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Paracetamol is commonly used to treat fever and pain in pregnant women, but there are growing concerns that this may cause attention deficit hyperactivity disorder and autism spectrum disorder in the offspring. A growing number of epidemiological studies suggests that relative risks for these disorders increase by an average of about 25% following intrauterine paracetamol exposure. The data analyzed point to a dose-effect relationship but cannot fully account for unmeasured confounders, notably indication and genetic transmission. Only few experimental investigations have addressed this issue. Altered behavior has been demonstrated in offspring of paracetamol-gavaged pregnant rats, and paracetamol given at or prior to day 10 of life to newborn mice resulted in altered locomotor activity in response to a novel home environment in adulthood and blunted the analgesic effect of paracetamol given to adult animals. The molecular mechanisms that might mediate these effects are unknown. Paracetamol has diverse pharmacologic actions. It reduces prostaglandin formation via competitive inhibition of the peroxidase moiety of prostaglandin H2 synthase, while its metabolite N -arachidonoyl-phenolamine activates transient vanilloid-subtype 1 receptors and interferes with cannabinoid receptor signaling. The metabolite N -acetyl-p-benzo-quinone-imine, which is pivotal for liver damage after overdosing, exerts oxidative stress and depletes glutathione in the brain already at dosages below the hepatic toxicity threshold. Given the widespread use of paracetamol during pregnancy and the lack of safe alternatives, its impact on the developing brain deserves further investigation.

Evidence for umbilical cord blood (UCB) cell therapies as a potential intervention for neurological diseases is emerging. To date, most existing trials worked with allogenic cells, as the collection of autologous UCB from high-risk patients is challenging. In obstetric emergencies the collection cannot be planned. In preterm infants, late cord clamping and anatomic conditions may reduce the availability. The aim of the present study was to assess the feasibility of UCB collection in neonates at increased risk of brain damage. Infants from four high-risk groups were included: newborns with perinatal hypoxemia, gestational age (GA) ≤30 + 0 weeks and/or birthweight <1,500 g, intrauterine growth restriction (IUGR), or monochorionic twins with twin-to-twin transfusion syndrome (TTTS). Feasibility of collection, quantity and quality of obtained UCB [total nucleated cell count (TNC), volume, sterility, and cell viability], and neonatal outcome were assessed. UCB collection was successful in 141 of 177 enrolled patients (hypoxemia n = 10; GA ≤30 + 0 weeks n = 54; IUGR n = 71; TTTS n = 6). Twenty-six cases were missed. The amount of missed cases per month declined over the time. Volume of collected UCB ranged widely (median: 24.5 ml, range: 5.0-102 ml) and contained a median of 0.77 × 10 8 TNC (range: 0.01-13.0 × 10 8 ). TNC and UCB volume correlated significantly with GA. A total of 10.7% (19/177) of included neonates developed brain lesions. To conclude, collection of UCB in neonates at high risk of brain damage is feasible with a multidisciplinary approach and intensive training. High prevalence of brain damage makes UCB collection worthwhile. Collected autologous UCB from mature neonates harbors a sufficient cell count for potential therapy. However, quality and quantity of obtained UCB are critical for potential therapy in preterm infants. Therefore, for extremely preterm infants alternative cell sources such as UCB tissue should be investigated for autologous treatment options because of the low yield of UCB.

