Supplementary Material for: Abnormal Magnetic Resonance Imaging in the Medial Prefrontal Cortex Following Prenatal Alcohol Exposure and Placental Insufficiency in a Preclinical Model

Introduction: The combination of Prenatal Alcohol Exposure (PAE) and Placental Insufficiency (PI) places infants at an increased risk for preterm birth and may worsen brain injury and neurobehavioral outcomes. In this preclinical study, the effect of PAE+PI on lateral, medial, and ventral prefrontal cortex (PFC), striatum and corpus callosum microstructure were investigated using diffusion tensor imaging (DTI). These brain regions are important for executive and higher cognitive functions, like cognitive flexibility. Methods: Pregnant Long-Evans rat dams voluntarily drank 5% ethanol in saccharin water or plain saccharin water until embryonic day 18 (E18) to mimic moderate PAE. On E19, an open laparotomy was completed, and the uterine arteries were transiently occluded for one hour. The dams in the sham group underwent the same procedure, but without uterine arteries occlusion. Offspring delivered normally on E22 and matured with their dams. At postnatal day 35 (P35), tissue was collected from male and female rat offspring from all four prenatal treatment groups (Sham, PAE, PI, and PAE+PI). Fixed brain tissue was then scanned ex vivo on a Bruker 11.7 T MRI. Fractional anisotropy (FA) and directional diffusion were measured in regions of interest (ROI). Two-way ANOVA with Tukey’s correction was used, with p<0.05 significant. Results: Diffusion Tensor Imaging (DTI) analyses of the PFC (n=14-30/group) revealed a significant impact of the prenatal exposure/insult on the FA (p<0.05), with sham having the lowest FA (0.24 ± 0.01) and PI having the highest FA (0.28 ± 0.02). The lateral PFC was significantly impacted by prenatal exposure/insult with sham having the highest radial diffusivity (RD; 4.97x10-4 ± 2.20x10-5 mm2/s; p<0.05) and mean diffusivity (MD; 4.41x10-4 ± 2.10x10-5 mm2/s; p<0.05) compared to the other groups. The striatum was sensitive to the prenatal exposure/insult, with the axial diffusivity (AD), RD, and MD all significantly increased in the PAE group and decreased in the PI group (p<0.05). In the corpus callosum, the prenatal exposure/insult significantly decreased the AD (p<0.05; PAE+PI AD: 5.00x10-4 ± 4.60x10-5 mm2/s). Conclusion: While all areas analyzed were impacted by the prenatal insults, the striatum, which consists primarily of efferent pathways, appears more vulnerable to injury compared to the PFC. Additional studies are needed to characterize the impact this may have on function related to these critical brain regions.