Effect of exogenous surfactants on viability and DNA synthesis in A549, immortalized mouse type II and isolated rat alveolar type II cells.

BACKGROUND: In mechanically ventilated preterm infants with respiratory distress syndrome (RDS), exogenous surfactant application has been demonstrated both to decrease DNA-synthesis but also and paradoxically to increase epithelial cell proliferation. However, the effect of exogenous surfactant has not been studied directly on alveolar type II cells (ATII cells), a key cell type responsible for alveolar function and repair. OBJECTIVE: The aim of this study was to investigate the effects of two commercially available surfactant preparations on ATII cell viability and DNA synthesis. METHODS: Curosurf® and Alveofact® were applied to two ATII cell lines (human A549 and mouse iMATII cells) and to primary rat ATII cells for periods of up to 24 h. Cell viability was measured using the redox indicator resazurin and DNA synthesis was measured using BrdU incorporation. RESULTS: Curosurf® resulted in slightly decreased cell viability in all cell culture models. However, DNA synthesis was increased in A549 and rat ATII cells but decreased in iMATII cells. Alveofact® exhibited the opposite effects on A549 cells and had very mild effects on the other two cell models. CONCLUSION: This study showed that commercially available exogenous surfactants used to treat preterm infants with RDS can have profound effects on cell viability and DNA synthesis.

Systemic hypothermia after neonatal encephalopathy: outcomes of neo.nEURO.network RCT.

OBJECTIVE: Mild hypothermia after perinatal hypoxic-ischemic encephalopathy (HIE) reduces neurologic sequelae without significant adverse effects, but studies are needed to determine the most-efficacious methods. METHODS: In the neo.nEURO.network trial, term neonates with clinical and electrophysiological evidence of HIE were assigned randomly to either a control group, with a rectal temperature of 37°C (range: 36.5-37.5°C), or a hypothermia group, cooled and maintained at a rectal temperature of 33.5°C (range: 33-34°C) with a cooling blanket for 72 hours, followed by slow rewarming. All infants received morphine (0.1 mg/kg) every 4 hours or an equivalent dose of fentanyl. Neurodevelopmental outcomes were assessed at the age of 18 to 21 months. The primary outcome was death or severe disability. RESULTS: A total of 129 newborn infants were enrolled, and 111 infants were evaluated at 18 to 21 months (53 in the hypothermia group and 58 in the normothermia group). The rates of death or severe disability were 51% in the hypothermia group and 83% in the normothermia group (P=.001; odds ratio: 0.21 [95% confidence interval [CI]: 0.09-0.54]; number needed to treat: 4 [95% CI: 3-9]). Hypothermia also had a statistically significant protective effect in the group with severe HIE (n=77; P=.005; odds ratio: 0.17 [95% CI: 0.05-0.57]). Rates of adverse events during the intervention were similar in the 2 groups except for fewer clinical seizures in the hypothermia group. CONCLUSION: Systemic hypothermia in the neo.nEURO.network trial showed a strong neuroprotective effect and was effective in the severe HIE group.

Role of p75NTR in NMDAR-mediated excitotoxic brain injury in neonatal mice.

