Disinhibition-Like Behavior Correlates with Frontal Cortex Damage in an Animal Model of Chronic Alcohol Consumption and Thiamine Deficiency

Abstract

Wernicke-Korsakoff syndrome (WKS) is induced by thiamine deficiency (TD) and mainly related to alcohol consumption. Frontal cortex dysfunction has been associated with impulsivity and disinhibition in WKS patients. The pathophysiology involves oxidative stress, excitotoxicity and inflammatory responses leading to neuronal death, but the relative contributions of each factor (alcohol and TD, either isolated or in interaction) to these phenomena are still poorly understood. A rat model was used by forced consumption of 20% (w/v) alcohol for 9 months (CA), TD hit (TD diet + pyrithiamine 0.25 mg/kg, i.p. daily injections the last 12 days of experimentation (TDD)), and both combined treatments (CA+TDD). Motor and cognitive performance and cortical damage were examined. CA caused hyperlocomotion as a possible sensitization of ethanol-induced excitatory effects and recognition memory deficits. In addition, CA+TDD animals showed a disinhibited-like behavior which appeared to be dependent on TDD. Additionally, combined treatment led to more pronounced alterations in nitrosative stress, lipid peroxidation, apoptosis and cell damage markers. Correlations between injury signals and disinhibition suggest that CA+TDD disrupts behaviors dependent on the frontal cortex. Our study sheds light on the potential disease-specific mechanisms, reinforcing the need for neuroprotective therapeutic approaches along with preventive treatments for the nutritional deficiency in WKS.

Keywords: Wernicke’s encephalopathy; apoptosis; cell damage; chronic alcohol; disinhibition; lipid peroxidation; nitrosative stress; nutritional deficit; recognition memory; thiamine deficiency.

Figure 5

Correlations between damage parameters and disinhibition measures of the EPM in the CA+TDD-treated animals. The trend lines on each graph show the combined regression analyses for the control (circles) and CA+TDD (triangles) groups. (A) NO⁻² levels in plasma were positively correlated with the EPM open entries ratio. Frontal cortical levels of (B) 4-HNE, (C) Caspase 9 and (D) HSP70, HMGB1 were positively correlated with the EPM open time and entry ratios. Pearson’s coefficient correlation r; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Figure A1

Decrement in plasma levels of total thiamine at the end of the protocol (36 weeks) versus intermediate status (week 12) in adult rats. Results normalized by body weight (ng/mL data divided by kilogram). Data are expressed as mean ± S.E.M.

Figure A2

Behavioral data including the oral thiamine-supplemented groups (C+T, CA+T). (A) Spontaneous alternation task (SAT). (B) Open field (OFT) testing locomotion and disinhibition. (C) Elevated plus maze (EPM). (D) Effect on memory abilities tested by NOT. Mean ± SEM (n = 6–10). Data analyzed by unpaired Student’s t-test or Mann–Whitney test to compare CA+T vs. CA group (p > 0.05, n.s.).

Figure A3

Biochemical data from the oral thiamine-supplemented CA group. Graphs indicate protein levels of markers in the frontal cortex and plasmatic nitrites. Western blot data of the respective proteins of interest were normalized by β-actin and expressed as a percentage of change versus the control group. (A) Nitrosative stress markers. Lipid peroxidation was measured by 4-Hydroxynonenal (4-HNE) accumulation. (B) Markers of apoptotic cell death: caspase 3, 8 and 9. (C) Damage-associated molecular patterns (DAMPS): HSP70, HSP60 and HMGB1. Mean ± SEM (n = 8–9). Unpaired Student’s t-test or Mann-Whitney were different from CA group at p < 0.05.

Figure 1

Experimental design, with timeline of treatments and behavioral testing. Body weight and water (H₂O) and ethanol (EtOH) consumption were recorded throughout the entire experiment and the TD diet intake during the last 12 days. BECs = blood ethanol concentrations; NSS/NBS = neurological examination; SAT = spontaneous alternation task; OFT = open field test; NOT = novel object recognition test; EPM: elevated plus maze.

Figure 2

Physiological parameters along the procedure. (A) Average weights at weekly intervals and at days 1 and 12 of the final TD diet protocol, along with the diet intake (g/Kg). All rats gained weight until week 34, but the CA animals were always below the controls. Weight loss in TDD and CA+TDD groups during TD diet treatment was consistent with the decreased intake. (B) Ethanol consumption (g/Kg) across weeks, along with blood ethanol concentrations (BECs (boxed numbers): mean ± SEM). The animals consumed more alcohol during their adolescent and young stages than in the adult stage. All data are expressed as mean ± S.E.M. Repeated measures ANOVAs; within–group differences: a p < 0.05; b p < 0.001; c p < 0.0001; between–group (different vs control group): ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 3

Behavioral changes as a function of the treatment condition. (A) Spontaneous alternation task (SAT). There was an increase in the total arm entries by the CA groups independent of TDD treatment, interpreted as hyperlocomotion, and no effect on spontaneous alternation reflecting spatial working memory. (B) Open field testing (OFT) locomotion and disinhibition. Here, we also found motor agitation by alcohol. Rats typically avoid open areas. CA+TDD-treated rats explored the inner zone for more time than the rest of group, reflecting a trend of disinhibition. (C) Elevated plus maze (EPM). Time spent and entries in open arms showed a remarkable disinhibited behavior just in rats with CA combined with TDD treatment. (D) Effect on memory abilities tested by NOT. Rats exposed to CA were impaired in their ability to discriminate the new object, thus showing a deficit in recognition memory. Mean ± SEM (n = 8–10). Two-way ANOVA or nonparametric Kruskal–Wallis test. Overall alcohol effect: ^& p < 0.05, ^&& p < 0.01. Interaction (CA × TDD) followed by post hoc test, different from control group: * p < 0.05, ** p < 0.01; different from CA: ^# p < 0.05, ^## p < 0.01.

Figure 4

Biochemical changes as a function of treatment condition. Graphs indicate protein levels of markers in the frontal cortex and plasmatic nitrites. Western blot data of the respective bands of interest (upper bands) were normalized by β–actin (lower band) and expressed as a percentage of change versus the control group. (A) Nitrosative stress markers. Nitrites (NO−₂) increased in plasma for all conditions of exposure, but the combination of CA and TDD enhanced this elevation. Lipid peroxidation was measured by 4–Hydroxynonenal (4–HNE) accumulation. There was a significant effect of CA and CA+TDD groups versus control group. (B) Markers of apoptotic cell death: caspase 3, 8 and 9. All treatments led to an elevation of caspase 9 relative to the controls. (C) Damage-associated molecular patterns (DAMPS): HSP70, HSP60 and HMGB1. CA+TDD treatment increased the HSP70 and HMGB1 expression with respect to the controls. Mean ± SEM (n = 8–10). Two-way ANOVA or nonparametric Kruskal–Wallis test. Overall alcohol effect: ^& p < 0.05, ^&&&& p < 0.0001; overall TDD effect: ^$ p < 0.05, ^$$ p < 0.01; and interaction (CA × TDD) followed by post hoc test, different from control group: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.