The antiapoptotic effect of remifentanil on the immature mouse brain: an ex vivo study.

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.

High t-PA release by neonate brain microvascular endothelial cells under glutamate exposure affects neuronal fate.

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.

Prenatal alcohol exposure affects vasculature development in the neonatal brain.

OBJECTIVE: In humans, antenatal alcohol exposure elicits various developmental disorders, in particular in the brain. Numerous studies focus on the deleterious effects of alcohol on neural cells. Although recent studies suggest that alcohol can affect angiogenesis in adults, the impact of prenatal alcohol exposure on brain microvasculature remains poorly understood. METHODS: We used a mouse model to investigate effects of prenatal alcohol exposure on the cortical microvascular network in vivo and ex vivo and the action of alcohol, glutamate, and vascular endothelial growth factor A (VEGF) on activity, plasticity, and survival of microvessels. We used quantitative reverse transcriptase polymerase chain reaction, Western blot, immunohistochemistry, calcimetry, and videomicroscopy. We characterized the effect of prenatal alcohol exposure on the cortical microvascular network in human controls and fetal alcohol syndrome (FAS)/partial FAS (pFAS) patients at different developmental stages. RESULTS: In mice, prenatal alcohol exposure induced a reduction of cortical vascular density, loss of the radial orientation of microvessels, and altered expression of VEGF receptors. Time-lapse experiments performed on brain slices revealed that ethanol inhibited glutamate-induced calcium mobilization in endothelial cells, affected plasticity, and promoted death of microvessels. These effects were prevented by VEGF. In humans, we evidenced a stage-dependent alteration of the vascular network in the cortices of fetuses with pFAS/FAS. Whereas no modification was observed from gestational week 20 (WG20) to WG22, the radial organization of cortical microvessels was clearly altered in pFAS/FAS patients from WG30 to WG38. INTERPRETATION: Prenatal alcohol exposure affects cortical angiogenesis both in mice and in pFAS/FAS patients, suggesting that vascular defects contribute to alcohol-induced brain abnormalities.

NO-dependent protective effect of VEGF against excitotoxicity on layer VI of the developing cerebral cortex.

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).

Influence of gestational age on fibrinolysis from birth to postnatal day 10.

OBJECTIVE: To compare components of the fibrinolytic cascade in newborns of gestational age ranging from extreme prematurity to full term, at birth and for the next 10 days, and in their mothers at delivery. STUDY DESIGN: We studied 10 extremely preterm neonates, 10 very preterm neonates, 10 moderately preterm neonates, 10 full-term neonates, and their mothers (n = 40). We measured the antigen levels of tissue-type plasminogen activator (t-PA), plasminogen activator inhibitors 1 (PAI-1) and 2 (PAI-2), and thrombin-activatable fibrinolysis inhibitor, as well as PAI-1 activity, in neonates at birth and on postnatal days 3 and 10 and in mothers at delivery. RESULTS: On day 10, both PAI-1 antigen and activity were higher in extremely preterm neonates than in full-term neonates (P = .004 and <.0006, respectively), and the t-PA/PAI-1 activity ratio was lower in the extremely preterm and very preterm neonates compared with the full-term neonates (P = .002 and .017, respectively). No significant differences in the fibrinolytic system components were seen among the 4 groups of mothers. CONCLUSIONS: The development of fibrinolysis suppression in extremely preterm infants within 10 days after birth may contribute to the increased risk of periventricular hemorrhagic infarction in these infants.

Effects of antenatal uteroplacental hypoperfusion on neonatal microvascularisation and excitotoxin sensitivity in mice.

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.

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.

Conditions requiring hospitalisations, more than general anaesthesia itself, are associated with diagnosis of learning disorders in children.