BACKGROUND: Perinatal brain injury in preterm infants is a major cause of neurological handicap. The role of the neurotrophin receptor p75 (p75(NTR)) in the pathogenesis and repair of neonatal excitotoxic brain injury is unknown. Depending on a complex interplay of neurotrophin signalling, p75(NTR) can, in addition to its trophic function, also induce apoptosis. HYPOTHESIS: We hypothesised that excitotoxicity increases p75(NTR) expression and p75(NTR) knockout (KO) mice have a significantly smaller lesion size upon excitotoxicity as compared to wild-type (WT) mice. METHODS: We used an established animal model of neonatal excitotoxic brain injury mimicking several key aspects of human preterm brain damage. We subjected five-day-old WT and KO mice to excitotoxic injury by means of a single intracranial ibotenate injection (N-methyl-D-aspartate receptor agonist, NMDAR) into one brain hemisphere. Lesion size, number of activated caspase-3- and apoptosis-inducing factor (AIF)-positive cells were determined as outcome parameters. Gender analyses were taken into account retrospectively. RESULTS: NMDAR-mediated excitotoxicity induced an upregulation of p75(NTR) expression in the peri-lesion area. Lesion size was significantly increased in female KO as compared to male KO animals. Knockout of p75(NTR) reduced the number of activated caspase-3 but not AIF-positive cells after NMDAR-mediated excitotoxic injury independently of gender. CONCLUSION: Since NMDAR-mediated excitotoxic brain injury induced p75(NTR) expression and caspase-3-activated apoptosis in p75(NTR) KO animals was decreased, we conclude that activation of p75(NTR) contributes to NMDAR-mediated apoptosis in the neonatal brain. An increase in lesion size in female animals after excitotoxic brain injury suggests that in females p75(NTR) seems to play a dual role.

Policy benchmarking report on neonatal health and social policies in 13 European countries.

BACKGROUND AND AIM: Preterm birth is the major cause of infant mortality and morbidity in both developed and developing countries. In Europe, the prevalence rate of premature birth ranges from 5.5 to 11.4% - an average of 7.1% of all live births. In this report, we aim to compare the current health and social policies, as well as practices in 13 EU member states. MATERIALS AND METHODS: Using desk research, relevant information was gathered from each of the 13 European countries with regard to the prevalence of preterm birth, the cost of preterm birth to healthcare budgets, and the relevant policies, guidelines and practices in place at the national and, in some cases, regional level. The information comes from a range of sources, including government and parent association websites, published scientific literature and media reports. RESULTS: Despite the growing prevalence and increasing costs, neonatal and preterm infant health rank low on the policy agendas of EU member states. CONCLUSION: Based on the findings, there are a number of recommendations that should be considered. The European Union should (i) recognize the growing challenge of prematurity in Europe and its significant impact on infant morbidity and mortality, (ii) improve neonatal health through the development and implementation of coordinated EU health and social policies, (iii) address the lack of comparable European data on prematurity, including prevalence, mortality, acute morbidity and long-term impairment, (iv) also increase the standard of neonatal care across Europe by supporting the development and implementation of European medical guidelines and quality standards, (v) support the development of European postgraduate training programmes in Peri- and Neonatology in order to increase the quality and availability of trained healthcare professionals.

Therapeutic hypothermia in neonates. Review of current clinical data, ILCOR recommendations and suggestions for implementation in neonatal intensive care units.

Recent evidence suggests that the current ILCOR guidelines regarding hypothermia for the treatment of neonatal encephalopathy need urgent revision. In 2005 when the current ILCOR guidelines were finalised one large (CoolCap trial, n=235) and one small RCT (n=67), in addition to pilot trials, had been published, and demonstrated that therapeutic hypothermia after perinatal asphyxia was safe. The CoolCap trial showed a borderline overall effect on death and disability at 18 months of age, but significant improvement in a large subset of infants with less severe electroencephalographic changes. Based on this and other available evidence, the 2005 ILCOR guidelines supported post-resuscitation hypothermia in paediatric patients after cardiac arrest, but not after neonatal resuscitation. Subsequently, a whole body cooling trial supported by the NICHD reported a significant overall improvement in death or disability. Further large neonatal trials of hypothermia have stopped recruitment and their final results are likely to be published 2009-2011. Many important questions around the optimal therapeutic use of hypothermia remain to be answered. Nevertheless, independent meta-analyses of the published trials now indicate a consistent, robust beneficial effect of therapeutic hypothermia for moderate to severe neonatal encephalopathy, with a mean NNT between 6 and 8. Given that there is currently no other clinically proven treatment for infants with neonatal encephalopathy we propose that an interim advisory statement should be issued to support and guide the introduction of therapeutic hypothermia into routine clinical practice.

Pharmacological and genetic inhibition of NADPH oxidase does not reduce brain damage in different models of perinatal brain injury in newborn mice.

