Search

This review explores the usefulness retinoic acid (RA) related physiological factors as possible biomarkers of embryotoxicity. RA is involved in the morphogenesis of the early embryo as well as in the development and maturation of a wide variety of organ anlagen. The region-specific homeostasis of RA in the embryo is in many ways the driving force determining developmental cell proliferation versus differentiation. As a consequence, RA concentrations are carefully controlled in time and space in the developing embryo. RA deficiency and overdosing both result in characteristic patterns of malformations that may involve many different organ systems. The central role of RA in embryo development provides us with a set of sensitive biomarkers that may be employed in developmental toxicity testing. This includes the synthesizing and metabolizing enzymes of RA, but also a myriad of related morphogenetic factors and their genes, of which the expression may be affected by changes in RA balance. Several examples of embryotoxicants interfering with the homeostasis of RA and related parameters have been described. A preliminary adverse outcome pathway framework for RA mediated malformations has been published. Expansion of this framework and its application in developmental toxicity testing may allow the detection of a large variety of embryotoxicants with diverse modes of action. RA homeostasis therefore provides a promising set of molecular tools that may be employed in the advancement of mode of action driven animal-free developmental toxicity testing., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Safety and potency assessment for batch release testing of established vaccines still relies partly on animal tests. An important avenue to move to batch release without animal testing is the consistency approach. This approach is based on thorough characterization of the vaccine, and the principle that the quality of subsequent batches is the consequence of the application of consistent production of batches monitored by a GMP quality system. Efforts to implement the consistency approach are supported by several drivers from industry, government, and research, but there are also several barriers that must be overcome. A workshop entitled "Consistency Approach, Drivers and Barriers" was organized, which aimed to discuss and identify drivers and barriers for the implementation of the 3Rs in the consistency approach from three different perspectives/domains (industry, regulatory and science frameworks). The workshop contributed to a better understanding of these drivers and barriers and resulted in recommendations to improve the overall regulatory processes for the consistency approach. With this report, we summarise the outcome of this workshop and intend to offer a constructive contribution to the international discussion on regulatory acceptance of the consistency approach., (Copyright © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Febrile seizures (FS) are the most common seizure type in children. Recurrent FS are a risk factor for developing temporal lobe epilepsy later in life and are known to have a strong genetic component. Experimental FS (eFS) can be elicited in mice by warm-air induced hyperthermia. We used this model to screen the chromosome substitution strain (CSS) panel derived from C57BL/6J and A/J for FS susceptibility and identified C57BL/6J-Chr2 A /NaJ (CSS2), as the strain with the strongest FS susceptibility phenotype. The aim of this study was to map FS susceptibility loci and select candidate genes on mouse chromosome 2. We generated an F 2 population by intercrossing the hybrids (F 1 ) that were derived from CSS2 and C57BL/6J mice. All CSS2-F 2 individuals were genotyped and phenotyped for eFS susceptibility, and QTL analysis was performed. Candidate gene selection was based on bioinformatics analyses and differential brain expression between CSS2 and C57BL/6J strains determined by microarray analysis. Genetic mapping of the eFS susceptibility trait identified two significant loci: FS-QTL2a (LOD-score 3.6) and FS-QTL2b (LOD-score 6.2). FS-QTL2a contained 44 genes expressed in the brain at post natal day 14. Four of these (Arl6ip6, Cytip, Fmnl2 Ifih1) contained a non-synonymous SNP comparing CSS2 and C57BL/6J, six genes (March7, Nr4a2, Gpd2, Grb14, Scn1a, Scn3a) were differentially expressed between these strains. A region within FS-QTL2a is homologous to the human FEB3 locus. The fact that we identify mouse FS-QTL2a with high FEB3 homology is strong support for the validity of the eFS mouse model to study genetics of human FS., (© 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Humans are exposed to bisphenol A (BPA) mainly through the diet, air, dust, skin contact and water. There are concerns about adverse health effects in humans due to exposure to bisphenol A (BPA). The European Food Safety Authority (EFSA) has extensively reviewed the available literature to establish a temporary Tolerable Daily Intake (t-TDI). This exposure level was based on all available literature published before the end of 2012. Since then, new experimental animal studies have emerged, including those that identified effects of BPA on the immune system after developmental exposure. These studies indicate that developmental immunotoxicity might occur at lower dose levels than previously observed and on which the current EFSA t-TDI is based. The Dutch National Institute for Public Health and the Environment (RIVM) organized an expert workshop in September 2015 to consider recently published studies on the developmental immunotoxicity of bisphenol A (BPA). Key studies were discussed in the context of other experimental studies. The workshop concluded that these new experimental studies provide credible evidence for adverse immune effects after developmental exposure to BPA at 5μg/kg BW/day from gestation day 15 to postnatal day 21. Supportive evidence for adverse immune effects in similar dose ranges was obtained from other publications that were discussed during the workshop. The dose level associated with adverse immune effects is considerably lower than the dose used by EFSA for deriving the t-TDI. The workshop unanimously concluded that the current EFSA t-TDI warrants reconsideration in the context of all currently available data., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Around 25% of the children in developed countries are affected with immune-based diseases. Juvenile onset diseases such as allergic, inflammatory and autoimmune diseases have shown increasing prevalences in the last decades. The role of chemical exposures in these phenomena is unclear. It is thought that the developmental immune system is more susceptible to toxicants than the mature situation. Developmental immunotoxicity (DIT) testing is nowadays not or minimally included in regulatory toxicology requirements. We reviewed whether developmental immune parameters in rodents would provide relatively sensitive endpoints of toxicity, whose inclusion in regulatory toxicity testing might improve hazard identification and risk assessment of chemicals. For each of the nine reviewed toxicants, the developing immune system was found to be at least as sensitive or more sensitive than the general (developmental) toxicity parameters. Functional immune (antigen-challenged) parameters appear more affected than structural (non-challenged) immune parameters. Especially, antibody responses to immune challenges with keyhole limpet hemocyanine or sheep red blood cells and delayed-type hypersensitivity responses appear to provide sensitive parameters of developmental immune toxicity. Comparison with current tolerable daily intakes (TDI) and their underlying overall no observed adverse effect levels showed that for some of the compounds reviewed, the TDI may need reconsideration based on developmental immune parameters. From these data, it can be concluded that the developing immune system is very sensitive to the disruption of toxicants independent of study design. Consideration of including functional DIT parameters in current hazard identification guidelines and wider application of relevant study protocols is warranted.

