Prenatal Alcohol Exposure Impairs the Placenta-Cortex Transcriptomic Signature, Leading to Dysregulation of Angiogenic Pathways.

Although alcohol consumption during pregnancy is a major cause of behavioral and learning disabilities, most FASD infants are late- or even misdiagnosed due to clinician's difficulties achieving early detection of alcohol-induced neurodevelopmental impairments. Neuroplacentology has emerged as a new field of research focusing on the role of the placenta in fetal brain development. Several studies have reported that prenatal alcohol exposure (PAE) dysregulates a functional placenta-cortex axis, which is involved in the control of angiogenesis and leads to neurovascular-related defects. However, these studies were focused on PlGF, a pro-angiogenic factor. The aim of the present study is to provide the first transcriptomic "placenta-cortex" signature of the effects of PAE on fetal angiogenesis. Whole mouse genome microarrays of paired placentas and cortices were performed to establish the transcriptomic inter-organ "placenta-cortex" signature in control and PAE groups at gestational day 20. Genespring comparison of the control and PAE signatures revealed that 895 and 1501 genes were only detected in one of two placenta-cortex expression profiles, respectively. Gene ontology analysis indicated that 107 of these genes were associated with vascular development, and String protein-protein interaction analysis showed that they were associated with three functional clusters. PANTHER functional classification analysis indicated that "intercellular communication" was a significantly enriched biological process, and 27 genes were encoded for neuroactive ligand/receptors interactors. Protein validation experiments involving Western blot for one ligand-receptor couple (Agt/AGTR1/2) confirmed the transcriptomic data, and Pearson statistical analysis of paired placentas and fetal cortices revealed a negative correlation between placental Atg and cortical AGTR1, which was significantly impacted by PAE. In humans, a comparison of a 38WG control placenta with a 36WG alcohol-exposed placenta revealed low Agt immunolabeling in the syncytiotrophoblast layer of the alcohol case. In conclusion, this study establishes the first transcriptomic placenta-cortex signature of a developing mouse. The data show that PAE markedly unbalances this inter-organ signature; in particular, several ligands and/or receptors involved in the control of angiogenesis. These data support that PAE modifies the existing communication between the two organs and opens new research avenues regarding the impact of placental dysfunction on the neurovascular development of fetuses. Such a signature would present a clinical value for early diagnosis of brain defects in FASD.

Effect of Neuroprotective Magnesium Sulfate Treatment on Brain Transcription Response to Hypoxia Ischemia in Neonate Mice.

MgSO4 is widely used in the prevention of preterm neurological disabilities but its modes of action remain poorly established. We used a co-hybridization approach using the transcriptome in 5-day old mice treated with a single dose of MgSO4 (600 mg/kg), and/or exposed to hypoxia-ischemia (HI). The transcription of hundreds of genes was altered in all the groups. MgSO4 mainly produced repressions culminating 6 h after injection. Bio-statistical analysis revealed the repression of synaptogenesis and axonal development. The putative targets of MgSO4 were Mnk1 and Frm1. A behavioral study of adults did not detect lasting effects of neonatal MgSO4 and precluded NMDA-receptor-mediated side effects. The effects of MgSO4 plus HI exceeded the sum of the effects of separate treatments. MgSO4 prior to HI reduced inflammation and the innate immune response probably as a result of cytokine inhibition (Ccl2, Ifng, interleukins). Conversely, MgSO4 had little effect on HI-induced transcription by RNA-polymerase II. De novo MgSO4-HI affected mitochondrial function through the repression of genes of oxidative phosphorylation and many NAD-dehydrogenases. It also likely reduced protein translation by the repression of many ribosomal proteins, essentially located in synapses. All these effects appeared under the putative regulatory MgSO4 induction of the mTORC2 Rictor coding gene. Lasting effects through Sirt1 and Frm1 could account for this epigenetic footprint.

