Elsevier

Neurotoxicology and Teratology

Volume 39, September–October 2013, Pages 77-83
Neurotoxicology and Teratology

Magnetic resonance microscopy-based analyses of the neuroanatomical effects of gestational day 9 ethanol exposure in mice

https://doi.org/10.1016/j.ntt.2013.07.009Get rights and content

Highlights

  • Early gestational ethanol exposure induces stage-dependent brain abnormalities

  • An acute GD 9 ethanol exposure reduces the volume of the cerebellum

  • Ethanol exposure can result in shape abnormalities in the absence of volume changes

  • The right side of the mouse brain is more severely affected by ethanol than the left

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.

Introduction

The full range of abnormalities that result from prenatal ethanol exposure, termed fetal alcohol spectrum disorder (FASD), includes craniofacial defects and growth retardation as well as damage to a variety of organ systems including the central nervous system (CNS). Associated with the latter are variable patterns and degrees of structural and functional alterations including cognitive and behavioral deficits (Mattson et al., 2013, Ware et al., 2013).

This variability of effect is a product of differing maternal ethanol dosages and timing of intake during pregnancy as well as concurrent exposure to other environmental agents, nutritional status, and genetic profiles (Jones, 2011). Of particular interest for the current report is the developmental stage-dependency of CNS insult; an issue that lends itself well to assessment in animal models. This information regarding the stage-dependent effects of ethanol is aimed at improving the postnatal and prenatal diagnosis of ethanol-exposed subjects.

Previous studies of prenatal rodents have shown that acute teratogen exposure occurring at times during embryogenesis that are separated by as little as half a day result in profoundly varying patterns of brain damage (Dunty et al., 2001, Godin et al., 2010, Kotch and Sulik, 1992, Parnell et al., 2009, Shenefelt, 1972). Notable in this regard are data illustrating stage-dependent patterns of ethanol-induced cell death in tissues including the developing brain, patterns that appear to reflect subsequent malformations (Dunty et al., 2001, Kotch and Sulik, 1992). Following up on these pathogenesis studies, the application of advanced imaging methodologies including magnetic resonance microscopy (MRM — high-resolution magnetic resonance imaging) and diffusion tensor imaging (DTI) has greatly enhanced our understanding of the gross structural brain defects that result from developmental stage-specific ethanol insult. To date, the results of imaging-based studies of the brains of mice acutely exposed to ethanol on gestational day (GD) 7, 8, and 10 have been reported (Godin et al., 2010, O'Leary-Moore et al., 2010, Parnell et al., 2009). Features unique to each of these exposure times were found. GD7 (early gastrulation stage) ethanol exposure-induced defects include a wide range of median forebrain deficiencies that fall within the holoprosencephaly (HPE) spectrum and entail cerebro-cortical, striatal, septal, and pituitary abnormalities. Ethanol exposure at this time point also resulted in neuronal migration defects presenting as leptomeningeal heterotopias and cortical dysplasia. Acute maternal ethanol treatment on GD 8, a time during which neurulation is beginning does not yield holoprosencephaly, but results in olfactory bulb, hippocampal and cerebellar volume reductions along with ventricular enlargement (Parnell et al., 2009). Similarly, acute ethanol exposure on GD 10 results in altered ventricular size and morphology. This was particularly pronounced in the third ventricle, a finding that may be indicative of alterations in the surrounding thalamus and hypothalamus (O'Leary-Moore et al., 2010). Also notable were significant volumetric reductions in the cerebral cortex.

Filling a void in the study of brain defects resulting from acute ethanol insult occurring at developmental stages present in the human during the third through the fourth week of gestation, the current investigation is directed toward imaging-based analyses of GD9 ethanol exposure in mice. In addition to methodologically extending the previous investigations of ethanol-induced volumetric changes, this study employs advanced shape analysis algorithms to localize regional brain changes. Overall, the information acquired through this work provides a foundation for understanding the variable outcomes associated with prenatal ethanol exposure and promises to be of value in helping to recognize and treat alcohol-affected individuals by enhancing our knowledge of the full spectrum of ethanol's teratogenesis.

Section snippets

Animal husbandry and maternal ethanol exposure

C57Bl/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and housed under reverse light/dark cycle conditions in a room that was held at constant temperature and humidity. Animals had water and standard laboratory chow available ad libitum. At the beginning of the dark cycle, 1–2 female mice were placed in the home cage of a singly-housed male for two hours. The female mice were examined for the presence of a copulation plug, and if present, the time of introduction of the

Growth retardation

When comparing GD 17 ethanol-exposed fetuses to GD 16.5 controls, no statistically significant differences in either whole body volume (Control = 478.6 ± 21.4 mm3, Ethanol = 420.3 ± 36.9 mm3 [mean ± standard error]) or crown-rump length (Control = 16.16 ± 0.26 mm, Ethanol = 15.42 ± 0.44 mm) were found, confirming previous findings of approximately a half day ethanol-induced developmental delay. In comparison to the stage-matched controls, the brains of the ethanol-exposed animals were reduced in size. More

Discussion

The results of this study are in keeping with former pathogenesis studies illustrating that acute GD 9 ethanol insult in mice selectively impacts the progenitors of the cerebral cortex, hippocampus, and cerebellum (Dunty et al., 2001). Regarding the latter, as assessed from 3-D reconstructions of MRM scans, a major finding from the acute GD 9 exposure employed for the current study is significant reduction in cerebellar volume. Using similar methodology, this end point had also been observed

Conflict of interest statement

No conflicts to disclose.

