Figure 5
CDDO-Me inhibits intracellular ROS accumulation induced by LPS and fibrillar AÎČ42 in neuronal cultures from E14 rat basal forebrain cholinergic mixed neuron-glia cultures (BFCs) and in dopaminergic cells. (A) Fold-change in intracellular ROS. Integrated optical density was measured from digital images of treated BFCs. Results are expressed as the mean ± S.E.M. of each condition relative to vehicle treatment. Values were analyzed by one-tailed Student's t-test, * denotes significance at p < 0.005. (B) BFCs loaded with the fluorescent indicator DCFDA. Intracellular ROS accumulation is evident after 24-hour treatment with 10 ng/mL LPS + 1 ÎŒM fAÎČ-42, but co-treatment with 100 nM CDDO-Me attenuated neuronal ROS accumulation. Scale bar = 50 ÎŒm. (C) Target-effector ROS accumulation assays of MN9D dopaminergic cells 30 minutes after transfer of conditioned medium (CM) from saline- or LPS-treated (24 hrs) BV2 microglia pre-incubated with CDDO-Me (10 nM) or vehicle (DMSO). (D) Direct treatment of MN9D dopaminergic cells with TNF (5 ng/mL) for 30 minutes after pre-incubation with CDDO-Me (10 nM) or vehicle (DMSO). (C, D) Values shown represent group means ± S.E.M of fold increase in DCFDA positive cell bodies relative to DMSO vehicle, saline treated control conditions and are averages of two independent experiments. Values were analyzed by two way ANOVA followed by Tukey's post hoc test. Groups denoted by different letters are significantly different at p < 0.05.