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ObjectiveTo explore clinical effect modifiers of systemic hydrocortisone in ventilated very preterm infants for survival and neurodevelopmental outcome at 2 years’ corrected age (CA).DesignSecondary analysis of a randomised placebo-controlled trial.SettingDutch and Belgian neonatal intensive care units.PatientsInfants born <30 weeks’ gestational age (GA), ventilator-dependent in the second week of postnatal life.InterventionInfants were randomly assigned to systemic hydrocortisone (cumulative dose 72.5 mg/kg; n=182) or placebo (n=190).Main outcome measuresThe composite of death or neurodevelopmental impairment (NDI) at 2 years’ CA and its components. Candidate effect modifiers (GA, small for GA, respiratory index, sex, multiple births, risk of moderate/severe bronchopulmonary dysplasia or death) were analysed using regression models with interaction terms and subpopulation treatment effect pattern plots.ResultsThe composite outcome was available in 356 (96.0%) of 371 patients (one consent withdrawn). For this outcome, treatment effect heterogeneity was seen across GA subgroups (<27 weeks: hydrocortisone (n=141) vs placebo (n=156), 54.6% vs 66.2%; OR 0.61 (95% CI 0.38 to 0.98); ≥27 weeks: hydrocortisone (n=30) vs placebo (n=31), 66.7% vs 45.2%; OR 2.43 (95% CI 0.86 to 6.85); p=0.02 for interaction). This effect was also found for the component death (<27 weeks: 20.1% vs 32.1%; OR 0.53 (95% CI 0.32 to 0.90); ≥27 weeks: 28.1% vs 16.1%; OR 2.04 (95% CI 0.60 to 6.95); p=0.049 for interaction) but not for the component NDI. No differential treatment effects were observed across other subgroups.ConclusionThis secondary analysis suggests that in infants <27 weeks’ GA, systemic hydrocortisone may improve the outcome death or NDI, mainly driven by its component death. There was insufficient evidence for other selected candidate effect modifiers.

Duchenne muscular dystrophy (DMD) is a degenerative X-linked muscle disease. Death frequently results from complications in cardiopulmonary systems. Preclinical/early diagnosis of cardiac autonomic abnormalities may aid initiate cardioprotective therapy and enhance prognosis. A cross sectional, prospective study of 38 DMD boys compared with 37 age-matched healthy controls was conducted. Lead II electrocardiography and beat-to-beat blood pressure were recorded to assess heart rate variability (HRV), blood pressure variability (BPV), and baroreceptor sensitivity (BRS) in a standardized environment. Data were analysed and correlated with disease severity and genotype. In the DMD group, the median age at assessment was 8 years [IQR 7–9 years], the median age at disease onset was 3 years [IQR, 2–6 years], and the mean duration of illness was 4 years [IQR, 2.5–5]. DNA sequencing showed deletions in 34/38 (89.5 %) and duplications in 4/38 (10.5%) patients. The median heart rate in DMD children was significantly higher [101.19 (Range, 94.71–108.49)] /min compared to controls [81 (Range, 76.2–92.76)] /min (p <  0.05). All the assessed HRV and BPV parameters were significantly impaired in DMD cases except for the coefficient of variance of systolic blood pressure. Further, BRS parameters were also significantly reduced in DMD, excluding alpha-LF. A positive correlation was found between alpha HF with age at onset and duration of illness. This study demonstrates a distinct early impairment of neuro-cardio-autonomic regulation in DMD. Simple yet effective non-invasive techniques such as HRV, BPV, and BRS may help identify cardiac dysfunction in a pre-clinical state, paving the way for early cardio-protective therapies and limiting disease progression in DMD patients.

Involvement of the cerebellum in the pathophysiology of hypoxic‐ischemic encephalopathy (HIE) in preterm infants is increasingly recognized. We aimed to assess the neuroprotective potential of intravenously administered multipotent adult progenitor cells (MAPCs) in the preterm cerebellum. Instrumented preterm ovine fetuses were subjected to transient global hypoxia‐ischemia (HI) by 25 minutes of umbilical cord occlusion at 0.7 of gestation. After reperfusion, two doses of MAPCs were administered intravenously. MAPCs are a plastic adherent bone‐marrow‐derived population of adult progenitor cells with neuroprotective potency in experimental and clinical studies. Global HI caused marked cortical injury in the cerebellum, histologically indicated by disruption of cortical strata, impeded Purkinje cell development, and decreased dendritic arborization. Furthermore, global HI induced histopathological microgliosis, hypomyelination, and disruption of white matter organization. MAPC treatment significantly prevented cortical injury and region‐specifically attenuated white matter injury in the cerebellum following global HI. Diffusion tensor imaging (DTI) detected HI‐induced injury and MAPC neuroprotection in the preterm cerebellum. This study has demonstrated in a preclinical large animal model that early systemic MAPC therapy improved structural injury of the preterm cerebellum following global HI. Microstructural improvement was detectable with DTI. These findings support the potential of MAPC therapy for the treatment of HIE and the added clinical value of DTI for the detection of cerebellar injury and the evaluation of cell‐based therapy. Systemic multipotent adult progenitor cells protected the preterm cerebellum in a preclinical animal model of perinatal asphyxia. Diffusion tensor imaging detected cerebellar injury and repair underlining the added clinical value of this imaging technique.

