Real-time analysis of microglial activation and motility in hepatic and hyperammonemic encephalopathy

Hepatic encephalopathy (HE) is a potentially fatal complication of acute liver failure, associated with severe neurological dysfunction and coma. The brain's innate immune cells, microglia, have recently been implicated in the pathophysiology of HE. To date, however, only ex vivo studies have been used to characterize microglial involvement. Our study uses in vivo two-photon imaging of awake-behaving mice expressing enhanced green fluorescent protein (eGFP) under the Cx3cr1 promoter to examine microglial involvement in two different models of encephalopathy - a slower, fatal model of azoxymethane-induced HE and a rapid, reversible acute hyperammonemic encephalopathy (AHE) induced by an ammonia load. To investigate the potential contribution of microglia to the neurological deterioration seen in these two models, we developed a software to analyze microglial activation and motility in vivo. In HE, we found that microglia do not become activated prior to the onset of neurological dysfunction, but undergo activation with mildly impaired motility during the terminal stage IV. We demonstrate that this microglial activation coincides with blood-brain barrier (BBB) opening and brain edema. Conversely, both microglial activation and motility are unchanged during AHE, despite the mice developing pathologically increased plasma ammonia and severe neurological dysfunction. Our study indicates that microglial activation does not contribute to the early neurological deterioration observed in either HE or AHE. The late microglial activation in HE may therefore be associated with terminal BBB opening and brain edema, thus exacerbating the progression to coma and increasing mortality.

► We developed a novel approach to measure microglial activation in vivo. ► Changes in microglial activation were followed over time in hepatic and hyperammonemic encephalopathies. ► Microglia are activated in the final stage of hepatic encephalopathy, not hyperammonemia. ► Microglial motility is also reduced in the final stage of hepatic encephalopathy. ► Blood-brain barrier opening and brain edema coincide with microglial changes in hepatic encephalopathy.