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In term neonates with hypoxic-ischemic encephalopathy (HIE), cerebellar injury is becoming more and more acknowledged. Animal studies demonstrated that Purkinje cells (PCs) are especially vulnerable for hypoxic-ischemic injury. In neonates, however, the extent and pattern of PC injury has not been investigated. The aim of this study was to characterize the morphology and distribution of PCs in the cerebellar vermis of term born neonates with HIE. Twenty-two term born neonates with severe HIE, several of which received therapeutic hypothermia, who underwent post-mortem autopsy of the brain including cerebellar vermis within three to five days after birth were included. Haematoxylin & Eosin (H&E) stained sections of the vermis were used to determine total PC count and morphology (normal, abnormal or non-classified) at the bases and crowns of the folia and of the lobules in both the anterior and posterior lobes. Differences in PC count and PC morphology between the anterior and posterior lobe and between the bases and crowns were compared. The total number of PCs was significantly higher at the crowns compared to the bases (p < 0.001) irrespective of the precise location. Besides, PCs at the bases more often had an abnormal morphology. Also, a significant difference between the injury in the anterior and posterior lobe was observed, notably at specific microscopic locations with more abnormal PCs in the posterior lobe. The number of PCs scored as abnormal was increased in the bases compared to the crowns, which might resemble supratentorial ulegyria., Competing Interests: Declarations. Ethical Approval: The Medical Ethical Committee (MEC) of UMC Utrecht confirmed that the ‘Medical Research Involving Human Subjects Act’ did not apply to this study and therefore an official approval of the MEC and informed consent was not required (MEC # 18–167). The UMC Utrecht biobank approved the use of the residual tissue (biobank # 18–284). This study was conducted in accordance with the Declaration of Helsinki. Competing Interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Perinatal brain injury following hypoxia-ischemia (HI) is characterized by high mortality rates and long-term disabilities. Previously, we demonstrated that depletion of Annexin A1, an essential mediator in BBB integrity, was associated with a temporal loss of blood-brain barrier (BBB) integrity after HI. Since the molecular and cellular mechanisms mediating the impact of HI are not fully scrutinized, we aimed to gain mechanistic insight into the dynamics of essential BBB structures following global HI in relation to ANXA1 expression. Global HI was induced in instrumented preterm ovine fetuses by transient umbilical cord occlusion (UCO) or sham occlusion (control). BBB structures were assessed at 1, 3, or 7 days post-UCO by immunohistochemical analyses of ANXA1, laminin, collagen type IV, and PDGFRβ for pericytes. Our study revealed that within 24 h after HI, cerebrovascular ANXA1 was depleted, which was followed by depletion of laminin and collagen type IV 3 days after HI. Seven days post-HI, increased pericyte coverage, laminin and collagen type IV expression were detected, indicating vascular remodeling. Our data demonstrate novel mechanistic insights into the loss of BBB integrity after HI, and effective strategies to restore BBB integrity should potentially be applied within 48 h after HI. ANXA1 has great therapeutic potential to target HI-driven brain injury.