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Prenatal inflammation is a major risk for preterm birth and neonatal morbidity, but its effects on postnatal immunity and organ functions remain unclear. Using preterm pigs as a model for preterm infants, we investigated whether prenatal intra-amniotic (IA) inflammation modulates postnatal systemic immune status and organ functions. Preterm pigs exposed to IA lipopolysaccharide (LPS) for 3 days were compared with controls at birth and postnatal day 5 after formula feeding. IA LPS induced mild chorioamnionitis but extensive intra-amniotic inflammation. There were minor systemic effects at birth (increased blood neutrophil counts), but a few days later, prenatal LPS induced delayed neonatal arousal, systemic inflammation (increased blood leukocytes, plasma cytokines, and splenic bacterial counts), altered serum biochemistry (lower albumin and cholesterol and higher iron and glucose values), and increased urinary protein and sodium excretion. In the gut and lungs, IA LPS–induced inflammatory responses were observed mainly at birth (increased LPS, CXCL8, and IL-1β levels and myeloperoxidase-positive cell density, multiple increases in innate immune gene expressions, and reduced villus heights), but not on postnatal day 5 (except elevated lung CXCL8 and diarrhea symptoms). Finally, IA LPS did not affect postnatal gut brush-border enzymes, hexose absorption, permeability, or sensitivity to necrotizing enterocolitis on day 5. Short-term IA LPS exposure predisposes preterm pigs to postnatal systemic inflammation after acute fetal gut and lung inflammatory responses.

BackgroundGeneral anesthetics could protect key neurotransmitter systems, such as the dopaminergic system, from hypoxic–ischemic encephalopathy (HIE) by limiting excessive glutamatergic neurotransmission. However, anesthetics may adversely affect inflammation and tau phosphorylation.MethodsA near-term sheep model of HIE by umbilical cord occlusion (UCO) under anesthesia was used. The effect of propofol and isoflurane on the dopaminergic neurotransmitter phenotype in the substantia nigra (SN) was studied using tyrosine hydroxylase immunohistochemistry. The overall microglial response and tau phosphorylation were also measured in the SN, surrounding the midbrain gray matter structures and the hippocampal white matter.ResultsThe isoflurane-treated UCO group had fewer tyrosine hydroxylase-expressing neurons in the SN at 8 h after the insult than the propofol-treated UCO or sham-operated groups (P<0.05). The microglial response was unchanged in the SN region. In the thalamus and the hippocampal stratum moleculare layer, the propofol-treated UCO group had a lower microglial response than the corresponding sham-operated group. Both UCO and the use of anesthetics additively increased tau phosphorylation in the SN region, thalamus, and hippocampus.ConclusionThe choice of anesthetics is important for an emergency C-section. Propofol could potentially protect the dopaminergic neurotransmitter phenotype within the SN at the cost of a widespread increase in tau phosphorylation.

BackgroundThe aim of this study was to evaluate the potential of fecal volatile organic compounds (VOCs), obtained by means of an electronic nose device (Cyranose 320), as early non-invasive biomarker for BPD.MethodsIn this nested case–control study performed at three Neonatal Intensive Care Units, fecal samples obtained at postnatal age of 7, 14, 21, and 28 days from preterm infants with severe bronchopulmonary dysplasia (BPD) were compared with fecal VOC profiles from matched controls. Microbiota analysis was performed by means of IS-pro technique on fecal samples collected at 28 days postnatally.ResultsVOC profiles of infants developing severe BPD (n=15) could be discriminated from matched controls (n=15) at postnatal age of 14 days (area under the curve (±95% confidence interval), P-value, sensitivity, specificity; 0.72 (0.54–0.90), 0.040, 60.0%, 73.3%), 21 days (0.71 (0.52–0.90), 0.049, 66.7%, 73.3%) and 28 days (0.77 (0.59–0.96), 0.017, 69.2%, 69.2%) but not at 7 days. Intestinal microbiota did not differ between BPD subjects and controls.ConclusionFecal VOC profiles of infants developing BPD could be differentiated from controls at postnatal day 14, 21, and 28. VOC differences could not be directed to intestinal microbiota alterations but presumably reflect local and systemic metabolic and inflammatory pathways associated with BPD.

Several studies associated altered gut microbiota composition in preterm infants with late-onset sepsis (LOS), up to days before clinical onset of sepsis. Microbiota analysis as early diagnostic biomarker is, however, in clinical practice currently not feasible because of logistic aspects and high costs. Therefore, we hypothesized that analysis of fecal volatile organic compounds (VOCs) may serve as noninvasive biomarker to predict LOS at a preclinical stage, because VOC reflect the composition and activity of intestinal microbial communities. In a prospective multicenter study, fecal samples were collected daily from infants with a gestational age of <30 weeks. VOC signatures of fecal samples from infants with LOS, collected up to 5 days before diagnosis, were analyzed by means of an electronic nose technology (Cyranose 320) and compared to matched controls. Fecal VOC profiles of infants with LOS (n = 36) could be discriminated from controls (n = 40) at 3 days (area under the curve [±95% confidence interval], Pvalue, sensitivity, specificity; 70.2 [52.2–88.3], 0.033, 57.1%, 61.5%), 2 days (77.7 [62.7–92.7], 0.050, 75.0%, 70.8%), and 1 day (70.4 [49.6–91.3], 0.037, 64.3%, 64.3%) before the onset of LOS. Fecal VOC profiles of preterm infants with LOS could be discriminated from matched controls, up to 3 days before clinical onset of the disease, underlining the hypothesis that intestinal microbiota may play an etiological role in LOS. Notably, VOC profiling is clinically feasible and the potential of this technique in the early detection of LOS needs to be confirmed in future studies.

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BackgroundPentoxifylline (PTX), a methylxanthine derivate with immunomodulating properties, has been used as adjunctive treatment in severe neonatal sepsis. The aim of the study was to investigate the anti-inflammatory effects of PTX on Lipopolysaccharides (LPS)-stimulated monocytes of preterm neonates in vitro compared with monocytes of term infants and adult controls.MethodsWhole cord blood samples and control adult blood samples were incubated with LPS and PTX. The expression of surface markers, phagocytosis, cytokine secretion, and Toll-like receptor (TLR)4 signaling of monocytes were assessed by flow cytometry. Changes of TLR4-messenger RNA (mRNA) levels were confirmed by reverse-transcriptase PCR.ResultsThe expression of CD14, CD11b, CD64, CD71, and CD80 was downregulated by PTX in a dose-dependent manner; the greatest effect was observed on CD14 and CD11b in preterm infants. PTX markedly downregulated LPS-induced tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 levels in all age groups. Early IL-10 production was significantly downregulated by PTX in term and preterm neonates, while remaining unchanged in adults. Moreover, PTX downregulated TLR4 expression of monocytes on cellular and mRNA level, decreased signaling, and suppressed phagocytosis.ConclusionPTX downregulated TLR4 expression and signaling, thereby leading to strong anti-inflammatory properties in monocytes. Age-dependent differences were identified for CD14 and CD11b expression and IL-10 production.

