Clare A. Berry

Chronic Fetal Exposure to Ureaplasma parvum Suppresses Innate Immune Responses in Sheep

The chorioamnionitis associated with preterm delivery is often polymicrobial with ureaplasma being the most common isolate. To evaluate interactions between the different proinflammatory mediators, we hypothesized that ureaplasma exposure would increase fetal responsiveness to LPS. Fetal sheep were given intra-amniotic (IA) injections of media (control) or Ureaplasma parvum serovar 3 either 7 or 70 d before preterm delivery. Another group received an IA injection of Escherichia coli LPS 2 d prior to delivery. To test for interactions, IA U. parvum-exposed animals were challenged with IA LPS and delivered 2 d later. All animals were delivered at 124 ± 1-d gestation (term = 150 d). Compared with the 2-d LPS exposure group, the U. parvum 70 d + LPS group had 1) decreased lung pro- and anti-inflammatory cytokine expression and 2) fewer CD3+ T lymphocytes, CCL2+, myeloperoxidase+, and PU.1+ cells in the lung. Interestingly, exposure to U. parvum for 7 d did not change responses to a subsequent IA LPS challenge, and exposure to IA U. parvum alone induced mild lung inflammation. Exposure to U. parvum increased pulmonary TGF-β1 expression but did not change mRNA expression of either the receptor TLR4 or some of the downstream mediators in the lung. Monocytes from fetal blood and lung isolated from U. parvum 70 d + LPS but not U. parvum 7 d + LPS animals had decreased in vitro responsiveness to LPS. These results are consistent with the novel finding of downregulation of LPS responses by chronic but not acute fetal exposures to U. parvum. The findings increase our understanding of how chorioamnionitis-exposed preterm infants may respond to lung injury and postnatal nosocomial infections.

Positive end-expiratory pressure and surfactant decrease lung injury during initiation of ventilation in fetal sheep

The initiation of ventilation in preterm, surfactant-deficient sheep without positive end-expiratory pressure (PEEP) causes airway injury and lung inflammation. We hypothesized that PEEP and surfactant treatment would decrease the lung injury from initiation of ventilation with high tidal volumes. Fetal sheep at 128-day gestational age were randomized to ventilation with: 1) no PEEP, no surfactant; 2) 8-cmH(2)O PEEP, no surfactant; 3) no PEEP + surfactant; 4) 8-cmH(2)O PEEP + surfactant; or 5) control (2-cmH(2)O continuous positive airway pressure) (n = 6-7/group). After maternal anesthesia and hysterotomy, the head and chest were exteriorized, and the fetus was intubated. While maintaining placental circulation, the fetus was ventilated for 15 min with a tidal volume escalating to 15 ml/kg using heated, humidified, 100% nitrogen. The fetus then was returned to the uterus, and tissue was collected after 30 min for evaluation of early markers of lung injury. Lambs receiving both surfactant and PEEP had increased dynamic compliance, increased static lung volumes, and decreased total protein and heat shock proteins 70 and 60 in bronchoalveolar lavage fluid compared with other groups. Ventilation, independent of PEEP or surfactant, increased mRNA expression of acute phase response genes and proinflammatory cytokine mRNA in the lung tissue compared with controls. PEEP decreased mRNA for cytokines (2-fold) compared with groups receiving no PEEP. Surfactant administration further decreased some cytokine mRNAs and changed the distribution of early growth response protein-1 expression. The use of PEEP during initiation of ventilation at birth decreased early mediators of lung injury. Surfactant administration changed the distribution of injury and had a moderate additive protective effect.