Mesiotemporal sclerosis (MTS), the most frequent form of drug-resistant temporal lobe epilepsy, often develops after an initial precipitating injury affecting the immature brain. To analyse early processes in epileptogenesis we used the juvenile pilocarpine model to study status epilepticus (SE)-induced changes in expression of key components in the glutamate-glutamine cycle, known to be affected in MTS patients. SE was induced by Li(+) /pilocarpine injection in 21-day-old rats. At 2-19 weeks after SE hippocampal protein expression was analysed by immunohistochemistry and neuron damage by FluoroJade staining. Spontaneous seizures occurred in at least 44% of animals 15-18 weeks after SE. As expected in this model, we did not observe loss of principal hippocampal neurons. Neuron damage was most pronounced in the hilus, where we also detected progressive loss of parvalbumin-positive GABAergic interneurons. Hilar neuron loss (or end-folium sclerosis), a common feature in patients with MTS, was accompanied by a progressively decreased glutamine synthetase (GS)-immunoreactivity from 2 (-15%) to 19 weeks (-33.5%) after SE. Immunoreactivity for excitatory amino-acid transporters, vesicular glutamate transporter 1 and glial fibrillary acidic protein was unaffected. Our data show that SE elicited in 21-day-old rats induces a progressive reduction in hilar GS expression without affecting other key components of the glutamate-glutamine cycle. Reduced expression of glial enzyme GS was first detected 2 weeks after SE, and thus clearly before spontaneous recurrent seizures occurred. These results support the hypothesis that reduced GS expression is an early event in the development of hippocampal sclerosis in MTS patients and emphasize the importance of astrocytes in early epileptogenesis., (© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Aims: Early-life seizures, particularly when prolonged, may be harmful to the brain. Current pharmacotherapy is often ineffective; therefore, novel neuro- and/or glio-transmitter systems should be explored for targeting. The P2X7 receptor is a cation-permeable channel with trophic and excitability effects on neurons and glia which is activated by high amounts of ATP that may be released in the setting of injury after severe seizures. Here, we tested the effects of A-438079, a potent and selective P2X7 receptor antagonist in a lesional model of early-life status epilepticus., Methods: Seizures were induced by intra-amygdala kainic acid in 10-day-old rat pups. Electrographic seizure severity, changes to P2X7 receptor expression, inflammatory responses and histological effects were evaluated., Results: Seizures induced by intra-amygdala kainic acid increased levels of P2X7 receptor protein and interleukin-1β and caused significant cell death within the ipsilateral hippocampus. A-438079 rapidly reached the brain following systemic injection in P10 rats. Intraperitoneal injection of A-438079 (5 and 15 mg/kg) 60 min after triggering seizures reduced seizure severity and neuronal death within the hippocampus. A-438079 had superior neuroprotective effects compared with an equally seizure-suppressive dose of phenobarbital (25 mg/kg)., Conclusions: These results suggest P2X7 receptor antagonists may be suitable as frontline or adjunctive treatments of pediatric status epilepticus or other early-life seizures, particularly when associated with brain damage., (© 2014 John Wiley & Sons Ltd.)