Background:Dexamethasone-phosphate (Dex-PO4) and the combination betamethasone-phosphate (Beta-PO4) + betamethasone-acetate (Beta-Ac) are the most used antenatal corticosteroids to promote fetal lung maturation. We compared fetal lung maturation induced by Beta-Ac+Beta-PO4, Dex-PO4, or Beta-PO4alone.Methods:Pregnant ewes received two intramuscular doses 24 h apart of 0.25 mg/kg/dose of Beta-Ac+Beta-PO4, Dex-PO4 or Beta-PO4; or 2 doses of 0.125 mg/kg/dose of Beta-PO4at 6, 12, or 24 h intervals. Fetuses were delivered 48 h after the first dose and ventilated for 30 min. We assessed ventilatory variables, vital signs, and blood gas. After ventilation pressure-volume curves were measured and lungs were sampled for analysis.Results:All treatments improved lung compliance and ventilation efficiency. Only Beta-Ac + Beta-PO4required lower positive inspiratory pressure compared with control. Beta-Ac + Beta-PO4and Beta-PO4alone, but not Dex-PO4, increased the mRNA of surfactant proteins compared with control. Low-dose Beta-PO4did not increase mRNA of surfactant proteins. There were no differences among Beta-PO4treatment intervals.Conclusion:Beta-Ac + Beta-PO4given as two doses 24 h apart was more effective in promoting fetal lung maturation than Dex-PO4or Beta-PO4alone, consistent with a prolonged exposure provided by the Beta-Ac + Beta-PO4. These results support the clinical use of combined Beta-Ac + Beta-PO4preparations over phosphate corticosteroids alone for fetal lung maturation.

Background:Current methods for assessing perinatal hypoxic conditions did not improve infant outcomes. Various waveform-based and interval-based ECG markers have been suggested, but not directly compared. We compare performance of ECG markers in a standardized ovine model for fetal hypoxia.Methods:Sixty-nine fetal sheep of 0.7 gestation had ECG recorded 4?h before, during, and 4?h after a 25-min period of umbilical cord occlusion (UCO), leading to severe hypoxia. Various ECG markers were calculated, among which were heart rate (HR), HR-corrected ventricular depolarization/repolarization interval (QTc), and ST-segment analysis (STAN) episodic and baseline rise markers, analogue to clinical STAN device alarms. Performance of interval- and waveform-based ECG markers was assessed by correlating predicted and actual hypoxic/normoxic state.Results:Of the markers studied, HR and QTcdemonstrated high sensitivity (=86%), specificity (=96%), and positive predictive value (PPV) (=86%) and detected hypoxia in =90% of fetuses at 4?min after UCO. In contrast, STAN episodic and baseline rise markers displayed low sensitivity (=20%) and could not detect severe fetal hypoxia in 65 and 28% of the animals, respectively.Conclusion:Interval-based HR and QTcmarkers could assess the presence of severe hypoxia. Waveform-based STAN episodic and baseline rise markers were ineffective as markers for hypoxia.

The protective effects of mesenchymal stromal cell‐derived extracellular vesicles (MSC‐EVs) were studied in an ovine preclinical model of preterm hypoxic‐ischemic brain injury. Systemic in utero administration of MSC‐EVs improved brain function by reducing the total number and duration of seizures and by preserving baroreceptor reflex sensitivity. These functional protections were accompanied by a tendency to prevent hypomyelination. Cerebral inflammation remained unaffected by the MSC‐EV treatment. Preterm neonates are susceptible to perinatal hypoxic‐ischemic brain injury, for which no treatment is available. In a preclinical animal model of hypoxic‐ischemic brain injury in ovine fetuses, we have demonstrated the neuroprotective potential of systemically administered mesenchymal stromal cells (MSCs). The mechanism of MSC treatment is unclear but suggested to be paracrine, through secretion of extracellular vesicles (EVs). Therefore, we investigated in this study the protective effects of mesenchymal stromal cell‐derived extracellular vesicles (MSC‐EVs) in a preclinical model of preterm hypoxic‐ischemic brain injury. Ovine fetuses were subjected to global hypoxia‐ischemia by transient umbilical cord occlusion, followed by in utero intravenous administration of MSC‐EVs. The therapeutic effects of MSC‐EV administration were assessed by analysis of electrophysiological parameters and histology of the brain. Systemic administration of MSC‐EVs improved brain function by reducing the total number and duration of seizures, and by preserving baroreceptor reflex sensitivity. These functional protections were accompanied by a tendency to prevent hypomyelination. Cerebral inflammation remained unaffected by the MSC‐EV treatment. Our data demonstrate that MSC‐EV treatment might provide a novel strategy to reduce the neurological sequelae following hypoxic‐ischemic injury of the preterm brain. Our study results suggest that a cell‐free preparation comprising neuroprotective MSC‐EVs could substitute MSCs in the treatment of preterm neonates with hypoxic‐ischemic brain injury, thereby circumventing the potential risks of systemic administration of living cells. Bone marrow‐derived mesenchymal stromal cells (MSCs) show promise in treating hypoxic‐ischemic injury of the preterm brain. Study results suggest administration of extracellular vesicles, rather than intact MSCs, is sufficient to exert therapeutic effects and avoids potential concerns associated with administration of living cells. The therapeutic efficacy of systemically administered mesenchymal stromal cell‐derived extracellular vesicles (MSC‐EVs) on hypoxia‐ischemia‐induced injury was assessed in the preterm ovine brain. Impaired function and structural injury of the fetal brain was improved following global hypoxia‐ischemia. A cell‐free preparation of MSC‐EVs could substitute for the cellular counterpart in the treatment of preterm neonates with hypoxic‐ischemic brain injury. This may open new clinical applications for “off‐the‐shelf” interventions with MSC‐EVs.

