Gestational age at time of in utero lipopolysaccharide exposure influences the severity of inflammation-induced diaphragm weakness in lambs.

The preterm diaphragm is functionally immature compared with its term counterpart. In utero inflammation further exacerbates preterm diaphragm dysfunction. We hypothesized that preterm lambs are more vulnerable to in utero inflammation-induced diaphragm dysfunction compared with term lambs. Pregnant ewes received intra-amniotic (IA) injections of saline or 10 mg lipopolysaccharide (LPS) 2 or 7 days before delivery at 121 days (preterm) or ∼145 days (term) of gestation. Diaphragm contractile function was assessed in vitro. Plasma cytokines, diaphragm myosin heavy chain (MHC) isoforms, and oxidative stress were evaluated. Maximum diaphragm force in preterm control lambs was significantly lower (22%) than in term control lambs ( P < 0.001). Despite similar inflammatory cytokine responses to in utero LPS exposure, diaphragm function in preterm and term lambs was affected differentially. In term lambs, maximum force after a 2-day LPS exposure was significantly lower than in controls (by ~20%, P < 0.05). In preterm lambs, maximum forces after 2-day and 7-day LPS exposures were significantly lower than in controls (by ~30%, P < 0.05). Peak twitch force after LPS exposure was significantly lower in preterm than in controls, but not in term lambs. In term lambs, LPS exposure increased the proportion of MHC-I fibers, increased twitch contraction times, and increased fatigue resistance relative to controls. In preterm diaphragm, the cross-sectional area of embryonic MHC fibers was significantly lower after 7-day versus 2-day LPS exposures. We conclude that preterm lambs are more vulnerable to IA LPS-induced diaphragm dysfunction than term lambs. In utero inflammation exacerbates diaphragm dysfunction and may increase susceptibility to postnatal respiratory failure.

Influence of antenatal glucocorticoid on preterm lamb diaphragm.

BackgroundPregnant women at a high risk of preterm delivery receive glucocorticoids to accelerate fetal lung maturation and surfactant synthesis. However, the effect of antenatal steroids on the developing diaphragm remains unclear. We hypothesized that maternal betamethasone impairs the fetal diaphragm, and the magnitude of the detrimental effect increases with longer duration of exposure. We aimed to determine how different durations of fetal exposure to maternal betamethasone treatment influence the fetal diaphragm at the functional and molecular levels.MethodsDate-mated merino ewes received intramuscular injections of saline (control) or two doses of betamethasone (5.7 mg) at an interval of 24 h commencing either 2 or 14 days before delivery. Preterm lambs were killed after cesarean delivery at 121-day gestational age. In vitro contractile measurements were performed on the right hemidiaphragm, whereas molecular/cellular analyses used the left costal diaphragm.ResultsDifferent durations of fetal exposure to maternal betamethasone had no consistent effect on the protein metabolic pathway, expression of glucocorticoid receptor and its target genes, cellular oxidative status, or contractile properties of the fetal lamb diaphragm.ConclusionThese data suggest that the potential benefits of betamethasone exposure on preterm respiratory function are not compromised by impaired diaphragm function after low-dose maternal intramuscular glucocorticoid exposure.

Gestational age at initial exposure to in utero inflammation influences the extent of diaphragm dysfunction in preterm lambs.

BACKGROUND AND OBJECTIVE: In utero infection may critically influence diaphragm development and predispose preterm infants to postnatal respiratory failure. We aimed to determine how frequency and gestational age (GA) at time of intra-amniotic (IA) lipopolysaccharide (LPS) exposure affects preterm diaphragm function. METHODS: Pregnant ewes received IA injections of saline or 10-mg LPS at 7 days or 21 days or weekly injections 21, 14 and 7 days before delivery at 121-day GA. Foetal lambs were killed with pentobarbitone (150 mg/kg; intravenous). Diaphragm contractile function was measured in vitro. Muscle fibre type, activation of protein synthesis and degradation pathways, pro-inflammatory signalling and oxidative stress were evaluated using immunofluorescence staining, RT-qPCR, ELISA, Western blotting and biochemical assay. RESULTS: In utero LPS exposure significantly impaired diaphragm contractile function. LPS exposure 7 days before delivery caused maximum specific twitch and tetanic forces 30% lower than controls. When the initial LPS exposure occurred 21 days before delivery maximum specific forces were 40% lower than controls. Earlier LPS exposure also prolonged twitch contraction time, increased fatigue resistance and elevated protein carbonyl content. Despite increased white blood cell counts and interleukin-6 mRNA expression following weekly LPS exposure, there were no significant differences in contractile properties between exposure 21 days before delivery and repeated LPS groups suggesting that frequency of inflammatory exposure does not influence the severity of contractile dysfunction. CONCLUSIONS: GA at time of initial LPS exposure, rather than frequency of exposure, determines the extent of inflammation-induced diaphragm dysfunction.

