade, unless A2A receptors were concurrently blocked with ZM241385. Data indicate that the paradoxical neostigmine-induced fade involves presynaptic mechanisms that regulate transmitter release and synaptic adenosine accumulation, including the activation of adenosine A2A and muscarinic M2 receptors., (© 2013 Elsevier B.V. All rights reserved.)

Objective: To explore the diaphragmatic toxicity in doxorubicin (DOX)-treated rats and the related mechanisms, as well as the effects of Shengmai Injection (SMI, ) on the diaphragmatic dysfunction., Methods: Thirty Sprague-Dawley male rats were randomly divided into three groups: control, DOX-treated and DOX+SMI treated groups. DOX was given to rats in DOX and DOX+SMI groups in 6 equal doses [2.5 mg/kg, intraperitoneal injection (i.p.)], on alternate days, over a period of 2 weeks for a cumulative dose of 15 mg/kg. SMI was given to DOX+SMI rats in 12 doses (3 mL/kg, i.p.) for a period of 2 weeks before the administration of DOX and 2 weeks during the administration of DOX. The rats in the control group received equal volume of normal saline. Subsequently, the twitch and tetanic characteristics and force-frequency relationships, and the malondialdehyde (MDA) levels and the superoxide dismutase (SOD) activities, as well as the mRNA content and proteins of inducible nitric oxide synthase (iNOS) were determined., Results: The DOX-treated rats had decreased the peak twitch tension (Pt), maximal tetanic tension (P0) and force-frequency relationship as compared with the control rats (P<0.01), while the diaphragm contractility in rats treated with SMI were significantly higher than that in DOX-treated rats (P<0.01). The DOX-treated rats had increased MAD levels and decreased SOD activities (P<0.05), and SMI decreased the MDA levels and increased the SOD activities in DOX-treated rats (P<0.05). Ultrastructure of diaphragm in the DOX-treated rats revealed typical alterations including fracture of diaphragm fibers, and edema and degeneration of mitochondria; these changes were relieved by SMI treatment. The mRNA content and protein of iNOS in DOX-treated rats were remarkably higher than those in control rats (P<0.01), while SMI decreased the mRNA expression level of iNOS in DOX-treated rats (P<0.05)., Conclusions: Lipid peroxidation is responsible for DOX-induced diaphragm toxicity. SMI protects diaphragm muscles and their function from DOX impairment, and these beneficial effects may be somehow correlated with the decrease in expression of iNOS and lipid peroxidation.

Background: Little is known about the effect of anesthetic technique on postoperative diaphragmatic function, which is associated with postoperative morbidity and recovery in patients undergoing laparoscopic pelvic surgery. The aim of this trial was to study the effect of combined general and epidural anesthesia versus general anesthesia on postoperative diaphragmatic function measured by ultrasonography in patients undergoing robot-assisted laparoscopic radical prostatectomy (RALRP)., Methods: Fifty-four patients undergoing RALRP were enrolled prospectively. Study population was randomized to receive general (group G, N.=27) or combined general and epidural (group GE, N.=27) anesthesia. Diaphragmatic inspiratory amplitude (DIA), and inspiration and expiration time (Ti and Te, respectively) were measured by M-mode ultrasonography during quiet/deep breathing and sniffing before the surgery and on postoperative days (POD) 1 and 2. Diaphragmatic inspiratory and expiratory velocities (DIV and DEV) were also calculated (DIA/Ti and DIA/Te, respectively). Spirometry was performed in addition to ultrasonography., Results: DIA during deep breathing and sniffing was significantly decreased on POD 1 in group G, while it was preserved in group GE. These reductions in diaphragmatic function were restored to preoperative values on POD 2 in both groups. Vital capacity and peak expiratory flow were diminished in group G on POD 1 and 2. However, spirometry revealed no impairment in group GE except for vital capacity on POD 1. The correlation coefficients (R2) between diaphragmatic function and spirometry variables ranged from 0.231 to 0.286. Postoperaitve pain was comparable., Conclusion: Combined general and epidural anesthesia may attenuate the severity of postoperative diaphragmatic dysfunction after RALRP compared to conventional general anesthesia.

Introduction: Sepsis often causes diaphragm contractile dysfunction. Dihydropyridine receptors (DHPRα1s and DHPRα1c) and ryanodine receptors (RyR1, RyR2, and RyR3) are essential for excitation-contraction coupling in striated muscles. However, their expression in diaphragm during sepsis have not been explored., Methods: Eight rats received endotoxin, and 8 more rats received placebo. After 24 hours, 3) diaphragm isometric contractile force was measured. The mRNA and protein levels of DHPRs and RyRs in diaphragm muscles were determined., Results: Sepsis weakened diaphragm contractile function. The expression levels of DHPRα1s and RyR1 were significantly lower in septic rats than in control rats. The expression levels of DHPRα1c and RyR3 were unaffected by sepsis. RyR2 was undetectable at both mRNA and protein levels in the control and sepsis groups., Conclusions: Weakened diaphragm contraction in the septic rats was associated with reduced mRNA and protein expression of DHPRα1s and RyR1, the isoforms of skeletal muscles., (Copyright © 2013 Wiley Periodicals, Inc.)

