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Persistent pulmonary hypertension of the newborn (PPHN) is a common neonatal condition in newborns admitted to the neonatal intensive care units (NICUs). PPHN has still a high mortality and morbidity. Inhaled nitric oxide (iNO) is the first line vasodilator therapy for PPHN in high income countries. In low-to-middle income countries (LMICs), availability of iNO remains scarce and expensive. The purpose of this scoping review was to evaluate the current existing literature for milrinone therapy in PPHN and to identify the knowledge gaps in milrinone use in infants with PPHN. The available evidence for milrinone remains limited both as monotherapy and as an adjuvant to iNO. The studies were heterogeneous, conducted in different settings, with different populations and more importantly the endpoints of these trials were short-term outcomes such as changes in oxygenation and blood pressure. Large prospective studies investigating long-term outcomes, mortality, and the need for Extracorporeal membrane oxygenation (ECMO) are warranted. Randomized controlled trials with milrinone as monotherapy are needed in LMICs where iNO availability remains limited. IMPACT: Milrinone has a potential role in the management of PPHN both as an adjuvant to iNO as well as a monotherapy. This scoping review identified the problems existing in the published literature on milrinone and the barriers to generalization of these results. Multi-centre randomized controlled trials on milrinone, especially involving centers from low- and middle-income countries are needed, where it can be evaluated as first-line pulmonary vasodilator therapy., (© 2024. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Background: Perinatal inflammation combined with hypoxia-ischemia (HI) exacerbates injury in the developing brain. Therapeutic hypothermia (HT) is standard care for neonatal encephalopathy; however, its benefit in inflammation-sensitized HI (IS-HI) is unknown., Methods: Twelve newborn piglets received a 2 µg/kg bolus and 1 µg/kg/h infusion over 52 h of Escherichia coli lipopolysaccharide (LPS). HI was induced 4 h after LPS bolus. After HI, piglets were randomized to HT (33.5 °C 1-25 h after HI, n = 6) or normothermia (NT, n = 6). Amplitude-integrated electroencephalogram (aEEG) was recorded and magnetic resonance spectroscopy (MRS) was acquired at 24 and 48 h. At 48 h, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive brain cell death, microglial activation/proliferation, astrogliosis, and cleaved caspase-3 (CC3) were quantified. Hematology and plasma cytokines were serially measured., Results: Two HT piglets died. aEEG recovery, thalamic and white matter MRS lactate/N-acetylaspartate, and TUNEL-positive cell death were similar between groups. HT increased microglial activation in the caudate, but had no other effect on glial activation/proliferation. HT reduced CC3 overall. HT suppressed platelet count and attenuated leukocytosis. Cytokine profile was unchanged by HT., Conclusions: We did not observe protection with HT in this piglet IS-HI model based on aEEG, MRS, and immunohistochemistry. Immunosuppressive effects of HT and countering neuroinflammation by LPS may contribute to the observed lack of HT efficacy. Other immunomodulatory strategies may be more effective in IS-HI., Impact: Acute infection/inflammation is known to exacerbate perinatal brain injury and can worsen the outcomes in neonatal encephalopathy. Therapeutic HT is the current standard of care for all infants with NE, but the benefit in infants with coinfection/inflammation is unknown. In a piglet model of inflammation (LPS)-sensitized HI, we observed no evidence of neuroprotection with cooling for 24 h, based on our primary outcome measures: aEEG, MRS Lac/NAA, and histological brain cell death. Additional neuroprotective agents, with beneficial immunomodulatory effects, require exploration in IS-HI models., (© 2021. The Author(s).)

