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Background: Propofol is a frequently used drug in pediatric anesthesia and neonatology. We have recently shown that exposure of rodent pups by propofol induces neuronal apoptosis in short term.Aim: The aim of the current investigation is to evaluatea) mechanisms of propofol induced neurotoxicity andb) whether the neurotoxic effect can be reduced by ErythropoietinMethods: Newborn rats were randomized into following groups i) sham ii) Propofol iii) Propofol with Erythropoietin intraperitoneal injections. Animals were killed after 6, 12, and 24 hours and the brains were harvested for gene (Affymetrix Chip) and protein expression (Western Blot) analyses.Results: Propofol induces caspase-3 activation which was inhibited by systemic treatment with EPO at 12 hours. Primary results of gene expression array analyses indicate that systemic Propofol administration induces a reduction in genes involved in Ca2+-regulatory mechanism as well as phosphoinositide-3-kinase signaling. In western blots analyses Propofol induced a transient activation of pAkt, which was increased by co-administration of EPO. In addition Propofol administration decraesed cytokine IL-18 brain levels.Conclusion: Administration of Propofol does not only induces neuronal cell death but also induces major modification in cellular signalling in the developing brain. Cell death induced by Propofol is reduced by co-administration of EPO. Additional investigations analysing the effect of propofol on long-term behavioural outcome and mechanisms of action are mandatory and ongoing.

The risk of dying is highest in the time around birth - a time when safe childbirth and effective neonatal care are essential to the survival of the newborn baby. Four million neonatal deaths occur every year around the world - that is 450 every hour. 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 compare the current health and social policies, as well as practices in 13 EU member states. Despite the growing prevalence and increasing costs, neonatal and preterm infant health rank low on the policy agendas of EU member states. Based on our findings we propose important issues to be considered for policies at EU and national levels.

Background and aims: Experimental studies show that oxygen, which is widely used in neonatal medicine for resuscitation and treatment of pulmonary hypertension, triggers a disruption of the maintenance of intracellular redox homeostasis. This disturbance can lead to oxidative stress and furthermore to neuronal apoptosis in the developing brain. The role of caspase-2 in apoptosis is poorly defined. Many in vitro studies of caspase-2 knockdown in cultured cells have implicated this caspase in cell death in response to different signaling pathways. To elucidate mechanisms of the caspase-2 inhibitor TRP601 and its mode of functioning in the developing brain in the context of hyperoxia, we investigated its impact on the levels of APAF-1, AIF, cytochrome c, caspase-9 and -3.Methods: Six-day old rats were exposed together with their mothers to 80% oxygen in the presence or absence of the caspase-2 inhibitor TRP601 (1 mg/kg) and were sacrificed after 12 or 24 hrs of hyperoxia following treatment. Dissected brains were either examined histologically to visualize degenerating cells or were subjected to protein studies.Results: Oxygen exposure triggered cell death at 12 to 24 hrs, which was attenuated by TRP601 treatment. Our protein studies demonstrated an upregulation of APAF-1, AIF, cytochrome c, caspase-9 and -3 in the cytosolic fraction of brain homogenates after hyperoxia, which reached control levels following TRP601 treatment.Conclusion: These findings suggest a protective role for the caspase-2 inhibitor TRP601 in the prevention of neonatal oxygen-induced apoptotic brain damage.Supported by the European Commission (Sixth Framework Program, contract no LSHM- CT-2006-036534).

