Fig. 6
The effect of opioid agonists and antagonists and immunoneutralization of inflammatory cytokines TNF-α and IL-6 on the ability of ethanol-or opioid-activated microglial conditioned media to induce apoptosis of POMC neurons. POMC neurons were differentiated from neural stem cells in culture and maintained in T25 flasks (1 × 106/well) for 2 days and then treated for 24 h with microglial conditioned media exposed to opioidergic agents for 24 h before determining neuronal apoptosis using a nucleosome assay. Bar graphs are showing the apoptotic effects of ethanol (50 mM) with or without DAMGO (50 μM), naltrexone (10 ng/ml), or DAMGO and naltrexone (a); ethanol (50 mM) with or without DPDPE (10 nM), naltrindole (50 μM), or DPDPE and naltrindole (b). Microglial conditioned media from ethanol with or without opioidergic agonist-treated cultures were mixed with antibody to TNF-α (1 ng/ml (c)), antibody to IL-6 (0.5 ng/ml (d)), antibody to IL-4 (1 ng/ml (e)), or antibody to IL-13 (5 ng/ml (f)) and added to POMC neuron cultures for 24 h to determine neuronal apoptosis. The effects of immunoneutralization of inflammatory and anti-inflammatory cytokines on ethanol with or without opioid-activated POMC neuronal apoptosis are shown in bar graphs. Each bar represents mean ± SEM of 5–8 samples. Data were compared by one-way analysis of variance (ANOVA) and the Newman-Keuls posttest. Differences between groups are shown by lines with p values on the top of bar graphs