Background: Chorioamnionitis, an intrauterine infection of the placenta and fetal membranes, is a common risk factor for adverse pulmonary outcomes in premature infants including BPD, which is characterized by an arrest in alveolar development. As endogenous epithelial stem/progenitor cells are crucial for organogenesis and tissue repair, we examined whether intrauterine inflammation negatively affects these essential progenitor pools., Methods: In an ovine chorioamnionitis model, fetuses were intra-amniotically exposed to LPS, 2d or 7d (acute inflammation) before preterm delivery at 125d of gestation, or to intra-amniotic Ureaplasma parvum for 42d (chronic inflammation). Lung function, pulmonary endogenous epithelial stem/progenitor pools, and downstream functional markers were studied., Results: Lung function was improved in the 7d LPS and 42d Ureaplasma groups. However, intrauterine inflammation caused a loss of P63+ basal cells in proximal airways and reduced SOX-9 expression and TTF-1+ Club cells in distal airways. Attenuated type-2 cell numbers were associated with lower proliferation and reduced type-1 cell marker Aqp5 expression, indicative for impaired progenitor function. Chronic Ureaplasma infection only affected distal airways, whereas acute inflammation affected stem/progenitor populations throughout the lungs., Conclusions: Acute and chronic prenatal inflammation improve lung function at the expense of stem/progenitor alterations that potentially disrupt normal lung development, thereby predisposing to adverse postnatal outcomes., Impact: In this study, prenatal inflammation improved lung function at the expense of stem/progenitor alterations that potentially disrupt normal lung development, thereby predisposing to adverse postnatal outcomes. Importantly, we demonstrate that these essential alterations can already be initiated before birth. So far, stem/progenitor dysfunction has only been shown postnatally. This study indicates that clinical protocols to target the consequences of perinatal inflammatory stress for the immature lungs should be initiated as early as possible and ideally in utero. Within this context, our data suggest that interventions, which promote function or repair of endogenous stem cells in the lungs, hold great promise., (© 2021. International Pediatric Research Foundation, Inc.)

Chorioamnionitis, inflammation of fetal membranes, is an important cause of preterm birth and a risk factor for the development of adverse neonatal outcomes including sepsis and intestinal pathologies. Intestinal bile acids (BAs) accumulation and hepatic cytokine production are involved in adverse intestinal outcomes. These findings triggered us to study the liver and enterohepatic circulation (EHC) following intra-amniotic (IA) lipopolysaccharide (LPS) exposure. An ovine chorioamnionitis model was used in which circulatory cytokines and outcomes of the liver and EHC of preterm lambs were longitudinally assessed following IA administration of 10 mg LPS at 5, 12 or 24h or 2, 4, 8 or 15d before preterm birth. Hepatic inflammation was observed, characterized by increased hepatic cytokine mRNA levels (5h - 2d post IA LPS exposure) and increased erythropoietic clusters (at 8 and 15 days post IA LPS exposure). Besides, 12h after IA LPS exposure, plasma BA levels were increased, whereas gene expression levels of several hepatic BA transporters were decreased. Initial EHC alterations normalized over time. Concluding, IA LPS exposure induces significant time-dependent changes in the fetal liver and EHC. These chorioamnionitis induced changes have potential postnatal consequences and the duration of IA LPS exposure might be essential herein.

Background: Exposure to inflammation exacerbates injury in neonatal encephalopathy (NE). We hypothesized that brain biomarker mRNA, cytokine mRNA and microRNA differentiate inflammation (E. coli LPS), hypoxia (Hypoxia), and inflammation-sensitized hypoxia (LPS+Hypoxia) in an NE piglet model., Methods: Sixteen piglets were randomized: (i) LPS 2 μg/kg bolus; 1 μg/kg infusion (LPS; n = 5), (ii) Saline with hypoxia (Hypoxia; n = 6), (iii) LPS commencing 4 h pre-hypoxia (LPS+Hypoxia; n = 5). Total RNA was acquired at baseline, 4 h after LPS and 1, 3, 6, 12, 24, 48 h post-insult (animals euthanized at 48 h). Quantitative PCR was performed for cytokines (IL1A, IL6, CXCL8, IL10, TNFA) and brain biomarkers (ENO2, UCHL1, S100B, GFAP, CRP, BDNF, MAPT). MicroRNA was detected using GeneChip (Affymetrix) microarrays. Fold changes from baseline were compared between groups and correlated with cell death (TUNEL) at 48 h., Results: Within 6 h post-insult, we observed increased IL1A, CXCL8, CCL2 and ENO2 mRNA in LPS+Hypoxia and LPS compared to Hypoxia. IL10 mRNA differentiated all groups. Four microRNAs differentiated LPS+Hypoxia and Hypoxia: hsa-miR-23a, 27a, 31-5p, 193-5p. Cell death correlated with TNFA (R = 0.69; p < 0.01) at 1-3 h and ENO2 (R = -0.69; p = 0.01) at 48 h., Conclusions: mRNA and miRNA differentiated hypoxia from inflammation-sensitized hypoxia within 6 h in a piglet model. This information may inform human studies to enable triage for tailored neuroprotection in NE., Impact: Early stratification of infants with neonatal encephalopathy is key to providing tailored neuroprotection. IL1A, CXCL8, IL10, CCL2 and NSE mRNA are promising biomarkers of inflammation-sensitized hypoxia. IL10 mRNA levels differentiated all three pathological states; fold changes from baseline was the highest in LPS+Hypoxia animals, followed by LPS and Hypoxia at 6 h. miR-23, -27, -31-5p and -193-5p were significantly upregulated within 6 h of a hypoxia insult. Functional analysis highlighted the diverse roles of miRNA in cellular processes.