Objective: Febrile seizures (FS) are the most common seizure type in young children. Complex FS are a risk factor for mesial temporal lobe epilepsy (mTLE). To identify new FS susceptibility genes we used a forward genetic strategy in mice and subsequently analyzed candidate genes in humans., Methods: We mapped a quantitative trait locus (QTL1) for hyperthermia-induced FS on mouse chromosome 1, containing the signal recognition particle 9 (Srp9) gene. Effects of differential Srp9 expression were assessed in vivo and in vitro. Hippocampal SRP9 expression and genetic association were analyzed in FS and mTLE patients., Results: Srp9 was differentially expressed between parental strains C57BL/6J and A/J. Chromosome substitution strain 1 (CSS1) mice exhibited lower FS susceptibility and Srp9 expression than C57BL/6J mice. In vivo knockdown of brain Srp9 reduced FS susceptibility. Mice with reduced Srp9 expression and FS susceptibility, exhibited reduced hippocampal AMPA and NMDA currents. Downregulation of neuronal Srp9 reduced surface expression of AMPA receptor subunit GluA1. mTLE patients with antecedent FS had higher SRP9 expression than patients without. SRP9 promoter SNP rs12403575(G/A) was genetically associated with FS and mTLE., Interpretation: Our findings identify SRP9 as a novel FS susceptibility gene and indicate that SRP9 conveys its effects through endoplasmic reticulum (ER)-dependent synthesis and trafficking of membrane proteins, such as glutamate receptors. Discovery of this new FS gene and mechanism may provide new leads for early diagnosis and treatment of children with complex FS at risk for mTLE.

Synthetic glucocorticoids are administered to pregnant women at risk for preterm delivery, to enhance fetal lung maturation. The benefit of this treatment is well established, however caution is necessary because of possible unwanted side effects on development of different organ systems, including the brain. Actions of glucocorticoids are mediated by corticosteroid receptors, which are highly expressed in the hippocampus, a brain structure involved in cognitive functions. Therefore, we analyzed the effects of a single antenatal dexamethasone treatment on the development of the mouse hippocampus. A clinically relevant dose of dexamethasone (0.4 mg/kg) was administered to pregnant mice at embryonic day 15.5 and the hippocampus was analyzed from embryonic day 16 until adulthood. We investigated the effects of dexamethasone treatment on anatomical changes, apoptosis and proliferation in the hippocampus, hippocampal volume and on total body weight. Our results show that dexamethasone treatment reduced body weight and hippocampal volume transiently during development, but these effects were no longer detected at adulthood. Dexamethasone treatment increased the number of apoptotic cells in the hippocampus until birth, but postnatally no effects of dexamethasone treatment on apoptosis were found. During the phase with increased apoptosis, dexamethasone treatment reduced the number of proliferating cells in the subgranular zone of the dentate gyrus. The number of proliferative cells was increased at postnatal day 5 and 10, but was decreased again at the adult stage. This latter long-term and negative effect of antenatal dexamethasone treatment on the number of proliferative cells in the hippocampus may have important implications for hippocampal network function.

