Ocular measurements in fetal alcohol spectrum disorders.

Fetal alcohol spectrum disorders (FASD) describe a range of physical, behavioral, and neurologic deficits in individuals exposed to alcohol prenatally. Reduced palpebral fissure length is one of the cardinal facial features of FASD. However, other ocular measurements have not been studied extensively in FASD. Using the Fetal Alcohol Syndrome Epidemiologic Research (FASER) database, we investigated how inner canthal distance (ICD), interpupillary distance (IPD), and outer canthal distance (OCD) centiles differed between FASD and non-FASD individuals. We compared ocular measurement centiles in children with FASD to non-FASD individuals and observed reductions in all three centiles for ICD, IPD, and OCD. However, when our non-FASD children who had various forms of growth deficiency (microcephaly, short-stature, or underweight) were compared to controls, we did not observe a similar reduction in ocular measurements. This suggests that reductions in ocular measurements are a direct effect of alcohol on ocular development independent of its effect on growth parameters, which is consistent with animal models showing a negative effect of alcohol on developing neural crest cells. Interpupillary distance centile appeared to be the most significantly reduced ocular measure we evaluated, suggesting it may be a useful measure to be considered in the diagnosis of FASD.

A systems biology approach to predictive developmental neurotoxicity of a larvicide used in the prevention of Zika virus transmission.

The need to prevent developmental brain disorders has led to an increased interest in efficient neurotoxicity testing. When an epidemic of microcephaly occurred in Brazil, Zika virus infection was soon identified as the likely culprit. However, the pathogenesis appeared to be complex, and a larvicide used to control mosquitoes responsible for transmission of the virus was soon suggested as an important causative factor. Yet, it is challenging to identify relevant and efficient tests that are also in line with ethical research defined by the 3Rs rule (Replacement, Reduction and Refinement). Especially in an acute situation like the microcephaly epidemic, where little toxicity documentation is available, new and innovative alternative methods, whether in vitro or in silico, must be considered. We have developed a network-based model using an integrative systems biology approach to explore the potential developmental neurotoxicity, and we applied this method to examine the larvicide pyriproxyfen widely used in the prevention of Zika virus transmission. Our computational model covered a wide range of possible pathways providing mechanistic hypotheses between pyriproxyfen and neurological disorders via protein complexes, thus adding to the plausibility of pyriproxyfen neurotoxicity. Although providing only tentative evidence and comparisons with retinoic acid, our computational systems biology approach is rapid and inexpensive. The case study of pyriproxyfen illustrates its usefulness as an initial or screening step in the assessment of toxicity potentials of chemicals with incompletely known toxic properties.

Pyriproxyfen and the microcephaly epidemic in Brazil - an ecological approach to explore the hypothesis of their association.

The microcephaly epidemic in Brazil generated intense debate regarding its causality, and one hypothesised cause of this epidemic, now recognised as congenital Zika virus syndrome, was the treatment of drinking water tanks with pyriproxyfen to control Aedes aegypti larvae. We present the results of a geographical analysis of the association between the prevalence of microcephaly confirmed by Fenton growth charts and the type of larvicide used in the municipalities that were home to the mothers of the affected newborns in the metropolitan region of Recife in Pernambuco, the state in Brazil where the epidemic was first detected. The overall prevalence of microcephaly was 82 per 10,000 live births in the three municipalities that used the larvicide Bti (Bacillus thuringiensis israelensis) instead of pyriproxyfen, and 69 per 10,000 live births in the eleven municipalities that used pyriproxyfen. The difference was not statistically significant. Our results show that the prevalence of microcephaly was not higher in the areas in which pyriproxyfen was used. In this ecological approach, there was no evidence of a correlation between the use of pyriproxyfen in the municipalities and the microcephaly epidemic.

Fetal Growth Outcomes in a Cohort of Polydrug- and Opioid-Dependent Patients.