Background: The addition of budesonide (Bud) 0.25 mg/kg to surfactant decreased the lung and systemic responses to mechanical ventilation in preterm sheep and the rates and severity of bronchopulmonary dysplasia (BPD) in preterm infants. We hypothesized that lower budesonide concentrations in surfactant will decrease injury while decreasing systemic corticosteroid exposure., Methods: Preterm lambs received either (1) protective tidal volume (V T ) ventilation with surfactant from birth or (2) injurious V T ventilation for 15 min and then surfactant treatment. Lambs were further assigned to surfactant mixed with (i) Saline, (ii) Bud 0.25 mg/kg, (iii) Bud 0.1 mg/kg, or (iv) Bud 0.04 mg/kg. All lambs were then ventilated with protective V T for 6 h., Results: Plasma Bud levels were proportional to the dose received and decreased throughout ventilation. In both protective and injurious V T ventilation, <4% of Bud remained in the lung at 6 h. Some of the improvements in physiology and markers of injury with Bud 0.25 mg/kg were also found with 0.1 mg/kg, whereas 0.04 mg/kg had only minimal effects., Conclusions: Lower doses of Bud were less effective at decreasing lung and systemic inflammation from mechanical ventilation. The plasma Bud levels were proportional to dose given and the majority left the lung.

With the current practice of therapeutic hypothermia for neonatal encephalopathy, disability rates and the severity spectrum of cerebral palsy are reduced. Nevertheless, safe and effective adjunct therapies are needed to optimize outcomes. This study's objective was to assess if 18 mg/kg melatonin given rapidly over 2 h at 1 h after hypoxia-ischemia with cooling from 1-13 h was safe, achieved therapeutic levels within 3 h and augmented hypothermic neuroprotection. Following hypoxia-ischemia, 20 newborn piglets were randomized to: (i) Cooling 1-13 h (HT; n = 6); (ii) HT+ 2.5% ethanol vehicle (HT+V; n = 7); (iii) HT + Melatonin (HT+M; n = 7). Intensive care was maintained for 48 h; aEEG was acquired throughout, brain MRS acquired at 24 and 48 h and cell death (TUNEL) evaluated at 48 h. There were no differences for insult severity. Core temperature was higher in HT group for first hour after HI. Comparing HT+M to HT, aEEG scores recovered more quickly by 19 h (p < 0.05); comparing HT+V to HT, aEEG recovered from 31 h (p < 0.05). Brain phosphocreatine/inorganic phosphate and NTP/exchangeable phosphate were higher at 48 h in HT+M versus HT (p = 0.036, p = 0.049 respectively). Including both 24 h and 48 h measurements, the rise in Lactate/N-acetyl aspartate was reduced in white (p = 0.030) and grey matter (p = 0.038) after HI. Reduced overall TUNEL positive cells were observed in HT+M (47.1 cells/mm 2 ) compared to HT (123.8 cells/mm 2 ) (p = 0.0003) and HT+V (97.5 cells/mm 2 ) compared to HT (p = 0.012). Localized protection was seen in white matter for HT+M versus HT (p = 0.036) and internal capsule for HT+M compared to HT (p = 0.001) and HT+V versus HT (p = 0.006). Therapeutic melatonin levels (15-30mg/l) were achieved at 2 h and were neuroprotective following HI, but ethanol vehicle was partially protective.

Autologous fat transfer (AFT) is limited by post-operative volume loss due to ischemia-induced cell death in the fat graft. Previous studies have demonstrated that electrical stimulation (ES) promotes angiogenesis in a variety of tissues and cell types. In this study we investigated the effects of ES on the angiogenic potential of adipose-derived stem cells (ASC), important progenitor cells in fat grafts with proven angiogenic potential. Cultured human ASC were electrically stimulated for 72 hours after which the medium of stimulated (ES) and non-stimulated (control) ASC was analysed for angiogenesis-related proteins by protein array and ELISA. The functional effect of ES on angiogenesis was then assessed in vitro and in vivo. Nine angiogenesis-related proteins were detected in the medium of electrically (non-)stimulated ASC and were quantified by ELISA. The pro-angiogenic proteins VEGF and MCP-1 were significantly increased following ES compared to controls, while the anti-angiogenic factor Serpin E1/PAI-1 was significantly decreased. Despite increased levels of anti-angiogenic TSP-1 and TIMP-1, medium of ES-treated ASC significantly increased vessel density, total vessel network length and branching points in chorio-allantoic membrane assays. In conclusion, our proof-of-concept study showed that ES increased the angiogenic potential of ASC both in vitro and in vivo.

