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RESEARCH REPORT |
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;
* corresponding author, dentane{at}snu.ac.kr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: apoptosis • bupivacaine • neurotoxicity • reactive oxygen species • Schwann cell
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Schwann cells, the important component of the peripheral nervous system, ensheath the axon and play an important part in axonal growth and regeneration, myelinization, and normal electrophysiological conductivity. Therefore, damage to Schwann cells is likely to have a direct effect on the conductivity of the axon and cause pathologic changes. Some pathologic conditions, such as diabetic peripheral neuropathy, have been associated with degenerative changes in Schwann cells (Zhu et al., 2002). The Schwann cell line, RT4-D6P2T, is a well-characterized subclone of a glial cell line, initially established from an ethylnitrosourea-induced rat peripheral neurotumor (Imada and Sueoka, 1978). This cell line was demonstrated to exhibit several morphological and functional properties of Schwann cells in a regulated manner.
Increasing numbers of studies have found that neurotoxicity of local anesthetics occurs through apoptosis (Tan et al., 2002; Johnson et al., 2004). Therefore, we hypothesized that local anesthetics cause neural complications through Schwann cell apoptosis, and investigated the apoptotic effects of commonly used local anesthetics—i.e., procaine, lidocaine, mepivacaine, ropivacaine, bupivacaine, and levobupivacaine—for peripheral neural blocks, in the Schwann cell line RT4-D6P2T.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Cytotoxicity Assay [Methyl Tetrazolium (MTT) Assay]
The cells were seeded into 96-well plates at a concentration of 2 X 104 cells/well. For the cytotoxicity assay, after exposure to local anesthetics, DMEM containing 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (Sigma) was changed in each well of the plates. Following incubation for 2 hrs at 37°C, the medium was removed, and 100 µL of DMSO (Sigma) was added. The absorbance at 570 nm was measured spectrophotometrically (Bio-Tek, Winooski, VT, USA), and the results were expressed as a percentage of the untreated control (% of control).
Apoptosis Experiments
Hoechst 33258 Nuclear Staining
Staining with the fluorescent dye, Hoechst 33258, allows for the discrimination of apoptotic cells on the basis of nuclear morphology and evaluation of membrane integrity. Hoechst 33258 (Sigma) was added to a final concentration of 5 µg/mL, and the cells were kept at 37°C for 30 min. Then the cells were visualized under a fluorescence microscope (BX60; Olympus, Tokyo, Japan) with UV excitation at 300–500 nm.
DNA Fragmentation on Agarose Gels
The formation of oligonucleosome-sized fragments of multiples of ~ 200 bp, producing typical DNA ladders on agarose gels, is the characteristic biochemical hallmark of apoptosis. Treated cells were trypsinized and subjected to lysis in 500 µL lysis buffer (100 mM NaCl, 10 mM Tris [pH 8.0], 25 mM EDTA, and 0.5% SDS). Cell lysates were incubated with 200 µg/mL DNase-free proteinase K (Sigma) for 1 hr at 42°C. Following phenol/chloroform extraction, the DNA was precipitated with isopropyl alcohol and 3 M sodium acetate. The precipitated DNA was suspended in phosphate-buffered saline (PBS; Gibco) and was incubated with DNase-free RNase (Sigma) for 30 min at 37°C. Following final ethanol precipitation, a 10-µg quantity of DNA, derived from each treatment, was fractionated on 1.8% agarose gel, and the DNA was viewed after the gel was stained with ethidium bromide.
Reactive Oxygen Species (ROS) Measurement
We determined intracellular ROS levels by staining cells with 2', 7'-dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes, Eugene, OR, USA), which was oxidized to highly fluorescent dichlorofluorescein (DCF) by ROS. The cells were loaded with 20 µM H2DCFDA at 37°C during the last 30 min of local anesthetic treatment. The cells were trypsinized and centrifuged at 300 g for 5 min. The pellet was washed, re-suspended in PBS, and analyzed by flow cytometry (FACSort analyzer; Becton Dickinson, San Jose, CA, USA) with a 488-nm laser line for excitation, and the data were processed with Cell Quest software (Becton Dickinson).
For anti-oxidant treatments, we confirmed the non-toxic effects of anti-oxidants at various doses of N-acetyl cysteine (NAC; Calbiochem, San Diego, CA, USA) and Trolox (Calbiochem) by MTT assay. When exposed to local anesthetics, the cells were treated with each anti-oxidant simultaneously. After incubation, the intracellular ROS level and cell viability were measured as described.
