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.

Effects of MgSO4 Alone or Associated with 4-PBA on Behavior and White Matter Integrity in a Mouse Model of Cerebral Palsy: A Sex- and Time-Dependent Study.

Cerebral palsy (CP) is defined as permanent disorders of movement and posture. Prematurity and hypoxia-ischemia (HI) are risk factors of CP, and boys display a greater vulnerability to develop CP. Magnesium sulfate (MgSO4) is administered to mothers at risk of preterm delivery as a neuroprotective agent. However, its effectiveness is only partial at long term. To prolong MgSO4 effects, it was combined with 4-phenylbutyrate (4-PBA). A mouse model of neonatal HI, generating lesions similar to those reported in preterms, was realized. At short term, at the behavioral and cellular levels, and in both sexes, the MgSO4/4-PBA association did not alter the total prevention induced by MgSO4 alone. At long term, the association extended the MgSO4 preventive effects on HI-induced motor and cognitive deficits. This might be sustained by the promotion of oligodendrocyte precursor differentiation after HI at short term, which led to improvement of white matter integrity at long term. Interestingly, at long term, at a behavioral level, sex-dependent responses to HI were observed. This might partly be explained by early sex-dependent pathological processes that occur after HI. Indeed, at short term, apoptosis through mitochondrial pathways seemed to be activated in females but not in males, and only the MgSO4/4-PBA association seemed to counter this apoptotic process.

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.

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.

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.

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.

In Utero Alcohol Exposure Impairs Retinal Angiogenesis and the Microvessel-Associated Positioning of Calretinin Interneurons.

In addition to brain disorders, which constitute a devastating consequence of prenatal alcohol exposure (PAE), eye development is also significantly affected. Given that the retina is a readily accessible part of the central nervous system, a better understanding of the impact of ethanol on retinal development might provide ophthalmological landmarks helpful for early diagnosis of fetal alcohol syndrome. This study aimed to provide a fine morphometric and cellular characterization of the development of retinal microvasculature and neurovascular interactions in a mouse model of fetal alcohol spectrum disorder (FASD). The data revealed that PAE impaired superficial vascular plexus development. In particular, progression of the vascular migration front was significantly decreased in PAE retinas, supporting a delay in plexus progression. Moreover, a significant decrease in the vessel density and number of perforating vessels was quantified in PAE mice, supporting less angiogenesis. The present study provides also the first evidence of a close interaction between migrating calretinin-positive interneurons and perforating microvessels in the inner nuclear layer of the developing retina. This neurovascular association was significantly impaired by PAE. Moreover, projections of amacrine cells were abnormally distributed and densified in stratum S1 and S2. In humans, comparison of a five-month-old control infant with a three-month-old alcohol-exposed case revealed a similar mispositioning of calretinin-positive interneurons. This opens new research avenues regarding a neurovascular contribution in the deleterious effects of alcohol in the developing retina and support that ophthalmological examination could become a promising approach for early detection of alcohol-exposed infants presenting with neurovascular brain defects.

Expression of placental CD146 is dysregulated by prenatal alcohol exposure and contributes in cortical vasculature development and positioning of vessel-associated oligodendrocytes.

Recent data showed that prenatal alcohol exposure (PAE) impairs the "placenta-brain" axis controlling fetal brain angiogenesis in human and preclinical models. Placental growth factor (PlGF) has been identified as a proangiogenic messenger between these two organs. CD146, a partner of the VEGFR-1/2 signalosome, is involved in placental angiogenesis and exists as a soluble circulating form. The aim of the present study was to investigate whether placental CD146 may contribute to brain vascular defects described in fetal alcohol spectrum disorder. At a physiological level, quantitative reverse transcription polymerase chain reaction experiments performed in human placenta showed that CD146 is expressed in developing villi and that membrane and soluble forms of CD146 are differentially expressed from the first trimester to term. In the mouse placenta, a similar expression pattern of CD146 was found. CD146 immunoreactivity was detected in the labyrinth zone and colocalized with CD31-positive endothelial cells. Significant amounts of soluble CD146 were quantified by ELISA in fetal blood, and the levels decreased after birth. In the fetal brain, the membrane form of CD146 was the majority and colocalized with microvessels. At a pathophysiological level, PAE induced marked dysregulation of CD146 expression. The soluble form of CD146 decreased in both placenta and fetal blood, whereas it increased in the fetal brain. Similarly, the expression of several members of the CD146 signalosome, such as VEGFR2 and PSEN, was differentially impaired between the two organs by PAE. At a functional level, targeted repression of placental CD146 by in utero electroporation (IUE) of CRISPR/Cas9 lentiviral plasmids resulted in (i) a decrease in cortical vessel density, (ii) a loss of radial vascular organization, and (iii) a reduced density of oligodendrocytes. Statistical analysis showed that the more the vasculature was impaired, the more the cortical oligodendrocyte density was reduced. Altogether, these data support that placental CD146 contributes to the proangiogenic "placenta-brain" axis and that placental CD146 dysfunction contributes to the cortical oligo-vascular development. Soluble CD146 would represent a promising placental biomarker candidate representative of alcohol-induced neurovascular defects in neonates, as recently suggested by PlGF (patents WO2016207253 and WO2018100143).