Background:Little is known about the effects of propofol on oxidative stress and its effect on key structures of the contractile apparatus as the myosin light chain 2 (MLC2) and the p38MAPK survival pathway in the preterm heart. We hypothesized that propofol administration could attenuate the hypoxic myocardial injury after birth asphyxia.Methods:Pregnant ewes were randomized to receive either propofol or isoflurane anesthesia. A total of 44 late-preterm lambs were subjected to in utero umbilical cord occlusion (UCO), resulting in asphyxia and cardiac arrest, or sham treatment. After emergency cesarean delivery, each fetus was resuscitated, mechanically ventilated, and supported under anesthesia for 8?h using the same anesthetic as the one received by its mother.Results:At 8?h after UCO, occurrence of reactive oxygen species and activation of inducible nitric oxide synthase in the heart were lower in association with propofol anesthesia than with isoflurane. This was accompanied by less degradation of MLC2 but higher p38MAPK level and in echocardiography with a trend toward a higher median left ventricular fractional shortening.Conclusion:The use of propofol resulted in less oxidative stress and was associated with less cytoskeletal damage of the contractile apparatus than the use of isoflurane anesthesia.

Chorioamnionitis, caused by intra-amniotic exposure to bacteria and their toxic components, is associated with fetal gut inflammation and mucosal injury. In a translational ovine model, we have shown that these adverse intestinal outcomes to chorioamnionitis were the combined result of local gut and pulmonary-driven systemic immune responses. Chorioamnionitis-induced gut inflammation and injury was largely prevented by inhibiting interleukin-1 (IL-1) signaling. Therefore, we investigated whether local (gut-derived) IL-1αsignaling or systemic IL-1α-driven immune responses (lung or chorioamnion/skin-derived) were sufficient for intestinal inflammation and mucosal injury in the course of chorioamnionitis. Fetal surgery was performed in sheep to isolate the lung, gastrointestinal tract, and chorioamnion/skin, and IL-1αor saline was given into the trachea, stomach, or amniotic cavity 1 or 6 days before preterm delivery. Selective IL-1αexposure to the lung, gut, or chorioamnion/skin increased the CD3+ cell numbers in the fetal gut. Direct IL-1αexposure to the gut impaired intestinal zonula occludens protein-1 expression, induced villus atrophy, changed the expression pattern of intestinal fatty acid-binding protein along the villus, and increased the CD68, IL-1, and TNF-αmRNA levels in the fetal ileum. With lung or chorioamnion/skin exposure to IL-1α, intestinal inflammation was associated with increased numbers of blood leukocytes without induction of intestinal injury or immaturity. We concluded that local IL-1αsignaling was required for intestinal inflammation, disturbed gut maturation, and mucosal injury in the context of chorioamnionitis.

Chorioamnionitis, caused by intra-amniotic exposure to bacteria and their toxic components, is associated with fetal gut inflammation and mucosal injury. In a translational ovine model, we have shown that these adverse intestinal outcomes to chorioamnionitis were the combined result of local gut and pulmonary-driven systemic immune responses. Chorioamnionitis-induced gut inflammation and injury was largely prevented by inhibiting interleukin-1 (IL-1) signaling. Therefore, we investigated whether local (gut-derived) IL-1α signaling or systemic IL-1α-driven immune responses (lung or chorioamnion/skin-derived) were sufficient for intestinal inflammation and mucosal injury in the course of chorioamnionitis. Fetal surgery was performed in sheep to isolate the lung, gastrointestinal tract, and chorioamnion/skin, and IL-1α or saline was given into the trachea, stomach, or amniotic cavity 1 or 6 days before preterm delivery. Selective IL-1α exposure to the lung, gut, or chorioamnion/skin increased the CD3+ cell numbers in the fetal gut. Direct IL-1α exposure to the gut impaired intestinal zonula occludens protein-1 expression, induced villus atrophy, changed the expression pattern of intestinal fatty acid-binding protein along the villus, and increased the CD68, IL-1, and TNF-α mRNA levels in the fetal ileum. With lung or chorioamnion/skin exposure to IL-1α, intestinal inflammation was associated with increased numbers of blood leukocytes without induction of intestinal injury or immaturity. We concluded that local IL-1α signaling was required for intestinal inflammation, disturbed gut maturation, and mucosal injury in the context of chorioamnionitis.

Background:Surfactant replacement therapy is the gold standard treatment of neonatal respiratory distress (RDS). Nebulization is a noninvasive mode of surfactant administration. We administered Poractant alfa (Curosurf) via a vibrating perforated membrane nebulizer (eFlow Neonatal Nebulizer) to spontaneously breathing preterm lambs during binasal continuous positive pressure ventilation (CPAP).Methods:Sixteen preterm lambs were operatively delivered at a gestational age of 133?±?1 d (term ~150 d), and connected to CPAP applied via customized nasal prongs. Nebulization was performed (i) with saline or (ii) with surfactant for 3?h in humidified or (iii) nonhumidified air, and with surfactant (iv) for 60?min or (v) for 30?min. We measured arterial oxygenation, lung gas volumes and surfactant pool size and deposition.Results:Nebulization of surfactant in humidified air for 3?h improved oxygenation and lung function, and surfactant was preferentially distributed to the lower lung lobes. Shorter nebulization times and 3?h nebulization in dry air did not show these effects. Nebulized surfactant reached all lung lobes, however the increase of surfactant pool size missed statistical significance.Conclusion:Positive effects of surfactant nebulization to spontaneously breathing preterm lambs depend on treatment duration, surfactant dose, air humidity, and surfactant distribution within the lung.