Co-existing infection/inflammation and birth asphyxia potentiate the risk of developing neonatal encephalopathy (NE) and adverse outcome. In a newborn piglet model we assessed the effect of E. coli lipopolysaccharide (LPS) infusion started 4 h prior to and continued for 48 h after hypoxia on brain cell death and systemic haematological changes compared to LPS and hypoxia alone. LPS sensitized hypoxia resulted in an increase in mortality and in brain cell death (TUNEL positive cells) throughout the whole brain, and in the internal capsule, periventricular white matter and sensorimotor cortex. LPS alone did not increase brain cell death at 48 h, despite evidence of neuroinflammation, including the greatest increases in microglial proliferation, reactive astrocytosis and cleavage of caspase-3. LPS exposure caused splenic hypertrophy and platelet count suppression. The combination of LPS and hypoxia resulted in the highest and most sustained systemic white cell count increase. These findings highlight the significant contribution of acute inflammation sensitization prior to an asphyxial insult on NE illness severity.

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.

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-PO 4 ) and the combination betamethasone-phosphate (Beta-PO 4 ) + 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-PO 4 , Dex-PO 4 , or Beta-PO 4 alone., Methods: Pregnant ewes received two intramuscular doses 24 h apart of 0.25 mg/kg/dose of Beta-Ac+Beta-PO 4 , Dex-PO4 or Beta-PO 4 ; or 2 doses of 0.125 mg/kg/dose of Beta-PO 4 at 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-PO 4 required lower positive inspiratory pressure compared with control. Beta-Ac + Beta-PO 4 and Beta-PO 4 alone, but not Dex-PO 4 , increased the mRNA of surfactant proteins compared with control. Low-dose Beta-PO 4 did not increase mRNA of surfactant proteins. There were no differences among Beta-PO 4 treatment intervals., Conclusion: Beta-Ac + Beta-PO 4 given as two doses 24 h apart was more effective in promoting fetal lung maturation than Dex-PO 4 or Beta-PO 4 alone, consistent with a prolonged exposure provided by the Beta-Ac + Beta-PO 4 . These results support the clinical use of combined Beta-Ac + Beta-PO 4 preparations over phosphate corticosteroids alone for fetal lung maturation.

Chorioamnionitis is caused by intrauterine infection with microorganisms including Candida albicans (C.albicans). Chorioamnionitis is associated with postnatal intestinal pathologies including necrotizing enterocolitis. The underlying mechanisms by which intra-amniotic C.albicans infection adversely affects the fetal gut remain unknown. Therefore, we assessed whether intra-amniotic C.albicans infection would cause intestinal inflammation and mucosal injury in an ovine model. Additionally, we tested whether treatment with the fungistatic fluconazole ameliorated the adverse intestinal outcome of intra-amniotic C.albicans infection. Pregnant sheep received intra-amniotic injections with 10(7) colony-forming units C.albicans or saline at 3 or 5 days before preterm delivery at 122 days of gestation. Fetuses were given intra-amniotic and intra-peritoneal fluconazole treatments 2 days after intra-amniotic administration of C.albicans. Intra-amniotic C.albicans caused intestinal colonization and invasive growth within the fetal gut with mucosal injury and intestinal inflammation, characterized by increased CD3(+) lymphocytes, MPO(+) cells and elevated TNF-α and IL-17 mRNA levels. Fluconazole treatment in utero decreased intestinal C.albicans colonization, mucosal injury but failed to attenuate intestinal inflammation. Intra-amniotic C.albicans caused intestinal infection, injury and inflammation. Fluconazole treatment decreased mucosal injury but failed to ameliorate C.albicans-mediated mucosal inflammation emphasizing the need to optimize the applied antifungal therapeutic strategy.

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 QTc demonstrated 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 QTc markers could assess the presence of severe hypoxia. Waveform-based STAN episodic and baseline rise markers were ineffective as markers for hypoxia.

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.

