Microglial activation is inhibited by selective anti-seizure medications

Abstract

Objective: To investigate the anti-inflammatory properties of anti-seizure medications (ASMs) administered to patients with drug-resistant epilepsy (DRE) and the role of sodium channels in microglial activation.

Material: Primary microglia monocultures from mice brains.

Treatment: Microglia were activated with 10 μg/mL lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (poly I:C) and pre- (45 min ASM then 2 h ASM plus stimulus) or post- (2 h stimulus then 24 h only ASM) treated with ASMs. Microglia were treated with cannabidiol (10 μM), stiripentol (250 μM), fenfluramine (50 μM), phenytoin (8 and 40 μM), cenobamate (300 and 900 μM), or the small molecule sodium channel blocker GS967 (10 and 30 μM). The sodium channel modulators tetrodotoxin (1 μM), µ-conotoxin KIIIA (1 μM), and β-pompilidotoxin (0.5 μM) were also applied.

Methods: Microglia activation was quantified through measurements of Ptgs2 (Cox2), Tnf-α, and Ifn-β induction by RT-qPCR and of cell morphology by immunocytochemistry. Expression of sodium channels in microglia was studied using PCR, RT-qPCR, immunohisto- and immunocytochemistry. Mann Whitney test and the Kruskal-Wallis test with Dunn's multiple comparisons post-test were used.

Results: ASMs have a differential effect on microglial activation. Uniquely, cenobamate inhibited the induction of Ifn-β and made the cells less amoeboid. The voltage gated sodium channel Na_v1.2 is expressed by microglial cells and its expression levels change with microglial inflammatory response. Toxins that block sodium channels modulated microglial activation.

Conclusions: ASMs, applied to patients with DRE, have a differential ability to reduce microglial activation and pro-inflammatory microglial morphology in vitro. Moreover, sodium channel blockage modulates inflammation through microglia activation. Taken together these results suggest, that further investigation of patient's immune response to ASMs could be important.

Keywords: Anti-seizure medication; Epilepsy; Inflammation; Microglia.

Fig. 1

Effect of ASMs on inflammation markers in microglia. Effect of CBD (A-C), FFA (D-F) and STP (G-I) on the mRNA levels of Ptgs2, Tnf-α or Ifn-β on microglial cells stimulated with LPS or poly I:C. Levels of Ptgs2 (A, D and G), Tnf-α (B, E and H) and Ifn-β (C, F and I) normalized to vehicle control in microglia pretreated with the drug for 45 min, followed by LPS (10 μg/mL) (A, B, D, E, G and H) or poly I:C (10 μg/mL) (C, F and I) for 6 h. Data are expressed as mean ± SEM (n = 3) *p < 0.05. CBD cannabidiol, DMSO dimethyl sulfoxide, FFA fenfluramine, STP stiripentol

Fig. 2

Effect of sodium channel blockers on inflammation markers in microglia. Effect of PHT (A-C), CNB (D-F) and GS967 (G-L) on the mRNA levels for Ptgs2, Tnf-α or Ifn-β on microglial cells stimulated with LPS or poly I:C. Levels of Ptgs2 (A, D, G, J) and Tnf-α (B, E, H, K) and Ifn-β (C, F, I, L) normalized to vehicle control in microglia pretreated with drug for 45 min, followed by LPS (10 μg/mL) (A, B, D, E, G, H, J, K) or poly I:C (10 μg/mL) (C, F, I, L) for 6 h. Data are expressed as mean ± SEM (n = 3) *p < 0.05; **p < 0.01. CNB cenobamate, DMSO dimethyl sulfoxide, PHT phenytoin

Fig. 3

Effect of sodium channel blockers on cyto- and chemokines in microglia. Effect of CBD (A), GS967 (B), PHT (C) and CNB (D) on the release of selected cyto- and chemokines on microglial cells, pretreated with the drug for 45 min, followed by LPS (10 μg/mL) (A, B and C) or poly I:C (10 μg/mL) (D) for 6 h. Cyto- and chemokine levels are expressed relative to their respective vehicle controls, which were normalized to a value of 1. (n = 3) (Exemplary pictures of the membranes can be found in supplementary Fig. 2). CBD cannabidiol, CNB cenobamate, PHT phenytoin