In preterm infants, phenobarbital is the first-line antiepileptic drug for neonatal seizures while caffeine is used for the treatment of apnea. Data from experimental animals suggest that phenobarbital and other anticonvulsants are toxic for the developing brain, while neuroprotective effects have been reported for caffeine both in newborn rodents and preterm human infants. To characterize the interaction of phenobarbital and caffeine in the hippocampus of the developing rodent brain, we examined the effects of both drugs given separately or together on postnatal neurogenesis after administration to neonatal rats throughout postnatal day (P) 4 to P6. Phenobarbital treatment (50 mg/kg) resulted in a significant decrease of proliferative capacity in the dentate gyrus. Phenobarbital also reduced expression of neuronal markers (doublecortin (DCX), calretinin, NeuN), neuronal transcription factors (Pax6, Sox2, Tbr1/2, Prox1), and neurotrophins (NGF, BDNF, NT-3) up to 24 h after the last administration. The phenobarbital-mediated impairment of neurogenesis was largely ameliorated by preconditioning with caffeine (10 mg/kg). In contrast, caffeine alone reduced proliferative capacity and expression of the neuronal markers DCX and NeuN at 6 h, but increased expression of neurotrophins and neuronal transcription factors at 6 and 12 h. These results indicate that administration of phenobarbital during the vulnerable phase of brain development negatively interferes with neuronal development, which can be prevented in part by co-administration of caffeine.

Phenobarbital is the most commonly used drug for the treatment of neonatal seizures but may induce neurodegeneration in the developing brain. Methylxanthine caffeine is used for the treatment of apnea in newborn infants and appears to be neuroprotective, as shown by antiapoptotic and anti-inflammatory effects in oxidative stress models in newborn rodents and reduced rates of cerebral palsy in human infants treated with caffeine. We hypothesized that caffeine may counteract the proapoptotic effects of phenobarbital in newborn rats. Postnatal day 4 (P4) rats received phenobarbital (50 mg/kg) +/- caffeine (10 mg/kg) for three consecutive days. Brains examined at 6, 12, and 24 h after last injection of phenobarbital showed a drastic increase of apoptotic cell death (TUNEL+), which was attenuated by co-treatment with caffeine at 6 and 24 h but not at 12 h. Phenobarbital also increased protein levels of apoptosis inducing factor (AIF) and cleaved caspase-3, which was reduced by caffeine co-administration at all time points investigated. RNA expression of the pro-inflammatory cytokines TNFα, IFNγ, and IL-1β, but not IL-18, was upregulated by phenobarbital. Co-treatment with caffeine significantly decreased these upregulations at all time points investigated, while caffeine without phenobarbital resulted in increased expression of TNFα, IL-1β, and IL-18, but not IFNγ at 6 h. Downregulation of the adenosine A1 and A2a receptors, both of which bind caffeine, by 24 h of phenobarbital exposure was partly antagonized by caffeine. These results raise the possibility that the phenobarbital-induced adverse effects could be reduced by a co-treatment with caffeine.

Perinatal infections have a negative impact on brain development. However, the underlying mechanisms leading to neurological impairment are not completely understood and reliable models of inflammation are urgently needed. Using phorbol-myristate-acetate as an activator of inflammation, we investigated the effect on the developing rodent brain. Neonatal rats and mice deficient in IL-18 or IRAK-4 were exposed to PMA. Brains were assessed for regulation of pro- and anti-inflammatory cytokines and cell death 24 hrs, 7 and 14 days after treatment. PMA induced an inflammatory response and caused widespread neurodegeneration in the brains of 3- and 7-day-old rats. In contrast, 14-day-old rats were resistant to the neurotoxic effect of PMA. Histological evaluation at the age of 14 and 21 days revealed a destruction of the cortical microstructure with decreased numerical density of neuronal cells. Mice deficient in IL-18 or IRAK-4 were protected against PMA induced brain injury. PMA treatment during a vulnerable period can alter brain development. IL-18 and IRAK-4 appear to be important for the development of PMA induced injury.