In utero LPS exposure impairs preterm diaphragm contractility.

Preterm birth is associated with inflammation of the fetal membranes (chorioamnionitis). We aimed to establish how chorioamnionitis affects the contractile function and phenotype of the preterm diaphragm. Pregnant ewes received intra-amniotic injections of saline or 10 mg LPS, 2 days or 7 days before delivery at 121 days of gestation (term = 150 d). Diaphragm strips were dissected for the assessment of contractile function after terminal anesthesia. The inflammatory cytokine response, myosin heavy chain (MHC) fibers, proteolytic pathways, and intracellular molecular signaling were analyzed using quantitative PCR, ELISA, immunofluorescence staining, biochemical assays, and Western blotting. Diaphragm peak twitch force and maximal tetanic force were approximately 30% lower than control values in the 2-day and 7-day LPS groups. Activation of the NF-κB pathway, an inflammatory response, and increased proteasome activity were observed in the 2-day LPS group relative to the control or 7-day LPS group. No inflammatory response was evident after a 7-day LPS exposure. Seven-day LPS exposure markedly decreased p70S6K phosphorylation, but no effect on other signaling pathways was evident. The proportion of MHC IIa fibers was lower than that for control samples in the 7-day LPS group. MHC I fiber proportions did not differ between groups. These results demonstrate that intrauterine LPS impairs preterm diaphragmatic contractility after 2-day and 7-day exposures. Diaphragm dysfunction, resulting from 2-day LPS exposure, was associated with a transient activation of proinflammatory signaling, with subsequent increased atrophic gene expression and enhanced proteasome activity. Persistently impaired contractility for the 7-day LPS exposure was associated with the down-regulation of a key component of the protein synthetic signaling pathway and a reduction in the proportions of MHC IIa fibers.

Interleukin-1 receptor antagonist protects against lipopolysaccharide induced diaphragm weakness in preterm lambs.

Chorioamnionitis (inflammation of the fetal membranes) is strongly associated with preterm birth and in utero exposure to inflammation significantly impairs contractile function in the preterm lamb diaphragm. The fetal inflammatory response to intra-amniotic (IA) lipopolysaccharide (LPS) is orchestrated via interleukin 1 (IL-1). We aimed to determine if LPS induced contractile dysfunction in the preterm diaphragm is mediated via the IL-1 pathway. Pregnant ewes received IA injections of recombinant human IL-1 receptor antagonist (rhIL-1ra) (Anakinra; 100 mg) or saline (Sal) 3 h prior to second IA injections of LPS (4 mg) or Sal at 119d gestational age (GA). Preterm lambs were killed after delivery at 121d GA (term = 150 d). Muscle fibres dissected from the right hemi-diaphragm were mounted in an in vitro muscle test system for assessment of contractile function. The left hemi-diaphragm was snap frozen for molecular and biochemical analyses. Maximum specific force in lambs exposed to IA LPS (Sal/LPS group) was 25% lower than in control lambs (Sal/Sal group; p=0.025). LPS-induced diaphragm weakness was associated with higher plasma IL-6 protein, diaphragm IL-1ß mRNA and oxidised glutathione levels. Pre-treatment with rhIL-1ra (rhIL-1ra/LPS) ameliorated the LPS-induced diaphragm weakness and blocked systemic and local inflammatory responses, but did not prevent the rise in oxidised glutathione. These findings indicate that LPS induced diaphragm dysfunction is mediated via IL-1 and occurs independently of oxidative stress. Therefore, the IL-1 pathway represents a potential therapeutic target in the management of impaired diaphragm function in preterm infants.