Etomidate is an intravenous anesthetic used during anesthesia induction. This agent induces spontaneous movements, especially myoclonus after its administration suggesting a putative primary effect at the central nervous system or the periphery. Therefore, the aim of this study was to investigate the presynaptic and postsynaptic effects of etomidate at the mouse neuromuscular junction (NMJ). Diaphragm nerve muscle preparations were isolated and stained with the styryl dye FM1-43, a fluorescent tool that tracks synaptic vesicles exo-endocytosis that are key steps for neurotransmission. We observed that etomidate induced synaptic vesicle exocytosis in a dose-dependent fashion, an effect that was independent of voltage-gated Na(+) channels. By contrast, etomidate-evoked exocytosis was dependent on extracellular Ca(2+) because its effect was abolished in Ca(2+)-free medium and also inhibited by omega-Agatoxin IVA (30 and 200nM) suggesting the participation of P/Q-subtype Ca(2+) channels. Interestingly, even though etomidate induced synaptic vesicle exocytosis, we did not observe any significant difference in the frequency and amplitude of miniature end-plate potentials (MEPPs) in the presence of the anesthetic. We therefore investigated whether etomidate could act on nicotinic acetylcholine receptors labeled with α-bungarotoxin-Alexa 594 and we observed less fluorescence in preparations exposed to the anesthetic. In conclusion, our results suggest that etomidate exerts a presynaptic effect at the NMJ inducing synaptic vesicle exocytosis, likely through the activation of P-subtype voltage gated Ca(2+) channels without interfering with MEPPs frequency. The present data contribute to a better understanding about the effect of etomidate at the neuromuscular synapse and may help to explain some clinical effects of this agent., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The effect of insulin on contractility of directly stimulated skeletal muscles was studied in experiments in isolated preparations of rat fast, extensor digitorum longus (m. EDL), slow, soleus (m. SOL) and mixed, diaphragm muscles. In addition (diaphragm only) characteristics of extracellularly recorded muscle fiber action potentials (APs) were evaluated before and after addition of insulin to a bath solution. Insulin (0.5-10 nM) decreased muscle twitch force. This negative inotropic effect of insulin was dose-dependent, with m. SOL appearing to be more sensitive to insulin than either m. EDL or diaphragm. Insulin did not affect strength of isotonic KCL- or caffeine-induced muscle contractures, but decreased second and increased first and third phases of extracellularly recorded muscle fiber APs. The analysis of the data obtained in this study and the data from the literature suggests changes in electrogenesis of the muscle fiber's t-tubular plasma membrane as a key element of the negative inotropic effect of the hormone on contractility of mammalian skeletal musculature. Possible mechanisms of such putative changes are discussed.