Background:Hyperoxia and hypoxia influence morbidity and mortality of preterm infants. Automated closed-loop control of the fraction of inspired oxygen (FiO2) has been shown to facilitate oxygen supplementation in the neonatal intensive care unit (NICU), but has not yet been tested during preterm resuscitation. We hypothesized that fully automated FiO2control based on predefined oxygen saturation (SpO2) targets was applicable in both preterm resuscitation and ventilation.Methods:Twenty-two preterm lambs were operatively delivered and intubated in a modified EXIT procedure. They were randomized to receive standardized resuscitation with either automated or manual FiO2control, targeting SpO2according to the Dawson curve in the first 10?min and SpO290–95% hereafter. Automated FiO2control also was applied during surfactant replacement therapy and subsequent ventilation.Results:Time within target range did not differ significantly between manual and automated FiO2control during resuscitation, however automated FiO2control significantly avoided hyperoxia. Automated FiO2control was feasible during surfactant replacement and kept SpO2within target range significantly better than manual control during subsequent ventilation.Conclusion:In our model, fully automated FiO2control was feasible in rapidly changing physiologic conditions during postnatal resuscitation and prevented hyperoxia. We conclude that closed loop FiO2control is a promising tool for the delivery room.

Background:Intrauterine Candida albicans infection causes severe fetal inflammatory responses and fetal injury in an ovine model. We hypothesized that intra-amniotic antifungal therapy with fluconazole would decrease the adverse fetal effects of intra-amniotic C. albicans in sheep.Methods:Sheep received an intra-amniotic injection of 107colony-forming units C. albicans. After 2 d, animals were then randomized to: (i) intra-amniotic and fetal intraperitoneal saline with delivery after 24?h (3 d C. albicans group); (ii) intra-amniotic and fetal intraperitoneal injections of fluconazole with delivery after either 24?h (3 d C. albicans plus 1 d fluconazole group) or 72?h (5 d C. albicans plus 3 d fluconazole group). Controls received intra-amniotic injections of saline followed by intra-amniotic and fetal intraperitoneal fluconazole injections.Results:Intra-amniotic C. albicans caused severe fetal inflammatory responses characterized by decreases in lymphocytes and platelets, an increase in posterior mediastinal lymph node weight and proinflammatory mRNA responses in the fetal lung, liver, and spleen. Fluconazole treatment temporarily decreased the pulmonary and chorioamnion inflammatory responses.Conclusion:The severe fetal inflammatory responses caused by intra-amniotic C. albicans infection were transiently decreased with fluconazole. A timely fetal delivery of antimicrobial agents may prevent fetal injury associated with intrauterine infection.

Background:Intrauterine inflammation activates the fetal immune system and can result in organ injury and postnatal complications in preterm infants. As the spleen is an important site for peripheral immune activation, we asked how the fetal spleen would respond to intrauterine inflammation over time. We hypothesized that intraamniotic lipopolysaccharide (IA LPS) exposure induces acute and persistent changes in the splenic cytokine profile and T-cell composition that may contribute to the sustained fetal inflammatory response after chorioamnionitis.Methods:Fetal sheep were exposed to IA LPS 5, 12, and 24?h and 2, 4, 8, or 15 d before delivery at 125 d of gestational age (term = 150 d). Splenic cytokine mRNA levels and cleaved caspase-3, CD3, and Foxp3 expression were evaluated.Results:IA LPS increased interleukin (IL)1, IL4, IL5, and IL10 mRNA by twofold 24?h after injection. Interferon gamma increased by fivefold, whereas IL23 decreased 15 d post-LPS exposure. Cleaved caspase-3–positive cells increased 2 and 8 d after LPS exposure. CD3 immunoreactivity increased within 5?h with increased Foxp3-postive cells at 12?h.Conclusion:Intrauterine inflammation induced a rapid and sustained splenic immune response with persistent changes in the cytokine profile. This altered immune status may drive sustained inflammation and injury in other fetal organs.

The incidence of necrotizing enterocolitis (NEC) is higher in formula-fed babies than in breast-fed babies, which may be caused by breast-feeding–induced gut maturation. The effect of breast-feeding on gut maturation has been widely studied in animal models. This study aimed to assess the effects of breast-feeding on intestinal maturation in prematurely born babies by evaluating postnatal changes in urinary intestinal fatty acid binding protein (I-FABP) levels, a specific enterocyte marker. Gut maturation in 40 premature babies (<37 weeks of gestation) without gastrointestinal morbidity was studied, of whom 21 were exclusively breast-fed and 19 were formula-fed infants. Urinary I-FABP levels as the measure of gut maturation were measured at 5, 12, 19, and 26 days after birth. In breast-fed infants, there was a significant increase in median urinary I-FABP levels between 5 and 12 days after birth (104 [78–340] pg/mL to 408 [173–1028] pg/mL, P= 0.002), whereas I-FABP concentration in formula-fed infants increased between 12 and 19 days after birth (105 [44–557] pg/mL, 723 [103–1670] pg/mL, P= 0.004). Breast-fed babies had significantly higher median urinary I-FABP levels at postnatal day 12 (P= 0.01). The time course of the postnatal increase in urinary I-FABP levels reflecting gut maturation was significantly delayed in formula-fed babies, suggesting a delayed physiological response in formula-fed compared with breast-fed infants.

The incidence of necrotizing enterocolitis (NEC) is higher in formula-fed babies than in breast-fed babies, which may be caused by breast-feeding–induced gut maturation. The effect of breast-feeding on gut maturation has been widely studied in animal models. This study aimed to assess the effects of breast-feeding on intestinal maturation in prematurely born babies by evaluating postnatal changes in urinary intestinal fatty acid binding protein (I-FABP) levels, a specific enterocyte marker.

