Abstracts

Rationale: Central infection with Theiler’s murine encephalomyelitis virus (TMEV) in C57Bl/6J mice is a useful model of infection-induced acute seizures and epileptogenesis. Diet sterilization alone can modify the presentation of acute seizures in TMEV-infected mice (Libbey et al, 2016); however, no study has yet defined the extent to which antibiotic-induced intestinal dysbiosis influences the activity profile of antiseizure medicines (ASM) in the TMEV model. Whether the gut microbiome influences the phenotype of acute seizures after TMEV infection is also unclear. We thus sought to demonstrate whehther antibiotic administration in a specific pathogen-free vivarium influences acute seizure presentation, the activity of ASMs, and the pharmacokinetic profile of ASMs in this mouse model.

Methods: Male C57BL/6J mice (four to five weeks old) received a broad-spectrum antibiotic cocktail (ABX) containing ampicillin, metronidazole, neomycin sulfate, and vancomycin (n=25) or vehicle (n=25) by oral gavage once daily beginning at arrival (Day 3) to Day 7 post-TMEV infection. Mice were infected with either intracerebral (i.c.) TMEV or PBS on Day 0. Mice received carbamazepine (CBZ; 20 mg/kg, i.p.) or vehicle (0.5% MC) twice daily Days 3-7 post-TMEV infection and were assessed for handling-induced seizures twice daily 30 min after CBZ dosing. Plasma samples were collected on Day 7 post-infection at 15 and 60 min post-CBZ administration to quantify the extent to which ABX-induced intestinal dysbiosis influences ASM pharmacokinetics.

Results: TMEV infection induced acute handling-induced seizures, regardless of pretreatment and ASM history as 9/10 (90%) ABX-CBZ mice, 6/10 (60%) ABX-VEH mice, 3/10 (30%) SAL-CBZ mice, and 10/10 (100%) SAL-VEH mice presented with seizures during the seven day monitoring period (X 2 =14.29, p=0.0025). Average seizure burden was: 15.7 in ABX-CBZ, 8.4 in ABX-VEH, 5.9 in SAL-CBZ, and 21.5 in SAL-VEH mice. There was a significant pretreatment x ASM interaction (F (1, 36) = 12.62, p=0.0011), with post-hoc tests revealing marked differences in seizure burden in VEH- versus ABX-pretreated mice (p=0.033). Further, the latency to Stage 5 seizure was substantially increased by CBZ during days three to seven post-infection; an effect absent in ABX-treated mice similarly administered CBZ. Spleens were 0.32% of body weight in ABX-CBZ mice, 0.35% in ABX-VEH mice, 0.34% in SAL-CBZ mice, and 0.37 % in SAL-VEH mice. In sham-infected mice, spleens were 0.51% of body weight in ABX-CBZ mice and 0.89% in SAL-CBZ mice. Plasma CBZ concentrations in TMEV-infected mice receiving ABX were 9.78±2.00 mg/mL 15 min post-dosing and 3.96±0.51 at 60 min post-dosing, consistent with published CBZ plasma concentrations in mice (Bialer et al, 2004) suggesting no pharmacokinetic differences because of ABX history.

Conclusions: Intestinal dysbiosis substantially alters the presentation of acute, handling-induced seizures and acute disease burden in the TMEV model of infection-induced seizures, potentially confounding evidence of ASM activity across labs. Care should be taken in the interpretation of anticonvulsant activity of investigational agents using the TMEV model.

Funding: University of Washington Plein Center for Geriatric Pharmacy.