Background & Aims: In Duchenne muscular dystrophy (DMD) and in the mdx mouse model of DMD, the lack of dystrophin leads to muscle degeneration and inflammation contributes to progression of the disease. In this study, we evaluated the effects of commercially available fish oil containing EPA and docosahexaenoic acid (DHA) on mdx., Methods: Mdx mice (14 days old) were treated with fish oil (FDC Vitamins; 0.002 g EPA and 0.001 g DHA) for 16 days by gavage. Control mdx mice received mineral oil (Nujol). Grip strength measurement was used for functional evaluation. The sternomastoid, diaphragm and biceps brachii muscles were removed and processed for histopathology and Western blot analysis., Results: Fish oil decreased creatine kinase and myonecrosis. In all muscles studied, the inflammatory area was significantly reduced after treatment (18.0 ± 3.0% inflammatory area in untreated mdx mice versus 4.0 ± 1% in treated mdx mice). Fish oil protected against the loss of muscle strength. Fish oil significantly reduced the levels of TNF-α and the levels of 4-HNE-protein adducts (30-34% reduction for both) in all muscles studied., Conclusions: Commercially available fish oil may be potentially useful to ameliorate dystrophic progression of skeletal muscles, deserving further clinical trials in DMD patients., (Copyright © 2012 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Theraphosid spider venoms can block neurotransmission in vertebrate nerve-muscle preparations in vitro, but few of the components involved have been characterized. In this work, we describe the neuromuscular activity of venom from the Brazilian theraphosid Vitalius dubius and report the purification and pharmacological characterization of VdTX-1, a 728 Da toxin that blocks nicotinic receptors. Neuromuscular activity was assayed in chick biventer cervicis preparations and muscle responses to exogenous ACh and KCl were determined before and after incubation with venom or toxin. Changes in membrane resting potential were studied in mouse diaphragm muscle. The toxin was purified by a combination of filtration through Amicon® filters, cation exchange HPLC and RP-HPLC; toxin purity and mass were confirmed by mass spectrometry. Venom caused progressive neuromuscular blockade and muscle contracture; the blockade but not the contracture was reversible by washing. Venom attenuated contractures to exogenous ACh and KCl. Filtration yielded low (LM, <5 kDa) and high (HM, >5 kDa) fractions, with the latter reproducing the contracture seen in venom but with a slight and progressive twitch blockade. The LM fraction caused reversible blockade and attenuated contractures to ACh, but had no effect on contractures to KCl. VdTX-1 (728 Da) purified from the LM fraction was photosensitive and reduced the E(max) to ACh in biventer cervicis muscle without affecting the EC₅₀; VdTX-1 also abolished carbachol-induced depolarizations. V. dubius venom contains at least two components that affect vertebrate neurotransmission. One component, VdTX-1, blocks nicotinic receptors non-competitively to produce reversible blockade without muscle contracture., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In the present work we investigated the toxic activities of two Bothrops snake venoms using in vivo and in vitro experimental protocols in mice and tested the protective effect of dexamethasone (DEXA) in different conditions, comparing it with the polyvalent antivenom. We also expanded the investigations on the antiophidic effect of the Eclipta prostrata (EP) crude extract. The administration of Bothrops jararaca and Bothrops jararacussu snake venoms induced muscle damage demonstrated in vivo by the elevation on plasma creatine kinase (CK) activity in mice and by the decrease in CK content in the extensor digitorum longus (EDL) muscle of these animals, and in vitro by the increase in the rate of CK release from the isolated EDL muscle. We also observed inflammatory response following perimuscular injection of B. jararacussu venom (1.0 mg/kg). Treatment with DEXA (1.0 mg/kg) preserved over 50% of the EDL muscle CK content in vivo when evaluated 24 and 72 h after the injection of B. jararacussu venom in mice, and likewise reduced about 20% of the edema induced by this venom. DEXA reduced in 50% the presence of inflammatory cells and their activity in EDL muscle. The EP extract (50 mg/kg) showed similar ability in preventing the induction of edema and the decrease in muscle CK content, and its association with DEXA showed additive effect. EP reduced over 77% of the plasma CK activity induced by the B. jararacussu venom. In the in vitro experiments, DEXA was not able to change the rate of CK release from EDL muscles exposed to 25 μg/mL of B. jararacussu venom, neither to prevent the fall in the amplitude of the indirectly evoked twitch at the phrenic-diaphragm preparation. EP extract showed otherwise a protective effect on these protocols, reaching up to 100% of protection when concentrations of 50.0 and 100.0 μg/mL were used. Altogether our results show that inflammation is at least in part responsible for the tissue damage induced by Bothrops snake venoms, once the steroidal anti-inflammatory drug dexamethasone was able to decrease the myotoxic effects of these venoms, by reducing the inflammatory response to the venom injection., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The neuromuscular activity of Bbil-TX, a PLA2 with catalytic activity isolated from Bothriopsis bilineata smargadina venom, was examined in chick biventer cervicis (BC) and mouse phrenic nerve-diaphragm (PND) preparations. In BC preparations, Bbil-TX (0.5-10 μg/ml) caused time- and concentration-dependent blockade that was not reversed by washing; the times for 50% blockade were 87 ± 7, 41 ± 7 and 19 ± 2 min (mean ± SEM; n = 4-6) for 1, 5 and 10 μg/ml, respectively. Muscle contractures to exogenous ACh and KCl were unaffected. The toxin (10 μg/ml) also did not affect the twitch-tension of directly-stimulated, curarized (10 μg/ml) BC preparations. However, Bbil-TX (10 μg/ml) produced mild morphological alterations (edematous and/or hyperchromic fibers) in BC; there was also a progressive release of CK (from 116 ± 17 IU/ml (basal) to 710 ± 91 IU/ml after 45 min). Bbil-TX (5 μg/ml)-induced blockade was markedly inhibited at 22-24 °C and pretreatment with p-bromophenacyl bromide (p-BPB) abolished the neuromuscular blockade. Bbil-TX (3-30 μg/ml, n = 4-6) caused partial time- and concentration-dependent blockade in PND preparations (52 ± 2% at the highest concentration). Bbil-TX (30 μg/ml) also markedly reduced the MEPPs frequency [from 26 ± 2.5 (basal) to 10 ± 1 after 60 min; n = 5; p < 0.05] and the quantal content [from 94 ± 14 (basal) to 24 ± 3 after 60 min; n = 5; p < 0.05] of PND preparations, but caused only minor depolarization of the membrane resting potential [from -80 ± 1 mV (basal) to -66 ± 2 mV after 120 min; n = 5; p < 0.05], with no significant change in the depolarizing response to exogenous carbachol. These results show that Bbil-TX is a presynaptic PLA2 that contributes to the neuromuscular blockade caused by B. b. smargadina venom., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background & Aims: Duchenne muscular dystrophy (DMD) is a genetic muscle disease caused by the absence of dystrophin. An established animal model of DMD is the mdx mouse, which is unable to express dystrophin. Inflammation, particularly the proinflammatory cytokine tumor necrosis factor alpha (TNF-α), strongly contributes to necrosis in the dystrophin-deficient fibers of the mdx mice and in DMD. In this study we investigated whether the antioxidant N-acetylcysteine (NAC) decreases TNF-α levels and protects the diaphragm muscle of mdx mice against necrosis., Methods: Mdx mice (14 days old) received daily intraperitoneal injections of NAC for 14 days, followed by removal of the diaphragm muscle. Control mdx mice were injected with saline., Results: NAC reduced TNF-α and 4-HNE-protein adducts levels, inflammation, creatine kinase levels, and myonecrosis in diaphragm muscle., Conclusions: NAC may be used as a complementary treatment for dystrophinopathies. However, clinical trials are needed to determine the appropriate dose for patients with Duchenne muscular dystrophy., (Copyright © 2012 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

