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Comparison of toxicity effects of ropivacaine, bupivacaine, and lidocaine on rabbit intervertebral disc cells in vitro
The Spine Journal 14 (2014) 483–490
Basic Science
Comparison of toxicity effects of ropivacaine, bupivacaine, and lidocaine on rabbit intervertebral disc cells in vitro Xian-Yi Cai, MDa, Li-Ming Xiong, MD, PhDa, Shu-Hua Yang, MD, PhDa,*, Zeng-Wu Shao, MD, PhDa, Mao Xie, MD, PhDa, Fei Gao, MD, PhDa, Fan Ding, MD, PhDb a
Department of Orthopaedic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 430022, Wuhan, China b Department of Orthopaedic Surgery, The First People’s Hospital of Jingmen, 448000, Jingmen, China Received 29 August 2012; revised 27 March 2013; accepted 15 June 2013
Abstract
BACKGROUND CONTEXT: It has been shown that bupivacaine, the most commonly used local anesthetic to relieve or control pain in interventional spine procedures, is cytotoxic to intervertebral disc (IVD) cells in vitro. However, some other common local anesthetics, such as ropivacaine and lidocaine, are also frequently used in the treatment of spine-related pain, and the potential effects of these agents remain unclear. PURPOSE: The purpose of this study was to evaluate the effect of various local anesthetics on rabbit IVD cells in vitro and further compare the cytotoxicity of ropivacaine, bupivacaine, lidocaine, and saline solution control. STUDY DESIGN: Controlled laboratory study. SUBJECTS: Rabbit annulus fibrosus (AF) and nucleus pulposus (NP) cells were isolated from Japanese white rabbits. METHODS: Both AF and NP cells at the second generation maintained in monolayer were exposed to various concentrations of local anesthetics (eg, bupivacaine) or different durations of exposure and evaluated for cell viability by use of cell counting kit-8 (CCK-8). In addition, to compare the cytotoxicity of ropivacaine, bupivacaine, lidocaine, and saline solution control in commercial concentration, the viability was analyzed by flow cytometry after 60-minute exposure, and the morphologic changes were observed by the phase-contrast microscopy. Apoptosis and necrosis of IVD cells were confirmed by using fluorescence microscopy with double staining of Hoechst 33342 and propidium iodide. RESULTS: Rabbit IVD cell death demonstrated a time and dose dependence in response to bupivacaine and lidocaine. However, ropivacaine only exerted a significant time-dependent effect on IVD cells. There was no significant difference in IVD viability after treatment with different doses of ropivacaine. In addition, the results showed that lidocaine was the most toxic of the three local anesthetics and that ropivacaine presented less cytotoxicity than lidocaine and bupivacaine. Fluorescence microscopy also confirmed that the short-term toxic effect of local anesthetics on both AF and NP cells was mainly caused by necrosis rather than apoptosis. CONCLUSIONS: Results show that bupivacaine and lidocaine decrease cell viability in rabbit IVD cells in a dose- and time-dependent manner. All local anesthetics should be avoided if at all possible. Ropivacaine may be a choice if necessary, but it is also toxic. The increase in cell death is more related with cell necrosis rather than cell apoptosis. If these results can be corroborated in tissue explant models or animal studies, caution regarding diagnosing, treating, and controlling spine-related pain with local anesthetics is prompted. Ó 2014 Elsevier Inc. All rights reserved.
Keywords:
Intervertebral disc; Bupivacaine; Lidocaine; Ropivacaine; Necrosis
FDA device/drug status: Not applicable. Author disclosures: X-YC: Nothing to disclose. L-MX: Nothing to disclose. S-HY: Nothing to disclose. Z-WS: Nothing to disclose. MX: Nothing to disclose. FG: Nothing to disclose. FD: Nothing to disclose. X-YC and L-MX contributed equally to this work. 1529-9430/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.spinee.2013.06.041
* Corresponding author. Department of Orthopaedic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 430022, Wuhan, China. Tel.: þ(86)027-85351627. E-mail address: [email protected] (S.-H. Yang)
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Introduction Low back pain is the most common cause of limited activity in people younger than 45 years and the second most frequent reason for visits to the physician [1]. Because of its advantages of minimal invasion and simplicity, injection therapy is adopted by many patients who are unwilling to undergo surgery. Indeed, the use of local anesthetic injection as a tool in diagnosing and treating spinal pain has sharply increased from 1994 to 2001 [2]. Currently, bupivacaine is frequently used in interventional spine procedures and is often administered intraoperatively and postoperatively to control the pain by local, epidural, or spinal injection [3,4]. Extensive use of bupivacaine in a myriad of diagnostic procedures and pain management has been largely based on the assumption that it is safe. Recently, the effects of bupivacaine on intervertebral disc (IVD) cell viability were investigated, and four studies have suggested that bupivacaine may be toxic to IVD cells. Lee et al. [5] reported that bupivacaine could decrease viability of rabbit and human disc cells at a time and dose dependence. Similarly, application of bupivacaine in clinically relevant concentrations such as 0.25% or 0.5% was also toxic to human IVD cells [6]. Specifically, in vitro exposure of organotypic cultures of the murine functional spine units to bupivacaine solution also dramatically decreased cell viability and matrix protein synthesis in a doseand time-dependent manner [7]. Moon et al. [8] recently confirmed that bupivacaine has dose- and time-dependent cytotoxic effects on human nucleus pulposus (NP) cells. Although aforementioned studies have shown cytotoxic effects of bupivacaine on IVD cells, it was still unclear whether this was specific to bupivacaine or could be seen with other local anesthetics. Furthermore, because both disc cell senescence and cell loss have been implicated in the development of IVD degeneration [9], an alternative, less cytotoxic local anesthetic could then be considered for diagnosis and treatment of low back pain. A variety of other local anesthetics, such as ropivacaine and lidocaine, are also used, both alone and in combination, in diagnosing and treating spinal pain with little information regarding potential effects of these agents on IVD cells [10]. A promising alternative to bupivacaine for spinerelated pain relief is ropivacaine. Ropivacaine is a longacting aminoamide member of the pipecoloxylidide group of local anesthetics that differs from bupivacaine only by the replacement of the butyl group on the piperidine nitrogen atom of the molecule with a propyl group [11]. As a result, ropivacaine is less lipid-soluble than bupivacaine, and then together with its stereoselective properties, contributes to the property that has fewer cardiotoxicity and neurotoxicity than bupivacaine [12–14]. At the same time, lidocaine is another commonly used agent for interventional spinal procedures [15]. Lidocaine and bupivacaine are both members of the amide group in the local anesthetic family, but the action duration of lidocaine is
