Glia and epilepsy: Experimental investigation of antiepileptic drugs in an astroglia/microglia co‐culture model of inflammation

• Published in: Epilepsia (Copenhagen) 2014-01, Vol.55 (1), p.184-192
• Main Authors: Dambach, Hannes, Hinkerohe, Daniel, Prochnow, Nora, Stienen, Martin N., Moinfar, Zahra, Haase, Claus G., Hufnagel, Andreas, Faustmann, Pedro M.
• Format: Article
• Language: English
• Subjects: Amines - pharmacology; Amines - therapeutic use; Animals; Anticonvulsants - pharmacology; Anticonvulsants - therapeutic use; Antiepileptic agents; Antiepileptic drugs; Astrocytes - drug effects; Astrocytes - physiology; Astroglia; Blotting, Western; Carbamazepine - pharmacology; Carbamazepine - therapeutic use; Cells, Cultured; Cellular viability; Coculture Techniques; Connexin 43; Connexin 43 - biosynthesis; Cyclohexanecarboxylic Acids - pharmacology; Cyclohexanecarboxylic Acids - therapeutic use; Cytokines; Dose-Response Relationship, Drug; Drug dosages; Enzyme-Linked Immunosorbent Assay; Epilepsy - drug therapy; Epilepsy - etiology; Epilepsy - physiopathology; Gabapentin; gamma-Aminobutyric Acid - pharmacology; gamma-Aminobutyric Acid - therapeutic use; Gap Junctions - drug effects; Gap Junctions - physiology; Glia; Inflammation; Inflammation - drug therapy; Inflammation - physiopathology; Microglia; Microglia - drug effects; Microglia - physiology; Neuroglia - drug effects; Neuroglia - physiology; Phenytoin - pharmacology; Phenytoin - therapeutic use; Rats; Rats, Wistar; Rodents; Transforming Growth Factor beta1 - biosynthesis; Tumor necrosis factor-TNF; Valproic Acid - pharmacology; Valproic Acid - therapeutic use
• ISSN: 0013-9580; 1528-1167
• DOI: 10.1111/epi.12473

Summary Purpose The contribution of glial cells, mainly astrocytes and microglia, to the pathophysiology of epilepsy is increasingly appreciated. Glia play a pivotal role in the initiation and maintenance of the central nervous system (CNS) immune response and neuronal metabolic and trophic supply. Recent clinical and experimental evidence suggests a direct relationship between epileptic activity and CNS inflammation, which is characterized by accumulation, activation, and proliferation of microglia and astrocytes. Concomitant glia‐mediated mechanisms of action of several antiepileptic drugs (AEDs) have been proposed. However, their direct effects on glial cells have been rarely investigated. We aimed to investigate the effect of commonly used AEDs on glial viability, the gap junctional network, the microglial activation, and cytokine expression in an in vitro astroglia/microglia co‐culture model. Methods Primary astrocytic cultures were prepared from brains of postnatal (P0–P2) Wistar rats and co‐cultured with a physiologic amount of 5%, as well as 30% microglia in order to mimic inflammatory conditions. Co‐cultures were treated with valproic acid (VPA), carbamazepine (CBZ), phenytoin (PHE), and gabapentin (GBT). Viability and proliferation were measured using the tetrazolium (MTT) assay. The microglial activation state was determined by immunocytochemical labeling. The astroglial connexin 43 (Cx43) expression was measured by Western blot analysis. The transforming growth factor‐β1 (TGF‐β1) and tumor necrosis factor‐α (TNF‐α) cytokine levels were measured by the quantitative sandwich enzyme immunosorbent assay (ELISA). Key Findings Astrocytes, co‐cultured with 5% microglia (M5 co‐cultures), showed a dose‐dependent, significant reduction in glial viability after incubation with PHE and CBZ. Furthermore, VPA led to highly significant microglial activation at all doses examined. The antiinflammatory cytokine TGF‐β1 release was induced by high doses of GBT and PHE. Astrocytes co‐cultured with 30% microglia (M30 co‐cultures) revealed a dose‐dependent significant reduction in glial viability after incubation with PHE, accompanied by increased TGF‐β1 and TNF‐α levels. However, CBZ significantly reduced the amount of activated microglial cells and increased the total number of inactivated microglia. Finally, CBZ resulted in reduced viability at all doses examined. Significance CNS inflammation is characterized by a disturbance of glial cell functions. Strong micr