After the first 16 weeks of intermittent alcohol exposure (MRS 2), two rats died prematurely; the remaining eight rats in the alcohol group reached average BALs of 292.98±42.12mg per 100 ml (range 240.5–385.6 mg per 100 ml) and lost 1.2% body weight (alcohol: 606±83.17 g), whereas controls gained 13.4% of their baseline body weight (control: 713.69±93.97 g, p=0.02). After 8 more weeks of alcohol exposure (MRS 3), the alcohol group achieved average BALs of 444.63±24.14 mg per 100 ml (range 324.3–514.1mg per 100 ml) and weighed 567.1±107.58 g (lost 6.8% more of their body weight) whereas controls weighed 733.47±96.94 g, having gained 2.9% more of their body weight since MRS 2 (p=0.003).
For metabolite signals normalized to tissue water, a two-group repeated-measures (three MRS sessions and seven metabolite signals) ANOVA revealed a significant group-by-metabolite- by-time interaction (p=0.0002). Follow-up analysis of metabolites considered in our primary hypothesis identified significant effects of group or group-by-session interactions for Cho, Glx, and Glu. The groups did not differ at baseline, but after 16 weeks of alcohol exposure, the alcohol group had significantly higher Cho (p=0.0002) levels than controls, and Glx (p=0.05) showed a trend in the same direction. After 8 more weeks of greater alcohol exposure, the alcohol group had higher Cho (p=0.001), Glx (p=0.0035), and Glu (p=0.0129) than the control group ( and ; ). A family-wise Bonferroni correction for four metabolite signals examined with pair-wise tests would require a one-tailed significance of p≤0.025, and a two-tailed significance of p≤0.0125. Thus, even with a two-tailed Bonferroni correction, the increase in Cho was significant at MRS 2, and increases in Cho, Glx, and Glu were significant at MRS 3.
Mean±SEM of the 7 metabolite signals quantified relative to tissue water for each of the 3 MRS acquisitions for the control (open circles) and alcohol-exposed (closed circles) rats. *p≤0.05, 1-tailed. **p≤0.05, 2-tailed.
Metabolite Levels in Basal Ganglia Voxel: Means and Standard Deviations
Ratios relative to tCr yielded a similar pattern of alcohol effects as did ratios relative to water. In particular, a repeated-measures ANOVA revealed a significant group-by-metabolite- by-time interaction (p=0.0001). Again, the groups did not differ at baseline, but after 16 weeks of alcohol exposure, the alcohol group had higher Cho/tCr (p=0.0001), Glx/tCr (p=0.0155), and Glu/tCr (p=0.0176) ratios, which persisted at 24 weeks of escalating alcohol exposure (Cho/tCr, p=0.0001; Glx/tCr, p=0.0004; Glu/tCr, p=0.0027; ). The ratio values and patterns across the three time points were similar whether Cho was analyzed relative to tCr or to NAA, ie in both cases, ratio differences across the three time points in controls were not significant, whereas Cho was higher in the alcohol group compared with controls at MRS 2 and MRS 3. In the alcohol group, Cho/tCr ratios demonstrated an increase across all times points such that MRS 1<MRS 2<MRS 3, and all three time comparisons were significant. Cho/NAA ratios followed the same pattern as Cho/tCr ratios; however, Cho/NAA levels at MRS 3 were not significantly higher than those at MRS 2.
Analysis of the remaining metabolites using two-tailed t-tests revealed no significant differences between groups, except for an elevation in mI relative to tissue water in the control compared to the alcohol group at MRS 3 (p=0.0216) that may be inaccurate because quantitation of mI in the presence of alcohol is hampered by spectral overlap with the methylene signal of ethanol at 3.6 ppm.
A final set of analyses took advantage of the sibling pair design (), which can reduce variance but, in this case, also reduced power because for the two rats that died, both they and their sibling pairs had to be removed from the analysis. The results yielded a significant difference (p≤0.05) in the Fisher’s protected least-significant difference test for Glx between MRS 1 and MRS 3 only.
Metabolite levels plotted by sibling pairs (alcohol-exposed is in black; control is in gray) at each MRI session for the 8 pairs that were available for the 3 scanning sessions. Thiamine and TMP levels at MRS 3, by sibling pairs, are also plotted.