This study looked at how toxic proteins in venoms of adult Australian eastern Brown snakes Pseudonaja textilis from South Australian and Queensland populations interact with physiological functions of the lab SD rat Rattus norvegicus. Circulatory collapse and incoagulable blood occurred instantly after injection of venom under the dorsal skin of anaesthetised and mechanically ventilated rats in an imitation of a P. textilis bite. Intravenous injection of purified P. textilis (Mackay, QLD) venom prothrombin activator proteins caused instant failure of circulation, testifying of high toxicity of these proteins and suggesting their role in rapid incapacitation of rodent prey. The hypothesis is further supported by circulatory collapse occurring instantly despite artificial respiration in envenomed rats and the finding of extremely high venom procoagulant potency in rat plasma. LC-MS and physiology assays revealed divergent venom composition and biological activity of South Australian (Barossa locality) and Queensland (Mackay locality) populations, which may be driven by selection for different prey. The Queensland venom of P. textilis was found to be more procoagulant and to exhibit predominately presynaptic neurotoxicity, while the South Australian venom contained diverse postsynaptic type II and III α-neurotoxins in addition to the presynaptic neurotoxins and caused significantly faster onset of neuromuscular blockade in the rat phrenic nerve-diaphragm preparation. LC-MS analysis found evidence of multiple coagulation factor X-like proteins in P. textilis venoms, including a match to P. textilis coagulation factor X isoform 2, previously known to be expressed only in the liver.

Pulmonary hypertension (PH) causes loss of body weight and inspiratory (diaphragm) muscle dysfunction. A model of PH induced by drug (monocrotaline, MCT) has been extensively used in mice to examine the etiology of PH. However, it is unclear if PH induced by MCT in mice reproduces the loss of body weight and diaphragm muscle dysfunction seen in patients. This is a pre-requisite for widespread use of mice to examine mechanisms of cachexia and diaphragm abnormalities in PH. Thus, we measured body and soleus muscle weight, food intake, and diaphragm contractile properties in mice after 6-8 weeks of saline (control) or MCT (600 mg/kg) injections. Body weight progressively decreased in PH mice, while food intake was similar in both groups. PH decreased (P<0.05) diaphragm maximal isometric specific force, maximal shortening velocity, and peak power. Protein carbonyls in whole-diaphragm lysates and the abundance of select myofibrillar proteins were unchanged by PH. Our findings show diaphragm isometric and isotonic contractile abnormalities in a murine model of PH induced by MCT. Overall, the murine model of PH elicited by MCT mimics loss of body weight and diaphragm muscle weakness reported in PH patients.

Background: The molecular mechanisms underlying the diaphragmatic defect in congenital diaphragmatic hernia (CDH) are still poorly understood. The transcription factor GATA4 is essential for normal development of the diaphragm. Recently, mutations in the GATA4 gene have been linked to human and rodent CDH. We hypothesized that diaphragmatic GATA4 expression is downregulated in the nitrofen CDH model., Methods: Pregnant rats received Nitrofen or vehicle on day 9 of gestation (D9). Fetuses were sacrificed on D13, D18, or D21. Pleuroperitoneal folds (n=20) and fetal diaphragms (n=40) were (micro) dissected and divided into CDH group and controls. RNA and protein were extracted. GATA4 mRNA levels were determined by real-time PCR. Protein levels were determined by ELISA and Immunohistochemistry., Results: mRNA levels and Protein levels were significantly decreased in the CDH group compared to controls on D13 (mRNA 15.96±6.99 vs. 38.10±5.01, p<0.05), D18 (mRNA 10.45±1.84 vs. 17.68±2.11, Protein 2.59±0.06 vs. 4.58±0.35 p<0.05) and D21 (mRNA 4.31±0.83 vs. 6.87±0.88, Protein 0.16±0.08 vs. 1.26±0.49, p<0.05). Immunoreactivity of GATA4 was markedly decreased in CDH-diaphragms on D13, D18, and D21., Conclusions: We provide evidence for the first time that diaphragmatic expression of GATA4 is downregulated in the nitrofen model, suggesting that decreased expression of GATA4 may impair diaphragmatic development in nitrofen-induced CDH., (© 2013 Wiley Periodicals, Inc.)

