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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.)

In industrialized countries, cerebral palsy affects 2.5‰ of preterm and term infants. At a neurochemical level, the massive release of glutamate constitutes a major process leading to excitotoxicity and neonatal brain lesions. Previous studies, conducted in the laboratory, revealed that, in (δ/δ)VEGF(A) transgenic mice, glutamate-induced brain lesions are exacerbated suggesting that VEGF(A) could play a protective action against excitotoxicity. Using a model of cultured cortical brain slices, the aim of the study was to characterize the central effects of VEGF against glutamate-induced excitotoxicity in neonates. Exposure of brain slices to glutamate induced a strong increase of necrotic cell death in the deep cortical layer VI and a decrease of apoptotic death in superficial layers II-IV. When administered alone, a 6-h treatment with VEGF(A) had no effect on both apoptotic and necrotic deaths. In contrast, VEGF(A) abolished the glutamate-induced necrosis observed in layer VI. While MEK and PI3-K inhibitors had no effect on the protective action of VEGF(A), L-NAME, a pan inhibitor of NOS, abrogated the effect of VEGF(A) and exacerbated the excitotoxic action of glutamate. Calcimetry experiments performed on brain slices revealed that VEGF(A) reduced the massive calcium influx induced by glutamate in layer VI and this effect was blocked by L-NAME. Neuroprotective effect of VEGF(A) was also blocked by LNIO and NPLA, two inhibitors of constitutive NOS, while AGH, an iNOS inhibitor, had no effect. Nitrite measurements, electron paramagnetic resonance spectroscopy and immunohistochemistry indicated that glutamate was a potent inducer of NO production via activation of nNOS in the cortical layer VI. In vivo administration of nNOS siRNA promoted excitotoxicity and mimicked the effects of L-NAME, LNIO and NPLA. A short-term glutamate treatment increased nNOS Ser1412 phosphorylation, while a long-term exposure inhibited nNOS/NR2B protein-protein interactions. Altogether, these findings indicate that, in deep cortical layers of mice neonates, glutamate stimulates nNOS activity. Contrasting with mature brain, NO production induced by high concentrations of glutamate is neuroprotective and is required for the anti-necrotic effect of VEGF(A)., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that belongs to the secretin/glucagon/vasoactive intestinal polypeptide superfamily. The PACAPergic system is actively expressed in the developing cerebellum of mammals. In particular, PACAP receptors are expressed by granule cell precursors suggesting a role of the peptide in neurogenesis of this cell type. Consistent with this hypothesis, several studies reported antiapoptotic effects of PACAP in the developing cerebellum. On the other hand, the sphingomyelin metabolites ceramides are recognized as important signaling molecules that play pivotal roles during neuronal development. Ceramides, which production can be induced by death factors such as FasL or TNFalpha, are involved in the control of cell survival during brain development through activation of caspase-dependent mechanisms. The present review focuses on the interactions between PACAP and ceramides in the control of granule cell survival and on the transduction mechanisms associated with the anti- and proapoptotic effects of PACAP and ceramides, respectively.

Cisplatin is a chemotherapeutic agent whose use is limited by side effects including neuropathies. In proliferating cells, toxic action of cisplatin is based on DNA interactions, while, in quiescent cells, it can induce apoptosis by interacting with proteins. In the present study, we compared cytotoxic mechanisms activated by cisplatin in primate and rodent neurons and in ovary cells in order to determine whether the anti-apoptotic peptide PACAP could selectively reduce neurotoxicity. In quiescent neurons, JNK and sphingomyelinase inhibitors blocked cisplatin-induced cell death. Toxicity was associated with DNA laddering, caspase-3 and -9 activations and Bax induction. These effects were prevented by PACAP. In proliferating cells, cisplatin activated caspase-8 but had no effect on caspase-9. PACAP exerted no protective effect. These data indicate that cisplatin activates distinct apoptotic pathways in quiescent neurons and proliferating cells and that PACAP may reduce neurotoxicity of cisplatin without affecting its chemotherapeutic efficacy.

Transplantation of neuronal precursor cells (NPCs) into the central nervous system could represent a powerful therapeutical tool against neurodegenerative diseases. Unfortunately, numerous NPCs die shortly after transplantation, predominantly due to caspase-dependent apoptosis. Using a culture of cerebellar neuronal precursors, we have previously demonstrated protective effect of the neuropeptide PACAP, which suppresses ceramide-induced apoptosis by blockade of the mitochondrial apoptotic pathway. The main objective of this study was to determine whether Bax repression can promote survival of NPCs allotransplanted into a host animal. In vivo and ex vivo experiments revealed that C2-ceramide increases Bax expression, while PACAP reverses this effect. In vitro tests using cerebellar NPCs demonstrated that the Bax-specific small interfering RNA (siRNA) could reduce their death and caspase-3 cleavage within the first 24 h. BrdU-labelled NPCs were subjected to transfection procedure with or without siRNA introduction before using for in vivo transplantation. Twenty-four hours after, the allografted NPCs containing siRNA showed significantly reduced level of caspase-3 cleavage, and the volume of their implants was almost twofold higher than in the case of empty-transfected precursors. These data evidence an important role of Bax in life/death decision of grafted NPCs and suggest that RNA interference strategy may be applicable for maintaining NPCs survival within the critical first hours after their transplantation.Cell Death and Differentiation (2008) 15, 1042–1053; doi:10.1038/cdd.2008.29; published online 7 March 2008 [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

Abstract: Experiments performed in mammals, including non-human primates, have demonstrated an increase in neuronal death rates normally seen in normal brain development. Such an increase is encountered in diseases but also after exposure of the brain to various class of anaesthetics. In living animals, it can (but not always) result in persistent cognitive impairment. Most of the experiments have been conducted in animals which were never exposed to any pain, which questions their relevancy. On the clinical side, all data comes from retrospective studies. Given the multiple bias, they cannot definitely state that a protocol, if toxic, is more or less when compared to another. Until now, prospective follow-up of children exposed to anaesthetics in utero or during the first months of life do not suggest a major deleterious effect. Yet, a minor one, if existing, would be hard to detect among polluting variables (e.g. pathology requiring anaesthesia, long hospitalization after birth, preterm birth, environmental stress…). For sure, when surgery is mandatory during pregnancy, it is generally for maternal indication and should not be a motif strong enough for foetal extraction, especially in terms where the baby has few chances to survive. Second, it is known for years than anaesthesia before 1year of age is much riskier than after 1year, whatever the theorical neurotoxicity is. Third, this enforces the need to develop tools enhancing the precision of anaesthesia as much as possible. Meanwhile, when an infant has undergone numerous general anaesthesias, we strongly recommend a long-time neurological follow-up. [Copyright &y& Elsevier]