Acknowledgments

This study was conducted at the UNC Bowles Center for Alcohol Studies as part of the Collaborative Initiative on Fetal Alcohol Spectrum Disorders and as part of the Carolina Institute for Developmental Disabilities. It was funded by grant nos. U01-AA017124, U01-AA0216521 and P60-AA011605 to KKS and grant K99/R00-AA018697 to SEP from the National Institute on Alcohol Abuse and Alcoholism/NIH; and by NIBIB grant U54-EB005149-01 and NICHD grant P30-HD03110 to MAS. MRM scanning was performed at the

References (41)

  • S.J. Astley et al.

    Magnetic resonance imaging outcomes from a comprehensive magnetic resonance study of children with fetal alcohol spectrum disorders

    Alcohol Clin Exp Res

    (2009)
  • S.Y. Chen et al.

    Protection from ethanol-induced limb malformations by the superoxide dismutase/catalase mimetic, EUK-134

    FASEB J

    (2004)
  • C.S. Cook et al.

    Fetal alcohol syndrome. Eye malformations in a mouse model

    Arch Ophthalmol

    (1987)
  • S.L. Davenport et al.

    The spectrum of clinical features in CHARGE syndrome

    Clin Genet

    (1986)
  • W.C. Dunty et al.

    Selective vulnerability of embryonic cell populations to ethanol-induced apoptosis: implications for alcohol-related birth defects and neurodevelopmental disorder

    Alcohol Clin Exp Res

    (2001)
  • E.A. Godin et al.

    Magnetic resonance microscopy defines ethanol-induced brain abnormalities in prenatal mice: effects of acute insult on gestational day 7

    Alcohol Clin Exp Res

    (2010)
  • P. Hammond et al.

    Face–brain asymmetry in autism spectrum disorders

    Mol Psychiatry

    (2008)
  • M.R. Herbert et al.

    Brain asymmetries in autism and developmental language disorder: a nested whole-brain analysis

    Brain

    (2005)
  • I.T. Jackson et al.

    Craniofacial and oral manifestations of fetal alcohol syndrome

    Plast Reconstr Surg

    (1990)
  • K.L. Jones

    The effects of alcohol on fetal development

    Birth Defects Res C Embryo Today

    (2011)
  • Cited by (40)

    • Short-term transcriptomic changes in the mouse neural tube induced by an acute alcohol exposure

      2023, Alcohol
      Citation Excerpt :

      Fetal alcohol spectrum disorders (FASD) are caused by in utero exposure to alcohol and are a leading cause of intellectual disabilities. Alcohol exposure during neurulation (embryonic day [E] 8–10 in mice, ∼4th week of pregnancy in humans) disrupts development of midline brain structures (Fish et al., 2016, 2018; Parnell et al., 2009, 2013). The regions of the brain most vulnerable to alcohol exposure during this developmental window are derived from the rostroventral neural tube (RVNT), including hypothalamic, septal, and pituitary structures.

    • Prenatal alcohol exposure disrupts Sonic hedgehog pathway and primary cilia genes in the mouse neural tube

      2021, Reproductive Toxicology
      Citation Excerpt :

      Alcohol exposure during gastrulation, the stage when the embryo first forms distinct cell layers (3rd week in humans, embryonic day [E] 7 in mice), induces widespread cell death in the neuroectoderm [5], and subsequent diminished Sonic hedgehog (Shh) signaling as a pathogenic mechanism for gastrulation-stage alcohol exposure [6–10]. Alcohol exposure during neurulation, as the neural tube forms and closes (∼4th-5th weeks in humans, E8−10 in mice), produces midline structural defects in brain regions such as the hypothalamus, ventricles, pituitary, and septal regions [11–14]. However, while prenatal alcohol exposure during neurulation causes cell death in regions such as the rhombencephalon, alcohol-induced apoptosis is not as pronounced in the rostroventral neural tube [5], the portion of the neural tube that gives rise to ventral midline brain structures.

    • The contributions of Dr. Kathleen K. Sulik to fetal alcohol spectrum disorders research and prevention

      2018, Alcohol
      Citation Excerpt :

      Ethanol administration on GD 7 produced craniofacial abnormalities characteristic of FAS and distinctive brain abnormalities indicative of midline hypoplasia (Lipinski et al., 2012). In contrast, ethanol administration on GD 8–9 produced a very different pattern of face (and brain) abnormalities, consistent with midline hyperplasia (Lipinski et al., 2012; Parnell et al., 2009, 2013). These findings reinforced Dr. Sulik's earlier conclusion that the “classic” FAS face was the consequence of ethanol exposure at a very specific period of gestation, prompting a search by CIFASD for the spectrum of craniofacial abnormalities that result from ethanol exposure at different developmental periods (Suttie et al., 2017).

    View all citing articles on Scopus
    View full text