Hypoxia-Ischemia Induced Age-Dependent Gene Transcription Effects at Two Development Stages in the Neonate Mouse Brain.

Human brain lesions in the perinatal period result in life-long neuro-disabilities impairing sensory-motor, cognitive, and behavior functions for years. Topographical aspects of brain lesions depend on gestational age at the time of insult in preterm or term infants and impaired subsequent steps of brain development and maturation. In mice, the Rice-Vannucci procedure of neonate hypoxia-ischemia (HI) was used at 5 days (P5) or P10, mimicking the development of 30 week-gestation fetus/preterm newborn, or full-term infant, respectively. Transcription response to HI was assessed at 3, 6, 12, and 24 h after insult, using micro-array technology. Statistical Pathway and Gene Ontology terms enrichments were investigated using DAVID®, Revigo® and Ingenuity Pathway Analysis (IPA®) to identify a core of transcription response to HI, age-specific regulations, and interactions with spontaneous development. Investigations were based on direction, amplitude, and duration of responses, basal expression, and annotation. Five major points deserve attention; (i) inductions exceeded repressions (60/40%) at both ages, (ii) only 20.3% (393/1938 records) were common to P5 and P10 mice, (iii) at P5, HI effects occurred early and decreased 24 h after insult whereas they were delayed at P10 and increased 24 h after insult, (iv) common responses at P5 and P10 involved inflammation, immunity, apoptosis, and angiogenesis. (v) age-specific effects occurred with higher statistical significance at P5 than at P10. Transient repression of 12 genes encoding cholesterol biosynthesis enzymes was transiently observed 12 h after HI at P5. Synaptogenesis appeared inhibited at P5 while induced at P10, showing reciprocal effects on glutamate receptors. Specific involvement of Il-1 (interleukin-1) implicated in the firing of inflammation was observed at P10. This study pointed out age-differences in HI responses kinetics, e.g., a long-lasting inflammatory response at P10 compared to P5. Whether the specific strong depression of cholesterol biosynthesis genes that could account for white matter-specific vulnerability at P5 or prevent delayed inflammation needs further investigation. Determination of putative involvement of Il-1 and the identification of upstream regulators involved in the delayed inflammation firing at P10 appears promising routes of research in the understandings of age-dependent vulnerabilities in the neonatal brain.

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.

In utero alcohol exposure exacerbates endothelial protease activity from pial microvessels and impairs GABA interneuron positioning.

In utero alcohol exposure can induce severe neurodevelopmental disabilities leading to long-term behavioral deficits. Because alcohol induces brain defects, many studies have focused on nervous cells. However, recent reports have shown that alcohol markedly affects cortical angiogenesis in both animal models and infants with fetal alcohol spectrum disorder (FASD). In addition, the vascular system is known to contribute to controlling gamma-aminobutyric acid (GABA)ergic interneuron migration in the developing neocortex. Thus, alcohol-induced vascular dysfunction may contribute to the neurodevelopmental defects in FASD. The present study aimed at investigating the effects of alcohol on endothelial activity of pial microvessels. Ex vivo experiments on cortical slices from mouse neonates revealed that in endothelial cells from pial microvessels acute alcohol exposure inhibits both glutamate-induced calcium mobilization and activities of matrix metalloproteinase-9 (MMP-9) and tissue plasminogen activator (tPA). The inhibitory effect of alcohol on glutamate-induced MMP-9 activity was abrogated in tPA-knockout and Grin1flox/VeCadcre mice suggesting that alcohol interacts through the endothelial NMDAR/tPA/MMP-9 vascular pathway. Contrasting with the effects from acute alcohol exposure, in mouse neonates exposed to alcohol in utero during the last gestational week, glutamate exacerbated both calcium mobilization and endothelial protease activities from pial microvessels. This alcohol-induced vascular dysfunction was associated with strong overexpression of the N-methyl-d-aspartate receptor subunit GluN1 and mispositioning of the Gad67-GFP interneurons that normally populate the superficial cortical layers. By comparing several human control fetuses with a fetus chronically exposed to alcohol revealed that alcohol exposure led to mispositioning of the calretinin-positive interneurons, whose density was decreased in the superficial cortical layers II-III and increased in deepest layers. This study provides the first mechanistic and functional evidence that alcohol impairs glutamate-regulated activity of pial microvessels. Endothelial dysfunction is characterized by altered metalloproteinase activity and interneuron mispositioning, which was also observed in a fetus with fetal alcohol syndrome. These data suggest that alcohol-induced endothelial dysfunction may contribute in ectopic cortical GABAergic interneurons, that has previously been described in infants with FASD.

