, 2010). Ouabain, a digitalis-derived glycoside is a well-recognized Na+/K+-ATPase inhibitor, especially pronounced at high concentrations. It also enhances LPS down-regulated iNOS activity in peritoneal macrophages (Sowa and Przewlocki, 1997). Ouabain, PD 332991 at extremely low concentrations, nanomolar and picomolar, stimulates

Na+/K+-ATPase activity (Zhang et al., 2008), and activates complex signalling cascades in kidney cells (Holthouser et al., 2010), and in cardiac and smooth muscle cells (Manunta et al., 2010). Ouabain also decreases the release of IL-1β in astrocytes (Forshammar et al., 2011). Bupivacaine is known to block Na+ channels at high concentrations and change the excitability of action potentials. Clinically, this drug has been used in the treatment of various inflammation-related conditions and diseases (Cassuto et al., 2006), and to treat long-term pain in both cancer and noncancer patients (Deer et al., 2002). Later it has been observed that local anaesthetics possess anti-inflammatory properties through their effects on cells of the immune system. These agents also attenuate the release of the pro-inflammatory cytokines TNF-α and IL-1β from intestinal cells (Lahav et al., 2002 and Bedirli et al., 2011). The purpose with the present study was to investigate if a number of non-steroid anti-inflammatory substances, administered within a wide dose range (10−12 M to 10−6 M) influence microglial release

of pro-inflammatory cytokines. The MAPK inhibitor microglial cells were stained with antibodies against the microglial specific antigen OX42, and with antibodies against TLR4, revealing that these cells do express TLR4 receptors (Fig. 1(A)). Microglia express TLR4 visualised with Western blot (Fig. 1(B)). Cultures were incubated with LPS for 0.5, 1, 4, 8, or 24 h. TLR4 is seen as a band at approximately 70 kDa. Full length TLR4 is observed at approximately 90 kDa, but cleavage fragments have been observed at 30 and 60 kDa (Zager et al., 2007). Since no other bands were present on the membrane the TLR4 antibody was considered specific even though it did not match the full size TLR4. The TLR4 is measured as integrated density and presented

as % of 0 h (Fig. 1(C)). Microglia incubated with LPS for 0.5, 1, 4, 8, or 24 h released TNF-α and IL-1β in accumulating Parvulin amount over time. After 4 h incubation a small release of TNF-α was observed, but it was not significant until 8 h of incubation (P<0.01; n=8), with increasing release after 24 h incubation (P>0.001; n=8) ( Fig. 2(A)). IL-1β release was small after 1, 4, and 8 h, and was significantly accumulated after 24 h ( Fig. 2(B)). Microglia were treated with dexamethasone or corticosterone 30 min before the cells were incubated with a cocktail of LPS and dexamethasone or corticosterone. TNF-α release was attenuated after both dexamethasone and corticosterone treatment (P<0.001; n=9) ( Fig. 3(A)). IL-1β release was attenuated after both dexamethasone and corticosterone treatment (P<0.001; n=9) ( Fig. 3(B)).