Georg Simbruner

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.

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

BACKGROUNDnInflammation 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.nnnHYPOTHESISnNADPH oxidase contributes to ROS formation and development of injury in the immature brain and inhibition of NADPH oxidase attenuates perinatal brain injury.nnnMETHODSnWe 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.nnnRESULTSnHypoxia-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.nnnCONCLUSIONnNADPH 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.

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

Background and aim:u2002 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.

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 5u2003µg/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‐Jadeu2003B staining and caspase‐3 activation. Pre‐treatment with interleukin‐1β 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‐Jadeu2003B 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.

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 μg/kg SCF or 200 μg/kg G-CSF. Dose-response studies indicated that G-CSF could increase grey matter lesions even at lower dosages (22 and 66 μg/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.

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

BackgroundIn 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.ObjectiveThe aim of this study was to investigate the effects of two commercially available surfactant preparations on ATII cell viability and DNA synthesis.MethodsCurosurf® 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.ResultsCurosurf® 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.ConclusionThis study showed that commercially available exogenous surfactants used to treat preterm infants with RDS can have profound effects on cell viability and DNA synthesis.

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

BACKGROUNDnPerinatal 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.nnnHYPOTHESISnWe 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.nnnMETHODSnWe 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.nnnRESULTSnNMDAR-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.nnnCONCLUSIONnSince 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.

265 Too Little, Too Late: Policy Benchmarking Report on Neonatal Health and Social Policies in 13 European Countries

265 Too Little, Too Late: Policy Benchmarking Report on Neonatal Health and Social Policies in 13 European Countries

68 CALORIMETRIC BIKINIS FOR THE UNHINDERED, LONGTERM ASSESSMENT OF DRY HEAT LOSS IN NEWBORN INFANTS

Neither indirect nor direct calorimetry are suitable for assessing metabolic rate in each newborn infant unhindered for an unlimited period of time. As an alternative, metabolic rate could be assessed as the sum of components of the energy balance equation. Among those, dry heat loss (HL) is the most important component. We investigated, whether dry heat loss determined by 17 heatflux transducers (HL tot) was related to dry heat loss determined from 12 or 8 heatflux tranducers and correction factors, a set-up nicknamed calorimetric bikini (HL-bik 12 or HL-bik 8). We studied twenty healthy newborns infants, attached five heatflux transducers to the head, six to the torso and six to the limbs of each infant and determined HL tot from the heatflux and the surface area, it represented, during a half hour period for prone and supine position respectively. The HL-bik 12 and HL-bik 8 correlated statistically significant with HL tot in the infants lying prone or supine (HL tot vs. HL-bik12: y =0.01+0.99x (Watt/kg), r=0.97, p<0.001 and HL tot vs HL-bik 8: y=0.16+0.91x, r=0.92, p<0.001). The mean and 1 S.D of differences between HL tot and HL-bikl2 or HL-bik 8 were < 1 % and < 6.5% of HL tot respectively. In conclusion, dry heat loss can be fairly accurately measured by a few calorimetric elements on the surface of the newborn infant and these calorimetric bikinis are a first step towards a method allowing unhindered monitoring of metabolic rate for an unlimited period of time.

91: BIOCHEMICAL VERSUS MORPHOLOGICAL EFFECTS OF HUMAN HEPATIC ALKALINE PHOSPHATASE IN A NEONATE WITH HYPOPHOSPHATASIA

Enzyme replacement therapy for a severely affected premature boy (birthweight: 2380 g, GA:36 weeks) with hypophosphatasia was attempted by infusions of purified human hepatic alkaline phosphatase(AP). Treatment started at age of two weeks and was repeated in weekly intervals until death (10 weeks). Samples of AP were diluted with 10 ml of physiological saline and infused over a time span of 30 minutes via an umbilical arterial catheter. No toxic or allergic side effects were observed. Serum-AP increased from 3 mU/ml before treatment to a maximum level of 195 mU/ml with a half-life time between 37 and 62 hours. Urinary excretion of phosphoethanolamine (PEA) decreased under therapy from a maximal value of 9.5 to 5.5 μmol/mg creatinine (normal: < 0.4). Calcium, phosphorus, parathormone and 1-25 dihydroxyvitamin D levels were within the normal range during therapy. Sequential radiographic studies showed no improvement of bone mineralization under therapy. Bone morphology was studied by light and electron microscopy before treatment and post mortem. In contrast to previous studies an unusually woven bundle bone structure was found with abrupt mineralization fronts without osteoblast-like cells. We conclude that this enzyme, substituted for the first time, altered PEA concentrations but failed to influence the iniiial abnormal bone structure of this infant.