BACKGROUND: Anaesthesia is neurotoxic in developing primates. Retrospective clinical studies show a correlation between exposure to anaesthesia during infancy and the occurrence of learning disorders (LD). Prospective studies failed to detect any influence of a single exposure to anaesthesia on neurodevelopment. We hypothesised that some specific populations of children were electively sensitive to anaesthesia-related neurotoxicity. METHODS: Using a case-control design, we analysed the medical histories of children with LD, compared to those of their normally reading siblings. Interviews were conducted and medical records were reviewed. The numbers of hospitalisations and anaesthesia exposures before the age of five years were determined. RESULTS: Four hundred fourteen dyslexic children were screened over a one-year period. Two hundred and seventy patients were excluded due to confounding variables (single child, all siblings showing LD or any condition placing the neurological prognosis at risk (N = 107/414 for the latter)) or inability to accurately collect evaluation criteria. In the 144 case-control pairs studied, the mean number of hospitalisations was significantly different (N = 1.097 ± 0 .135/case versus 0.667 ± 0.097/control, p = 0.0052), as was the proportion of hospitalised patients (54.2% versus 38.9%, p = 0.0031). The mean number of anaesthesia exposures per individual was not statistically different (N = 0.958 ± 0.183/case versus 0.569 ± 0.107/control, p = 0.0732), but the proportion of children anaesthetised at least once was (43.8% (cases) versus 33.3% (controls), p = 0.0301). DISCUSSION: One or more hospitalisation(s) may reflect a health status and/or have an iatrogenic effect disrupting the normal setting up of learning abilities. Anaesthesia may play a role, but a correlation between LD and anaesthesia is of a lower magnitude than between LD and hospitalisation.

PACAP prevents toxicity induced by cisplatin in rat and primate neurons but not in proliferating ovary cells: involvement of the mitochondrial apoptotic pathway.

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.

Detection of Neisseria meningitidis DNA from skin lesion biopsy using real-time PCR: usefulness in the aetiological diagnosis of purpura fulminans.

OBJECTIVE: The present study evaluated the usefulness of a real-time polymerase chain reaction (rtPCR) assay for the detection of Neisseria meningitidis (Nm) and genogrouping on skin lesion biopsies in patients with purpura fulminans (PF). DESIGN: Retrospective single-centre study. SETTING: Adult and paediatric intensive care units at the University Hospital of Rouen. PATIENTS: All patients admitted between January 2000 and January 2006, with a final diagnosis of PF and for which a skin biopsy and blood cultures were performed, were included. INTERVENTIONS: Skin biopsy and blood cultures were used for culture and rtPCR. MEASUREMENTS AND MAIN RESULTS: Thirty-four patients fulfilled the criteria (27 children and 7 adults). Nm rtPCR performed on skin biopsy was positive in 100% (34/34) of cases, compared with only 14.7% (5/34) for skin culture (p=0.0001). rtPCR genogrouping on skin biopsy was positive in 58.8% (20/34) of the cases compared with 14.7% (5/34) for skin culture (p=0.0013). For patients (n=17) in whom rtPCR was performed both on blood and skin biopsy, skin biopsy gave a significantly higher rate of Nm detection [100% (17/17) vs. 58.8% (10/17); p=0.023] and genogroup characterisation [76.5% (13/17) vs. 35.3% (6/17); p=0.045] than blood. We encountered no specimen with culture-positive and rtPCR-negative results (negative predictive value of rtPCR 100%). CONCLUSION: In suspected PF cases, skin biopsy is more reliable to identify Nm and its genogroup than blood or, probably, CSF, especially when PCR methods are used. This could help the implementation of public health interventions, especially concerning a vaccination policy.

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 N-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, alpha4beta2 (heteropentameric) and alpha7 (homopentameric). Inhibition of alpha4beta2, either pharmacological (i.e., an alpha4beta2 nAChR antagonist) or molecular (beta2-/- knockout mice), abolished the protective effect of nicotine in vivo and in vitro, suggesting the involvement of alpha4beta2 nAChR in neonatal nicotine-related neuroprotection. In contrast, activation of alpha7 nAChR, which is protective in adult animals, was deleterious in our neonatal model, whereas its blockade, either pharmacological or molecular (alpha7-/- knockout mice) provided neuroprotection. Neuroprotective strategies must consider these opposite properties of distinct nAChR isoforms in neonates.

Effect of acamprosate on neonatal excitotoxic cortical lesions in in utero alcohol-exposed hamsters.

Alcohol exposition during pregnancy has irreversible effects on the fetus brain. In hamsters, intrapallial injection of the glutamate receptor agonist ibotenic acid (100ng) on the day of birth (P0) induced neuronal migration disorders. In utero alcohol (7%) exposure from day 5 of gestation to P5, enhanced lesions size measured in pups' brain at P5. The administration for the same period of the taurine derivative acamprosate together with alcohol or in water to pregnant females reduced the rate of occurrence of nodular heterotopia, sub-pial ectopia and microgyria in non-alcohol-exposed pups. In addition acamprosate diminished lesion size in alcohol-exposed and non-exposed pups. A significant dose-related effect of acamprosate was observed. In addition, acamprosate rescued 27% of the pups injected with 10 microg ibotenic acid, a lethal dose in alcohol-exposed animals.

