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Background: The use of remifentanil in a context of potential prematurity led us to explore ex vivo the opioid effects on the immature mouse brain. Remifentanil enhances medullary glutamatergic N-methyl-D-aspartate (NMDA) receptor activity. Furthermore, in neonatal mouse cortex, NMDA was previously shown to exert either excitotoxic or antiapoptotic effects depending on the cortical layers. With the use of a model of acute cultured brain slices, we evaluated the potential necrotic and apoptotic effects of remifentanil, alone or associated with its glycine vehicle (commercial preparation of remifentanil, C.P. remifentanil), on the immature brain., Methods: Cerebral slices from postnatal day 2 mice were treated up to 5 hours with the different compounds, incubated alone or in the presence of NMDA. The necrotic effect was studied by measuring lactate dehydrogenase activity and 7-Aminoactinomycin D labeling. Apoptotic death was evaluated by measurement of caspase-3 activity and cleaved caspase-3 protein levels, using Western blot and immunohistochemistry. Extrinsic and intrinsic apoptotic pathways were investigated by measuring caspase-8, caspase-9 activities, Bax protein levels, and mitochondrial integrity., Results: C.P. remifentanil was ineffective on necrotic death, whereas it significantly reduced caspase-3 activity and cortical cleaved caspase-3 levels. C.P. remifentanil inhibited cortical Bax protein expression, caspase-9 activity, and preserved mitochondrial integrity, whereas it had no effect on caspase-8 activity. Its action targeted the neocortex superficial layers, and it was reversed by the opioid receptors antagonist naloxone and the NMDA antagonist MK801. Remifentanil and glycine acted synergistically to inhibit apoptotic death. In addition, C.P. remifentanil enhanced the antiapoptotic effect of NMDA, whereas it did not improve NMDA excitotoxicity in brain slices., Conclusion: The present data indicate that at a supraclinical concentration C.P. remifentanil had no pronecrotic effect but exerted ex vivo antiapoptotic action on the immature mouse brain, involving the opioid and NMDA receptors, and the mitochondrial-dependent apoptotic pathway. Assessment of the impact of the antiapoptotic effect of remifentanil in in vivo neonatal mouse models of brain injury will also be essential to measure its consequences on the developing brain.

