No benefit from chronic lithium dosing in a sibling-matched, gender balanced, investigator-blinded trial using a standard mouse model of familial ALS

Abstract

Background: In any animal model of human disease a positive control therapy that demonstrates efficacy in both the animal model and the human disease can validate the application of that animal model to the discovery of new therapeutics. Such a therapy has recently been reported by Fornai et al. using chronic lithium carbonate treatment and showing therapeutic efficacy in both the high-copy SOD1G93A mouse model of familial amyotrophic lateral sclerosis (ALS), and in human ALS patients.

Methodology/principal findings: Seeking to verify this positive control therapy, we tested chronic lithium dosing in a sibling-matched, gender balanced, investigator-blinded trial using the high-copy (average 23 copies) SOD1G93A mouse (n = 27-28/group). Lithium-treated mice received single daily 36.9 mg/kg i.p. injections from 50 days of age through death. This dose delivered 1 mEq/kg (6.94 mg/kg/day lithium ions). Neurological disease severity score and body weight were determined daily during the dosing period. Age at onset of definitive disease and survival duration were recorded. Summary measures from individual body weight changes and neurological score progression, age at disease onset, and age at death were compared using Kaplan-Meier and Cox proportional hazards analysis. Our study did not show lithium efficacy by any measure.

Conclusions/significance: Rigorous survival study design that includes sibling matching, gender balancing, investigator blinding, and transgene copy number verification for each experimental subject minimized the likelihood of attaining a false positive therapeutic effect in this standard animal model of familial ALS. Results from this study do not support taking lithium carbonate into human clinical trials for ALS.

Figure 1. Daily average body weight change and neurological severity score in SOD1^G93A mice.

Group average change from initial body weight (top panels) and neurological disease severity score (bottom panels) are compared over time from age at study start to age at last death. Left panels show data from all animals (All); middle and right panels show data from females (F) and males (M), respectively. Vehicle control (CTRL) animals received daily 0.2 mL i.p. injections of isotonic saline. Lithium-treated (DRUG) animals received daily 36.9 mg/kg i.p. injections of lithium carbonate dissolved in isotonic saline. Because animals died at different ages, group means were computed after carrying forward last values for each animal until the last animal died. Results of statistical analysis for summary measures taken from these data are given in Table 1.

Figure 2. Average number of days spent at each neurological severity score level in SOD1^G93A mice.

Left panel shows data from all animals (All); middle and right panels show data from females (F) and males (M), respectively. Vehicle control (CTRL) animals received daily 0.2 mL i.p. injections of isotonic saline. Lithium-treated (DRUG) animals received daily 36.9 mg/kg i.p. injections of lithium carbonate dissolved in isotonic saline. Data are treatment group means±s.e.m.. Scores advance in severity from 0 to 4. Results of statistical analysis for these data are given in Table 2.

Figure 3. Kaplan-Meier time-to-failure plot for onset of symptomatic neurological disease in SOD1^G93A mice.

The age at which mice attain a neurological severity score of 2 is taken to be definitive onset of symptomatic disease. Left panel shows data from all animals (All); middle and right panels show data from females (F) and males (M), respectively. Vehicle control (CTRL) animals received daily 0.2 mL i.p. injections of isotonic saline. Lithium-treated (DRUG) animals received daily 36.9 mg/kg i.p. injections of lithium carbonate dissolved in isotonic saline. Results of statistical analysis for these data are given in the upper portion of Table 3.

Figure 4. Kaplan-Meier survival plot for age at death in SOD1^G93A mice.

Left panel shows data from all animals (All); middle and right panels show data from females (F) and males (M), respectively. Vehicle control (CTRL) animals received daily 0.2 mL i.p. injections of isotonic saline. Lithium-treated (DRUG) animals received daily 36.9 mg/kg i.p. injections of lithium carbonate dissolved in isotonic saline. Results of statistical analysis for these data are given in the lower portion of Table 3.