Abstracts

Rationale: Epilepsy resulting from CNS infection is often refractory to currently available anti-seizure drugs. C57BL/6J mice infected with Theiler's murine encephalomyelitis virus (TMEV) show acute behavioral seizures between 3-7 days post-infection (dpi), exhibit clinically relevant pathological and physiological changes in the hippocampus, survive the infection and later develop chronic epilepsy after about 2-3 months of infection. Therefore, this is an appropriate model to study mechanism(s) of epileptogenesis for infection-induced epilepsy. Several studies have implicated brain-derived neurotrophic factor (BDNF) signaling in the development of epilepsy. BNDF is known to affect neuronal transmission by modulating the expression and cellular trafficking of excitatory and inhibitory receptors. BDNF may also cause hyperexcitation by impairing chloride homeostasis and GABAergic inhibition by downregulating K+-Cl- cotransporter (KCC2). Therefore, we hypothesized that increased BDNF signaling impairs GABAergic inhibition via cation chloride cotransporters that may underlie network hyperexcitability and seizures following viral infection. Methods: Mice were injected with either TMEV or PBS (sham) and monitored for acute seizures. TMEV-infected mice with or without acute seizures and sham-treated mice were sacrificed at various time points post-infection (n=4-6 mice per time point). The hippocampi samples were used to quantify the protein level of BDNF by immunoassay and of chloride transporters (KCC2 and NKCC1) by western blot. In another study, acute brain slices (n=4-6 mice) from TMEV-infected mice with seizures at 5 dpi were incubated for 4.5 hours with 250 ng/ml of rhTrkB-Fc (scavenging bodies of BDNF to inhibit BDNF signaling) or rhIgG1-Fc (control) in artificial cerebrospinal fluid with continuous oxygenation. Then, the hippocampi were isolated to measure the protein level of KCC2 by western blot. Results: We found significant increase in the level of BDNF in the hippocampus of TMEV-infected mice with seizures at the onset of acute seizures. BDNF level continued to increase during disease progression and more than doubled during acute and chronic seizures (p<0.001). There was no change in the expression of NKCC1, whereas the expression of KCC2 was significantly decreased at 5 dpi (p<0.01) in the hippocampi from TMEV-infected mice. Therefore the ratio of NKCC1 to KCC2 was reduced (p<0.05), which may cause intracellular accumulation of chloride and may contribute to hyperexcitability by reversing GABA-mediated inhibition. Interestingly, inhibiting BDNF signaling by rhTrkB-Fc restored the level of KCC2 in hippocampus of TMEV-infected mice with seizures. Conclusions: Our data suggest that increased level of BDNF causes downregulation of KCC2 and that may contribute to epileptogenesis in this model. Funding: This work was funded by NIH/NINDS 7R01NS036692-16 (HWS) and NIH/NINDS 7R01NS082851-04 (HWS).