Glutamate excitotoxicity is a consolidated hypothesis in neonatal brain injuries and tissue plasminogen activator (t-PA) participates in the processes through proteolytic and receptor mediated effects. In brain microvascular endothelial cell (nBMEC) cultures from neonates, t-PA content and release upon glutamate are higher than in adult (aBMECs) cultures. Owing to the variety of t-PA substrates and receptor targets, the study was aimed at determining the putative roles of endothelial t-PA in the neonatal brain parenchyma under glutamate challenge. Basal t-PA release was 4.4 fold higher in nBMECs vs aBMECs and glutamate was 20 fold more potent to allow Evans blue vascular permeability in neonate microvessels indicating that, under noxious glutamate (50 μM) exposure, high amounts of endothelial t-PA stores may be mobilized and may access the nervous parenchyma. Culture media from nBMECS or aBMECs challenged by excitotoxic glutamate were applied to neuron cultures at DIV 11. While media from adult cells did not evoke more LDH release in neuronal cultures that under glutamate alone, media from nBMECs enhanced 2.2 fold LDH release. This effect was not observed with media from t-PA(-/-) nBMECs and was inhibited by hr-PAI-1. In Cortical slices from 10 day-old mice, hrt-PA associated with glutamate evoked neuronal necrosis in deeper (more mature) layers, an effect reversed by NMDA receptor GluN1 amino-terminal domain antibody capable of inhibiting t-PA potentiation of the receptor. In superficial layers (less mature), hrt-PA alone inhibited apoptosis, an effect reversed by the EGF receptor antagonist AG1478. Applied to immature neurons in culture (DIV5), media from nBMEC rescued 85.1% of neurons from cell death induced by serum deprivation. In cortical slices, the anti-apoptotic effect of t-PA fitted with age dependent localization of less mature neurons. These data suggest that in the immature brain, propensity of vessels to release high amounts of t-PA may not only impact vascular integrity but may also influence neuronal fate, via regulation of apoptosis in immature cells and, as in adult by potentiating glutamate toxicity in mature neurons. The data point out putative implication of microvessels in glutamate neurotoxicity in the development, and justify research towards vessel oriented neuroprotection strategies in neonates., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Vascular intrauterine growth restriction (IUGR) occurs in about 5% of pregnancies and may reduce the incidence of periventricular leukomalacia in preterm newborns. We evaluated neonatal excitotoxicity in a murine model of vascular IUGR involving unilateral uterine ligation on embryonic day (E)13.5. Birth weight was significantly decreased in the ligation group compared with the sham group (p < 0.001). VEGFs, VEGF receptors (VEGFRs), and NMDA receptor subunit mRNAs in brain extracts were assayed using quantitative RT-PCR. Ligation was associated with increased mRNAs for the vascular marker PECAM-1 on postnatal day (PD)2 and VEGFR-3 on PD2 and PD10, contrasting with decreased VEGFA and VEGFC on PD10. Microvessel density was increased on PD7. Ligated and sham pups received intracerebral ibotenate (NMDA agonist) on PD2 or PD10. Cortical and white matter (WM) lesions after 5 d were reduced in ligated versus sham pups injected on PD2 (p < 0.001 and p < 0.01, respectively); this effect persisted on PD42 (p < 0.01 and p < 0.05, respectively). With ibotenate on PD10, lesions were exacerbated after 5 d in the ligated group in the cortex (p < 0.05) and WM (p < 0.05) and on PD42 in the cortex (p < 0.05). In conclusion, vascular IUGR offered only transient protection against neonatal excitotoxic lesions, possibly via angiogenesis.

Objective: To expose the current knowledge about the anaesthetic effects on the developing brain., Data Sources: Publications (original articles and reviews) in English and in French language from 1980 were obtained from the Medline database using alone or in combination following keywords: anaesthetics, developing brain, neurodevelopment, neurogenesis, synaptogenesis, neurotoxicity, apoptosis., Data Synthesis: Several lines of evidence resulting from animal experiments conducted in rodents and non-human primates have suggested that exposing the developing brain to anaesthetic drugs may elicit an increase a physiological programmed neuronal death (i.e. apoptosis). This neuronal death is not only seen at the cellular level but also results in alterations in some behavioural abilities in the adult animal. However, the vast majority of experiments reported have been conducted in animals not exposed to any surgical or painful stimulation. Moreover, the literature raises contradictory results, some authors not confirming this neurotoxic effect of anaesthetic drugs. Last, available clinical data are scarce and do not allow to claim that exposure to general anaesthesia definitely alters the cognitive development of children., Conclusion: This review raises the question of the innocuity of anaesthetic agents on the developing brain; further clinical trials are required in order to test this effect on human babies., (2010 Elsevier Masson SAS. All rights reserved.)

Few data are available on the involvement of brain microvascular endothelial cells (BMECs) in excitotoxic neonatal brain lesions. Therefore, we developed an original approach for investigating mouse-derived BMECs in vitro. We hypothesized that newborn and adult BMEC cultures would show age-related differences in phenotype and sensitivity to glutamate. Expression of the monocarboxylate transporter, MCT1, was higher in neonatal than in adult BMECs, whereas expression of the glucose transporter, GLUT1, was higher in adult than in neonatal BMECs that overexpressed the N-methyl-D-aspartate receptor NR1 subunit (NMDAR1) compared with adult BMECs. The ability of neonatal and adult BMECs to be activated by glutamate was confirmed through intracellular calcium ([Ca2+]i) recording. The glutamate-induced [Ca2+]i increase was blocked by the selective NMDAR antagonist, MK-801. Significant glutamate-evoked concentration-dependent release of tissue-type plasminogen activator (t-PA) and matrix metalloproteinases (MMPs) activities was found in supernatants of neonatal, but not in adult BMECs. The glutamate-mediated release of t-PA, MMP-2, and MMP-9 proteolytic activities in neonatal BMECs was blocked by MK-801. Conceivably, this protease release from neonatal BMECs may participate in neonatal brain lesions.

