[Show abstract][Hide abstract] ABSTRACT: Potent synthetic cannabinoids (SCBs) are illegally distributed drugs of abuse that are frequently consumed in spite of their adverse consequences. This study was designed to determine if the toxicity observed in adults also extends to the prenatal period by examining the developmental toxicity/teratogenicity of one of these SCBs, CP-55,940, in a mammalian model. First, immunohistochemistry was employed for cannabinoid receptor 1 (CB1) localization within gestational day (GD) 8 mouse embryos; this receptor was identified in the cranial neural plate, suggesting that the endogenous cannabinoid system may be involved in normal development. Based on this information and on previous avian teratogenicity studies, the current investigation focused on cannabinoid exposure during neurulation. The treatment paradigm involved acute i.p. administration of vehicle, 0.0625, 0.125, 0.25, 0.5, 1.0, or 2.0mg/kg CP-55,940 to time-mated C57Bl/6J mice on their 8th day of pregnancy (n>10 litters per treatment group). On GD 17, litters were harvested and examined for numbers of live, dead, or resorbed fetuses, as well as for fetal weight, length, and gross morphological abnormalities. No effect on litter size, fetal weight, or crown rump length was seen at any of the CP-55,940 dosages tested. Major malformations involving the craniofacies and/or eyes were noted in all drug-treated groups. Selected fetuses with craniofacial malformations were histologically sectioned and stained, allowing investigation of brain anomalies. Observed craniofacial, ocular, and brain abnormalities in drug-treated fetuses included lateral and median facial clefts, cleft palate, microphthalmia, iridial coloboma, anophthalmia, exencephaly, holoprosencephaly, and cortical dysplasia. With the most commonly observed defects involving the eyes, the incidence and severity of readily identifiable ocular malformations were utilized as a basis for dose-response analyses. Ocular malformation ratings revealed dose-dependent CP-55,940 teratogenicity within the full range of dosages tested. While examination of additional critical periods and in depth mechanistic studies is warranted, the results of this investigation clearly show the dose-dependent teratogenicity of this SCB.
Full-text · Article · Dec 2015 · Neurotoxicology and Teratology
[Show abstract][Hide abstract] ABSTRACT: This chapter provides an overview of animal model-based studies that have generated information critical to our understanding of the pathogenesis and mechanisms underlying alcohol-induced birth defects, in particular those involving the brain. Focus is placed on the developing organism itself, rather than the mother, placenta, or other extraembryonic tissues. Components of the cascades of alcohol-induced damage that are considered herein are excessive cell death, changes in the cell cycle and proliferation, cell migration, cell morphogenesis, and gene expression as well as free radical damage and interference with cell signaling. The roles played by one or more of these various factors in the genesis of structural and functional birth defects are dependent upon alcohol exposure patterns and dosage, the involved tissue, and the prenatal stage(s) at the time of exposure. Technologic advances and rapidly increasing knowledge in the fields of genetics, cell, developmental, and neurobiology are critical to accurately piecing together experimental evidence in refining our understanding of the genesis of alcohol-induced birth defects, to the planning and execution of future studies, and to applying the knowledge gained to diminish the severity or occurrence of fetal alcohol spectrum disorder.
No preview · Article · Oct 2014 · Handbook of Clinical Neurology
[Show abstract][Hide abstract] ABSTRACT: Prenatal alcohol exposure can result in long-term cognitive and behavioral deficits. Fetal alcohol spectrum disorder (FASD) refers to a range of permanent birth defects caused by prenatal alcohol exposure, and is the most common neurodevelopmental disorder in the US. Studies by autopsy and conventional structural MRI indicate that the midline structures of the brain are particularly vulnerable to prenatal alcohol exposure. Diffusion tensor imaging (DTI) has shown that abnormalities in brain white matter especially the corpus callosum are very common in FASD. Quantitative susceptibility mapping (QSM) is a novel technique that measures tissue's magnetic property. Such magnetic property is affected by tissue microstructure and molecular composition including that of myelin in the white matter. In this work, we studied three major white matter fiber bundles of a mouse model of FASD and compared it to control mice using both QSM and DTI. QSM revealed clear and significant abnormalities in anterior commissure, corpus callosum, and hippocampal commissure, which were likely due to reduced myelination. Our data also suggested that QSM may be even more sensitive than DTI for examining changes due to prenatal alcohol exposure. Although this is a preclinical study, the technique of QSM is readily translatable to human brain.
