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Bupivacaine Induces Apoptosis via ROS in the Schwann Cell Line

¹ Department of Dental Anesthesiology and Dental Research Institute, Seoul National University College of Dentistry, 28 Yongon-dong Chongno-gu, Seoul 110-744, Korea;
² Department of Anesthesiology, Seoul National University College of Medicine, Seoul, Korea; and
³ Department of Craniomaxillofacial Structure and Functional Biology, Seoul National University College of Dentistry, Seoul, Korea;

View larger version (54K): [in a new window]   Figure 1. Analysis of cytotoxicity of local anesthetics in the Schwann cell line, RT4-D6P2T. (A) Among cell samples treated with procaine, lidocaine, mepivacaine, ropivacaine, bupivacaine, and levobupivacaine for 18 hrs, bupivacaine and levobupivacaine induced Schwann cell death at a concentration of 1 mM, compared with untreated control. However, the cytotoxicity of bupivacaine was more obvious than that of levobupivacaine. (B) Dose-responsiveness was observed during the 18-hour bupivacaine treatment. The 50% lethal dose (LD50) was calculated statistically as 476 µM. (C) The time-course of cell death was obtained after the treatment with 500 µM bupivacaine. Therefore, bupivacaine-induced cell death occurred in a dose- and time-dependent manner. (D) The cell morphology was examined by phase-contrast microscopy (100x) in the untreated control (D-a) and in the presence of 500 µM bupivacaine for 6-hour (D-b), 9-hour (D-c), and 18-hour (D-d) incubation. After 9 hrs, immortalized Schwann cells were transformed to round and shrunken shapes. In some Schwann cells, the disappearance of dendrites was observed. Almost all Schwann cells lost their cellular integrity or were detached from the bottom of the plates after 24 hrs (scale bar = 100 µm). The diagrams show the results of 5 independent experiments. Values are mean ± SD. *p < 0.05, **p < 0.01 statistically different from the control.

View larger version (47K): [in a new window]   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.

View larger version (23K): [in a new window]   Figure 3. ROS involvement in bupivacaine-induced Schwann cell apoptosis. (A) After 500 µM bupivacaine treatment, the levels of intracellular ROS, measured by DCF fluorescence with flow cytometry, were increased and maintained in Schwann cells from 2 to 4 hrs. Control was overlaid (thick line), and numerals indicate increased mean value of DCF fluorescence intensity of treated cells. Direct inhibition of intracellular ROS production by simultaneous treatment of 5 mM NAC and 0.1 mM Trolox with 500 µM bupivacaine is shown in (B). (C) No significant change of NO level, another potent signaling molecule in apoptosis, was found in the early period of bupivacaine-induced Schwann cell death. The diagrams show the results of 5 independent experiments. Values are mean ± SD. *p < 0.05 statistically different from the 500 µM bupivacaine treatment group (B) or the untreated control (C).

View larger version (31K): [in a new window]   Figure 4. The inhibition of bupivacaine-induced Schwann cell apoptosis by anti-oxidant treatment. (A) Treatment with 5 mM NAC and 0.1 mM Trolox, which inhibited the production of intracellular ROS, significantly reversed the Schwann cell death induced by 500 µM bupivacaine treatment, as determined by MTT assay. (B) The blocking effects of the anti-oxidants on caspase-3 and PARP expressions were detected by Western blotting at 9-hour incubation with 500 µM bupivacaine, and these data represent a typical experiment conducted three times with similar results. (–), untreated; (+), treated. The diagrams show the results of 5 independent experiments. Values are mean ± SD. *p < 0.05 statistically different from 500 µM bupivacaine treatment group.