The pathological mechanisms underlying neurological deficits observed in individuals born prematurely are not completely understood. A common form of injury in the preterm population is periventricular white matter injury (PWMI), a pathology associated with impaired brain development. To mitigate or eliminate PWMI, there is an urgent need to understand the pathological mechanism(s) involved on a neurobiological, structural, and functional level. Recent clinical data suggest that a percentage of premature infants experience relative hyperoxia. Using a hyperoxic model of premature brain injury, we have previously demonstrated that neonatal hyperoxia exposure in the mouse disrupts development of the white matter (WM) by delaying the maturation of the oligodendroglial lineage. In the present study, we address the question of how hyperoxia-induced alterations in WM development affect overall WM integrity and axonal function. We show that neonatal hyperoxia causes ultrastructural changes, including: myelination abnormalities (i.e., reduced myelin thickness and abnormal extramyelin loops) and axonopathy (i.e., altered neurofilament phosphorylation, paranodal defects, and changes in node of Ranvier number and structure). This disruption of axon-oligodendrocyte integrity results in the lasting impairment of conduction properties in the adult WM. Understanding the pathology of premature PWMI injury will allow for the development of interventional strategies to preserve WM integrity and function.

The cholinergic anti-inflammatory pathway is a neural mechanism that suppresses the innate inflammatory response and controls inflammation employing acetylcholine as the key endogenous mediator. In this study, we investigated the effects of the cholinergic agonists, physostigmine and donepezil, on neurodegeneration, inflammation and oxidative stress during oxygen toxicity in the developing rat brain. The aim of this study was to investigate the level of neurodegeneration, expression of proinflammatory cytokines, glutathione and lipid peroxidation after hyperoxia and treatment with the acetylcholinesterase (AChE) inhibitors, physostigmine and donepezil in the brain of neonatal rats. Six-day-old Wistar rats were exposed to 80% oxygen for 12-24 h and received 100 μg/kg physostigmine or 200 μg/kg donepezil intraperitoneally. Sex-matched littermates kept in room air and injected with normal saline, physostigmine or donepezil served as controls. Treatment with both inhibitors significantly reduced hyperoxia-triggered activity of AChE, neural cell death and the upregulation of the proinflammatory cytokines IL-1β and TNF-α in the immature rat brain on the mRNA and protein level. In parallel, hyperoxia-induced oxidative stress was reduced by concomitant physostigmine and donepezil administration, as shown by an increased reduced/oxidized glutathione ratio and attenuated malondialdehyde levels, as a sign of lipid peroxidation. Our results suggest that a single treatment with AChE inhibitors at the beginning of hyperoxia attenuated the detrimental effects of oxygen toxicity in the developing brain and may pave the way for AChE inhibitors, which are currently used for the treatment of Alzheimer's disease, as potential candidates for adjunctive neuroprotective therapies to the immature brain., (Copyright © 2013 S. Karger AG, Basel.)

Intrauterine infection and inflammation are major reasons for preterm birth. The switch from placenta-mediated to lung-mediated oxygen supply during birth is associated with a sudden rise of tissue oxygen tension that amounts to relative hyperoxia in preterm infants. Both infection/inflammation and hyperoxia have been shown to be involved in brain injury of preterm infants. Hypothesizing that they might be additive or synergistic, we investigated the influence of a systemic lipopolysaccharide (LPS) application on hyperoxia-induced white matter damage (WMD) in newborn rats. Three-day-old Wistar rat pups received 0.25 mg/kg LPS i.p. and were subjected to 80% oxygen on P6 for 24 h. The extent of WMD was assessed by immunohistochemistry, western blots, and diffusion tensor (DT) magnetic resonance imaging (MRI). In addition, the effects of LPS and hyperoxia were studied in an in vitro co-culture system of primary rat oligodendrocytes and microglia cells. Both noxious stimuli, hyperoxia, and LPS caused hypomyelination as revealed by western blot, immunohistochemistry, and altered WM microstructure on DT-MRI. Even so, cellular changes resulting in hypomyelination seem to be different. While hyperoxia induces cell death, LPS induces oligodendrocyte maturity arrest without cell death as revealed by TUNEL-staining and immunohistological maturation analysis. In the two-hit scenario cell death is reduced compared with hyperoxia treated animals, nevertheless white matter alterations persist. Concordantly with these in vivo findings we demonstrate that LPS pre-incubation reduced premyelinating-oligodendrocyte susceptibility towards hyperoxia in vitro. This protective effect might be caused by upregulation of interleukin-10 and superoxide dismutase expression after LPS stimulation. Reduced expression of transcription factors controlling oligodendrocyte development and maturation further indicates oligodendrocyte maturity arrest. The knowledge about mechanisms that triggered hypomyelination contributes to a better understanding of WMD in premature born infants.

