Experimental Investigations of Monomethyl and Dimethyl Fumarate in an Astrocyte-Microglia Co-Culture Model of Inflammation.

INTRODUCTION: Multiple sclerosis (MS) is the most common chronic inflammatory, demyelinating disease of the central nervous system. Dimethyl fumarate (DMF) and monomethyl fumarate (MMF) belong to the disease-modifying drugs in treatment of MS. There is evidence that astrocytes and microglia are involved in MS pathology, but few studies are available about MMF and DMF effects on astrocytes and microglia. The aim of this study was to investigate the effects of MMF and DMF on microglial activation and morphology as well as potential effects on glial viability, Cx43, and AQP4 expressions in different set-ups of an in vitro astrocyte-microglia co-culture model of inflammation.

METHODS: Primary rat glial co-cultures of astrocytes containing 5% (M5, mimicking "physiological" conditions) or 30% (M30, mimicking "pathological, inflammatory" conditions) of microglia were treated with different concentrations of MMF (0.1, 0.5, and 2 μg/mL) or DMF (1.5, 5, and 15 μM) for 24 h. Viability, proliferation, and cytotoxicity of glial cells were examined using MTT assay. Immunocytochemistry was performed to analyze the microglial phenotypes. Connexin 43 (Cx43) and aquaporin 4 (AQP4) expressions were quantified by immunoblot analysis.

RESULTS: Treatment with different concentrations of MMF or DMF for 24 h did not change the glial cell viability in M5 and M30 co-cultures. Microglial phenotypes were not altered by DMF under physiological M5 conditions, but treatment with higher concentration of DMF (15 μM) induced microglial activation under inflammatory M30 conditions. Incubation with different concentrations of MMF had no effects on microglial phenotypes. The Cx43 expression in M5 and M30 co-cultures was not changed significantly by immunoblot analysis after incubation with different concentrations of DMF or MMF for 24 h. The AQP4 expression was significantly increased in M5 co-cultures after incubation with 5 μm DMF. Under the other conditions, AQP4 expression was not affected by DMF or MMF.

DISCUSSION: In different set-ups of the astrocyte-microglia co-culture model of inflammation, MMF has not shown significant effects. DMF had only limited effects on microglia phenotypes and AQP4 expression. In summary, mechanisms of action of fumarates probably do not involve direct effects on microglia phenotypes as well as Cx43 and AQP4 expression.