approximately one-half that of bupivacaine [16]. It was shown that only 0.5% lidocaine has been effective in cervical epidural injections for managing chronic neck pain with disc herniation [17]. More importantly, recent studies also demonstrated that there are a number of detrimental effects of ropivacaine and lidocaine on cartilage and chondrocytes [18,19]. Lo et al. [20] observed that bupivacaine, ropivacaine, and lidocaine have a negative effect in a dose- and duration-dependent manner on chondrocyte viability. Park et al. [21] reported that bupivacaine and lidocaine have toxic effects on equine chondrocytes, and bupivacaine is the most toxic of the three local anesthetics in commercial concentration (ie, 2% lidocaine, 2% mepivacaine, and 0.5% bupivacaine). However, the results of Grishko et al. [22] suggested that 2% lidocaine may be the more toxic than 0.5% ropivacaine and 0.5% bupivacaine. In light of these observations, it would be prudent to examine the effects of three common local anesthetics on IVD cell viability, a subpopulation of which are chondrocytic in nature. It is also important to determine which local anesthetic may have the lowest cytotoxic effects on IVD cells, and then it could be a safer alternative drug than the others. This study aimed to determine whether short-term exposures to bupivacaine, lidocaine, and ropivacaine decreased directly the viability of both rabbit annulus fibrosus (AF) and NP cells in a time- and dose-dependent manner in vitro and, furthermore, compare the toxic effects of three common local anesthetics with equipotent doses on rabbit IVD cells. Materials and methods As there is limited availability of normal nondegenerated human disc tissue, the normal and healthy IVD cells isolated from adult Japanese white rabbits were used in this study. Isolation and culture of primary IVD cells Experimental studies were conducted under the protocol approved by the animal experimentation committee of Huazhong University of Science and Technology. Annulus fibrosus and NP were surgically dissected from the thoracolumbar spine (L5–T10) of skeletally mature Japanese white rabbits (3 months, male) immediately after killing by air embolism. Cells were isolated as described previously [23,24]. Briefly, tissue from each level was collected, minced, and followed by enzymatic digestion. The resulting cells were seeded in six-well culture plates in Dulbecco’s modified Eagle’s medium/ham’s F-12 (DMEM/F-12; Gibco, USA) containing 10% (for AF cells) or 20% (for NP cells) fetal bovine serum (Gibco, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco, Grand Island, NY, USA) at 37 C, 5% CO2 atmosphere. Culture medium was changed every other day. After about
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one or two weeks, cells were passaged by 0.25% trypsinization (Amresco, Solon, OH, USA) when they reached confluence of 80% to 90%. The IVD cells at the second generation maintained in a monolayer were used throughout the following experiments. Experimental groups To examine the time-dependent effect of different local anesthetics, both rabbit AF and NP cells were divided into four treatment groups, and each group was exposed to one of the following experimental agents: 0.9% normal saline solution; 0.5% ropivacaine (Ropivacaine HCl; AstraZeneca AB, Sweden); 0.5% bupivacaine (Bupivacaine HCl; Zhaohui Pharm, China); and 2% lidocaine (Lidocaine HCl; Zhaohui Pharm, China) for 30, 60, 90, and 120 minutes. To determine the dose-dependent effect, both AF and NP cells were cultured in ropivacaine (0.5%, 0.25%, 0.125%), bupivacaine (0.5%, 0.25%, 0.125%), lidocaine (2%, 1%, 0.5%), and 0.9% saline as a control for 60 minutes. The concentrations used were those of the commercial local anesthetic solution, and their twofold serially diluted solutions were prepared with normal saline. Cell viability tests CCK-8 assay At designated time points, cell viability was determined using cell counting kit-8 (CCK-8; Beyotime, China) colorimetric assay in six independent experiments. Rabbit IVD cells were seeded in 96-well culture plates at a density of 1104 cells per well and incubated for 48 hours in a humidified 5% CO2 incubator at 37 C. The growth medium was then removed from each well, the wells washed once with phosphate-buffered saline (PBS) and 100 mL local anesthetic added to each well. After exposure, local anesthetics were removed by inspiration, and all the wells filled with 100 mL of DMEM/F12 and 10 mL CCK-8 solution. After incubated for 4 hours at 37 C according to the manufacturer’s instructions, cell proliferation was assessed by absorbance detection at A450 nm with a microplate reader (Biotek, Winooski, VT, USA). Flow cytometry At designated time points, the IVD cells from each treatment group were labeled using Annexin V/propidium iodide (PI) (KeyGen Biotech, China) double staining according to the manufacturer’s instructions as described previously. Briefly, after release from culture conditions by trypsinization and centrifugation, cells were washed with cold PBS twice and resuspended in 500 mL binding buffer. To each suspension, 5 mL Annexin V-FITC (fluoresceinconjugated Annexin V) and 5 mL PI were added to stain the cells. Cells were incubated in the dark for 15 minutes at room temperature. Samples were applied to flow cytometry (BD LSR II, Becton Dickinson) using FlowJo V 7.6.1 software (Tree Star, Olten, Switzerland). Propidium
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iodide–positive and Annexin V–positive/negative staining represent necrotic cells, PI-negative and Annexin V–positive staining apoptotic cells, and both PI- and Annexin V–negative staining live cells, respectively. Fluorescence microscopy Samples for fluorescence microscopy were stained with Hoechst 33342 and PI to distinguish apoptotic and necrotic cells. Propidium iodide can only enter the affected plasma membrane just as trypan blue dye can. The Hoechst 33342 dye can display the remaining cells, which are not stained with PI, and the morphologic changes of apoptosis can be distinguished from the normal nucleus from this staining [21]. At designated time points, morphological changes of the cells were examined using an Apoptosis and Necrosis Assay Kit (Beyotime, China). Briefly, after treatment, cells in six-well plates were cultured in 1 mL binding buffer, 5 mL Hoechst 33342, and 5 mL PI added and further incubated in the dark for 20 minutes at 4 C. After staining, the cells were washed once with PBS (pH 7.4), and the stained nuclei were observed using a fluorescence microscope (Olympus IX71) with ultraviolet filter (excitation spectrum: 350 nm, emission wavelength: 460 nm). Both red (PI) and blue (Hoechst) colors could be visualized through this filter; however, red nuclei were recounted more precisely using green filter (excitation spectrum: 460–550 nm, emission wavelength: 590 nm). Statistical analysis Experimental values were obtained from at least three independent experiments. The results are represented as means6standard deviation. Statistical analyses were performed by using the Student t test or one or two-way analysis of variance (GraphPad Prism; GraphPad Software Inc., La Jolla, CA, USA), where appropriate. The Bonferroni post hoc test was used to determine the source of observed differences. Significance was set at p!.05. Results Evaluation of time- and dose-dependent effects of local anesthetics on the viability of rabbit IVD cells To test the effects of three common local anesthetics on both AF and NP cell viability, cells grown in monolayer culture were exposed to local anesthetics at different concentrations for various periods of time, and cell viability was measured using CCK-8 assay. For cytotoxicity, results from untreated cells were set to 100%. Values less than 100% represented cell death caused by local anesthetics. The results of AF and NP cells treated with various doses of ropivacaine, bupivacaine, and lidocaine were summarized in Fig. 1. As compared with the IVD cells treated with normal saline, those treated with local anesthetics all showed significant reduction in viability (all p!.05) except 0.125% and 0.25% ropivacaine (not significant). Assessment of the effect of individual local anesthetics showed a statistically
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Fig. 1. CCK-8 assay dose-course. To determine dose-dependent effects of the local anesthetics, CCK-8 assay was carried out after 60-minute exposure to various concentrations of local anesthetics and the group of 0.9% saline served as control. Cell viability was significantly reduced in a dose-dependent manner, except for ropivacaine. Data are expressed as mean6SD from four independent experiments (*p!.05 vs. control; oneway ANOVA/Bonferroni). AF, annulus fibrosus; NP, nucleus pulposus; SD, standard deviation; ANOVA, analysis of variance.
