This work illustrates the fetal brain dysmorphology that results from acute GD10 ethanol exposure in mice and the utility of MRM-based methodologies for this type of investigation. In particular, regional brain segmentation and 3-D reconstruction have allowed assessment of volumetric as well as morphological alterations. In distinguishing ethanol-induced dysmorphology from generalized developmental delay, normal regional brain growth trajectories as established from MRM data have been especially valuable. As expected regarding the latter, raw volumetric data from GD16, GD16.5 and GD17 fetal brains showed age-related increases for the entire brain and for each of its sub-regions that were analyzed. Notably, the overall 38% brain volume increase that occurred between GD16 and GD17 was accompanied by a 16% reduction in the total ventricular volume, with the reduction in the volume of the 3rd ventricle over this period of time being particularly pronounced. In comparing GD17 ethanol-exposed fetuses to controls, the GD16.5 control group was found to be developmentally most comparable; i.e. the ethanol-exposed fetuses were delayed in their development by about a half day. Importantly however, compared to the GD16.5 control group, prenatal ethanol exposure significantly increased both the lateral and 3rd ventricular volumes; volumes that should be decreasing with time. In addition to the volumetric changes, the morphology of the 3rd ventricle following GD10 maternal ethanol treatment was abnormal as compared to that in all control stages examined.
Ventricular enlargement has also been noted in other FASD models (eg. Mattson et al, 1994
; Sakata-Haga et al, 2004
; Zhou et al, 2003
) as well as in clinical studies of prenatal alcohol exposure (Johnson et al, 1996
; Swayze et al, 1997
). In humans, ventriculomegaly can be diagnosed prenatally and is associated with subsequent developmental delays and a high risk of other types of brain anomalies including lissencephaly, absent corpus callosum, septo-optic dysplasia, hydrocephalus and aqueductal stenosis (reviewed by Laskin et al, 2005
; Gaglioti et al, 2009
). Of these, agenesis of the corpus callosum is the most frequently detected co-occurring anomaly (Griffiths et al, 2010). In fetuses with prenatally-diagnosed ventriculomegaly, further prenatal examination with MR is, therefore, indicated (Manfredi et al, 2009). Based on the current findings, additionally indicated is acquisition of maternal drinking history.
In addition to ventricular changes, regional brain abnormalities were also noted among the ethanol-exposed fetuses that were examined. In particular, the volume of the cerebral cortex was found to be reduced. While exposure paradigms have varied, several studies in animal models have shown that the cerebral cortex is especially sensitive to gestational ethanol exposure (eg. Miller & Potempa, 1990
; Mihalick et al, 2001
, Burke et al, 2009
; Godin et al, 2010). Human MRI data has also indicated that ethanol exposure during gestation has a disproportionate effect on the developing cerebral cortex (reviewed by Spadoni et al, 2007
; Norman et al, 2009
). An over-abundance of gray matter and not enough white matter in cortical regions around the left posterior parietal cortex has been reported in prenatally ethanol-exposed individuals (Sowell et al, 2001
). Additionally, alterations in gray matter asymmetry in the posterior/inferior temporal lobe regions (Sowell et al, 2002
), as well as an excess in cortical thickness in bilateral temporal, inferior parietal and frontal regions (Sowell et al, 2008
) have been found. Further examination utilizing sophisticated image analyses tools in animal models could provide additional insight as to subregions of the cortex that are especially sensitive to specific ethanol exposure periods.
That the 3rd
ventricle was enlarged and dysmorphic in a large proportion of the ethanol-exposed fetuses examined may be suggestive of insult to the surrounding brain tissue; the diencephalon/thalamus and hypothalamus. However, thalamic volumes were unchanged in the analysis of raw volume data and actually increased when taking into account total brain size following prenatal ethanol exposure. The latter is likely explained by the substantial decrease in cortical volumes after prenatal ethanol exposure. In regard to the former, given the limited size of the 3rd
ventricle, even a small increase in the number of voxels translates to a large change in the overall 3rd
ventricular volume. A reduction of this same magnitude in a large region such as the thalamus would not necessarily result in any statistically identified result. More detailed examination and segmentation of sub-regions of the thalamus and hypothalamus could potentially uncover volume deficits. Supporting this expectation is a previous report by Dunty and colleagues (2001)
who examined patterns of cell death following acute ethanol exposure at varying time points and found that the hypothalamus was particularly affected following acute ethanol exposure on GD10.5 (Dunty et al, 2001
). As discussed in a review by Weinberg et al, (2008)
, that alterations in the hypothalamic-pituitary-adrenal (HPA) axis can be induced following maternal administration of an ethanol-containing liquid diet throughout pregnancy is a well-established finding. Additionally, Park et al, (2004)
showed that in C57Bl/6J mice, acute early gestational exposure to ethanol enhances a corticosterone-mediated response to stress. Clearly, along with more detailed histological analyses, postnatal functional analyses with particular attention to perturbations of hormonal cascades are needed as a follow up to the current observations.