In the immature human brain, periventricular leukomalacia (PVL) is the predominant white matter injury underlying the development of cerebral palsy. PVL has its peak incidence during a well-defined period in human brain development (23-32 weeks postconceptional age) characterized by extensive oligodendrocyte migration and maturation. We hypothesized that the dramatic rise of oxygen tissue tension associated with mammalian birth and additional oxygen exposure of the preterm infant during intensive care may be harmful to immature oligodendrocytes (OLs). We therefore investigated the effects of hyperoxia on rat oligodendroglia cells in vitro and in vivo. Immature OLs (OLN-93), their progenitors [preoligodendrocytes (pre-OL)], and mature OLs were subjected to 80% hyperoxia (24-96 hr). Flow cytometry was used to assess cell death. Cell viability was measured by metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT). In addition, 6-day-old rat pups were subjected to 80% oxygen (24 hr) and then sacrificed, and their brains were processed for immunfluorescence staining. Apoptosis was detected at various stages (annexin-V, activated caspase-3) after 24-48 hr of incubation in 80% oxygen in pre- and immature OLs. Mature OLs were resistant to oxygen exposure. These results were confirmed by MTT assay. This cell death was blocked by administration of the pan-caspase inhibitor zVAD-fmk. Degeneration of OLs was confirmed in 7-day-old rat brains by positive staining for activated caspase-3. Hyperoxia triggers maturation-dependent apoptosis in immature and pre-OLs and involves caspase activation. This mechanism may be relevant to the white matter injury observed in infants born preterm.

Background: Elevated serum concentrations of S-100B, a 21-kDa protein expressed in astroglial cells, has been used to assess cerebral damage after head trauma, infection, ischemia, and perinatal asphyxia., Objective: As S-100B is eliminated by the kidneys, we investigated the feasibility of measuring S-100B in urine of newborns with severe perinatal asphyxia, and in very low birth weight (VLBW) preterm infants at risk for neurodevelopmental impairment., Methods: We first analyzed urine samples of 8 term or near-term newborns without major medical problems, followed by urine samples of 2 term newborns with severe birth asphyxia, and finally urine samples of 8 VLBW (gestational age 24-28 weeks) infants collected every 4 h for up to 10 days., Results: Urinary S-100B concentrations in 8 term or near-term newborns without major medical problems were consistently <1 microg/l. In 2 term newborns with severe asphyxia (Apgar 0/0/0 and 0/2/4) who subsequently had widespread cerebral damage on magnetic resonance imaging, peak urinary S-100B concentrations on the first day of life were 28.1 and 28.4 microg/l, respectively. In 5/8 VLBW infants, urinary S-100B was> microg/l in samples obtained on the first day of life (range 1.2-44.9 microg/l, median 6.8 microg/l). Peak S-100B in urine samples collected during the first 12 h of life were negatively related to gestational age (R(s)=-0.882, p=0.009). Three of the 8 preterm infants had peak urinary concentrations>0 microg/l but neither ultrasound signs of brain damage nor neurodevelopmental delay at 1 year corrected age., Conclusions: The determination of urinary S-100B concentrations might be helpful in term infants with severe asphyxia, while high urinary S-100B concentrations in preterm infants are to be attributed to immaturity., (Copyright (c) 2006 S. Karger AG, Basel.)