Introduction: Early-onset fetal growth restriction (FGR) requires timely, often preterm, delivery to prevent fetal hypoxia causing stillbirth or neurologic impairment. Antenatal corticosteroids (CCS) administration reduces neonatal morbidity and mortality following preterm birth, most effectively when administered within 1 week preceding delivery. Optimal timing of CCS administration is challenging in early-onset FGR, as the exact onset and course of fetal hypoxia are unpredictable. International guidelines do not provide a directive on this topic. In the Netherlands, two timing strategies are commonly practiced: administration of CCS when the umbilical artery shows (A) a pulsatility index above the 95th h centile and (B) absent or reversed end-diastolic velocity (a more progressed disease state). This study aims to (1) use practice variation to compare CCS timing strategies in early-onset FGR on fetal and neonatal outcomes and (2) develop a dynamic tool to predict the time interval in days until delivery, as a novel timing strategy for antenatal CCS in early-onset FGR., Methods and Analysis: A multicentre, retrospective cohort study will be performed including pregnancies complicated by early-onset FGR in six tertiary hospitals in the Netherlands in the period between 2012 and 2021 (estimated sample size n=1800). Main exclusion criteria are multiple pregnancies and fetal congenital or genetic abnormalities. Routinely collected data will be extracted from medical charts. Primary outcome for the comparison of the two CCS timing strategies is a composite of perinatal, neonatal and in-hospital mortality. Secondary outcomes include the COSGROVE core outcome set for FGR. A multivariable, mixed-effects model will be used to compare timing strategies on study outcomes. Primary outcome for the dynamic prediction tool is 'days until birth'., Ethics and Dissemination: The need for ethical approval was waived by the Ethics Committee (University Medical Center Utrecht). Results will be published in open-access, peer-reviewed journals and disseminated by presentations at scientific conferences., Trial Registration Number: ClinicalTrials.gov: NCT05606497., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Perinatal arterial ischaemic stroke (PAIS) is an important cause of neurodevelopmental disabilities. In this first-in-human study, we aimed to assess the feasibility and safety of intranasally delivered bone marrow-derived allogeneic mesenchymal stromal cells (MSCs) to treat PAIS in neonates., Methods: In this open-label intervention study in collaboration with all neonatal intensive care units in the Netherlands, we included neonates born at full term (≥36 weeks of gestation) with MRI-confirmed PAIS in the middle cerebral artery region. All eligible patients were transferred to the neonatal intensive care unit of the Wilhelmina Children's Hospital. Neonates received one dose of 45-50 × 10 6 bone-marrow derived MSCs intranasally within 7 days of presenting signs of PAIS. The primary endpoints were acute and subacute safety outcomes, including vital signs, blood markers, and the occurrence of toxicity, adverse events, and serious adverse events. The occurrence of unexpected cerebral abnormalities by a repeat MRI at 3 months of age was a secondary endpoint. As part of standard clinical follow-up at Wilhelmina Children's Hospital, we assessed corticospinal tract development on MRI and performed motor assessments at 4 months of age. This study is registered with ClinicalTrials.gov, NCT03356821., Findings: Between Feb 11, 2020, and April 29, 2021, ten neonates were enrolled in the study. Intranasal administration of MSCs was well tolerated in all ten neonates. No serious adverse events were observed. One adverse event was seen: a mild transient fever of 38°C without the need for clinical intervention. Blood inflammation markers (C-reactive protein, procalcitonin, and leukocyte count) were not significantly different pre-administration versus post-administration and, although thrombocyte levels increased (p=0·011), all were within the physiological range. Follow-up MRI scans did not show unexpected structural cerebral abnormalities. All ten patients had initial pre-Wallerian changes in the corticospinal tracts, but only four (40%) patients showed asymmetrical corticospinal tracts at follow-up MRI. Abnormal early motor assessment was found in three (30%) infants., Interpretation: This first-in-human study demonstrates that intranasal bone marrow-derived MSC administration in neonates after PAIS is feasible and no serious adverse events were observed in patients followed up until 3 months of age. Future large-scale placebo-controlled studies are needed to determine the therapeutic effect of intranasal MSCs for PAIS., Funding: Netherlands Organization for Health Research and Development (ZonMw)., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Chorioamnionitis is a major risk factor for preterm birth and an independent risk factor for postnatal morbidity for which currently successful therapies are lacking. Emerging evidence indicates that the timing and duration of intra-amniotic infections are crucial determinants for the stage of developmental injury at birth. Insight into the dynamical changes of organ injury after the onset of chorioamnionitis revealed novel therapeutic windows of opportunity. Importantly, successful development and implementation of therapies in clinical care is currently impeded by a lack of diagnostic tools for early (prenatal) detection and surveillance of intra-amniotic infections. In the current study we questioned whether an intra-amniotic infection could be accurately diagnosed by a specific volatile organic compound (VOC) profile in exhaled breath of pregnant sheep. For this purpose pregnant Texel ewes were inoculated intra-amniotically with Ureaplasma parvum and serial collections of exhaled breath were performed for 6 days. Ureaplasma parvum infection induced a distinct VOC-signature in expired breath of pregnant sheep that was significantly different between day 0 and 1 vs. day 5 and 6. Based on a profile of only 15 discriminatory volatiles, animals could correctly be classified as either infected (day 5 and 6) or not (day 0 and 1) with a sensitivity of 83% and a specificity of 71% and an area under the curve of 0.93. Chemical identification of these distinct VOCs revealed the presence of a lipid peroxidation marker nonanal and various hydrocarbons including n-undecane and n-dodecane. These data indicate that intra-amniotic infections can be detected by VOC analyses of exhaled breath and might provide insight into temporal dynamics of intra-amniotic infection and its underlying pathways. In particular, several of these volatiles are associated with enhanced oxidative stress and undecane and dodecane have been reported as predictive biomarker of spontaneous preterm birth in humans. Applying VOC analysis for the early detection of intra-amniotic infections will lead to appropriate surveillance of these high-risk pregnancies, thereby facilitating appropriate clinical course of action including early treatment of preventative measures for pre-maturity-associated morbidities., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ophelders, Boots, Hütten, Al-Nasiry, Jellema, Spiller, van Schooten, Smolinska and Wolfs.)

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., (© 2020 The Authors. STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Periodicals LLC on behalf of AlphaMed Press.)

With a worldwide incidence of 15 million cases, preterm birth is a major contributor to neonatal mortality and morbidity, and concomitant social and economic burden Preterm infants are predisposed to life-long neurological disorders due to the immaturity of the brain. The risks are inversely proportional to maturity at birth. In the majority of extremely preterm infants (<28 weeks' gestation), perinatal brain injury is associated with exposure to multiple inflammatory perinatal triggers that include antenatal infection (i.e., chorioamnionitis), hypoxia-ischemia, and various postnatal injurious triggers (i.e., oxidative stress, sepsis, mechanical ventilation, hemodynamic instability). These perinatal insults cause a self-perpetuating cascade of peripheral and cerebral inflammation that plays a critical role in the etiology of diffuse white and grey matter injuries that underlies a spectrum of connectivity deficits in survivors from extremely preterm birth. This review focuses on chorioamnionitis and hypoxia-ischemia, which are two important antenatal risk factors for preterm brain injury, and highlights the latest insights on its pathophysiology, potential treatment, and future perspectives to narrow the translational gap between preclinical research and clinical applications.