OBJECTIVES: To evaluate the effects of prenatal polydrug and exclusive opioid use on fetal growth outcomes. METHODS: This analysis relied on the data obtained from two prospective cohorts at the University of New Mexico. For both cohorts, pregnant women were recruited during one of their prenatal care visits and followed up to delivery. The merged sample included 59 polydrug users, 22 exclusive opioid users, and 278 abstinent controls. Continuous growth measures (birth weight, height, occipital frontal circumference [OFC], and corresponding sex-specific percentiles) were compared by ANOVA and ANCOVA in bivariate and multivariable analyses, respectively. Categorical outcomes (prevalence of small-for-gestational age [SGA] for weight, length, and OFC) were compared among groups by Chi-square and multivariable logistic regression analyses.. RESULTS: The sample included a large proportion of ethnic minorities (78.8% Hispanic) and patients with low educational attainment (68% ≤ high school). The risk of microcephaly (OFC<10th percentile) was significantly greater in the polydrug (OR=4.7; 95% CI: 2.0; 10.8) and exclusive opioid (OR=2.8; 95% CI: 1.0; 8.1) groups compared to abstinent controls. CONCLUSION: Given that microcephaly is often associated with serious neurocognitive and behavioral deficits later in life, our finding of 49.2% incidence of microcephaly among polydrug users is alarming and requires further investigation.

Prenatal alcohol exposure patterns and alcohol-related birth defects and growth deficiencies: a prospective study.

BACKGROUND: The physical features of fetal alcohol syndrome include smooth philtrum, thin vermillion border, short palpebral fissures, microcephaly, and growth deficiencies on weight and height. However, little is known about the specific quantities of alcohol exposure, pattern of drinking, timing of exposure, and magnitude of risk for each of these features. METHODS: Using data on 992 subjects collected prospectively in California between 1978 and 2005, we examined the patterns and timing of alcohol exposure in relation to these features. Structural features were assessed by a dysmorphologist who performed a blinded physical examination of all infants. Patterns of drinking were evaluated by drinks per day, number of binge episodes, and maximum number of drinks. Timing of exposure was evaluated 0 to 6 weeks postconception, 6 to 12 weeks postconception, first trimester, second trimester, and third trimester. RESULTS: Higher prenatal alcohol exposure in every pattern was significantly associated with the incidence of smooth philtrum but not with short palpebral fissures. The strongest associations were with timing of exposure in the second half of the first trimester (RR 1.25, 95% CI 1.14 to 1.36 for average number of drinks per day; RR 1.17, 95% CI 1.09 to 1.26 for maximum number of drinks in 1 episode). Similarly, thin vermillion border was most strongly associated with exposure in the second half of the first trimester. Findings with respect to timing of exposure were similar for microcephaly and reduced birth weight. However, reduced birth length was increased with exposure in any trimester. These associations were linear, and there was no evidence of a threshold. CONCLUSIONS: Reduced birth length and weight, microcephaly, smooth philtrum, and thin vermillion border are associated with specific gestational timing of prenatal alcohol exposure and are dose-related without evidence of a threshold. Women should continue to be advised to abstain from alcohol consumption from conception throughout pregnancy.

Differential effects of antiepileptic drugs on neonatal outcomes.

Offspring of women with epilepsy (WWE) on AEDs are at increased risks for major congenital malformations and reduced cognition. They may be at risk for other adverse neonatal outcomes. Women with epilepsy on carbamazepine (CBZ), lamotrigine (LTG), phenytoin (PHT), or valproate (VPA) monotherapy were enrolled in a prospective, observational, multicenter study of the neurodevelopmental effects of AEDs. The odds ratio for small for gestational age (SGA) was higher for VPA vs. PHT, VPA vs. LTG, and CBZ vs. PHT. Microcephaly rates were elevated to 12% for all newborns and at 12 months old, but normalized by age 24 months. Reduced Apgar scores occurred more frequently in the VPA and PHT groups at 1 min, but scores were near normal in all groups at 5 min. This study demonstrates increased risks for being born SGA in the VPA and CBZ groups, and transiently reduced Apgar scores in the VPA and PHT groups. Differential risks among the AEDs can help inform decisions about AED selection for women during childbearing years.