[Show abstract][Hide abstract] ABSTRACT: Subtle behavioral and cognitive deficits have been documented in patient cohorts with orofacial clefts (OFCs). Recent neuroimaging studies argue that these traits are associated with structural brain abnormalities but have been limited to adolescent and adult populations where brain plasticity during infancy and childhood may be a confounding factor. Here, we employed high resolution magnetic resonance microscopy to examine primary brain morphology in a mouse model of OFCs. Transient in utero exposure to the Hedgehog (Hh) signaling pathway antagonist cyclopamine resulted in a spectrum of facial dysmorphology, including unilateral and bilateral cleft lip and palate, cleft of the secondary palate only, and a non-cleft phenotype marked by midfacial hypoplasia. Relative to controls, cyclopamine-exposed fetuses exhibited volumetric differences in several brain regions, including hypoplasia of the pituitary gland and olfactory bulbs, hyperplasia of the forebrain septal region, and expansion of the third ventricle. However, in affected fetuses the corpus callosum was intact and normal division of the forebrain was observed. This argues that temporally-specific Hh signaling perturbation can result in typical appearing OFCs in the absence of holoprosencephaly-a condition classically associated with Hh pathway inhibition and frequently co-occurring with OFCs. Supporting the premise that some forms of OFCs co-occur with subtle brain malformations, these results provide a possible ontological basis for traits identified in clinical populations. They also argue in favor of future investigations into genetic and/or environmental modulation of the Hh pathway in the etiopathogenesis of orofacial clefting.
[Show abstract][Hide abstract] ABSTRACT: Background:
The first trimester of human development and the equivalent developmental period in animal models is a time when teratogenic ethanol (EtOH) exposure induces the major structural birth defects that fall within fetal alcohol spectrum disorder (FASD). Previous FASD research employing an acute high dose maternal intraperitoneal EtOH treatment paradigm has identified sensitive periods for a number of these defects. Extending this work, this investigation utilized high resolution magnetic resonance microscopy (MRM)-based analyses to examine the dysmorphology resulting from maternal dietary EtOH intake occurring during selected first trimester-equivalent time periods.
Female C57Bl/6J mice were acclimated to a liquid 4.8% EtOH (v/v)-containing diet, then bred while on standard chow. Dams were again provided the EtOH-containing liquid diet for a period that extended either from the beginning of gestational day (GD) 7 to the end of GD 11 or from the beginning of GD 12 to the end of GD 16. On GD 17, a subset of fetuses was selected for MRM-based analyses. Group comparisons were made for litter characteristics and gross dysmorphology, as well as whole and regional brain volumes.
EtOH-induced stage of exposure-dependent structural brain abnormalities were observed. The GD 7 to 11 EtOH-exposed group presented with a significant decrease in cerebellar volume and an increase in septal volume, while GD 12 to 16 EtOH treatment resulted in a reduction in right hippocampal volume accompanied by enlarged pituitaries. Additionally, the GD 12 to 16 EtOH exposure caused a high incidence of edema/fetal hydrops.
These results illustrate the teratogenic impact of maternal dietary EtOH intake occurring at time periods approximately equivalent to weeks 3 through 6 (GD 7 to 11 in mice) and weeks 7 through 12 (GD 12 to 16 in mice) of human gestation, further documenting EtOH's stage of exposure-dependent neuroteratogenic end points and highlighting the vulnerability of selected brain regions during the first trimester. Additionally they suggest that clinical attention should be paid to fetal hydrops as a likely component of FASD.