Background:Damage-associated molecular patterns (DAMPs) and antimicrobial peptides (AMPs) are components of pulmonary innate immunity and tissue repair. We hypothesized that DAMPs and AMPs would increase in response to fetal pulmonary inflammation caused by chorioamnionitis in a time-dependent manner.Methods:Fetal sheep were exposed to intra-amniotic saline or lipopolysaccharide (LPS) (10?mg) between 5?h and 15 d prior to preterm delivery at 125?±?2 d. Lung tissue mRNAs for proinflammatory cytokines; AMPs: myeloid AMP-29 (MAP29), dodecapeptide, sheep ß-defensin-1 (SBD1), and sheep ß-defensin-2 (SBD2); and DAMPs: interleukin (IL)-1a, lactoferrin, heat-shock protein-70 (HSP70), high-mobility group box protein-B1 (HMGB1), and receptor for advanced glycation endproducts (RAGE) were measured by reverse-transcriptase quantitative polymerase chain reaction. Immunohistochemistry of DAMPs and in situ hybridization of AMPs was performed.Results:IL-1a, IL-1ß, IL-6, IL-8, IL-10, MCP-1, and tumor necrosis factor (TNF)-a mRNA increased after LPS exposure. MAP29, dodecapeptide, SBD1, and SBD2 mRNA were suppressed at 24?h. MAP29 and dodecapeptide mRNA then increased at 8 d. Lactoferrin increased at 24?h. There were no changes for HMGB1, HSP70, or RAGE. MAP29 and dodecapeptide localized to alveolar cells, increased 8 d after exposure to LPS.Conclusion:AMPs are initially suppressed in the fetal lung by LPS-induced chorioamnionitis. The late induction of MAP29 and dodecapeptide may be related to lung repair.

Background:A new technique was proposed to administer surfactant to spontaneous breathing preterm infants by placing a thin catheter through the vocal cords. This technique was not studied with respect to oxygenation, gas exchange, surfactant distribution, and lung mechanics. We tested the technique of less-invasive surfactant administration (LISA) in a spontaneous breathing preterm lamb model.Methods:Preterm lambs (n = 12) of 133–134 d gestational age were randomized to the following three groups: (i) continuous positive airway pressure (CPAP) only, (ii) CPAP + LISA, and (iii) intubation and mechanical ventilation with surfactant administration. Surfactant was labeled with samarium oxide. During the next 180 min, blood gas analyses were performed. Postmortem, lungs were removed and surfactant distribution was assessed, and pressure–volume curves were performed.Results:Pao2in the LISA-treated lambs was significantly higher than in the lambs that exclusively received CPAP. Moreover, Pao2values were similar between the LISA-treated and the intubated lambs. Overall, surfactant deposition was less in the LISA lambs, with significantly less surfactant distributed to the right upper lobe. Lung compliance was better in the intubated lambs compared with the LISA-treated lambs, although this did not reach significance.Conclusion:LISA improved oxygenation, similar to conventional surfactant application techniques, despite lower surfactant deposition and lung compliance.

Background:Preventing preterm birth and subsequent adverse neonatal sequelae is among the greatest clinical challenges of our time. Recent studies suggest a role for Candida spp. in preterm birth and fetal injury, as a result of their colonization of either the vagina and/or the amniotic cavity. We hypothesized that intraamniotic Candida albicans would cause a vigorous, acute fetal inflammatory response.Methods:Sheep carrying singleton pregnancies received single intraamniotic injections of either saline (control) or 107colony-forming units C. albicans 1 or 2 d prior to surgical delivery and euthanasia at 124?±?2 d gestation.Results:Colonization of the amniotic cavity by C. albicans resulted in a modest inflammatory response at 1 d and florid inflammation at 2 d, characterized by fetal thrombocytopenia, lymphopenia, and significant increases of inflammatory cytokines/chemokines in the fetal membranes skin, lung, and the amniotic fluid.Conclusion:Acute colonization of the amniotic cavity by C. albicans causes severe intrauterine inflammation and fetal injury. C. albicans is a potent fetal pathogen that can contribute to adverse pregnancy outcomes.

Background:Intra-amniotic lipopolysaccharide (LPS) exposure may affect neonatal outcome by altering fetal lung and immune system development. We hypothesized that intra-amniotic LPS exposure would cause persistent fetal pulmonary responses as the lungs develop in utero.Methods:Fetal lambs were exposed to intra-amniotic LPS at 118 or at 118 and 123 d of gestational age (GA) with delivery at 125, 133, or 140 d (term = 147 d). Immune responses, PU.1 expression, Toll-like receptor (TLR)-1,2,4,6 mRNA levels, mast cell levels, and pulmonary elastin deposition were evaluated.Results:After a single dose of LPS, pulmonary inflammatory responses were observed with increases of (i) PU.1 and TLR1 at 125 d GA and (ii) monocytes, lymphocytes, TLR2, and TLR6 at 133 d GA. Repetitive LPS exposure resulted in (i) increases of neutrophils, monocytes, PU.1, and TLR1 at 125 d GA; (ii) increases of neutrophils, PU.1, and TLR2 at 133 d GA; and (iii) decreases of mast cells, elastin foci, TLR4, and TLR6 at early gestation. At 140 d GA, only PU.1 was increased after repetitive LPS exposure.Conclusion:The preterm fetal lung can respond to a single exposure or repeated exposures from intra-amniotic LPS in multiple ways, but the absence of inflammatory and structural changes in LPS-exposed fetuses delivered near term suggest that the fetus can resolve an inflammatory stimulus in utero with time.

