An Alternative Cuprizone-Induced Demyelination and Remyelination Mouse Model

Abstract

The cuprizone model is a well-established and investigated paradigm to study demyelination and remyelination in rodents. Cuprizone is usually administrated by mixing in the powdered or pelleted rodent chow. However, since cuprizone is sensitive to the environment and the consumption of it varies between different animals, the major issue is the discrepancy in demyelination of the animals. This study reports the development of the cuprizone model by gavage administrations in mice. Following testing a series of doses of cuprizone, 400 mg/kg/day was found to be the best dosage to induce dramatic and consistent demyelination after 5 weeks of administration; while remyelination quickly occurred after 9 days of cuprizone withdrawal. The advantage of this alternative model is that the consumption of cuprizone could be well controlled, and the mice were exposed to the same dose of cuprizone. Thus, the variation in demyelination was minimized. This alternative cuprizone dosing regime minimizes the interanimal variability on demyelination and hence provides a consistent model for pharmacological evaluations, in addition to reducing the number of animals used in the experiments.

Keywords: animal model; cuprizone; demyelination; oral administration; remyelination.

Figure 1.

The body weight and survival of mice treated with different gavage dosages of cuprizone. The body weight was measured 3 times/week during 5-week cuprizone administrations (n = 4). The body weight showed that CPZ 1000 mg/kg group was toxic to mice, and all mice died after 12 days of cuprizone administrations. The CPZ 100 and CPZ 300 mg/kg groups had no mortality. ANOVA with Dunnett’s test was performed to analyze the difference between the groups, and p value < .05 was considered statistically significant, **p < .01, ***p < .001. g = gram; CPZ = cuprizone.

Figure 2.

Demyelination induced by cuprizone oral gavage administrations. (a) The body weight was measured 3 times/week during 5-week cuprizone administrations for different dosage groups (vehicle group: n = 4; oral gavage groups: n = 7). After the cuprizone oral dosing, the body weight of all oral gavage groups decreased. All mice died after 20 days of cuprizone administrations in 800 mg/kg group. ANOVA with Dunnett’s test was performed to analyze the difference between the groups, and p value < .05 was considered statistically significant, ***p < .001. Representative images of Black-Gold II staining in the forebrain (b) and hindbrain (c) of corpus callosum region from the mice in each group. Scale bar = 400 µm ((b) and (c)). Quantification of mean density of Black-Gold II staining in corpus callosum region from forebrain (d) and hindbrain (e) reveals prominent demyelination in cuprizone oral dose groups. ANOVA with Dunnett’s test was performed to analyze the difference between the groups. Error bars represent standard errors of the mean (SEM), and p value < .05 was considered statistically significant, ****p < .0001, ns indicates no significant difference. g = gram; CPZ = cuprizone.

Figure 3.

Optimization of cuprizone oral dosage for demyelination induction. (a) The body weight was measured 3 times/week during 5-week cuprizone (CPZ) administrations for different groups of animals (vehicle and 400 mg/kg groups: n = 4; 200 mg/kg and 300 mg/kg groups: n = 5). After the cuprizone oral dosing, the body weight of all oral gavage groups decreased. ANOVA with Dunnett’s test was performed to analyze the difference between the groups, and p value < .05 was considered statistically significant, ***p < .001, ****p < .0001. (b) and (c) Representative images of Black-Gold II staining in the forebrain (b) and hindbrain (c) of corpus callosum region from the mice. Scale bar = 400 µm ((b) and (c)). Quantification of mean density of Black-Gold II staining in corpus callosum region from forebrain (d) and hindbrain (e) reveals prominent demyelination in CPZ oral dose administration groups. ANOVA with Dunnett’s test was performed to analyze the difference between groups. Error bars represent standard error of the mean (SEM), and p value < .05 was considered statistically significant. ****p < .0001. g = gram; CPZ = cuprizone; Q.d. = once a day; b.i.d. = twice day; MPK = mg/kg.

Figure 4.

Remyelination after cuprizone withdrawal. (a) The body weight was measured 3 times/week during 5-week cuprizone administrations which was then followed by 9 days of cuprizone withdrawal (vehicle group: n = 3; 400 mg/kg group: n = 4; 300 mg/kg group: n = 5; cuprizone withdrawal groups: n = 4). During the cuprizone oral dosing, the body weight of oral gavage group decreased. After cupriozne withdrawal, cuprizone mice gained body weight rapidly. ANOVA with Dunnett’s test was performed to analyze the difference between the groups, and p value < .05 was considered statistically significant, ****p < .0001. (b) and (c) Representative images of Black-Gold II staining in the forebrain (b) and hindbrain (c) of corpus callosum region from mice in each group show the demyelination and remyelination. Scale bar = 400 µm ((b) and (d)). (d) and (e) Quantification of mean density of Black-Gold II staining signals in corpus callosum region from forebrain (d) and hindbrain (e) reveals successful remyelination after cuprizone withdrawal. ANOVA with Sidek’s test was performed to analyze the difference between groups. Error bars represent standard error of the mean (SEM), and p value < .05 was considered statistically significant. **p < .01, ****p < .0001. g = gram; CPZ = cuprizone; Q.d. = once a day; MPK = mg/kg; w = weeks; d = days.