Interestingly it was observed that both IL-1 receptor antagonists

Interestingly it was observed that both IL-1 receptor antagonists and intrathecal administration of IL-1α prevent neuronal apoptosis, while such actions were not seen after IL-1β administration ( Mika, 2008). Both IL-1α and IL-1β act on the IL-1 receptor. The mechanisms behind IL-1β and its receptor are still unclear regarding responses to inflammation and provision of any form of protection. Another substance of interest is the local anaesthetic bupivacaine, which can block neural activity and prevent nerve-injury-induced spinal microglia activation (Wen et al., 2011). In our microglial cultures,

pre-stimulation with bupivacaine Cobimetinib prior to cell activation by LPS did not suppress the TNF-α or IL-1β releases. However, bupivacaine decreases the IL-1β release at ultralow concentration in astrocyte primary cultures (Block et al., in press). As none of the tested substances, which have been shown to have anti-inflammatory Sunitinib cost effects on astrocytes in extremely low concentrations, decreased the cytokine release, we extended the study to include also treatment of microglial with high concentrations of these substances. Naloxone and ouabain both attenuated the increased TNF-α release at high concentration, but showed no ability

to decrease the cytokine release compared with controls. All chemicals were obtained from Sigma-Aldrich (St Louis, MO, USA) if not stated otherwise. The experimental protocols were approved by the Ethical Committee in Gothenburg for Laboratory Animals (Nos. 205-2010). Purified microglial cells were obtained from astroglial-enriched Farnesyltransferase cultures, rat cerebral cortex, grown as previously described by Hansson et al. (1984). Confluent astroglial-enriched cultures, no more than 6 weeks old, were shaken for 30 min at 37 °C and 240 rpm on an orbital shaker in an incubator with a humidified atmosphere of 95% air and 5% CO2. Culture medium, minimum essential medium (MEM), with suspended microglial cells was collected. The microglial cell suspension was plated in six well plates (NUNC), both

with or without glass coverslips, and cells were allowed to adhere for 30 min in the incubator. Together with nonadherent cells, the culture medium was removed from the wells and discarded. Additional medium containing microglia was added. This was repeated until a satisfactory amount (~50 μg total protein per well) of microglia was obtained in each well. The culture medium was then replaced with fresh, pre-warmed (37 °C) supplemented MEM, and microglial cells were kept in the incubator and allowed to rest overnight (Persson et al., 2006). Cells were incubated with lipopolysaccharide (LPS, Escherichia coli O111:B4) (1 ng/ml) for 0.5, 1, 4, 8, or 24 h. Some cells were treated with dexamethasone or corticosterone (10−6 M) for 30 min before they were incubated with LPS in a cocktail in un-supplemented MEM.

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