Neonatal cerebral hypoxia-ischemia in mice triggers age-dependent vascular effects and disabilities in adults; implication of tissue plasminogen activator (tPA).

Neonatal encephalopathy frequently results from hypoxia-ischemia (HI) or inflammation in preterm or term neonates. Neuropathology depends on cerebral development at insult time, but the poor correlation of neuromotor, cognitive, and behavioral disabilities in infancy with initial imaging and clinical records precludes early prognosis. The Rice-Vannucci HI procedure was applied to wild type and tissue plasminogen activator knockout (tPA-KO) mice as surrogates for human preterm (with five-day-old postnatal (P5) mice) or human term (with ten-day-old postnatal (P10) mice). Acute and delayed T2-magnetic resonance imaging (T2-MRI) signals and cognitive deficits in adulthood (spatial memory and social interaction) were investigated in the same animals. Early vascular tPA and matrix metalloproteinase-9 (MMP-9) activities, blood-brain barrier permeability to water or IgG, and microglial activation were assessed separately. HI in P5 or P10 mice induced early hemisphere swelling in T2-MRI scans, and a delayed atrophy of the cortex and hippocampus, but affected white matter in the P5 group only, irrespective of the wild type or tPA-KO genotype. Adults had no motor disabilities, but we did find HI-induced age-dependent deficits, preferentially social interaction and activity in P5 mice, and spatial learning in P10 mice. In P5 mice, tPA-KO prevented MMP-9 activation, IgG extravasation, microglial activation, and behavior impairments. In P10 mice, MMP-9 activation and inflammatory processes remained in the hippocampus of the tPA-KO group, and also contributed to persistent spatial learning deficits. Perinatal HI in mice mimicked the unpredictability of outcomes from imaging in human clinics. Delayed deficits appeared associated to vascular dysfunction-induced inflammation, which recalls our previous work showing major vascular maturation between P5 and P10 stages. Using omics to explore neural, glial, or brain vessel markers in neonate blood may be a promising perspective to identify pertinent prognostic tools.

Glutamate controls vessel-associated migration of GABA interneurons from the pial migratory route via NMDA receptors and endothelial protease activation.

During cortex development, fine interactions between pyramidal cells and migrating GABA neurons are required to orchestrate correct positioning of interneurons, but cellular and molecular mechanisms are not yet clearly understood. Functional and age-specific expression of NMDA receptors by neonate endothelial cells suggests a vascular contribution to the trophic role of glutamate during cortical development. Associating functional and loss-of-function approaches, we found that glutamate stimulates activity of the endothelial proteases MMP-9 and t-PA along the pial migratory route (PMR) and radial cortical microvessels. Activation of MMP-9 was NMDAR-dependent and abrogated in t-PA-/- mice. Time-lapse recordings revealed that glutamate stimulated migration of GABA interneurons along vessels through an NMDAR-dependent mechanism. In Gad67-GFP mice, t-PA invalidation and in vivo administration of an MMP inhibitor impaired positioning of GABA interneurons in superficial cortical layers, whereas Grin1 endothelial invalidation resulted in a strong reduction of the thickness of the pial migratory route, a marked decrease of the glutamate-induced MMP-9-like activity along the PMR and a depopulation of interneurons in superficial cortical layers. This study supports that glutamate controls the vessel-associated migration of GABA interneurons by regulating the activity of endothelial proteases. This effect requires endothelial NMDAR and is t-PA-dependent. These neurodevelopmental data reinforce the debate regarding safety of molecules with NMDA-antagonist properties administered to preterm and term neonates.

