Influence of innate immune activation on endocrine and metabolic pathways in infancy.

Prematurity is the leading cause of neonatal morbidity and mortality worldwide. Premature infants often require extended hospital stays, with increased risk of developing infection compared with term infants. A picture is emerging of wide-ranging deleterious consequences resulting from innate immune system activation in the newborn infant. Those who survive infection have been exposed to a stimulus that can impose long-lasting alterations into later life. In this review, we discuss sepsis-driven alterations in integrated neuroendocrine and metabolic pathways and highlight current knowledge gaps in respect of neonatal sepsis. We review established biomarkers for sepsis and extend the discussion to examine emerging findings from human and animal models of neonatal sepsis that propose novel biomarkers for early identification of sepsis. Future research in this area is required to establish a greater understanding of the distinct neonatal signature of early and late-stage infection, to improve diagnosis, curtail inappropriate antibiotic use, and promote precision medicine through a biomarker-guided empirical and adjunctive treatment approach for neonatal sepsis. There is an unmet clinical need to decrease sepsis-induced morbidity in neonates, to limit and prevent adverse consequences in later life and decrease mortality.

Sex differences in murine sternohyoid muscle tolerance of acute severe hypoxic stress.

Given that sex differences inherent to muscle might at least contribute to male risk for obstructive sleep apnoea syndrome (OSAS), our objective was to test the hypothesis that male sternohyoid muscle exhibits greater susceptibility to severe hypoxic stress compared with female muscle. Adult male and female C57Bl6/J mouse sternohyoid isometric and isotonic functional properties were examined ex vivo at 35 °C in tissue baths under control and severe hypoxic conditions. Hypoxia was detrimental to peak force (Fmax), work (Wmax) and power (Pmax), but not shortening velocity (Vmax). Two-way analysis of variance revealed a significant sex x gas interaction for Fmax (p<0.05), revealing inferior hypoxic tolerance in male sternohyoid muscle. However, increases in male shortening velocity in severe hypoxia preserved power-generating capacity which was equivalent to values determined in female muscle. Fmax decline in hypoxic female sternohyoid was considerably less than in male muscle, illustrating an inherent tolerance of force-generating capacity mechanisms to hypoxic stress in female airway dilator muscle. We speculate that this could confer a distinct advantage in vivo in terms of the defense of upper airway caliber.

In vivo neutralization of IL-6 receptors ameliorates gastrointestinal dysfunction in dystrophin-deficient mdx mice.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a fatal disease characterized by progressive deterioration and degeneration of striated muscle. A mutation resulting in the loss of dystrophin, a structural protein which protects cells from contraction-induced damage, underlies DMD pathophysiology. Damage to muscle fibers results in chronic inflammation and elevated levels of proinflammatory cytokines such as interleukin-6 (IL-6). However, loss of cellular dystrophin also affects neurons and smooth muscle in the gastrointestinal (GI) tract with complaints such as hypomotility, pseudo-obstruction, and constipation reported in DMD patients. METHODS: Using dystrophin-deficient mdx mice, studies were carried out to examine colonic morphology and function compared with wild-type mice. Treatment with neutralizing IL-6 receptor antibodies (xIL-6R) and/or the corticotropin-releasing factor (CRF) 2 receptor agonist, urocortin 2 (uro2) was tested to determine if they ameliorated GI dysfunction in mdx mice. KEY RESULTS: Mdx mice exhibited thickening of colonic smooth muscle layers and delayed stress-induced defecation. In organ bath studies, neurally mediated IL-6-evoked contractions were larger in mdx colons. In vivo treatment of mdx mice with xIL-6R normalized defecation rates and colon lengths. Uro2 treatment did not affect motility or morphology. The potentiated colonic contractile response to IL-6 was attenuated by treatment with xIL-6R. CONCLUSIONS & INFERENCES: These findings confirm the importance of dystrophin in normal GI function and implicate IL-6 as an important regulator of GI motility in the mdx mouse. Inhibition of IL-6 signaling may offer a potential new therapeutic strategy for treating DMD-associated GI symptoms.

The ß2 -adrenoceptor agonist terbutaline recovers rat pharyngeal dilator muscle force decline during severe hypoxia.

