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Anesth Analg 2008; 106:1456-1464
© 2008 International Anesthesia Research Society
doi: 10.1213/ane.0b013e318168514b
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ANESTHETIC PHARMACOLOGY

In Vitro, Inhibition of Mitogen-Activated Protein Kinase Pathways Protects Against Bupivacaine- and Ropivacaine-Induced Neurotoxicity

Philipp Lirk, MD, MSc*, Ingrid Haller, MD*, Hans Peter Colvin*, Leopold Lang{dagger}, Bettina Tomaselli, PhD{ddagger}, Lars Klimaschewski, MD{dagger}, and Peter Gerner, MD*§

From the *Department of Anesthesiology and Critical Care, {dagger}Division of Neuroanatomy, and {ddagger}Biocenter/Division of Neurobiochemistry, Medical University of Innsbruck, Austria; and §Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women's Hospital/Harvard Medical School, Boston, Massachusetts.

Address correspondence and reprint requests to Dr. Philipp Lirk, Department of Anesthesiology and Critical Care Medicine, Innsbruck Medical University, Anichstr. 35, 6020 Innsbruck, Austria. Address e-mail to philipp.lirk{at}i-med.ac.at.


   Abstract
Top
 Abstract
Introduction
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
BACKGROUND: Animal models show us that specific activation of the p38 mitogen-activated protein kinase (MAPK) may be a pivotal step in lidocaine neurotoxicity, but this has not been investigated in the case of two very widely used local anesthetics, bupivacaine and ropivacaine. We investigated the hypotheses that these drugs (A) are less neurotoxic than the prototype local anesthetic, lidocaine (B) are selectively toxic for subcategories of dorsal root ganglion neurons and (C) induce activation of either p38 MAPK or related enzymes, such as the c-jun terminal N-kinase (JNK) and extracellular signal-regulated kinase (ERK).

METHODS: We incubated primary sensory neuron cultures with doses of lidocaine, bupivacaine, and ropivacaine equipotent at blocking sodium currents. Next, we sought to determine potential selectivity of bupivacaine and ropivacaine toxicity on neuron categories defined by immunohistochemical staining, or size. Subsequently, the involvement of p38 MAPK, JNK, and ERK was tested using enzyme-linked immunosorbent assays. Finally, the relevance of MAPK pathways in bupivacaine- and ropivacaine-induced neurotoxicity was determined by selectively inhibiting activity of p38 MAPK, JNK, and ERK.

RESULTS: We found that the neurotoxic potency of bupivacaine and ropivacaine is dose-dependent and similar in vitro, but is not selective for any of the investigated subgroups of neurons. Neurotoxicity of bupivacaine and ropivacaine was mediated, at least in part, by MAPKs. Specifically, we demonstrated the relevance of both p38 MAPK and JNK pathways for the neurotoxicity of bupivacaine and characterized the involvement of the p38 MAPK pathway in the neurotoxicity of ropivacaine.

CONCLUSIONS: Given equipotent doses, the neurotoxic potential of lidocaine does not appear to be significantly different from that of bupivacaine and ropivacaine in vitro. Moreover, bupivacaine and ropivacaine do not exert their neurotoxicity differently on specific subsets of dorsal root ganglion neurons. Their neurotoxic effects are brought about through the activation of specific MAPKs; the specific pharmacologic inhibition of these kinases attenuates toxicity in vitro.


   Introduction
Top
 Abstract
 Introduction
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Local anesthetics (LAs) are indispensable in regional anesthesia and pain management. However, epidemiological studies suggest that complications such as transient radicular irritation may follow up to 30% of spinal anesthetics,1 and devastating complications such as cauda equina syndrome affect roughly 1 in 8000 patients.2,3

Different LAs exhibit variable propensities to induce neuronal damage. For example, it has been suggested that bupivacaine and ropivacaine are less toxic towards neurons than lidocaine both in vitro4 and in vivo.5,6 Ropivacaine has not been in clinical use as long as bupivacaine, but case reports of local neurotoxicity have already appeared.7,8 Thus, it is unclear which LA should be recommended when local neurotoxicity is likely, e.g., ulnar nerve block.