Why considering sexual differences is necessary when studying encephalopathy of prematurity through rodent models.

Preterm birth is a high-risk factor for the development of gray and white matter abnormalities, referred to as "encephalopathy of prematurity," that may lead to life-long motor, cognitive, and behavioral impairments. The prevalence and clinical outcomes of encephalopathy of prematurity differ between sexes, and elucidating the underlying biological basis has become a high-priority challenge. Human studies are often limited to assessment of brain region volumes by MRI, which does not provide much information about the underlying mechanisms of lesions related to very preterm birth. However, models using KO mice or pharmacological manipulations in rodents allow relevant observations to help clarify the mechanisms of injury sustaining sex-differential vulnerability. This review focuses on data obtained from mice aged P1-P5 or rats aged P3 when submitted to cerebral damage such as hypoxia-ischemia, as their brain lesions share similarities with lesion patterns occurring in very preterm human brain, before 32 gestational weeks. We first report data on the mechanisms underlying the development of sexual brain dimorphism in rodent, focusing on the hippocampus. In the second part, we describe sex specificities of rodent models of encephalopathy of prematurity (RMEP), focusing on mechanisms underlying differences in hippocampal vulnerability. Finally, we discuss the relevance of these RMEP. Together, this review highlights the need to systematically search for potential effects of sex when studying the mechanisms underlying deficits in RMEP in order to design effective sex-specific medical interventions in human preterms.

Time- and sex-dependent efficacy of magnesium sulfate to prevent behavioral impairments and cerebral damage in a mouse model of cerebral palsy.

Cerebral lesions acquired in the perinatal period can induce cerebral palsy (CP), a multifactorial pathology leading to lifelong motor and cognitive deficits. Several risk factors, including perinatal hypoxia-ischemia (HI), can contribute to the emergence of CP in preterm infants. Currently, there is no international consensus on treatment strategies to reduce the risk of developing CP. A meta-analysis showed that magnesium sulfate (MgSO4) administration to mothers at risk of preterm delivery reduces the risk of developing CP (Crowther et al., 2017). However, only a few studies have investigated the long-term effects of MgSO4 and it is not known whether sex would influence MgSO4 efficacy. In addition, the search for potential deleterious effects is essential to enable broad use of MgSO4 in maternity wards. We used a mouse model of perinatal HI to study MgSO4 effects until adolescence, focusing on cognitive and motor functions, and on some apoptosis and inflammation markers. Perinatal HI at postnatal day 5 (P(5)) induced (1) sensorimotor deficits in pups; (2) increase in caspase-3 activity 24 h after injury; (3) production of proinflammatory cytokines from 6 h to 5 days after injury; (4) behavioral and histological alterations in adolescent mice with considerable interindividual variability. MgSO4 prevented sensorimotor alterations in pups, with the same efficacy in males and females. MgSO4 displayed anti-apoptotic and anti-inflammatory effects without deleterious side effects. Perinatal HI led to motor coordination impairments in female adolescent mice and cognitive deficits in both sexes. MgSO4 tended to prevent these motor and cognitive deficits only in females, while it prevented global brain tissue damage in both sexes. Moreover, interindividual and intersexual differences appeared regarding the lesion size and neuroprotection by MgSO4 in a region-specific manner. These differences, the partial prevention of disorders, as well as the mismatch between histological and behavioral observations mimic clinical observations. This underlines that this perinatal HI model is suitable to further analyze the mechanisms of sex-dependent perinatal lesion susceptibility and MgSO4 efficacy.

Magnesium Sulfate Prevents Neurochemical and Long-Term Behavioral Consequences of Neonatal Excitotoxic Lesions: Comparison Between Male and Female Mice.