The conclusion of our recent paper that performance of the STAN device in clinical practice is potentially limited by high false-negative and high false-positive STAN-event rates and loss of ST waveform assessment capacity during severe hypoxemia, evoked comments by Kjellmer, Lindecrantz and Rosén. These comments can be summarized as follows: 1) STAN analysis is based on a unipolar lead but the authors used a negative aVF lead, and they did not validate this methodology; 2) The fetuses used in the study were too young to display the signals that the authors were trying to detect. In response to these comments we now provide both a theoretical and an experimental underpinning of our approach. In an in vivo experiment in human we placed several electrodes over the head (simulating different places of a scalp electrode), simultaneously recorded Einthoven lead I and II, and constructed -aVF from these two frontal leads. Irrespective of scalp electrode placement, the correlation between any of unipolar scalp electrode-derived signals and constructed-aVF was excellent (≥ 0.92). In response to the second comment we refer to a study which demonstrated that umbilical cord occlusion resulted in rapid increase in T/QRS ratio that coincided with initial hypertension and bradycardia at all gestational ages which were tested from 0.6-0.8 gestation. The animals of our study were in this gestational range and, hence, our experimental setup can be used to assess STAN's quality to detect fetal hypoxia. In conclusion, we have clearly demonstrated the appropriateness of using-aVF as a proxy for a scalp electrode-derived signal in STAN in these preterm lambs. Investigation why STAN could not detect relevant ST-changes and instead produced erroneous alarms in our experimental setup is hampered by the fact that the exact STAN algorithm (signal processing and analysis) is not in the public domain., Competing Interests: The authors reconfirm and declare that no competing interests exist. Although Rik Vullings is scientific director and shareholder of Nemo Healthcare (Veldhoven, the Netherlands), a company that develops innovative technology for improved pregnancy monitoring, this does not alter our adherence to PLOS ONE policies on sharing data and materials.

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.

Introduction: The inconclusive clinical results for ST-waveform analysis (STAN) in detecting fetal hypoxemia may be caused by the signal processing of the STAN-device itself. We assessed the performance of a clinical STAN device in signal processing and in detecting hypoxemia in a fetal sheep model exposed to prolonged umbilical cord occlusion (UCO)., Methods: Eight fetal lambs were exposed to 25 minutes of UCO. ECG recordings were analyzed during a baseline period and during UCO. STAN-event rates and timing of episodic T/QRS rise, baseline T/QRS rise and the occurrence of biphasic ST-waveforms, as well as signal loss, were assessed., Results: During baseline conditions of normoxemia, a median of 40 (IQR, 25-70) STAN-events per minute were detected, compared to 10 (IQR, 2-22) during UCO. During UCO STAN-events were detected in five subjects within 10 minutes and in six subjects after 18 minutes, respectively. Two subjects did not generate any STAN-event during UCO. Biphasic ST event rate was reduced during UCO (median 0, IQR 0-5), compared to baseline (median 32, IQR, 6-55). ST-waveforms could not be assessed in 62% of the recording time during UCO, despite a good quality of the ECG signal., Conclusions: The STAN device showed limitations in detecting hypoxemia in fetal sheep after prolonged UCO. The STAN device produced high false positive event rates during baseline and did not detect T/QRS changes adequately after prolonged fetal hypoxemia. During 14% of baseline and 62% of the UCO period, the STAN-device could not process the ECG signal, despite its good quality. Resolving these issues may improve the clinical performance of the STAN device.

Perinatal asphyxia, a condition of impaired gas exchange during birth, leads to fetal hypoxia-ischemia (HI) and is associated with postnatal adverse outcomes including intestinal dysmotility and necrotizing enterocolitis (NEC). Evidence from adult animal models of transient, locally-induced intestinal HI has shown that inflammation is essential in HI-induced injury of the gut. Importantly, mesenchymal stem cell (MSC) treatment prevented this HI-induced intestinal damage. We therefore assessed whether fetal global HI induced inflammation, injury and developmental changes in the gut and whether intravenous MSC administration ameliorated these HI-induced adverse intestinal effects. In a preclinical ovine model, fetuses were subjected to umbilical cord occlusion (UCO), with or without MSC treatment, and sacrificed 7 days after UCO. Global HI increased the number of myeloperoxidase positive cells in the mucosa, upregulated mRNA levels of interleukin (IL)-1β and IL-17 in gut tissue and caused T-cell invasion in the intestinal muscle layer. Intestinal inflammation following global HI was associated with increased Ki67+ cells in the muscularis and subsequent muscle hyperplasia. Global HI caused distortion of glial fibrillary acidic protein immunoreactivity in the enteric glial cells and increased synaptophysin and serotonin expression in the myenteric ganglia. Intravenous MSC treatment did not ameliorate these HI-induced adverse intestinal events. Global HI resulted in intestinal inflammation and enteric nervous system abnormalities which are clinically associated with postnatal complications including feeding intolerance, altered gastrointestinal transit and NEC. The intestinal histopathological changes were not prevented by intravenous MSC treatment directly after HI, indicating that alternative treatment regimens for cell-based therapies should be explored.

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.

Unlabelled: 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., Significance: 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., (©AlphaMed Press.)