significant decrease in cell viability with increasing concentration of bupivacaine (p!.05) and lidocaine (p!.05), except ropivacaine (not significant). The results from both AF and NP cells treated with 0.5% ropivacaine, 0.5% bupivacaine, and 2% lidocaine at different time points were summarized in Fig. 2, Top and Bottom, respectively. There was statistically significant difference in the percentage of cell death among different time points for all the local anesthetics (all p!.05). Comparison of ropivacaine, bupivacaine, and lidocaine toxicity on rabbit IVD cells To compare the toxic effects of 0.5% ropivacaine, 0.5% bupivacaine, and 2% lidocaine on IVD cells, cell viability was measured using flow cytometry after treatment for 1 hour with local anesthetics (Fig. 3, Top and Middle). Cell viability was decreased to 67.15%, 53.26%, and 39.12%,
Fig. 2. CCK-8 assay time-course. To elucidate time-dependent effects of the local anesthetics, CCK-8 assay was carried out after 30, 60, 90, and 120 minutes and the group of 0.9% saline served as control, respectively. Cell viability was significantly reduced in a time-dependent manner. Data are expressed as mean6SD from four independent experiments (*p!.05 vs. control; two-way ANOVA/Bonferroni). AF, annulus fibrosus; NP, nucleus pulposus; SD, standard deviation; ANOVA, analysis of variance.
respectively, after 1 hour exposure of rabbit AF cells to 0.5% ropivacaine, 0.5% bupivacaine, and 2% lidocaine as compared with 0.9% saline (78.64%) and that of NP cells was 51.09% (ropivacaine), 42.44% (bupivacaine), 33.02% (lidocaine), and 64.18% (saline), respectively. It was suggested that lidocaine may be the most toxic anesthetic and ropivacaine the least on both AF and NP cells. The CCK8 assay (Figs. 1 and 2) and Hoechst 33342 and PI double staining (Fig. 4) also showed similar results after treatment of local anesthetics for 1 hour. As seen in Fig. 3, Bottom, rabbit IVD cells grown on monolayer exposed to bupivacaine and lidocaine revealed greater alteration in cell morphology such as loss of cell contact and increased cell detachment than ropivacaine after treatment for 1 hour. Characterization of cytotoxic effects of local anesthetics on viability of rabbit IVD cells The results of flow cytometry can also be used to assess relative contribution of apoptosis and necrosis to total cell death (Fig. 3, Top and Middle). Most AF cell death appeared because of necrosis (Annexin-Vneg/pos/PIpos) rather than apoptosis (Annexin-Vpos/PIneg). Necrosis was significantly induced after treatment of AF cells with 0.5%
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Fig. 3. Cytotoxic effects of local anesthetics on the viability of rabbit IVD cells grown in monolayer. Cell viability was determined by flow cytometry. (Top) Representative dot plot of cell viability by flow cytometry analysis after Annexin V/PI dual staining. Quadrant 4 (Q4) shows live cells, Quadrants 1 and 2 (Q1 and Q2) show necrotic cells, and Quadrant 3 (Q3) shows apoptotic cells. (Middle) Histogram for statistical analysis shows the results of the living, necrosis, and apoptosis assays after 1 hour of incubation with 0.5% ropivacaine, 0.5% bupivacaine, 2% lidocaine, and 0.9% saline as control. The values are expressed as mean6SD from three independent experiments (*p!.05 vs. control, one-way ANOVA/Bonferroni). (Bottom) Phase-contrast photomicrograph of rabbit IVD cells treated with local anesthetics for 1 hour (magnification, 100). AF, annulus fibrosus; NP, nucleus pulposus; PI, propidium iodide; SD, standard deviation; ANOVA, analysis of variance; IVD, intervertebral disc.
bupivacaine (39.91%) and 2% lidocaine (56.28%). Treatment with 0.5% ropivacaine led to a mild induction of necrosis (24.73%) as compared with control (17.27%). In contrast, the apoptosis rate was significantly reduced after treatment with 0.5% ropivacaine (8.12%), 0.5% bupivacaine (3.54%), and 2% lidocaine (4.92%), respectively, as
compared with necrosis rate (all p!.05). The cytotoxic effects of the local anesthetics on NP cells showed similar trends with the necrosis rate being 41.68% (0.5% ropivacaine), 49.56% (0.5% bupivacaine), and 52.75% (2% lidocaine), respectively (all p!.05), and correspondingly the apoptosis rate being only 7.21%, 8.00%, and 14.24%.
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Fig. 4. Effects of different local anesthetics for 1 hour on viability of rabbit IVD cells. Each photograph shows a group of cells treated with saline, 0.5% ropivacaine, 0.5% bupivacaine, or 2% lidocaine. The necrotic cells are red, and the nuclei of intact cells are blue. The photographs of blue and red fluorescence were taken under a same field and then were merged (magnification, 100). AF, annulus fibrosus; NP, nucleus pulposus; IVD, intervertebral disc.
To confirm that the cytotoxic effects of local anesthetics mainly results from necrosis rather than apoptosis, we do Hoechst 33342 and PI double staining to recheck cell viability. The results were quite consistent with those from the flow cytometry (ie, regarding the population of necrotic cells) (Fig. 4). Apparent apoptotic morphologic changes, such as shrunken or fragmented nuclei, were not observed under fluorescence microscopy. Images showed that the percentages of necrotic cells in bupivacaine and lidocaine groups were significantly higher than those of control and ropivacaine groups in both AF and NP cells. Discussion This study showed that the commonly used local anesthetics bupivacaine, lidocaine, and ropivacaine had a negative effect on rabbit IVD cells in vitro. Using the cell culture model, our studies demonstrated that the effect of exposure to bupivacaine and lidocaine occurred in both dose- and duration-dependent fashions. Although the detrimental effects of ropivacaine increased with prolonged duration of exposure, there was no significant dose-dependent response to ropivacaine in both AF and NP cells. The observed cytotoxicity of bupivacaine to IVD cells was similar to that of recently published articles [5–8]. In addition, our study is particularly relevant in light of recent evidence of the chondrotoxicity of lidocaine and ropivacaine. Miyazaki et al. [25] reported that lidocaine decreases the viability and proteoglycan production of bovine articular chondrocytes as the concentration of lidocaine increases. Jacobs et al. [26] similarly evaluated human chondrocytes viability after exposure to various concentrations of lidocaine and found that lidocaine exhibited both time- and dose-dependent effects. As compared with the chondrocytes treated with normal saline, those treated with ropivacaine also showed significant reductions in cell viability [18]. At the same time, it was also a confirmation of some decrease in viability in response to normal saline alone, a finding consistent with previous reports using both human and rabbit cell lines [5].
