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. 2003 Feb 15;23(4):1228–1236. doi: 10.1523/JNEUROSCI.23-04-01228.2003

Fig. 6.

Fig. 6.

Role of ROS in the induction of synergistic neurotoxicity by rotenone and LPS. A, Measurement of release of superoxide and levels of intracellular ROS. To measure superoxide release, rat neuron–glia or neuron-enriched cultures were stimulated with vehicle, 0.5 nm rotenone, and/or 0.5 ng/ml LPS. The release of superoxide was measured by the SOD-inhibitable reduction of cytochrome c. The levels of intracellular ROS were detected with CM-H2-DCFDA. The relative fluorescence intensities in the control cultures were 300–450 and 450–600 arbitrary units per well for N/N andN/G, respectively. The results are expressed as the percentage of control and are the mean ± SEM of two to three experiments performed in triplicate. *p < 0.05 and **p < 0.005 compared with the control cultures, respectively; ++p < 0.05 compared with the cultures treated with either rotenone or LPS alone.B, Measurement of the release of superoxide from microglia. Microglia-enriched cultures were pretreated for 30 min with 5 μm DPI or 0.25 mm apocynin before stimulation with vehicle, 0.5 nm rotenone, and/or 0.5 ng/ml LPS. Superoxide released from microglia was determined by measuring the SOD-inhibitable reduction of cytochrome c. The results are the mean ± SEM of three experiments performed in triplicate. *p < 0.05 compared with the vehicle control;+p < 0.05 compared with the rotenone and LPS-treated cultures. C, Effect of apocynin,l-NAME, or SOD–catalase on the rotenone and LPS-induced synergistic neurotoxicity. Rat neuron–glia cultures were pretreated for 30 min with vehicle, 0.25 mm apocynin, or 1 mml-NAME before treatment with 0.5 nm rotenone and 0.5 ng/ml LPS. SOD–catalase (100 and 150 U/ml, respectively) were added at the same time with rotenone and LPS. Seven days later, neurotoxicity was determined by [3H]DA uptake assay. The results are the mean ± SEM of three experiments performed in triplicate. **p < 0.005 compared with the vehicle control;+p < 0.05 compared with the rotenone and LPS-treated cultures. N/N, Neuron-enriched cultures;N/G, neuron–glia cultures; C, control;R, rotenone; L, LPS; D, DPI; A, apocynin; S/C, SOD–catalase;L-N, l-NAME.