Chorioamnionitis, an inflammatory reaction of the fetal membranes to microbes, is an important cause of preterm birth and associated with inflammation-driven lung injury. However, inflammation in utero overcomes immaturity of the premature lung by inducing surfactant lipids and lung gas volume. Previously, we found that lipopolysaccharide (LPS)-induced chorioamnionitis resulted in pulmonary inflammation with increased effector T cells and decreased regulatory T cell (Treg) numbers. Because Tregs are crucial for immune regulation, we assessed the effects of interleukin (IL)-2-driven selective Treg expansion on the fetal lung in an ovine chorioamnionitis model. Instrumented fetuses received systemic prophylactic IL-2 treatment [118 days gestational age (dGA)] with or without subsequent exposure to intra-amniotic LPS (122 dGA). Following delivery at 129 dGA (term 147 dGA), pulmonary and systemic inflammation, morphological changes, lung gas volume, and phospholipid concentration were assessed. IL-2 pretreatment increased the FoxP3(+)/CD3(+) ratio, which was associated with reduced CD3-positive cells in the fetal lungs of LPS-exposed animals. Prophylactic IL-2 treatment did not prevent pulmonary accumulation of myeloperoxidase- and PU.1-positive cells or elevation of bronchoalveolar lavage fluid IL-8 and systemic IL-6 concentrations in LPS-exposed animals. Unexpectedly, IL-2 treatment improved fetal lung function of control lambs as indicated by increased disaturated phospholipids and improved lung gas volume. In conclusion, systemic IL-2 treatment in utero preferentially expanded Tregs and improved lung gas volume and disaturated phospholipids. These beneficial effects on lung function were maintained despite the moderate immunomodulatory effects of prophylactic IL-2 in the course of chorioamnionitis., (Copyright © 2016 the American Physiological Society.)

Background: Preterm infants are at risk for hypoxic-ischemic encephalopathy. No therapy exists to treat this brain injury and subsequent long-term sequelae. We have previously shown in a well-established pre-clinical model of global hypoxia-ischemia (HI) that mesenchymal stem cells are a promising candidate for the treatment of hypoxic-ischemic brain injury. In the current study, we investigated the neuroprotective capacity of multipotent adult progenitor cells (MAPC®), which are adherent bone marrow-derived cells of an earlier developmental stage than mesenchymal stem cells and exhibiting more potent anti-inflammatory and regenerative properties., Methods: Instrumented preterm sheep fetuses were subjected to global hypoxia-ischemia by 25 min of umbilical cord occlusion at a gestational age of 106 (term ~147) days. During a 7-day reperfusion period, vital parameters (e.g., blood pressure and heart rate; baroreceptor reflex) and (amplitude-integrated) electroencephalogram were recorded. At the end of the experiment, the preterm brain was studied by histology., Results: Systemic administration of MAPC therapy reduced the number and duration of seizures and prevented decrease in baroreflex sensitivity after global HI. In addition, MAPC cells prevented HI-induced microglial proliferation in the preterm brain. These anti-inflammatory effects were associated with MAPC-induced prevention of hypomyelination after global HI. Besides attenuation of the cerebral inflammatory response, our findings showed that MAPC cells modulated the peripheral splenic inflammatory response, which has been implicated in the etiology of hypoxic-ischemic injury in the preterm brain., Conclusions: In a pre-clinical animal model MAPC cell therapy improved the functional and structural outcome of the preterm brain after global HI. Future studies should establish the mechanism and long-term therapeutic effects of neuroprotection established by MAPC cells in the developing preterm brain exposed to HI. Our study may form the basis for future clinical trials, which will evaluate whether MAPC therapy is capable of reducing neurological sequelae in preterm infants with hypoxic-ischemic encephalopathy.

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: Chorioamnionitis results from an infection of the fetal membranes and is associated with fetal adverse outcomes notably in the intestine. Using a translational ovine model, we showed that intra-amniotic exposure to inflammatory stimuli decreased the regulatory/effector T (Treg/Teff) cell balance in the gut, which was accompanied by intestinal inflammation and mucosal injury. We thus aimed to augment the Treg/Teff cell ratio in the fetal gut by prophylactic IL-2 treatment and evaluate whether it is sufficient to prevent chorioamnionitis-induced intestinal inflammation and mucosal injury., Methods: Fetal sheep (122 d of gestation) were intra-amniotically exposed to lipopolysaccharide for 2 or 7 days with or without prophylactic IL-2 treatment (4 d). We evaluated the infiltration of inflammatory cells in the ileum and mesenteric lymph nodes. Cytokine gene expression was analyzed in fetal ileum and the inflammatory changes were correlated with gut wall integrity., Results: IL-2 administration preferentially increased intestinal Treg cells and thus the Treg/Teff cell ratio. Prophylactic IL-2 treatment reduced the lipopolysaccharide-induced influx of neutrophils and CD3(+) T cells and decreased the messenger RNA levels of proinflammatory cytokines including IL-6 and IL-17 in the fetal ileum. Importantly, prophylactic IL-2 treatment prevented mucosal damage without inducing fetal adverse treatment outcomes., Conclusions: Our data show that prophylactic IL-2 treatment prevents fetal intestinal inflammation and mucosal injury in the context of experimental chorioamnionitis. Modulation of the Treg/Teff cell balance may contribute to the protective effects of IL-2.

