Fig. 5. Dimethyl thiazol diphenyl tetrazolium bromide (MTT) reduction is decreased by bupivacaine and was partially recovered by recombinant human erythropoietin (rhEPO) pretreatment and cotreatment. ( A ) Time course of bupivacaine-induced MTT reduction. MTT tests were performed, and different bupivacaine concentrations were evaluated (control [•], 0.001 mm bupivacaine [○], 1 mm [▾], 3 mm [Δ], and 5 mm [▪]). Bupivacaine induced time-dependent and concentration-dependent MTT reduction; P = 0.021 with one-way analysis of variance and * P < 0.05 versus control and 0.001 at the same time, † P < 0.05 versus 1 mm at the same time with Student–Newman–Keuls post hoc test. ( B ) Determination of rhEPO pretreatment concentration with 3 mm bupivacaine treatment for a 48-h period. MTT reduction was measured using an MTT test. The rhEPO protective concentration was around 1 U/ml, which was used for the subsequent experiments. ( C ) Both bupivacaine concentration–dependent (3–5 mm) and rhEPO pretreatment duration–dependent (no rhEPO for white bars , 8 h of 1 U/ml rhEPO cotreatment for gray bars , and 24 h of 1 U/ml rhEPO cotreatment for dark gray bars ) effects were evaluated for human skeletal muscle myoblast MTT reduction for a 48-h bupivacaine treatment. P < 0.001 with two-way analysis of variance (α= 0.05) and * P < 0.05 versus control, † P < 0.5 versus bupivacaine at the same concentration; § versus 8 h of rhEPO treatment with bupivacaine at the same concentration, with Student–Newman–Keuls post hoc test. Tests were performed in quadruplicate and repeated six times. Results are reported as mean ± SD.