RATIONALE: Obstructive sleep apnoea syndrome (OSAS) is a debilitating condition characterized by recurrent occlusions of the pharyngeal airway during sleep accompanied by arterial hypoxaemia. Upper airway muscle dysfunction is implicated in the pathophysiology of OSAS. Pharmacological agents that improve muscle contractile and endurance properties may have therapeutic value. AIM: We tested the hypothesis that the ß(2) -adrenoceptor agonist terbutaline improves rat sternohyoid muscle performance especially during hypoxic stress. METHODS: Isometric contractile and endurance properties were examined ex vivo in Krebs solution at 35°C. Muscles were incubated in tissue baths under hyperoxic (95% O(2) /5% CO(2)) conditions in the absence (control) or presence of the ß(2) -adrenoceptor agonist terbutaline (1 µM). In additional experiments under hypoxic (95% N(2) /5% CO(2)) conditions, the effects of terbutaline were examined in the presence of the ß-adrenoceptor antagonist propranolol (1 µM). RESULTS: Hypoxia significantly impaired sternohyoid force production. Terbutaline completely recovered hypoxic depression of force, an effect that was blocked by co-application with propranolol. CONCLUSION: The ß(2) -adrenoceptor agonist terbutaline completely recovers hypoxic depression of upper airway muscle force. ß(2) -adrenoceptor agonists warrant investigation in animal models of OSAS reporting upper airway and diaphragm muscle dysfunction.

Hydrogen peroxide alters sternohyoid muscle function.

Upper airway (UA) dilator muscles are critical for the maintenance of airway patency. Injury or fatigue to this group of muscles, as observed in patients with obstructive sleep apnoea (OSA) and animal models of OSA, may leave the UA susceptible to collapse. Although the mechanisms underlying respiratory muscle dysfunction are not completely understood, there is strong evidence suggesting a link between increased production of reactive oxygen species and altered muscle function. The aim of this study was to examine the effects of H2O2 on rat sternohyoid muscle function in vitro. Sternohyoid contractile and endurance properties were examined at 35 °C under control or hypoxic conditions. Studies were conducted in the presence of varying concentrations of H2O2 (0, 0.01, 0.1 and 1 mM). Muscle function was also examined in the presence of antioxidants [desferoxamine (DFX), catalase] and the reducing agent dithiothreitol (DTT). H2O2 decreased muscle endurance in a concentration-dependent manner. This was partially reversed by catalase, DFX and DTT. Our results suggest that oxidants may contribute to UA respiratory muscle dysfunction with implications for the control of UA patency in vivo.

Sternohyoid and diaphragm muscle form and function during postnatal development in the rat.

NEW FINDINGS: What is the central question of this study? Co-ordinated activity of the thoracic pump and pharyngeal dilator muscles is critical for maintaining airway calibre and respiratory homeostasis. Whilst postnatal maturation of the diaphragm has been well characterized, surprisingly little is known about the developmental programme in the airway dilator muscles. What is the main finding and its importance? Developmental increases in force-generating capacity and fatigue in the sternohyoid and diaphragm muscles are attributed to a maturational shift in muscle myosin heavy chain phenotype. This maturation is accelerated in the sternohyoid muscle relative to the diaphragm and may have implications for the control of airway calibre in vivo. The striated muscles of breathing, including the thoracic pump and pharyngeal dilator muscles, play a critical role in maintaining respiratory homeostasis. Whilst postnatal maturation of the diaphragm has been well characterized, surprisingly little is known about the developmental programme in airway dilator muscles given that co-ordinated activity of both sets of muscles is needed for the maintenance of airway calibre and effective pulmonary ventilation. The form and function of sternohyoid and diaphragm muscles from Wistar rat pups [postnatal day (PD) 10, 20 and 30] was determined. Isometric contractile and endurance properties were examined in tissue baths containing Krebs solution at 35°C. Myosin heavy chain (MHC) isoform composition was determined using immunofluorescence. Muscle oxidative and glycolytic capacity was assessed by measuring the activities of succinate dehydrogenase and glycerol-3-phosphate dehydrogenase using semi-quantitative histochemistry. Sternohyoid and diaphragm peak isometric force and fatigue increased significantly with postnatal maturation. Developmental myosin disappeared by PD20, whereas MHC2B areal density increased significantly from PD10 to PD30, emerging earlier and to a much greater extent in the sternohyoid muscle. The numerical density of fibres expressing MHC2X and MHC2B increased significantly during development in the sternohyoid. Diaphragm succinate dehydrogenase activity and sternohyoid glycerol-3-phosphate dehydrogenase activity increased significantly with age. Developmental increases in force-generating capacity and fatigue in the sternohyoid and diaphragm muscles are attributed to a postnatal shift in muscle MHC phenotype. The accelerated maturation of the sternohyoid muscle relative to the diaphragm may have implications for the control of airway calibre in vivo.

Upper airway dilator muscle weakness following intermittent and sustained hypoxia in the rat: effects of a superoxide scavenger.