MDMA (3,4-methylenedioxymethamphetamine) causes damage to fine serotonergic fibers and apoptotic neurodegeneration in the adult rat brain. But little is known about the toxic effects of MDMA in the developing brain. The incidence of MDMA abuse is particularly high in adolescents. But the prevalence of MDMA use in woman of child-bearing age and the consequences on their offsprings are unknown. For this reason we administered MDMA (20 – 60 mg/kg) i.p. To 7-day-old rats. Rats treated with normal saline alone served as controls. Pups were sacrificed 24 hrs after injection and brain sections were examined histologically by means of De Olmos cupric silver staining to visualize degenerating cells. MDMA doses of 60 mg/kg induced significant apoptotic neurodegeneration. To explore pathogenetic mechanisms, we investigated the impact of MDMA on the neurotrophine BDNF (brain derived neurotrophic factor). Pups were sacrificed at defined time points (2, 6, 12 and 24 hrs) after injection and brains were processed for RT-PCR, real-time PCR and Western-Blot analysis. mRNA levels for BDNF were upregulated at 6 hrs in animals receiving MDMA compared to controls and even increased at 12 and 24 hours. real-time PCR analysis of the thalamus revealed similar pattern of upregulation of BDNF mRNA transcription. Determined by immunoblot protein expression of BDNF also showed an increase at 12 and 24 hours. These preliminary data suggest that a single injection of MDMA causes neurodegeneration in the neonatal rat brain and that higher doses are required for similar toxicity compared to adult rats. The upregulation in the expression of brain derived neurotrophic factor (BDNF) is may be an important mechanism that lead to survival of neuronal cells in the developing brain.

Objective: Vascular endothelial growth factor (VEGF) expression promotes angiogenesis and neuronal repair. The aim of this study was to investigate VEGF expression in relation to neurodegeneration, either caused by hyperoxia, a frequent problem in the treatment of preterm infants with respiratory dysfunction, or caused by brain injury, an important complication (e. g. In case of intracerebral haemorrhage) in the early neonatal period of immature newborns.Methods: 6 d old Wistar rat pups were either subjected to 80 % oxygen or treated with phorbolester. After 0, 2, 6, 12 and 24 h of exposition animals (n = 5 per group) were sacrificed and the brain cortices were processed for molecular experiments on RNA (quantitative RT-PCR) and protein (Western blot) levels using VEGF specific primers and antibodies.Results: In association with hyperoxia a decrease in VEGF mRNA expression down to about 25 % of the basal level of the control animals (no additional oxygen exposition) was noted. This effect was already evident at 2 h with a minimum of VEGF mRNA expression at 2 h and 6 h (p < 0.01). The protein expression also showed a decrease of VEGF signal associated with hyperoxia. The weakest signal appeared at 12 h (p < 0.01). In the case of phorbolester treatment we have seen an increase of VEGF expression up to about 200 % compared to the controls, with a maximum of mRNA at 6 h (p < 0.01) and a maximum of protein expression at 12 h and 24 h (p < 0.05).Conclusion: Exposure of newborn rats to hyperoxia induces a statistically significant time-dependent decrease, treatment with phorbolester a statistically significant time-dependent increase in VEGF expression in the cortices of the animals. These effects are evident on the transcriptional and translational level.

Background: In neonatal medicine, oxygen is frequently used in the treatment of respiratory diseases, and preterm infants are generally exposed to relative hyperoxia compared to in-utero conditions. Recently, oxygen has been identified to cause apoptosis in the brain of newborn rats and rodents. Therefore, it may contribute to the development of cerebral palsy and neurocognitive defects often seen in preterm infants. Pro-inflammatory cytokines, i.e. IL-1b and IL-18, have been proven to be involved in neuronal apoptosis caused by hyperoxia. In contrast, IL-10 is an anti-inflammatory and anti-apoptotic cytokine that may have neuro-protective properties.Methods: We analysed expression of IL-10 receptor (IL-10R) and IL-10 in cortex and thalamus of six day old newborn Wistar rats (P6) exposed to 80% oxygen for 0, 2, 6, 12, 24 and 48 hours. mRNA levels were determined by semiquantitative reverse transcription polymerase chain reaction, and protein expression was analysed by immunoblotting.Results: Compared to rats not exposed to hyperoxia, mRNA of IL-10 and IL-10R increased markedly during oxygen exposure with peak levels at 6 and 24 hours, respectively (IL-10R: p<0.01, IL-10: p=0.01). A four-fold upregulation of IL-10R protein was found at 24 hours.Conclusions: Expression of anti-inflammatory IL-10 and IL-10R in the brain of newborn rats is upregulated during exposure to neurotoxic doses of oxygen. IL-10 may act as an internal neuroprotective agent against hyperoxia-induced cell death.

