Hb metabolite–induced TNFα, IL-1β, and HO-1 mRNA expression in astrocyte cell cultures. mRNA expression of TNFα (A), IL-1β (B) and HO-1 (C) in primary rabbit astrocyte cell cultures exposed to oxyHb, metHb, cyan-Hb, and hemin for four hours at concentrations of 1 μM (white bars), 5 μM (light shaded bars) and 15 μM (dark shaded bars), was determined using real-time PCR, as described in the Methods section. The mRNA expression of TNFα, IL-1β and HO-1 was normalized against GAPDH and is given as fold change. The fold-change values were calculated by normalizing against control samples from untreated cells. Results are from triplicate experiments and presented as mean ± SEM. Differences between the respective exposures and control conditions were analyzed using Mann–Whitney U. * P <0.05, ** <0.01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Hb, hemoglobin; HO-1, heme oxygenase; metHb, methemoglobin; oxyHb, oxyhemoglobin; SEM, standard error of the mean.