Protective Effects of Probiotics on Cognitive and Motor Functions, Anxiety Level, Visceral Sensitivity, Oxidative Stress and Microbiota in Mice with Antibiotic-Induced Dysbiosis

Abstract

Accumulating clinical and preclinical data indicate a prominent role of gut microbiota in regulation of physiological functions. The gut-brain axis imbalance due to gut dysbiosis is associated with a range of neurodegenerative diseases. Probiotics were suggested not only to restore intestinal dysbiosis but also modulate stress response and improve mood and anxiety symptoms. In this study, we assessed the effects of probiotic lactobacilli on behavioral reactions, the level of oxidative stress and microbiota content in mice administered to broad-spectrum antibiotics. Our study demonstrates that antibiotic treatment of adolescent mice for two weeks resulted in higher mortality and lower weight gain and induced significant changes in behavior including lower locomotor and exploratory activity, reduced muscle strength, visceral hypersensitivity, higher level of anxiety and impaired cognitive functions compared to the control group. These changes were accompanied by decreased diversity and total amount of bacteria, abundance of Proteobacteria and Verrucomicrobia phyla, and reduced Firmicutes/Bacteroides ratio in the gut microbiota. Moreover, a higher level of oxidative stress was found in brain and skeletal muscle tissues of mice treated with antibiotics. Oral administration of two Lactobacillus strains prevented the observed changes and improved not only microbiota content but also the behavioral alterations, suggesting a neuroprotective and antioxidant role of probiotics.

Keywords: antibiotic-induced dysbiosis; anxiety level; cognitive functions; locomotor and exploratory activity; microbiota; motor coordination; muscle strength; oxidative stress; probiotic lactobacilli; visceral and mechanical sensitivity.

Figure 1

The scheme of the experiments.

Figure 2

Mortality and weight gain of the mice during administration of antibiotics and lactobacilli treatment. (A) Mortality (grey sector) in control, AB and AB + LB groups. (B) The weight gain before (unshaded boxes) and after two weeks treatment (shaded boxes) in the control (white); AB (dark grey); AB + LB (light grey) groups. Boxes—SEM, black line—median, the circle inside—mean value, whiskers—5–95 percentiles of dates. * p < 0.05 compared to the initial values, # p < 0.05 compared to the control group.

Figure 3

Alpha-diversity box plots. Effects of the broad-spectrum antibiotics alone (AB) and in combination with two Lactobacillus strains (AB + LB) on bacterial diversity and richness measures in cecal contents of mice on conclusion of experiments; including phylogenetic diversity (A), Chao1 (B), Shannon (C) and Simpson (D) indices, and number of observed bacterial species (E). n = 3 per group.

Figure 4

Alterations of cecal microbiota in mice treated with the broad-spectrum antibiotics alone (AB) and in combination with two Lactobacillus strains (AB + LB) as compared to the control (Control) group (n = 3 per group). (A) Average phylum distribution. (B) The most abundant taxa at the family level.

Figure 5

Locomotor and exploratory activity and anxiety of mice with administration of antibiotics and lactobacilli treatment in the open field. Horizontal activity (A), rearings (B), exploratory activity (C), grooming (D), latency to exit from the central zone (E) and defecation score (F) of mice from the control (white columns); AB (grey columns); AB + LB (light grey columns) groups. Data are expressed as mean ± SEM. * p < 0.05 compared to the control group, # p < 0.05 compared to the AB group.

Figure 6

The anxiety level of mice with administration of antibiotics and treatment with lactobacilli. (A) Integral anxiety value of mice from control (white circles), AB (dark grey circle) and AB + LB (light grey circle) groups before (0) and during injections of antibiotics for 14 days. (B,C) Time spent in the light box and number of transitions between the chambers of mice from the control (white columns); AB (grey columns); AB + LB (light grey columns) groups. Data are expressed as mean ± SEM. * p < 0.05 compared to the control group.

Figure 7

Motor coordination and muscle strength of mice after administration of antibiotics and treatment with lactobacilli. Latency to fall in Rotarod test (A) and the time spent on the grip (before falling) in paw grip endurance test (B) of mice from the control (white columns); AB (grey columns); AB + LB (light grey columns) groups. Data are expressed as mean ± SEM. * p < 0.05 compared to the control group, # p < 0.05 compared to the AB group.

Figure 8

Cognitive dysfunctions and visceral nociception of mice treated with antibiotics and lactobacilli. Percentage of alternation in the T maze (A), novel object recognition (NOR) score (B) and mechanical withdrawal threshold (C) of mice from control (white columns); AB (grey columns); AB + LB (light grey columns) groups. Data are expressed as mean ± SEM. * p < 0.05 compared to the control group, # p < 0.05 compared to the AB group.

Figure 9

The level of oxidative stress in the brain and skeletal muscle tissue of mice after administration of antibiotics and lactobacilli. MDA level (A), total glutathione (B), and activity of glutathione peroxidase (C) in muscle and brain tissue of mice from the control (white columns); AB (grey columns); AB + LB (light grey columns) groups. Boxes—SEM, black line—median, the circle inside—mean value, whiskers—5–95 percentiles of dates. * p < 0.05 compared to the control group, # p < 0.05 compared to the AB group.