Vincent Laudenbach

Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration

Hypoxia stimulates angiogenesis through the binding of hypoxia-inducible factors to the hypoxia-response element in the vascular endothelial growth factor (Vegf) promotor. Here, we report that deletion of the hypoxia-response element in the Vegf promotor reduced hypoxic Vegf expression in the spinal cord and caused adult-onset progressive motor neuron degeneration, reminiscent of amyotrophic lateral sclerosis. The neurodegeneration seemed to be due to reduced neural vascular perfusion. In addition, Vegf165 promoted survival of motor neurons during hypoxia through binding to Vegf receptor 2 and neuropilin 1. Acute ischemia is known to cause nonselective neuronal death. Our results indicate that chronic vascular insufficiency and, possibly, insufficient Vegf-dependent neuroprotection lead to the select degeneration of motor neurons.

Effects of alpha(2)-adrenoceptor agonists on perinatal excitotoxic brain injury: comparison of clonidine and dexmedetomidine.

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. &agr;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 &mgr;g/kg), dexmedetomidine (1–30 &mgr;g/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 &mgr;m 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 &mgr;m;P < 0.01) and clonidine (−37 ± 19% at 100 &mgr;m;P < 0.01) as compared with controls. In both models, the selective &agr;2-adrenoceptor antagonist yohimbine abolished the neuroprotective effect of clonidine and dexmedetomidine. Conclusions Clonidine and dexmedetomidine are potent neuroprotectors that act by stimulating the &agr;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.

Selective activation of central subtypes of the nicotinic acetylcholine receptor has opposite effects on neonatal excitotoxic brain injuries

The incidence of neurological disabilities ascribable to perinatal injury is rising in Western countries, raising ethical and financial problems. No curative treatments are available. The pathophysiology of brain lesions of hypoxic‐ischemic or inflammatory origin involves various neurotransmitters or neuromodulators. Among these, glutamate plays a key role. By overactivating V‐methyl‐d‐aspartate receptors, it triggers the excitotoxic cascade. Although addictive, nicotine prevents excitotoxic neuronal death in adult animals. Its potential neuroprotective effects have not been evaluated in neonates. We found that nicotine is neuroprotective in vivo, in a murine model of neonatal excitotoxic brain injury, and in vitro, in primary cultures of cortical neurons. We investigated the respective roles in nicotine‐related neuroprotection of the two dominant nicotinic acetylcholine receptor (nAChR) isoforms, namely, α4β2 (heteropentameric) and α7 (homopentameric). Inhibition of α4β2, either pharmacological (i.e., an α4β2 nAChR antagonist) or molecular (β2‐/‐ knockout mice), abolished the protective effect of nicotine in vivo and in vitro, suggesting the involvement of α4β2 nAChR in neonatal nicotine‐related neuroprotection. In contrast, activation of α7 nAChR, which is protective in adult animals, was deleterious in our neonatal model, whereas its blockade, either pharmacological or molecular (α7‐/‐ knockout mice) provided neuroprotection. Neuroprotective strategies must consider these opposite properties of distinct nAChR isoforms in neonates.

Neurotoxic Effects of Fluorinated Glucocorticoid Preparations on the Developing Mouse Brain: Role of Preservatives

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.

Nociceptin/orphanin FQ exacerbates excitotoxic white-matter lesions in the murine neonatal brain

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.

Anesthetics Affect the Uptake but Not the Depolarization-evoked Release of GABA in Rat Striatal Synaptosomes

Background Numerous classes of anesthetic agents have been shown to enhance the effects mediated by the postsynaptic gamma‐aminobutyric acid A (GABAA) receptor‐coupled chloride channel in the mammalian central nervous system. However, presynaptic actions of anesthetics potentially relevant to clinical anesthesia remain to be clarified. Therefore, in this study, the effects of intravenous and volatile anesthetics on both the uptake and the depolarization‐evoked release of GABA in the rat stratum were investigated. Methods Assay for specific GABA uptake was performed by measuring the radioactivity incorporated in purified striatal synaptosomes incubated with3 H‐GABA (20 nM, 5 min, 37 degrees Celsius) and increasing concentrations of anesthetics in either the presence or the absence of nipecotic acid (1 mM, a specific GABA uptake inhibitor). Assay for GABA release consisted of superfusing3 H‐GABA preloaded synaptosomes with artificial cerebrospinal fluid (0.5 ml *symbol* min sup 1, 37 degrees Celsius) and measuring the radioactivity obtained from 0.5 ml fractions over 18 min, first in the absence of any treatment (spontaneous release, 8 min), then in the presence of either KCl alone (9 mM, 15 mM) or with various concentrations of anesthetics (5 min), and finally, with no pharmacologic stimulation (5 min). The following anesthetic agents were tested: propofol, etomidate, thiopental, ketamine, halothane, enflurane, isoflurane, and clonidine. Results More than 95% of3 H‐GABA uptake was blocked by a 10 sup 3 ‐M concentration of nipecotic acid. Propofol, etomidate, thiopental, and ketamine induced a dose‐related, reversible, noncompetitive, inhibition of3 H‐GABA uptake: IC50 = 4.6 plus/minus 0.3 x 105 M, 5.8 plus/minus 0.3 x 10 sup ‐5 M, 2.1 plus/minus 0.4 x 10 sup ‐3 M, and 4.9 plus/minus 0.5 x 10 sup ‐4 M for propofol, etomidate, thiopental, and ketamine, respectively. Volatile agents and clonidine had no significant effect, even when used at concentrations greater than those used clinically. KCl application induced a significant, calcium‐dependent, concentration‐related, increase from basal3 H‐GABA release, +34 + 10% (P < 0.01) and +61 plus/minus 13% (P < 0.001), respectively, for 9 mM and 15 mM KCl. The release of3 H‐GABA elicited by KCl was not affected by any of the anesthetic agents tested. Conclusions These results indicate that most of the intravenous but not the volatile anesthetics inhibit the specific high‐affinity3 H‐GABA uptake process in vitro in striatal nerve terminals. However, this action was observed at clinically relevant concentrations only for propofol and etomidate. In contrast, the depolarization‐evoked3 H‐GABA release was not affected by anesthetics. Together, these data suggest that inhibition of GABA uptake, which results in synaptic GABA accumulation, might contribute to propofol and etomidate anesthesia.