Magnesium sulfate (MgSO4) administration to mothers at risk of preterm delivery is proposed as a neuroprotective strategy against neurological alterations such as cerebral palsy in newborns. However, long-term beneficial or adverse effects of MgSO4 and sex-specific sensitivity remain to be investigated. We conducted behavioral and neurochemical studies of MgSO4 effects in males and females, from the perinatal period to adolescence in a mouse model of cerebral neonatal lesion. The lesion was produced in 5-day-old (P5) pups by ibotenate intracortical injection. MgSO4 (600 mg/kg, i.p.) prior to ibotenate prevented lesion-induced sensorimotor alterations in both sexes at P6 and P7. The lesion increased glutamate level at P10 in the prefrontal cortex, which was prevented by MgSO4 in males. In neonatally lesioned adolescent mice, males exhibited more sequelae than females in motor and cognitive functions. In the perirhinal cortex of adolescent mice, the neonatal lesion induced an increase in vesicular glutamate transporter 1 density in males only, which was negatively correlated with cognitive scores. Long-term sequelae were prevented by neonatal MgSO4 administration. MgSO4 never induced short- or long-term deleterious effect on its own. These results also strongly suggest that sex-specific neuroprotection should be foreseen in preterm infants.

Experimental and clinical evidence of differential effects of magnesium sulfate on neuroprotection and angiogenesis in the fetal brain.

Clinical studies showed beneficial effects of magnesium sulfate regarding the risk of cerebral palsy. However, regimen protocols fluctuate worldwide and risks of adverse effects impacting the vascular system have been reported for human neonates, keeping open the question of the optimal dosing. Using clinically relevant concentrations and doses of magnesium sulfate, experiments consisted of characterizing, respectively, ex vivo and in vivo, the effects of magnesium sulfate on the nervous and vascular systems of mouse neonates by targeting neuroprotection, angiogenesis, and hemodynamic factors and in measuring, in human fetuses, the impact of a 4-g neuroprotective loading dose of magnesium sulfate on brain hemodynamic parameters. Preclinical experiments using cultured cortical slices from mouse neonates showed that the lowest and highest tested concentrations of magnesium sulfate were equally potent to prevent excitotoxic-induced cell death, cell edema, cell burst, and intracellular calcium increase, whereas no side effects were found regarding apoptosis. In contrast, in vivo data revealed that magnesium sulfate exerted dose-dependent vascular effects on the fetal brain. In particular, it induced brain hypoperfusion, stabilization of Hif-1α, long-term upregulation of VEGF-R2 expression, impaired endothelial viability, and altered cortical angiogenesis. Clinically, in contrast to 6-g loading doses used in some protocols, a 4-g bolus of magnesium sulfate did not altered fetal brain hemodynamic parameters. In conclusion, these data provide the first mechanistic evidence of double-sword and dose-dependent actions of magnesium sulfate on nervous and vascular systems. They strongly support the clinical use of neuroprotection protocols validated for the lowest (4-g) loading dose of magnesium sulfate.

Proteomic and transcriptomic study of brain microvessels in neonatal and adult mice.

