Background Hepatic encephalopathy (HE) is a neurological complication resulting from acute or chronic liver disease. Hyperammonemia leading to astrocyte swelling and cerebral edema in combination with neuroinflammation including microglia activation, mainly contribute to the pathogenesis of HE. However, little is known about microglia and their inflammatory response, as well as their influence on astrocytic channels and astrocyte swelling under hyperammonemia.
Objective To investigate the effects of ammonia on the microglial activation, glial viability, connexin 43 (Cx43) and aquaporin 4 (AQP4) expression in astrocytes in in vitro astrocyte-microglia co-culture model.
Methods Primary rat glial cocultures of astrocytes containing 5% (M5, representing "physiological" conditions) or 30% (M30, representing "pathological" conditions) of microglia were incubated with 3 mM, 5 mM, 10 mM and 20 mM ammonium chloride (NH4Cl) for 6 h and 24 h in order to mimic the conditions of HE. An MTT assay was performed to measure the viability, proliferation and cytotoxicity of cells. The microglial phenotypes were analysed by immunocytochemistry. The expression of Cx43 and AQP4 were quantified by immunoblot analysis.
Results A significant reduction of glial viability was observed in M30 co-cultures after incubation with 20 mM NH4Cl for 6 h, whereas in M5 co-cultures the viability remained unchanged. Microglial activation was detected by immunocytochemistry after incubation with 3 mM, 5 mM and 10 mM NH4Cl for 6 h and 24 h in M5 as well as in M30 co-cultures. The Cx43 expression was increased significantly in M30 co-cultures after 6 h incubation with 5 mM NH4Cl. The AQP4 expression was increased significantly in M5 co-cultures treated with 10 mM NH4Cl for 6 h.
Conclusions Our findings showed a significant microglial activation, decrease of viability and increase in Cx43 and AQP4 expression after NH4Cl incubation in astrocyte-microglia co-culture model. Based on previous in vitro studies suggesting that microglia activation influences astrocytic networks, it can be assumed that the microglial activation under hyperammonemia can modulate the Cx43 and AQP4 expression in astrocytes in a dynamic way and this can contribute to astrocytic dysfunction in HE.