Fig. 4

Effect of ASMs on inflammation markers on preactivated microglia. Effect of CBD (A), GS967 (B), PHT (C and H), STP (D), FFA (E) and CNB (F and G) on the mRNA levels of Ptgs2 (A-E) on microglial cells pre-stimulated with LPS (10 μg/mL) for 2 h and Ifn-β mRNA levels (F–H) after pretreatment with poly I:C (10 μg/mL) for 2 h. RT-qPCR analysis was performed 24 h after change to only drug. Data are expressed as mean ± SEM (n = 3) *p < 0.05; **p < 0.01. CBD cannabidiol, CNB cenobamate, DMSO dimethyl sulfoxide, FFA fenfluramine, PHT phenytoin, STP stiripentol

Fig. 5

Effect of ASMs on microglia morphology. Quantitation of area (A left) and circularity (A right) and representative field for CTL (B) LPS (C) poly I:C (D) after cells were stimulated with either LPS or poly I:C. Quantiation of area (E left) and circularity (E right) and representative field for vehicle (F) and CBD (G). Quantiation of area (H left) and circularity (H right) and representative field for vehicle (I) and CNB (J). Quantiation of area (K left) and circularity (K right) and representative field for vehicle (L) and FFA (M). Quantiation of area (N left) and circularity (N right) and representative field for vehicle (O) and STP (P). Quantiation of area (Q left) and circularity (Q right) and representative field for vehicle (R), GS967 (S). Quantiation of area (T left) and circularity (T right) and representative field for vehicle (U), 8 µM PHT (V) and 40 µM PHT (W). Treatment initiated 2 h after microglial activation. All except for CNB were activated with LPS 10 µg/mL. For CNB activation was by poly I:C 10 µg/mL. Cells were fixiated with PFA 24 h after treatment. Immunocytochemistry for IBA1 (red), nuclei DAPI (blue). Merged images shown. Graphs represent mean ± S.E.M. ***p < 0.001; ****p < 0.0001. CBD cannabidiol, CNB cenobamate, CTL control, DMSO dimethyl sulfoxide, FFA fenfluramine, LPS lipopolysaccharide, PHT Phenytoin, PIC poly I: C polyinosinic:polycytidylic acid, STP stiripentol, IBA1 ionized calcium-binding adapter molecule 1

Fig. 6

Effect of stiripentol on cyto- and chemokines in microglia. Effect of STP on the release of selected cyto- and chemokines on microglial cells pre-stimulated with LPS (10 μg/mL) for 2 h followed with only STP for 24 h. Cyto- and chemokine levels are expressed relative to their respective vehicle controles, which were normalized to a value of 1. (n = 3) STP stiripentol

Fig. 7

Sodium channel modulator effect on microglia. A Effect of TTX (A1) and β-PMTX (A2) on the mRNA levels of Tnf-α on microglial cells stimulated with LPS. Levels of Tnf-α normalized to LPS treated microglia, microglia were pretreated with toxin for 45 min, followed by LPS (10 µg/mL) for 6 h. Data are expressed as mean ± SEM (n = 3) *p < 0.05. B Spearman Rho between Scn8a and Ptgs2 mRNA levels (B). C expression of Na_v1.6 in activated microglia, LPS (1 µg/mL) treated for 24 h (C). Immunocytochemistry for IBA1 (green), Na_v1.6 (red), nuclei DAPI (blue). D Brain (control) and microglia untreated (-LPS) or treated (+ LPS (1 µg/mL)) for 24 h. 564 bp = Scn1a transcript with poison exon and 498 bp without inclusion of the poison exon (D1). Brain (control) and microglia untreated (-LPS) or treated (+ LPS (1 µg/ml)) for 24 h. 364 bp = Scn8a full length transcript, 309 bp = Scn8a transcript with poison exon, 241 bp = Scn8a transcript that skips exon (D2). E Comparison of Scn2a and Scn3a mRNA levels in microglia (E). F Expression of Na_v1.2 in mouse in vivo in microglia (F), P14 (F1–F3), P56 (F4–F6). G Expression of Na_v1.2 in vitro in microglia (G). F and G Immunohisto- and immunocytochemistry for IBA1 (red), Na_v1.2 (green), nuclei DAPI (blue). H Scn2a mRNA levels after 24 h of activation by LPS (1 µg/mL) (H). I and J Microglia pretreated with either µ-Cono (I) or Phrixo-3 (J) for 45 min followed by LPS (10 μg/mL) for 6 h. Data are expressed as mean ± SEM (n = 3) *p < 0.05; **p < 0.01. CTL control, LPS lipopolysaccharide, µ-Cono µ-conotoxin KIIIA, Phrixo-3 phrixotoxin-3, TTX tetrodotoxin, β-PMTX β-pompilidotoxin, IBA1 ionized calcium-binding adapter molecule 1