Epilepsy comprises several syndromes, amongst the most common being mesial temporal lobe epilepsy with hippocampal sclerosis. Seizures in mesial temporal lobe epilepsy with hippocampal sclerosis are typically drug-resistant, and mesial temporal lobe epilepsy with hippocampal sclerosis is frequently associated with important co-morbidities, mandating the search for better understanding and treatment. The cause of mesial temporal lobe epilepsy with hippocampal sclerosis is unknown, but there is an association with childhood febrile seizures. Several rarer epilepsies featuring febrile seizures are caused by mutations in SCN1A, which encodes a brain-expressed sodium channel subunit targeted by many anti-epileptic drugs. We undertook a genome-wide association study in 1018 people with mesial temporal lobe epilepsy with hippocampal sclerosis and 7552 control subjects, with validation in an independent sample set comprising 959 people with mesial temporal lobe epilepsy with hippocampal sclerosis and 3591 control subjects. To dissect out variants related to a history of febrile seizures, we tested cases with mesial temporal lobe epilepsy with hippocampal sclerosis with (overall n = 757) and without (overall n = 803) a history of febrile seizures. Meta-analysis revealed a genome-wide significant association for mesial temporal lobe epilepsy with hippocampal sclerosis with febrile seizures at the sodium channel gene cluster on chromosome 2q24.3 [rs7587026, within an intron of the SCN1A gene, P = 3.36 × 10(-9), odds ratio (A) = 1.42, 95% confidence interval: 1.26-1.59]. In a cohort of 172 individuals with febrile seizures, who did not develop epilepsy during prospective follow-up to age 13 years, and 6456 controls, no association was found for rs7587026 and febrile seizures. These findings suggest SCN1A involvement in a common epilepsy syndrome, give new direction to biological understanding of mesial temporal lobe epilepsy with hippocampal sclerosis with febrile seizures, and open avenues for investigation of prognostic factors and possible prevention of epilepsy in some children with febrile seizures.

Mesial temporal lobe epilepsy (mTLE) is a chronic neurological disorder characterized by recurrent seizures. The pathogenic mechanisms underlying mTLE may involve defects in the post-transcriptional regulation of gene expression. MicroRNAs (miRNAs) are non-coding RNAs that control the expression of genes at the post-transcriptional level. Here, we performed a genome-wide miRNA profiling study to examine whether miRNA-mediated mechanisms are affected in human mTLE. miRNA profiles of the hippocampus of autopsy control patients and two mTLE patient groups were compared. This revealed segregated miRNA signatures for the three different patient groups and 165 miRNAs with up- or down-regulated expression in mTLE. miRNA in situ hybridization detected cell type-specific changes in miRNA expression and an abnormal nuclear localization of select miRNAs in neurons and glial cells of mTLE patients. Of several cellular processes implicated in mTLE, the immune response was most prominently targeted by deregulated miRNAs. Enhanced expression of inflammatory mediators was paralleled by a reduction in miRNAs that were found to target the 3'-untranslated regions of these genes in reporter assays. miR-221 and miR-222 were shown to regulate endogenous ICAM1 expression and were selectively co-expressed with ICAM1 in astrocytes in mTLE patients. Our findings suggest that miRNA changes in mTLE affect the expression of immunomodulatory proteins thereby further facilitating the immune response. This mechanism may have broad implications given the central role of astrocytes and the immune system in human neurological disease. Overall, this work extends the current concepts of human mTLE pathogenesis to the level of miRNA-mediated gene regulation.

Purpose: Febrile seizures (FS) are the most common seizure type in children between the age of 6 months and 5 years. Although FS are largely benign, recurrent FS are a major risk factor for developing temporal lobe epilepsy (TLE) later in life. The mechanisms underlying FS are largely unknown; however, family and twin studies indicate that FS susceptibility is under complex genetic control. We have recently developed a phenotypic screen to study the genetics of FS susceptibility in mice. Using this screen in a phenotype-driven genetic strategy we analyzed the C57BL/6J-Chr #(A)/NaJ chromosome substitution strain (CSS) panel. In each CSS line one chromosome of the A/J strain is substituted in a genetically homogeneous C57BL/6J background. The analysis of the CSS panel revealed that A/J chromosomes 1, 2, 6, 10, 13, and X carry at least one quantitative trait locus (QTL) for heat-induced FS susceptibility. The fact that many X-linked genes are highly expressed in the brain and have been implicated in human developmental disorders often presenting with seizures (like fragile X mental retardation) prompted us to map the chromosome X QTL., Methods: C57BL/6J mice were mated with C57BL/6J-Chr X(A) /NaJ (CSSX) to generate F(2)-generations-CXBL6 and BL6CX-originating from CSSX or C57BL/6J mothers, respectively. Heat-induced FS were elicited on postnatal day 14 by exposure to a controlled warm airstream of 50°C. The latency to heat-induced FS is our phenotype. This phenotype has previously been validated by video-electroencephalography (EEG) monitoring. After phenotyping and genotyping the F(2)-population, QTL analysis was performed using R/QTL software., Key Findings: QTL analysis revealed a significant peak with an LOD-score of 3.25. The 1-LOD confidence interval (149,886,866-158,836,462 bp) comprises 52 protein coding genes, of which 34 are known to be brain expressed. Two of these brain-expressed genes have previously been linked to X-linked epilepsies, namely Cdkl5 and Pdha1., Significance: Our results show that the mouse genetics of X-linked FS susceptibility is complex, and that our heat-induced FS-driven genetic approach is a powerful tool for use in unraveling the complexities of this trait in mice. Fine-mapping and functional studies will be required to further identify the X-linked FS susceptibility genes., (Wiley Periodicals, Inc. © 2012 International League Against Epilepsy.)