BACKGROUND: Inflammation and reactive oxygen species (ROS) are important in the development of perinatal brain injury. The ROS-generating enzyme NADPH oxidase (Nox2) is present in inflammatory cells and contributes to brain injury in adult animal models. HYPOTHESIS: NADPH oxidase contributes to ROS formation and development of injury in the immature brain and inhibition of NADPH oxidase attenuates perinatal brain injury. METHODS: We used animal models of term hypoxia-ischemia (HI) (P9 mice) as well as ibotenate-induced excitotoxic injury (P5 mice) mimicking features of periventricular leukomalacia in preterm infants. In vitro microglia cell cultures were used to investigate NADPH oxidase-dependent ROS formation. In vivo we determined the impact 1) of HI on NADPH oxidase gene expression 2) of genetic (gp91-phox/Nox2 knock-out) and 3) of pharmacological NADPH oxidase inhibition on HI-induced injury and NMDA receptor-mediated excitotoxic injury, respectively. Endpoints were ROS formation, oxidative stress, apoptosis, inflammation and extent of injury. RESULTS: Hypoxia-ischemia increased NADPH oxidase subunits mRNA expression in total brain tissue in vivo. In vitro ibotenate increased NADPH oxidase-dependent formation of reactive oxygen species in microglia. In vivo the inhibition of NADPH oxidase did not reduce the extent of brain injury in any of the animal models. In contrast, the injury was increased by inhibition of NADPH oxidase and genetic inhibition was associated with an increased level of galectin-3 and IL-1beta. CONCLUSION: NADPH oxidase is upregulated after hypoxia-ischemia and activated microglia cells are a possible source of Nox2-derived ROS. In contrast to findings in adult brain, NADPH oxidase does not significantly contribute to the pathogenesis of perinatal brain injury. Results obtained in adult animals cannot be transferred to newborns and inhibition of NADPH oxidase should not be used in attempts to attenuate injury.

Dextromethorphan is protective against sensitized N-methyl-D-aspartate receptor-mediated excitotoxic brain damage in the developing mouse brain.

Enhanced glutamate release and inflammation play an important role in the pathogenesis of developmental brain injury. Although N-methyl-d-aspartate receptor (NMDAR) antagonists potently attenuate neonatal brain damage in several animal models, they can also impact trophic functions in the developing brain. As a consequence, high-affinity NMDAR antagonists have been shown to trigger widespread apoptotic neurodegeneration in the newborn brain. Dextromethorphan (DM), a low-affinity NMDAR antagonist with anti-inflammatory properties, may be neuroprotective against excitotoxic and inflammation-enhanced excitotoxic brain injury, without the associated stimulation of apoptotic degeneration. Using an established newborn mouse model of excitotoxic brain damage, we determined whether systemic injection of DM significantly attenuates excitotoxic lesion size. We investigated several doses and time regimens; a dose of 5 microg/g DM given in a combination of both pre-injury and repetitive post-injury treatment proved most effective. DM treatment significantly reduced lesion size in gray and white matter by reducing cell death as shown by a decreased Fluoro-Jade B staining and caspase-3 activation. Pre-treatment with interleukin-1beta and lipopolysaccharide enhanced NMDAR-mediated excitotoxic brain injury and microglial cell activation. This sensitizing effect was abolished by DM treatment, as the effectiveness of DM in reducing lesion size and microglial cell activation was similar to phosphate-buffered saline-pre-treated controls. In all cases, no gender-specific differences were detected. DM treatment did not trigger any apoptotic neurodegeneration (caspase-3 cleavage, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, Fluoro-Jade B staining). Although functional parameters were not measured, our data corroborate reports that DM is neuroprotective and that it may therefore improve functional outcome following perinatal brain injury.

T3 replacement does not prevent excitotoxic cell death but reduces developmental neuronal apoptosis in newborn mice.