Prevention of retinoic acid-induced early craniofacial abnormalities by vitamin B12 in mice.

OBJECTIVE: The purpose of the present study was to identify the potential effect of prenatal vitamin B12 administration on retinoic acid (RA)-induced early craniofacial abnormalities in mice and to investigate the possible mechanisms by which vitamin B12 reduces malformations. DESIGN: In our study, whole embryo culture was used to explore the effect of vitamin B12 on mouse embryos during the critical period of organogenesis. All embryos were exposed to 0.4 µM RA and different concentrations of vitamin B12 and scored for their growth in the branchial region at the end of a 48-hour culture period. The endothelin-1 (ET-1)/dHAND protein expression levels in the first branchial arch were investigated using an immunohistochemical method. RESULTS: In the whole embryo culture, 100 and 10 µM vitamin B12 dose-dependently prevented branchial region malformations and decreased craniofacial defects by 90.5% and 77.3%, respectively. ET-1 and dHAND protein levels were significantly increased in vitamin B12-supplemented embryos compared to the RA-exposed group in embryonic branchial region. CONCLUSIONS: These results suggest that vitamin B12 may prevent RA-induced craniofacial abnormalities via prevention of an RA-induced decrease of ET-1 and dHAND protein levels in the branchial region during the organogenic period. This study may shed new light on preventing craniofacial abnormalities.

Effects of methylazoxymethanol-induced micrencephaly on parvalbumin-positive GABAergic interneurons in the rat rostral basolateral amygdala.

The amygdala plays a crucial role in anxiety-related behavior and various neuropsychiatric disorders. The offspring of dams, administered methylazoxymethanol acetate (MAM) intraperitoneally at gestational day 15, exhibit micrencephaly and anxiety-related behavior, such as hyperactivity in rearing and crossing behavior, alongside a distinct Fos expression profile in the basolateral (BLA) and central amygdala. However, the histochemical underpinnings of these changes remain to be elucidated. To determine the histochemical alterations in MAM-induced model rats, we performed Nissl staining, immunohistochemistry for parvalbumin (PV) or calbindin (Calb), and immunohistochemistry for PV in conjunction with in situ hybridization for glutamate decarboxylase (GAD). We compared immunoreactivity in the BLA between normal and MAM-induced model rats and observed a significant decrease in the number of PV-positive neurons in MAM-induced model rats; however, no significant differences in the number of Nissl- and Calb-positive neurons were observed. We did not detect any significant between-group differences with regards to the effects of environmental enrichment on the number of PV-positive neurons in the BLA. Double-labeling for GAD and PV revealed that many PV-positive neurons colocalized with digoxigenin-GAD65/67 signals. In addition, GAD/PV double-positive neurons and the total number of GAD-positive neurons in the BLA were lower in the MAM-induced model rats. These results indicate that histochemical alterations observed in the BLA of the MAM-induced model rats may attribute to an aberrant GABAergic inhibitory system.

Methylnitrosourea induces neural progenitor cell apoptosis and microcephaly in mouse embryos.

BACKGROUND: Prenatal exposure to methylnitrosourea (MNU), an alkylating agent, induces microcephaly in mice. However, its pathogenetic mechanism has not been clarified, especially that in the development of the cerebral cortex. METHODS: ICR mice were treated with MNU at 10 mg/kg intraperitoneally on day 13.5 or 15.5 of gestation, and the embryos were observed histologically 24 hr after treatment with MNU or at term. To clarify the pathogenesis of microcephaly and histological changes, especially apoptosis, neurogenesis, and neural migration/positioning, we performed histological analysis employing a cell-specific labeling experiment using thymidine-like substances (BrdU, CldU, and IdU) and markers of neurons/neural stem cells. RESULTS: Histological abnormalities of the dorsal telencephalon, and the excessive cell death of proliferative neural progenitor/stem cells were noted in the MNU-treated embryos. The highest frequencies of cell death occurred at 36 hr after MNU treatment, and little or no neurogenesis was observed in the ventricular zone of the dorsal telencephalon. Abnormality of the radial migration was caused by the reduction of radial fibers in the radial glias. Birth-date analysis revealed the abnormal positioning of neurons and aberrant lamination of the cerebral cortex. CONCLUSIONS: Our data suggest that prenatal exposure to MNU induces the excessive cell death of neural precursor/stem cells, and the defective development of the cerebral cortex, resulting in microcephalic abnormalities.