The finding in this study of a periventricular heterotopias extending into the 3rd
ventricular space of one of the ethanol-exposed fetuses also illustrates insult to the diencephalon. A similar 3rd
ventricle heterotopias was previously reported to result from acute ethanol exposure on GD7 in mice (Sulik et al, 1984
). In a previous MRM-based study, acute GD7 ethanol exposure was shown to cause (leptomeningeal) cerebral cortical heterotopias in mice (Godin et al, 2010) and both cortical heterotopias and heterotopias localized to the lateral ventricle and interventricular foramen after gestational ethanol exposure have been reported in rats (Komatsu et al, 2001
; Sakata-Haga et al, 2004
). Both cerebral cortical heterotopias and ventricular heterotopias have been found in humans exposed to ethanol during gestation (eg. Jones & Smith, 1973
; Coulter et al, 1993
; Clarren et al, 1978
; Peiffer et al, 1979
; Clarren et al, 1981
). Heterotopias are highly linked to seizure activity (Verrotti et al, 2009). Given the heightened prevalence of seizure activity among individuals with prenatal alcohol exposure as compared to the general population (eg. Sun et al, 2009
, Bell et al, 2010), and considering the occurrence of low seizure thresholds in FASD rodent models (Bonthius et al, 2001a
; Bonthius et al, 2001b
), it is likely that as imaging technologies advance more heterotopias will be discovered in both humans and animal models. Indeed, a very recent case report describes MRI-based discovery of polymicrogyria and two periventricular lesions in a 16 year old girl with FAS who presented with seizures (Reinhardt et al, 2010).
As compared to the MRM-based findings reported for acute ethanol-induced GD7 and GD8 fetal brain changes (Parnell et al, 2009
; Godin et al, 2010), the GD10 pattern of insult differs. The holoprosencephaly spectrum characterizes the GD7 ethanol exposure time, but did not result from GD10 treatment. Notable following GD8 insult was a reduction in hippocampal, olfactory bulb, and cerebellar volumes in which the right side of the brain tended to be preferentially affected, a finding not evident in the current study (Parnell et al, 2009
). Further, GD8 treatment increased 4th
ventricular volume, while GD10 treatment has a major affect on the 3rd
ventricle. Analyses of data from other days of acute ethanol insult to the mouse embryo (esp. GD9 & GD11) are currently being finalized, with preliminary results indicating unique dysmorphology patterns for each (unpublished observations).
Despite its limitations (e.g. cost, scanning time, access to sophisticated imaging systems), MRM affords unprecedented opportunities to define teratogenic endpoints. In addition to examination of brain dysmorphology as described herein, the application of MRM to the study of other organ systems holds significant promise. To date, MRM-based studies of normal and abnormal cardiovascular system development have proven particularly informative (Smith, 2001
; Petiet et al, 2008
), further highlighting the potential of this technology for teratology investigations. Although MRM cannot replace standard teratological techniques such as routine histology and Wilson’s razor sections, with advancing technology that will enable more rapid imaging and lower costs for each specimen, and with more imaging centers becoming available, undoubtedly, MRM will be more widely employed to assess whole body morphology.
In conclusion, recognition that GD10 ethanol exposure in mice yields not only growth retardation, but a stage-dependent pattern of CNS defects was facilitated by the generation of, and comparison to, MRM-based data illustrating normal regional brain growth trajectories. A major effect on ventricular and cortical volume was shown. Clinical application of these findings rests, in part, in appreciation of the fact that these defects can arise from insult at a period in development corresponding to times prior to the middle of the first human trimester and the potential of even early human prenatal imaging to identify these types of CNS changes (eg. Kfir et al, 2009