Background. Hyperbilirubinemia in newborn infants and especially in premature infants enhances the risk for developing cerebral palsy. In the developing rat brain and cell culture hyperoxia has been identified to cause neurodegeneration. We therefore examined the effects of bilirubin in combination with hyperoxia in cultured rat neuronal cultures.Methods. Primary cortical neuronal cultures were prepared from Wistar rat embryos at 18 days gestation and exposed to bilirubin (0,1–250 µM) in combination with hyperoxia (80%) for 48 h. Control cells were kept in normoxia. Cell viability was assessed by the methyltetrazolium method (MTT) and lactate dehydrogenase release (LDH). Cells showed morphologic changes consistent with apoptosis.Results. In normoxia bilirubin reduced cell viability by 58 % vs controls (p< 0,05) above a concentration of 5 µM whereas LDH release signifficantly increased from 20% to 94% above bilirubin concentration from 25 µM. Combination with hyperoxia caused additional loss of MTT cleavage and LDH release was increased in all concentrations of bilirubin from 0.1–250 µM.Conclusion. We conclude that a combination of bilirubin and hyperoxia results in stronger detrimental effects on neurons than bilirubin or hyperoxia alone. Our results suggest that in premature infants exposed to higher oxygen concentrations hyperbilirubinemia may exacerbate neuronal damage and contribute to neurological impairment.

Objective: Pontosubicular neuron necrosis (PSN) represents an age-specific response to severe hypoxic-ischemic injury (HII) occurring in human neonates but not in older children or adults. Histologically, PSN is characterised by acute neuronal death in the pontine nuclei and the hippocampal subiculum bearing the hallmarks of apoptosis. The expression of Rbm3, a glycine-rich RNA-binding protein, is enhanced under hypoxic conditions and independent of HIF (hypoxia inducible factor). It is well known, that proteins which are dependant on HIF, e.g. Erythropoetin play an important role in HII. This study aims to determine whether the HIF-independent activation of Rbm3 is also a significant factor in the pathogenesis of PSN. Methods: We have investigated the expression of Rbm3 in human autopsy material consisting of 12 PSN cases and 10 age-matched controls without PSN. Immunohistochemistry and double labeling for Rbm3 and the astrocyte marker glial fibrillary acid protein (GFAP), the microglia/macrophage specific marker KiM1P and the neuronal marker NeuN was performed on formalin-fixed, paraffin-embedded brain specimens.Results: In PSN cases and controls, mainly neuronal cells expressed Rbm3. The number of immunopositive cells was significantly increased (p=0.001) in PSN cases. Predominantly degenerating cells with signs of later apoptotic stages showed Rbm3 expression. In earlier stages of apoptosis immunopositivity for Rbm3 was increased compared to contols, but less prominent.Conclusion: In addition to HIF-dependant proteins, the induction of the HIF-independent protein RMB3 is observed in response to human hypoxic-ischemic injury.

Background: In the developing human brain, periventricular leukomalacia (PVL) is the predominant white matter injury underlying the development of cerebral palsy. Our group recently showed that a rise of oxygen tissue tension is a powerful trigger to initiate apoptosis in premature and immature, but not in mature oligodendrocytes (OL). Estrogen plays an important role in the development and function of the cerebral nervous system, and estrogen receptors show a specific distribution with high density arround the third ventricle, the region where OL progenitors are generated. We therefore examined the effects of estrogen on hyperoxia-induced cell death in cultured rat immature oligodendroglia cells.Methods: OLN-93 cells, derived from spontaneously transformed cells of 5- to 10-day-old primary rat brain oligodendrocytes were subjected to 24–72 h of hyperoxia (80% O2) in the presence or absence of estrogen (17beta-estradiol) at various concentrations. These immature oligodendrocytes resemble an intermediate stage between oligodendrocyte precursors and mature oligodendrocytes. Flow cytometry was used to assess apoptosis via annexin-V, anti-activated caspase-3 antibody, and propidium iodide staining. Oligodendrocyte maturation status and caspase-3 activation were confirmed by immunochemistry.Results: In immature rat oligodendrocytes (OLN-93), apoptosis was detected at various stages (early: annexin-V, effector: caspase-3) after 24–72 h of hyperoxia (80% O2). Cell death was reduced up to 30% by preincubation with estrogen at a concentration optimum of 10–7 M estrogen.Conclusion: Estrogen reduces apoptosis in immature oligodendrocytes exposed to hyperoxia. The sudden drop in circulating estrogen of placental origin after mammalian birth may aggravate the susceptibility of immature oligodendrocytes to increased oxygen tension. Supported by grants from the German Federal Department of Education and Research (BMBF, # 01 ZZ 0101), Bonn, and the Ernst Schering Research Foundation, Berlin.