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).

Oligodendrocyte lineage is severely affected in human alcohol-exposed foetuses.

Prenatal alcohol exposure is a major cause of neurobehavioral disabilities. MRI studies in humans have shown that alcohol is associated with white matter microstructural anomalies but these studies focused on myelin abnormalities only after birth. Only one of these studies evaluated oligodendrocyte lineage, but only for a short period during human foetal life. As data are lacking in humans and alcohol is known to impair oligodendrocyte differentiation in rodents, the present study aimed to compare by immunohistochemistry the oligodendrocyte precursor cells expressing PDGFR-α and immature premyelinating/mature oligodendrocytes expressing Olig2 in the ganglionic eminences and the frontal cortex of 14 human foetuses exposed to alcohol from 15 to 37 weeks' gestation with age-matched controls. The human brains used in this study were obtained at the time of foetal autopsies for medical termination of pregnancy, in utero or post-natal early death. Before birth, PDGFR-α expression was strongly increased in the ganglionic eminences and the cortex of all foetuses exposed to alcohol except at the earliest stage. No massive generation of Olig2 immunoreactive cells was identified in the ganglionic eminences until the end of pregnancy and the density of Olig2-positive cells within the cortex was consistently lower in foetuses exposed to alcohol than in controls. These antenatal data from humans provides further evidence of major oligodendrocyte lineage impairment at specific and key stages of brain development upon prenatal alcohol exposure including defective or delayed generation and maturation of oligodendrocyte precursors.

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.

Neuropathological hallmarks of fetal hydrocephalus linked to CCDC88C pathogenic variants.

The prevalence of congenital hydrocephalus has been estimated at 1.1 per 1000 infants when including cases diagnosed before 1 year of age after exclusion of neural tube defects. Classification criteria are based either on CSF dynamics, pathophysiological mechanisms or associated lesions. Whereas inherited syndromic hydrocephalus has been associated with more than 100 disease-causing genes, only four genes are currently known to be linked to congenital hydrocephalus either isolated or as a major clinical feature: L1CAM, AP1S2, MPDZ and CCDC88C. In the past 10 years, pathogenic variants in CCDC88C have been documented but the neuropathology remains virtually unknown. We report the neuropathology of two foetuses from one family harbouring two novel compound heterozygous pathogenic variants in the CCDC88C gene: a maternally inherited indel in exon 22, c.3807_3809delinsACCT;p.(Gly1270Profs*53) and a paternally inherited deletion of exon 23, c.3967-?_c.4112-?;p.(Leu1323Argfs*10). Medical termination of pregnancy was performed at 18 and 23 weeks of gestation for severe bilateral ventriculomegaly. In both fetuses, brain lesions consisted of multifocal atresia-forking along the aqueduct of Sylvius and the central canal of the medulla, periventricular neuronal heterotopias and choroid plexus hydrops. The second fetus also presented lumbar myelomeningocele, left diaphragmatic hernia and bilateral renal agenesis. CCDC88C encodes the protein DAPLE which contributes to ependymal cell planar polarity by inhibiting the non-canonical Wnt signaling pathway and interacts with MPDZ and PARD3. Interestingly, heterozygous variants in PARD3 result in neural tube defects by defective tight junction formation and polarization process of the neuroepithelium. Besides, during organ formation Wnt signalling is a prerequisite for planar cell polarity pathway activation, and mutations in planar cell polarity genes lead to heart, lung and kidney malformations. Hence, candidate variants in CCDC88C should be carefully considered whether brain lesions are isolated or associated with malformations suspected to result from disorders of planar cell polarity.

