Lacosamide intake during pregnancy increases the incidence of foetal malformations and symptoms associated with schizophrenia in the ofspring of mice

The use of frst and second generation antiepileptic drugs during pregnancy doubles the risk of major congenital malformations and other teratogenic defects. Lacosamide (LCM) is a third-generation antiepileptic drug that interacts with collapsing response mediator protein 2, a protein that has been associated with neurodevelopmental diseases like schizophrenia. The aim of this study was to test the potential teratogenic efects of LCM on developing embryos and its efects on behavioural/histological alterations in adult mice. We administered LCM to pregnant mice, assessing its presence, and that of related compounds, in the mothers’ serum and in embryonic tissues using liquid chromatography coupled to quadrupole/time of fight mass spectrometry detection. Embryo morphology was evaluated, and immunohistochemistry was performed on adult ofspring. Behavioural studies were carried out during the frst two postnatal weeks and on adult mice. We found a high incidence of embryonic lethality and malformations in mice exposed to LCM during embryonic development. Neonatal mice born to dams treated with LCM during gestation displayed clear psychomotor delay and behavioural and morphological alterations in the prefrontal cortex, hippocampus and amygdala that were associated with behaviours associated with schizophrenia spectrum disorders in adulthood. We conclude that LCM and its metabolites may have teratogenic efects on the developing embryos, refected in embryonic lethality and malformations, as well as behavioural and histological alterations in adult mice that resemble those presented by patients with schizophreniaWe are very thankful to Mark Sefton and Miguel Ruiz-Veguilla for revising the manuscript and valuable comments on the manuscript. We also gratefully acknowledge Servicio de Microanalisis de la Universidad de Sevilla (CITIUS) for the support ofered with UPLC-QTOF/MS. Tis work was supported by the Andalusian Regional Ministry of Economy, Science and Innovation (P11-cts-7634); the Andalusian Regional Ministry of Health (PI-0438-2010) and the Instituto de Salúd Carlos III (PS09/00050, CP08/00111, CPII14/00033) cofnanced by European Development Regional Fund “A way to achieve Europe” ERDF to PYG; Fundación Ramón Areces, DGICYT BFU2011-27207, and the Junta de Andalucía CTS-2257 to AMC; Funding FEDER Project UNSE10–1E-429 to RFT and MVN; Francisco José de Caldas fellowship by COLCIENCIAS to V.V-LDepartamento de Biología Ambiental y Salud Públic

Evaluation of the looptail mutation efects on neural tube closure in early mouse embryo.

Motivation: Neural tube defects (NTDs), such as spina bifida, are defined as congenital malformations that impair proper neural tube closure in early embryonic development. There are deeply complex mechanisms behind NTDs, of which we can stand out the role of the Wnt/PCP pathway, that through cytoskeleton reorganization and cell-cell interactions drives tissue rearrangements which finally lead to neural tube closure. In this project, cellular morphology and dynamics in node cells, an essential organizer in early development, have been studied in both, its dorsal and ventral regions, to determine how affected it is in Wnt/PCP-mutant mouse embryos . It is the great importance of NTDs in current population what has motivated this study as it has been estimated that NTDs prevalence varies between 0.5-0.8/1000 births in most European countries and the USA, and it is even 20 times higher in countries such as China. Taking into account this data, it seems to exist a clear need to investigate the physiopathology of these malformations and initiate the run for further solutions.Methods: In pursuit of valuable information, the looptail (Lp) mouse strain, carrying a mutation in one of the WNT/PCP pathway core proteins, Vangl2, has been selected as it is a great NTDs model. Whole-mount inmunohistochemistry against Zonulla Occludens-1 (ZO-1), a tight junction protein, as well as phalloidin staining, have been performed on mice embryos. Afterwards, z-images have been obtained through scannig laser confocal microscopy. Finally, segmentation has been done using the python software "SeedWaterSegmenter" and the "DVRosettes" and "CellRois" macros, both from Fiji/ImageJ.Results: The aforementioned macros offer personalized morphological information for each cell and its interaction with neighbouring cells according to the pre-selected parameters: apical area, apical elongation, cell apical orientation and vertex order (number of cells sharing a vertex). In the previous months, this group has been working with CD1 mouse embryos in order to optimize the complex methodology and results concerning looptail embryos are expected to be available soon.Conclusions: Morphological parameters will be crucial as we can estimate how apical constriction and cell deviation from the midline axis are affected in Lp embryos. As well as cell dynamics parameters, especially rosettes formation (5-cell unions), which play a key role in driving coordinated cell movements