To our knowledge, the present study is the first to directly quantify and compare the effects of bupivacaine, ropivacaine, and lidocaine on the viability of healthy IVD cells. The concentrations of local anesthetics in our experiments were established according to those that were used in clinic or recent studies of cytotoxicity of ropivacaine, bupivacaine, and lidocaine on IVD cells or chondrocytes [5,11,15,19]. Our data showed that 2% lidocaine is the most toxic agent on both AF and NP cells, followed by 0.5% bupivacaine, and 0.5% ropivacaine is the least toxic local anesthetic on IVD cells. These findings are consistent with previous in vitro studies of comparing the cytotoxic effects of common local anesthetics on human articular chondrocytes. Piper and Kim [11] reported that 0.5% ropivacaine is significantly less chondrotoxic than 0.5% bupivacaine on both intact human articular cartilage and chondrocyte culture. Grishko et al. [22] suggested that exposure to 2% lidocaine caused massive necrosis and toxicity more significantly than 0.5% bupivacaine and 0.5% ropivacaine on primary human chondrocytes in vitro. However, a study using equine chondrocytes found that bupivacaine was the most toxic of 2% lidocaine, 2% mepivacaine, and 0.5% bupivacaine [21]. The contradictions among the studies may be contributed to the differences in model systems; thus, homogeneity regarding equine chondrocytes used in studies of human chondrocytes or rabbit IVD cells may not be possible. The exact mechanism involved in local anesthetic toxicity to IVD cells has not been well understood. Local anesthetics have been shown to probably affect mitochondrial energetics, and mitochondrial insults can induce either apoptosis or necrosis, with less severe injuries leading to apoptosis [27–29]. In the present study, necrosis may be considered to be the main mechanism of death of rabbit IVD cells after 60-minute exposure to local anesthetics, according to flow cytometric analysis and Hoechst 33342 nuclear staining. However, we cannot completely rule out a correlation between apoptosis of IVD cells and local anesthetics. Grishko et al. [22] reported that exposure of human chondrocytes to lidocaine, bupivacaine,
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or ropivacaine in vitro causes an increase in the induction of apoptosis after the drugs have been removed, suggesting a mitochondrial pathway may be involved in this induction of apoptosis. Johnson et al. [30] also found that lidocaine induced both necrosis and apoptosis in neuronal cells, with the mechanism of cell death dependent on anesthetic dose. The results of Karpie and Chu also suggested that the apoptosis rate increases with the extension of time after local anesthetic removed [19,31]. In addition, Grouselle et al. [32] reported potential restoration of mitochondrial transmembrane potential after removal of the local anesthetic. It appears likely that the toxicity of local anesthetics is related to mitochondrial dysfunction, with the mechanism of cell death dependent on the dose of anesthetics and the time for restoring. Thus, how local anesthetics causes death of IVD cells via different mechanisms between the immediate or short-term effects and the long-term effects remains to be investigated. Further studies are also needed to explain the different mechanisms that operate in lower or higher concentration of local anesthetics to IVD cells. Our results were highly reproducible, with little variation between experiments; however, limitations of our study include the following. First, our studies were performed in vitro, and the observations are not necessarily indicative of what happens in vivo. Whether the same concentrations or durations of exposure of local anesthetics used in this in vitro study would simulate the in vivo exposure in a clinical setting is unclear because of potential dilution effects. In particular, it is possible that damage to the IVD cells is limited by the fact they are surrounded by the abundant extracellular matrix. Therefore, further studies in an animal model are warranted. Rather, the in vitro studies provide a reproducible, quantitative means of assessing the viability of cells within a controlled environment, and then we could compare the direct effects of different local anesthetics. Second, we chose to examine rabbit IVD cells to establish the toxicity of local anesthetics in healthy cells. Because of hardness to get healthy human IVD tissue, most of the studies finished their researches by using IVD from patients with severe IVD degeneration. However, IVD cells from patients were a mixed population of less and more damaged IVD cells and so that studies did not test the effects of local anesthetics alone, making it difficult to apply the results to healthy human IVD cells. Third, monolayer NP cells were used in our study to determine the direct effect of local anesthetics, although many studies demonstrated that NP cells cultured in three-dimensional alginate bead environment can maintain the physiologic phenotype, which may affect the cellular response. However, bupivacaine exposure resulted in similar levels of NP cell death in different microenvironments, whether grown on monolayer or threedimensional alginate beads [5]. In addition, alginate does not entirely mimic the complex matrix structure of the disc, and the diffusion of drugs through alginate may differ from that of IVD tissue.
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Conclusions This study demonstrated dose- and time-dependent cytotoxic effects of bupivacaine and lidocaine on rabbit IVD cells in vitro, whereas ropivacaine only exerted a significant time-dependent toxic effect. We also have shown that, in vitro, 0.5% bupivacaine and 2% lidocaine are significantly toxic to rabbit IVD cells after only 60-minute exposure and 0.5% ropivacaine is significantly less toxic than them are on both AF and NP cells. These results show all local anesthetics should be avoided if at all possible. Ropivacaine may be a choice if necessary, but it is also toxic. In addition, the trends of three commonly used local anesthetics all demonstrate that necrosis appears to be a greater contributor to cell death than apoptosis in short-time after exposure. Although the conclusions from this study are limited to the in vitro environment and long-term clinical effects need to be evaluated, these data suggest a possible difference of local anesthetics in accelerating the cell death that occurs in disc degeneration. It should be considered as a topic for future in vivo investigations. Acknowledgments This work was supported by a grant from National Natural Sciences Foundation of China (No. 30700841). The authors are grateful to the members of the IVD degeneration research team at the Institute of Orthopedic Surgery of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, the People’s Republic of China for their help. References [1] Andersson GB. Epidemiological features of chronic low-back pain. Lancet 1999;354:581–5. [2] Friedly J, Chan L, Deyo R. Increases in lumbosacral injections in the Medicare population: 1994–2001. Spine 2007;32:1754–60. [3] Sice PJ, Chan D, MacIntyre PA. Epidural analgesia after spinal surgery via intervertebral foramen. Br J Anaesth 2005;94:378–80. [4] Kotilainen E, Muittari P, Kirvel€a O. Intradiscal glycerol or bupivacaine in the treatment of low back pain. Acta Neurochir (Wien) 1997;139:541–5. [5] Lee H, Sowa G, Vo N, et al. Effect of bupivacaine on intervertebral disc cell viability. Spine J 2010;10:159–66. [6] Quero L, Klawitter M, Nerlich AG, et al. Bupivacaine—the deadly friend of intervertebral disc cells? Spine J 2011;11:46–53. [7] Wang D, Vo NV, Sowa GA, et al. Bupivacaine decreases cell viability and matrix protein synthesis in an intervertebral disc organ model system. Spine J 2011;11:139–46. [8] Moon JH, Kuh SU, Park HS, et al. Triamcinolone decreases bupivacaine toxicity to intervertebral disc cell in vitro. Spine J 2012;12: 665–73. [9] Zhao CQ, Wang LM, Jiang LS, Dai LY. The cell biology of intervertebral disc aging and degeneration. Ageing Res Rev 2007;6:247–61. [10] Staal JB, de Bie RA, de Vet HC, et al. Injection therapy for subacute and chronic low back pain: an updated Cochrane review. Spine 2009;34:49–59. [11] Piper SL, Kim HT. Comparison of ropivacaine and bupivacaine toxicity in human articular chondrocytes. J Bone Joint Surg Am 2008; 90:986–91.
