Abstracts

Rationale: A dysregulated innate immune response, peripheral inflammatory cell infiltration and breakdown of the blood-brain barrier have been implicated in the initiation, progression and perpetuation of seizures. To detect and quantify CNS-infiltrating and CNS resident immune cells, we used an unbiased multi-color flow cytometric analysis of inflammatory cells and tested the hypothesis that early-life seizures prime the brain and allow the infiltration of leukocytes, causing exaggerated microglia activation, increased seizure susceptibility and exacerbation of neuronal injury following a second-hit kainic acid (KA)-induced status epilepticus (SE). Methods: Wild-type C57BL/6 mice were subjected to KA-SE on P14 followed by KA-SE on P28 (KA/KA). Controls included mice injected with KA only at P28 (PBS/KA) and mice not experiencing any seizures (PBS/PBS) (n=6/group). Latency to seizure onset after KA-SE was measured and compared between PBS/KA and KA/KA animals. 24 h after KA-SE (or PBS) at P28, temporal lobes containing hippocampus, amygdala and pyriform cortices were harvested from saline perfused animals and CNS mononuclear cells, isolated using enzymatic digestion and Percoll gradients. To detect the presence of CNS-resident microglia and CNS-infiltrating leukocytes, the absolute total percentages, cell numbers and activation status of microglia and infiltrating leukocytes were quantified using multicolor flow cytometry; data were analyzed using FlowJo software. Results: CNS-resident and infiltrating cells were identified as microglia (CD11b CD45LOW); CD4 T cells (CD45HIGH CD3 CD4 ); conventional dendritic cells (cDC) (CD45HIGHCD11b CD11c ) and macrophages (M?) (CD45HIGHCD11b CD11c-). Following KA-SE at P28, we detected infiltration of significant number of peripheral leukocytes in the temporal lobes of mice. Moreover, mice with prior experience of KA-SE (KA/KA) had increased percentages and cell numbers of infiltrating dendritic cells and macrophages compared to PBS/KA or PBS/PBS mice. Notably, there were increased numbers of CD4 and CD8 T cells in the brains of KA/KA mice compared to PBS/KA or PBS/PBS controls. Mice with prior experience of KA-SE had significantly shorter latency to subsequent seizures compared to controls (1202 sec 65 (KA/KA) vs. 1695 sec 129 (PBS/KA), n=10, p<0.02). Conclusions: We detected and quantified peripherally derived innate and adaptive immune inflammatory cells following KA-SE. We show significant increases in CNS-infiltrating peripheral inflammatory lymphocytes, macrophages and dendritic cells after KA-SE in the temporal lobes of mature animals sensitized by early-life seizures. In parallel, there was an increase in later seizure susceptibility in animals exposed to early-life convulsions. Our results suggest that recurrent seizures may exacerbate BBB leakage and infiltration of peripheral leukocytes in the brain and potentiate seizures and seizure-induced changes. Priming effects of early-life convulsions may be mediated by involvement of both innate and adaptive immunity.