Magnetic Resonance Microscopy Defines Ethanol‐Induced Brain Abnormalities in Prenatal Mice: Effects of Acute Insult on Gestational Day 7

Abstract

Background This magnetic resonance microscopy (MRM)-based report is the 2nd 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 3rd week post-fertilization 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 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 GD 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 3rd week of human prenatal development.

Radiological studies of fetal alcohol spectrum disorders in humans and animal models: An updated comprehensive review

“…The three weeks of gestation in mice corresponds approximately to the first and second trimester in humans in terms of brain development. Mouse brains continue to develop ten to twelve days postnatally, equivalent to the last trimester of human fetal brain development [151]. Therefore, mouse models designed to simulate third trimester PAE effects in…”

“…The only QSM study in rodents examined white matter tracts of ex vivo brains at postnatal day (PD) 45 in the offspring of C57Bl/6J mice that were intraperitoneally injected with acute high doses of ethanol at GD7 (same model as in [151]) [163]. Following PAE, the volume of the anterior commissure was reduced, and three white matter regions including the corpus callosum, anterior commissure and hippocampal commissure showed lower susceptibility contrast with the surrounding gray matter compared to control mice.…”

Perspectives on R.E. Shenefelt's 1972 Teratology publication entitled “Morphogenesis of malformations in hamsters caused by retinoic acid: relation to dose and stage at treatment”

“…Exemplary of a teratogenic agent for which the critical periods appear virtually the same as RA is alcohol (ethanol). Like RA, when administered acutely on gestational day (GD) 7 in the mouse, alcohol causes holoprosencephaly and microphthalmia (Cook et al, 1987;Godin et al, 2010;Johnston, 1982, 1983); on GD 8, it typically yields exencephaly ; on GD 8.5, it yields heart defects including transposition of the great vessels (Daft et al, 1986); and on GD 9, in mice, alcohol exposure causes forelimb defects (Johnson et al, 2007;. While the mechanism(s) of ethanol's teratogenic effects remain incompletely defined, a number of studies suggest a role for ethanol-mediated alterations in RA concentrations (Duester, 1991;Johnson et al, 2007;Pullarkat, 1991).…”

A study of the consequences of early gestational ethanol and/or nicotine exposure in the mouse

“…Following from this, in the field of DOHaD it is common to sample only a few offspring per litter, with the assumption that the findings from these individuals are representative of the characteristics of their littermates (Haseman and Hogan, 1975). However, there is an increasing body of evidence to suggest that there is greater intra-litter variability than inter-litter variability in fetal outcomes following many prenatal exposures (van der Schoot, 1992; Howdeshell and Saal, 2000; Barlow et al, 2003;Godin et al, 2010;Kietzman et al, 2014;Han et al, 2016).…”