As selective serotonin reuptake inhibitors have an inhibitory effect on nicotinic acetylcholine receptors, they may affect the neuromuscular transmission and interact with neuromuscular blockers. This study was designed to observe the effect of fluoxetine on neuromuscular transmission and its interaction with rocuronium using the rat phrenic nerve hemidiaphragm and rabbit head drop methods. Rat phrenic nerve hemidiaphragms were mounted and stimulated using a train of four pulses (TOF). The effect of fluoxetine was studied on both indirectly and directly stimulated basal twitch responses by plotting cumulative dose response curves (DRCs). DRCs of rocuronium were obtained in the absence, and presence of 5 μm and 20 μm fluoxetine to study its interaction. ED5 , ED50 and ED95 values of rocuronium DRCs in absence and presence of fluoxetine were calculated. Fluoxetine significantly inhibited twitch responses in both indirect and directly stimulated preparations. Fluoxetine (20 μm) caused an increase in the potency of rocuronium such that the ED50 and ED95 values of rocuronium DRCs were significantly decreased. Partially inhibited twitch responses by fluoxetine (100 μm) were not reversed by neostigmine (3.3 μm) or 3,4 diaminopyridine (0.25 μm). Rabbits were given fluoxetine 0.25 mg kg(-1) and 1 mg kg(-1) orally for 15 days, and on 15th day, rocuronium infusion was given, and time for head drop was recorded. The time of head drop was significantly reduced in fluoxetine pretreated as compared to control group. Fluoxetine blocks the neuromuscular transmission and increases the potency of rocuronium-induced neuromuscular block., (© 2013 Blackwell Publishing Ltd.)

Postsynaptic inhibition is a key element of neural circuits underlying behavior, with 20-50% of all mammalian (nongranule) neurons considered inhibitory. For rhythmic movements in mammals, e.g., walking, swimming, suckling, chewing, and breathing, inhibition is often hypothesized to play an essential rhythmogenic role. Here we study the role of fast synaptic inhibitory neurotransmission in the generation of breathing pattern by blocking GABA(A) and glycine receptors in the preBötzinger complex (preBötC), a site essential for generation of normal breathing pattern, and in the neighboring Bötzinger complex (BötC). The breathing rhythm continued following this blockade, but the lung inflation-induced Breuer-Hering inspiratory inhibitory reflex was suppressed. The antagonists were efficacious, as this blockade abolished the profound effects of the exogenously applied GABA(A) receptor agonist muscimol or glycine, either of which under control conditions stopped breathing in vagus-intact or vagotomized, anesthetized, spontaneously breathing adult rats. In vagotomized rats, GABA(A)ergic and glycinergic antagonists had little, if any, effect on rhythm. The effect in vagus-intact rats was to slow the rhythm to a pace equivalent to that seen after suppression of the aforementioned Breuer-Hering inflation reflex. We conclude that postsynaptic inhibition within the preBötC and BötC is not essential for generation of normal respiratory rhythm in intact mammals. We suggest the primary role of inhibition is in shaping the pattern of respiratory motor output, assuring its stability, and in mediating reflex or volitional apnea, but not in the generation of rhythm per se.

Background: Interscalene brachial plexus block (ISBPB) provides excellent analgesia after rotator cuff surgery but is associated with diaphragm dysfunction. In this study, ISBPB with 20 mL of 0.125% or 0.25% bupivacaine were compared to assess the effect on diaphragm function, oxygen saturation, pain control, opioid requirements, and patient satisfaction., Materials and Methods: In this prospective, randomized, double-blind study, 30 adults undergoing outpatient arthroscopic rotator cuff repair were enrolled to receive ultrasound-guided interscalene brachial plexus catheter placement with 20 mL of 0.125% (n = 15) or 0.25% bupivacaine (n = 15). Diaphragm function and oxygen saturation were assessed before ISBPB placement and on discharge from the postanesthesia care unit. Postoperative pain scores, opioid requirements, and patient satisfaction were compared., Results: Diaphragm function and oxygen saturation were superior in the low concentration group. Absent or paradoxic motion of the diaphragm was present in 78% of the 0.25% group compared with 21% of patients in the 0.125% group (P = .008). Oxygen saturation decreased 4.3% in the 0.25% group compared with a decrease of 2.6% in the 0.125% group (P = .04). Pain scores averaged 1 of 10 in the 0.25% group and 0 of 10 in the 0.125% group (P = .02). Opioid requirements and patient satisfaction were not different between the two groups., Conclusions: In this randomized, double-blind comparison of ISBPB performed with 20 mL of 0.125% or 0.25% bupivacaine, diaphragm function and oxygen saturation were superior in patients treated with more dilute bupivacaine. Furthermore, there were no clinically significant differences in pain scores, and no statistically significant differences in opioid requirements and patient satisfaction., (Copyright © 2013 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Mosby, Inc. All rights reserved.)

