• Energy Research

Background:  This magnetic resonance microscopy (MRM)‐based report is the second in a series designed to illustrate the spectrum of craniofacial and central nervous system (CNS) dysmorphia resulting from single‐ and multiple‐day maternal ethanol treatment. The study described in this report examined the consequences of ethanol exposure on gestational day (GD) 7 in mice, a time in development when gastrulation and neural plate development begins; corresponding to the mid‐ to late third week postfertilization in humans. Acute GD 7 ethanol exposure in mice has previously been shown to result in CNS defects consistent with holoprosencephaly (HPE) and craniofacial anomalies typical of those in Fetal Alcohol Syndrome (FAS). MRM has facilitated further definition of the range of GD 7 ethanol‐induced defects.Methods:  C57Bl/6J female mice were intraperitoneally (i.p.) administered vehicle or 2 injections of 2.9 g/kg ethanol on day 7 of pregnancy. Stage‐matched control and ethanol‐exposed GD 17 fetuses selected for imaging were immersion fixed in a Bouins/Prohance solution. MRM was conducted at either 7.0 Tesla (T) or 9.4 T. Resulting 29 μm isotropic spatial resolution scans were segmented and reconstructed to provide 3D images. Linear and volumetric brain measures, as well as morphological features, were compared for control and ethanol‐exposed fetuses. Following MRM, selected specimens were processed for routine histology and light microscopic examination.Results:  Gestational day 7 ethanol exposure resulted in a spectrum of median facial and forebrain deficiencies, as expected. This range of abnormalities falls within the HPE spectrum; a spectrum for which facial dysmorphology is consistent with and typically is predictive of that of the forebrain. In addition, other defects including median facial cleft, cleft palate, micrognathia, pituitary agenesis, and third ventricular dilatation were identified. MRM analyses also revealed cerebral cortical dysplasia/heterotopias resulting from this acute, early insult and facilitated a subsequent focused histological investigation of these defects.Conclusions:  Individual MRM scans and 3D reconstructions of fetal mouse brains have facilitated demonstration of a broad range of GD 7 ethanol‐induced morphological abnormality. These results, including the discovery of cerebral cortical heterotopias, elucidate the teratogenic potential of ethanol insult during the third week of human prenatal development.

AbstractBACKGROUND: The application of magnetic resonance microscopy (MRM) to the study of normal and abnormal prenatal mouse development has facilitated discovery of dysmorphology following prenatal ethanol insult. The current analyses extend this work, providing a regional brain volume‐based description of normal braingrowth and illustrating the consequences of gestational day (GD) 10 ethanol exposure in the fetal mouse. METHODS: To assess normal growth, control C57Bl/6J fetuses collected on GD 16, GD 16.5, and GD 17 were scanned using a 9.4‐T magnet, resulting in 29‐μm isotropic resolution images. For the ethanol teratogenicity studies, C57Bl/6J dams were administered intraperitoneal ethanol (2.9 g/kg) at 10 days, 0 hr, and 10 days, 4 hr, after fertilization, and fetuses were collected for analyses on GD 17. From individual MRM scans, linear measurements and regional brain volumes were determined and compared. RESULTS: In control fetuses, each of the assessed brain regions increased in volume, whereas ventricular volumes decreased between GD 16 and GD 17. Illustrating a global developmental delay, prenatal ethanol exposure resulted in reduced body volumes, crown‐rump lengths, and a generalized decrease in regional brain volumes compared with GD 17 controls. However, compared with GD 16.5, morphologically matched controls, ethanol exposure resulted in volume increases in the lateral and third ventricles as well as a disproportionate reduction in cortical volume. CONCLUSIONS: The normative data collected in this study facilitate the distinction between GD 10 ethanol‐induced developmental delay and frank dysmorphology. This work illustrates the utility of MRM‐based analyses for developmental toxicology studies and extends our knowledge of the stage‐dependency of ethanol teratogenesis. Birth Defects Research (Part A), 2010. © 2010 Wiley‐Liss, Inc.

Ethanol treatment on postnatal day seven (P7) causes robust brain cell death and is a model of late gestational alcohol exposure (Ikonomidou et al., 2000). To investigate the long-term effects of P7 ethanol treatment on adult brain, mice received either two doses of saline or ethanol on P7 (2.5g/kg, s.c., 2 hours apart) and were assessed as adults (P82) for brain volume (using postmortem MRI) and cellular architecture (using immunohistochemistry). Adult mice that received P7 ethanol had reduced MRI total brain volume (4%) with multiple brain regions being reduced in both males and females. Immunohistochemistry indicated reduced frontal cortical parvalbumin immunoreactive (PV+IR) interneurons (18-33%) and reduced Cux1+IR layer II pyramidal neurons (15%) in both sexes. Interestingly, markers of adult hippocampal neurogenesis differed between sexes, with only ethanol treated males showing increased doublecortin and Ki67 expression (52 and 57% respectively) in the dentate gyrus, consistent with increased neurogenesis compared to controls. These findings suggest that P7 ethanol treatment causes persistent reductions in adult brain volume and frontal cortical neurons in both males and females. Increased adult neurogenesis in males, but not females, is consistent with differential adaptive responses to P7 ethanol toxicity between the sexes. One day of ethanol exposure, e.g. P7, causes persistent adult brain dysmorphology.