Intellectual deficit associated with transplacentally induced microcephaly in the rat.

Fischer rats injected with methylazoxymethanol late in pregnancy produce young with considerably reduced cerebral hemispheres. They appear normal otherwise. As adults these animals make many more errors in the Hebb-Williams maze than do control animals.

Effects of environmental enrichment on the activity of the amygdala in micrencephalic rats exposed to a novel open field.

Environmental enrichment (EE) mediates recovery from sensory, motor, and cognitive deficits and emotional abnormalities. In the present study, we examined the effects of EE on locomotor activity and neuronal activity in the amygdala in control and methylazoxymethanol acetate (MAM)-induced micrencephalic rats after challenge in a novel open field. Control rats housed in EE (CR) showed reduced locomotor activity compared to rats housed in a conventional cage (CC), whereas hyperactivity was seen in MAM rats housed in a conventional cage (MC) and in MAM rats housed in EE (MR). Novel open field exposure in both CC and MC resulted in a marked increase in Fos expression in the anterior and posterior parts of the basolateral amygdaloid nucleus, basomedial nucleus, and medial nucleus, whereas these increases in expression were not observed in CR. The effect of EE on Fos expression in the amygdala was different in MR exposed to a novel open field compared to CR. Furthermore, we observed a quite different pattern of Fos expression in the central nucleus of the amygdala between control and MAM rats. The present results suggest that neuronal activity in the amygdala that responds to anxiety is altered in MAM rats, especially when the rats are reared in EE. These alterations may cause behavioral differences between control and MAM rats.

Indole-3-acetic acid induces microencephaly in mouse fetuses.

To determine the effect of indole-3-acetic acid (IAA), known as natural auxin, on developing fetus, pregnant mice were injected with 500 or 1000 mg/kg on various gestation days (Days). With the repeated treatment during Days 7-15, the fetal brains exhibited a reduction in size and weight in a dose-dependent manner on Day 18. Histopathologically, hypoplasia of the cortical plate, piriform cortex, hippocampus and thalamus were observed. From the single treatment on 1 day during Days 9-14, the sensitive period of IAA-induced microencephaly was found to be during Days 10-13 and the most significant response in the fetuses was seen on Day 11 or 12. With the repeated treatment during Days 11-13, apoptotic cells mainly increased in the medial and dorsal layer of the neuroepithelium and prepalate with a reduction in cell density in the telencephalon, diencephalon, mesencephalon and metencephalon on Day 12.5. p53-positve cells were detected associated with apoptotic cells in neuroepithelium. Therefore, IAA administration to pregnant mice induces apoptosis mediated by p53 in the embryo's neuroepithelium, decreases formation of neurons and leads to microencephaly in the fetuses.

The larvicide pyriproxyfen blamed during the Zika virus outbreak does not cause microcephaly in zebrafish embryos.

Although the zika virus (ZIKV) has now been strongly correlated with emerging cases of microcephaly in the Americas, suspicions have been raised regarding the use of pyriproxyfen, a larvicide that prevents mosquito development, in drinking water. The effects of this compound on neurodevelopment have not yet been addressed specifically in vertebrates. As a result, we aimed at addressing the effects, if any, of pyriproxyfen on neurodevelopment in the zebrafish embryo as a vertebrate model. Using zebrafish transgenic lines expressing GFP in different cell populations (elavl3 in newborn neurons, gfap and nestin in neural stem cells), we focused on the analysis of whole embryonic brain volume after confocal 3D-reconstruction and the quantification of purified neural stem cells during early neurodevelopment by FACS-cell sorting from whole in vivo embryos. Interestingly, though lethal at very high doses, pyriproxyfen did not cause brain malformation nor any significant changes in the number of observed stem cells in the developing central nervous system. Our data indicate that pyriproxyfen does not affect central nervous system development in zebrafish, suggesting that this larvicide on its own, may not be correlated with the increase in microcephaly cases reported recently.