BACKGROUND: Periventricular leukomalacia (PVL) is a major cause of neurological handicap in pre-term infants. At present, there are no effective or causal therapies available. Thyroid hormones play an essential role in brain development and are reported to be decreased in pre-terms and following brain injury in adults. HYPOTHESIS: Excitotoxic brain damage of newborn mice decreases thyroid hormone concentrations. Exogenous T3 administration restores thyroid hormone levels and reduces perinatal brain damage in an animal model of PVL. DESIGN AND METHOD: To create white and gray matter (WM/GM) lesion mimicking several key aspects of PVL, we injected ibotenic acid (Ibo), a glutamate analog, into the right hemisphere (intracranially (i.c.)) of 5-day-old mice. T3 (10 microg/kg body weight (bw)) was injected intraperitoneally (i.p.) 1 h or repeatedly 1/24/48/72/96 h post-insult. We determined lesion size, number of apoptotic cells in WM/GM and serum T3/T4 concentration at 24 and 120 h after injury. Serum T3/T4 concentration was also determined before and 1 and 2h after T3 administration. RESULTS: Excitotoxic brain damage did not alter serum T3/T4 concentrations within 120 h of injury. Serum T3 levels were distinctly elevated within 1 h of T3 injection; however, this elevation was relatively short-lived (half-life estimated to be less than 12 h). Neither single nor repetitive T3 treatment regimen reduced excitotoxic lesion size, but it did reduce apoptosis. CONCLUSIONS: T3 replacement does not prevent excitotoxic cell death, but it does reduce developmental neuronal apoptosis, which could participate to the beneficial neuropsychological effects of hormone therapy. Further study is therefore warranted.

Erythropoietin is neuroprotective against NMDA-receptor-mediated excitotoxic brain injury in newborn mice.

Using an established mouse model of human periventricular leukomalacia, we investigated whether EPO could reduce excitotoxic damage. When administered 1 h following intracerebral injection of 10 microg ibotenic acid at day 5 of life, both a single injection of EPO (5000 IU/kg bw) and repetitive administrations of EPO reduced white and gray matter lesion size. The therapeutic window for protection was small as the protective effect of EPO was lost when EPO administration was delayed to 4 h post-insult. EPO-mediated upregulation of EPO-R, but not EPO, mRNA was observed within 4 h of the excitotoxic insult. The EPO effect was gender independent. Minor hematopoetic effects were observed following EPO treatment. We conclude that a single dose of EPO is sufficient to reduce excitotoxic brain injury and may therefore possess therapeutic relevance in the clinical setting.

Systemic application of granulocyte-colony stimulating factor and stem cell factor exacerbates excitotoxic brain injury in newborn mice.

Granulocyte-colony stimulating factor (G-CSF) has been shown to reduce brain lesion size and mortality in adult mice after hypoxic-ischemic injury. Another hematopoietic growth factor, stem cell factor (SCF), has been shown to be up-regulated in the brains of adult rodents following brain damage, where it stimulates postlesional neurogenesis. Injection of the excitotoxic agent ibotenate into the brain of newborn mice produces a brain lesion characterized by neuronal death and white matter cysts, which is similar to periventricular leucomalacia. The aim of the present study was to investigate whether administration of SCF and G-CSF is neuroprotective against ibotenate lesions in neonatal mice. Contrary to our expectations, cortical and white matter brain lesions induced by ibotenate were enhanced following the administration of 50 microg/kg SCF or 200 microg/kg G-CSF. Dose-response studies indicated that G-CSF could increase grey matter lesions even at lower dosages (22 and 66 microg/kg). Administration of SCF and G-CSF in combination also increased cortical and white matter lesions, to 133 +/- 8% and 187 +/- 12%. In the undamaged brain, G-CSF or G-CSF+SCF treatment had no effect on apoptosis in the grey or white matter; however, these treatments significantly increased apoptosis in the damaged brain. Our data clearly demonstrate that G-CSF and SCF are not neuroprotective and result in deleterious enhancement of excitotoxic brain damage in newborn mice. We conclude that G-CSF and SCF should be used cautiously in newborn infants with brain lesions; if they are used, long term neurologic and neurodevelopmental follow-up is warranted.