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lidocaine, bupivacaine, and ropivacaine. J Bone Joint Surg Am 2010;92:609–18. Rannou F, Poiraudeau S, Foltz V, et al. Monolayer anulus fibrosus cell cultures in a mechanically active environment: local culture condition adaptations and cell phenotype study. J Lab Clin Med 2000;136:412–21. Ding F, Shao ZW, Yang SH, et al. Role of mitochondrial pathway in compression-induced apoptosis of nucleus pulposus cells. Apoptosis 2012;17:579–90. Miyazaki T, Kobayashi S, Takeno K, et al. Lidocaine cytotoxicity to the bovine articular chondrocytes in vitro: changes in cell viability and proteoglycan metabolism. Knee Surg Sports Traumatol Arthrosc 2011;19:1198–205. Jacobs TF, Vansintjan PS, Roels N, et al. The effect of lidocaine on the viability of cultivated mature human cartilage cells: an in vitro study. Knee Surg Sports Traumatol Arthrosc 2011;19:1206–13. Irwin W, Fontaine E, Agnolucci L, et al. Bupivacaine myotoxicity is mediated by mitochondria. J Biol Chem 2002;277:12221–7. Dabadie P, Bendriss P, Erny P, Mazat JP. Uncoupling effects of local anesthetics on rat liver mitochondria. FEBS Lett 1987;226: 77–82. Kroemer G, Dallaporta B, Resche-Rigon M. The mitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol 1998;60:619–42. Johnson ME, Uhl CB, Spittler KH, et al. Mitochondrial injury and caspase activation by the local anesthetic lidocaine. Anesthesiology 2004;10:1184–94. Chu CR, Izzo NJ, Coyle CH, et al. The in vitro effects of bupivacaine on articular chondrocytes. J Bone Joint Surg Br 2008;90: 814–20. Grouselle M, Tueux O, Dabadie P, et al. Effect of local anaesthetics on mitochondrial membrane potential in living cells. Biochem J 1990;271:269–72.
Basic Science
Comparison of toxicity effects of ropivacaine, bupivacaine, and lidocaine on rabbit intervertebral disc cells in vitro Xian-Yi Cai, MDa, Li-Ming Xiong, MD, PhDa, Shu-Hua Yang, MD, PhDa,*, Zeng-Wu Shao, MD, PhDa, Mao Xie, MD, PhDa, Fei Gao, MD, PhDa, Fan Ding, MD, PhDb a
Department of Orthopaedic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 430022, Wuhan, China b Department of Orthopaedic Surgery, The First People’s Hospital of Jingmen, 448000, Jingmen, China Received 29 August 2012; revised 27 March 2013; accepted 15 June 2013
Abstract
BACKGROUND CONTEXT: It has been shown that bupivacaine, the most commonly used local anesthetic to relieve or control pain in interventional spine procedures, is cytotoxic to intervertebral disc (IVD) cells in vitro. However, some other common local anesthetics, such as ropivacaine and lidocaine, are also frequently used in the treatment of spine-related pain, and the potential effects of these agents remain unclear. PURPOSE: The purpose of this study was to evaluate the effect of various local anesthetics on rabbit IVD cells in vitro and further compare the cytotoxicity of ropivacaine, bupivacaine, lidocaine, and saline solution control. STUDY DESIGN: Controlled laboratory study. SUBJECTS: Rabbit annulus fibrosus (AF) and nucleus pulposus (NP) cells were isolated from Japanese white rabbits. METHODS: Both AF and NP cells at the second generation maintained in monolayer were exposed to various concentrations of local anesthetics (eg, bupivacaine) or different durations of exposure and evaluated for cell viability by use of cell counting kit-8 (CCK-8). In addition, to compare the cytotoxicity of ropivacaine, bupivacaine, lidocaine, and saline solution control in commercial concentration, the viability was analyzed by flow cytometry after 60-minute exposure, and the morphologic changes were observed by the phase-contrast microscopy. Apoptosis and necrosis of IVD cells were confirmed by using fluorescence microscopy with double staining of Hoechst 33342 and propidium iodide. RESULTS: Rabbit IVD cell death demonstrated a time and dose dependence in response to bupivacaine and lidocaine. However, ropivacaine only exerted a significant time-dependent effect on IVD cells. There was no significant difference in IVD viability after treatment with different doses of ropivacaine. In addition, the results showed that lidocaine was the most toxic of the three local anesthetics and that ropivacaine presented less cytotoxicity than lidocaine and bupivacaine. Fluorescence microscopy also confirmed that the short-term toxic effect of local anesthetics on both AF and NP cells was mainly caused by necrosis rather than apoptosis. CONCLUSIONS: Results show that bupivacaine and lidocaine decrease cell viability in rabbit IVD cells in a dose- and time-dependent manner. All local anesthetics should be avoided if at all possible. Ropivacaine may be a choice if necessary, but it is also toxic. The increase in cell death is more related with cell necrosis rather than cell apoptosis. If these results can be corroborated in tissue explant models or animal studies, caution regarding diagnosing, treating, and controlling spine-related pain with local anesthetics is prompted. Ó 2014 Elsevier Inc. All rights reserved.
Keywords:
Intervertebral disc; Bupivacaine; Lidocaine; Ropivacaine; Necrosis
FDA device/drug status: Not applicable. Author disclosures: X-YC: Nothing to disclose. L-MX: Nothing to disclose. S-HY: Nothing to disclose. Z-WS: Nothing to disclose. MX: Nothing to disclose. FG: Nothing to disclose. FD: Nothing to disclose. X-YC and L-MX contributed equally to this work. 1529-9430/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.spinee.2013.06.041
* Corresponding author. Department of Orthopaedic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 430022, Wuhan, China. Tel.: þ(86)027-85351627. E-mail address: [email protected] (S.-H. Yang)
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Introduction Low back pain is the most common cause of limited activity in people younger than 45 years and the second most frequent reason for visits to the physician [1]. Because of its advantages of minimal invasion and simplicity, injection therapy is adopted by many patients who are unwilling to undergo surgery. Indeed, the use of local anesthetic injection as a tool in diagnosing and treating spinal pain has sharply increased from 1994 to 2001 [2]. Currently, bupivacaine is frequently used in interventional spine procedures and is often administered intraoperatively and postoperatively to control the pain by local, epidural, or spinal injection [3,4]. Extensive use of bupivacaine in a myriad of diagnostic procedures and pain management has been largely based on the assumption that it is safe. Recently, the effects of bupivacaine on intervertebral disc (IVD) cell viability were investigated, and four studies have suggested that bupivacaine may be toxic to IVD cells. Lee et al. [5] reported that bupivacaine could decrease viability of rabbit and human disc cells at a time and dose dependence. Similarly, application of bupivacaine in clinically relevant concentrations such as 0.25% or 0.5% was also toxic to human IVD cells [6]. Specifically, in vitro exposure of organotypic cultures of the murine functional spine units to bupivacaine solution also dramatically decreased cell viability and matrix protein synthesis in a doseand time-dependent manner [7]. Moon et al. [8] recently confirmed that bupivacaine has dose- and time-dependent cytotoxic effects on human nucleus pulposus (NP) cells. Although aforementioned studies have shown cytotoxic effects of bupivacaine on IVD cells, it was still unclear whether this was specific to bupivacaine or could be seen with other local anesthetics. Furthermore, because both disc cell senescence and cell loss have been implicated in the development of IVD degeneration [9], an alternative, less cytotoxic local anesthetic could then be considered for diagnosis and treatment of low back pain. A variety of other local anesthetics, such as ropivacaine and lidocaine, are also used, both alone and in combination, in diagnosing and treating spinal pain with little information regarding potential effects of these agents on IVD cells [10]. A promising alternative to bupivacaine for spinerelated pain relief is ropivacaine. Ropivacaine is a longacting aminoamide member of the pipecoloxylidide group of local anesthetics that differs from bupivacaine only by the replacement of the butyl group on the piperidine nitrogen atom of the molecule with a propyl group [11]. As a result, ropivacaine is less lipid-soluble than bupivacaine, and then together with its stereoselective properties, contributes to the property that has fewer cardiotoxicity and neurotoxicity than bupivacaine [12–14]. At the same time, lidocaine is another commonly used agent for interventional spinal procedures [15]. Lidocaine and bupivacaine are both members of the amide group in the local anesthetic family, but the action duration of lidocaine is