We investigated whether changes in perinatal outcomes occurred following introduction of key tobacco control policies in the Netherlands: smoke-free legislation in workplaces plus a tobacco tax increase and mass media campaign (January-February 2004); and extension of the smoke-free law to the hospitality industry, accompanied by another tax increase and mass media campaign (July 2008). This was a national quasi-experimental study using Netherlands Perinatal Registry data (2000-2011; registration: ClinicalTrials.gov NCT02189265). Primary outcome measures were: perinatal mortality, preterm birth, and being small-for-gestational age (SGA). The association with timing of the tobacco control policies was investigated using interrupted time series logistic regression analyses with adjustment for confounders. Among 2,069,695 singleton births, there were 13,027 (0.6%) perinatal deaths, 116,043 (5.6%) preterm live-births and 187,966 (9.1%) SGA live-births. The 2004 policies were not associated with significant changes in the odds of developing any of the primary outcomes. After the 2008 policy change, a -4.4% (95% CI -2.4; -6.4, p < 0.001) decrease in odds of being SGA was observed. A reduction in SGA births, but not preterm birth or perinatal mortality, was observed in the Netherlands after extension of the smoke-free workplace law to bars and restaurants in conjunction with a tax increase and mass media campaign.

Background: Hyperoxia and hypoxia influence morbidity and mortality of preterm infants. Automated closed-loop control of the fraction of inspired oxygen (FiO(2)) 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 FiO(2) control based on predefined oxygen saturation (SpO(2)) 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 FiO(2) control, targeting SpO(2) according to the Dawson curve in the first 10 min and SpO(2) 90-95% hereafter. Automated FiO(2) control also was applied during surfactant replacement therapy and subsequent ventilation., Results: Time within target range did not differ significantly between manual and automated FiO(2) control during resuscitation, however automated FiO(2) control significantly avoided hyperoxia. Automated FiO(2) control was feasible during surfactant replacement and kept SpO(2) within target range significantly better than manual control during subsequent ventilation., Conclusion: In our model, fully automated FiO(2) control was feasible in rapidly changing physiologic conditions during postnatal resuscitation and prevented hyperoxia. We conclude that closed loop FiO(2) control is a promising tool for the delivery room.

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: 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 10(7) colony-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-positive 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.

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 β-defensin-1 (SBD1), and sheep β-defensin-2 (SBD2); and DAMPs: interleukin (IL)-1α, 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-1α, IL-1β, IL-6, IL-8, IL-10, MCP-1, and tumor necrosis factor (TNF)-α 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: Gut immaturity is linked with postnatal intestinal disorders. However, biomarkers to assess the intestinal developmental stage around birth are lacking. The aim of this study was to gain more insight on intestinal fatty acid-binding protein (I-FABP) as an indicator of gut maturity., Methods: Antenatal I-FABP distribution and release was investigated in extremely premature, moderately premature, and term lambs, and these findings were verified in human urinary samples. Ileal I-FABP distribution was confirmed in autopsy material within 24 h postnatally., Results: Median (range) serum I-FABP levels were lower in extremely premature lambs compared with moderately premature lambs (156 (50.0-427) vs. 385 (100-1,387) pg/ml; P = 0.02). Contrarily, median early postnatal urine I-FABP levels in human infants were higher in extremely premature compared with moderately premature and term neonates (1,219 (203-15,044) vs. 256 (50-1,453) and 328 (96-1,749) pg/ml; P = 0.008 and P = 0.04, respectively). I-FABP expression was most prominent in nonvacuolated enterocytes and increased with rising gestational age (GA) in ovine and human tissue samples. The epithelial distribution pattern changed from a phenotype displaying I-FABP-positive enterocytes merely in the crypts early in gestation into a phenotype with I-FABP expressing cells exclusively present in the villus tips at term in ovine and human tissue., Conclusion: In this ovine and human study, increasing GA is accompanied by an increase in I-FABP tissue content. Cord I-FABP levels correlate with gestation in ovine fetuses, identifying I-FABP as a marker for gut maturation. Raised postnatal urine I-FABP levels in preterm human infants may indicate intestinal injury and/or inflammation in utero.

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: Pao2 in the LISA-treated lambs was significantly higher than in the lambs that exclusively received CPAP. Moreover, Pao2 values 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 10(7) colony-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 β-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: 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.

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.

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(+4) to 36(+3) wk 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-α (HIF-1α), 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-1α 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 via cardiac iNOS.