Possible preventive effect of inositol in folate- resistant spina bifida occulta mouse model

Motivation: Neural tube is the embryonic structure that develops into the brain and spinal cord. Failure in the process of neural tube closure during embryonic development results in severe birth defects called neural tube defects (NTDs), including anencephaly and spina bifida. Susceptibility to NTDs is influenced by genetic and environmental factors including maternal nutrition. Clinical trials demonstrate that up to 70% of NTDs can be prevented by folic acid supplementation in early pregnancy whereas the remaining NTDs are resistant to folate. Spina bifida occulta (SBO) is included in these folic acid non-responsive NTDs. Previous studies demonstrate that inositol, a water-soluble vitamin, prevents NTDs in genetic mice models of folate-resistant NTDs, such as curly tail mutants mice. Moreover, D-chiro-inositol isomer is more effective than myo-inositol reducing the frecuency of spina bifida in curly tail mice. Loop-tail mutants mice are also resistant to supplementation with folic acid. These Loop-tail (Lp) mice are mutants for Vangl2 gene implicated in the non-canonical Wnt signalling pathway (Wnt-PCP), which have a crucial role in the beginning of the closure of the neural tube. At embryonic day 11.5 (E11.5) is evident the failure of dorsal fusion of neural folds in Lp+/- embryos and a cellular aggregate appears covering this defect. This aggregate resembles the lipoma characteristic of lipomyelomeningocele, the most common form of SBO. In this project, we are going to study the possible effect of D-chiro-inositol maternal supplementation to reduce the incidence of dorsal fusion failure and the formation of the cellular aggregate in Lp+/- embryos and in Lp+/-: Daam1+/gt double mutant embryos (both genes are members of the Wnt-PCP pathway), in which the cellular aggregate also develops.Methods: Oral administration of D-chiro-inositol to pregnant mice was performed twice daily intervals from E8.5 to E10.5. These females were paired either with Lp+/- or with Lp+/-: Daam1+/gt double mutants male mice and they were sacrified at E12.5. Embryos were genotyped and the results were compared to the macroscopic phenotype that was previosly registered. Histological studies allowed us to verify the existence of the cellular aggregate in Lp+/- and Lp+/-: Daam1+/gt embryos. In addition, hybridization in situ and immunohistochemistry were carried out to analyze the expression of certain genes to characterize the cellular aggregate

Implication of the non-canonical Wnt pathway in neural tube defects

Motivation: Neurulation is the embryonic process that leads to the development of the neural tube, precursor of the brain and spinal cord. Failures in this process cause neural tube defects (NTD), such as spina bifida and other lethal defects. There are two types of spina bifida, open spina bifida and spina bifida occulta (SBO). The most important form of SBO is lipomyelomeningocele, which is characterized by a subcutaneous lipoma that is generally located in the lumbar or sacral region. A major knowledge in understanding the genetic basis of neurulation has been the discovery of the crucial role of the non-canonical Wnt signalling pathway (Wnt-PCP). In this project, we are going to investigate the role of the Wnt-PCP pathway in the final step of the closing of the posterior neuropore. For this purpose, we will use mutant mice for Vangl-2 gene (Loop-tail), a member of the Wnt-PCP pathway. In parallel, we will carry on genetic studies to correlate these genetic alterations in lipomyelomeningocele patients.Methods: In the mouse part of this study, standard histological techniques is been used to determine the incidence of caudal malformations in wild-type and Loop-tail heterozygous embryos. In addition, gene expression by in situ hybridization in the whole embryos is been done to analyse candidate genes, as well as at the protein distribution by immunostaining on cryostat sections. In the human study, we are analysing Vangl-2 gene in lipomyelomeningocele patients to detect possible genetic alterations compared to control patients