Interleukin 1 in Lipopolysaccharide Induced Chorioamnionitis in the Fetal Sheep

We tested the hypothesis that interleukin 1 (IL-1) mediates intra-amniotic lipopolysaccharide (LPS)-induced chorioamnionitis in preterm fetal sheep. Time-mated Merino ewes with singleton fetuses received IL-1α, LPS, or saline (control) by intra-amniotic injection 1 to 2 days before operative delivery at 124 ± 1 days gestational age (N = 5-9/group; term = 150 days). Recombinant human IL-1 receptor antagonist (rhIL-1ra) was given into the amniotic fluid 3 hours before intra-amniotic LPS or saline to block IL-1 signaling. Inflammation in the chorioamnion was determined by histology, cytokine messenger RNA (mRNA), protein expression, and by quantitation of activated inflammatory cells. Intra-amniotic IL-1 and LPS both induced chorioamnionitis. However, IL-1 blockade with IL-1ra did not decrease intra-amniotic LPS-induced increases in pro-inflammatory cytokine mRNAs, numbers of inflammatory cells, myeloperoxidase, or monocyte chemotactic protein-1-expressing cells in the chorioamnion. We conclude that IL-1 and LPS both can cause chorioamnionitis, but IL-1 is not an important mediator of LPS-induced chorioamnionitis in fetal sheep.

Lipopolysaccharide-induced weakness in the preterm diaphragm is associated with mitochondrial electron transport chain dysfunction and oxidative stress.

Diaphragmatic contractility is reduced in preterm lambs after lipopolysaccharide (LPS) exposure in utero. The mechanism of impaired fetal diaphragm contractility after LPS exposure is unknown. We hypothesise that in utero exposure to LPS induces a deficiency of mitochondrial complex activity and oxidative damage in the fetal diaphragm. To test this hypothesis, we used a well-established preterm ovine model of chorioamnionitis: Pregnant ewes received intra-amniotic (IA) saline or 10 mg LPS, at 2 d or 7 d prior to surgical delivery at 121 d GA (term = 150 d). The fetus was killed humanely immediately after delivery for tissue sampling. Mitochondrial fractions were prepared from the isolated diaphragm and mitochondrial electron transfer chain activities were evaluated using enzymatic assays. Oxidative stress was investigated by quantifying mitochondrial oxidative protein levels and determining antioxidant gene and protein (catalase, superoxide dismutase 2 and glutathione peroxidase 1) expression. The activity of the erythroid 2-related factor 2 (Nrf2)-mediated antioxidant signalling pathway was examined by quantifying the Nrf2 protein content of cell lysate and nuclear extract. A 2 d LPS exposure in utero significantly decreased electron transfer chain complex II and IV activity (p<0.05). A 7 d LPS exposure inhibited superoxide dismutase 2 and catalase expression at gene and protein levels, and Nrf2 pathway activity (p<0.05) compared with control and 2 d LPS groups, respectively. Diaphragm mitochondria accumulated oxidised protein after a 7 d LPS exposure. We conclude that intrauterine exposure to LPS induces mitochondrial oxidative stress and electron chain dysfunction in the fetal diaphragm, that is further exacerbated by impairment of the antioxidant signalling pathway and decreased antioxidant activity.

Pressure-limited sustained inflation vs. gradual tidal inflations for resuscitation in preterm lambs

Support of the mechanically complex preterm lung needs to facilitate aeration while avoiding ventilation heterogeneities: whether to achieve this gradually or quickly remains unclear. We compared the effect of gradual vs. constant tidal inflations and a pressure-limited sustained inflation (SI) at birth on gas exchange, lung mechanics, gravity-dependent lung volume distribution, and lung injury in 131-day gestation preterm lambs. Lambs were resuscitated with either 1) a 20-s, 40-cmH2O pressure-limited SI (PressSI), 2) a gradual increase in tidal volume (Vt) over 5-min from 3 ml/kg to 7 ml/kg (IncrVt), or 3) 7 ml/kg Vt from birth. All lambs were subsequently ventilated for 15 min with 7 ml/kg Vt with the same end-expiratory pressure. Lung mechanics, gas exchange and spatial distribution of end-expiratory volume (EEV), and tidal ventilation (electrical impedance tomography) were recorded regularly. At 15 min, early mRNA tissue markers of lung injury were assessed. The IncrVt group resulted in greater tissue hysteresivity at 5 min (P = 0.017; two-way ANOVA), higher alveolar-arterial oxygen difference from 10 min (P < 0.01), and least uniform gravity-dependent distribution of EEV. There were no other differences in lung mechanics between groups, and the PressSI and 7 ml/kg Vt groups behaved similarly throughout. EEV was more uniformly distributed, but Vt least so, in the PressSI group. There were no differences in mRNA markers of lung injury. A gradual increase in Vt from birth resulted in less recruitment of the gravity-dependent lung with worse oxygenation. There was no benefit of a SI at birth over mechanical ventilation with 7 ml/kg Vt.

