Effects of intrauterine alcohol exposure on three-week-old Sprague-Dawley Rat distal tibia: an immunohistochemistry and three-dimensional micro-focus x-ray computed tomography (3d-μCT) investigation

Abstract

Children exposed to alcohol during gestation are at risk of developing a spectrum of neurodevelopmental disorders known as fetal alcohol spectrum disorders (FASD). The most severe form of this disorder is fetal alcohol syndrome (FAS), which is characterised by neurological deficits, facial dysmorphogenesis and growth retardation. Most research is on neurological deficits and facial dysmorphogenesis, with less attention given to the effects of gestational alcohol exposure on the skeletal system. Therefore, this study investigated the effects of prenatal alcohol exposure on bone growth and development in a postnatal three week old Sprague-Dawley rats tibia using immunohistochemical and three dimensional microfocus x-ray computed tomography (μCT) investigations. Female Sprague-Dawley rats (n=15) were time-mated and divided in to three groups as follows: (i) the ethanol group (n=6) received 0.015 ml/g body weight of 25.2% alcohol for 19 days, (ii) the saline control group (n=6) received the same dose of 0.9% saline for the same period and (iii) the untreated control group (n=3) received neither ethanol nor saline. Two Pups (n=30) from each dam (n=30} were randomly selected and terminated on day 21 after delivery and bilateral tibiae (n=60) were dissected, fixed, decalcified, processed and then subjected to histological and immunohistochemistry staining as well as μCT. The growth plate surface area was significantly smaller with fewer cell number in proliferative and hypertrophic zones in the ethanol group. There were also fewer Ki-67 labelled cells recorded in the ethanol group. However, the full and shaft length of the

tibia were similar among all three groups in the study. We found both lower bone volume to total volume (BV/TV) and trabecular thickness (TbTh) to be lower in the ethanol group in comparison with the controls. The trabecular number (TbN) was not affected in our study. However, there was more trabecular spacing in the ethanol group. This, considered together with the lower BV/TV and TbTh suggests that, although the bone length was similar in all groups, the internal morphology was not the same among the study groups. This indicates that prenatal alcohol exposure at our dosage may affect internal architecture while sparing the external bone length. This disruption of the internal bone morphology may also explain why FAS children are prone to osteoporosis and fracture as they may have less bony material internally. Employing a binary logistic regression showed that the distal medullary canal area and trabecular spacing were the main parameters that determined group membership into either ethanol treated or saline control. This means that these two are affected the most in gestational alcohol exposure. Our finding of a positive correlation between medullary canal area and cortical thickness indicates that the smaller medullary canal was coupled with thinner cortical bone. We propose that this may potentially explain weaker bones observed in FAS children. In the ethanol group, trabecular thickness and spacing had negative loadings to the principal component analysis model. These two parameters were also negatively correlated. This suggests that trabecular formation may have been delayed in the ethanol group.

Gestational alcohol exposure had an adverse effect on the growth plate with respect to its general size, respective zone sizes and the number of cells in each zone. This may be how diminished stature of the offspring occurs. Fewer proliferative cells were found using the anti-Ki67 antibody, indicating that in utero alcohol exposure slows cell proliferation, contributing to the small stature. Logistic regression showed that the distal medullary canal area and trabecular separation were the main parameters affected the most in gestational alcohol. The negative correlation of trabecular thickness and spacing in the ethanol group may be a contributor to bone weakness. These findings add new knowledge to how in utero alcohol exposure affects the offspring.