Interleukin (IL)-18 is a potent inflammatory cytokine of the IL-1 family. It is synthesized as an inactive precursor (pro-IL-18), which is cleaved into its functionally active form by caspase-1. Resident cells of the CNS express IL-18 and caspase-1 constitutively, thus providing a local IL-18-dependent immune response. Recent studies have highlighted a crucial role for IL-18 in mediating neuroinflammation and neurodegeneration in the CNS under pathological conditions, such as bacterial and viral infection, autoimmune demyelinating disease, and hypoxic-ischemic, hyperoxic and traumatic brain injuries. This review provides a synopsis of the current knowledge of IL-18-dependent mechanisms of action during acute neurodegeneration in immature and adult brains.

Infants born prematurely may develop neurocognitive deficits without an obvious cause. Oxygen, which is widely used in neonatal medicine, constitutes one possible contributing neurotoxic factor, because it can trigger neuronal apoptosis in the developing brain of rodents. We hypothesized that two caspase-1-processed cytokines, interleukin (IL)-1beta and IL-18, are involved in oxygen-induced neuronal cell death. Six-day-old Wistar rats or C57/BL6 mice were exposed to 80% oxygen for various time periods (2, 6, 12, 24, and 48 hours). Neuronal cell death in the brain, as assessed by Fluoro-Jade B and silver staining, peaked at 12 to 24 hours and was preceded by a marked increase in mRNA and protein levels of caspase 1, IL-1beta, IL-18, and IL-18 receptor alpha (IL-18Ralpha). Intraperitoneal injection of recombinant human IL-18-binding protein, a specific inhibitor of IL-18, attenuated hyperoxic brain injury. Mice deficient in IL-1 receptor-associated kinase 4 (IRAK-4), which is pivotal for both IL-1beta and IL-18 signal transduction, were protected against oxygen-mediated neurotoxicity. These findings causally link IL-1beta and IL-18 to hyperoxia-induced cell death in the immature brain. These cytokines might serve as useful targets for therapeutic approaches aimed at preserving neuronal function in the immature brain, which is exquisitely sensitive to a variety of iatrogenic measures including oxygen.

Substantial neurologic morbidity occurs in survivors of premature birth. Premature infants are exposed to partial oxygen pressures that are fourfold higher compared to intrauterine conditions, even if no supplemental oxygen is administered. Here we report that short exposures to nonphysiologic oxygen levels can trigger apoptotic neurodegeneration in the brains of infant rodents. Vulnerability to oxygen neurotoxicity is confined to the first 2 weeks of life, a period characterized by rapid growth, which in humans expands from the sixth month of pregnancy to the third year of life. Oxygen caused oxidative stress, decreased expression of neurotrophins, and inactivation of survival signaling proteins Ras, extracellular signal-regulated kinase (ERK 1/2), and protein kinase B (Akt). The synRas-transgenic mice overexpressing constitutively activated Ras and phosphorylated kinases ERK1/2 in the brain were protected against oxygen neurotoxicity. Our findings reveal a mechanism that could potentially damage the developing brain of human premature neonates.

Pharmacological blockade of NMDA receptor function induces apoptotic neurodegeneration in the developing rat brain. However, the use of NMDA receptor antagonists as anesthetics and sedatives represents a difficult-to-avoid clinical practice in pediatrics. This warrants the search for adjunctive neuroprotective measures that will prevent or ameliorate neurotoxicity of NMDA receptor antagonists. The NMDA receptor antagonist MK801 triggered apoptosis in the neonatal rat forebrain, most notably in cortex and thalamus. MK801 exposure reduced mRNA levels of erythropoietin (EPO) and the EPO receptor, suggesting that loss of endogenous EPO activity may contribute to MK801-induced apoptosis. Coadministration of recombinant EPO (rEPO) conferred 50% neuroprotection, partially restored MK801-induced reduction of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) mRNA, and prevented decreased phosphorylation levels of extracellular signal-regulated protein kinase-1/2 (ERK1/2) and Akt. These observations indicate that rEPO partly rescues newborn rats from MK801-mediated brain damage by enhancing neurotrophin-associated signaling pathways.