Similarly, it has not been established whether bupivacaine and ropivacaine neurotoxicity affects certain cell types selectively. As high concentrations of LAs are used clinically for neurolyses for the therapy of certain pain syndromes, LAs that demonstrate higher toxicity towards nociceptive neurons could be used for lytic blocks when motor block is not desirable.

Moreover, the detailed pathways leading to bupivacaine- and ropivacaine-induced neuronal damage have not been described. Recently, we demonstrated that inhibition of the p38 mitogen-activated protein kinase (MAPK), reduces neurotoxicity elicited by the LA lidocaine in vitro and in vivo.9 In contrast, closely related mitogen-activated pathways, such as the c-jun N-terminal kinase (JNK) pathway, are not relevant in lidocaine-induced nerve injury10 but contribute substantially to the neurotoxicity of the LA tetracaine.11 Similarly, apoptosis has been suggested as a major pathogenic mechanism induced by lidocaine9,12 and bupivacaine13 but not ropivacaine.12 Therefore, the pathways leading to nerve damage vary among LAs. The relative contributions of MAPKs to bupivacaine- and ropivacaine-induced neurotoxicity have not been investigated.

Methods to identify the underlying mechanism and develop methods to actively prevent LA-induced local neurotoxicity would constitute a major breakthrough in regional anesthesia. LAs might be coinjected with specific neuroprotective drugs, depending on the pathway of toxicity caused by a specific LA.

Therefore, using primary sensory neuron cultures, the present project was designed to test the hypotheses that bupivacaine and ropivacaine (A) are less neurotoxic than the prototype LA lidocaine (B) are selectively toxic for subcategories of dorsal root ganglion (DRG) neurons and (C) induce activation of either p38 MAPK or related enzymes such as the JNK and extracellular signal-regulated kinase (ERK).


   METHODS
Top
 Abstract
 Introduction
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Drugs
Unless stated otherwise, drugs were purchased from Sigma Aldrich (Vienna, Austria, or St. Louis, MO). For the in vitro experiments, the pH of the bupivacaine stock solution in dimethyl sulfoxide (DMSO) (100 mM) was 5.15 and of the ropivacaine stock solution in DMSO (100 mM) 4.95. The pH of the final drug solutions (10 mM) added to neuron cultures was 7.64 for bupivacaine and 7.6 for ropivacaine. For lidocaine, the pH of stock solution in DMSO (1M) was 4.65 and the pH of the final solution (40 mM) was 7.38. The osmolality of the medium was 305 mosm/kg, and the addition of experimental compounds at the specified concentrations did not significantly change the osmolality compared with controls. Controls were incubated with the same concentration of DMSO as the experimental cultures.

Electrophysiology
As a first step, we sought to calculate concentrations of bupivacaine and ropivacaine equipotent to 40 mM (approximately 1%) lidocaine for cell culture experiments. Electrophysiologic experiments were performed to determine IC50 values for neuronal Nav1.1–3 and Nav1.6 Na+ channel blockade in rat pituitary GH3 cells.14

We used the whole-cell configuration of the patch clamp technique15 to record macroscopic Na+ currents at room temperature (21°C–23°C). The resistance of the pipette electrodes ranged from 0.8 to 1.2 M{Omega}. Command voltages were controlled by pCLAMP software (Axons Instruments, Inc., Foster City, CA) and were delivered by a List-EPC7 patch clamp amplifier (List-Electronic, Darmstadt-Eberstadt, Germany). After the whole-cell configuration was established, cells were dialyzed for 30 min before data were acquired. Data were filtered at 3 kHz, sampled at 50 kHz, collected, and stored with pCLAMP software. Leak and capacitance currents were subtracted by the P/-4 protocol. Whole-cell recordings were maintained for more than 1 h in this preparation with little or no rundown of the Na+ current. Pipette electrodes were filled with an internal solution containing 100 mM NaF, 30 mM NaCl, 10 mM EGTA, and 10 mM HEPES titrated with CsOH to a pH of 7.2. The external solution consisted of 85 mM choline Cl, 2 mM CaCl2, 65 mM NaCl, and 10 mM HEPES titrated with tetramethyl ammonium hydroxide to a pH of 7.4. These solutions create an outward Na+ current at +30 mV,16 further reducing potential problems associated with series resistance errors.