The potency of three newly developed bispyridinium compounds (K454, K456, K458) to reactivate tabun-inhibited acetylcholinesterase and reduce tabun-induced lethal toxic effects was compared with the oxime K203 and trimedoxime using in vivo methods. The study determining percentage of reactivation of tabun-inhibited diaphragm and brain acetylcholinesterase in poisoned rats showed that the reactivating efficacy of all newly developed oximes is comparable with K203 but lower than the reactivating potency of trimedoxime in diaphragm. In the brain, their potency to reactivate tabun-inhibited acetylcholinesterase is lower compared with trimedoxime and the oxime K203. All three newly developed oximes were also found to be relatively effective in reducing lethal toxic effects in tabun-poisoned mice. Their therapeutic efficacy is consistent with the therapeutic potency of the oxime K203. On the other hand, their potency to reduce acute toxicity of tabun is significantly lower compared with trimedoxime. In conclusion, the reactivating and therapeutic potency of all three newly developed oximes does not prevail the effectiveness of the oxime K203 and trimedoxime and, therefore, they are not suitable for their replacement of commonly used oximes for the treatment of acute tabun poisoning.

Ethnopharmacological Relevance: Chromolaena odorata Linn., is used in traditional Indian medicine in the treatment of diabetes and eye problems., Aim of the Study: The present study was designed to investigate the effect of the ethanol extract Chromolaena odorata leaves (ACO) in streptozotocin (STZ; 45 mg/kg, i.v) induced diabetes and cataract in rats., Materials and Methods: Different doses of ACO (200 and 400mg/kg) was administered once daily for eight weeks to STZ-induced diabetic rats. To know the mechanism of action of title plant, AUC(glucose), AUC(insulin), Homeostatic Model Assessment (HOMA), insulin tolerance test (ITT) and glucose uptake by rat hemi-diaphragms were carried out. Further, cataract score was taken once in a week upto eight weeks and opacity index was measured. HPLC fingerprinting profiling of ACO was also carried out., Results: Administration of ACO exhibited significant reduction in glucose, HOMA, lipid profiles and significantly improved glucose and insulin tolerance, glycogen content, glucose uptake by skeletal muscle, serum insulin and HDL-c levels. In addition, ACO also decreased oxidative stress by improving endogenous antioxidants. Further, treatment of ACO showed significantly reduced onset and extent of cataract., Conclusion: The present data suggested that the treatment of ACO reversed the STZ-induced diabetes and cataract in rats. The observed beneficial effects may be mediated by interacting with multiple targets operating in diabetes mellitus and its complication. Taken together, this study provided the scientific evidence for the traditional use of Chromolaena odorata., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

This work was undertaken to investigate the toxic activity of the post-synaptic neurotoxic fraction isolated from the venom of the Egyptian sand viper (Cerastes cerastes cerastes), and the ability of grape seed extract to antagonize this effect produced in sublethally intoxicated mice, with an emphasis on ultrastructural features. Light and transmission electron microscopy of diaphragms of intoxicated mice showed myonecrosis, myofiber hypercontraction, sarcomere disorganization, and mitochondrial damage. Alterations in motor neurons and axon terminals were also observed. The toxic activities of C. cerastes cerastes neurotoxin were inhibited by administrating grape seed extract, either before or after intoxication, showing that grape seed extract has protective and therapeutic potential to be used as antivenom., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The neuroprotection induced by Hypericum brasiliense Choisy extract (HBE) and its main active polyphenol compound quercetin, against Crotalus durissus terrificus (Cdt) venom and crotoxin and crotamine, was enquired at both central and peripheral mammal nervous system. Cdt venom (10 μg/mL) or crotoxin (1 μg/mL) incubated at mouse phrenic nerve-diaphragm preparation (PND) induced an irreversible and complete neuromuscular blockade, respectively. Crotamine (1 μg/mL) only induced an increase of muscle strength at PND preparations. At mouse brain slices, Cdt venom (1, 5, and 10 μg/mL) decreased cell viability. HBE (100 μg/mL) inhibited significantly the facilitatory action of crotamine (1 μg/mL) and was partially active against the neuromuscular blockade of crotoxin (1 μg/mL) (data not shown). Quercetin (10 μg/mL) mimicked the neuromuscular protection of HBE (100 μg/mL), by inhibiting almost completely the neurotoxic effect induced by crotoxin (1 μg/mL) and crotamine (1 μg/mL). HBE (100 μg/mL) and quercetin (10 μg/mL) also increased cell viability in mice brain slices. Quercetin (10 μg/mL) was more effective than HBE (100 μg/mL) in counteracting the cell lysis induced by Cdt venom (1 and 10 μg/mL, resp.). These results and a further phytochemical and toxicological investigations could open new perspectives towards therapeutic use of Hypericum brasiliense standardized extract and quercetin, especially to counteract the neurotoxic effect induced by snake neurotoxic venoms.