Context: Birth triggers a large fetal neuroendocrine response, which is more pronounced in infants born vaginally than in those born by elective cesarean section (ECS). The two related peptides arginine vasopressin (AVP) and oxytocin (OT) play an essential role in peripheral and central stress adaptation and have a shared receptor mediating their function. Elevated cord blood levels of AVP and its surrogate marker copeptin, the C-terminal part of AVP prohormone, have been found after vaginal delivery (VD) as compared to ECS, while release of OT in response to birth is controversial. Moreover, AVP, copeptin and OT have not yet been measured simultaneously at birth. Objective: To test the hypothesis that AVP but not OT levels are increased in infants arterial umbilical cord blood in response to birth stress and to characterize AVP secretion in direct comparison with plasma copeptin. Methods: In a prospective single-center cross-sectional study, we recruited healthy women with a singleton pregnancy and more than 36 completed weeks of gestation delivering via VD or ECS (cesarean without prior uterine contractions or rupture of membranes). Arterial umbilical cord blood samples were collected directly after birth, centrifuged immediately and plasma samples were frozen. Concentrations of AVP and OT were determined by radioimmunoassay and that of copeptin by ultrasensitive immunofluorescence assay. Results: A total of 53 arterial umbilical cord blood samples were collected, n = 29 from VD and n = 24 from ECS. Ten venous blood samples from pregnant women without stress were collected as controls. AVP and copeptin concentrations were significantly higher in the VD group than in the ECS group (both p < 0.001), median (range) AVP 4.78 (2.38–8.66) vs. 2.38 (1.79–3.88) (pmol/L), copeptin 1692 (72.1–4094) vs. 5.78 (3.14–17.97), respectively, (pmol/L). In contrast, there was no difference in OT concentrations (pmol/L) between VD and ECS, 6.00 (2.71–7.69) vs. 6.14 (4.26–9.93), respectively. AVP and copeptin concentrations were closely related (Rs = 0.700, p < 0.001) while OT did not show any correlation to either AVP or copeptin. In linear regression models, vaginal delivery and biochemical stress indicators, base deficit and pH, were independent predictors for both AVP and copeptin. OT was not linked to base deficit or pH. Conclusion: Vaginal birth causes a profound secretion of AVP and copeptin in infants. Whereas AVP indicates acute stress events, copeptin provides information on cumulative stress events over a longer period. In contrast, fetal OT is unaffected by birth stress. Thus, AVP signaling but not OT mediates birth stress response in infants. This unique hormonal activation in early life may impact neurobehavioral development in whole life. [ABSTRACT FROM AUTHOR]

Abstract: Induced hypothermia is the only therapy with proven efficacy to reduce brain damage after perinatal asphyxia. While hypothermia down-regulates global protein synthesis and cell metabolism, low temperature induces a small subset of proteins that includes the RNA-binding protein RBM3 (RNA-binding motif protein 3), which has recently been implicated in cell survival. Here, immunohistochemistry of the developing postnatal murine brain revealed a spatio-temporal neuronal RBM3 expression pattern very similar to that of doublecortin, a marker of neuronal precursor cells. Mild hypothermia (32°C) profoundly promoted RBM3 expression and rescued neuronal cells from forced apoptosis as studied in primary neurons, PC12 cells, and cortical organotypic slice cultures. Blocking RBM3 expression in neuronal cells by specific siRNAs significantly diminished the neuroprotective effect of hypothermia while vector-driven RBM3 over-expression reduced cleavage of PARP, prevented internucleosomal DNA fragmentation, and LDH release also in the absence of hypothermia. Together, neuronal RBM3 up-regulation in response to hypothermia apparently accounts for a substantial proportion of hypothermia-induced neuroprotection. [Copyright &y& Elsevier]

Abstract: Both mild hypoxia and exogenous 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. [Copyright &y& Elsevier]