The balance between tissue-type plasminogen activator (tPA) and one of its inhibitors, neuroserpin, has crucial roles in the central nervous system, including the control of neuronal migration, neuronal plasticity and neuronal death. In the present study, we demonstrate that the activation of the transforming growth factor-beta (TGFbeta)-related BMPR-IB (also known as BMPR1B and Alk6)- and Smad5-dependent signalling pathways controls neuroserpin transcription. Accordingly, we demonstrate for the first time that anti-Mullerian hormone (AMH), a member of the TGFbeta family, promotes the expression of neuroserpin in cultured neurons but not in astrocytes. The relevance of these findings is confirmed by the presence of both AMH and AMH type-II receptor (AMHR-II) in brain tissues, and is supported by the observation of reduced levels of neuroserpin in the brain of AMHR-II-deficient mice. Interestingly, as previously demonstrated for neuroserpin, AMH protects neurons against N-methyl-D-aspartate (NMDA)-mediated excitotoxicity both in vitro and in vivo. This study demonstrates the existence of an AMH-dependent signalling pathway in the brain leading to an overexpression of the serine-protease inhibitor, neuroserpin, and neuronal survival.

Alcohol is one of the most common noxious substance to which fetuses are exposed. The aim of the study was to determine the effects of in utero alcohol exposure on excitotoxin-induced neuronal migration disorders. Female hamsters received alcohol (7%) for 3-5 mo or for the last 9-12 d of gestation. Alcohol diet was continued for 5 d during lactation in both groups. Drinking behavior was monitored. Peak plasma alcohol levels were 104+/-12 mg/dL and 225+/-6 mg/dL after 30 min for hamsters receiving an intragastric dose of 3 mL or 5 mL alcohol, respectively. At birth, pups received intrapallial injections ibotenic acid (1 ng, 100 ng, or 10 microg). Histology and N-methyl-D-aspartic acid (NMDA) receptor labeling by 3H-MK-801 in the pups cortices were studied. Short-term-alcohol-exposed pups had normal body and brain weights at birth, but their body growth was retarded postnatally. Ibotenic acid induced similar neuronal migration impairments in control and alcohol-exposed pups (nodular heterotopia in the white matter and/or deep cortical layers, subpial ectopia, and micro- or polymicrogyria). The size of lesions induced by 100 ng ibotenic acid was increased in alcohol-exposed pups; the 10 microg dose was lethal. The density of 3H-MK-801 binding sites was similar in the three groups, indicating that exacerbated ibotenic acid excitotoxicity in alcohol-exposed pups did not result from increased NMDA receptor density. This study shows that alcohol exposure at levels that do not induce neuron migration disorders is sufficient to enhance the effects of the hypoxia-ischemia mimicking effects of ibotenic acid.

Maternal smoking is associated with an increased risk of intra-uterine growth retardation and conduct disorder in their children, and is a major risk factor for sudden infant death syndrome. Among the numerous compounds present in cigarette smoke, carbon monoxide and nicotine have been largely studied. They can both affect the fetal brain by inducing intra-uterine hypoxia or by acting directly on the developing brain.

