Glia and epilepsy: Experimental investigation of antiepileptic drugs in an astroglia/microglia co‐culture model of inflammation
Authors: Dambach Hannes , Hinkerohe Daniel , Prochnow Nora , Stienen Martin N. , Moinfar Zahra , Haase Claus G. , Hufnagel Andreas , Faustmann Pedro M. 
Published: December 2013

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 andCNSinflammation, 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 usedAEDs 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 byWestern 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 withPHEandCBZ. Furthermore,VPAled to highly significant microglial activation at all doses examined. The antiinflammatory cytokineTGF‐β1 release was induced by high doses ofGBTandPHE.

Astrocytes co‐cultured with 30% microglia (M30 co‐cultures) revealed a dose‐dependent significant reduction in glial viability after incubation withPHE, accompanied by increasedTGF‐β1 andTNF‐α levels. However,CBZsignificantly reduced the amount of activated microglial cells and increased the total number of inactivated microglia. Finally,CBZresulted in reduced viability at all doses examined.

Significance

CNSinflammation is characterized by a disturbance of glial cell functions. Strong microglial activation, a typical hallmark of inflammation, was induced byVPAinM5 and continued inM30 co‐cultures. With regard to the direct relation betweenCNSinflammation and seizures,VPAseems to be unsuitable for reducing inflammatory conditions. The reverse effect was achieved afterCBZ. We noticed significant microglial inactivation, after incubation of theM30 co‐cultures. In conclusion, we suggest thatAEDs with antiinflammatory glial features are beneficial for seizures caused by persistent brain inflammation.