Obstructive sleep apnoea syndrome (OSAS) is a common disorder associated with upper airway muscle dysfunction. Agents that improve respiratory muscle performance may have considerable therapeutic value. We examined the effects of acute exposure to sustained and intermittent hypoxia on rat pharyngeal dilator muscle function. Additionally, we sought to test the efficacy of antioxidant treatment in ameliorating or preventing hypoxia-related muscle dysfunction. Isometric contractile and endurance properties of isolated rat sternohyoid muscle bundles were examined at 35 °C in vitro. Muscle bundles were exposed to one of four gas treatments: hyperoxia (control), sustained hypoxia (SH), intermittent hypoxia (IH) or hypoxia/re-oxygenation (HR), in the absence or presence of the superoxide scavenger--Tempol (10 mM). Stress-frequency relationship was determined in response to electrical stimulation (10-100 Hz in increments of 10-20 Hz, train duration: 300 ms). Muscle performance was also assessed during repetitive muscle stimulation (40 Hz, 300 ms every 2 s for 2.5 min). Compared to control, IH and HR treatments significantly decreased sternohyoid muscle force. The negative inotropic effect of the two gas protocols was similar, but both were of lesser magnitude than the effects of SH. SH, but not IH and HR, increased muscle fatigue. Tempol significantly increased sensitivity to stimulation in all muscle preparations and caused a leftward shift in the stress-frequency relationship of IH and SH treated muscles. Tempol did not ameliorate sternohyoid muscle fatigue during SH. We conclude that Tempol increases upper airway muscle sensitivity to stimulation but only modestly ameliorates respiratory muscle weakness during intermittent and sustained hypoxic conditions in vitro. Respiratory muscle fatigue during sustained hypoxia appears unrelated to oxidative stress.

Chronic hypobaric hypoxia increases isolated rat fast-twitch and slow-twitch limb muscle force and fatigue.

Chronic hypoxia alters respiratory muscle force and fatigue, effects that could be attributed to hypoxia and/or increased activation due to hyperventilation. We hypothesized that chronic hypoxia is associated with phenotypic change in non-respiratory muscles and therefore we tested the hypothesis that chronic hypobaric hypoxia increases limb muscle force and fatigue. Adult male Wistar rats were exposed to normoxia or hypobaric hypoxia (PB=450 mm Hg) for 6 weeks. At the end of the treatment period, soleus (SOL) and extensor digitorum longus (EDL) muscles were removed under pentobarbitone anaesthesia and strips were mounted for isometric force determination in Krebs solution in standard water-jacketed organ baths at 25 °C. Isometric twitch and tetanic force, contractile kinetics, force-frequency relationship and fatigue characteristics were determined in response to electrical field stimulation. Chronic hypoxia increased specific force in SOL and EDL compared to age-matched normoxic controls. Furthermore, chronic hypoxia decreased endurance in both limb muscles. We conclude that hypoxia elicits functional plasticity in limb muscles perhaps due to oxidative stress. Our results may have implications for respiratory disorders that are characterized by prolonged hypoxia such as chronic obstructive pulmonary disease (COPD).

Comparison of the motor discharge to the voluntary sphincters of continence in the rat.

BACKGROUND: The rat external anal sphincter (EAS) and external urethral sphincter (EUS) are voluntary muscles of continence that can display similar synchronous electromyographic (EMG) activity patterns. However, the two sphincters are quite different in structure and function. The EUS is a fast twitch muscle and contains fibers expressing type 2B myosin. In contrast, the EAS exhibits slower kinetics and lacks type 2B fibers. This striking contrast in kinetics and fiber type profiles may be shaped by differences in the basal motor drive that each sphincter receives. METHODS: A double EMG approach was used to obtain spontaneously active single motor unit action potentials from the EUS and EAS simultaneously and compare their basal discharge frequencies in urethane anaesthetized rats. KEY RESULTS: The basal firing rates of motor units of the EUS and EAS were not significantly different (3.9 ± 0.9 Hz vs. 3.1 ± 1.6 Hz, respectively, n = 7 animals, P = 0.32, paired Student's t-test). However, auto-correlogram analysis showed that EUS is driven by neurons with faster instantaneous firing frequencies: 30.5 ± 2.4 Hz vs 14.3 ± 0.9 Hz in the EAS (P = 0.03, paired Student's t-test). CONCLUSIONS & INFERENCES: The oscillator(s) driving the EUS operate(s) at a frequency twice that of the EAS. This may explain the presence of type 2B fibers in the EUS. In the inter-micturition periods no cross correlation was found in motor discharge to the sphincters suggesting that the two muscles do not share a common central drive to sustain the continent tonus of the two outlet tracts.

