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

Abstract

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 x 512 x 1024 image arrays were acquired using 3-D spin warp encoding. The resulting 29 microm (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.

Fig. 1

MRM scans illustrating sites of linear measurements made for GD 17 mouse fetuses. On the sagittal view shown in (A) the vertical line is placed at the location of the crown-rump measures, while the lines labeled b–e are at the sites of the corresponding horizontal scan images. The line in (B) is at the site used for cerebellar diameter determinations. In (C) the lines depict the following sites of measurement: third ventricle width (line #1); septal width (line #2); brain width (biparietal diameter) (line #3); frontothalamic distance (line #4); midsagittal brain length (line #5); head length (line #6). The lines in (D) illustrate sites of olfactory bulb width and length measures, and in (E) the line is located at the site of measurement for the optic globe diameter. Bar in (A) = 2 mm; bars in (B–E) = 1 mm.

Fig. 2

Regional segmentation of MRM scans. Images in A, B, and C are representative coronal, sagittal, and horizontal scans, respectively. Illustrated are color-coded regions that were manually segmented for each of the brains examined for this study. Computer-generated 3-D reconstruction of these regions provides a basis for volume determinations and for visualization of normal and abnormal morphology. Illustrated in (D) is a brain reconstruction with the upper portion of the right side removed, allowing visualization of interior structures.

Fig. 3

Graphic illustration of ethanol-induced proportional volume changes in GD 17 fetal mouse brain regions. Significant average volume reductions are indicated by (*), while increases are indicated by (#).

Fig. 4

3-D reconstructions of control and ethanol-exposed GD 17 fetal mouse brain regions. As compared to the images of a normal brain (A–D), those from a fetus that had been acutely exposed to ethanol on GD 8 (E–F) illustrate a visibly small right olfactory bulb (arrow in E) and excessive space between the bulbs, a relatively large ventral midline view of the septal region (arrow in F); notable enlargement of the third ventricle (arrow in G) as observed from a ventral view of the reconstructed ventricular system; and narrowing of the cerebral aqueduct (arrow in H) as observed in a posterior view of the ventricular system.

Fig. 5

Horizontal MRM scans illustrate abnormalities in an ethanol exposed fetal brain (B) as compared with that of a control (A). At the level of the anterior commissure (arrowheads in A and B), the third ventricle (arrow in B) is notably larger in the ethanol-exposed fetus. Also notable is the abnormal right olfactory bulb.

Fig. 6

Optic abnormalities and choanal stenosis in alcohol-exposed fetuses. Microphthalmia and irideo-retinal coloboma (arrow in B) are evident in GD 17 ethanol-exposed fetuses. [Compare the affected left eye in (B) to the normal left eye of a control GD 17 fetus in (A).] Additionally, MRM revealed an optic nerve coloboma in the right eye of an ethanol-exposed fetus (arrow in D). Of particular note is unilateral choanal stenosis in an alcohol-exposed fetus. A coronal MRM scan (C) at the level indicated by the line in (B) illustrates a pinhole-like opening (arrow), representing the extreme narrowing of the right choana. A horizontal scan (E) of the fetus in (C) illustrates the simple contours of the small right nasal cavity (arrow). This fetus also presented with anophthalmia on its right side.

Fig. 7

Scanning electron micrographs illustrate face and brain defects in GD 10 embryos following GD 8 maternal ethanol treatment. Comparison to a control embryo (A, B) illustrates abnormal upper midface and forebrain contours in the 2 affected embryos shown (C–F). In both affected embryos the medial nasal prominences (M) are abnormally positioned, being directed away from the lateral nasal prominences (L) and allowing full view of the developing vomeronasal organs (arrow). As compared with control, the interior of the forebrain in each of the affected embryos appears hypoplastic, with the degree of affect being consistent with that for the face. C = corpus striatum, curved arrow = optic stalk. Bars = 100 lm (modified from Sulik et al., 1986).