Seizures below one minute in duration are difficult to assess correctly using seizure detection algorithms. We aimed to improve neonatal detection algorithm performance for short seizures through the use of trend templates for seizure onset and end. Bipolar EEG were recorded within a transiently asphyxiated ovine model at 0.7 gestational age, a common experimental model for studying brain development in humans of 30-34 weeks of gestation. Transient asphyxia led to electrographic seizures within 6-8 h. A total of 3159 seizures, 2386 shorter than one minute, were annotated in 1976 h-long EEG recordings from 17 foetal lambs. To capture EEG characteristics, five features, sensitive to seizures, were calculated and used to derive trend information. Feature values and trend information were used as input for support vector machine classification and subsequently post-processed. Performance metrics, calculated after post-processing, were compared between analyses with and without employing trend information. Detector performance was assessed after five-fold cross-validation conducted ten times with random splits. The use of trend templates for seizure onset and end in a neonatal seizure detection algorithm significantly improves the correct detection of short seizures using two-channel EEG recordings from 54.3% (52.6-56.1) to 59.5% (58.5-59.9) at FDR 2.0 (median (range); p < 0.001, Wilcoxon signed rank test). Using trend templates might therefore aid in detection of short seizures by EEG monitoring at the NICU.

Rationale: Chorioamnionitis has been associated with increased risk for fetal brain damage. Although, it is now accepted that synaptic dysfunction might be responsible for functional deficits, synaptic densities/numbers after a fetal inflammatory challenge have not been studied in different regions yet. Therefore, we tested in this study the hypothesis that LPS-induced chorioamnionitis caused profound changes in synaptic densities in different regions of the fetal sheep brain., Material and Methods: Chorioamnionitis was induced by a 10 mg intra-amniotic LPS injection at two different exposure intervals. The fetal brain was studied at 125 days of gestation (term = 150 days) either 2 (LPS2D group) or 14 days (LPS14D group) after LPS or saline injection (control group). Synaptophysin immunohistochemistry was used to quantify the presynaptic density in layers 2-3 and 5-6 of the motor cortex, somatosensory cortex, entorhinal cortex, and piriforme cortex, in the nucleus caudatus and putamen and in CA1/2, CA3, and dentate gyrus of the hippocampus., Results: There was a significant reduction in presynaptic bouton densities in layers 2-3 and 5-6 of the motor cortex and in layers 2-3 of the entorhinal and the somatosensory cortex, in the nucleus caudate and putamen and the CA1/2 and CA3 of the hippocampus in the LPS2D compared to control animals. Only in the motor cortex and putamen, the presynaptic density was significantly decreased in the LPS14 D compared to the control group. No changes were found in the dentate gyrus of the hippocampus and the piriforme cortex., Conclusion: We demonstrated that LPS-induced chorioamnionitis caused a decreased density in presynaptic boutons in different areas in the fetal brain. These synaptic changes seemed to be region-specific, with some regions being more affected than others, and seemed to be transient in some regions.

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: 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.

The artificial placenta as a fascinating treatment alternative for neonatal lung failure has been the subject of clinical research for over 50 years. Pumpless systems have been in use since 1986. However, inappropriate dimensioning of commercially available oxygenators has wasted some of the theoretical advantages of this concept. Disproportional shunt fractions can cause congestive heart failure. Blood priming of large oxygenators and circuits dilutes fetal hemoglobin (as the superior oxygen carrier), is potentially infectious, and causes inflammatory reactions. Flow demands of large extracorporeal circuits require cannula sizes that are not appropriate for use in preterm infants. NeonatOx, a tailored low-volume oxygenator for this purpose, has proven the feasibility of this principle before. We now report the advances in biological performance of a refined version of this specialized oxygenator., (© 2013 Wiley Periodicals, Inc. and International Center for Artificial Organs and Transplantation.)

Rationale: Chorioamnionitis and antenatal glucocorticoids are common exposures for preterm infants and can affect the fetal brain, contributing to cognitive and motor deficits in preterm infants. The effects of antenatal glucocorticoids on the brain in the setting of chorioamnionitis are unknown. We hypothesized that antenatal glucocorticoids would modulate inflammation in the brain and prevent hippocampal and white matter injury after intra-amniotic lipopolysaccharide (LPS) exposure., Methods: Time-mated ewes received saline (control), an intra-amniotic injection of 10 mg LPS at 106d GA or 113d GA, maternal intra-muscular betamethasone (0.5 mg/kg maternal weight) alone at 113d GA, betamethasone at 106d GA before LPS or betamethasone at 113d GA after LPS. Animals were delivered at 120d GA (term=150d). Brain structure volumes were measured on T2-weighted MRI images. The subcortical white matter (SCWM), periventricular white matter (PVWM) and hippocampus were analyzed for microglia, astrocytes, apoptosis, proliferation, myelin and pre-synaptic vesicles., Results: LPS and/or betamethasone exposure at different time-points during gestation did not alter brain structure volumes on MRI. Betamethasone alone did not alter any of the measurements. Intra-amniotic LPS at 106d or 113d GA induced inflammation as indicated by increased microglial and astrocyte recruitment which was paralleled by increased apoptosis and hypomyelination in the SCWM and decreased synaptophysin density in the hippocampus. Betamethasone before the LPS exposure at 113d GA prevented microglial activation and the decrease in synaptophysin. Betamethasone after LPS exposure increased microglial infiltration and apoptosis., Conclusion: Intra-uterine LPS exposure for 7d or 14d before delivery induced inflammation and injury in the fetal white matter and hippocampus. Antenatal glucocorticoids aggravated the inflammatory changes in the brain caused by pre-existing intra-amniotic inflammation. Antenatal glucocorticoids prior to LPS reduced the effects of intra-uterine inflammation on the brain. The timing of glucocorticoid administration in the setting of chorioamnionitis can alter outcomes for the fetal brain.