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.

Developmental changes in diaphragm muscle function in the preterm and postnatal lamb.

The preterm diaphragm is structurally and functionally immature, potentially contributing to an increased risk of respiratory distress and failure. We investigated developmental changes in contractile function and susceptibility to fatigue of the costal diaphragm in the fetal lamb to understand factors contributing to the risk of developing diaphragm dysfunction and respiratory disorders. We hypothesized that the functional capacity of the diaphragm will vary with maturational stage as will its susceptibility to fatigue.

Variable ventilation enhances ventilation without exacerbating injury in preterm lambs with respiratory distress syndrome

Background:As compared with constant respiratory rate (RR) and tidal volume (VT) during controlled conventional mechanical ventilation (CV), variable ventilation (VV) using the same breath-to-breath minute volume but variable VT and RRs enhances ventilation efficiency in preterm lambs. We hypothesized that if VT was adjusted to target permissive hypercarbia, VV would result in more efficient gas exchange without increasing inflammatory and injurious responses in the lung.Methods:Preterm lambs at 129 d gestation were anesthetized, tracheotomized, and randomized to either CV (n = 8) or VV (n = 8) using the same initial average VT and RR. Lung mechanics and gas exchange were measured intermittently, and average VT was adjusted to target partial pressure of arterial carbon dioxide (PaCO2) of 40–50 mm Hg for 3 h. Lung injury and inflammation were assessed from bronchoalveolar lavage fluid, lung tissue, and peripheral blood.Results:VV achieved permissive hypercarbia using a lower average VT, peak inspiratory pressure, and elastance (increased compliance) as compared with CV. Oxygenation and markers of lung tissue inflammation or injury were not different apart from a lower wet:dry tissue ratio in the VV lungs.Conclusions:VV improves ventilation efficiency and in vivo lung compliance in the ovine preterm lung without increasing lung inflammation or lung injury.

Nebulised surfactant to reduce severity of respiratory distress: a blinded, parallel, randomised controlled trial

Objective To evaluate if nebulised surfactant reduces intubation requirement in preterm infants with respiratory distress treated with nasal continuous positive airway pressure (nCPAP). Design Double blind, parallel, stratified, randomised control trial. Setting Sole tertiary neonatal unit in West Australia. Patients Preterm infants (290–336 weeks’ gestational age, GA) less than 4 hours of age requiring 22%–30% supplemental oxygen, with informed parental written consent. Interventions Infants were randomised within strata (290–316 and 320–336 weeks’ GA) to bubble nCPAP or bubble nCPAP and nebulised surfactant (200 mg/kg: poractant alfa) using a customised vibrating membrane nebuliser (eFlow neonatal). Surfactant nebulisation (100 mg/kg) was repeated after 12 hours for persistent supplemental oxygen requirement. Main outcome measures The primary outcomes were requirement for intubation and duration of mechanical ventilation at 72 hours. Data analysis followed the intention-to-treat principle. Results 360 of 606 assessed infants were eligible; 64 of 360 infants were enrolled and randomised (n=32/group). Surfactant nebulisation reduced the requirement for intubation within 72 hours: 11 of 32 infants were intubated after continuous positive airway pressure (CPAP) and nebulised surfactant compared with 22 of 32 infants receiving CPAP alone (relative risk (95% CI)=0.526 (0.292 to 0.950)). The reduced requirement for intubation was limited to the 320–336 weeks’ GA stratum. The median (range) duration of ventilation in the first 72 hours was not different between the intervention (0 (0–62) hours) and control (9 (0–64) hours; p=0.220) groups. There were no major adverse events. Conclusions Early postnatal nebulised surfactant may reduce the need for intubation in the first 3 days of life compared with nCPAP alone in infants born at 290–336 weeks’ GA with mild respiratory distress syndrome. Confirmation requires further adequately powered studies. Trial registration number ACTRN12610000857000.