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.

Binge-drinking is common in human adolescents. The adolescent brain is undergoing structural maturation and has a unique sensitivity to alcohol neurotoxicity. Therefore, adolescent binge ethanol may have long-term effects on the adult brain that alter brain structure and behaviors that are relevant to alcohol use disorders.

Adolescents binge drink more than any other age group, increasing risk of disrupting the development of the frontal cortex. We hypothesized that adolescent binge drinking would lead to persistent alterations in adulthood. In this study, we modeled adolescent weekend underage binge-drinking, using adolescent mice (post-natal days [P] 28-37). The adolescent intermittent binge ethanol (AIE) treatment includes 6 binge intragastric doses of ethanol in an intermittent pattern across adolescence. Assessments were conducted in adulthood following extended abstinence to determine if there were persistent changes in adults. Reversal learning, open field and other behavioral assessments as well as brain structure using magnetic imaging and immunohistochemistry were determined. We found that AIE did not impact adult Barnes Maze learning. However, AIE did cause reversal learning deficits in adults. AIE also caused structural changes in the adult brain. AIE was associated with adulthood volume enlargements in specific brain regions without changes in total brain volume. Enlarged regions included the orbitofrontal cortex (OFC, 4%), cerebellum (4.5%), thalamus (2%), internal capsule (10%) and genu of the corpus callosum (7%). The enlarged OFC volume in adults after AIE is consistent with previous imaging studies in human adolescents. AIE treatment was associated with significant increases in the expression of several extracellular matrix (ECM) proteins in the adult OFC including WFA (55%), Brevican (32%), Neurocan (105%), Tenacin-C (25%), and HABP (5%). These findings are consistent with AIE causing persistent changes in brain structure that could contribute to a lack of behavioral flexibility.

Background:  Magnetic resonance microscopy (MRM), magnetic resonance imaging (MRI) at microscopic levels, provides unprecedented opportunities to aid in defining the full spectrum of ethanol’s insult to the developing brain. This is the first in a series of reports that, collectively, will provide an MRM‐based atlas of developmental stage‐dependent structural brain abnormalities in a Fetal Alcohol Spectrum Disorders (FASD) mouse model. The ethanol exposure time and developmental stage examined for this report is gestational day (GD) 8 in mice, when the embryos are at early neurulation stages; stages present in humans early in the fourth week postfertilization.Methods:  For this study, pregnant C57Bl/6J mice were administered an ethanol dosage of 2.8 g/kg intraperitoneally at 8 days, 0 hour and again at 8 days, 4 hours postfertilization. On GD 17, fetuses that were selected for MRM analyses were immersion fixed in a Bouin’s/Prohance® solution. Control fetuses from vehicle‐treated dams were stage‐matched to those that were ethanol‐exposed. The fetal mice were scanned ex vivo at 7.0 T and 512 × 512 × 1024 image arrays were acquired using 3‐D spin warp encoding. The resulting 29 μm (isotropic) resolution images were processed using ITK‐SNAP, a 3‐D segmentation/visualization tool. Linear and volume measurements were determined for selected brain, head, and body regions of each specimen. Comparisons were made between control and treated fetuses, with an emphasis on determining (dis)proportionate changes in specific brain regions.Results:  As compared with controls, the crown‐rump lengths of stage‐matched ethanol‐exposed GD 17 fetuses were significantly reduced, as were brain and whole body volumes. Volume reductions were notable in every brain region examined, with the exception of the pituitary and septal region, and were accompanied by increased ventricular volumes. Disproportionate regional brain volume reductions were most marked on the right side and were significant for the olfactory bulb, hippocampus, and cerebellum; the latter being the most severely affected. Additionally, the septal region and the pituitary were disproportionately large. Linear measures were consistent with those of volume. Other dysmorphologic features noted in the MR scans were choanal stenosis and optic nerve coloboma.Conclusions:  This study demonstrates that exposure to ethanol occurring in mice at stages corresponding to the human fourth week postfertilization results in structural brain abnormalities that are readily identifiable at fetal stages of development. In addition to illustrating the utility of MR microscopy for analysis of an FASD mouse model, this work provides new information that confirms and extends human clinical observations. It also provides a framework for comparison of structural brain abnormalities resulting from ethanol exposure at other developmental stages and dosages.