Both mild hypoxia and exogenonus erythropoietin may protect the brain against subsequent severe hypoxia, and the conditioning effect of transient hypoxia is partly mediated by hypoxia-induced endogenous erythropoietin. We now observed in several experimental models that combining transient hypoxia and exogenous erythropoietin may cause neuronal damage. High-dose erythropoietin (40 IU/mL) profoundly impeded synaptic transmission of rat hippocampal slice cultures when used in conjunction with moderate hypoxia (10% O2 for two 8-h periods). Addition of erythropoietin increased viability of cultured rat embryonic cortical neurons at 21% O2 but decreased viability under hypoxic conditions (2% O2) in a dose-dependent fashion. Death of human neuronal precursor cells challenged by oxygen and glucose deprivation was increased by erythropoietin when cells were cultured under hypoxic but not under normoxic conditions. In neonatal rats exposed to moderate hypoxia plus erythropoietin, numbers of degenerating cerebral neurons were increased, as compared to controls or rats subjected to either hypoxia or erythropoietin alone. Thus, erythropoietin may aggravate rather than ameliorate neuronal damage when administered during transient hypoxia. This work was supported in parts by grants from the Bundesministerium für Bildung und Forschung, Bonn (01ZZ0101), the Wilhelm Sander Stiftung, Munich (2000.091.1), the Sonnenfeld-Stiftung, Berlin, and the Förderverein für Frühgeborene im Virchow-Klinikum, Berlin, Germany.

Neurologic morbidity often occurs in survivors of premature birth. Clinical studies have identified hyperoxia as a risk factor for cerebral palsy. Short exposure to high oxygen levels can trigger neurodegeneration in the developing rat brain. Toxicity of oxygen is associated with inactivation of intracellular proteins that promote survival and increased expression of the proapoptotic Fas-receptor. In this animal model of neonatal brain damage we tested Estradiol against the detrimental effect of oxygen. This hormone has neuroprotective properties that result from activation of estrogen receptors and cross-talk with signaling pathways that are activated by neurotrophins. 6-day old rats were subjected to 80% oxygen and Estradiol was co-administered (600 µg/kg i.p.) at beginning of exposure. Pups were sacrificed at defined time points and brains were either examined histologically to visualize degenerating cells or were snap frozen for molecular studies. Estradiol conferred significant protection against oxygen-induced neurodegeneration. To explore further possible molecular mechanisms, we investigated the impact of Estradiol on the expression of the death receptor Fas and Fas-Ligand. Following hyperoxia for 12 and 24h increased levels of Fas and Fas-Ligand mRNA were found. In the presence of Estradiol mRNA levels of the Fas-system were reduced. Immunoblot analysis on two signaling pathways (MAPK, PI3-kinase) that control neuronal survival, revealed that co-administration of Estradiol counteracts inactivation of the two key players ERK1/2 and AKT. The downregulation in the expression of Fas and Fas-Ligand and increased levels of active forms of ERK1/2 and AKT may represent important mechanisms that lead to survival of neuronal cells. Our results suggest that Estradiol might be suitable as a neuroprotective agent in preterm and term infants who need to be exposed to oxygen. Especially in preterm infants, who are prematurely deprived of maternal intrauterine estrogen, maintaining physiological plasma levels of 17ß-Estradiol may help to protect from potentially neurotoxic triggers.