Infants born before 29 weeks gestation incur a major risk of preterm encephalopathy and subependymal/intracerebral/intraventricular haemorrhage. In mice, an ontogenic window of haemorrhage risk was recorded up to 5 days after birth in serpine1 knock-out animals. Using proteome and transcriptome approaches in mouse forebrain microvessels, we previously described the remodelling of extracellular matrix and integrins likely strengthening the vascular wall between postnatal day 5 (P5) and P10. Haemorrhage is the ultimate outcome of vessel damage (i.e., during ischaemia), although discreet vessel insults may be involved in the aetiology of preterm encephalopathy. In this study, we examined proteins identified by mass spectrometry and segregating in gene ontology pathways in forebrain microvessels in P5, P10, and adult wild type mice. In parallel, comparative transcript levels were obtained using RNA hybridization microarrays and enriched biological pathways were extracted from genes exhibiting at least a two-fold change in expression. Five major biological functions were observed in those genes detected both as proteins and mRNA expression undergoing at least a two-fold change in expression in one or more age comparisons: energy metabolism, protein metabolism, antioxidant function, ion exchanges, and transport. Adult microvessels exhibited the highest protein and mRNA expression levels for a majority of genes. Energy metabolism-enriched gene ontology pathways pointed to the preferential occurrence of glycolysis in P5 microvessels cells versus P10 and adult preparations enriched in aerobic oxidative enzymes. Age-dependent levels of RNA coding transport proteins at the plasma membrane and mitochondria strengthened our findings based on protein data. The data suggest that immature microvessels have fewer energy supply alternatives to glycolysis than mature structures. In the context of high energy demand, this constraint might account for vascular damage and maintenance of the high bleeding occurrence in specific areas in immature brain.

Major remodeling of brain microvessels during neonatal period in the mouse: A proteomic and transcriptomic study.

Preterm infants born before 29 gestation weeks incur major risk of subependymal/intracerebral/intraventricular hemorrhage. In mice, neonate brain endothelial cells are more prone than adult cells to secrete proteases under glutamate challenge, and invalidation of the Serpine 1 gene is accompanied by high brain hemorrhage risk up to five days after birth. We hypothesized that the structural and functional states of microvessels might account for age-dependent vulnerability in mice up to five days after birth and might represent a pertinent paradigm to approach the hemorrhage risk window observed in extreme preterms. Mass spectrometry proteome analyses of forebrain microvessels at days 5, 10 and in adult mice revealed 899 proteins and 36 enriched pathways. Microarray transcriptomic study identified 5873 genes undergoing at least two-fold change between ages and 93 enriched pathways. Both approaches pointed towards extracellular matrix, cell adhesion and junction pathways, indicating delayed microvascular strengthening after P5. Furthermore, glutamate receptors, proteases and their inhibitors exhibited convergent evolutions towards excitatory aminoacid sensitivity and low proteolytic control likely accounting for vascular vulnerability in P5 mice. Thus, age vascular specificities must be considered in future therapeutic interventions in preterms. Data are available on ProteomeXchange (identifier PXD001718) and NCBI Gene-Expression-Omnibus repository (identification GSE67870).

Age-dependent neonatal intracerebral hemorrhage in plasminogen activator inhibitor 1 knockout mice.

Intracerebral-intraventricular hemorrhages (ICH/IVH) in very preterm neonates are responsible for high mortality and subsequent disabilities. In humans, tissue plasminogen activator (t-PA) initiates fibrinolysis and activates endoluminal-endothelial receptors; dysfunction of the t-PA inhibitor (PAI-1) results in recurrent hemorrhages. We used PAI-1 knockout (PAI-1) mice to examine the role of t-PA in age-dependent intracranial hemorrhages as a possible model of preterm ICH/IVH. Intracortical injection of 2 µL of phosphate-buffered saline produced a small traumatic injury and a high rate of hemorrhage in PAI-1 pups at postnatal day 3 (P3) or P5, whereas it had no effect in wild-type neonates. This resulted in white matter and cortical lesions, ventricle enlargement, hyperlocomotion, and altered cortical levels of serotonin and dopamine in the adult PAI mice. N-methyl-D-aspartate receptor blockers, plasmin- and matrix metalloproteinases inhibitors reduced hemorrhage and tissue lesions. In contrast to P3 to P5, no significant hemorrhages were induced in P10 PAI-1 pups and there were no behavioral or neurochemical alterations in adulthood. These data suggest that microvascular immaturity up to P5 in mice is a determinant factor required for t-PA-dependent vascular rupture. Neonatal PAI-1 mice could be a useful ICH/IVH model for studying the ontogenic window of vascular immaturity and vascular protection against later neurodisabilities.

The efficiency of glutamate uptake differs between neonatal and adult cortical microvascular endothelial cells.