Chorioamnionitis frequently induces a fetal inflammatory response syndrome (FIRS), characterized by an elevation of proinflammatory mediators and systemic inflammation. Although there is increasing evidence that inflammation and lipid metabolism influence each other, the effects of chorioamnionitis-induced FIRS on fetal lipid homeostasis are currently not known. Accordingly, we hypothesize that chorioamnionitis induces an inflammatory response in the fetal liver, consequently leading to metabolic disturbances. Chorioamnionitis was induced by intra-amniotic injection of 10 mg endotoxin (control) for 2 d or 2 wk before delivery. Saline injections were given to controls. The effect of chorioamnionitis on hepatic inflammation and metabolic parameters was analyzed in ovine fetuses at the GA of 125 d (normal GA = 150 d). We found that 2 d after the endotoxin injections, inflammatory markers were significantly higher compared with controls. In addition, lipid and glucose metabolism were disturbed in response to endotoxin. Moreover, the antioxidant state capacity was reduced, and hepatic damage was apparent. Two weeks after the endotoxin injections, the fetal livers were still inflamed and had higher glucose concentrations in the blood. In addition, the levels of markers for hepatic damage (alanine aminotransferase and aspartate aminotransferase) were increased. In conclusion, chorioamnionitis induces liver inflammation leading to metabolic disturbances in the fetus.

A systemic inflammatory response of the fetus, reflected by histologic funisitis, is a risk factor for bronchopulmonary dysplasia (BPD). Impaired pulmonary angiogenesis accompanied by simplification and rarification of alveoli is a histologic hallmark of BPD. Angiopoietin-1 mediates vascular development, maturation, and stabilization. Endostatin mainly acts as an angiostatic factor. We hypothesized that funisitis was associated with changes of endostatin and angiopoietin-1 concentrations in the airways and that an imbalance between the factors might be associated with BPD or death. We measured concentrations of angiopoietin-1 and endostatin by enzyme-linked immunosorbent assay in tracheobronchial aspirate fluid samples of 42 ventilated preterm infants during postnatal days 1 through 15. The secretory component for IgA served as reference protein. A standardized histologic examination was used to distinguish three groups: chorioamnionitis, funisitis, and controls without inflammation. Concentrations of the mediators steadily decreased. Funisitis was associated with lower concentrations of both proteins, which might impair their physiologic activities in pulmonary angiogenesis. An increase of the ratio angiopoietin-1/endostatin until day 7 of life indicated a shift of the mediators potentially favoring angiogenesis. However, infants, who developed BPD or died, had a decreased ratio on days 1, 3, and 15, suggesting an imbalance toward inhibition of pulmonary angiogenesis.

Intratracheal lipopolysaccharide (LPS) causes acute inflammation and injurious mechanical ventilation results in pulmonary and systemic inflammation. We aimed to determine in preterm lungs if continuous positive airway pressure (CPAP) protects against pulmonary and systemic inflammation, compared with conventional mechanical ventilation (CMV) after intratracheal LPS. Preterm fetuses were exposed to maternal betamethasone and Epostane 36 h before delivery at 133 d gestational age (term = 150 d). Lambs were intubated and randomized to receive gentle CMV (tidal volume 8 mL/kg) or CPAP with 8 cm H2O pressure. Surfactant (10 mg/kg) mixed with 1 mg LPS or saline was instilled into the trachea at 15 min. Blood gas status, ventilation variables, and arterial pressures were recorded for 3 h. Static pressure-volume curves and lung and systemic inflammation were assessed postmortem. CPAP lambs had elevated Paco2 and minute ventilation compared with the CMV lambs. Cytokine mRNA was increased in the lungs and liver of CPAP and CMV lambs relative to unventilated controls. Intratracheal LPS amplified the cytokine mRNA responses of IL-1beta, IL-6, and IL-8 in the lung and liver. Blood neutrophils decreased similarly after LPS in CPAP and CMV groups. Cytokine markers of lung injury or the systemic response to intratracheal LPS were not decreased by CPAP relative to CMV, in preterm lambs

Positive end-expiratory pressure (PEEP) protects the lung from injury during sustained ventilation, but its role in protecting the lung from injury during the initiation of ventilation in the delivery room is not established. We aimed to evaluate whether PEEP and/or tidal volume (VT) within the first 15-min of ventilation are protective against lung injury. Operatively delivered preterm lambs (133 +/- 1 d gestation) were randomly assigned to unventilated controls or to one of four 15 min ventilation interventions: 1) VT15 mL/kg, PEEP 0 cm H2O; 2) VT15 mL/kg, PEEP 5 cm H2O; 3) VT8 mL/kg, PEEP 0 cm H2O; and 4) VT8 mL/kg, PEEP 5 cm H2O. Each group was subsequently ventilated with VT 10 mL/kg, PEEP 5 cm H2O for 1 h 45 min. Lung function was assessed and measurements of lung injury were evaluated postmortem. After the 15 min ventilation maneuver, the VT15 groups were hypocarbic, had higher oxygenation, and required lower pressures than the VT8 groups; no consistent effect of PEEP was found. Markers of lung injury were significantly elevated in all ventilation groups compared with unventilated controls; no effect of PEEP was found. Ventilation resulted in localization of IL-6 to the small airways. Initial ventilation of preterm lambs with PEEP and/or VT of 8 mL/kg did not prevent an inflammatory injury to the lung.