Voltage-dependent blockade by lidocaine, bupivacaine, and ropivacaine was determined by applying a prepulse or conditioning pulse long enough to permit the drug-channel binding interaction to reach steady-state. The potencies for the resting state were determined by constructing a dose–response curve at a conditioning potential of –150 mV and a test pulse of 30 mV for 4.4 ms.

Primary Sensory Neuron Culture
DRG cultures were obtained as described previously.9,17 Neurons were acutely harvested from adult (8–9 weeks) female Sprague-Dawley rats, which were euthanized by CO2 narcosis according to the institutional protocol (Animal Committee of the Austrian Federal Ministry of Education, Science and Culture, Vienna, Austria). DRGs were desheathed and incubated in 5000 U/mL collagenase for 60 min at 37°C, followed by incubation in 0.25% trypsin/EDTA for 15 min. After inactivation of trypsin with Roswell Park Memorial Institute (RPMI) medium containing 10% horse/5% fetal bovine serum, neurons were triturated and plated in RPMI medium supplemented with N-2 additives (1:100; N-2 medium appears to be selective for neuronal cell lines and does not support the growth of non-neuronal cells) and antibiotics, including penicillin, 1000 U/mL; streptomycin, 1000 µg/mL; and amphotericin B, 25 µg/mL in 0.85% saline, all purchased from Invitrogen (Vienna, Austria). Neurons were allowed to adhere to the glass floor of dishes coated with poly-d-lysine/laminin for 24 h. Poly-d-lysine was applied at a concentration of 0.1 mg/mL in distilled H2O and laminin at 7 µg/mL in RPMI solution. Cell cultures were kept at 37°C in a humidified atmosphere containing 5% CO2.

Determination of Bupivacaine- and Ropivacaine-Induced Neurotoxicity and Its Dose-Dependency in DRG Cell Cultures
To compare the neurotoxicity of lidocaine, bupivacaine, and ropivacaine, cultures were incubated with 40 mM lidocaine, 8 mM bupivacaine, or 11.7 mM ropivacaine for 24 h. This concentration of lidocaine caused approximately 50% cell death in pilot studies. The corresponding equipotent concentrations of bupivacaine and ropivacaine were calculated in relation to the IC50 values for sodium channel blockade.

To determine dose-dependency of neurotoxicity, cultures were treated for 24 h with different concentrations of either bupivacaine (2.5, 10, 15, and 25 mM) or ropivacaine (1, 2.5, 5, 10, 15, and 25 mM).

After incubation with drugs for 24 h, cultures were fixed with 4% paraformaldehyde at 4°C for 30 min, followed by permeabilization with Tween 20 (0.5% in phosphate-buffered saline, PBS) for 10 min at room temperature. Subsequently, monoclonal antibodies against neurofilament (N52m, 1:1000, Sigma) were added for 2 h at 37°C or overnight at 4°C. After two washes with PBS, the cultures were treated with fluorescent anti-mouse IgGs (Alexa Fluor 488 goat anti-mouse IgG, 1:2000, Molecular Probes) for 1 h at room temperature in darkness and subsequently washed twice with PBS.

The outcome variable and marker for drug neurotoxicity was the number of adherent and morphologically healthy neurons (well-defined cell body and nucleus; homogeneous, not grained cytoplasm). The neurons were counted along two passes through the diameters of each well at 20x magnification. DRG neurons typically have a large and distinctly spherical cell body with a clearly visible nucleus and are thereby distinguished from non-neuronal cells, such as glial cells. In all experiments, control cultures were incubated with vehicle DMSO corresponding to the highest concentration of the test drug.

Determination of Size-Selectivity of Bupivacaine and Ropivacaine Neurotoxicity
To determine the selective effect of bupivacaine and ropivacaine neurotoxicity on dissociated rat primary sensory neurons of varying sizes, histograms were performed in cultures before and after incubation with drugs at 10 mM or with vehicle. Images of neurons in 25 defined fields (20x magnification) were taken, and histograms of cell sizes were created with MetamorphTM software (version 6.2r5, Visitron Systems, Munich, Germany). The size of neurons was determined by measuring two diameters (perpendicular to each other), and the mean of these two measurements was taken as the average cell diameter. Cells were grouped arbitrarily into 1 of 3 groups, i.e., mean diameter smaller than 35 µm, between 35 and 65 µm, and larger than 65 µm.