Neuronal apoptosis and gray matter heterotopia in microcephaly produced by cytosine arabinoside in mice.

Primary microcephaly can be accompanied by numerous migration anomalies. This experiment was undertaken to examine the pathogenesis of gray matter heterotopia and microcephaly that is produced after administering cytosine arabinoside (Ara-C) to mice. Pregnant mice were intraperitoneally injected with Ara-C at 30 mg/kg body weight on days 13.5 and 14.5 of gestation, and then their offspring were examined. On embryonic day 15.5, in the ventricular zone of the cingulate cortex, the neuroepithelial cells lacked BrdU immunoreactivity. Nestin-immunoreactive radial glial fibers and calretinin-positive subplate fibers were disrupted. TUNEL reaction was remarkable throughout the cerebral hemisphere. Subcortical heterotopia in the cingulate cortex and subependymal nodular heterotopia in the dorsolateral part of the lateral ventricles became detectable by the first day after birth. Thirty-two days after birth, microcephaly was apparent; subcortical heterotopia was observed to have increased in size while it was still located in the frontal and cingulate cortices. This experiment demonstrated that Ara-C induces neuronal apoptosis throughout the cerebral hemisphere. The immunohistochemical characteristics in the gray matter heterotopia suggest that both the subcortical and the subependymal heterotopias were formed by neurons originally committed to the neocortex. We conclude that the gray matter heterotopia that accompanies the microcephaly was produced by a disturbance of radial, tangential, and interkinetic neuronal migrations due to the toxicity of Ara-C in the immature developing brain.

Ascorbic acid inhibits ROS production, NF-kappa B activation and prevents ethanol-induced growth retardation and microencephaly.

In this study, we established an embryo model to study the effects of ethanol on fetal development. When embryos of Xenopus laevis (the African clawed frog) were exposed to ethanol, the resultant tadpoles had significantly reduced brain sizes (microencephaly) and retarded growth rates. These effects, similar to those observed in human fetal alcohol syndrome (FAS), were dose- and time-dependent. We further showed that the antioxidant ascorbic acid (vitamin C) could inhibit the ethanol-induced reactive oxygen species (ROS) production and NF-kappaB activation and protect the ethanol-treated embryos against microencephaly and growth retardation. These results suggest the involvement of NF-kappaB and oxidative stress in ethanol-mediated developmental defects, and the potential use of ascorbic acid as a new and effective protective agent for FAS.

ENU mutagenesis identifies mice modeling Warburg Micro Syndrome with sensory axon degeneration caused by a deletion in Rab18.

Mutations in the gene of RAB18, a member of Ras superfamily of small G-proteins, cause Warburg Micro Syndrome (WARBM) which is characterized by defective neurodevelopmental and ophthalmological phenotypes. Despite loss of Rab18 had been reported to induce disruption of the endoplasmic reticulum structure and neuronal cytoskeleton organization, parts of the pathogenic mechanism caused by RAB18 mutation remain unclear. From the N-ethyl-N-nitrosourea (ENU)-induced mutagenesis library, we identified a mouse line whose Rab18 was knocked out. This Rab18(-/-) mouse exhibited stomping gait, smaller testis and eyes, mimicking several features of WARBM. Rab18(-/-) mice were obviously less sensitive to pain and touch than WT mice. Histological examinations on Rab18(-/-) mice revealed progressive axonal degeneration in the optic nerves, dorsal column of the spinal cord and sensory roots of the spinal nerves while the motor roots were spared. All the behavioral and pathological changes that resulted from abnormalities in the sensory axons were prevented by introducing an extra copy of Rab18 transgene in Rab18(-/-) mice. Our results reveal that sensory axonal degeneration is the primary cause of stomping gait and progressive weakness of the hind limbs in Rab18(-/-) mice, and optic nerve degeneration should be the major pathology of progressive optic atrophy in children with WARBM. Our results indicate that the sensory nervous system is more vulnerable to Rab18 deficiency and WARBM is not only a neurodevelopmental but also neurodegenerative disease.