A simple method for short-term controlled anesthesia in newborn mice.

In this study, we describe a simple and inexpensive method for inducing short-term anesthesia and rapid recovery in newborn mice. Litters of Swiss mice pups were randomly allocated to testing on postnatal days 2, 5, and 8. Anesthesia was induced by placing the pup in a syringe and adding a volume of isoflurane-saturated gas that produced an estimated level of 32% isoflurane. Exposure to isoflurane lasted 30 s. All the pups survived the anesthesia. At all study ages, this method abolished the nociceptive response to tail clamp without inducing mortality, thus showing effective anesthesia. Recovery from anesthesia was assessed immediately after isoflurane exposure, based on two nonnoxious behavioral tests: the defensive response to a drop of water (10 tests, 1 min apart) and 10 min later the righting reflex, i.e., the time to recovery of the prone position (five tests, 10 min apart). The water drop test scores increased during the recovery phase toward the control values in all age groups. Treatment and time had no significant effect on righting reflex scores. The initial volume in the syringe, the volume of added isoflurane-saturated gas, and the duration of exposure may be adjusted according to postnatal age and specific strains or species (e.g., rats). This method is well suited to behavioral or physiological phenotype studies in developing mice, in which noxious procedures must precede functional testing, making rapid recovery from anesthesia a key requirement.

Vascular endothelial growth factor and its high-affinity receptor (VEGFR-2) are highly expressed in the human forebrain and cerebellum during development.

Vascular endothelial growth factor (VEGF) is an angiogenic and neurotrophic factor in both adult and neonatal animals, but its expression and role have been incompletely studied in the developing human brain. We analyzed the distribution of VEGF and its high-affinity receptor VEGFR-2 in the human forebrain and cerebellum at developmental stages from 14 weeks' gestation (WG) to the13th postnatal month. Tissue samples free of detectable neuropathologic abnormalities were assessed by immunohistochemistry and confocal microscopy using anti-human VEGF and VEGFR-2 antibodies. The VEGFR-2 was first expressed in the whole cerebral mantle and in migrating cells in the intermediate zone, whereas VEGFwas found in superficial layers of the cortical plate, in radial glia, and in the cerebellar external germinal cell layer. From 23 WG, temporospatial VEGFR-2 expression was superimposable on that ofVEGF in the cortical plate, intermediate zone, basal ganglia, limbicstructures, and external germinal cell layer. The VEGF/VEGFR-2-positive astrocytes were observed during their generation and migration from 23 WG to the first postnatal month. The VEGF-positive mature oligodendrocytes were observed in myelinating structures in the forebrain from birth and in the cerebellum from 24WG. These data suggest that VEGF and VEGFR-2 are likely involved in several aspects of human brain development.

Newborn- and adult-derived brain microvascular endothelial cells show age-related differences in phenotype and glutamate-evoked protease release.

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.

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-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.

Neonatal hypoxic preconditioning involves vascular endothelial growth factor.

We studied hypoxic preconditioning (HxP) in the murine developing brain, focusing on the role for vascular endothelial growth factor (VEGF). Newborn mice were used as follows: (1) HxP (or normoxia) then intracerebral (i.c.) NMDA or AMPA-kainate agonist; (2) HxP then intraperitoneal (i.p.) anti-VEGFR2/Flk1 or anti-VEGFR1/Flt1 monoclonal blocking antibody (mAb) then i.c. NMDA/AMPA-kainate agonist; (3) i.p. VEGF then i.c. NMDA/AMPA-kainate agonist; and (4) in mutants lacking the hypoxia-responsive element (HRE) of the VEGF-A gene (VEGF( partial differential/ partial differential)) and their wild-type littermates (VEGF(+/+)), HxP followed by i.c. NMDA agonist. HxP reduced the size of NMDA-related cortical and AMPA-kainate-related cortical and white matter excitotoxic lesions. Anti-VEGFR2/Flk1 mAb prevented HxP-induced neuroprotection. VEGF produced dose-dependent reduction in cortical lesions. HxP did not prevent, but instead exacerbated, brain lesions in VEGF( partial differential/ partial differential) mutants. Thus, exogenous as well as endogenous VEGF reduces excitotoxic brain lesions in the developing mouse. The VEGF/VEGFR2/Flk1 pathway is involved in the neuroprotective response to HxP.

Effect of perinatal alcohol exposure on ibotenic acid-induced excitotoxic cortical lesions in newborn hamsters.

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.