approximately one-half that of bupivacaine [16]. It was shown that only 0.5% lidocaine has been effective in cervical epidural injections for managing chronic neck pain with disc herniation [17]. More importantly, recent studies also demonstrated that there are a number of detrimental effects of ropivacaine and lidocaine on cartilage and chondrocytes [18,19]. Lo et al. [20] observed that bupivacaine, ropivacaine, and lidocaine have a negative effect in a dose- and duration-dependent manner on chondrocyte viability. Park et al. [21] reported that bupivacaine and lidocaine have toxic effects on equine chondrocytes, and bupivacaine is the most toxic of the three local anesthetics in commercial concentration (ie, 2% lidocaine, 2% mepivacaine, and 0.5% bupivacaine). However, the results of Grishko et al. [22] suggested that 2% lidocaine may be the more toxic than 0.5% ropivacaine and 0.5% bupivacaine. In light of these observations, it would be prudent to examine the effects of three common local anesthetics on IVD cell viability, a subpopulation of which are chondrocytic in nature. It is also important to determine which local anesthetic may have the lowest cytotoxic effects on IVD cells, and then it could be a safer alternative drug than the others. This study aimed to determine whether short-term exposures to bupivacaine, lidocaine, and ropivacaine decreased directly the viability of both rabbit annulus fibrosus (AF) and NP cells in a time- and dose-dependent manner in vitro and, furthermore, compare the toxic effects of three common local anesthetics with equipotent doses on rabbit IVD cells. Materials and methods As there is limited availability of normal nondegenerated human disc tissue, the normal and healthy IVD cells isolated from adult Japanese white rabbits were used in this study. Isolation and culture of primary IVD cells Experimental studies were conducted under the protocol approved by the animal experimentation committee of Huazhong University of Science and Technology. Annulus fibrosus and NP were surgically dissected from the thoracolumbar spine (L5–T10) of skeletally mature Japanese white rabbits (3 months, male) immediately after killing by air embolism. Cells were isolated as described previously [23,24]. Briefly, tissue from each level was collected, minced, and followed by enzymatic digestion. The resulting cells were seeded in six-well culture plates in Dulbecco’s modified Eagle’s medium/ham’s F-12 (DMEM/F-12; Gibco, USA) containing 10% (for AF cells) or 20% (for NP cells) fetal bovine serum (Gibco, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco, Grand Island, NY, USA) at 37 C, 5% CO2 atmosphere. Culture medium was changed every other day. After about
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one or two weeks, cells were passaged by 0.25% trypsinization (Amresco, Solon, OH, USA) when they reached confluence of 80% to 90%. The IVD cells at the second generation maintained in a monolayer were used throughout the following experiments. Experimental groups To examine the time-dependent effect of different local anesthetics, both rabbit AF and NP cells were divided into four treatment groups, and each group was exposed to one of the following experimental agents: 0.9% normal saline solution; 0.5% ropivacaine (Ropivacaine HCl; AstraZeneca AB, Sweden); 0.5% bupivacaine (Bupivacaine HCl; Zhaohui Pharm, China); and 2% lidocaine (Lidocaine HCl; Zhaohui Pharm, China) for 30, 60, 90, and 120 minutes. To determine the dose-dependent effect, both AF and NP cells were cultured in ropivacaine (0.5%, 0.25%, 0.125%), bupivacaine (0.5%, 0.25%, 0.125%), lidocaine (2%, 1%, 0.5%), and 0.9% saline as a control for 60 minutes. The concentrations used were those of the commercial local anesthetic solution, and their twofold serially diluted solutions were prepared with normal saline. Cell viability tests CCK-8 assay At designated time points, cell viability was determined using cell counting kit-8 (CCK-8; Beyotime, China) colorimetric assay in six independent experiments. Rabbit IVD cells were seeded in 96-well culture plates at a density of 1104 cells per well and incubated for 48 hours in a humidified 5% CO2 incubator at 37 C. The growth medium was then removed from each well, the wells washed once with phosphate-buffered saline (PBS) and 100 mL local anesthetic added to each well. After exposure, local anesthetics were removed by inspiration, and all the wells filled with 100 mL of DMEM/F12 and 10 mL CCK-8 solution. After incubated for 4 hours at 37 C according to the manufacturer’s instructions, cell proliferation was assessed by absorbance detection at A450 nm with a microplate reader (Biotek, Winooski, VT, USA). Flow cytometry At designated time points, the IVD cells from each treatment group were labeled using Annexin V/propidium iodide (PI) (KeyGen Biotech, China) double staining according to the manufacturer’s instructions as described previously. Briefly, after release from culture conditions by trypsinization and centrifugation, cells were washed with cold PBS twice and resuspended in 500 mL binding buffer. To each suspension, 5 mL Annexin V-FITC (fluoresceinconjugated Annexin V) and 5 mL PI were added to stain the cells. Cells were incubated in the dark for 15 minutes at room temperature. Samples were applied to flow cytometry (BD LSR II, Becton Dickinson) using FlowJo V 7.6.1 software (Tree Star, Olten, Switzerland). Propidium
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iodide–positive and Annexin V–positive/negative staining represent necrotic cells, PI-negative and Annexin V–positive staining apoptotic cells, and both PI- and Annexin V–negative staining live cells, respectively. Fluorescence microscopy Samples for fluorescence microscopy were stained with Hoechst 33342 and PI to distinguish apoptotic and necrotic cells. Propidium iodide can only enter the affected plasma membrane just as trypan blue dye can. The Hoechst 33342 dye can display the remaining cells, which are not stained with PI, and the morphologic changes of apoptosis can be distinguished from the normal nucleus from this staining [21]. At designated time points, morphological changes of the cells were examined using an Apoptosis and Necrosis Assay Kit (Beyotime, China). Briefly, after treatment, cells in six-well plates were cultured in 1 mL binding buffer, 5 mL Hoechst 33342, and 5 mL PI added and further incubated in the dark for 20 minutes at 4 C. After staining, the cells were washed once with PBS (pH 7.4), and the stained nuclei were observed using a fluorescence microscope (Olympus IX71) with ultraviolet filter (excitation spectrum: 350 nm, emission wavelength: 460 nm). Both red (PI) and blue (Hoechst) colors could be visualized through this filter; however, red nuclei were recounted more precisely using green filter (excitation spectrum: 460–550 nm, emission wavelength: 590 nm). Statistical analysis Experimental values were obtained from at least three independent experiments. The results are represented as means6standard deviation. Statistical analyses were performed by using the Student t test or one or two-way analysis of variance (GraphPad Prism; GraphPad Software Inc., La Jolla, CA, USA), where appropriate. The Bonferroni post hoc test was used to determine the source of observed differences. Significance was set at p!.05. Results Evaluation of time- and dose-dependent effects of local anesthetics on the viability of rabbit IVD cells To test the effects of three common local anesthetics on both AF and NP cell viability, cells grown in monolayer culture were exposed to local anesthetics at different concentrations for various periods of time, and cell viability was measured using CCK-8 assay. For cytotoxicity, results from untreated cells were set to 100%. Values less than 100% represented cell death caused by local anesthetics. The results of AF and NP cells treated with various doses of ropivacaine, bupivacaine, and lidocaine were summarized in Fig. 1. As compared with the IVD cells treated with normal saline, those treated with local anesthetics all showed significant reduction in viability (all p!.05) except 0.125% and 0.25% ropivacaine (not significant). Assessment of the effect of individual local anesthetics showed a statistically
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Fig. 1. CCK-8 assay dose-course. To determine dose-dependent effects of the local anesthetics, CCK-8 assay was carried out after 60-minute exposure to various concentrations of local anesthetics and the group of 0.9% saline served as control. Cell viability was significantly reduced in a dose-dependent manner, except for ropivacaine. Data are expressed as mean6SD from four independent experiments (*p!.05 vs. control; oneway ANOVA/Bonferroni). AF, annulus fibrosus; NP, nucleus pulposus; SD, standard deviation; ANOVA, analysis of variance.
