Abstracts

Rationale: Dravet syndrome has long been considered a channelopathy due to confirmed mutations in the gene encoding the alpha 1 subunit of the voltage gated sodium channel (SCN1A), evidence has emerged of the consequences of SCN1A dysfunction that is beyond seizure related damage. We and others have established that recurrent seizures lead to activation of immune responses, which can further precipitate seizures and create a deleterious positive feedback loop. Ample evidence has shown effectiveness of immunomodulatory agents in treating epilepsy. Nevertheless, it remains controversial whether immune activation is the cause or consequence of recurrent seizures. We thus characterized detailed immune infiltration and activation in the brain of mouse, SCN1Atm1Kea, which models Dravet syndrome prior to seizure onset. The goal of this project is to understand whether overt immune activation can be detected in the well-established Dravet mouse model to further delineate the role of peripheral leukocytes in epileptogenesis. Methods: SCN1Atm1Kea global knockout was maintained on 129 strain background (129.SCN1A+/-) and was crossed to C57Bl/6 to generate F1 mice. Onset of clinical seizure was measured by video monitoring at post-natal day 18 (P18). Both female and male F1 knockout (KO) mice and wildtype (WT) littermate controls were euthanized and perfused with cold PBS at P17, one day before clinical seizure onset. Brains devoid of cerebellum and brain stem were harvested, grouped by gender, and processed to generate single cell suspension suitable for flow cytometric analyses. Results: We detected significantly increased brain infiltration of innate immune cells from the periphery, including monocytes, macrophages, dendritic cells, as well as adaptive immune cells, such as T lymphocytes, B cells, and natural killer cells in the KO mice compared with gender-matched littermate controls. Remarkably, frequency of brain-infiltrating T cells in the KO mice was at least 5-fold higher than the WT controls. Most importantly, more than 90% of these infiltrating T cells were CD8+ cytotoxic T cells. Interestingly, female KO mice exhibited higher cellularity in leukocyte infiltrates than male mice, consistent with gender discrepancies in classical autoimmune diseases. Comparable counts and proportions of different subsets of leukocytes were noted in the spleen of both KO and WT mice suggesting normal leukocyte development. Conclusions: Our preliminary data demonstrated for the first time that brain-infiltration of peripherally derived leukocytes are detected prior to seizure onset in a Dravet mouse model. Cytotoxic T cells are enriched in the cortex most likely in an antigen-dependent manner as they are predominatly memory cells that have been activated previously. These T cells are capable of directly ligating apoptotic receptors expressed by neurons and different glial cells to induce cell death. Funding: This work is supported by grant NIH/NINDS R01NS073768 and R21NS094999 to S.K. and S.D.M., and a post-doctoral fellowship 262243 from the Epilepsy Foundation of America to D.X.