Evaluating signs of hippocampal neurotoxicity induced by a revisited paradigm of voluntary ethanol consumption in adult male and female Sprague-Dawley rats

Abstract

Background: Binge alcohol drinking is considered a prominent risk factor for the development of alcohol-use disorders, and could be model in rodents through the standard two-bottle preference choice test. The goal was to recreate an intermittent use of alcohol during 3 consecutive days each week to ascertain its potential impact on hippocampal neurotoxicity (neurogenesis and other neuroplasticity markers), and including sex as a biological variable, given the well-known sex differences in alcohol consumption.

Methods: Ethanol access was granted to adult Sprague-Dawley rats for 3 consecutive days per week, followed by 4 days of withdrawal, during 6 weeks, mimicking the most common pattern of intake in people, drinking over the weekends in an intensive manner. Hippocampal samples were collected to evaluate signs of neurotoxicity.

Results: Female rats consumed significantly more ethanol than males, although intake did not escalate over time. Ethanol preference levels remained below 40% over time and did not differ between sexes. Moderate signs of ethanol neurotoxicity were observed in hippocampus at the level of decreased neuronal progenitors (NeuroD + cells), and these effects were independent of sex. No other signs of neurotoxicity were induced by ethanol voluntary consumption when measured through several key cell fate markers (i.e., FADD, Cyt c, Cdk5, NF-L) by western blot analysis.

Conclusions: Overall, the present results suggest that even though we modeled a situation where no escalation in ethanol intake occurred across time, mild signs of neurotoxicity emerged, suggesting that even the use of ethanol during adulthood in a recreational way could lead to certain brain harm.

Keywords: Alcohol voluntary drinking; Hippocampus; Neurogenesis; Rat; Sex differences.

Fig. 1

Experimental design. a Sprague-Dawley rats were exposed during 6 consecutive weeks to a weekly schedule consisting of a 3-day continued voluntary ethanol access (two-bottle choice: 20% ethanol vs. water; D1–D3) followed by a 4-day withdrawal period (two bottles of water). Rats were killed on the last day of ethanol exposure on week 6 (D38). b Changes in body weight across weeks (g). Groups of treatment: Control-male (n = 10); Ethanol-male (n = 11); Control-female (n = 10); Ethanol-female (n = 11). Columns represent mean ± SEM of change in body weight (g). Individual symbols are shown for each rat. #p < 0.05 when comparing the effect of sex (female vs. male rats; three-way RM ANOVA)

Fig. 2

Intermittent access to 20% ethanol in a two-bottle choice test for 3 consecutive days. a–c Total fluid (ml) and d–f Water (ml) intake daily (a–d), weekly (b–e) and expressed as the 18 days overall average (c–f). Groups of treatment: Control-male (n = 10); Ethanol-male (n = 11); Control-female (n = 10); Ethanol-female (n = 11). Columns represent mean ± SEM of the amount of liquid consumed in ml. Individual symbols are shown for each rat. Three-way RM ANOVAs showed no significant effects of Bottle Choice or Sex. g–i Ethanol preference (%) and j–l Ethanol (g/kg) consumption daily (g–j), weekly (h–k) and expressed as the 18 days overall average (i–l). Groups of treatment: Ethanol-male (n = 11); Ethanol-female (n = 11). Columns represent mean ± SEM of the preference for ethanol (expressed as a % value) or ethanol dose consumed (g/kg). Individual symbols are shown for each rat. #p < 0.05 when comparing the general effect of sex (two-way repeated-measures ANOVA); *p < 0.05 when comparing the dose consumed by female rats vs. male rats (Student’s t-test)

Fig. 3

Evaluating signs of ethanol-induced neurotoxicity in hippocampus. Quantitative analysis of a Ki-67 and b NeuroD + cells in the left dentate gyrus of the hippocampus by immunohistochemistry or of c FADD, d Cytochrome c (Cyt c) e Cdk5, f NF-L and g β-actin protein content by western blot analysis in the hippocampus of male and female rats exposed to the two-bottle choice test (Control vs. Ethanol) on D38. Groups of treatment: Control-male (n = 10); Ethanol-male (n = 11); Control-female (n = 10); Ethanol-female (n = 11). Columns represent mean ± SEM of the number of + cells quantified in 8 sections from the middle part of the hippocampus and expressed as % change vs. control-male rats or of n experiments per group and expressed as a percentage of Control-male-treated rats. Individual symbols are shown for each rat. Two-way ANOVAs evaluating the potential effects of Bottle Choice (ethanol vs. water) and Sex. #p < 0.05 when comparing the general effect of sex and **p < 0.01 when comparing the effects of having access to a bottle with ethanol vs. control (general effect independently of sex). Bottom panels: representative images showing individual Ki-67 (brown labeling in the blue granular layer) and NeuroD (dark blue labeling in the blue granular layer) cells taken with a light microscope (40 × objective lens) or representative immunoblots depicting labeling of FADD, Cyt c, Cdk5, NF-L and β-actin are shown for each set of experiments. Other representative images for Ki-67 and NeuroD labeling and/or full immunoblots from which images were taken could be found in Supplementary Materials