Advancing the understanding of behaviors associated with Bacille Calmette Guérin infection using multivariate analysis

Abstract

Behavioral indicators in the murine Bacille Calmette Guérin (BCG) model of inflammation have been studied individually; however, the variability of the behaviors across BCG levels and the mouse-to-mouse variation within BCG-treatment group are only partially understood. The objectives of this study were: (1) to gain a comprehensive understanding of sickness and depression-like behaviors in a BCG model of inflammation using multivariate approaches, and (2) to explore behavioral differences between BCG-treatment groups and among mice within group. Adult mice were challenged with either 0mg (saline), 5mg or 10mg of BCG (BCG-treatment groups: BCG0, BCG5, or BCG10, respectively) at Day 0 of the experiment. Sickness indicators included body weight changes between Day 0 and Day 2 and between Day 2 and Day 5, and horizontal locomotor activity and vertical activity (rearing) measured at Day 6. Depression-like indicators included duration of immobility in the forced swim test and in the tail suspension test at Day 6 and sucrose consumption in the sucrose preference test at Day 7. The simultaneous consideration of complementary sickness and depression-like indicators enabled a more precise characterization of behavioral changes associated with BCG-treatment and of mouse-to-mouse variation, relative to the analysis of indicators individually. Univariate and multivariate analyses confirmed differences between BCG-treatment groups in weight change early on the trial. Significant differences between BCG-treatment groups in depression-like behaviors were still measurable after Day 5. The potential for multivariate models to account for the correlation between behavioral indicators and to augment the analytical precision relative to univariate models was demonstrated both for sickness and for depression-like indicators. Unsupervised learning approaches revealed the complementary information provided by the sickness and depression-like indicators considered. Supervised learning approaches using cross-validation confirmed subtle differences between BCG-treatment groups and among mice within group identified by the consideration of sickness and depression-like indicators. These findings support the recommendation for multivariate and multidimensional analyses of sickness and depression-like indicators to augment the systemic understanding of the behavioral changes associated with infection.

Keywords: Cluster analysis; Depression-like indicator; Discriminant analysis; Principal component analysis; Sickness.

Figure 1

Body weight across days post injection (at Day 0) and across BCG-treatment level [BCG0 (n=7), BCG5 (n=6), and BCG10 (n=5)] estimated from a repeated-measurements longitudinal model. Estimates were adjusted for weight prior to the start of the experiment and for heterogeneity of variance. Whiskers denote 95% confidence limits of the estimates.

Figure 2

Dendrogram displaying the distance (branch length measured in semi-partial R² units) between mice across BCG treatment groups. Mouse identification includes the leftmost vertical axis label denoting the BCG-treatment group [10 = BCG10 (n=5), 5 = BCG5 (n=6), or 0 = BCG0, (n=7)] followed by “_” and the mouse unique identifier number.

Figure 3

Dendrogram displaying the distance (branch length measured in proportion of variance explained) between sickness indicators including change in weight between Day 0 and 2 (W20), change in weight between Day 2 and 5 (W52), horizontal locomotor activity (LMA) and rearing (REA), and depression-like indicators including immobility in the tail suspension test (TST), immobility in the forced swim test (FST) and sucrose preference test (SPT) based on n=18 mice.

Figure 4

Distribution of the mice in the BCG10 (red squares, n=5), BCG5 (orange diamonds, n=6) and BCG0 (green circles, n=7) across the first three principal components (Prin1, Prin2, Prin3).

Figure 5

Coefficients (or loadings) received by each sickness and depression-like indicator (based on n=18 mice) in the first three principal components (PC1, PC2, and PC3). Small circle: weight change between Day 0 and 2; large circle: weight change between Day 2 and 5; small square: horizontal locomotor activity; large square: rearing; small diamond: tail suspension immobility; medium diamond: forced swim immobility; and large diamond: sucrose preference.

Figure 6

Left. Distribution of the behavioral indicators across multidimensional scaling dimensions 1 and 2. Right. Distribution of mice denoted by the BCG-treatment group [_0 = BCG0 (n=7), _5 = BCG5 (n=6), _10 = BCG10 (n=5)] followed by “_” and by the mouse identifier number from multidimensional scaling dimensions 1 and 3.