The impact of binge alcohol consumption on the developing humerus of adolescent Sprague Dawley rats

Abstract

Binge drinking is the ingestion of alcohol in high volumes during a short period, raising blood alcohol concentration to ≥80 mg/dL. It is common among adolescents, which is concerning since this is a critical period for skeletal growth and development, processes that binge drinking may impair. Moreover, the question of whether alcohol abstinence can reverse any damage inflicted by binge drinking is also contemplated. Therefore, this study investigated how binge drinking affects bone structure, strength, and development in the adolescent humerus and whether these effects can be reversed through alcohol abstinence. The study included 48 adolescent Sprague Dawley rats (24 males and 24 females) aged 7 weeks (49 postnatal days) as the age corresponds to 14.5 human years which represents the human adolescent period (ages 10-19). They were divided into two major groups where Group 1 was the chronic binge consumption model, (four-week alcohol exposure) and Group 2 was the binge alcohol recovery model (four-week alcohol exposure followed by a 26-day abstinent period). Each group had an alcohol-exposed (A) and control (B) group resulting in a total of four groups (1A,1B,2A,2B) where each of the four groups consisted of n =12 rats (6 males and 6 females). A 20% (vol/vol) alcohol solution at a dose of 3g/kg was administered to the study animals, as a single dose via oral gavage. An isocaloric equivalent of maltose dextrin, via oral gavage, was given to the control animals. Alcohol/maltose dextrin was administered 3 days/week (on every alternative day) and no further administration was done during the remaining 4 days of the week. Humeral osteometry was performed and humeral weights were recorded to investigate the impact of binge drinking on the external structure. Following this, we used 3D micro-focus X-ray tomography (3D-μCT) allowing for detailed examination of humeral morphometry. To assess humeral strength, we performed a three-point bending test. Finally, we evaluated humeral development using Haematoxylin and Eosin (H&E) staining and immunohistochemistry (IHC). H&E staining facilitated a qualitative assessment of growth plate chondrocytes (cartilage cells) and IHC provided a quantitative measure of chondrocyte proliferation, using the anti-Ki-67 antibody. Statistical analysis included normality tests (such as the Shapiro Wilk test), T-tests and the Mann Whitney test (for non-parametric data). In the chronic binge consumption model, four weeks of alcohol exposure caused reduced humeral weight in females. In the binge alcohol recovery model, trabecular number (Tb.N), spacing (Tb.Sp), and fracture times remained significantly affected. Overall, the findings indicate that four weeks of binge alcohol exposure did not cause severe structural damage to the humeri in either sex, as most parameters showed minimal or no change across both models. The results of the binge alcohol recovery model suggest that recovery may be possible and that male humeri respond iii better to alcohol exposure than female humeri. However, significant alterations in select measures, such as reduced humeral weights in females (Group 1); delayed fracture times in females (Group 2) and changes in trabecular architecture in males (Group 2), suggest that alcohol exposure, while not overtly damaging, did exert measurable adverse effects. The research also showcased evidence of site-specific heterogeneity as the proximal and distal regions of the humeri had a differential response to alcohol exposure. Lastly, the research underscored the influence of sexual dimorphism, as both male and female humeri exhibited distinct responses to alcohol exposure. The findings of this study are relevant in the clinical setting, indicating that binge drinking in adolescents has an impact on the developing skeleton, more so in males than in females, and can lead to growth and skeletal complications including an increase in fracture rates in this vulnerable population.