Full-text · Article · Jun 2014 · Alcoholism Clinical and Experimental Research
[Show abstract][Hide abstract] ABSTRACT: Disruption of the Hedgehog signaling pathway has been implicated as an important molecular mechanism in the pathogenesis of fetal alcohol syndrome. In severe cases, the abnormalities of the face and brain that result from prenatal ethanol exposure fall within the spectrum of holoprosencephaly. Single allele mutations in the Hh pathway genes Sonic Hedgehog (SHH) and GLI2 cause holoprosencephaly with extremely variable phenotypic penetrance in humans. Here, we tested whether mutations in these genes alter the frequency or severity of ethanol-induced dysmorphology in a mouse model. Timed pregnancies were established by mating Shh(+/-) or Gli2(+/-) male mice backcrossed to C57BL/6J strain, with wildtype females. On gestational day 7, dams were treated with two ip doses of 2.9 g/kg ethanol (or vehicle alone), administered four hrs apart. Fetuses were then genotyped and imaged, and the severity of facial dysmorphology was assessed. Following ethanol exposure, mean dysmorphology scores were increased by 3.2- and 6.6-fold in Shh(+/-) and Gli2(+/-) groups, respectively, relative to their wildtype littermates. Importantly, a cohort of heterozygous fetuses exhibited phenotypes not typically produced in this model but associated with severe holoprosencephaly, including exencephaly, median cleft lip, otocephaly, and proboscis. As expected, a correlation between the severity of facial dysmorphology and medial forebrain deficiency was observed in affected animals. While Shh(+/-) and Gli2(+/-) mice have been described as phenotypically normal, these results illustrate a functional haploinsufficiency of both genes in combination with ethanol exposure. By demonstrating an interaction between specific genetic and environmental risk factors, this study provides important insights into the multifactorial etiology and complex pathogenesis of fetal alcohol syndrome and holoprosencephaly.
[Show abstract][Hide abstract] ABSTRACT: Animal model-based studies have shown that ethanol exposure during early gestation induces developmental stage-specific abnormalities of the face and brain. The exposure time-dependent variability in ethanol's teratogenic outcomes is expected to contribute significantly to the wide spectrum of effects observed in humans with fetal alcohol spectrum disorder (FASD). The work presented here employs a mouse FASD model and magnetic resonance microscopy (MRM; high resolution magnetic resonance imaging) in studies designed to further our understanding of the developmental stage-specific defects of the brain that are induced by ethanol. At neurulation stages, i.e. at the beginning of gestational day (GD) 9 and again 4 hours later, time-mated C57Bl/6J dams were intraperitoneally administered 2.9 g/kg ethanol or vehicle. Ethanol-exposed fetuses were collected on GD 17, processed for MRM analysis, and results compared to comparably staged controls. Linear and volume measurements as well as shape changes for numerous individual brain regions were determined. GD 9 ethanol exposure resulted in significantly increased septal region width, reduction of cerebellar volume, and enlargement of all of the ventricles. Additionally, the results of shape analyses showed that many areas of the ethanol-exposed brains including the cerebral cortex, hippocampus and right striatum were significantly misshapen. These data demonstrate that ethanol can induce dysmorphology that may not be obvious based on volumetric analyses alone, highlight the asymmetric aspects of ethanol-induced defects, and add to our understanding of ethanol's developmental stage-dependent neuroteratogenesis.
No preview · Article · Jul 2013 · Neurotoxicology and Teratology
[Show abstract][Hide abstract] ABSTRACT: Objective:
While SEX-determining region Y-Box 2 (SOX2) mutations are typically recognized as yielding ocular and central nervous system abnormalities, they have also been associated with other craniofacial defects. To elucidate the genesis of the latter, Sox2 hypomorphic (Sox2(HYP)) mice were examined, with particular attention to secondary palatal development.