Nitric Oxide (NO) Measurement
We identified the content of NO by determining the total concentration of nitrites by the Griess method. Briefly, after incubation with the local anesthetic, a 50-µL quantity of the cell supernatant was prepared and colormetrically analyzed according to the instructions provided with the total NO detection kit (R&D Systems, Minneapolis, MN, USA) to measure NO release.
Western Blot Analysis
We prepared cell lysates by extracting proteins with lysis buffer [40 mM Tris-HCl (pH 8.0), 120 mM NaCl, 0.1% triton X-100, 1 mM EDTA, 0.02% sodium azide], supplemented with protease inhibitors. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane. The membrane was blocked with 5% non-fat dried milk in Tris-buffered saline and then incubated with a 1:1000 dilution of primary antibodies [caspase-3, poly ADP-ribose polymerase (PARP), and actin; Santa Cruz, CA, USA] for 1 hr at room temperature. Blots were developed by a 1:3000 diluted secondary antibody of HRP-linked anti-rabbit or anti-mouse IgG (Cell Signaling, Beverly, MA, USA), and proteins were visualized by the use of the Amersham ECL system (Amersham, Buckinghamshire, UK). The protein blot was treated with stripping buffer (2% SDS, 62.5 mM Tris-HCl, 100 mM mercaptoethanol) and used again with the other antibodies for the detection of additional proteins.
Statistical Analysis
The data from the MTT assay were expressed as means ± SD in terms of percentage. Statistical analysis was performed by ANOVA (Tukey’s test and Dunnett’s multiple comparisons test), and p-values < 0.05 were considered significant. The dose-response data of bupivacaine were fitted to a logistic equation, yielding 50% lethal dose (LD50) and slopes.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). Moreover, the bupivacaine-induced cell death occurred in a dose- and time-dependent manner (Figs. 1B, 1C), and LD50 was statistically calculated to be approximately 476 µM.
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). Up to 3 hrs, the 500-µM-bupivacaine-treated Schwann cells showed no abnormal findings (Fig. 1D-b). However, after 9 hrs, they appeared to have condensed nuclei and were transformed to round and shrunken shapes, with the disappearance of dendrites noted in some Schwann cells (Fig. 1D-c). After 18 hrs, almost all Schwann cells lost their cellular integrity (Fig. 1D-d). For reference, the doubling time of the RT4-D6P2T was assessed as about 20 to 24 hrs (Imada and Sueoka, 1978). These observations were generally consistent with the data from the MTT assay.
Apoptosis Experiments
At both 6-hour and 9-hour incubations with 500 µM bupivacaine, Hoechst 33258 nuclear staining showed the nuclear alterations indicative of apoptosis—condensed, coalesced, and segmented nuclei with a brighter blue fluorescence (Fig. 2A). A DNA fragmentation pattern, one of the typical signs of late apoptosis, was definite at the 9-hour incubation with 500 µM bupivacaine (Fig. 2B). Bupivacaine caused the activation of caspase-3 and PARP degradation (Fig. 2C). The activation of caspase-3, reported as an "apoptotic effector signal" (Thorburn, 2004), was observed from 6-hour incubation with 500 µM bupivacaine. Also, the degradation of PARP, which appears to be involved in DNA repair and genome surveillance and is used as another hallmark of apoptosis (Decker and Muller, 2002), was evident from 9 hrs after bupivacaine treatment.
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). In addition, the anti-oxidants, 5 mM NAC and 0.1 mM Trolox, directly inhibited ROS production (Fig. 3B). We previously confirmed that the inhibitory effects of 5 mM NAC and 0.1 mM Trolox on ROS production were the most prominent. In contrast to the increase of ROS, no significant change of NO level was observed up to 6 hrs (Fig. 3C).