Background: A growing number of children have severe neurologic impairment related to very premature birth. Experimental data suggest that overstimulation of cerebral N-methyl-d-aspartate (NMDA) receptors caused by excessive glutamate release may be involved in the genesis of perinatal hypoxic-ischemic brain injury. alpha(2)-Adrenoceptor agonists are protective in models of brain ischemia in adults. The authors sought to determine whether they prevent perinatal excitotoxic neuronal damage., Methods: Five-day-old mice were allocated at random to clonidine (4-400 microg/kg), dexmedetomidine (1-30 microg/kg), or saline injected intraperitoneally before an intracerebral stereotactic injection of the NMDA receptor agonist ibotenate; cortical and white matter lesions were quantified 5 days later by histopathologic examination. Cortical neuron cultures exposed to 300 microm NMDA were used to evaluate the effects of clonidine or dexmedetomidine on neuronal death assessed by counting the number of pycnotic nuclei after fluorescent chromatin staining., Results: In vivo, both clonidine and dexmedetomidine induced significant concentration-dependent reductions in the size of ibotenate-induced lesions in the cortex and white matter. In vitro, the number of neurons damaged by NMDA exposure was significantly decreased by both dexmedetomidine (-28 +/- 12% at 10 microm; P < 0.01) and clonidine (-37 +/- 19% at 100 microm; P < 0.01) as compared with controls. In both models, the selective alpha2-adrenoceptor antagonist yohimbine abolished the neuroprotective effect of clonidine and dexmedetomidine., Conclusions: Clonidine and dexmedetomidine are potent neuroprotectors that act by stimulating the alpha(2) adrenoceptors. These results obtained in a murine model of perinatal excitotoxic injury may be relevant to some forms of neonatal brain damage in humans.

Prenatal betamethasone (Celestene) therapy reduces the incidence of brain damage, whereas prenatal or neonatal dexamethasone (Soludecadron) increases the risk of brain lesions or neuromotor deficits. To determine whether this increase is ascribable to the sulfites used as preservatives in Soludecadron, we investigated the effects of 12 h of exposure to pure dexamethasone, Soludecadron, pure betamethasone, Celestene, and sulfites on in vitro and in vivo death of neurons cultured under basal conditions or with excitotoxic agents (N-methyl-D-aspartate or (S)-5-bromowillardiine) or hypoxia. Apoptotic features were quantitated using a fluorescent chromatin stain (Hoechst 33258). Neuronal viability was unaffected by pure dexamethasone, pure betamethasone, or Celestene. Soludecadron or sulfites significantly increased neuronal loss. Pure dexamethasone or pure betamethasone produced a 40-50% decrease in neuronal death induced by N-methyl-D-aspartate, (S)-5-bromowillardiine, or hypoxia, whereas Soludecadron had no effect and sulfites significantly increased the neurotoxicity of excitotoxic agents. In in vivo experiments involving terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling after several i.p. injections of fluorinated glucocorticoids, Soludecadron, but not pure dexamethasone, significantly increased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-stained cells in neocortical layers and thalamus. These experimental findings suggest that injectable dexamethasone should be used with caution during the perinatal period.

Intracerebral administration of the excitotoxin ibotenate to newborn mice induces white-matter lesions, mimicking brain lesions that occur in human preterm infants. Nociceptin (NC), also called orphanin FQ, is the endogenous ligand of the opioid receptor-like 1 (ORL1) receptor and does not bind classical high-affinity opioid receptors. In the present study, administration of NC exacerbated ibotenate-induced white-matter lesions while coadministration of ibotenate with either of two NC antagonists reduced excitotoxic white-matter lesions by up to 64%. Neither ibotenate plus endomorphin I (a selective mu receptor agonist), nor ibotenate plus naloxone (a classical opioid receptor antagonist) modulated the excitotoxic lesion. Pretreatment with antisense oligonucleotides targeting the NC precursor peptide mRNA significantly reduced ibotenate-induced white-matter damage. Finally, high doses of fentanyl, which stimulates both classical mu-opioid receptors and ORL1, exacerbated excitotoxic white-matter lesion. This toxic effect was blocked by inhibiting ORL1 but not classical opioid receptors. Together, these findings show that endogenous or exogenous stimulation of the ORL1 receptor can be neurotoxic and that blocking NC signaling protects the white matter against excitotoxic challenge. These data point to potential new avenues for neuroprotection in human preterm infants at high risk of brain lesions.