Riluzole, a novel antiglutamate, blocks GABA uptake by striatal synaptosomes

The effect of riluzole (2-amino-6-trifluoro-methoxybenzothiazole, a novel antiglutamate agent) on the uptake of [3H]GABA (gamma-aminobutyric acid) by striatal synaptosomes was investigated in rats. Both nipecotic acid (a classical blocker of GABA uptake) and riluzole were found to inhibit [3H]GABA uptake in a dose-related fashion (IC50 = 3.6 x 10(-6) M and 4.3 x 10(-5) M for nipecotic acid and riluzole, respectively). These results indicate that, in addition to its antiglutamate properties, riluzole probably also promotes synaptic GABA accumulation, which might contribute to the anticonvulsant and/or anesthetic properties of this pharmacological agent.

Anti-Mullerian-hormone-dependent regulation of the brain serine-protease inhibitor neuroserpin

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-β (TGFβ)-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 TGFβ 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.

Dual Effect of Glutamate on GABAergic Interneuron Survival during Cerebral Cortex Development in Mice Neonates

In term and preterm neonates, massive glutamate release can lead to excitotoxic white-matter and cortical lesions. Because of its high permeability toward calcium, the N-methyl-D-aspartic acid (NMDA) receptor is thought to play an important role in excitotoxic lesions and NMDA antagonists therefore hold promise for neuroprotection. We found that, in neonatal mouse cortex, a given NMDA concentration exerted either excitotoxic or antiapoptotic effects depending on the cortical layers. In layer VI, NMDA led to excitotoxicity, sustained calcium mobilization, and necrosis of Gad67GFP neurons. In the immature layers II-IV, NMDA decreased apoptosis and induced transient calcium mobilization. The NMDA antagonist MK801 acted as a potent caspase-3 activator in immature layers II-IV and affected gamma aminobutyric acid (GABA)ergic interneurons. The apoptotic effect of MK801-induced BAX expression, mitochondrial potential collapse and caspase-9 activation. In vivo Bax small interfering ribonucleic acid and a caspase-9 inhibitor abrogated MK801-induced apoptosis and pyknotic nucleus formation. Ketamine, an anesthetic with NMDA antagonist properties, mimicked the apoptotic effects of MK801. These data indicate a dual effect of glutamate on survival of immature and mature GABAergic neurons and suggest that ketamine may induce apoptosis of immature GABAergic neurons.

Open vs thorascopic surgical management of bronchogenic cysts

BackgroundThe aim of this study was to compare the operative outcome in children undergoing open vs thoracoscopic resection of bronchogenic cysts.MethodsThe medical records of children who underwent the resection of bronchogenic cysts from 1990 through 2000 were reviewed. Four cyst resections were performed by the open technique and five using a thoracoscopic procedure. The age of the patients, length of hospital stay, duration of drainage, operating time, and outcome were investigated.ResultsThe mean age of patients undergoing the open procedure was 3 years and 3 months; the mean age for thoracoscopy patients was 7 years and 10 months (p < 0.05). The operating time for the open procedure was 70 ± 25 min; for the laparoscopic procedure, it was 78 ± 6 min (p, NS), except in one case with a main bronchial tail that required conversion (320 min). Duration of surgical drainage was 6.5 ± 3 days for the open procedure and 2.5 ± 1 days for the thoracoscopic one (p < 0.05). Hospital stay for open patients was 12 days ± 0 days; it was 6 ± 1.6 days for thoracoscopic patients (p < 0.01). There were no deaths. The thoracoscopic procedure failed once due to a main bronchial tail and had to be converted to an open procedure. Other early complications included a bronchopulmonary infection after an open cyst excision and an atelectasis after a thoracoscopic cyst excision. Late complications included one reoperation for incomplete excision in each of the two groups.ConclusionBronchogenic cyst resection can be performed safely. For complete treatment of these patients, total excision of the wall cyst is needed. In selected patients, the thoracoscopic procedure may decrease the duration of surgical drainage and length of hospital stay without increasing the operating time or MSK for complications.