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). At the 9-hour incubation with 500 µM bupivacaine, the activation of caspase-3 and PARP degradation, which strongly suggested that apoptosis was proceeding, was also blocked by anti-oxidants in Western blot analysis (Fig. 4B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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While procaine, lidocaine, mepivacaine, and ropivacaine did not affect the cell viability at doses within 1 mM, bupivacaine induced Schwann cell death in a dose- and time-dependent manner (LD50 was approximately 476 µM). Considering that common commercial dental cartridges of bupivacaine contain 0.5% (approximately 14.6 mM), bupivacaine might cause Schwann cell death even at a subclinical concentration. Moreover, in peripheral neural blocks, such as infiltration and block anesthesia, a much higher level of local anesthetics is predicted at the targeted nerve, compared with epidural and spinal anesthesia. There are many clinical reports concerning the cytotoxic effects of bupivacaine (Sadeh et al., 1985; Bergman et al., 2003; Groban, 2003). In addition, bupivacaine was found to be the most neuronal-membrane-toxic among lidocaine, ropivacaine, and bupivacaine (Pardo et al., 2002). These findings could be explained in parallel with the properties of high anesthetic potency of bupivacaine, e.g., the ability to inhibit Na+ channels (Nau et al., 1999). It is further interesting that levobupivacaine, an isomer of bupivacaine, was also cytotoxic to RT4-D6P2T, the peripheral glial cell line, as it was to the neuronal cell line (Tan et al., 2002). In contrast to the general agreement on the cytotoxic effects of bupivacaine, the neurotoxicity of levobupivacaine is controversial (Vladimirov et al., 2000).
In this study, we used an established Schwann cell line, RT4-D6P2T, to verify the direct cytotoxicity of local anesthetics on isolated Schwann cells. In particular, RT4-D6P2T was reported to express endogenous proteins for myelinization at levels that were equal to those of primary Schwann cell cultures (Hai et al., 2002).
After in vivo perineural injections of local anesthetics, Schwann cells underwent distinctive pathological changes (Powell et al., 1988). Within clinical concentrations, histopathologic and functional changes of Schwann cells, whether myelinated or not, were observed (Kalichman et al., 1989). With 0.5% bupivacaine, marked disruption and vacuolization of myelin sheaths were noted after either a twice daily block or a 3-hour infusion in the rat sciatic nerve (Kytta et al., 1986). Functional recovery, measured by compound action potential, was not complete at 3 wks. Thus, it is evident that damage to Schwann cells could result in conduction block, and that less severe injury to the myelin sheath or Schwann cell may cause slowing of conduction (Kalichman, 1993).
Apoptosis is an essential mechanism for cell integrity in development and survival. However, it is also triggered by non-physiologic stimulation and can lead to pathologic conditions. In the Schwann cell line, no significantly altered level of NO, reported to be related to apoptotic or inflammatory processes in Schwann cells (Conti et al., 1999), was observed, while the ROS level was elevated in the early period of bupivacaine-induced apoptosis. Our findings indicate that bupivacaine could be an apoptotic trigger in Schwann cells via ROS generation.
Oxidative stress, characterized by overwhelming ROS, is indispensable for the development and progression of peripheral neuropathy, because of the high content of phospholipids and relatively insufficient free-radical defense of peripheral nerves (Stevens et al., 2000). In neuronal cells, ROS results in membrane lipid peroxidation, nitration of proteins, and degradation of DNA, all of which are associated with the course of apoptosis (Fiskum, 2004). In addition, a study on diabetic neuropathy supports our results, in that oxidative stress induced Schwann cell apoptosis, and targeted therapies aimed at generating ROS might prove effective (Vincent et al., 2002). In this study, the block of ROS production by anti-oxidants inhibited the activation of caspase-3 and PARP degradation, and reversed bupivacaine-induced cell death. Accumulating evidence has formed the basis for our belief that the production of ROS triggered the release of cytochrome c from mitochondria, intracellular Ca2+ elevation, and caspase-3 activation, all of which lead to apoptosis in neuronal cells (Annunziato et al., 2003).
In conclusion, this study with a Schwann cell line demonstrated that bupivacaine could induce apoptosis, which is mediated not by NO but by an increase of intracellular ROS, and which proceeds through the activation of caspase-3 and PARP degradation. These results strongly suggest that the cytotoxicity of bupivacaine on Schwann cells may be associated with potential neurological complications after peripheral neural blocks. However, because these are in vitro experiments, with an immortalized cell line and theoretical stimulation, we are unable to simply extrapolate our ex vivo data to in vivo animal experiments or to human research. Further in vivo studies are required to assess ROS involvement in bupivacaine-treated native peripheral nerves.
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ACKNOWLEDGMENTS |
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Received February 17, 2004; Last revision June 8, 2005; Accepted June 18, 2005
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REFERENCES |
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