Sex Differences in the Behavioural Aspects of the Cuprizone-Induced Demyelination Model in Mice

Abstract

Multiple sclerosis is an autoimmune disease characterised by demyelination in the central nervous system. The cuprizone-induced demyelination model is often used in mice to test novel treatments for multiple sclerosis. However, despite significant demyelination, behavioural deficits may be subtle or have mixed results depending on the paradigm used. Furthermore, the sex differences within the model are not well understood. In the current study, we have sought to understand the behavioural deficits associated with the cuprizone-induced demyelination model in both male and female C57BL/6J mice. Using Black gold II stain, we found that cuprizone administration over 6 weeks caused significant demyelination in the corpus callosum that was consistent across both sexes. Cuprizone administration caused increased mechanical sensitivity when measured using an electronic von Frey aesthesiometer, with no sex differences observed. However, cuprizone administration decreased motor coordination, with more severe deficits seen in males in the horizontal bar and passive wire hang tests. In contrast, female mice showed more severe deficits in the motor skill sequence test. Cuprizone administration caused more anxiety-like behaviours in males compared to females in the elevated zero maze. Therefore, this study provides a better understanding of the sex differences involved in the behavioural aspects of cuprizone-induced demyelination, which could allow for a better translation of results from the laboratory to the clinic.

Keywords: anxiety; cuprizone; demyelination; motor coordination; multiple sclerosis; sex differences.

Figure 1

Cuprizone administration led to weight loss and demyelination in male and female C57BL/6J mice. The body weight of the mice was measured daily over the 42 days of cuprizone (CPZ) administration. (A) Male and (B) female mice administered cuprizone lost weight compared to the normal food diet, n = 8–10. (C) Area under the curve (AUC) analysis showed that all groups administered cuprizone lost weight, with the 0.3% cuprizone treatment in males showing more severe weight loss than the 0.2% dose in males and the 0.3% dose in females. (D) Representative images showing Black gold II myelin stain taken from day 42 of cuprizone administration. (E) Threshold analysis gave the percentage of area stained within a region of interest positioned at the midline of the corpus callosum (CC). In the male and female animals treated with cuprizone, there was a reduction in the area of myelin stained compared to mice given normal food. Analysed in 3–4 sections per animal (averaged), n = 5–10. The scale bar is 200 µm. # p < 0.05, ### p < 0.001, #### p < 0.0001 for 0.2% cuprizone vs. normal food. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 for 0.3% cuprizone vs. normal food, or as indicated. n.s. = not significant. Data presented as mean ± SEM.

Figure 2

Cuprizone administration decreased mechanical sensitivity threshold in male and female C57BL/6J mice. Mechanical sensitivity was measured using an electronic von Frey aesthesiometer. In (A) male and (B) female mice, cuprizone (CPZ) administration led to a decrease in the mechanical threshold. Two-way ANOVA with Bonferroni post-tests. (C) In a comparison between all mice on day 0, it was found that female mice have a lower threshold than male mice. (D) The z-score analysis normalised the results to the corresponding normal food control. All doses of cuprizone led to a decrease in mechanical threshold with no sex or dose-dependent effects. (A,B) Two-way repeated-measures ANOVA with Bonferroni post-tests. (C) Unpaired t-test. (D) One-way ANOVA with Bonferroni post-tests. ## p < 0.01, #### p < 0.0001 for 0.2% cuprizone vs. normal food. *** p < 0.001, **** p < 0.0001 for 0.3% cuprizone vs. normal food, or as indicated. n.s. = not significant. Data presented as mean ± SEM. n = 13–18.

