Abstracts

Rationale: Mounting evidence has implicated a critical role of immunity and nuroinflammation in the evolution of drug-resistant epilepsy. The abundance of peripherally derived immune cells in resected human epileptic tissue suggests that brain infiltrating lymphocytes may be a potential therapeutic target to prevent epilepsy. Global immunuosuppression often has untoward side effects and blocking T-cell responses have been shown to accelerate epileptogenesis in the intrahippocampal kainic acid (KA) model of temporal lobe epilepsy (TLE). We aimed to characterize infiltrating immune cells in the mouse hippocampus in the intrahippocampal KA model of epilepsy to optimize target selection for immunomodulation. Methods: We induced prolonged status epilepticus by stereotactically injecting KA (200nl x 1 mg/ml) into CA1 of the hippocampus in male C57BL/6J mice aged 6-7 weeks. Two weeks after the injection, KA-injected mice (n=11) and PBS-injected control mice (n=10) were perfused with PBS and individual hippocampi and spleens were dissected and pooled into ipsilateral KA, contralateral KA, ipsilateral control, contralateral control, KA spleen, and control spleen groups. Lymphocytes were isolated from the tissue and quantified with flow cytometry using a T cell panel including the following markers: CD3, CD4, CD44, gamma-delta TCR, CD69, CD28, CD25, NK1.1, CD11b, and CD8. Results: Intrahippocampal KA injection induced convulsive status epilepticus for over 3 hours in all mice (11/11). KA-injected hippocampi showed a nearly two-fold increase in CD8+ T cells, gamma delta T cells, natural killer cells, and natural killer T cells per gram tissue compared to PBS controls. There were more CD8+ than CD4+ T cells in the KA-injected hippocampi whereas the CD8+ to CD4+ T cell ratios were reversed in PBS-injected hippocampi. KA-injected hippocampi and the contralateral hippocampus had increased activated CD8+/CD44+ (1.5x) and CD8+/CD69+ (3x) T cells but a decrease in activated CD4+/CD44+ (1.5x) T cells as compared with controls. There was no significant difference in T cell prevalence between ipsilateral and contralateral hippocampi of PBS-injected controls. The immune response to the stereotaxic injection itself is likely negligible given the lack of difference in T cell prevalence between ipsilateral and contralateral PBS-injected hippocampi. Conclusions: The increase in cytotoxic and activated T cells, in addition to gamma-delta T cells, natural killer cells, and natural killer T cells in the hippocampus after the KA injection demonstrated the heightened inflammatory milieu after status epilepticus prior to occurrence of spontaneous recurrent seizures in this model of TLE. Our finding highlights the diversity of cell types that may be contributing to or preventing epileptogenesis and provides quantifiable outcome measures for future immunomodulatory therapy. Funding: NIH/NINDS R01 NS073768