Fig. 2.

Both caspase and calpain activity contribute toward hydrogen peroxide (H₂O₂)-induced toxicity in 1-week-old cultures, whereas toxicity in 3-week-old cultures is mediated through calpain. (a) MAP2 cell-based ELISA was performed in 1- or 3-week-old rat cortical cultures in response to H₂O₂. In 1-week-old rat cortical cultures, H₂O₂ induced a dose-dependent decrease in the fluorescence signal strength in MAP2 cell-based ELISA, and the decrease was blocked by the calpain inhibitor, MDL28170, and the caspase inhibitor, OPH. In 3-week-old rat cortical cultures, H₂O₂ induced a dose-dependent decrease in the fluorescence signal strength in MAP2 cell-based ELISA, and the decrease was blocked by MDL28170, but not by OPH (***P < 0.001, compared with veh; ^###P < 0.001, compared with cells treated with 300 μM H₂O₂). (b) Neuronal survival as determined by counts of MAP2-positive cells in 1- or 3-week-old rat cortical cultures in response to H₂O₂. Data represent the average ±SD counts of multiple wells (n = 6; ***P < 0.001, compared with veh; ^#P < 0.05, ^###P < 0.001, compared with cells treated with 300 μM H₂O₂). (c) One- or 3-week-old rat cortical cultures were treated with different concentrations of H₂O₂ and the expression levels of calpain-cleaved spectrin, caspase-3, and cleaved caspase-3 were determined (n = 3). The numerical values specified beneath the respective bands of western blots represent the fold change in protein expression as compared with that of the control (1.0). Each sample was subjected to SDS-PAGE and stained with coomassie blue to show equal loading of samples. ELISA, enzyme-linked immunosorbent; MAP2, microtubule-associate protein 2; NMDA, N-methyl-d-aspartate; PAGE, polyacrylamide gel electrophoresis; veh, vehicle.