Intestinal Dysbiosis Alters Seizure Burden and Antiseizure Medicine Activity Profile in the Theiler’s Virus Model of Acute Encephalitis

Abstract

Background: Epilepsy is a neurological disorder characterized by recurring, unprovoked seizures and chronic cognitive comorbidities. Epilepsy can arise due to numerous factors, including genetics, brain injury, and central nervous system infection. Brain infection with Theiler’s murine encephalomyelitis virus (TMEV) in C57BL/6J mice is thus a relevant animal model of infection-induced acute seizures and epileptogenesis. Acute seizures in this model are driven by brain infiltration of peripheral immune system macrophages, which secrete pro- inflammatory cytokines that further accelerate brain inflammation1. Diet formulation can dramatically modify the presentation of acute seizures in TMEV-infected mice and influence the diversity and composition of the intestinal microbiome (Zierath et al, Epilepsia 2024)2 that critically shapes immune system reactivity. However, whether antibiotic-induced gut microbiome dysbiosis influences the phenotype of symptomatic seizures after TMEV infection is unclear. Further, no study has assessed whether antibiotic-induced intestinal dysbiosis influences antiseizure medicine activity (ASM) in the TMEV model. We thus sought to define the extent towhich antibiotic administration influenced acute seizure presentation, the anticonvulsant activity of the prototype ASM, carbamazepine (CBZ), and pharmacokinetics of this ASM in this mouse seizure model. We hypothesized that there would be an effect of intestinal dysbiosis on seizure burden and ASM efficacy in the TMEV model and that this change would be independent of pharmacokinetic differences. Methods: Male C57BL/6J mice (4-5 weeks-old) received a broad-spectrum antibiotic cocktail (ABX) containing ampicillin, metronidazole, neomycin sulfate, and vancomycin (n=55) or vehicle (n=60) by oral gavage (p.o.) once daily beginning at arrival (Day -2) to Day 7 post- TMEV infection (p.i.) to induce intestinal dysbiosis. Mice were then infected with either intracerebral TMEV or PBS on Day 0. Mice received carbamazepine (CBZ; 20 mg/kg) or vehicle (VEH, 0.5% MC) intraperitoneally (i.p.) twice daily Days 3-7 p.i. and were assessed for handling-induced seizures 30 min after CBZ treatment. Plasma was collected on Day 7 p.i. at 15 and 60 min post-CBZ treatment to quantify the extent to which ABX-induced gut dysbiosis influences ASM pharmacokinetics. Results: TMEV infection induced acute symptomatic seizures, regardless of pretreatment and CBZ history. There were 18/25 (72%) ABX-CBZ mice, 7/20 (35%) ABX-VEH mice, 7/20 (35%) SAL-CBZ mice, and 15/20 (75%) SAL-VEH mice that presented with seizures during the 7-day monitoring period. Average seizure burden was: 12.5 in ABX-CBZ, 4.7 in ABX-VEH, 5.7 in SAL-CBZ, and 16.1 in SAL-VEH mice. There was a significant pretreatment x ASM interaction (F (1, 81) = 16.0, p=0.0001), with post-hoc tests revealing marked differences in seizure burden in SAL- versus ABX-pretreated mice (p=0.004). Further, CBZ administration significantly increased the latency to Stage 5 seizure during days 3-7 p.i.; an effect that was not present in ABX-treated mice similarly administered CBZ. In TMEV-infected mice, spleens were 0.32% of body weight in ABX-CBZ mice, 0.34% in ABX-VEH mice, 0.36% in SAL-CBZ mice, and 0.38% in SAL-VEH mice. In sham-infected mice, spleens were 0.45% of body weight in ABX-CBZ mice, 0.65% in SAL-CBZ mice, and 0.36% in SAL-VEH mice. Plasma CBZ concentrations did not differ between SAL and ABX pretreatment groups (F (3, 37) = 0.3468), suggesting that ABX history did not influence CBZ pharmacokinetics. Conclusion: Antibiotic-induced gut dysbiosis markedly altered the presentation of symptomatic seizures and acute disease burden in the TMEV mouse model, reflecting a novel therapeutic target for seizure control: the gut microbiome. ABX-induced gut dysbiosis also significantly changed acute seizure control by CBZ, but did not significantly influence plasma concentrations of CBZ. The gut-brain axis is thus a relevant contributor to the clinical course of TMEV infection. This study altogether demonstrates that the gut-brain axis is an understudied therapeutic target in epilepsy that may benefit from greater investigation.