Immune Response in Murine Brain Early After Seizure Induction by Frontal Electric Stimulation.

Abstract

Immune responses that are caused by seizures or that can contribute to epileptogenesis are poorly understood. Lymphocyte immune responses in brain after a single seizure are unknown. We developed a new murine model system to analyze early brain immune responses after a single generalized tonic clonic seizure. C57bl/6 mice (males, 6-8 wk old) received either sham (electrodes placed subcutaneously over frontal lobe but no current passed) or electrical stimulus (electrodes placed subcutaneously over frontal lobe, 15-30 mA, 100 Hz, 0.4ms burst duration, 1 sec train duration) that resulted in submaximal (no hind-limb extension) or maximal seizures (hind-limb extension). Pretreatment of mice with pentobarbital (i.p., 25mg/kg) or inhaled ethyl-chloride (for 20s) prevented convulsions. Mice were killed at 1-24 hr and 7 days, brains pooled, and single-cell suspensions prepared, after which cells were separated according to density (Percoll gradients) and cell numbers determined (flow cytometry). In some experiments, brain was dissected into neocortex, hippocampus, midbrain, and cerebellum before assay. This is the first report that a single maximal seizure induced by frontal electric stimulus results in increased lymphocyte numbers in brain. This effect was not obtained when similar frontal electric stimulus resulted in submaximal seizures, or when mice were pretreated with pentobarbital or ethyl-chloride. The increases in lymphocyte numbers after maximal seizure were predominantly in neocortex (CD4+ and CD8+ T and B cells) and in hippocampus (T cells). The T and B cell subsets also expressed high levels of the activation markers/adhesion receptors CD11a, CD29, and CD44, which are required for lymphocyte migration into brain. After seizure, these markers were upregulated on peripheral T and B cells before T and B cell numbers increased in brain, suggesting that the increases in these cells in brain are due to their entry from the periphery. CD4+ and CD8+ T cells in brain intracytoplasmically expressed IL-2, a cytokine that is a potent stimulator of lymphocyte proliferation and enhances the killing activity of cytotoxic T cells. CD4+ T cells also expressed IL-4, the cytokine required for IgE isotype switching by B cells. B cells in brain was IgG+ and IgE+, setting the stage for immune responses, and even allergic response in brain. CD8+ T cells expressed IFN-g and TNF-a, cytokines that activate microglia and brain macrophages. Taken together, the results indicate that there are early immune responses in brain after a single seizure, which may play a role in allergy/autoimmunity, synaptic remodeling, and in the development of epilepsy.