Figure 2. Bupivacaine-induced apoptosis in the Schwann cell line, RT4-D6P2T. (A) Hoechst 33258 nuclear staining showed major nuclear alterations in the Schwann cells after the 500-µM bupivacaine treatment (400x). Untreated control cultures (A-a), and the cells exposed to 500 µM bupivacaine for 9 hrs (A-b) and 18 hrs (A-c). With generalized shrinkage, arrows indicate bupivacaine-induced, condensed, coalesced, and segmented nuclei with a brighter blue fluorescence (scale bar = 20 µm). (B) Bupivacaine induced oligonucleosomal DNA fragmentation as shown on 1.8% agarose gel. Lanes from the left: DNA molecular markers, untreated control, and 3-, 6-, and 9-hour incubations after 500 µM bupivacaine treatment, respectively. Typical apoptotic DNA ladders were evident in the 9-hour incubation group. Similar results were obtained from 5 additional separate experiments. (C) In addition, bupivacaine caused the activation of caspase-3 and PARP degradation in a time-dependent manner. Compared with the untreated control, the activation of caspase-3 was observed from 6-hour incubation with 500 µM bupivacaine. The degradation of PARP was obvious from 9 hrs after the exposure to 500 µM bupivacaine. Taken together, these results demonstrated that bupivacaine triggered apoptosis through the involvement of caspase-3 and PARP. The data represent a typical experiment conducted three times with similar results.