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

Abstract

SUMMARYPurpose: 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-b1 (TGF-b1) and tumor necrosis factor-a (TNF-a) 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-b1 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-b1 and TNF-a 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 microglial activation, a typical hallmark of inflammation, was induced by VPA in M5 and continued in M30 co-cultures. With regard to the direct relation between CNS inflammation and seizures, VPA seems to be unsuitable for reducing inflammatory conditions. The reverse effect was achieved after CBZ. We noticed significant microglial inactivation, after incubation of the M30 co-cultures. In conclusion, we suggest that AEDs with antiinflammatory glial features are beneficial for seizures caused by persistent brain inflammation.

Autoimmunity and inflammation in status epilepticus: from concepts to therapies

“…They demonstrated important microglial activation induced by VPA at all doses. According to these results, and as stated by the authors, 'VPA seems to be unsuitable for reducing inflammatory conditions' in humans [32].…”

“…Here again, contradictory results exist for CBZ, which rather demonstrated anti-inflammatory properties in the previously mentioned study by Dambach and colleagues [32]. More surprisingly maybe, diazepam, the drug of choice for SE in childhood, has been demonstrated to inhibit T-cell function, and thereby decrease the production of IFN-g that possesses antiviral and immunomodulatory properties [34].…”

Prophylactic Use of Antiepileptic Drugs in Patients with Spontaneous Intracerebral Hemorrhage

“…25,26 In an in vitro model of inflammation, phenytoin reduced the glial viability and altered microglial activation. 27 In the present study, phenytoin was the most frequently used AED and appeared harmful in the unadjusted models, but this negative effect was completely attenuated after controlling for confounding factors.…”

Influence of drugs on gap junctions in glioma cell lines and primary astrocytes in vitro

“…The effect of some common AEDs including phenytoin (PHE), carbamazepine (CBZ), gabapentin (GBT), and VPA on the astroglial Cx43 expression in astroglia/microglia cultures of newborn rats was recently investigated (Dambach et al, 2014). In this study, astrocytes were co-cultured with different percentages of microglial cells (M5 or M30).…”