The effects of microinjections of the excitatory neurotoxin kainic acid (2 mg/ml; 49 ± 1 nl) on the mechanically induced tracheobronchial cough, sneeze, and solitary expulsions from the trachea were examined in 11 anesthetized rabbits. Kainic acid was injected into the medulla (1.6-2.8 mm rostral to the obex, 1.4-1.6 and 2.9-3.2 mm below the dorsal medullary surface). Blood pressure, esophageal pressure (EP), and electromyograms (EMGs) of the diaphragm (DIA) and abdominal muscles (ABD) were recorded. Kainic acid reduced the number of coughs (means ± SE) from 3.8 ± 2.0 to 0.9 ± 0.7 (p = 0.016), the amplitude of DIA cough from 90 ± 11 to 42 ± 13 % (p = 0.004), ABD EMG moving average from 103 ± 9 to 37 ± 15 % (p = 0.006), and inspiratory from 0.67 ± 0.13 to 0.36 ± 0.12 kPa (p = 0.013) and expiratory EP from 1.70 ± 0.54 to 0.89 ± 0.46 kPa (p = 0.008). Kainic acid had no effect on the number of sneeze reflexes nor did it affect solitary expulsions from the trachea. These effects were accompanied by significant increases in systemic blood pressure and respiratory rate. Spatiotemporal analysis of the cough and sneeze reflexes revealed increases in the duration of cough active expiratory phase, in the intervals between maxima of DIA and ABD EMG discharges, and in the active portion of total cough phase duration. Our findings suggest a diverse role of raphe neurons in the central control of motor airway responses such as coughing and sneezing. A complex function of raphe neurons in the generation of the cough motor pattern also is suggested.

Opioid receptors which are involved in cough generation are abundantly expressed in the brainstem. Codeine is a potent μ-opioid receptor agonist. In the present study we examined the effects of naloxone, a μ-opioid receptor antagonist, on mechanically-induced tracheobronchial cough and on the cough suppressing effect of codeine in six pentobarbitone anesthetized spontaneously breathing rabbits. A single dose of naloxone (0.4 mg/kg) followed by a single dose of codeine (7 mg/kg) were administered intravenously. The number and amplitude of cough and sneeze reflexes were examined sequentially; before and after naloxone, and then after codeine. We found that neither did naloxone alone nor codeine given after prior naloxone pretreatment appreciably affect coughing or sneezing. Likewise, there were no significant differences in the diaphragm and abdominal muscles electromyographic moving averages, or the inspiratory and expiratory esophageal pressure amplitudes. However, we detected a tendency for the rise in expiratory motor drive during coughing and sneezing after injection of naloxone. The respiratory rate was significantly higher after naloxone in comparison with control (P < 0.001). No significant differences in arterial blood pressure were observed. We conclude that the failure of codeine to suppress the cough reflex on the background of naloxone pretreatment confirms the involvement of μ-opioid mechanism in the central antitussive effect of codeine.

Understanding the biological activity profile of the snake venom components is fundamental for improving the treatment of snakebite envenomings and may also contribute for the development of new potential therapeutic agents. In this work, we tested the effects of BthTX-I, a Lys49 PLA(2) homologue from the Bothrops jararacussu snake venom. While this toxin induces conspicuous myonecrosis by a catalytically independent mechanism, a series of in vitro studies support the hypothesis that BthTX-I might also exert a neuromuscular blocking activity due to its ability to alter the integrity of muscle cell membranes. To gain insight into the mechanisms of this inhibitory neuromuscular effect, for the first time, the influence of BthTX-I on nerve-evoked ACh release was directly quantified by radiochemical and real-time video-microscopy methods. Our results show that the neuromuscular blockade produced by in vitro exposure to BthTX-I (1 μM) results from the summation of both pre- and postsynaptic effects. Modifications affecting the presynaptic apparatus were revealed by the significant reduction of nerve-evoked [(3)H]-ACh release; real-time measurements of transmitter exocytosis using the FM4-64 fluorescent dye fully supported radiochemical data. The postsynaptic effect of BthTX-I was characterized by typical histological alterations in the architecture of skeletal muscle fibers, increase in the outflow of the intracellular lactate dehydrogenase enzyme and progressive depolarization of the muscle resting membrane potential. In conclusion, these findings suggest that the neuromuscular blockade produced by BthTX-I results from transient depolarization of skeletal muscle fibers, consequent to its general membrane-destabilizing effect, and subsequent decrease of evoked ACh release from motor nerve terminals., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

We previously demonstrated that diaphragm muscle weakness is present in experimental pulmonary arterial hypertension (PH). However, the nature of this diaphragm weakness is still unknown. Therefore, the aim of this study was to investigate whether changes at the sarcomeric level contribute to diaphragm weakness in PH. For this purpose, in control rats and rats with monocrotaline-induced PH, contractile performance and myosin heavy chain content of demembranated single diaphragm fibers were determined. We observed a reduced maximal tension of 20% (P < 0.05), whereas tension cost was preserved in type 2X and 2B diaphragm fibers in PH compared with control. The reduced maximal tension was associated with a reduction of force generated per half-sarcomeric myosin heavy chain content. Additionally, reduced Ca(2+) sensitivity of force generation was found in type 2X fibers compared with control, which could exacerbate diaphragm muscle weakness at submaximal activation. No changes in maximal tension and Ca(2+) sensitivity of force generation were observed in fibers from the nonrespiratory extensor digitorum longus muscle. Together, these findings indicate that diaphragm weakness in PH is at least partly caused by sarcomeric dysfunction, which appears to be specific for the diaphragm.

