An in vitro model of drug-resistant seizures for selecting clinically effective antiseizure medications in Febrile Infection-Related Epilepsy Syndrome.

Introduction: FIRES is a rare epileptic encephalopathy induced by acute unremitting seizures that occur suddenly in healthy children or young adults after a febrile illness in the preceding 2 weeks. This condition results in high mortality, neurological disability, and drug-resistant epilepsy. The development of new therapeutics is hampered by the lack of validated experimental models. Our goal was to address this unmet need by providing a simple tool for rapid throughput screening of new therapies that target pathological inflammatory mechanisms in FIRES. The model was not intended to mimic the etiopathogenesis of FIRES which is still unknown, but to reproduce salient features of its clinical presentation such as the age, the cytokine storm and the refractoriness of epileptic activity to antiseizure medications (ASMs). Methods: We refined an in vitro model of mouse hippocampal/temporal cortex acute slices where drug-resistant epileptic activity is induced by zero Mg2+/100 µM 4-aminopirydine. Clinical evidence suggests that acute unremitting seizures in FIRES are promoted by neuroinflammation triggered in the brain by the preceding infection. We mimicked this inflammatory component by exposing slices for 30 min to 10 µg/ml lipopolysaccharide (LPS). Results: LPS induced a sustained neuroinflammatory response, as shown by increased mRNA levels of IL-1ß, CXCL1 (IL-8), TNF, and increased IL-1ß/IL-1Ra ratio. Epileptiform activity was exacerbated by neuroinflammation, also displaying increased resistance to maximal therapeutic concentrations of midazolam (100 µM), phenytoin (50 µM), sodium valproate (800 µM), and phenobarbital (100 µM). Treatment of LPS-exposed slices with two immunomodulatory drugs, a mouse anti-IL-6 receptor antibody (100 µM) corresponding to tocilizumab in humans, or anakinra (1.3 µM) which blocks the IL-1 receptor type 1, delayed the onset of epileptiform events and strongly reduced the ASM-resistant epileptiform activity evoked by neuroinflammation. These drugs were shown to reduce ASM-refractory seizures in FIRES patients. Discussion: The neuroinflammatory component and the pharmacological responsiveness of epileptiform events provide a proof-of-concept validation of this in vitro model for the rapid selection of new treatments for acute ASM-refractory seizures in FIRES.

A systems-level analysis highlights microglial activation as a modifying factor in common epilepsies.

AIMS: The causes of distinct patterns of reduced cortical thickness in the common human epilepsies, detectable on neuroimaging and with important clinical consequences, are unknown. We investigated the underlying mechanisms of cortical thinning using a systems-level analysis. METHODS: Imaging-based cortical structural maps from a large-scale epilepsy neuroimaging study were overlaid with highly spatially resolved human brain gene expression data from the Allen Human Brain Atlas. Cell-type deconvolution, differential expression analysis and cell-type enrichment analyses were used to identify differences in cell-type distribution. These differences were followed up in post-mortem brain tissue from humans with epilepsy using Iba1 immunolabelling. Furthermore, to investigate a causal effect in cortical thinning, cell-type-specific depletion was used in a murine model of acquired epilepsy. RESULTS: We identified elevated fractions of microglia and endothelial cells in regions of reduced cortical thickness. Differentially expressed genes showed enrichment for microglial markers and, in particular, activated microglial states. Analysis of post-mortem brain tissue from humans with epilepsy confirmed excess activated microglia. In the murine model, transient depletion of activated microglia during the early phase of the disease development prevented cortical thinning and neuronal cell loss in the temporal cortex. Although the development of chronic seizures was unaffected, the epileptic mice with early depletion of activated microglia did not develop deficits in a non-spatial memory test seen in epileptic mice not depleted of microglia. CONCLUSIONS: These convergent data strongly implicate activated microglia in cortical thinning, representing a new dimension for concern and disease modification in the epilepsies, potentially distinct from seizure control.

Microglia proliferation plays distinct roles in acquired epilepsy depending on disease stages.

OBJECTIVE: Microgliosis occurs in animal models of acquired epilepsy and in patients. It includes cell proliferation that is associated with seizure frequency and decreased neuronal cells in human epilepsy. The role of microglia proliferation in the development of acquired epilepsy is unknown; thus, we examined its contribution to spontaneous seizure, neurodegeneration, and cognitive deficits in different disease phases. METHODS: We used a model of acquired epilepsy triggered by intra-amygdala kainic acid in C57BL6N adult male mice. Mice were electroencephalographically (EEG) monitored (24/7) during status epilepticus and in early and chronic disease. Microglia proliferation was blocked by GW2580, a selective CSF1 receptor inhibitor, supplemented in the diet for 21 days from status epilepticus onset. Then, mice were returned to placebo diet until experiment completion. Control mice were exposed to status epilepticus and fed with placebo diet. Experimental mice were tested in the novel object recognition test (NORT) and in Barnes maze, and compared to control and sham mice. At the end of the behavioral test, mice were killed for brain histopathological analysis. Additionally, seizure baseline was monitored in chronic epileptic mice, then mice were fed for 14 days with GW2580 or placebo diet under 24/7 EEG recording. RESULTS: GW2580 prevented microglia proliferation in mice undergoing epilepsy, whereas it did not affect microglia or basal excitatory neurotransmission in the hippocampus of naive mice. Mice with occluded microglia proliferation during early disease development underwent status epilepticus and subsequent epilepsy similar to placebo diet mice, and were similarly impaired in NORT, with improvement in Barnes maze. GW2580-treated mice displayed neuroprotection in the hippocampus. In contrast, blockade of microglia proliferation in chronic epileptic mice resulted in spontaneous seizure reduction versus placebo mice. SIGNIFICANCE: Microglia proliferation during early disease contributes to neurodegeneration, whereas in late chronic disease it contributes to seizures. Timely pharmacological interference with microglia proliferation may offer a potential target for improving disease outcomes.