Toll-like receptors (TLRs) are pattern recognition molecules that initiate innate immune responses. Intra-amniotic exposure of fetal sheep to pro-inflammatory stimuli causes pulmonary inflammation and induced lung maturation. We examined TLR ontogeny and fetal lung responsiveness to three different TLR agonists. We cloned ovine TLRs 2, 3, and 4 and found 83-88% homology between these ovine and human TLRs. Lung TLR2 and 4 mRNAs increased throughout late gestation to 50% of adult level in the term newborn lamb. Doses of 10 mg of PAMCysK4 (TLR2 agonist), poly I:C dsRNA (TLR3 agonist), or E. coli O55:B5 lipopoysaccharide (LPS) (TLR4 agonist) were given by intra-amniotic injection 2 d or 7 d before operative delivery of preterm lambs at 123 d (n = 4-7/group). The TLR4 agonist induced lung inflammation and maturation, whereas the TLR2 agonist gave less consistent responses. Intra-amniotic LPS increased TLR2 mRNA expression primarily in the inflammatory cells and TLR4 mRNA diffusely in multiple cell types. The TLR3 agonist had no effects, and TLR3 mRNA in the fetal lung did not change after LPS exposure. We conclude that TLR2 and TLR4 mRNAs increase through gestation and expression of TLR2 and TLR4 are induced by LPS in the fetal sheep lung.

Chorioamnionitis and funisitis are associated with neonatal morbidity and mortality. We hypothesized that chorioamnionitis may stress fetal endothelium, activate proinflammatory gene transcription. and affect angiogenic homeostasis in fetal capillaries. Placentas from preterm infants were stained for heat-shock protein 70, nuclear factor-kappaB, hypoxia-inducible factor-1alpha, and vascular endothelial growth factor (VEGF). VEGF receptors (VEGF-R) 1 and 2 as well as the receptor tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (TIE-2), which is involved in vascular remodeling, were quantified. Immunohistochemistry was analyzed by counting positive capillaries in placental terminal villi. Staining intensity was quantified by a three-step semiquantitative scale. The samples were divided into three matched groups according to histology: chorioamnionitis with funisitis ("funisitis"), chorioamnionitis without funisitis ("chorioamnionitis"), and control group with no inflammation. In tissues from the funisitis or chorioamnionitis group, heat-shock protein 70 expression was increased over the control group. More nuclear factor-kappaB-positive nuclei of endothelial cells in capillaries were counted in the funisitis and chorioamnionitis groups. Expression of VEGF and VEGF-R1 and -R2 were reduced in cases of funisitis or chorioamnionitis in comparison with controls. Hypoxia-inducible factor-1alpha expression tended to be slightly lower in the funisitis and chorioamnionitis groups but did not reach statistical significance. We speculate that cellular stress and changes in angiogenic homeostasis induced by proinflammatory activation of fetal endothelium in chorioamnionitis may not be limited to the placenta but may also involve other fetal organs.

Chorioamnionitis and funisitis are associated with preterm labor and postnatal morbidity. Activation of endothelium resulting in up-regulation of adhesion molecules seems to be a key mechanism in development of organ damage. We investigated whether chorioamnionitis with or without funisitis in preterm infants induced expression and shedding of adhesion molecules in the umbilical cord and resulted in increased concentrations of E-selectin, intercellular adhesion molecule (ICAM)-1, IL-1beta, IL-6, and IL-8 in the cord blood. Data were obtained by using immunohistochemistry and ELISA. Thirty-two preterm infants were divided into three groups according to histology: chorioamnionitis with funisitis, chorioamnionitis without funisitis, and controls without signs of inflammation. ICAM-1 expression on arterial endothelium was higher with funisitis compared with chorioamnionitis alone or with the control group. Similar results for ICAM-1 expression were found in venous endothelium, vascular walls, Wharton's jelly, and amnion epithelium. Endothelial E-selectin and vascular cell adhesion molecule (VCAM)-1 expression was only induced significantly with funisitis. Serum-concentrations of soluble ICAM-1 were higher with funisitis compared with chorioamnionitis alone or control group. Similarly, concentrations of soluble E-selectin, IL-1beta, IL-6, and IL-8 were increased exclusively with funisitis. In conclusion, only chorioamnionitis with funisitis was associated with systemic inflammation and endothelial activation with up-regulation and shedding of umbilical cord adhesion molecules. We speculate that this activation of endothelium may not be limited to the umbilical cord but may also involve other organs resulting in neonatal morbidity. This underlines the importance of funisitis as a risk factor for adverse outcome.

