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

Abstract

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-1β, CXCL1 (IL-8), TNF, and increased IL-1β/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.

Keywords: antiseizure medications; cytokines; drug-refractory status epilepticus; immunomodulatory drugs; neuroinflammation.

Figure 1

LPS-induced neuroinflammation in hippocampus/temporal cortex slices. RT-qPCR analysis of cytokine mRNA (A–E) in hippocampal/temporal cortex slices incubated with aCSF alone or with 10 μg/ml LPS for 30 min, followed by 30 min or 60 min washout in aCSF. Reference genes were s2Vb and Actb. Data are presented as fold-increase vs. control value in aCSF incubated slices (mean ± SEM and single values). *p < 0.05, **p < 0.01 vs. aCSF by Kruskal–Wallis test followed by Dunn's multiple comparison test.

Figure 2

Lipopolysaccharide effect on epileptiform activity in hippocampus/temporal cortex slices. (A, B) Depict representative activity maps (first row; higher field potential, FP, frequency corresponds to lighter green color) and raster plots (second and third rows) of epileptiform activity recorded in the temporal cortex (CTX) or hippocampus (HPC) during 0 Mg²⁺/100 μM 4-AP perfusion alone (A) or in slices pre-incubated with LPS [(B); 10 μg/ml LPS for 30 min, followed by 5 min washout], then exposed to 0 Mg²⁺/100 μM 4-AP for 40 min. (C) Reports quantification of epileptiform activity (FP frequency, burst duration and amplitude) during T1 (0–10 min from the start of 0 Mg²⁺/4-AP perfusion) reckoned in the area of higher activity (as shown by activity map/raster plot) in each slice. (D, E) Depict representative activity maps and raster plots during T2 (20–30 min from the start of 0 Mg²⁺/4-AP perfusion). (F) Reports quantification of epileptiform activity during T2 reckoned in the area of higher activity in each slice. Data are presented as mean ± SEM and single values (0 Mg²⁺ + 4-AP, n = 9 slices; LPS + 0 Mg²⁺ + 4AP, n = 10 slices). *p < 0.05 vs. 0 Mg²⁺/4AP by Mann–Whitney test.

Figure 3

Incidence of status epilepticus in LPS-treated slices. (A) Depicts a representative activity map (higher FP frequency corresponds to lighter green color) and raster plot showing a status epilepticus (SE) event in hippocampus. Enlarged tracing depicts FPs from one representative electrode. (B) Shows the incidence of SE events in 0 Mg²⁺/4-AP exposed slices ± LPS (10 μg/ml LPS for 30 min, followed by 5 min washout). 0 Mg²⁺ + 4-AP, n = 9 slices; LPS+0 Mg²⁺ + 4AP, n = 10 slices. p < 0.05 vs. 0 Mg²⁺/4AP by Chi-square test.

Figure 4

Effect of ASMs on epileptiform activities. (A, B) Show the effect of 800 μM sodium valproate (SVP), 50 μM phenytoin (PHE), 100 μM phenobarbital (PB) and midazolam (MDZ, 100 μM) on field potential (FP) frequency in slices perfused with 0 Mg²⁺/4-AP (A) or pre-treated with LPS (B) (10 μg/ml LPS for 30 min, followed by 5 min washout), then exposed to 0 Mg²⁺/100 μM 4-AP for 40 min. Quantification of epileptiform activity was done during T2 (20–30 min from the start of 0 Mg²⁺/4-AP perfusion) in the area of higher activity in each slice. Data are presented as mean ± SEM and single values (n = 5–7 slices/experimental group) *p < 0.05; **p < 0.01 by Mann–Whitney test vs. respective control slices (aCSF, no ASMs added). (C) Shows the incidence of the combination of ictal and SE events in LPS-pretreated slices in the various experimental groups. *p < 0.05 vs. 0 Mg²⁺/4AP by Chi-square test. (D, E) Depict representative activity maps (higher FP frequency corresponds to lighter green color) and raster plots in temporal cortex (CTX) and hippocampus (HPC) after addition of the various ASMs to slices perfused with 0 Mg²⁺/4-AP ± LPS. SVP, sodium valproate; PHE, phenytoin; PB, phenobarbital, MDZ, midazolam.

Figure 5

Effects of anti-IL-6R antibody and anakinra on epileptiform activity in lipopolysaccharide-treated slices. (A, B) The onset of the first field potential (FP) event (A) and FP frequency (B) in the various experimental groups (n = 6 slices/group). FP frequency was calculated during T2 (20–30 min from the start of 0 Mg²⁺/4-AP perfusion). Slices were preincubated with aCSF containing LPS (10 μg/ml for 30 min, followed by 5 min aCSF washout), then perfused in aCSF ± anti-mouse IL-6R Ab (100 μM) or ± anakinra (1.3 μM) for 15 min followed by 0 Mg²⁺/4-AP ± drugs for 40 min. *p < 0.05; **p < 0.01 by Mann–Whitney test vs. respective control slices (aCSF, no added drugs). (C) Depicts representative activity maps (higher FP frequency corresponds to lighter green color) and raster plots in temporal cortex (CTX) and hippocampus (HPC) in slices perfused with 0 Mg²⁺/4-AP+LPS with or without the immunomodulatory drugs.