Glutamate transporters (excitatory amino-acid transporters (EAATs)) are essential for brain homeostasis. While previous studies indicate that the vascular endothelium contributes to glutamate efflux in the adult brain, little information is available regarding glutamate uptake in the immature brain. The present study shows a differential expression pattern of EAATs between cortical microvessels in adults and newborns. In addition, adult cortical endothelial cells take up glutamate more efficiently than neonatal cells. Our findings indicate age-specific changes in extracellular glutamate regulation by brain endothelial cells, suggesting differences in the efficiency of glutamate efflux during an excitotoxic process that, in turn, may contribute to age-specific brain vulnerability.

Hypoxia-ischemia or excitotoxin-induced tissue plasminogen activator- dependent gelatinase activation in mice neonate brain microvessels.

Hypoxia-ischemia (HI) and excitotoxicity are validated causes of neonatal brain injuries and tissue plasminogen activator (t-PA) participates in the processes through proteolytic and receptor-mediated pathways. Brain microvascular endothelial cells from neonates in culture, contain and release more t-PA and gelatinases upon glutamate challenge than adult cells. We have studied t-PA to gelatinase (MMP-2 and MMP-9) activity links in HI and excitotoxicity lesion models in 5 day-old pups in wild type and in t-PA or its inhibitor (PAI-1) genes inactivated mice. Gelatinolytic activities were detected in SDS-PAGE zymograms and by in situ fluorescent DQ-gelatin microscopic zymographies. HI was achieved by unilateral carotid ligature followed by a 40 min hypoxia (8%O2). Excitotoxic lesions were produced by intra parenchymal cortical (i.c.) injections of 10 µg ibotenate (Ibo). Gel zymograms in WT cortex revealed progressive extinction of MMP-2 and MMP-9 activities near day 15 or day 8 respectively. MMP-2 expression was the same in all strains while MMP-9 activity was barely detectable in t-PA⁻/⁻ and enhanced in PAI-1⁻/⁻ mice. HI or Ibo produced activation of MMP-2 activities 6 hours post-insult, in cortices of WT mice but not in t-PA⁻/⁻ mice. In PAI-1⁻/⁻ mice, HI or vehicle i.c. injection increased MMP-2 and MMP-9 activities. In situ zymograms using DQ-gelatin revealed vessel associated gelatinolytic activity in lesioned areas in PAI-1⁻/⁻ and in WT mice. In WT brain slices incubated ex vivo, glutamate (200 µM) induced DQ-gelatin activation in vessels. The effect was not detected in t-PA⁻/⁻ mice, but was restored by concomitant exposure to recombinant t-PA (20 µg/mL). In summary, neonatal brain lesion paradigms and ex vivo excitotoxic glutamate evoked t-PA-dependent gelatinases activation in vessels. Both MMP-2 and MMP-9 activities appeared t-PA-dependent. The data suggest that vascular directed protease inhibition may have neuroprotection potential against neonatal brain injuries.

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.

[How to manage gait and balance disorders among older adults aged 65 years and older with mild to moderate dementia in clinical practice?]. / Troubles de la marche et de l'équilibre de la personne âgée de 65 ans et plus ayant une démence légère à modérée : quelle prise en charge en pratique courante ?

Gait and balance disorders are frequent in demented older adults. Their management (i.e., diagnosis, assessment and treatment) is challenging in daily practice because of numerous evaluation tests available, difficulties to select the most adapted intervention, and the lack of knowledge of physicians and health professionals concerning adapted centers to refer their patients to those patients. Thus, a working group of experts was organized by the Gérontopôle of Pays de Loire, France, in December 2010 with the aim to provide clinical guidelines for the management of older adults aged 65 years and older with mild to moderate dementia with gait and balance disorders. These guidelines provide answers to the following questions: 1) Is there gait and/or balance disorders? 2) Which specific tests used? and 3) How to treat patients?

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.