Alcohol-induced facial dysmorphology in C57BL/6 mouse models of fetal alcohol spectrum disorder

Abstract

Alcohol consumption during pregnancy causes fetal alcohol spectrum disorder (FASD), which includes a range of developmental deficits. Fetal alcohol syndrome is the most severe form of FASD and can be diagnosed with pathognomonic facial features such as a smooth philtrum, short palpebral fissure, and thin upper vermilion. However, many children with developmental damage because of prenatal alcohol exposure exhibit none, or only a subset, of the above features, making diagnosis difficult. This study explored novel analyses to quantify the effect of a known dose of alcohol on specific facial measurements in substrains C57BL/B6J (B6J) and C57BL/6NHsd (B6N) mice. Mouse dams were provided alcohol (Alc) consisting of 4.8% (vol/vol) alcohol in a liquid diet for 16 days prepregnancy and chow and water diet during mating, and then the alcohol liquid diet was reinstated on gestational days 7 (E7) to gestational day 17 (E17). Treatment controls included a pair-fed (PF) group given matched volumes of an alcohol-free liquid diet made isocalorically and a group given ad lib access to lab chow and water (Chow). Maternal diet intake (Alc and PF), blood alcohol concentrations (BACs), embryo weights, and 15 morphometric facial measurements for E17 embryos were analyzed. B6N dams drank more alcohol during pregnancy and generated higher BAC than B6J dams. Both the Alc and PF treatments induced significant reductions in embryo weights relative to Chow in both substrains. Alcohol treatments produced significant changes, relative to controls, in 4 of the 15 facial measures for the B6N substrain but only in two measures for the B6J substrain. Discriminant analysis demonstrated successful classification of the alcohol-exposed versus nonalcohol-exposed B6N embryos, with a high sensitivity of 86%, specificity 80%, and overall classification (total correct 83%), whereas B6J mice yielded sensitivity of 80%, specificity 78%, and overall correct classification in 79%. In addition, B6N mice showed significantly more effects of pair feeding on these facial measures than did B6J mice, suggesting that the B6N substrain may be more vulnerable to nutritional stress during pregnancy. Overall, these data indicate that both B6N and B6J mice were vulnerable to alcohol but show differences in the severity and location of alcohol-induced dysmorphic facial features and may parallel findings from human studies comparing different ethnic groups. Furthermore, these findings suggest that discriminant analysis may be useful in predicting alcohol exposure in either mouse substrains.

Figure 1

Alcohol consumption for B6N and B6J animal groups. 1a and b. Alcohol consumption was recorded daily in both B6N and B6J, during pre-pregnancy 16 days (a) and post-pregnancy (b) E7-E17. Daily means -± SEM are reported in g/kg intake. N=30, B6N, and 45, B6J, pre-pregnancy, 15, B6N, 15, B6J, post pregnancy. Age of dams was 27 weeks prior to pre-pregnancy treatment. 1c and d. Daily means -± SEM of liquid diet volume consumed reported in ml/kg. Table (e) reflect differences in total g/kg intake both pre and post pregnancy. (*) Denotes significant difference. Table (f) reflects alcohol treated dam weights for each sub-strain reported as mean ± SEM.

Figure 2

A. Chart of blood alcohol concentrations (mg/dl) analized using gas chromatography. Data is reported as MEAN ± SEM with an N=8 for each sun-strain. (*) Denotes significant differences between sub-strain using student t-test. B. Histogram of E17 embryo weights from all treatment groups. Average weights for all embryos are reported in grams ± SEM. (*) Denotes significant difference by (ANOVA) within treatment groups compared to chow. (**) Denotes difference between groups for designated treatment. N = 7,11,12 B6N, Alc, PF, Chow, 12,9,7 B6J, Alc, PF Chow.

Figure 3

E17 embryos depicting 15 measurement landmarks used for 2D analysis. Distinct definitions for landmark selects are described in methods table 1. N= B6N; alc 6, chow 12, PF 11 and B6J: alc 12, chow 7, PF 9. Calibration scale is at 1000μm.

Figure 4

Embryo photos showing changes in facial measurements used as predictors within the two sub-strains. Red lines arrows indicate the type and direction of change for each sub-strain. In all cases alcohol induced decreased measurements. Calibration is at 1000μm.