significant decrease in cell viability with increasing concentration of bupivacaine (p!.05) and lidocaine (p!.05), except ropivacaine (not significant). The results from both AF and NP cells treated with 0.5% ropivacaine, 0.5% bupivacaine, and 2% lidocaine at different time points were summarized in Fig. 2, Top and Bottom, respectively. There was statistically significant difference in the percentage of cell death among different time points for all the local anesthetics (all p!.05). Comparison of ropivacaine, bupivacaine, and lidocaine toxicity on rabbit IVD cells To compare the toxic effects of 0.5% ropivacaine, 0.5% bupivacaine, and 2% lidocaine on IVD cells, cell viability was measured using flow cytometry after treatment for 1 hour with local anesthetics (Fig. 3, Top and Middle). Cell viability was decreased to 67.15%, 53.26%, and 39.12%,
Fig. 2. CCK-8 assay time-course. To elucidate time-dependent effects of the local anesthetics, CCK-8 assay was carried out after 30, 60, 90, and 120 minutes and the group of 0.9% saline served as control, respectively. Cell viability was significantly reduced in a time-dependent manner. Data are expressed as mean6SD from four independent experiments (*p!.05 vs. control; two-way ANOVA/Bonferroni). AF, annulus fibrosus; NP, nucleus pulposus; SD, standard deviation; ANOVA, analysis of variance.
respectively, after 1 hour exposure of rabbit AF cells to 0.5% ropivacaine, 0.5% bupivacaine, and 2% lidocaine as compared with 0.9% saline (78.64%) and that of NP cells was 51.09% (ropivacaine), 42.44% (bupivacaine), 33.02% (lidocaine), and 64.18% (saline), respectively. It was suggested that lidocaine may be the most toxic anesthetic and ropivacaine the least on both AF and NP cells. The CCK8 assay (Figs. 1 and 2) and Hoechst 33342 and PI double staining (Fig. 4) also showed similar results after treatment of local anesthetics for 1 hour. As seen in Fig. 3, Bottom, rabbit IVD cells grown on monolayer exposed to bupivacaine and lidocaine revealed greater alteration in cell morphology such as loss of cell contact and increased cell detachment than ropivacaine after treatment for 1 hour. Characterization of cytotoxic effects of local anesthetics on viability of rabbit IVD cells The results of flow cytometry can also be used to assess relative contribution of apoptosis and necrosis to total cell death (Fig. 3, Top and Middle). Most AF cell death appeared because of necrosis (Annexin-Vneg/pos/PIpos) rather than apoptosis (Annexin-Vpos/PIneg). Necrosis was significantly induced after treatment of AF cells with 0.5%
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Fig. 3. Cytotoxic effects of local anesthetics on the viability of rabbit IVD cells grown in monolayer. Cell viability was determined by flow cytometry. (Top) Representative dot plot of cell viability by flow cytometry analysis after Annexin V/PI dual staining. Quadrant 4 (Q4) shows live cells, Quadrants 1 and 2 (Q1 and Q2) show necrotic cells, and Quadrant 3 (Q3) shows apoptotic cells. (Middle) Histogram for statistical analysis shows the results of the living, necrosis, and apoptosis assays after 1 hour of incubation with 0.5% ropivacaine, 0.5% bupivacaine, 2% lidocaine, and 0.9% saline as control. The values are expressed as mean6SD from three independent experiments (*p!.05 vs. control, one-way ANOVA/Bonferroni). (Bottom) Phase-contrast photomicrograph of rabbit IVD cells treated with local anesthetics for 1 hour (magnification, 100). AF, annulus fibrosus; NP, nucleus pulposus; PI, propidium iodide; SD, standard deviation; ANOVA, analysis of variance; IVD, intervertebral disc.
bupivacaine (39.91%) and 2% lidocaine (56.28%). Treatment with 0.5% ropivacaine led to a mild induction of necrosis (24.73%) as compared with control (17.27%). In contrast, the apoptosis rate was significantly reduced after treatment with 0.5% ropivacaine (8.12%), 0.5% bupivacaine (3.54%), and 2% lidocaine (4.92%), respectively, as
compared with necrosis rate (all p!.05). The cytotoxic effects of the local anesthetics on NP cells showed similar trends with the necrosis rate being 41.68% (0.5% ropivacaine), 49.56% (0.5% bupivacaine), and 52.75% (2% lidocaine), respectively (all p!.05), and correspondingly the apoptosis rate being only 7.21%, 8.00%, and 14.24%.
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Fig. 4. Effects of different local anesthetics for 1 hour on viability of rabbit IVD cells. Each photograph shows a group of cells treated with saline, 0.5% ropivacaine, 0.5% bupivacaine, or 2% lidocaine. The necrotic cells are red, and the nuclei of intact cells are blue. The photographs of blue and red fluorescence were taken under a same field and then were merged (magnification, 100). AF, annulus fibrosus; NP, nucleus pulposus; IVD, intervertebral disc.
To confirm that the cytotoxic effects of local anesthetics mainly results from necrosis rather than apoptosis, we do Hoechst 33342 and PI double staining to recheck cell viability. The results were quite consistent with those from the flow cytometry (ie, regarding the population of necrotic cells) (Fig. 4). Apparent apoptotic morphologic changes, such as shrunken or fragmented nuclei, were not observed under fluorescence microscopy. Images showed that the percentages of necrotic cells in bupivacaine and lidocaine groups were significantly higher than those of control and ropivacaine groups in both AF and NP cells. Discussion This study showed that the commonly used local anesthetics bupivacaine, lidocaine, and ropivacaine had a negative effect on rabbit IVD cells in vitro. Using the cell culture model, our studies demonstrated that the effect of exposure to bupivacaine and lidocaine occurred in both dose- and duration-dependent fashions. Although the detrimental effects of ropivacaine increased with prolonged duration of exposure, there was no significant dose-dependent response to ropivacaine in both AF and NP cells. The observed cytotoxicity of bupivacaine to IVD cells was similar to that of recently published articles [5–8]. In addition, our study is particularly relevant in light of recent evidence of the chondrotoxicity of lidocaine and ropivacaine. Miyazaki et al. [25] reported that lidocaine decreases the viability and proteoglycan production of bovine articular chondrocytes as the concentration of lidocaine increases. Jacobs et al. [26] similarly evaluated human chondrocytes viability after exposure to various concentrations of lidocaine and found that lidocaine exhibited both time- and dose-dependent effects. As compared with the chondrocytes treated with normal saline, those treated with ropivacaine also showed significant reductions in cell viability [18]. At the same time, it was also a confirmation of some decrease in viability in response to normal saline alone, a finding consistent with previous reports using both human and rabbit cell lines [5].