Effects on the maternofetal unit of the rabbit model after substitution of the amniotic fluid with perfluorocarbons.

OBJECTIVES: Exchanging amniotic fluid (AF) with perfluorocarbon (PFC) may serve as a medium for fetoscopic surgery. This study evaluates the distribution and physiologic effects of intraamniotic PFC as a medium for fetoscopy. METHODS: Fetuses of 17 pregnant rabbits underwent either exchange of the AF with PFC, electrolyte solution (ES), or control. The quality of vision during fetoscopy was assessed in AF and PFC. After 6 h, we determined the distribution of PFC in the maternofetal unit. RESULTS: Quality of vision during fetoscopy was better in PFC than with AF. There was no difference in fetal survival between the study groups. PFC was demonstrated on X-ray in the pharynx of 4 fetuses, and the esophagus in 1. CONCLUSIONS: PFC provided an ideal medium for fetoscopy without fetal compromise.

Intrapulmonary perfluorooctyl bromide instillation in fetal rabbits.

BACKGROUND: Instilling perfluorooctyl bromide (PFOB) into the fetal lung may lead to alveolar distension. OBJECTIVE: The aim of the study was to evaluate the safety of PFOB instillation into fetal lungs and to determine the radiographic distribution and tissue concentration of PFOB in New Zealand white rabbits. METHODS: Sibling fetuses of pregnant (day 27) New Zealand white rabbits were randomized to intratracheal instillation of 1 mL PFOB with tracheal ligation, instillation without ligation, and unmanipulated controls. The maternal animals were killed directly after instillation, at 3 or 6 hours (n = 10 each). For each study cohort, we determined fetal lung/body weight (FLBW) ratios, the radiographic distribution of PFOB, as well as pulmonary PFOB and water content by tissue distillation. PFOB concentrations in maternal and fetal tissues were assessed by gas chromatography. RESULTS: The relative amount of fetal lung PFOB recovered by fractional distillation was highest in ligated (25%) and lower in unligated lungs (9%). Extrapulmonary PFOB was found in the fetal brain (2.0 +/- 0.7 ppm), but not in any other fetal or maternal tissues. Mean FLBW ratios were highest in ligated fetuses, followed by unligated fetuses and controls. PFOB partially displaced fetal lung water. PFOB was visible in the lungs of all treated fetuses. Fetal survival between manipulated and unmanipulated fetuses did not differ. CONCLUSIONS: After prenatal intrapulmonary instillation, some PFOB remains in the lung, even if the trachea is not ligated, and may exert distending pressure on the alveoli.

Postnatal lung mechanics, lung composition, and surfactant synthesis after tracheal occlusion vs prenatal intrapulmonary instillation of perfluorocarbon in fetal rabbits.

BACKGROUND/PURPOSE: Fetal tracheal occlusion (TO) accelerates lung growth but decreases surfactant production. We have previously shown that instillation of perfluorooctylbromide (PFOB) into fetal rabbit lungs leads to lung growth similar to TO. This study compares neonatal lung mechanics and surfactant production after prenatal intrapulmonary PFOB instillation vs TO. METHODS: In each of 18 pregnant rabbits on gestational day 27, sets of 4 fetuses underwent either (1) intrapulmonary instillation of 1 mL PFOB, (2) TO, (3) instillation of 1 mL 0.9% NaCl (saline), and (4) hysteroamniotomy without fetal manipulation (control). Fetuses were born by cesarean delivery after 48 hours. Fetuses of 12 rabbits were mechanically ventilated for 15 minutes to evaluate lung compliance and airway resistance. Pulmonary surfactant protein B (SP-B) was quantified by immunohistochemistry in fetuses of the remaining 6 rabbits. RESULTS: Compliance was decreased in the TO group after cesarean delivery (0.33 +/- 0.13 mL/cm H2O) compared with PFOB (0.59 +/- 0.12 mL/cm H2O), saline (0.50 +/- 0.12 mL/cm H2O), and control (0.52 +/- 0.10 mL/cm H2O) fetuses. Mean fetal lung to body weight ratio was higher in TO and PFOB fetuses compared with saline and control. Higher water content and lower numbers of surfactant protein B-positive cells were found in the TO-treated fetuses. CONCLUSIONS: Both prenatal intrapulmonary instillation of PFOB and TO accelerate lung growth, but TO is associated with decreased postnatal lung compliance, possibly influenced by decreased surfactant production and increased fluid retention. Conversely, instillation of PFOB preserved lung compliance and surfactant synthesis.