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.

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.

Prenatal alcohol exposure is a leading cause of interneuronopathy in humans.

Alcohol affects multiple neurotransmitter systems, notably the GABAergic system and has been recognised for a long time as particularly damaging during critical stages of brain development. Nevertheless, data from the literature are most often derived from animal or in vitro models. In order to study the production, migration and cortical density disturbances of GABAergic interneurons upon prenatal alcohol exposure, we performed immunohistochemical studies by means of the proliferation marker Ki67, GABA and calretinin antibodies in the frontal cortical plate of 17 foetal and infant brains antenatally exposed to alcohol, aged 15 weeks' gestation to 22 postnatal months and in the ganglionic eminences and the subventricular zone of the dorsal telencephalon until their regression, i.e., 34 weeks' gestation. Results were compared with those obtained in 17 control brains aged 14 weeks of gestation to 35 postnatal months. We also focused on interneuron vascular migration along the cortical microvessels by confocal microscopy with double immunolabellings using Glut1, GABA and calretinin. Semi-quantitative and quantitative analyses of GABAergic and calretininergic interneuron density allowed us to identify an insufficient and delayed production of GABAergic interneurons in the ganglionic eminences during the two first trimesters of the pregnancy and a delayed incorporation into the laminar structures of the frontal cortex. Moreover, a mispositioning of GABAergic and calretininergic interneurons persisted throughout the foetal life, these cells being located in the deep layers instead of the superficial layers II and III. Moreover, vascular migration of calretininergic interneurons within the cortical plate was impaired, as reflected by low numbers of interneurons observed close to the cortical perforating vessel walls that may in part explain their abnormal intracortical distribution. Our results are globally concordant with those previously obtained in mouse models, in which alcohol has been shown to induce an interneuronopathy by affecting interneuron density and positioning within the cortical plate, and which could account for the neurological disabilities observed in children with foetal alcohol disorder spectrum.

Ethanol-Induced Alterations in Placental and Fetal Cerebrocortical Annexin-A4 and Cerebral Cavernous Malformation Protein 3 Are Associated With Reductions in Fetal Cortical VEGF Receptor Binding and Microvascular Density.

Jegou et al. (2012) have reported prenatal alcohol exposure (PAE)-induced reductions of angiogenesis-related proteins in mouse placenta. These effects were associated with striking alterations in microvascular development in neonatal cerebral cortex. Here, we employed a rat model of moderate PAE to search for additional proteins whose placental and fetal cortical expression is altered by PAE, along with a subsequent examination of fetal cerebral cortical alterations associated with altered protein expression. Long-Evans rat dams voluntarily consumed either a 0 or 5% ethanol solution 4 h each day throughout gestation. Daily ethanol consumption, which resulted in a mean peak maternal serum ethanol concentration of 60.8 mg/dL, did not affect maternal weight gain, litter size, or placental or fetal body weight. On gestational day 20, rat placental: fetal units were removed by Caesarian section. Placental protein expression, analyzed by 2D-PAGE - tandem mass spectroscopy, identified a total of 1,117 protein spots, 20 of which were significantly altered by PAE. To date, 14 of these PAE-altered proteins have been identified. Western blotting confirmed the alterations of two of these placental proteins, namely, annexin-A4 (ANX-A4) and cerebral cavernous malformation protein 3 (CCM-3). Specifically, PAE elevated ANX-A4 and decreased CCM-3 in placenta. Subsequently, these two proteins were measured in fetal cerebral cortex, along with radiohistochemical studies of VEGF binding and histofluorescence studies of microvascular density in fetal cerebral cortex. PAE elevated ANX-A4 and decreased CCM-3 in fetal cerebral cortex, in a pattern similar to the alterations observed in placenta. Further, both VEGF receptor binding and microvascular density and orientation, measures that are sensitive to reduced CCM-3 expression in developing brain, were significantly reduced in the ventricular zone of fetal cerebral cortex. These results suggest that the expression angiogenesis-related proteins in placenta might serve as a biomarker of ethanol-induced alterations in microvascular development in fetal brain.

Paradoxical effect of ethanol on potassium channel currents and cell survival in cerebellar granule neurons.

