Abstracts

Rationale: Temporal lobe epilepsy (TLE) is the most common form of acquired epilepsy, which often results as a consequence of CNS infection and other insults. A high proportion of TLE patients are resistant to current anti-seizure drugs. As the majority of these drugs target neuronal mechanisms, the role of glial cells in epilepsy is now being actively investigated to identify alternative treatments and disease modifying therapies. The astrocyte-specific glutamate transporter GLT-1 accounts for approximately 90% of glutamate uptake in the brain. Reports have suggested that decreases in the expression and/or function of this transporter may contribute to epilepsy pathophysiology. Thus, modulation of GLT-1 could represent a compelling pharmacological target.  CEF (CEF) is a commonly used β-lactam antibiotic that has been shown to increase GLT-1 expression by increasing GLT-1 promoter activation and prevent neuronal injury in both in vivo and in vitro models. A novel mouse model of infection-induced TLE, which recapitulates clinical observations, offers a unique opportunity to study such astrocyte mechanisms underlying epileptogenesis as well as novel disease modifying strategies. Theiler's murine encephalomyelitis virus (TMEV) infected C57BL/6J mice exhibit acute seizures between 3 and 7 days post-infection (DPI), neurodegeneration, hippocampal atrophy and sclerosis, decreased levels of hippocampal GLT-1, and a proportion go on to develop epilepsy. As such, the purpose of this study was to examine the effects of CEF treatment during the period of acute TMEV-induced seizures on behavioral seizure phenotypes and GLT-1 expression. Methods: C57/BL6 mice were injected intracerebrally with TMEV. Animals were then separated into saline (n = 12) and drug (n = 13) treatment groups and injected daily from 0 DPI to 7 DPI with either saline or 200 mg/kg  CEF i.p., the treatment paradigm standardly used in the literature. During this period mice were monitored daily for behavioral seizures and sacrificed 24 hours after the last saline or CEF injection (8 DPI). Hippocampi were acutely dissected and frozen, processed to separate out the membrane fraction, and immunoblotted with GLT-1 and GAPDH antibodies. Immunostaining levels were quantified using ImageJ. Results: Seizures were evaluated twice daily in a blinded fashion using the Racine scale. 200 mg/kg CEF treatment had no significant effect on seizure incidence, with 12/12 animals seizing in the saline group and 12/13 animals seizing in the CEF group, or total number of seizures. A small but significant reduction in cumulative behavioral seizures was observed in the 200 mg/kg CEF treatment group. Analysis of hippocampal tissue revealed no significant difference in hippocampal mass between the two groups (an indicator of atrophy). Furthermore, CEF treatment failed to increase GLT-1 levels in TMEV-infected mice treated for 7 days. Conclusions: These results indicate that CEF had a small but significant effect on cumulative behavioral seizures, but had no effect on seizure incidence, total number of seizures, and failed to rescue the decrease in GLT-1 seen in the hippocampi of seizing TMEV-injected mice. These results follow recent reports that the drug’s clinical efficacy has been conflicting, exemplified most recently by the drugs failure to meet the predetermined criteria in a phase 3 trial for ALS despite promising preclinical results. We thus conclude that preclinical testing of novel astrocyte-targeted therapeutics in a range of disease models with different astrocyte pathophysiologies is essential for informing future preclinical and clinical investigations of epilepsy dugs. Funding: This work was supported by AFPE, NINDS.