Background:Antenatal inflammation and maternal corticosteroids induce fetal lung maturation but interfere with late lung development. Canonical Wingless-Int (Wnt) signaling directs lung development and repair. We showed that intra-amniotic (IA) lipopolysaccharide (LPS) exposure disrupted developmental signaling pathways in the preterm lamb lungs. Therefore, we hypothesized that pulmonary Wnt signaling was altered by exposure to IA LPS and/or antenatal corticosteroids.Methods:Ovine fetuses were exposed to IA LPS, maternal intramuscular betamethasone, a control saline injection, or a combination thereof at 107 and/or 114 d gestational age (term = 150 d gestational age) before delivery at 121 d gestational age.Results:IA LPS exposure decreased the lung expression of lymphoid enhancer-binding factor 1 (LEF1), a major Wnt pathway effector. WNT1, WNT4, and downstream messenger ß-catenin decreased after LPS exposure. WNT7b mRNA increased fourfold 14 d post–LPS exposure. Betamethasone treatment 7 d before LPS exposure prevented the reduction in LEF1 expression, whereas betamethasone administration after LPS normalized the LPS-induced increase in Wnt7b mRNA.Conclusion:IA LPS exposure decreased canonical Wnt signaling in the developing lung. Antenatal corticosteroids before or after IA inflammation had different effects on pulmonary Wnt signaling. This study provides new insights into possible mechanisms by which prenatal inflammation affects lung development and how corticosteroid can be beneficial in this setting.

Background:Sensorineural hearing loss (SNHL) is a common feature in the postasphyxial syndrome in newborns. Several anesthetic drugs have been proposed to attenuate secondary neuronal injury elicited by hypoxia–ischemia. We hypothesized that propofol anesthesia reduces auditory impairment after perinatal asphyxia in comparison with isoflurane.Methods:Twenty-three pregnant ewes were randomized to propofol or isoflurane anesthesia and sedation. The lambs underwent in utero umbilical cord occlusion (isoflurane n = 5; propofol n = 7) and were compared with sham-treated animals (isoflurane n = 5; propofol n = 6) at a gestational age of 133 d. For 8?h after delivery by cesarean section, repeated auditory brainstem responses (ABRs) were recorded to obtain hearing thresholds, peak amplitudes, latencies, and interpeak latencies.Results:Significantly elevated mean thresholds, diminished amplitudes, and elevated latencies were observed in the asphyxia group relative to the control group through the observation period. Comparison of anesthetic treatment in the asphyxia group revealed a significantly lower elevation in threshold and less impairment in the ABR amplitudes and latencies during propofol anesthesia as compared with isoflurane anesthesia.Conclusion:Our results support the hypothesis that anesthesia with propofol has a preventive effect on the functional changes to the auditory pathway in the event of perinatal asphyxia.

Background:Reintroduction of enteral nutrition in neonates with necrotizing enterocolitis (NEC) should take place when the gut is ready for its normal function. Too early a start of oral feeding might lead to disease relapse, whereas prolonged discontinuation of enteral nutrition is associated with impaired gut function and parenteral nutrition–related complications. This study evaluated whether noninvasive urinary measurement of intestinal fatty acid binding protein (I-FABP) at the time of refeeding can predict clinical outcome in neonates with NEC.Methods:Urinary I-FABP concentrations were measured in 21 infants with NEC just before reintroducing enteral nutrition. Poor outcome was defined as unsuccessful reintroduction of enteral feeding (EF), (re)operation for NEC, or death related to NEC after reintroduction of EF.Results:Median urinary I-FABP levels in neonates with poor outcome (n = 5) were significantly higher as compared with I-FABP levels in neonates with good outcome (n = 16) (P < 0.01). A clinically significant cutoff value of 963 pg/ml was found to discriminate between infants with poor outcome and those with good outcome (sensitivity 80%, specificity 94%).Conclusion:Noninvasive urinary I-FABP measurement at time of refeeding differentiates neonates with poor outcome from neonates with good outcome in NEC. Urinary I-FABP measurement may therefore be helpful in the timing of EF in neonates with NEC.

Background:Cardiac dysfunction is reported to occur after severe perinatal asphyxia. We hypothesized that anesthesia of the mother with propofol during emergency cesarean section (c-section) would result in less cardiac injury (troponin T) in preterm fetuses exposed to global severe asphyxia in utero than anesthesia with isoflurane. We tested whether propofol decreases the activity of proapoptotic caspase-3 by activating the antiapoptotic AKT kinase family and the signal transducer and activator of transcription-3 (STAT-3).Methods:Pregnant ewes were randomized to receive either propofol or isoflurane anesthesia. A total of 44 late-preterm lambs were subjected to in utero umbilical cord occlusion (UCO), resulting in asphyxia and cardiac arrest, or sham treatment. After emergency c-section, each fetus was resuscitated, mechanically ventilated, and supported under anesthesia for 8?h using the same anesthetic as the one received by its mother.Results:At 8?h after UCO, the fetuses whose mothers had received propofol anesthesia had lower plasma troponin T levels, and showed a trend toward a higher median left ventricular ejection fraction (LVEF) of 84% as compared with 74% for those whose mothers had received isoflurane. Postasphyxia activation of caspase-3 was lower in association with propofol anesthesia than with isoflurane. Postasphyxia levels of STAT-3 and the AKT kinase family rose 655% and 500%, respectively with the use of propofol anesthesia for the mother.Conclusion:The use of propofol for maternal anesthesia results in less cardiac injury in late-preterm lambs subjected to asphyxia than the use of isoflurane anesthesia. The underlying mechanism may be activation of the antiapoptotic STAT-3 and AKT pathways.