Determination of Phenotype Selectivity of Bupivacaine and Ropivacaine Neurotoxicity
To determine the proportion of neurons positive for calcitonin gene-related peptide (CGRP), indicative of a small-diameter peptidergic sensory neuron phenotype that transmits pain sensations and is central in the pathogenesis of neurogenic inflammation, we used rabbit polyclonal antiserum to CGRP in a dilution of 1:400 in PBS/bovine serum albumin 0.3% (Biomol). Alexa Fluor 488 goat anti-rabbit IgG, a rabbit-specific anti-IgG antibody that is conjugated to an Alexa Fluorescent dye, was used as secondary antibody (1:2000 in PBS/bovine serum albumin 0.3%, Molecular Probes). Omission of primary or secondary antibodies in parallel dishes served as control to assure specificity of the antibody staining.

We used fluorescein isothiocyanate-labeled Bandaireae simplicifolia isolectin B4 (IB4) to identify the subset of small-diameter nonpeptidergic sensory neurons, which are also involved in nociception. Neurons were incubated with 10 µg/mL fluorescein isothiocyanate-labeled IB4 in PBS for 2 h, rinsed, and analyzed.

Enzyme-Linked Immunosorbent Assay
Double-sandwich enzyme-linked immunosorbent assay (ELISA) for p38, phospho-p38, ERK1,2 phospho-ERK1,2, JNK, and phospho-JNK was performed with commercial kits from SuperArray Bioscience (Frederick, MD) according to the manufacturer's protocols. In brief, dissociated adult rat DRG cell cultures were prepared as described above, and cells were seeded into 96-well plates; 24 h after plating, the cultures underwent experimental treatment with either 10 mM bupivacaine or 10 mM ropivacaine for 4 h. Cells were then fixed with 4% paraformaldehyde to preserve any activation-specific protein modification, such as phosphorylation. Blank wells (not seeded with any cells), detection control wells (seeded with cells, only incubated with secondary but not primary antibody), and experimental control wells (seeded with cells but not experimentally treated) were included for control purposes. Each experimental condition was performed in triplicate as a control for systematic variation. After fixation, cells were washed twice with 200 µL washing buffer and incubated with 100 µL quenching buffer for 20 min at room temperature (both buffers were included in the kit). Cells were washed once with 200 µL washing buffer, incubated with 100 µL antigen retrieval buffer in a microwave oven at 30% power for 3 min, and, after cooling to room temperature, washed again with 100 µL washing buffer. After blocking buffer (100 µL) was added for a1-h incubation at room temperature, cells were washed with washing buffer, and 50 µL of diluted primary antibody (1:100) was added to each appropriate well. To the negative control wells only antibody dilution buffer was added. Cells were incubated for 1 h at room temperature, washed, and incubated with 100 µL of diluted secondary antibody for 1 h at room temperature. Colorimetric detection was performed by addition of developing solution to each well for 10 min at room temperature. Stop solution was added to stop the reaction and avoid over-development, and absorbance was read at 450 nm with a reference wavelength of 620 nm on an ELISA Plate reader.

Antibody reading was normalized to the respective cell numbers of each well, and the phosphoprotein-specific antibody ratio was normalized to the pan-protein-specific ratio for the same experimental condition.

Inhibition of p38, ERK, and JNK
To assess the putative p38, ERK1,2, and JNK activation during LA-induced neurotoxicity, we pharmacologically inhibited these kinases using a specific inhibitor of p38 MAPK (SB203580) or its inactive analog SB202474 (Calbiochem, Darmstadt, Germany); the specific inhibitor of ERK1/2, U0126 (Calbiochem); and the specific inhibitor of JNK, SP600125 (Calbiochem). Stock solutions (2.5 mM for SB203580, 3.6 mM for SB202474, 1 mM for U0126, and 18 mM for SP600125) of the inhibitors were prepared in DMSO. Bupivacaine and Ropivacaine was incubated with or without inhibitors dissolved in medium containing N2 additives and antibiotics at a concentration of 10 µM for 24 h, and neuron number in culture was chosen as the outcome variable. To detect a possible toxic influence of the inhibitors themselves, we incubated cultures with each of the MAPK inhibitors alone. Culture conditions in these experiments were the same as those of the main experiments.