In rodents, the mediobasal hypothalamus and the hypothalamic paraventricular nucleus (PVN) are implicated in leptin signaling. Surprisingly little data is available on the human hypothalamus. We set out to study the expression of suppressor-of-cytokine-signaling 3 (SOCS3), α-melanocyte stimulating hormone (αMSH) and agouti-related protein (AgRP) in the infundibular nucleus (IFN) and to investigate the relationship between these neuropeptide expressions and serum leptin concentrations in a blood sample taken within 24h before death. We studied post-mortem human brain material by means of quantitative immunocytochemistry. We found that SOCS3 immunoreactivity was widely distributed throughout the hypothalamus, and most prominent in the PVN, whereas expression levels in the IFN were low. Surprisingly, SOCS3 expression in the PVN was inversely related to serum leptin. A significant positive correlation was observed between AgRP and NPY expression in the IFN. The inverse correlation between SOCS3 expression in the PVN and serum leptin was unexpected and may be related to the hypothalamic adaptation to fatal illness rather than to nutritional status, or may represent an interspecies difference., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Febrile seizures (FS) are the most common seizure type in children and recurrent FS are a risk factor for developing temporal lobe epilepsy. Although the mechanisms underlying FS are largely unknown, recent family, twin and animal studies indicate that genetics are important in FS susceptibility. Here, a forward genetic strategy was used employing mouse chromosome substitution strains (CSS) to identify novel FS susceptibility quantitative trait loci (QTLs). FS were induced by exposure to warm air at postnatal day 14. Video electroencephalogram monitoring identified tonic-clonic convulsion onset, defined as febrile seizure latency (FSL), as a reliable phenotypic parameter to determine FS susceptibility. FSL was determined in both sexes of the host strain (C57BL/6J), the donor strain (A/J) and CSS. C57BL/6J mice were more susceptible to FS than A/J mice. Phenotypic screening of the CSS panel identified six strains(CSS1, -2, -6 -10, -13 and -X) carrying QTLs for FS susceptibility. CSS1, -10 and -13 were less susceptible (protective QTLs), whereas CSS2, -6 and -X were more susceptible (susceptibility QTLs) to FS than the C57BL/6J strain. Our data show that mouse FS susceptibility is determined by complex genetics, which is distinct from that for chemically induced seizures. This is the first dataset using CSS to screen for a seizure trait in mouse pups. It provides evidence for common FS susceptibility QTLs that serve as starting points to fine map FS susceptibility QTLs and to identify FS susceptibility genes. This will increase our understanding of human FS, working toward the identification of new therapeutic targets.

Febrile seizures (FS) are the most prevalent seizures in children. Although FS are largely benign, complex FS increase the risk to develop temporal lobe epilepsy (TLE). Studies in rat models for FS have provided information about functional changes in the hippocampus after complex FS. However, our knowledge about the genes and pathways involved in the causes and consequences of FS is still limited. To enable molecular, genetic and knockout studies, we developed and characterized an FS model in mice and used it as a phenotypic screen to analyze FS susceptibility. Hyperthermia was induced by warm air in 10- to 14-day-old mice and induced FS in all animals. Under the conditions used, seizure-induced behavior in mice and rats was similar. In adulthood, treated mice showed increased hippocampal Ih current and seizure susceptibility, characteristics also seen after FS in rats. Of the seven genetically diverse mouse strains screened for FS susceptibility, C57BL/6J mice were among the most susceptible, whereas A/J mice were among the most resistant. Strains genetically similar to C57BL/6J also showed a susceptible phenotype. Our phenotypic data suggest that complex genetics underlie FS susceptibility and show that the C57BL/6J strain is highly susceptible to FS. As this strain has been described as resistant to convulsants, our data indicate that susceptibility genes for FS and convulsants are distinct. Insight into the mechanisms underlying seizure susceptibility and FS may help to identify markers for the early diagnosis of children at risk for complex FS and TLE and may provide new leads for treatment.