To our knowledge, the present study is the first to directly quantify and compare the effects of bupivacaine, ropivacaine, and lidocaine on the viability of healthy IVD cells. The concentrations of local anesthetics in our experiments were established according to those that were used in clinic or recent studies of cytotoxicity of ropivacaine, bupivacaine, and lidocaine on IVD cells or chondrocytes [5,11,15,19]. Our data showed that 2% lidocaine is the most toxic agent on both AF and NP cells, followed by 0.5% bupivacaine, and 0.5% ropivacaine is the least toxic local anesthetic on IVD cells. These findings are consistent with previous in vitro studies of comparing the cytotoxic effects of common local anesthetics on human articular chondrocytes. Piper and Kim [11] reported that 0.5% ropivacaine is significantly less chondrotoxic than 0.5% bupivacaine on both intact human articular cartilage and chondrocyte culture. Grishko et al. [22] suggested that exposure to 2% lidocaine caused massive necrosis and toxicity more significantly than 0.5% bupivacaine and 0.5% ropivacaine on primary human chondrocytes in vitro. However, a study using equine chondrocytes found that bupivacaine was the most toxic of 2% lidocaine, 2% mepivacaine, and 0.5% bupivacaine [21]. The contradictions among the studies may be contributed to the differences in model systems; thus, homogeneity regarding equine chondrocytes used in studies of human chondrocytes or rabbit IVD cells may not be possible. The exact mechanism involved in local anesthetic toxicity to IVD cells has not been well understood. Local anesthetics have been shown to probably affect mitochondrial energetics, and mitochondrial insults can induce either apoptosis or necrosis, with less severe injuries leading to apoptosis [27–29]. In the present study, necrosis may be considered to be the main mechanism of death of rabbit IVD cells after 60-minute exposure to local anesthetics, according to flow cytometric analysis and Hoechst 33342 nuclear staining. However, we cannot completely rule out a correlation between apoptosis of IVD cells and local anesthetics. Grishko et al. [22] reported that exposure of human chondrocytes to lidocaine, bupivacaine,
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or ropivacaine in vitro causes an increase in the induction of apoptosis after the drugs have been removed, suggesting a mitochondrial pathway may be involved in this induction of apoptosis. Johnson et al. [30] also found that lidocaine induced both necrosis and apoptosis in neuronal cells, with the mechanism of cell death dependent on anesthetic dose. The results of Karpie and Chu also suggested that the apoptosis rate increases with the extension of time after local anesthetic removed [19,31]. In addition, Grouselle et al. [32] reported potential restoration of mitochondrial transmembrane potential after removal of the local anesthetic. It appears likely that the toxicity of local anesthetics is related to mitochondrial dysfunction, with the mechanism of cell death dependent on the dose of anesthetics and the time for restoring. Thus, how local anesthetics causes death of IVD cells via different mechanisms between the immediate or short-term effects and the long-term effects remains to be investigated. Further studies are also needed to explain the different mechanisms that operate in lower or higher concentration of local anesthetics to IVD cells. Our results were highly reproducible, with little variation between experiments; however, limitations of our study include the following. First, our studies were performed in vitro, and the observations are not necessarily indicative of what happens in vivo. Whether the same concentrations or durations of exposure of local anesthetics used in this in vitro study would simulate the in vivo exposure in a clinical setting is unclear because of potential dilution effects. In particular, it is possible that damage to the IVD cells is limited by the fact they are surrounded by the abundant extracellular matrix. Therefore, further studies in an animal model are warranted. Rather, the in vitro studies provide a reproducible, quantitative means of assessing the viability of cells within a controlled environment, and then we could compare the direct effects of different local anesthetics. Second, we chose to examine rabbit IVD cells to establish the toxicity of local anesthetics in healthy cells. Because of hardness to get healthy human IVD tissue, most of the studies finished their researches by using IVD from patients with severe IVD degeneration. However, IVD cells from patients were a mixed population of less and more damaged IVD cells and so that studies did not test the effects of local anesthetics alone, making it difficult to apply the results to healthy human IVD cells. Third, monolayer NP cells were used in our study to determine the direct effect of local anesthetics, although many studies demonstrated that NP cells cultured in three-dimensional alginate bead environment can maintain the physiologic phenotype, which may affect the cellular response. However, bupivacaine exposure resulted in similar levels of NP cell death in different microenvironments, whether grown on monolayer or threedimensional alginate beads [5]. In addition, alginate does not entirely mimic the complex matrix structure of the disc, and the diffusion of drugs through alginate may differ from that of IVD tissue.
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Conclusions This study demonstrated dose- and time-dependent cytotoxic effects of bupivacaine and lidocaine on rabbit IVD cells in vitro, whereas ropivacaine only exerted a significant time-dependent toxic effect. We also have shown that, in vitro, 0.5% bupivacaine and 2% lidocaine are significantly toxic to rabbit IVD cells after only 60-minute exposure and 0.5% ropivacaine is significantly less toxic than them are on both AF and NP cells. These results show all local anesthetics should be avoided if at all possible. Ropivacaine may be a choice if necessary, but it is also toxic. In addition, the trends of three commonly used local anesthetics all demonstrate that necrosis appears to be a greater contributor to cell death than apoptosis in short-time after exposure. Although the conclusions from this study are limited to the in vitro environment and long-term clinical effects need to be evaluated, these data suggest a possible difference of local anesthetics in accelerating the cell death that occurs in disc degeneration. It should be considered as a topic for future in vivo investigations. Acknowledgments This work was supported by a grant from National Natural Sciences Foundation of China (No. 30700841). The authors are grateful to the members of the IVD degeneration research team at the Institute of Orthopedic Surgery of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, the People’s Republic of China for their help. References [1] Andersson GB. Epidemiological features of chronic low-back pain. Lancet 1999;354:581–5. [2] Friedly J, Chan L, Deyo R. Increases in lumbosacral injections in the Medicare population: 1994–2001. Spine 2007;32:1754–60. [3] Sice PJ, Chan D, MacIntyre PA. Epidural analgesia after spinal surgery via intervertebral foramen. Br J Anaesth 2005;94:378–80. [4] Kotilainen E, Muittari P, Kirvel€a O. Intradiscal glycerol or bupivacaine in the treatment of low back pain. Acta Neurochir (Wien) 1997;139:541–5. [5] Lee H, Sowa G, Vo N, et al. Effect of bupivacaine on intervertebral disc cell viability. Spine J 2010;10:159–66. [6] Quero L, Klawitter M, Nerlich AG, et al. Bupivacaine—the deadly friend of intervertebral disc cells? Spine J 2011;11:46–53. [7] Wang D, Vo NV, Sowa GA, et al. Bupivacaine decreases cell viability and matrix protein synthesis in an intervertebral disc organ model system. Spine J 2011;11:139–46. [8] Moon JH, Kuh SU, Park HS, et al. Triamcinolone decreases bupivacaine toxicity to intervertebral disc cell in vitro. Spine J 2012;12: 665–73. [9] Zhao CQ, Wang LM, Jiang LS, Dai LY. The cell biology of intervertebral disc aging and degeneration. Ageing Res Rev 2007;6:247–61. [10] Staal JB, de Bie RA, de Vet HC, et al. Injection therapy for subacute and chronic low back pain: an updated Cochrane review. Spine 2009;34:49–59. [11] Piper SL, Kim HT. Comparison of ropivacaine and bupivacaine toxicity in human articular chondrocytes. J Bone Joint Surg Am 2008; 90:986–91.
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