Figure 3

Cuprizone administration decreased motor coordination in C57BL/6J mice. Motor coordination was measured using the horizontal bar test in (A) male and (B) female mice. Cuprizone (CPZ) administration led to a decrease in the score in both sexes. (C) Z-score analysis of the area under the curve data showed a sex and dose-dependent effect, with the 0.3% cuprizone treatment in males decreasing the horizontal bar score more than the other cuprizone treatment groups. The passive wire hang test in (D) male and (E) female mice showed a deficit in cuprizone-treated male mice but not female mice. (F) Z-score analysis showed the 0.3% dose of cuprizone affected males greater than females. (A,B,D,E) Two-way repeated-measures ANOVA with Bonferroni post-tests. (C,F) One-way ANOVA with Bonferroni post-tests. # p < 0.05, ## p < 0.01, for 0.2% cuprizone vs. normal food. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 for 0.3% cuprizone vs. normal food, or as indicated. Data presented as mean ± SEM. n = 12–18.

Figure 4

Cuprizone administration decreased complex motor coordination in C57BL/6J mice. (A) Timeline of motor skill sequence (MOSS) testing in the cuprizone-induced demyelination model. The training wheel (TW) is used on days 28–42 and the complex wheel (CW) on days 42–49. (B) Schematic diagram of MOSS wheel rung pattern, 38 rungs for the training wheel and 16 rungs for the complex wheel. The maximum velocity per day for the length of the experiment in (C) males and (D) females showed a deficit with cuprizone administration. Cumulative distance travelled was recorded on the (E,F) training wheel on days 28–41 and the (G,H) complex wheel on days 42–49. Mice on normal food exercised significantly more than cuprizone-intoxicated mice on the training and complex wheel. (I) Z-score analysis for the MOSS testing showed the 0.3% cuprizone affected females more severely than males. However, (J) z-score analysis of all measures from MOSS testing, horizontal bar and passive wire hang, overall showed that the 0.3% dose in males had the greatest deficit in motor coordination. (C–H) Two-way repeated-measures ANOVA with Bonferroni post-tests. (I,J) One-way ANOVA with Bonferroni post-tests. # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 for 0.2% cuprizone vs. normal food. ** p < 0.01, *** p < 0.001, **** p < 0.0001 for 0.3% cuprizone vs. normal food, or as indicated. ^ p < 0.05 for 0.2% cuprizone vs. 0.3% cuprizone. Data presented as mean ± SEM. n = 8–10.

Figure 5

Administration of cuprizone has anxiogenic effects in male mice but not female mice. The elevated zero maze tested mice for 5 min with (A,B) time spent in the open arm, (C,D) the number of open arm entries and (E,F) the number of head dips measured for both sexes. Cuprizone in male mice exhibited a dose-dependent reduction in the number of open arm entries and a reduction in the time spent in the open arms. (G) Z-score analysis of all behaviours on the elevated zero maze (EZM) showed that only the male 0.3% cuprizone treatment group had an anxiogenic effect. (H,I) The marble burying test was used to measure anxiogenic and repetitive–compulsive behaviour in the mice. The 0.3% cuprizone dose reduced the number of marbles buried in both sexes. (J) Z-score analysis of the marble burying behaviour confirmed there was no sex difference between the 0.3% cuprizone dose. (K) Z-score analysis of the elevated zero maze and marble burying data combined showed an increase in anxiety-like behaviours in male mice treated with cuprizone compared to female mice. Unpaired t-tests for female comparisons and one-way ANOVA with Bonferroni post-tests for the male and z-score comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. n.s. = not significant. Data presented as mean ± SEM. n = 10–16.

Figure 6

Cuprizone has more severe behavioural deficits in males compared to females. Z-score analysis was used to compare the treatment groups normalised to the healthy control for each sex. The total score was calculated from mechanical sensitivity, horizontal bar, passive wire hang, motor skill sequence, elevated zero maze and marble burying behavioural tests. In males, there was a dose-dependent effect with a more severe deficit in the 0.3% cuprizone group. When compared between sexes, the 0.3% cuprizone dose had more severe behavioural deficits in the males rather than females. One-way ANOVA with Bonferroni post-tests. **** p < 0.0001, n.s. = not significant. Stars below the bars indicate comparisons to the corresponding healthy control group. Data presented as mean ± SEM.