Magnetic resonance microscopy defines ethanol-induced brain abnormalities in prenatal mice: Effects of acute insult on gestational day 8

The impact of prenatal alcohol exposure on the brain is becoming increasingly evident (Abel, 2006; May et al., 2006). Indeed, within Fetal Alcohol Spectrum Disorder (FASD, the umbrella term encompassing the spectrum of ethanol-related developmental abnormalities), which is estimated to occur in at least 1% of all births, central nervous system (CNS) abnormalities predominate. In spite of its prevalence, much remains to be learned regarding the structural and functional damage to the brain caused by maternal alcohol use; knowledge that can be applied to diagnosis, treatment and prevention. The application of magnetic resonance imaging (MRI) to clinical FASD assessments is of great importance in filling the gaps in our understanding of alcohol-induced CNS dysmorphology. Results of MRI analyses of individuals with fullblown Fetal Alcohol Syndrome (FAS) were first reported in the early 1990s (Mattson et al., 1992, 1994; Riley et al., 1995). Reductions in the size of the basal ganglia, the corpus callosum, and lobules I–V of the cerebellar vermis were observed. In addition to abnormal corpora callosa Swayze and colleagues (1997) found that patients diagnosed with FAS may also exhibit other midline brain abnormalities, such asseptum cavi pellucidi and cavum vergae. Another MRI-based study of the corpus callosum illustrated that changes occur in the shape of this fiber tract even in individuals without the facial features of FAS (Bookstein et al., 2002). Children exposed prenatally (either with or without a diagnosis of full FAS) have also been shown to have hypoplastic cerebellar vermis and hemispheres, as well as small hippocampi (Autti-Ramo et al., 2002). In MRI studies by Cortese and colleagues (2006), deficiencies involving the caudate nucleus have been documented in the absence of the full FAS phenotype. Recently, MRI studies of children with heavy prenatal alcohol exposure have also illustrated abnormal cortical thickness in temporal, parietal, and frontal cortical regions (Sowell et al., 2008). Although human imaging studies continue to increase our knowledge of the range and pattern of ethanol-induced defects in humans, they cannot readily provide the controlled, rapid, and comprehensive analyses allowed by application of advanced imaging methodologies to studies of experimental animal models. Johnson and colleagues were the first to observe the utility of magnetic resonance microscopy (MRM) for studying anatomy in fixed tissues using MRI at microscopic levels (Johnson et al., 1993). These methods have recently been used to acquire a complete atlas of the mouse at gestational day (GD) 10.5 – postnatal day (PD) 32 (Petiet et al., 2008). Others have recently shown that a powerful use of MRM at a resolution of 25.4 · 25.4 · 26 lm is for high throughput, phenotype-driven screens for developmental malformations in mouse models of human birth defects (Schneider et al., 2004). This approach has, for example, provided sufficient detail to visualize and characterize abnormal cardiovascular anatomy in fetal mice, without the need for labor intensive histological sectioning. Herein, we present the results of a similar approach to the study of alcohol-induced structural brain abnormalities in an FASD model. Numerous studies of FASD animal models have collectively illustrated that alcohol induces CNS abnormalities in a developmental-stage and dosage-dependent manner [National Institute on Alcohol Abuse and Alcoholism (NIAAA), 2000; Randall, 1987]. Major structural abnormalities, including those of the brain, are typically the result of insult during embryonic stages of development. Consequently, this developmental period has been initially targeted for MRI investigations. This work employs a C57Bl/6J mouse model that, following acute maternal ethanol administration at selected embryonic stages, has previously been shown to present with CNS abnormalities and all of the salient craniofacial features of FAS, as well as other stage-dependent abnormalities in other organ systems (Cook and Sulik, 1986; Cook et al., 1987; Daft et al., 1986; Dunty et al., 2001; Gage and Sulik, 1991; Kotch and Sulik, 1992; Kotch et al., 1992; Schambra et al., 1990; Sulik, 1984; Sulik and Johnston, 1982; Sulik et al., 1981; Webster et al.,1983). Importantly, the presence of externally visible anomalies of the face and eyes has previously been shown to be indicative of concurrent CNS insult in this acute exposure FASD model (Cook et al., 1987; Sulik, 2005; Sulik and Johnston, 1983). Toward the long-term goal of identifying the full range of structural CNS defects that prenatal ethanol exposure can cause, we have initiated MRM analyses of animals that are expected to exhibit the severe end of the spectrum of insult. Identification of vulnerable brain regions in severely affected animals will, undoubtedly, provide clues that will facilitate discovery of more subtle manifestations of ethanol’s insult. This approach is similar to selecting children with known physical features of FAS for subsequent CNS studies. Even though C57Bl/6J mice are inbred, an acute maternal dose of ethanol commonly yields considerable intra-litter variability with respect to degree of effect. For example, among a litter whose mother is given ethanol on just day 7 of her pregnancy, it is common for some pups to present with apparently normal craniofacies, while others have the typical facies of FAS, or of holoprosencephaly (Sulik and Johnston, 1982). That within a single litter (even among controls), there is a range of embryonic stages representing as much as 12 hours of development, probably explains a great deal of this phenotypic variability. Frequently, the most severely affected pups in an ethanolexposed litter die (most likely, due to non-CNS-related defects) or are cannibalized by their mothers shortly after birth. With the hope of identifying even the most severe ethanol-induced structural brain abnormalities, it is, therefore, necessary to conduct prenatal examinations. Pilot feasibility studies utilizing a 7.0 T magnet have illustrated that GD 17, 2–3 days before birth, is an optimal time for MRM analyses of fetal mouse brains (Petiet et al., 2007). At this stage, the fetuses can be noninvasively fixed by immersion in a contrast-enhancing solution (Petiet et al., 2007) that affords very high-resolution (29 lm) imaging. Importantly, the imaging procedure is such that the scans are isotropic, thus allowing accurate 3-D reconstruction and subsequent volume assessments. For this report, which is the first in a series that, collectively, is designed to provide an MRM-based atlas of developmental stage-dependent structural brain abnormalities in a FASD mouse model, the ethanol exposure time examined is GD 8. At this time of development, the embryos are at early neurulation stages; stages present in humans early in the fourth week postfertilization. Ex vivo MRM was conducted on developmental stage-matched control and ethanol-exposed GD 17 fetuses. The degree of brain development on GD 17 in mice corresponds approximately to that in weeks 12–13 in human fetuses (Clancy et al., 2007); a time when prenatal diagnostic imaging is feasible and is becoming increasingly common. The images acquired for this study were utilized to provide both linear and volumetric measures and to define the ethanol-induced collection of structural brain abnormalities.