Clefts of the secondary palate were found to be highly penetrant in Sox2(HYP) mice. The palatal clefting occurred in the absence of mandibular hypoplasia and resulted from delayed or failed shelf elevation.
Sox2 hypomorphism can result in clefting of the secondary palate, an effect that appears to be independent of mandibular hypoplasia and is thus expected to result from an abnormality that is inherent to the palatal shelves and/or their progenitor tissues. Further clinical attention relative to SOX2 mutations as a basis for secondary palatal clefts appears warranted.
No preview · Article · May 2013 · The Cleft Palate-Craniofacial Journal
[Show abstract][Hide abstract] ABSTRACT: Objective: The interfrontal bone (IF) resides along the anterior region of the metopic suture in many strains of mice. Development of the IF can be influenced by mutations affecting neural tube development (DR Johnson, 1976), i.e. Gli3 (Xtbph) which is part of the sonic hedgehog (SHH) signaling pathway. The objective of this experiment was to determine the role of Shh and Gli2(negative regulator in SHH signaling) in the development of the IF bone.
Method: Heads from adult (>12wks old) male and female Shh and Gli2 haploinsufficient and genotype controls were dissected, microCT scanned, bleached and dried. IF length and width (mm) for each animal was measured in triplicate using a Leica GZ6 stereozoom microscope and a calibrated reticle. For each sex/genotype group, 10 Shh+/-, 10 Gli2+/-, 10 Shh+/+, and 10 Gli2+/+skulls were examined. Groups were compared using paired T-test and p ≤ 0.05 was considered significant. Measurements to determine the correlation of IF bone size and skull widths from microCT scans are underway.
Result: IF lengths (males) Shh+/- (1.28±0.97), Shh+/+ (3.35±1.14), Gli2+/- (3.29±0.56) and Gli2+/+ (3.32±0.47). IF lengths (females) Shh+/- (1.13±1.10), Shh+/+ (3.16±1.12), Gli2+/- (2.59±1.36) and Gli2+/+ (2.80±1.08). IF widths (males) Shh+/- (0.64±0.52), Shh+/+ (1.55±0.57), Gli2+/- (1.98±0.70) and Gli2+/+ (1.51±0.35). IF widths (females) Shh+/- (0.39±0.38), Shh+/+ (1.53±0.84), Gli2+/- (1.40±0.89) and Gli2+/+ (1.75±1.52). No significant differences in IF were observed between the genotype controls or between Gli2+/- and Gli2+/+. Male and female IF bones were smaller in Shh+/-vs. genotype controls, p < 0.01.
Conclusion: Haploinsufficiency of Shh leads to reduction in this midline structure, the interfrontal bone (IF). Haploinsufficiency of Gli2 does not alter IF development, even though GLI2 acts as a regulator in SHH signaling.
[Show abstract][Hide abstract] ABSTRACT: There is a genetic contribution to fetal alcohol spectrum disorders (FASD), but the identification of candidate genes has been elusive. Ethanol may cause FASD in part by decreasing the adhesion of the developmentally critical L1 cell adhesion molecule through interactions with an alcohol binding pocket on the extracellular domain. Pharmacologic inhibition or genetic knockdown of ERK2 did not alter L1 adhesion, but markedly decreased ethanol inhibition of L1 adhesion in NIH/3T3 cells and NG108-15 cells. Likewise, leucine replacement of S1248, an ERK2 substrate on the L1 cytoplasmic domain, did not decrease L1 adhesion, but abolished ethanol inhibition of L1 adhesion. Stable transfection of NIH/3T3 cells with human L1 resulted in clonal cell lines in which L1 adhesion was consistently sensitive or insensitive to ethanol for more than a decade. ERK2 activity and S1248 phosphorylation were greater in ethanol-sensitive NIH/3T3 clonal cell lines than in their ethanol-insensitive counterparts. Ethanol-insensitive cells became ethanol sensitive after increasing ERK2 activity by transfection with a constitutively active MAP kinase kinase 1. Finally, embryos from two substrains of C57BL mice that differ in susceptibility to ethanol teratogenesis showed corresponding differences in MAPK activity. Our data suggest that ERK2 phosphorylation of S1248 modulates ethanol inhibition of L1 adhesion by inside-out signaling and that differential regulation of ERK2 signaling might contribute to genetic susceptibility to FASD. Moreover, identification of a specific locus that regulates ethanol sensitivity, but not L1 function, might facilitate the rational design of drugs that block ethanol neurotoxicity.
