Effect of dietary supplementation with Lactobacillus helveticus R0052 on seizure thresholds and antiseizure potency of sodium valproate in mice

Abstract

Objective: Both animal and human studies, though limited, showed that multi-strain probiotic supplementation may reduce the number of seizures and/or seizure severity. Here, we evaluated the effect of a single strain probiotic supplementation on seizure susceptibility, antiseizure efficacy of sodium valproate, and several behavioral parameters in mice.

Methods: Lactobacillus helveticus R0052 was given orally for 28 days. Its influence on seizure thresholds was evaluated in the ivPTZ- and electrically-induced seizure tests. The effect on the antiseizure potency of valproate was assessed in the scPTZ test. We also investigated the effects of probiotic supplementation on anxiety-related behavior (in the elevated plus maze and light/dark box tests), motor coordination (in the accelerating rotarod test), neuromuscular strength (in the grip-strength test), and spontaneous locomotor activity. Serum and brain concentrations of valproate as well as cecal contents of SCFAs and lactate were determined using HPLC method.

Results: L. helveticus R0052 significantly increased the threshold for the 6 Hz-induced psychomotor seizure. There was also a slight increase in the threshold for myoclonic and clonic seizure in the ivPTZ test. L. helveticus R0052 did not affect the threshold for tonic seizures both in the maximal electroshock- and ivPTZ-induced seizure tests. No changes in the antiseizure potency of valproate against the PTZ-induced seizures were reported. Interestingly, L. helveticus R0052 increased valproate concentration in serum, but not in the brain. Moreover, L. helveticus R0052 did not produce any significant effects on anxiety-related behavior, motor coordination, neuromuscular strength, and locomotor activity. L. helveticus R0052 supplementation resulted in increased concentrations of total SCFAs, acetate, and butyrate.

Conclusions: Altogether, this study shows that a single-strain probiotic - L. helveticus R0052 may decrease seizure susceptibility and this effect can be mediated, at least in part, by increased production of SCFAs. In addition, L. helveticus R0052 may affect bioavailability of valproate, which warrants further investigations.

Keywords: Anticonvulsant; Excitation/inhibition balance; Gut-microbiota-brain axis; Microbiota-derived metabolites; Psychobiotic.

Fig. 1

Schematic illustration of the experimental design

Fig. 2

Effect of L. helveticus R0052 administration on the seizure threshold in the maximal electroshock seizure test (A) and the 6 Hz-induced seizure test (B) in mice. Mice were administered orally with 2 × 10⁹ cfu of L. helveticus R0052 for 28 days. Control animals received 200 μl of PBS. Seizure test was performed 120 min after last administration. Data are presented as CC₅₀ values (in mA) with upper and lower 95% confidence limits. Each experimental group consisted of 20 animals. The statistical significance was evaluated using Student’s t test. ****p < 0.0001 vs. control group

Fig. 3

Effect of L. helveticus R0052 administration on the threshold for the first myoclonic twitch (A), generalized clonus (B), and forelimb tonus (C) in the ivPTZ seizure threshold test in mice. Mice were administered orally with 2 × 10⁹ cfu of L. helveticus R0052 for 28 days. Control animals received 200 μl of PBS. Seizure test was performed 120 min after last administration. Data are presented as box plots showing: interquartile range (the width of the box), minimum and maximum values (whiskers), and median (horizontal line). Each experimental group consisted of 15 animals. The statistical significance was evaluated using Mann–Whitney test. *p < 0.05 vs. control group

Fig. 4

Effect of L. helveticus R0052 administration on the anticonvulsant potency of valproate in the scPTZ test (A), and concentrations of valproate in serum (B) and brain (C) Mice were administered orally with 2 × 10⁹ cfu of L. helveticus R0052 (for 28 days. Control animals received 200 μl of PBS. Seizure test or decapitation was performed 120 min after last administration. Valproate was administered orally, 30 before PTZ injection or decapitation. ED₅₀ (± SEM) values (determined in groups of 40 mice each) represent a dose of valproate predicted to protect 50% of mice tested against the scPTZ-induced seizure. Valproate concentrations (determined in groups of 10 mice each) are presented as box plots showing: interquartile range (the width of the box), minimum and maximum values (whiskers), and median (horizontal line). The statistical significance was evaluated using Student’s t test (A) or Mann–Whitney test (B and C). *p < 0.05 vs. control group

Fig. 5

Effect of L. helveticus R0052 administration on anxiety-like behavior in mice assessed in the elevated plus maze test (A–B) and the light/dark box test (C–D). Mice were administered orally with 2 × 10⁹ cfu of L. helveticus R0052 for 27 days. Control animals received 200 μl of PBS. Behavioral testing was performed 120 min after administration. Data are presented as box plots showing: interquartile range (the width of the box), minimum and maximum values (whiskers), and median (horizontal line). Each experimental group consisted of 12 animals. The statistical significance was evaluated using Mann–Whitney test

Fig. 6

Effect of L. helveticus R0052 administration on locomotor activity (A), neuromuscular strength (B), and motor coordination (C and D). Mice were administered orally with 2 × 10⁹ cfu of L. helveticus R0052 for 27 days. Control animals received 200 μl of PBS. Behavioral testing was performed 120 min after administration. Data are presented as box plots showing: interquartile range (the width of the box), minimum and maximum values (whiskers), and median (horizontal line). Each experimental group consisted of 12 animals. The statistical significance was evaluated using Mann–Whitney test

Fig. 7

Effect of L. helveticus R0052 administration on the concentrations of total SCFAs (A), acetate (B), propionate (C), butyrate (D), and lactate (E). Mice were administered orally with 2 × 10⁹ cfu of L. helveticus R0052 for 28 days. Control animals received 200 μl of PBS. Fecal samples were collected before and after treatment. Data are presented as box plots showing: interquartile range (the width of the box), minimum and maximum values (whiskers), and median (horizontal line). Each experimental group consisted of 10 animals. The differences between control and L. helveticus R0052-treated group were evaluated using Mann–Whitney test, while the Wilcoxon matched-pairs signed rank test was used for before-after treatment comparison in the same mice. *p < 0.05, **p < 0.01 vs. control group; ^#p < 0.05 vs. before treatment group