Chronic hypoxia increases rat diaphragm muscle endurance and sodium-potassium ATPase pump content.

The effects of chronic hypoxia (CH) on respiratory muscle are poorly understood. The aim of the present study was to examine the effects of CH on respiratory muscle structure and function, and to determine whether nitric oxide is implicated in respiratory muscle adaptation to CH. Male Wistar rats were exposed to CH for 1-6 weeks. Sternohyoid and diaphragm muscle contractile properties, muscle fibre type and size, the density of fibres expressing sarco/endoplasmic reticulum calcium-ATPase (SERCA) 2 and sodium-potassium ATPase (Na+,K+-ATPase) pump content were determined. Muscle succinate dehydrogenase (SDH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) dehydrogenase activities were also assessed. Acute and chronic blockade of nitric oxide synthase (NOS) was employed to determine whether or not NO is critically involved in functional remodelling in CH muscles. CH improved diaphragm, but not sternohyoid, fatigue tolerance in a time-dependent fashion. This adaptation was not attributable to increased SDH or NADPH dehydrogenase activities. The areal density of muscle fibres and relative area of fibres expressing SERCA2 were unchanged. Na+,K+-ATPase pump content was significantly increased in CH diaphragm. Chronic NOS inhibition decreased diaphragm Na+,K+-ATPase pump content and prevented CH-induced increase in muscle endurance. This study provides novel insight into the mechanisms involved in CH-induced muscle plasticity. The results may be of relevance to respiratory disorders characterised by CH, such as chronic obstructive pulmonary disease.

Ventilatory drive is enhanced in male and female rats following chronic intermittent hypoxia.

Obstructive sleep apnoea is characterized by chronic intermittent hypoxia (CIH) due to recurrent apnoea. We have developed a rat model of CIH, which shows evidence of impaired respiratory muscle function. In this study, we wished to characterize the ventilatory effects of CIH in conscious male and female animals. Adult male (n=14) and female (n=8) Wistar rats were used. Animals were placed in chambers daily for 8 h with free access to food and water. The gas supply to one half of the chambers alternated between air and nitrogen every 90 s, for 8 h per day, reducing ambient oxygen concentration in the chambers to 5% at the nadir (intermittent hypoxia; n=7 male, n=4 female). Air supplying the other chambers was switched every 90 s to air from a separate source, at the same flow rates, and animals in these chambers served as controls (n=7 male, n=4 female). Ventilatory measurements were made in conscious animals (typically sleeping) after 10 days using whole-body plethysmography. Normoxic ventilation was increased in both male and female CIH-treated rats compared to controls but this did not achieve statistical significance. However, ventilatory drive was increased in CIH-treated rats of both sexes as evidenced by significant increases in mean and peak inspiratory flow. Ventilatory responses to acute hypoxia (F(I)O(2) = 0.10; 6 min) and hyperoxic hypercapnia (F(I)CO(2) = 0.05; 6 min) were unaffected by CIH treatment in male and female rats (P>0.05, ANOVA). We conclude that CIH increases respiratory drive in adult rats. We speculate that this represents a form of neural plasticity that may compensate for respiratory muscle impairment that occurs in this animal model.

Upper airway pressure-flow relationships and pharyngeal constrictor EMG activity during prolonged expiration in awake goats.

We undertook the present investigation to establish whether narrowing/closure of the upper airway occurs during spontaneous and provoked respiratory rhythm disturbances and whether pharyngeal constrictor muscle recruitment occurs coincident with upper airway occlusion during prolonged expiratory periods. Upper airway pressure-flow relationships and middle pharyngeal constrictor (mPC) EMG activities were recorded in 11 adult female goats during spontaneous and provoked prolongations in expiratory time (Te). A total of 213 spontaneous prolongations of expiration were recorded. Additionally, 169 prolonged expiratory events preceded by an augmented breath were included in the analyses. In separate trials on different days, Te was prolonged by systemic administration of dopamine, by raising the inspired fraction of O(2) from 0.10 to 1.00 during poikilocapnic conditions or by systemic administration of clonidine. Continuous tonic activation of the mPC EMG was observed during each prolonged Te period regardless of the duration or initiating cause. However, significant increases in subglottic tracheal pressure, with expiratory airflow braking indicative of upper airway narrowing or closure, was only observed during spontaneous events without a preceding augmented breath and during clonidine-induced events. Tonic mPC activation proved an unreliable indicator of airway occlusion. Furthermore, mPC muscle activation alone is not sufficient to induce pharyngeal occlusion during prolonged expiration. Our data suggest that airway closure is not a common occurrence during provoked respiratory disturbances in awake goats. We propose that airway closure, when present during prolonged Te, is more likely dependent on activation of laryngeal adductor muscles with glottic braking independent of pharyngeal narrowing.