Statistics
Sample size was based on preliminary results. The distribution of data in pharmacologic experiments was determined by Kolmogorov-Smirnov analysis. Statistical analysis used one-way analysis of variance with post hoc Bonferroni correction. Unless otherwise stated, summarized data are presented as mean ± sd. Statistical significance was assumed at P < 0.05, results were regarded as highly significant at P < 0.001.


   RESULTS
Top
 Abstract
 Introduction
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Electrophysiology
The IC50 in blocking neuronal Nav1.1–3 and Nav1.6 Na+ channels in GH3 cells at a prepulse of –150 mV was 960 ± 54 µM for lidocaine, 190 ± 27 µM for bupivacaine, and 281 ± 2 for ropivacaine (Fig. 1). Therefore, concentrations equipotent to 40 mM lidocaine (approximately 1%) were calculated to be 8 mM for bupivacaine and 11.7 mM for ropivacaine (Fig. 1).


Figure 122
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  		Figure 1. Dose–response curves for hyperpolarized neuronal sodium channels (GH3 cells expressing Nav1.1, 1.2, and 1.3). The hyperpolarized state affinity for lidocaine (A), bupivacaine (B), and ropivacaine (C) on Na+ channels was measured with a prepulse of –150 mV for 10 s. Pulses were delivered at 30-s intervals. The peak amplitudes of Na+ currents, evoked by a test pulse to +30 mV for 4.4 ms (pulse protocol is inserted above the representative tracings), were measured at various drug concentrations, normalized with respect to the peak amplitude in control, and plotted against the drug concentration. Lines connecting data points represent fits to the data with the Hill equation.

 

Comparative Neurotoxicity of Lidocaine, Bupivacaine, and Ropivacaine
Adult rat DRG cell cultures were exposed to lidocaine, bupivacaine, and ropivacaine at equipotent concentrations for 24 h to determine their relative neurotoxic potencies. All three test substances induced a significant reduction of neuron numbers compared with control cultures. Specifically, incubation of cultures with 40 mM lidocaine resulted in a reduction of neuron count to 46 ± 10% (n = 10) as compared to control cultures, which were defined as 100% (n = 10). Furthermore, treatment with 8 mM bupivacaine yielded a neuron count of 54 ± 26% (n = 10), and incubation with 11.7 mM ropivacaine resulted in a neuron number of 50 ± 21% (n = 10). Among the three compounds tested, no significant difference in the extent of neurotoxicity could be detected (Fig. 2).


Figure 222
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  		Figure 2. Comparative neurotoxicity of lidocaine, bupivacaine, and ropivacaine. Dorsal root ganglion cultures were incubated with 40 mM lidocaine, 8 mM bupivacaine, and 11.7 mM ropivacaine for 24 h, followed by determination of neuron numbers. Neurotoxicity is represented by a reduction of neuron survival. Data are presented as mean ± sd of 10 cultures. ***P < 0.001 as compared to controls.

 

Dose-Dependency of Bupivacaine and Ropivacaine Neurotoxicity
Bupivacaine was added to cultures for 24 h at concentrations of 2.5, 10, 15, and 25 mM to determine dose-dependency of bupivacaine neurotoxicity. Neuron numbers in control cultures (124 ± 29, n = 3) were defined as 100%. While application of 2.5 mM bupivacaine did not show a significant neurotoxic effect on the neuron cultures (relative neuron number 81 ± 19%, n = 3), incubation with 10 mM bupivacaine significantly decreased neuron numbers to 53 ± 1 (n = 3, P < 0.01), as did incubation with 15 mM bupivacaine (neuron number 38 ± 1%, n = 3, P < 0.001) and 25 mM bupivacaine (neuron number 38 ± 6%, n = 3, P < 0.001).