Hypoxic-ischemic encephalopathy (HIE) is common in preterm infants, but currently no curative therapy is available. Cell-based therapy has a great potential in the treatment of hypoxic-ischemic preterm brain injury. Granulocyte-colony stimulating factor (G-CSF) is known to mobilize endogenous hematopoietic stem cells (HSC) and promotes proliferation of endogenous neural stem cells. On these grounds, we hypothesized that systemic G-CSF would be neuroprotective in a large translational animal model of hypoxic-ischemic injury in the preterm brain. Global hypoxia-ischemia (HI) was induced by transient umbilical cord occlusion in instrumented preterm sheep. G-CSF treatment (100μg/kg intravenously, during five consecutive days) was started one day before the global HI insult to ascertain mobilization of endogenous stem cells within the acute phase after global HI. Mobilization of HSC and neutrophils was studied by flow cytometry. Brain sections were stained for microglia (IBA-1), myelin basic protein (MBP) and myeloperoxidase (MPO) to study microglial proliferation, white matter injury and neutrophil invasion respectively. Electrographic seizure activity was analyzed using amplitude-integrated electroencephalogram (aEEG). G-CSF effectively mobilized CD34-positive HSC in the preterm sheep. In addition, G-CSF caused marked mobilization of neutrophils, but did not influence enhanced invasion of neutrophils into the preterm brain after global HI. Microglial proliferation and hypomyelination following global HI were reduced as a result of G-CSF treatment. G-CSF did not cause a reduction of the electrographic seizure activity after global HI. In conclusion, G-CSF induced mobilization of endogenous stem cells which was associated with modulation of the cerebral inflammatory response and reduced white matter injury in an ovine model of preterm brain injury after global HI. G-CSF treatment did not improve neuronal function as shown by seizure analysis. Our study shows that G-CSF treatment has neuroprotective potential following hypoxic-ischemic injury in the preterm brain., (© 2013.)

Hypoxic-ischemic encephalopathy (HIE) in preterm infants is a severe disease for which no curative treatment is available. Cerebral inflammation and invasion of activated peripheral immune cells have been shown to play a pivotal role in the etiology of white matter injury, which is the clinical hallmark of HIE in preterm infants. The objective of this study was to assess the neuroprotective and anti-inflammatory effects of intravenously delivered mesenchymal stem cells (MSC) in an ovine model of HIE. In this translational animal model, global hypoxia-ischemia (HI) was induced in instrumented preterm sheep by transient umbilical cord occlusion, which closely mimics the clinical insult. Intravenous administration of 2 x 10(6) MSC/kg reduced microglial proliferation, diminished loss of oligodendrocytes and reduced demyelination, as determined by histology and Diffusion Tensor Imaging (DTI), in the preterm brain after global HI. These anti-inflammatory and neuroprotective effects of MSC were paralleled by reduced electrographic seizure activity in the ischemic preterm brain. Furthermore, we showed that MSC induced persistent peripheral T-cell tolerance in vivo and reduced invasion of T-cells into the preterm brain following global HI. These findings show in a preclinical animal model that intravenously administered MSC reduced cerebral inflammation, protected against white matter injury and established functional improvement in the preterm brain following global HI. Moreover, we provide evidence that induction of T-cell tolerance by MSC might play an important role in the neuroprotective effects of MSC in HIE. This is the first study to describe a marked neuroprotective effect of MSC in a translational animal model of HIE.

Rationale: Chorioamnionitis induces preterm delivery and acute involution of the fetal thymus which is associated with postnatal inflammatory disorders. We studied the immune response, cell composition, and architecture of the fetal thymus following intraamniotic lipopolysaccharide (LPS) exposure., Methods: Time-mated ewes received an intraamniotic injection of LPS 5, 12, or 24 hours or 2, 4, 8, or 15 days before delivery at 125 days gestational age (term = 150 days)., Results: The LPS exposure resulted in decreased blood lymphocytes within 5 hours and decreased thymic corticomedullary ratio within 24 hours. Thymic interleukin 6 (IL6) and IL17 messenger RNA (mRNA) increased 5-fold 24 hours post-LPS exposure. Increased toll-like receptor 4 (TLR4) mRNA and nuclear factor κB positive cells at 24 hours after LPS delivery demonstrated acute thymic activation. Both TLR4 and IL1 mRNA increased by 5-fold and the number of Foxp3-positive cells (Foxp3+ cells) decreased 15 days after exposure., Conclusion: Intraamniotic LPS exposure caused a proinflammatory response, involution, and a persistent depletion of thymic Foxp3+ cells indicating disturbance of the fetal immune homeostasis.