The effects of breath-holds and Muller manoeuvres on upper airway carbon dioxide concentration in humans.

BACKGROUND: Obstructive sleep apnoea is caused by collapse of the upper airway. The presence of CO(2) in the upper airway lumen evokes a number of reflexes which favour upper airway re-opening, and we have proposed previously that CO(2) would build up in the upper airway following airway collapse and that this would contribute to reflex airway re-opening. However, it is not known if CO(2) can transfer from the alveoli to the anatomical dead space of the upper airway during apnoea. OBJECTIVES: To determine if alveolar CO(2) can enter the upper airway during breath-holds and Muller manoeuvres. MATERIAL AND METHODS: With local ethics committee approval, 6 male volunteers (aged 22-48 years), following a quiet inspiration, carried out breath-holds and Muller manoeuvres until breaking point. CO(2) was measured continuously in samples obtained from the hypopharynx using an infrared analyser with a sample rate of 50 ml/min. Muller manoeuvres (forced inspirations against a closed upper airway) mimic the respiratory efforts which occur during obstructive apnoeas. RESULTS: In all cases, CO(2) increased progressively during apnoeas. There was a much larger increase in Muller manoeuvres (3.78 +/- 0.51%, mean +/- SEM at breaking point) compared to breath-holds. DISCUSSION: These results show that upper airway CO(2) concentration rises substantially during apnoeas and suggest that transfer of CO(2) from the lungs to the upper airway may evoke a number of reflex effects which could affect breathing and upper airway re-opening during obstructive apnoeas.

Upper airway EMG responses to acute hypoxia and asphyxia are impaired in streptozotocin-induced diabetic rats.

Obstructive sleep apnoea (OSA) is a major clinical disorder that is characterised by multiple episodes of upper airway obstruction due to failure of the upper airway dilator muscles to maintain upper airway patency. The incidence of OSA is high in many endocrine disorders including both insulin-dependent and non-insulin-dependent diabetes but the reasons for this are not known. We wished to test the hypothesis that central respiratory motor output to the upper airway muscles is preferentially impaired in a rat model of diabetes mellitus. Sternohyoid (SH) and diaphragm (DIA) EMG activities were recorded in control and streptozotocin (STZ)-induced diabetic rats during normoxia, hypoxia (7.5% O2 in N2) and asphyxia (7.5% O2 and 3% CO2) under pentobarbitone anaesthesia. SH EMG responses to acute hypoxia and asphyxia were significantly impaired in STZ-induced diabetic rats compared to control animals (+47.1 +/- 5.7 vs. +11.7 +/- 1.9% during hypoxia in control and diabetic animals respectively and +56.5 +/- 7.9 vs. +15.7 +/- 5.0% during asphyxia). However, DIA EMG responses to hypoxia and asphyxia were not different for the two groups. We propose that the higher prevalence of OSA in diabetic patients is related to preferential impairment of cranial motor output to the dilator muscles of the upper airway in response to physiological stimuli.

Effects of chronic hypobaric hypoxia on contractile properties of rat sternohyoid and diaphragm muscles.

1. Chronic hypoxia occurs in a variety of circumstances, including respiratory disease and exposure to altitude, and is known to affect respiratory muscle structure. However, little is known about its effects on respiratory muscle contractile properties. 2. Rats were exposed to normoxia (n = 16) or hypobaric hypoxia (n = 16; barometric pressure 450 mmHg) for 6 weeks. Contractile properties were measured in isolated sternohyoid and diaphragm muscles in warmed, oxygenated Krebs' solution. Isometric twitch and tetanic tension, contraction time, half-relaxation time and tension-frequency relationship were determined using field stimulation with platinum electrodes. Fatigue was induced by stimulation at 40 Hz with 300 msec trains of 0.5 Hz for 5 min. 3. Chronic hypoxia had no effect on bodyweight, but did increase haematocrit. Chronic hypoxia increased specific force development in both muscles and increased sternohyoid fatigue. Chronic hypoxia had no effect on contractile kinetics in either muscle, but shifted the tension-frequency relationship to the left in the diaphragm. 4. Therefore, chronic hypoxia alters rat respiratory muscle force and fatigue, either due to the direct effects of hypoxia or to increased muscle activation.