Impact of Myo-inositol supplementation in the prevention of neural tube defects

Motivation: Neural tube defects (NTDs) occur during early development by failure of neural tube closure and cause severe birth problems like spina bifida. It has been demonstrated that the mechanisms required during neural tube closure are regulated by genetic and environmental factors, such as maternal diet. Folic acid supplementation during pregnancy prevents the appearance of NTDs in 70% of the cases and the remaining 30% are considered folate resistant. For that reason, there is a need to find new supplements that could help preventing NTDs. One compound that is being tested is inositol, a simple carbon six sugar alcohol that participates in a diverse range of cellular functions. In clinical trials, inositol was used to prevent NTDs and the offspring from mothers that included inositol and folic acid in their diet during pregnancy did not develop NTDs. In our laboratory we are using Loop-tail, mutant of the member of the Wnt-PCP Vangl2, that in heterozygosity presents an incidence of 6% spina bifida. Previous studies using Loop-tail, revealed that a cellular aggregate originates in neural tube dorsal zone of heterozygous embryos. This aggregate, Sox10 positive, is formed by cells from the neural crest which did not migrate correctly. Besides, this cellular aggregate shares similarities with lipomyelomeningocele, the most common type of spina bifida occulta. In order to prevent the appearance of this cellular aggregate our laboratory previously used D-Chiro-inositol in Loop-tail mice as a supplement. Although the aggregate size and prevalence was reduced, crown-rump length of heterozygous embryos was significantly shorter than control embryos. Therefore, we are currently testing Myo-inositol, the isomeric form of inositol used in the human trials designed to prevent NTDs. Methods: Myo-inositol was administered in the drinking water to pregnant females from day E1.5 of gestation until E11,5 and embryos were collected at stage E12,5. From each litter, data referring to number of implants, resorptions and genotype of the embryos was registered for possible effects on embryotoxicity. In situ hybridization using a Sox10 probe, neural crest marker, was later performed in order to study the presence and intensity of cellular aggregates

Prevention of neural tube defects through maternal supplementation

Neurulation is a crucial step in embryonic development that leads to the formation of the neural tube, a structure that ends up shaping the central nervous system. Neural tube defects (NTDs) appear when neurulation fails and the neural tube does not close completely. These diseases are usually lethal and, in the mildest cases, they are incurable and disabling; for this reason, research on NTDs focuses especially on their prevention. Currently, 70% of NTDs are preventable by maternal supplementation with folic acid, a vitamin necessary for the production of new cells. Our research group attends to find complementary compounds to folic acid for the prevention of folate-resistant NTDs. To that end, we studied the effect of inositol, an organic vitaminic compound with promising results in both human and mouse trials.Our laboratory uses the Looptail mouse model, a mutant strain for the VANGL2 gene, a member of the non-canonical Wnt pathway. In heterozygosity, these mice have a curly tail phenotype and a low incidence of spina bifida. We have also discovered that heterozygous mid-developed embryos have cell aggregates in the neural tube that resemble the most common form of spina bifida occulta in humans. In our research group, we carry out the in situ hybridization technique in these embryos using a probe that marks the aggregates. This allows us to compare the number, intensity and size of cell aggregates between embryos from untreated females and those from supplemented ones. Our preliminary results indicate that D-chiro-inositol combined with folic acid is the best combination for maternal supplementation, demonstrating higher levels of cellular aggregate reduction and no embryo toxicity.On the other hand, we have designed plasmids with the VANGL2 promoter, which together with CRISPR/Cas9 technology, it will allow us to study the effect on gene expression of possible mutations, as well as identify the most important regions of the promoter. Ultimately, our group aims to open new research routes for the prevention of NTDs, in order to avoid these serious and incurable diseases

Neuroprotective strategies against neonatal hypoxia-ischemia.