Numbers of neurons and glia in mature rat somatosensory cortex: effects of prenatal exposure to ethanol.

Stereological methods were used to examine the consequences of prenatal exposure to ethanol on the structure of area 3, primary somatosensory cortex, of the mature hooded rat. Pregnant rats were fed a liquid diet containing 6.7% (v/v) ethanol (Et), pair-fed an isocaloric liquid control diet (Ct), or fed a diet of chow and water (Ch). Cresyl violet-stained sections of 3-month-old pups were examined. The corrected mean size of the cell bodies of neurons in layers other than layer V was significantly smaller in the Et-treated rats; conversely, the mean somatic size of glia in each layer was significantly larger in the Et-treated rats. The laminar cell packing density for neurons and glia, however, was similar in rats from both treatment groups. The overall volume of area 3 and the volume of individual layers were about 33% smaller in Et-treated rats than in the pair-fed controls. Thus, the estimated total number of neurons in Et-treated rats (1.79 X 10(6] was significantly fewer than in Ch-treated rats (2.77 X 10(6] and in Ct-treated rats (2.66 X 10(6]. The total number of glia also was about 30% fewer in Et-treated rats than in the controls. Not all layers were affected equivalently. The space occupied by the neuropil was significantly greater in Et-treated rats, but only in layers II/III, IV, and VI; hence, the cell body/neuropil ratio in these layers was less in Et-treated rats than in the controls. Therefore, microcephaly caused by prenatal exposure to ethanol results not only from a miniaturization of the brain, but also from a permanent abnormal organization of cerebral cortex.

Amyloid precursor protein processing in vivo--insights from a chemically-induced constitutive overactivation of protein kinase C in Guinea pig brain.

Aberrant proteolytical processing of the amyloid precursor protein (APP) gives rise to beta-amyloid peptides, which form deposits characteristic for the brains of Alzheimer's disease patients. From in vitro studies, protein kinase C (PKC) is known for almost 20 years to be involved the secretory pathway of APP processing, resulting in the reduced generation of beta-amyloid peptides. However, the toxicity of activators of PKC, such as phorbol esters, has prevented to test the hypothesis of an inverse regulation of secretory APP processing and beta-amyloid generation in vivo. Here we present an animal model which allows to reveal the function of PKC in the proteolytical processing of APP in vivo. Studies by Johnstone and Coyle from the early 1980s have shown that treatment of pregnant rats with methylazoxymethanol acetate (MAM) results in the induction of neocortical microencephalopathy of the offsprings. Later on, the constitutive overactivation of PKC isoforms was described in affected brain structures of these animals. This led to the idea to study the APP processing under conditions of overactivated PKC in the brains of such animals in vivo. However, in mice and rats one can follow the generation of secretory APP products but the detection of rodent beta-amyloid peptides is delicate. Therefore, we adapted the MAM model to guinea pigs, which have a human beta-amyloid sequence, and investigated the relation between secretory APP processing and beta-amyloid generation in vivo. In the brains of microencephalic guinea pigs we observed increased levels of secretory APP fragments but no change in the concentration of beta-amyloid peptides. Our results indicate that both pathways of APP processing are differentially controlled under these experimental conditions in vivo.

Spectrum of disorders associated with enlarged sylvian fissures in infancy.

Disproportionate enlargement of the sylvian fissures (ESF) on MRI of an infant's CNS suggests underdevelopment of the cortical operculum. We reviewed charts of infants with isolated ESF. Conditions associated with ESF included feeding difficulties and facial dysmorphism (syndromic or nonsyndromic). There may be an embryologic link between growth of the opercular cortex and development of the face.