Abstract
Introduction: Foetal Alcohol Spectrum Disorder (FASD) is an array of disorders attributed to CNS damage, behavioural and social manifestations after varying degrees of prenatal exposure to alcohol. Due to the majority of effects being phenotypically subtle, many cases are not diagnosed, and subsequently do not receive support and treatment. Drinking during pregnancy is an on-going issue in New Zealand, in particular binge-like episodes resulting in high blood alcohol concentrations. These episodes have been shown to continue throughout pregnancy in some women, and a lack of evidence correlating specific structural and functional outcomes after a single alcohol binge during the third trimester leaves room for question about what timing and quantity of alcohol induces lasting abnormalities in early life and adulthood.
Methods: Two cohorts of animals were used – (1) for stereological studies and (2) for behavioural studies. On Post Natal day 6 (PN6), the human third-trimester equivalent, male and female Long Evans rats were randomly assigned into one of three treatment groups and administered an intragastric intubation; Ethanol 6.0g/kg (E6), 5.25g/kg (E5), 4.5g/kg (E4); and two control groups - Intubation control (IC) and Suckle Control (SC). Cohort 2 underwent social play behaviour analysis via video recording from P32-34, and again on PN80-82. Animals were later analysed on the Elevated T-maze (ETM) for anxiety-related responses to an aversive environment. Cohort 1 were deeply anaesthetised and perfused on PN365. Brains were removed, cryoprotected, frozen and sectioned in the coronal plane at 60μm. A random systematic set of sections were stained with thionin followed by unbiased stereological methods using the optical disector to determine the number of neurons in the Anterior Cingulate Cortex (Acc) and hippocampal CA1 area.
Results: Cohort 1 revealed a dose-dependent loss of hippocampal CA1 cells across all alcohol-exposed groups (P<0.01); along with E6 and E5 animals showing a 39.6% and 30.8% (P<0.05) mean decrease in Acc cells relative to IC animals. The E6 group also had significantly less hippocampal CA1 and Acc neurons than the E4 group (P<0.05). Play behaviour analysis in cohort 2 revealed increased rates of attack by the alcohol-exposed and IC groups compared with controls (P<0.05). SC animals did not display the normal reduction in attack frequency between adolescence and adulthood, while E6 animals preferred the evasive form of defence significantly more often than all other groups during adulthood (P<0.05). ETM testing did not reveal any definitive anxiogenic / anxiolytic effects of prenatal alcohol exposure (P = 0.058), however E6 animals showed a significant lack of response inhibition from trial 1 to trial 2 on the inhibitory avoidance task relative to IC and SC animals (P<0.05). The one-way escape task revealed E6 animals took significantly less time than E4 animals to escape into the closed arm, however as no difference was found with relation to control groups this result was equivocal.
Conclusion: It is clear that a single ethanol binge on PN6 induces significant neuronal death in both the hippocampal CA1 (E6 = 49%, E5 = 38%, E4 = 26%) and Acc (E6 = 28%, E5 = 24%, E4 = 5%) areas, in a dose dependent fashion. Both of these areas are involved with social functioning, and corresponding social play abnormalities were observed, however not in the fashion hypothesized. During adolescence, both ethanol-exposed and control groups were behaving abnormally and showing different rates of defensive patterns than previously reported in the literature. This may be due to a “rub off” effect of the alcohol animals being housed with the control animals. These social play behaviour anomalies seen in adolescence were not evident in adulthood, indicating some level of functional recovery to appropriate levels of attack and defence during a ten-minute play period. However E6 animals showed a significantly higher probability of evading an attack than SC animals, suggesting a preference for bout termination possibly due to intimidation by the more cognitively intact SC animals. Anxiety testing revealed anomalies in response inhibition in E6 animals, however further refined studies are needed to definitively elucidate these putatively subtle changes.
These data contribute to the growing pool of evidence that any amount of alcohol during any stage of pregnancy may have serious repercussions, and only through continuing research will we begin to build an evidence base for public health policy and guidelines.