Premature infants are less capable of maintaining thermal balance of head and body with increases of thermal environment than with decreases.

We investigated whether premature infants nursed at the upper range of normal body temperature are more capable of maintaining their nasopharyngeal and rectal temperature when exposed to a 1 degrees C increase or a 1 degrees C decrease of incubator temperature. In a randomized controlled trial, premature infants were exposed to a 1 degrees C increase (T + 1 degrees C; n = 10), or to a 1 degrees C decrease (T - 1 degrees C; n = 10) of incubator temperature. Nasopharyngeal, rectal, and skin temperatures as well as heat flux at various sites, heart rate, and activity were measured over a 6-hour period. The absolute changes in core temperatures, Tnasoph and Trectal, were significantly greater in the T + 1 degrees C compared with T - 1 degrees C (T + 1 degrees C versus T - 1 degrees C: Tnasoph 0.44 +/- 0.31 degrees C and 0.18 +/- 0.14 degrees C respectively; p < 0.001; T(rectal) 0.43 +/- 0.30 degrees C and 0.25 +/- 0.10 degrees C, respectively; p < 0.01) when exposed to the increase or decrease in incubator temperature. Premature infants are less able to cope with increases in incubator temperature given that rectal and nasopharyngeal temperature change more when environmental temperature is increased.

Lung growth induced by prenatal instillation of perfluorocarbon into the fetal rabbit lung.

The study's aim was to evaluate whether prenatal instillation of perfluorooctylbromide (PFOB, a perfluorocarbon) into the lungs of fetal rabbits leads to increased lung growth. Hysteroamniotomy was performed in eight pregnant New Zealand white rabbits on gestational day 27. In each mother, four fetuses were randomized to undergo either 1) endotracheal intubation and intrapulmonary instillation of 1 ml PFOB, 2) intrapulmonary instillation of 1 ml 0.9% NaCl solution (saline), 3) no fetal manipulation (control), or 4) tracheal occlusion (TO). The distribution of PFOB was documented radiographically. The fetuses were born by cesarean section after 48 h, sacrificed, weighed, and their lungs excised. Fetal lung to body weight ratios (FLBW) were determined, and the lungs were snap frozen for histomorphologic analysis and lung tissue distillation. On macroscopic inspection, PFOB-filled and tracheally-occluded lungs were markedly larger than saline-filled and control lungs. Mean FLBW was higher in fetuses treated with intrapulmonary instillation of PFOB (0.037+/-0.009), compared with fetuses receiving saline (0.027+/-0.008) or the unmanipulated controls (0.028+/-0.008). FLBW was highest after TO (0.049+/-0.008). After 48 h, in-vivo radiographs did not demonstrate any residual PFOB. Average dry fetal left lung weight (in g) was much higher in the TO (0.064+/-0.029) and PFOB (0.062+/-0.016) fetuses compared with the saline (0.054+/-0.017) and control (0.043+/-0.012) groups. Alveolar architecture on microscopy was similar between all groups, although the alveolar septae appeared thicker and more cellular after PFOB treatment and TO. We concluded that prenatal intrapulmonary PFOB instillation leads to increased lung growth in the late gestation rabbit model. Although PFOB instillation resulted in lower wet FLBW than TO, the increase in dry lung weight is comparable. This novel technique may be a less invasive and less noxious treatment strategy for pulmonary hypoplasia associated with diaphragmatic hernia.