Effect of bupivacaine on cell death, ROS generation and p38 MAPK activation. Neuro2a cells were treated with 2 mM bupivacaine for the time indicated. (A) Cell viability was measured with the MTT assay. (B) Caspase-3 activity was measured with a fluorometric substrate. (C) DNA extracted from the cells was analyzed by agarose gel electrophoresis. (D) Intracellular levels of ROS were measured with a fluorogenic probe. (E) Expression of p38 and phospho-p38 (P-p38) following cell exposure to bupivacaine (2 mM) or H2O2 (0.5 mM) was measured by Western blotting. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. control (not treated with bupivacaine or H2O2, n = 6).