Thiamine and Phosphate Derivatives
Plasma assay for thiamine and its phosphate derivates demonstrated that thiamine (p=0.0063) and TMP (p=0.0377) levels were significantly lower in the alcohol group than controls (). A follow-up set of analyses taking advantage of the sibling pair design () confirmed the significant difference for thiamine (p=0.02). The alcohol-exposed animals had thiamine and TMP levels approximately 30% below the level of the controls. Within the alcohol group, thiamine (r=−0.86, p=0.013; ρ=−0.96, p=0.0182) and TMP (r=−0.97, p=0.0004; ρ=−0.82, p=0.0442) levels correlated with BALs taken shortly before MRS 3, indicating the higher the BALs, the lower the thiamine and TMP levels (). TMP levels also correlated with the difference in alcohol rat weights between MRS 2 and MRS 3, indicating the lower TMP levels, the more weight loss (r=0.82, p=0.024; ρ=0.89, p=0.0287). Due to the death of two alcohol-exposed animals prior to the termination of the experiment, reduced statistical power mitigated against detecting significant correlations between thiamine levels and metabolite levels or neurological status. In the combined groups, Cho relative to tissue water at MRS 3 correlated with TMP (r=−0.50, p=0.0427; ρ=−0.62, p=0.0124) and Cho/tCr at MRS 3 correlated with thiamine (r=−0.54, p=0.0245; ρ=−0.51, p=0.0414) and TMP (r=−0.61, p=0.0089; ρ=−0.68, p=0.0062); Glu/tCr at MRS 3 correlated with TMP (r=−0.47, p=0.0542; ρ=−0.55, p=0.0267).
Thiamine and Its Phosphates (in nmol/l): Means and Standard Deviations
Scatter plots of a simple regression between alcohol rats’ BALs shortly before MRS 3 and plasma thiamine and TMP levels at the termination of the experiment. Higher BALs correlate with lower thiamine and TMP levels.
None of the rats exhibited liver morphological changes consistent with moderate (grade 3) or severe (grade 4) pathology. Indeed, none of the rats exhibited morphological hepatic changes consistent with alcoholic cirrhosis as they lacked observable hepatocellular parenchymal loss, macro-and micronodular hepatocellular regeneration, or bridging (centrilobular, portal–portal, and central–portal) fibrosis. Hepatocellular swelling and/or necrosis with neutrophilic inflammation () is potentially indicative of alcoholic hepatitis. Minimal to mild hepatocellular swelling and necrosis were noted in four of seven alcohol-exposed rats, two of which also displayed accompanying neutrophilic inflammation. Two of ten control rats demonstrated minimal to mild hepatocellular swelling and necrosis with secondary neutrophilic inflammation. Sinusoidal fibrosis was absent, but minimal to mild portocentric fibrosis was observed in five of seven alcoholic and five of ten control rats. Mallory bodies were not detected. The groups did not differ significantly in the presence or severity of hepatocellular swelling, necrosis, inflammation, or portocentric fibrosis.
Figure 5 Left lateral lobe liver specimens from alcohol-exposed animals (a, c, and d) and a control animal (b) were H&E stained. (a) Focal area of hepatocellular necrosis. (b) Hepatic glycogenosis. (c) Minimal microvesicular hepatic steatosis. (d) Mild (more ...)
Hepatic steatosis ranged in severity from minimal to mild and was noted in all seven alcohol-exposed rats but only four of ten control rats. In the alcohol rats, hepatic steatosis was microvesicular in six of seven (), and macrovesicular in six of seven () livers. In control animals, hepatic steatosis was microvesicular in 2 of 10 and macrovesicular in 3 of 10 livers. Alcohol and control rats differed significantly for the presence and severity of macrovesicular (p=0.0009) and microvesicular (p=0.0032) lipidosis. An additional significant difference (p=0.0007) between groups was the presence of minimal hepatic glycogenosis in two of seven alcohol rats, whereas all ten control animals exhibited glycogenosis. Hepatic glycogenosis was subgrossly midzonal in distribution, and morphologically appeared as clear, negatively staining floccular material that displaced the normal eosinophilic cytoplasm of hepatocytes ().
Simple regression analysis included all rats to test relations between changes in liver morphology, metabolite levels at MRS 3, and thiamine status. Greater presence and severity of microvesicular lipidosis correlated with higher levels of Glu (ρ=0.65, p=0.0095; ) at MRS 3. Greater presence and severity of macrovesicular lipidosis correlated with higher levels of Cho at MRS 3 (ρ=0.51, p=0.0408; ). Higher Cho (ρ=−0.51, p=0.0412), Glx (ρ=−0.64, p=0.0105), and Glu (ρ=−0.53, p=0.0333) at MRS 3 also correlated with absent or minimal glycogenosis. Macro- and microvesicular lipidosis each correlated with thiamine (macrovesicular, ρ=−0.76, p=0.0025; microvesicular, ρ=−0.51, p=0.041) and TMP (macrovesicular, ρ=−0.63, p=0.0117; microvesicular, ρ=−0.61, p=0.0142) levels.
Rotarod testing of balance maintenance did not differentiate the groups at any experimental test session. By contrast, neurological examination revealed more signs in the alcohol than control group. At MRS 2, six of eight of the alcohol rats and none of the ten controls exhibited neurological signs on 2 consecutive days. Neurological signs persisted when rats were again tested just before MRS 3, with all eight of the alcohol rats and none of the controls exhibiting signs on 2 consecutive testing days (). Frequently observed neurological signs included altered motor (loss of righting reflex, stereotypy, tremor) and autonomic (palpebral closure, excessive lacrimation, and nasal discharge) functions.
Frequency of neurological signs for 8 alcohol rats before MRS 2 and MRS 3.