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