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: 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: 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: Hypoxic-ischemic encephalopathy (HIE) is one of the most important causes of brain injury in preterm infants. Preterm HIE is predominantly caused by global hypoxia-ischemia (HI). In contrast, focal ischemia is most common in the adult brain and known to result in cerebral inflammation and activation of the peripheral immune system. These inflammatory responses are considered to play an important role in the adverse outcomes following brain ischemia. In this study, we hypothesize that cerebral and peripheral immune activation is also involved in preterm brain injury after global HI., Methods: Preterm instrumented fetal sheep were exposed to 25 minutes of umbilical cord occlusion (UCO) (n = 8) at 0.7 gestation. Sham-treated animals (n = 8) were used as a control group. Brain sections were stained for ionized calcium binding adaptor molecule 1 (IBA-1) to investigate microglial proliferation and activation. The peripheral immune system was studied by assessment of circulating white blood cell counts, cellular changes of the spleen and influx of peripheral immune cells (MPO-positive neutrophils) into the brain. Pre-oligodendrocytes (preOLs) and myelin basic protein (MBP) were detected to determine white matter injury. Electro-encephalography (EEG) was recorded to assess functional impairment by interburst interval (IBI) length analysis., Results: Global HI resulted in profound activation and proliferation of microglia in the hippocampus, periventricular and subcortical white matter. In addition, non-preferential mobilization of white blood cells into the circulation was observed within 1 day after global HI and a significant influx of neutrophils into the brain was detected 7 days after the global HI insult. Furthermore, global HI resulted in marked involution of the spleen, which could not be explained by increased splenic apoptosis. In concordance with cerebral inflammation, global HI induced severe brain atrophy, region-specific preOL vulnerability, hypomyelination and persistent suppressed brain function., Conclusions: Our data provided evidence that global HI in preterm ovine fetuses resulted in profound cerebral inflammation and mobilization of the peripheral innate immune system. These inflammatory responses were paralleled by marked injury and functional loss of the preterm brain. Further understanding of the interplay between preterm brain inflammation and activation of the peripheral immune system following global HI will contribute to the development of future therapeutic interventions in preterm HIE.

Chorioamnionitis and fetal sepsis can induce a fetal inflammatory response syndrome (FIRS) which is closely related to the development of white matter injury in the fetal brain. Large epidemiological studies support the link between FIRS and fetal brain injury with a clear association between the presence of in utero inflammation and neurodevelopmental complications such as cerebral palsy, autism and cognitive impairments later in life. Translational animal models of chorioamnionitis and fetal sepsis are essential in understanding the underlying pathophysiological mechanisms of fetal brain injury after exposure to intra-uterine inflammation. Concerning this aspect, ovine models have high translational value since neurodevelopment in sheep closely resembles the human situation. In this article, we will review clinical and experimental evidence for the link between FIRS and white matter injury in the fetal brain. With respect to experimental findings, we will particularly focus on the lessons learned from ovine models of chorioamnionitis and fetal sepsis. We also highlight two key players implied in the pathophysiology of white matter injury after in utero exposure to inflammation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The concept of an artificial placenta has been pursued in experimental research since the early 1960s. The principle has yet to be successfully implemented in neonatal care despite the constant evolution in extracorporeal life support technology and advancements in neonatal intensive care in general. For more than three decades, the physical dimensions of the required equipment necessitated pump-driven circuits; however, recent advances in oxygenator technology have allowed exploration of the simpler and physiologically preferable concept of pumpless arteriovenous oxygenation. We expect that further miniaturization of the extracorporeal circuit will allow the implementation of the concept into clinical application as an assist device. To this end, NeonatOx (Fig. 1), a custom-made miniaturized oxygenator with a filling volume of 20 mL, designed by our own group, has been successfully implemented with a preterm lamb model of less than 2000 g body weight as an assist device. We provide an overview of milestones in the history of extracorporeal membrane oxygenation of the preterm newborn juxtaposed against current and future technological advancements. Key limitations, which need to be addressed in order to make mechanical gas exchange a clinical treatment option of prematurity-related lung failure, are also identified., (© 2012, Copyright the Authors. Artificial Organs © 2012, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.)

Chorioamnionitis which results from microbial invasion of the amniotic cavity is the most frequent cause of preterm birth. Chorioamnionitis is associated with an increased risk of early-onset sepsis but the mechanisms underlying this association remain largely unknown. We hypothesize that developmental alterations of fetal organs and the immune system in the course of chorioamnionitis determine the risk of development of early onset sepsis. The purpose of this review is therefore to summarize the consequences of chorioamnionitis on fetal development and speculate how those antenatal changes might predispose to early onset sepsis.

Background: Chorioamnionitis is a major risk factor for preterm birth in multifetal pregnancies. However, there is little clinical data whether chorioamnionitis is restricted to one amniotic compartment in multifetal pregnancies., Objective: To explore whether chorioamnionitis is confined to the exposed compartment and does not cross to the unaffected fetus in twin pregnancy., Methods: In twin pregnant sheep, one of the twins was exposed to either 2 or 14 days of intra-amniotic lipopolysaccharide (LPS) while the co-twin was exposed to either 2 or 14 days of intra-amniotic saline (n = 3 for each exposure). Singletons were included in this study to compare the grade of inflammation with twins. All fetuses were delivered at 125 days of gestation (term = 150 days). Chorioamnionitis was confirmed by histological examination. Lung inflammation was assessed by cell count in bronchoalveolar lavage. Lung compliance was assessed at 40 cm H(2)O. Results were compared using analysis of variance (ANOVA) with a post-hoc Tukey analysis., Results: Inflammation in placenta, membranes and lung of LPS-exposed twins was significantly higher after 2 and 14 days of exposure when compared to the saline-exposed co-twins. Lung compliance in LPS-exposed twins was significantly increased after 14 days when compared to saline-exposed co-twins. Intrauterine LPS exposure increased lung compliance and inflammation in the membranes, placenta and lung to the same extent in twins as in singletons., Conclusion: In twin pregnant sheep, inflammation of the membranes, placenta and fetal lung was strictly limited to the exposed fetus in the amniotic compartment in which the LPS was injected., (Copyright © 2012 S. Karger AG, Basel.)