Motivation: Neonatal hypoxia ischemia (HI) is a brain damage caused by oxygen deprivation in newborns. Nowadays, it continues to be a major cause of neonatal mortality and lifelong neurodevelopmental disabilities worldwide.Neonatal HI physiopathology includes oxidative stress, inflammation, and apoptosis. Some studies have shown that nutraceuticals that have antioxidant, anti-inflammatory or anti-apoptotic properties can prevent neonatal HI by reducing brain damage. Thus, the aim of this work is to evaluate the neuroprotective potential of a plant-derived phenolic compound (PDPC) administered as a pre-treatment before the HI event, with focus on myelination and astroglia activation.Methods: Our group used the Rice-Vannucci mouse model of neonatal HI by ligating the left common carotid artery and then subjecting 7-day-old pups to hypoxia for 90 min. Pups were administered PDPC (at 20 or 100 mg/kg) or saline intraperitoneally 20 min before the intervention. Two days later, the brains were dissected, homogenized and stored at -80ºC for future experiments to evaluate the antioxidant potential of the PDPC. In a parallel study, seven days later, the brains were dissected to study myelination and astroglia activation by immunohistochemistry using MBP (myelin basic protein) and GFAP (glial fibrillary acidic protein) to assess the neuroprotective potential of PDPC.Results: Preliminary results indicate a loss of myelination in the ipsilateral hemispheres (where the carotid artery was ligated) of mice subjected to HI, but this loss seemed to be significantly reduced in a dose dependent manner in those mice pretreated with the PDPC. In the case of astroglia staining, experiments are still ongoing but these suggest an overexpression of astroglia in certain areas of the affected hemisphere of the brain in HI mice, such as the cortex or thalamus. We are currently stablishing an image analysis protocol to evaluate the overexpression of GFAP+ cells and observe if it lowers in the treated versus the non-treated group.Conclusions: To conclude, we can highlight that ongoing experiments suggest that pretreatment with PDPC, specially at 100 mg/kg, preserves myelination and may reduce astroglial activation. Future experiments will also evaluate the antioxidant potential of PDPC pretreatment by measuring the ROS production and the activity of antioxidant enzymes in brain homogenates

Impact of maternal dietary supplementation in the prevention of neuropediatric diseases

Motivation: A balanced maternal diet during pregnancy is crucial for the proper development of the embryo. In this project, we will study the impact of maternal dietary supplementation in the prevention of the development of neonatal strokes and neural tube defects (NTDs) using two mouse models.1. Neonatal stroke is a pathology with a heterogeneous etiology, leading to important sequelae. Currently, hypothermia is the only clinical intervention used in full-term newborns with perinatal asphyxia. However, the influence of diet has recently begun to be considered relevant in reducing brain damage in possible future injuries. Thus, maternal diet supplementation with omega-3 fatty acids as a tool for neuroprotection has been proposed. In order to test this, we will start with the development of a neonatal mouse model of hypoxia-ischemia (HI) and characterize the type and severity of brain injury.2. Neurulation refers to the neural tube formation process. When neural tube fails to close completely, NTDs are originated, including craniorachischisis or spina bifida. While 70% of NTDs can be prevented with folic acid supplementation during pregnancy, the rest remains resistant. Loop-tail mouse is a folic acid resistant model of NTDs who carries a mutation for the Vangl2 gene (which is involved in the Wnt-PCP pathway). Previous studies have shown that inositol prevents NTDs in folate-resistant models. Preliminary studies of our group have suggested that maternal supplementation with D-chiro-inositol during embryonic days (E)8.5-E10.5 has a positive effect on the dorsal fusion of the neural folds and on the distribution of actin present in these. Therefore, we propose to extend the period of supplementation (E1.5-E11.5) to determine whether such effect is more pronounced.Methods: We used the Rice-Vannucci model of HI modified to postnatal day (P)8 CD1 mice. Pups were subjected to unilateral left carotid artery ligation and subsequently exposed to 9% O2 for 45’. Infarct size measurement was done 24h post HI, evaluation of brain damage was done 3 days post-HI and behavioural outcomes were assessed at P8, P9 and P12. To assess the impact of maternal dietary supplementation in the prevention of NTD we provided water supplemeted with D-chiro-inositol to pregnant mice Vangl2+/Lp from E1.5 to E11.5. Embryos were obtained and genotyped at E12.5 for later phenotype analysis. In addition, in situ hybridization studies and fluorescent phalloidin stainings were performe

