Veterinary session

Hepatic encephalopathy (HE) is a neuropsychiatric syndrome that has importance in both human and veterinary medicine and is most commonly the consequence of portosystemic shunt and/or hepatic failure. Among the several pathomechanisms of HE, neuroinflammation is of paramount importance and plays a significant role in the pathogenesis of HE, still many details of this process is unknown. Although the inflammatory response present in the disease is believed to be primarily caused my microglia cells, data from scientific literature proves that the astrocyte cells also have the ability to produce cytokins and thus induce inflammation. The aim of our work is to examine the pathologic role of astrocytes in neuroinflammation in HE. During our study, we examined the effects of different relevant factors in HE on the IL-6 and IL-1ß production of primary rat astrocyte cultures.

Generation of an applicable in vitro model was necessary for our research, the brain tissue of 1-2-day old Sprague-Dawley rats were used for this purpose. The primary astrocyte monolayer was decontaminated of microglia cells with a mitotic inhibitor, cytosine β-d-arabinofuranoside (Ara-C), then a lysosomotrop agent, leucin-methylesther (LME). The success of the treatment was checked by immunofluorescence. After creating the pure primary rat astrocyte cultures, they were treated with agents proven to have a role in the pathogenesis of HE in doses that were based on data from the literature, and we measured the subsequent IL-6 and IL-1ß production by ELISA method. The utilised agents were bacterial lipopolysaccharide, manganese, ammonia and hydrogen-peroxide (H2O2), the latter was used to trigger oxidative stress. The optimal dose of H2O2 used in our experiment was determined by data from the literature and results from our previous studies. During these studies, we managed to determine the H2O2 concentration that could provoke the state of oxidative stress without causing a significant extent of cell death. For this we treated the astrocyte culture with an increasing concentration of H2O2, then we measured the reactive oxygen species (ROS) formed within the cells using an indicator molecule, the 5-6-chloromethyl 2’,7’-dichlorodihydrofluorescein-diacetate (CM-DCFH-DA), and we examined the extent of cell death using a fluorescent staining method (propidium-iodide).

Our results have shown that although the astrocytes are able to produce a detectable amout of IL-1ß and IL-6, the studied factors, which all contribute to the pathogenesis of HE, do not cause a significant increase in IL-1ß and IL-6 production. These controversial findings imply that further examination of the role of astrocyte cells in inflammatory processes is required.