Background: Respiratory distress syndrome in preterm babies is caused by a pulmonary surfactant deficiency, but also by its inactivation due to various conditions, including plasma protein leakage. Surfactant replacement therapy is well established, but clinical observations and in vitro experiments suggested that its efficacy may be impaired by inactivation. A new synthetic surfactant (CHF 5633), containing synthetic surfactant protein B and C analogs, has shown comparable effects on oxygenation in ventilated preterm rabbits versus Poractant alfa, but superior resistance against inactivation in vitro. We hypothesized that CHF 5633 is also resistant to inactivation by serum albumin in vivo., Methodology/principal Findings: Nineteen preterm lambs of 127 days gestational age (term = 150 days) received CHF 5633 or Poractant alfa and were ventilated for 48 hours. Ninety minutes after birth, the animals received albumin with CHF 5633 or Poractant alfa. Animals received additional surfactant if P(a)O(2) dropped below 100 mmHg. A pressure volume curve was done post mortem and markers of pulmonary inflammation, surfactant content and biophysiology, and lung histology were assessed. CHF 5633 treatment resulted in improved arterial pH, oxygenation and ventilation efficiency index. The survival rate was significantly higher after CHF 5633 treatment (5/7) than after Poractant alfa (1/8) after 48 hours of ventilation. Biophysical examination of the surfactant recovered from bronchoalveolar lavages revealed that films formed by CHF 5633-treated animals reached low surface tensions in a wider range of compression rates than films from Poractant alfa-treated animals., Conclusions: For the first time a synthetic surfactant containing both surfactant protein B and C analogs showed significant benefit over animal derived surfactant in an in vivo model of surfactant inactivation in premature lambs.

Rationale: Chorioamnionitis is associated with preterm delivery and involution of the fetal thymus. Women at risk of preterm delivery receive antenatal corticosteroids which accelerate fetal lung maturation and improve neonatal outcome. However, the effects of antenatal corticosteroids on the fetal thymus in the settings of chorioamnionitis are largely unknown. We hypothesized that intra-amniotic exposure to lipopolysaccharide (LPS) causes involution of the fetal thymus resulting in persistent effects on thymic structure and cell populations. We also hypothesized that antenatal corticosteroids may modulate the effects of LPS on thymic development., Methods: Time-mated ewes with singleton fetuses received an intra-amniotic injection of LPS 7 or 14 days before preterm delivery at 120 days gestational age (term = 150 days). LPS and corticosteroid treatment groups received intra-amniotic LPS either preceding or following maternal intra-muscular betamethasone. Gestation matched controls received intra-amniotic and maternal intra-muscular saline. The fetal intra-thoracic thymus was evaluated., Results: Intra-amniotic LPS decreased the cortico-medullary (C/M) ratio of the thymus and increased Toll-like receptor (TLR) 4 mRNA and CD3 expression indicating involution and activation of the fetal thymus. Increased TLR4 and CD3 expression persisted for 14 days but Foxp3 expression decreased suggesting a change in regulatory T-cells. Sonic hedgehog and bone morphogenetic protein 4 mRNA, which are negative regulators of T-cell development, decreased in response to intra-amniotic LPS. Betamethasone treatment before LPS exposure attenuated some of the LPS-induced thymic responses but increased cleaved caspase-3 expression and decreased the C/M ratio. Betamethasone treatment after LPS exposure did not prevent the LPS-induced thymic changes., Conclusion: Intra-amniotic exposure to LPS activated the fetal thymus which was accompanied by structural changes. Treatment with antenatal corticosteroids before LPS partially attenuated the LPS-induced effects but increased apoptosis in the fetal thymus. Corticosteroid administration after the inflammatory stimulus did not inhibit the LPS effects on the fetal thymus.

Gas exchange in premature neonates is regularly impaired by structural and functional immaturity of the lung. Mechanical ventilation, which is vitally important to sustain oxygenation and CO(2) elimination, causes, at the same time, mechanical and inflammatory destruction of lung tissue. To date, extracorporeal oxygenation is not a treatment option, one reason among others being the size of available oxygenators and cannulas. We hypothesized that a substantial improvement in gas exchange can be achieved by maintenance of the fetal cardiopulmonary bypass and interposition of a suitable passively driven (arteriovenous) membrane oxygenator. In close cooperation between engineers and neonatologists, we developed a miniaturized oxygenator and adapted cannulas to be used as a pumpless extracorporeal lung support that is connected to the circulation via cannulation of the umbilical cord vessels. First in vitro and in vivo studies show promising results. We regard this as one step on the way to clinical application of the artificial placenta., (© 2011, Copyright the Authors. Artificial Organs © 2011, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.)

Targeted gene delivery is a promising strategy to cure disease on its basic level at the site of interest. The ultrastructure, internalization, and transfection efficiency of lipoplexes was investigated. We found that at a charge ratio (rho) of 4.0 lipoplexes had optimum characteristics for gene delivery in vitro. To decrease the size of lipoplexes, we used a method of continuous-flow microfluidics. PEGylation of lipoplexes did not hinder internalization, but was found to hamper transfection. To discriminate between uptake and transfection efficiency of lipoplexes, we used fluorescence-based approaches: microscopy and FACS. To this end, GFP plasmid was labeled with Alexa 594, and, in parallel experiments, GFP plasmid was combined with rhodamine-labeled lipid. Our studies confirm that cellular uptake does not imply transfection efficiency, and that hurdles in cellular processing have to be taken before targeted gene delivery becomes an established therapeutic option.