The effect of maternal diabetes on the Wnt-PCP pathway during embryogenesis as reflected in the developing mouse eye

Embryopathies that develop as a consequence of maternal diabetes have been studied intensely in both experimental and clinical scenarios. Accordingly, hyperglycaemia has been shown to downregulate the expression of elements in the non-canonical Wnt-PCP pathway, such as the Dishevelled-associated activator of morphogenesis 1 (Daam1) and Vangl2. Daam1 is a formin that is essential for actin polymerization and for cytoskeletal reorganization, and it is expressed strongly in certain organs during mouse development, including the eye, neural tube and heart. Daam1gt/gt and Daam1gt/+ embryos develop ocular defects (anophthalmia or microphthalmia) that are similar to those detected as a result of hyperglycaemia. Indeed, studying the effects of maternal diabetes on the Wnt-PCP pathway demonstrated that there was strong association with the Daam1 genotype, whereby the embryopathy observed in Daam1gt/+ mutant embryos of diabetic dams was more severe. There was evidence that embryonic exposure to glucose in vitro diminishes the expression of genes in the Wnt-PCP pathway, leading to altered cytoskeletal organization, cell shape and cell polarity in the optic vesicle. Hence, the Wnt-PCP pathway appears to influence cell morphology and cell polarity, events that drive the cellular movements required for optic vesicle formation and that, in turn, are required to maintain the fate determination. Here, we demonstrate that the Wnt-PCP pathway is involved in the early stages of mouse eye development and that it is altered by diabetes, provoking the ocular phenotype observed in the affected embryos

Prenatal Stroke: Detection and Neuroprotection

Neonatal stroke is the most common cause of death and disability in newborns and is often associated with persistent motor, sensory and cognitive impairments. Recently, great efforts have been made towards neuroprotection with bioactive compounds and because of to the promising results found in adult stroke patients; we consider evaluating it in the pediatric population. In this line of research, our group has developed a neonatal hypoxic-ischemic (HI) mouse model in which the supplementation of pregnant females with olive leaf extract (OLE) is being tested as a neuroprotection tool against the potencial ischemic damage. Through studies of histology, immunohistochemistry and behavior test, we are evaluating the degree of damage between the control group and the one subjected to HI in the presence and absence of supplementation with OLE. On the other hand, the discovery of biomarkers is key for the early diagnosis of neonatal stroke, which would favor the immediate application of neuroprotective therapy with the aim of minimizing the possible sequelae. Based on this, the second part of the project is the collection and analysis of plasma and dried blood samples (as a minimally invasive sample) from each of the different groups under study. Neonatal stroke is a disease with a high emotional burden for patients as well as their families, aside from severe repercussions that have a significant impact on the quality of life of both patients and their families. The design of a neuroprotective strategy and an early diagnosis protocol for perinatal stroke will represent a great advance in the prevention of brain damage and help to increase the life expectancy and the standard of living of this population