Multi-modal imaging reveals differential brain volumetric, biochemical, and white matter fiber responsivity to repeated intermittent ethanol vapor exposure in male and female rats

Abstract

A generally accepted framework derived predominately from animal models asserts that repeated cycles of chronic intermittent ethanol (EtOH; CIE) exposure cause progressive brain adaptations associated with anxiety and stress that promote voluntary drinking, alcohol dependence, and further brain changes that contribute to the pathogenesis of alcoholism. The current study used CIE exposure via vapor chambers to test the hypothesis that repeated episodes of withdrawals from chronic EtOH would be associated with accrual of brain damage as quantified using in vivo magnetic resonance imaging (MRI), diffusion tensor imaging (DTI), and MR spectroscopy (MRS). The initial study group included 16 male (~325g) and 16 female (~215g) wild-type Wistar rats exposed to 3 cycles of 1-month in vapor chambers + 1 week of abstinence. Half of each group (n = 8) was given vaporized EtOH to blood alcohol levels approaching 250 mg/dL. Blood and behavior markers were also quantified. There was no evidence for dependence (i.e., increased voluntary EtOH consumption), increased anxiety, or an accumulation of pathology. Neuroimaging brain responses to exposure included increased cerebrospinal fluid (CSF) and decreased gray matter volumes, increased Choline/Creatine, and reduced fimbria-fornix fractional anisotropy (FA) with recovery seen after one or more cycles and effects in female more prominent than in male rats. These results show transient brain integrity changes in response to CIE sufficient to induce acute withdrawal but without evidence for cumulative or escalating damage. Together, the current study suggests that nutrition, age, and sex should be considered when modeling human alcoholism.

Keywords: Diffusion tensor imaging; Liver; Magnetic resonance spectroscopy; White matter; behavior; repeated withdrawal.

Fig. 1.

a) Weight (mean ± SD) of animals across the course of the experiment. b) Blood alcohol levels (BALs) over the course of the experiment. BALs (mean ± SD) for each EtOH cycle are presented separately for female (red) and male (blue) EtOH-exposed rats. [EtOH cycle I: average of BALs at times 3, 4; EtOH cycle II: average of times 8, 9, 10; EtOH cycle II average of times 13, 14, 15.].

Fig. 2.

Two-bottle choice of a) EtOH or b) water consumption over the course of the experiment. Consumption is in the negative direction because data presented is total grams consumed starting from 0 (i.e., consumption decreased weight of the bottle). A red asterisk indicates t-test significance (p ≤ .05) between female EtOH and control groups.

Fig. 3.

Behavioral measures: a) open field average velocity; b) neurological signs. Red asterisk indicates t-test significance (p ≤ .05) between female EtOH and control groups; blue cross indicates t-test significance (p ≤ .05) between male EtOH and control groups.

Fig. 4.

Means and standard deviations (unadjusted for baseline values) of control (gray) and females (red) animals across 3 cycles for AST and ALT.

Fig. 5.

Means and standard deviations of control (gray) and exposed animals by sex (males = blue, females = red) for 7 scan sessions across 3 cycles for total brain volume (i.e, growth), and volumes of CSF, gray matter and white matter. Left column is unadjusted values; right column are data adjusted for baseline volume. * indicate statistically significant difference between exposed and control animals. Also see Supplementary Table 1.

Fig. 6.

Top: MRS voxel placement. Middle: representative spectrum before (left) and after (right) spectral baseline removal. Bottom: Means and standard deviations of control (gray) and exposed animals by sex (males = blue, females = red) for 7 scan sessions across 3 cycles for MRS measured Choline/Creatine. Left column is unadjusted values; right column are data adjusted for baseline volume. * indicate statistically significant difference between exposed and control animals. Also see Supplementary Table 2.

Fig. 7.

Means and standard deviations of control (gray) and exposed animals by sex (males = blue, females = red) for 7 scan sessions across 3 cycles for DTI FA in genu, splenium, and fimbria-fornix. Left column is unadjusted values; right column are data adjusted for baseline volume. * indicates statistically significant difference between exposed and control animals. Also see Supplementary Table 3.