Microglia were treated with ultralow (10−12 M) or high (10−6 M)

concentrations of naloxone, ouabain, or bupivacaine, 30 min before the cells were incubated with a cocktail of LPS and naloxone, ouabain, or bupivacaine for 24 h, respectively. Naloxone, ouabain, or bupivacaine were not able to attenuate the TNF-α release after LPS incubation (n=9). Instead naloxone and ouabain at ultralow concentration increased the TNF-α release ( Fig. 3(A)). None of the different substances were able to decrease the IL-1β release (n=9) ( Fig. 3(B)). The selection of choosing one ultralow and one high concentration of the anti-inflammatory substances are due to results obtained from concentration curves, and results obtained from astrocytes. LPS-induced TNF-α release from microglia after

stimulation with bupivacaine, www.selleckchem.com/products/blz945.html 10−18–10−3 M, shows that bupivacaine was not able to decrease the TNF-α release after find more LPS incubation, except at 10−3 M, where the cells died (Fig. 4). The other concentration curves for naloxone and ouabain showed similar results, (not shown). LPS-induced IL-1β release from astrocytes after naloxone and ouabain stimulation with different concentrations has earlier been published by our group (Forshammar et al., 2011), as well as with bupivacaine stimulation (Block et al., in press). The TNF-α release is very small in our astrocytes (Andersson et al., 2005). After nerve Ureohydrolase injury a course of events takes place where the microglial

receptor TLR4 has been implicated (Tanga et al., 2005). Signals from the surrounding milieu trigger microglial activation through this receptor, where after the cells will be activated and release pro-inflammatory cytokines. Activation of TLR4 by the inflammatory stimulus LPS (Neher et al., 2011) results in increased expression of TNF-α in microglia (Zhou et al., 2010). In our microglial cell model we see increases of both TNF-α and IL-1β after 8 h and 24 h, respectively of LPS incubation. The cells express TLR4, even at a high level before they were stimulated with LPS, which can be due to a high TLR4 protein content already at time point zero. TNF-α is released in response to inflammation or other types of insult where it can act protective to neurons (Fontaine et al., 2002), and astrocytes (Kuno et al., 2006) because it is able to encourage the expression of anti-apoptopic and anti-oxidative proteins and peptides. It has also been demonstrated that microglia protect neurons against ischaemia through the synthesis of TNF-α (Lambertsen et al., 2009). As we demonstrate, inflammatory activated microglial cells are stimulated by signals, which activate TLR4 and the cells change their release of pro-inflammatory cytokines. One tentative target to restore these processes would be to inhibit the inflammation activating cellular changes and to decrease the pro-inflammatory cytokine release.