Purpose: Rocuronium has been associated with muscle weakness when administered in prolonged infusions. The effect of sugammadex and rocuronium together on muscle is unknown. In this study, we examined the effects of rocuronium and sugammadex, and the complex formed by these agents, on cardiac and diaphragmatic muscle cells., Methods: Forty-two Sprague-Dawley male rats were divided into six groups. Group I received only rocuronium at a dose of 1 mg/kg and groups II and III received sugammadex alone at doses of 16 and 96 mg/kg, respectively. Groups IV and V received 1 mg/kg rocuronium plus 16 mg/kg sugammadex and 1 mg/kg rocuronium plus 96 mg/kg sugammadex, respectively. Group six was the control group and received only 0.9 % NaCl without any drug., Results: Histopathological examination demonstrated that rocuronium and high doses of sugammadex accumulated in both cardiac and diaphragm muscle tissues. We also observed intense edema and degeneration in diaphragmatic and myocardial cells when the rocuronium-sugammadex complex was used. Rocuronium and sugammadex remain in the circulation for a long time and they may cause skeletal muscle myopathy, vacuolization, pyknotic nuclear clumps, and hypertrophy, and weaken the muscle fibers., Conclusion: Rocuronium, sugammadex, and rocuronium-sugammadex complexes cause histopathological changes and immunoreactivity to calcineurin in muscle cells.

Background: Respiratory difficulties are sometimes reported by patients with cranial-cervical and other forms of dystonia, but the nature or mechanisms of the breathing problems have not been well characterized., Case Report: We review 13 dystonic patients with various respiratory complaints and describe their symptoms and response to treatment, including botulinum toxin (BoNT) injections., Discussion: Gasping, stridor, interrupted flow of speech, paradoxical breathing, dyspnea on exertion, and other respiratory symptoms reported by patients suggest involvement of the upper airways, chest, and diaphragm. BoNT injections may be, at least partially, beneficial in some patients., Conclusion: This series of patients draws attention to respiratory distress as a potentially serious, even life-threatening, complication of dystonia., (Copyright © 2012 Movement Disorder Society.)

Botulinum neurotoxins (BoNTs) comprise a family of neurotoxic proteins synthesized by anaerobic bacteria of the genus Clostridium. Each neurotoxin consists of two polypeptide chains: a 100kDa heavy chain, responsible for binding and internalization into the nerve terminal of cholinergic motoneurons and a 50kDa light chain that mediates cleavage of specific synaptic proteins in the host nerve terminal. Exposure to BoNT leads to cessation of voltage- and Ca(2+)-dependent acetylcholine (ACh) release, resulting in flaccid paralysis which may be protracted and potentially fatal. There are no approved therapies for BoNT intoxication once symptoms appear, and specific inhibitors of the light chain developed to date have not been able to reverse the consequences of BoNT intoxication. An alternative approach for treatment of botulism is to focus on compounds that act by enhancing ACh release. To this end, we examined the action of the K(+) channel blocker 3,4-diaminopyridine (3,4-DAP) in isolated mouse hemidiaphragm muscles intoxicated with 5pM BoNT/A. 3,4-DAP restored tension within 1-3min of application, and was effective even in totally paralyzed muscle. The Ca(2+) channel activator (R)-roscovitine (Ros) potentiated the action of 3,4-DAP, allowing for use of lower concentrations of the K(+) channel blocker. In the absence of 3,4-DAP, Ros was unable to augment tension in BoNT/A-intoxicated muscle. This is the first report demonstrating the efficacy of the combination of 3,4-DAP and Ros for the potential treatment of BoNT/A-mediated muscle paralysis., (Published by Elsevier Ltd.)

Introduction: Glucocorticoids are the only drugs available for the treatment of Duchenne muscular dystrophy (DMD), but it is unclear whether their efficacy is dependent on their anti-inflammatory activity., Methods: To address this issue, mdx mice were treated daily with methylprednisolone and non-steroidal anti-inflammatory drugs (NSAIDs: aspirin, ibuprofen, parecoxib)., Results: NSAID treatment was effective in ameliorating muscle morphology and reducing macrophage infiltration and necrosis. The percentage of regenerating myofibers was not modified by the treatments. The drugs were effective in reducing COX-2 expression and inflammatory cytokines, but they did not affect utrophin levels. The effects of the treatments on contractile performance were analyzed. Isometric tension did not differ in treated and untreated muscle, but the resistance to fatigue was decreased by treatment with methylprednisolone and aspirin., Conclusions: NSAIDs have a beneficial effect on mdx muscle morphology, pointing to a crucial role of inflammation in the progression of DMD., (Copyright © 2012 Wiley Periodicals, Inc.)