Background:The understanding of hypoxemia-induced changes in baroreflex function is limited and may be studied in a fetal sheep experiment before, during, and after standardized hypoxic conditions.Methods:Preterm fetal lambs were instrumented at 102 d gestation (term: 146 d). At 106 d, intrauterine hypoxia–­ischemia was induced by 25?min of umbilical cord occlusion (UCO). Baroreflex-related fluctuations were calculated at 30-min intervals during the first week after UCO by transfer function (cross-spectral) analysis between systolic blood pressure (SBP) and R–R interval fluctuations, estimated in the low-frequency (LF, 0.04–0.15 Hz) band. LF transfer gain (baroreflex sensitivity) and delay (s) reflect the baroreflex function.Results:Baseline did not differ in LF transfer gain and delay between controls and the UCO group. In controls, LF gain showed postnatal increase. By contrast, LF gain gradually decreased in the UCO group, resulting in significantly lower values 4–7 d after UCO. In the UCO group, LF delay increased and differed significantly from controls.Conclusion:Our results show that intrauterine hypoxia–ischemia results in reduced baroreflex sensitivity over a period of 7 d, indicating limited efficacy to buffer BP changes by adapting heart rate. Cardiovascular dysregulation may augment already present cerebral damage after systemic hypoxia–ischemia in the reperfusion period.

Bacterial re‐programming of PBMC‐derived monocytes induces the emergence of a distinct monocytic phenotype with profound phagocytic impairments but a preserved ability for naïve T cell stimulation. Septic diseases are characterized by an initial systemic, proinflammatory phase, followed by a period of anti‐inflammation. In the context of the latter, monocytes have been described to display altered functions, including reduced TNF secretion and T cell‐stimulating capacities in response to recall antigens. This hyporesponsiveness is supposed to be detrimental for coping with secondary infections. We here characterize bacterially reprogrammed PBMC‐derived monocytes with special focus on their phagocytic activity. Hence, we have implemented a surrogate model of the early, postinflammatory period by exposing PBMCs to Escherichia colion d0 and rechallenging them with bacteria on d2. This induced the emergence of a distinct monocytic phenotype with profound phagocytic impairments but a preserved ability for naïve T cell stimulation. The compromising effects on phagocytosis required the presence of bacteria and were not mimicked by TLR4 ligation or exposure to isolated cytokines alone. Moreover, the impairments were specific for the engulfment of bacteria and were coupled to a selective down‐regulation of FcγR and SR expression. Intriguingly, this monocytic phenotype contributed to the stimulation of a TH17‐polarized adaptive immune response in the context of secondary infection. Our findings extend the current knowledge of monocytic reprogramming and identify the phagocytic capacity of monocytes as a putative sepsis biomarker.

Our study aimed at automated power spectral analysis of the EEG in preterm infants to identify changes of spectral measures with maturation. Weekly (10–20 montage) 4-h EEG recordings were performed in 18 preterm infants with GA <32 wk and normal neurological follow-up at 2 y, resulting in 79 recordings studied from 274to 363wk of postmenstrual age (PMA, GA postnatal age). Automated spectral analysis was performed on 4-h EEG recordings. The frequency spectrum was divided in delta 1 (0.5–1 Hz), delta 2 (1–4 Hz), theta (4–8 Hz), alpha (8–13 Hz), and beta (13–30 Hz) band. Absolute and relative power of each frequency band and spectral edge frequency were calculated. Maturational changes in spectral measures were observed most clearly in the centrotemporal channels. With advancing PMA, absolute powers of delta 1 to 2 and theta decreased. With advancing PMA, relative power of delta 1 decreased and relative powers of alpha and beta increased, respectively. In conclusion, with maturation, spectral analysis of the EEG showed a significant shift from the lower to the higher frequencies. Computer analysis of EEG will allow an objective and reproducible analysis for long-term prognosis and/or stratification of clinical treatment.

Our study aimed at automated power spectral analysis of the EEG in preterm infants to identify changes of spectral measures with maturation. Weekly (10–20 montage) 4-h EEG recordings were performed in 18 preterm infants with GA <32 wk and normal neurological follow-up at 2 y, resulting in 79 recordings studied from 27+4to 36+3wk of postmenstrual age (PMA, GA + postnatal age). Automated spectral analysis was performed on 4-h EEG recordings. The frequency spectrum was divided in delta 1 (0.5–1 Hz), delta 2 (1–4 Hz), theta (4–8 Hz), alpha (8–13 Hz), and beta (13–30 Hz) band. Absolute and relative power of each frequency band and spectral edge frequency were calculated. Maturational changes in spectral measures were observed most clearly in the centrotemporal channels. With advancing PMA, absolute powers of delta 1 to 2 and theta decreased. With advancing PMA, relative power of delta 1 decreased and relative powers of alpha and beta increased, respectively. In conclusion, with maturation, spectral analysis of the EEG showed a significant shift from the lower to the higher frequencies. Computer analysis of EEG will allow an objective and reproducible analysis for long-term prognosis and/or stratification of clinical treatment.

Exposure of the fetus to antenatal inflammation can occur from chorioamnionitis, which may progress to a fetal inflammatory response syndrome (FIRS) and to fetal sepsis. We tested whether the fetal myocardium responded to systemic Gram-negative endotoxinaemia. We hypothesized that the myocardium would respond to inflammation by changes in hypoxia-inducible factor- (HIF-1), inducible NO-synthase (iNOS), Toll-like receptors 2 and 4 (TLR2 and TLR4), IL-6, and phosphorylated signal transducer and activator of transcription-3 (pSTAT3). To model systemic endotoxinaemia, fetal sheep were exposed to Gram-negative endotoxin or saline i.v. 3 d before preterm delivery at 113 d of gestation (term 147 d). All endotoxin-exposed animals developed cardiac dysfunction within these 72 h. Cardiac mRNA and protein levels of HIF-1 and TLR2 and TLR4 mRNA increased, whereas STAT3 phosphorylation decreased significantly. IL-6 and iNOS mRNA remained unchanged. Fetal systemic endotoxinaemia induced myocardial inflammation by activating TLR2 and 4. The following cardiac dysfunction seems not to be mediated viacardiac iNOS.