Preview · Article · Feb 2013 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Linear brain measurements of entire populations reflect volumetric and DSM results. Linear measurements were produced from transverse MRM sections at the level of the anterior commissure in all scanned animals. Biparietal diameter, spanning the widest distance across the cerebrum, third ventricular width (TVW), and septal region width (SRW) were measured. For each group, individual litters are represented by separate columns of data points, which are superimposed on box and whisker plots. Box parameters represent the 25th and 75th percentile of the population, while error bars represent the 10th and 90th percentile. Inside the boxes, a solid line represents the population median, while the mean is represented by a dashed line. Letters above each bar indicate group classes; the same letter above a subset of bars denotes lack of statistical difference, whereas different letters represent statistically different classes (p<0.05). The septal region was absent (%) at the level of the anterior commissure in three animals exposed to ethanol at GD7. These values are not plotted, nor included in population statistics. H&E stained sections from an animal in each treatment group illustrate the septal region area corresponding to the boxed region in the MRM section.
[Show abstract][Hide abstract] ABSTRACT: Volumetric analysis of individual brain regions in control and ethanol exposure groups. Individual brain region volumes were derived from manual segmentation. To determine disproportionate differences, the volume of each region was calculated as a percentage of total brain volume in each animal. Paired structures are shown individually. Letters above each bar indicate group classes; the same letter above a subset of bars denotes lack of statistical difference, whereas different letters represent statistically different classes (p<0.05).
[Show abstract][Hide abstract] ABSTRACT: Stage-specific ethanol exposure causes varying degrees of ocular defects. Prior to fixation of vehicle and ethanol exposed fetuses for MRM, both eyes were imaged by bright-field microscopy. Ocular defects were rated on a scale from 1–5 as follows: (1) normal; (2) slight microphthalmia or slight pupil shape abnormality; (3) slight microphthalmia and slight pupil shape abnormality, (4) moderate microphthalmia; and (5) severe microphthalmia and as previously described . Representative images of each defect category are shown below. Analysis was performed in the entire study population.
[Show abstract][Hide abstract] ABSTRACT: Morphing illustrates unique facial phenotypes in each ethanol exposure group. Rapidly interpolated images provide dynamic morphs between mean control (WT) and mean ethanol-exposed facial surfaces in portrait and profile view.
[Show abstract][Hide abstract] ABSTRACT: Morphing illustrates opposing changes in brain region shape between ethanol exposure groups. Rapidly interpolated images provide dynamic morphs between the mean GD7 exposure group and the mean GD8.5 exposure group from a posterior-oblique view.
[Show abstract][Hide abstract] ABSTRACT: 3D visualization of face-brain dysmorphology resulting from stage-specific ethanol exposure. For the animals shown in Figure 7, the brain and face are visualized concurrently in 3D by reducing head surface opacity using Slicer3, an open source image visualization platform (www.slicer.org).
[Show abstract][Hide abstract] ABSTRACT: Morphing illustrates unique brain and facial phenotypes in each ethanol exposure group. Rapidly interpolated images provide dynamic morphs between the mean control (WT) and mean ethanol-exposed brain and facial surfaces from a superior (downward at the snout) and inferior (upward at the mandible) view.