Corticosteroids are routinely administered to women at risk for preterm delivery to induce fetal lung maturation. Antenatal corticosteroids have immunomodulatory effects on fetal immune cells that are poorly understood. We hypothesized that maternal betamethasone would alter in fetal monocytes both the initiation of inflammation in response to pro-inflammatory stimuli and the resolution of inflammation by phagocytosis of apoptotic neutrophils. Preterm lambs at 124 d gestation were delivered 15 h, 1 d, 2 d, or 7 d after 0.5 mg/kg maternal intramuscular betamethasone. Monocytes from cord blood were isolated and cultured and results were compared with monocytes from preterm lambs exposed to maternal saline or monocytes from adult sheep. Phagocytosis of Escherichia coli was not changed, however, phagocytosis of apoptotic neutrophils was low in fetal monocytes but increased after 7 d exposure to maternal betamethasone to the level found in adult monocytes. Hydrogen peroxide production after endotoxin stimulus was significantly reduced to 7.1 +/- 2.2 micromol at 5 h, 8.7 +/- 2.9 micromol at 24 h, and 4.1 +/- 1.9 micromol at 48 h versus 16.4 +/- 3.6 micromol in control animals; at 7 d, the hydrogen peroxide production increased to 74.3 +/- 19.7 micromol (p < 0.05, per 10(6) monocytes). IL-6 production was reduced at 15 h after maternal betamethasone but at no other time point. Maternal betamethasone initially suppressed several fetal monocyte functions, however, at 7 d, measurements of initiation and resolution of inflammation were increased to levels similar to monocytes from adult sheep. The time-dependent changes in maternal betamethasone modulation of the responses of fetal monocytes may influence immune function of the preterm lamb after delivery.

The preterm infant has functionally immature monocytes. The effects of common clinical interventions and exposures that might modulate inflammation were evaluated using monocytes isolated from blood of preterm lambs [130 d gestational age (GA)], near-term lambs (141 d GA), and adult sheep. Endotoxin stimulated hydrogen peroxide production by adult monocytes, but monocytes from 130-d and 141-d GA lambs had a reduced and delayed hydrogen peroxide production. Endotoxin did not decrease apoptosis of monocytes from 130-d and 141-d GA lambs but decreased apoptosis of adult monocytes. Dexamethasone increased the phagocytosis of bacteria and apoptotic cells by adult monocytes by 35% but not by monocytes from 130-d and 141-d GA lambs. Synthetic and natural surfactants and dipalmitoylphosphatidylcholine increased phagocytosis of apoptotic cells by monocytes from preterm, term, and adult sheep. Monocytes from preterm and term lambs differ from adult monocytes in tests of both the initiation and the resolution of inflammation. The reduced phagocytosis of apoptotic cells by monocytes from the preterm may contribute to prolonged inflammation in diseases such as bronchopulmonary dysplasia.

Continuous positive airway pressure (CPAP) is being used clinically to avoid mechanical ventilation of preterm infants as a strategy to minimize lung injury. There is little experimental information about how CPAP might minimize lung injury after preterm birth. We induced preterm labor in antenatal glucocorticoid-treated sheep carrying twins at 133 d gestation with an inhibitor of progesterone synthesis. The lambs delivered spontaneously approximately 2 d later and were randomized to three groups: no ventilation (n = 4), conventional mechanical ventilation to a target PCO(2) of 40 mm Hg (n = 5), or CPAP using a bubble CPAP device set to deliver 5 cm H(2)O pressure (n = 6). The CPAP lambs breathed without distress and maintained PCO(2) values of approximately 60 mm Hg. At 2 h of age, the lungs of the CPAP lambs held 74 +/- 4 mL/kg air at 40 cm H(2)O pressure, which was more than the 60 +/- 3 mL/kg for the ventilated lambs (p < 0.05). Lymphocyte and monocyte numbers in alveolar washes were equivalent in the unventilated, ventilated, and CPAP lambs. However, no neutrophils were found in the unventilated lambs, and the ventilated lambs had 6.6 times more neutrophils in alveolar washes than did the CPAP lambs (p < 0.05). The cells in alveolar wash from CPAP lambs contained less hydrogen peroxide than did the cells from ventilated lambs (p < 0.05). In this model in preterm lambs CPAP results in lower indicators of acute lung injury than does mechanical ventilation during the first 2 h of life.