Figure 1. Effects of molecular hydrogen in drinking water on sickness behaviour induced by systemic administration of LPS.

(A) Changes in bodyweight monitored for 2 weeks after the administration of LPS. Baseline bodyweight is calculated as average of the last 3 days preceding the administration of LPS. Mice drinking molecular hydrogen-enriched H-ERW drop slightly less than control mice, and recover faster to baseline bodyweight (N = 8 mice per group; * p<0.05, repeated measures ANOVA, followed by contrast analysis). (B) Spontaneous locomotor activity in a novel environment. The baseline activity was recorded 22 h prior to LPS and was used as reference for activity recorded 2 h after the injection of either vehicle (sham), or LPS. Note that the habituation to the testing conditions accounts for 25% decrease in locomotor activity. LPS administration decreases the locomotor activity to 25% in control mice, and to 45% in H-ERW mice (sham-control: N = 15; sham-H-ERW: N = 15; LPS-controls: N = 13; LPS-H-ERW: N = 14; * p<0.05, factorial ANOVA followed by contrast analysis). (C) Spontaneous locomotion in the homecage expressed as average number of visits per hour (see text for details) for 6 consecutive light-dark cycles (light and dark phases indicated at the bottom of the graph). The injection of LPS is marked by arrow. Arrowheads indicate the peaks in locomotor activity induced by handling for measuring bodyweight. Note that the pattern with 2 or 3 distinct peaks of activity is preserved in H-ERW mice even during the first dark phase following the LPS injection (although at lower amplitude than at baseline), and is restored earlier than in control mice. “baseline” indicates the two light-dark cycles that were used for deriving baseline values for all parameters analysed. (D) Spontaneous locomotor activity resting time in the homecage (light and dark phases are indicated at the top of the graph). Systemic administration of LPS (arrow) induces a significant decrease in spontaneous locomotion during both light and dark phases of the following circadian cycle, followed by a transient hyperactive period in control, but not in H-ERW mice. Resting time is significantly increased only during the dark phase following the LPS injection in H-ERW mice, compared to 3 consecutive phases in the control mice. Arrowheads indicate the timepoints where a significant change in spontaneous locomotion or resting time occurs in only one group (i.e. either LPS-control, or LPS-H-ERW). (E) Circadian distribution of locomotor activity and resting time. The circadian distribution of both activity and resting time was restored earlier in mice drinking H-ERW (* p<0.05, repeated measures ANOVA followed by contrast analysis). C, D, E–N = 8 mice per group, recorded in parallel in groups of 4 LPS-control and 4 LPS-H-ERW mice.