Transient exposure to ethanol (EtOH) results in a massive neurodegeneration in the developing brain leading to behavioral and cognitive deficits observed in fetal alcohol syndrome. There is now compelling evidence that K+ channels play an important role in the control of programmed cell death. The aim of the present work was to investigate the involvement of K+ channels in the EtOH-induced cerebellar granule cell death and/or survival. At low and high concentrations, EtOH evoked membrane depolarization and hyperpolarization, respectively. Bath perfusion of EtOH (10 mM) depressed the I(A) (transient K+ current) potassium current whereas EtOH (400 mM) provoked a marked potentiation of the specific I(K) (delayed rectifier K+ current) current. Pipette dialysis with GTPgammaS or GDPbetaS did not modify the effects of EtOH (400 mM) on both membrane potential and I(K) current. In contrast, the reversible depolarization and slowly recovering inhibition of I(A) induced by EtOH (10 mM) became irreversible in the presence of GTPgammaS. EtOH (400 mM) induced prodeath responses whereas EtOH (10 mM) and K+ channel blockers promoted cell survival. Altogether, these results indicate that in cerebellar granule cells, EtOH mediates a dual effect on K+ currents partly involved in the control of granule cell death.

Role of complement anaphylatoxin receptors (C3aR, C5aR) in the development of the rat cerebellum.

There is now strong evidence for non-immune or inflammatory functions of complement, notably in the central nervous system. In particular, it has been recently reported that the anaphylatoxin receptors C3aR and C5aR are transiently expressed in the cerebellar cortex of newborn rat, suggesting that anaphylatoxins are involved in the histogenesis of the cerebellum. In the present study, we have investigated the effects of C3aR and C5aR agonists and antagonists on the development of the cerebellum of 11-12-day-old rats in vivo and in vitro. Sub-dural injection of C3aR and C5aR agonists at the surface of the cerebellum transiently modified the thickness of the cortical layers. The C5aR agonist provoked an enlargement of the external granule cell layer (EGL) that was due to increased proliferation of immature granule neurons. Conversely, the C3aR agonist decreased the thickness of the EGL and increased the thickness of the internal granule cell layer (IGL), suggesting that C3a accelerates the migration process of granule cells from the EGL to the IGL. Video-microscopy examination of cultured granule neurons confirmed the role of C3aR in cell motility. These results provide clear evidence for the involvement of anaphylatoxin receptors in the histogenesis of the cerebellar cortex.

[Fetal alcohol exposure: when placenta would help to the early diagnosis of child brain impairments]. / Alcoolisation fœtale - Le placenta au secours du diagnostic précoce des troubles du développement cérébral de l'enfant.

Alcohol consumption during pregnancy constitutes a major cause of neurodevelopmental and behavioral disabilities. Whereas it is possible for clinicians to establish a perinatal diagnosis of fetal alcohol syndrome, the more severe expression of fetal alcohol spectrum disorder (FASD), most FASD children are late or mis-diagnosed due to a lack of clear morphological and neurodevelopmental abnormalities. Several precious years of care are consequently lost. Recent data revealed a functional placenta-brain axis involved in the control of the fetal brain angiogenesis which is impaired by in utero alcohol exposure. Because in the developing fetal brain a correct angiogenesis is required for a correct neurodevelopment, these preclinical and clinical advances pave the way for a new generation of placental biomarkers for early diagnosis of FASD.

TITLE: Alcoolisation fœtale - Le placenta au secours du diagnostic précoce des troubles du développement cérébral de l'enfant. ABSTRACT: La consommation d'alcool au cours de la grossesse constitue une cause majeure de troubles du comportement et de handicap. Alors qu'il est possible pour un clinicien d'établir un diagnostic néonatal du syndrome d'alcoolisation fœtale, l'atteinte la plus sévère des troubles causés par l'alcoolisation fœtale (TCAF), une grande majorité des enfants échappe à un diagnostic précoce en raison de l'absence d'anomalies morphologiques évidentes. Plusieurs années de prise en charge sont alors perdues. Des avancées récentes ont permis d'établir l'existence d'un axe fonctionnel placenta-cerveau impliqué dans le contrôle de l'angiogenèse cérébrale, qui se trouve dérégulé chez les enfants exposés in utero à l'alcool. Une angiogenèse cérébrale normale étant un prérequis à l'établissement d'un neurodéveloppement correct, ces avancées ouvrent la voie à l'identification d'une nouvelle génération de biomarqueurs placentaires d'atteinte cérébrale pour le diagnostic précoce des enfants TCAF.