Cultures were treated for 24 h with different concentrations of ropivacaine (1, 2.5, 5, 10, 15, and 25 mM). Compared with control cultures (n = 8), incubation with 1 mM ropivacaine did not result in a significant reduction in number of neurons (76 ± 3%, n = 6), whereas concentrations ≥2.5 mM highly significantly decreased survival of neurons. Specifically, incubation with 2.5 mM ropivacaine resulted in a relative neuron number of 57 ± 2% (n = 6), and incubation with 5, 10, 15, and 25 mM ropivacaine yielded neuron counts of 55 ± 4% (n = 8), 32 ± 3% (n = 8), 28 ± 9% (n = 6), and 13 ± 6% (n = 6) relative to controls, respectively (Fig. 3).


Figure 322
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  		Figure 3. Dose-dependency of bupivacaine and ropivacaine neurotoxicity. A, Incubation of dorsal root ganglion cultures with 2.5, 10, 15, and 25 mM bupivacaine for 24 h revealed neurotoxic effects, as represented by decreased neuron survival, at concentrations of 10 mM and higher. B, Incubation of cultures with ropivacaine at concentrations of 2.5, 10, 20, and 40 mM for 24 h showed neurotoxicity at concentrations of 2.5 mM or higher. Data are presented as mean ± sd of ≥3 cultures. *P < 0.05, **P < 0.01, ***P < 0.001, ns not significant.

 

Bupivacaine and Ropivacaine Neurotoxicity Is Not Selective for Cell Size or Phenotype
Cell size histograms of control cultures showed a roughly bimodal size distribution of the total neuronal population in the DRG. These histograms, created before and after incubation with 10 mM bupivacaine or 10 mM ropivacaine for 24 h, showed that although both compounds clearly exerted neurotoxic effects, they were not selectively toxic for specific cell sizes (size distributions were about the same as those of control cultures). Specifically, in control cultures (n = 12) 65% of all cells had an average diameter of 0–35 µm, 30% were within the range of 35–65 µm, and 5% had an average diameter of more than 65 µm. In bupivacaine-treated cultures (n = 12), 61% of cells had an average diameter between 0 and 35 µm, 35% between 35 and 65 µm, and 4% between of 65 and 95 µm. Cultures treated with 10 mM ropivacaine (n = 12) contained 47% of cells with an average diameter of 0–35 µm, 49% of cells between 35 and 65 µm, and 4% of cells with an average diameter of 65 µm or more.

Moreover, immunohistochemistry showed that bupivacaine and ropivacaine did not preferentially affect distinct neuron phenotypes, such as CGRP- or IB4-positive neuron populations. In control cultures (n = 4), 49% of all cells were IB4-positive and 32% showed CGRP-positive staining, whereas in bupivacaine-treated cultures (n = 4) 26% of all cells were IB4-positive and 22% were positive for CGRP staining. Cultures incubated with ropivacaine (n = 4) contained 25% IB4-positive cells and 32% CGRP-positive cells (Fig. 4).


Figure 422
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  		Figure 4. Size and phenotype selectivity of adult dorsal root ganglion (DRG) cultures treated with either bupivacaine or ropivacaine. DRGs were pooled from all levels. A, The size distribution of control cultures (48 h postseeding) and cultures incubated with 10 mM bupivacaine or 10 mM ropivacaine, respectively, shows that the neurotoxic effect induced by either bupivacaine or ropivacaine is not selective for specific cell sizes. Cell size categories are plotted against relative share of cells. B, Distribution of IB4- and calcitonin gene-related peptide (CGRP)-positive neuron populations in control cultures and cultures incubated for 24 h with 10 mM bupivacaine or 10 mM ropivacaine, respectively. Neither bupivacaine- nor ropivacaine-induced neurotoxicity was selective for distinct phenotypes. Data are presented as mean ± sd of four cultures.

 

Incubation with Bupivacaine and Ropivacaine Induces Activation of p38 MAPK and JNK
ELISA (n = 3) detected activation of both p38MAPK and JNK in adult rat DRG neurons after bupivacaine or ropivacaine treatment for 4 h. Both compounds induced phosphorylation of p38MAPK and JNK, indicated by significant increases in their respective ratios[active/total] compared with controls. The phosphorylation state of p42/44 MAPK (ERK1,2) remained unaffected (Fig. 5).


Figure 522
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  		Figure 5. The effect of bupivacaine and ropivacaine on mitogen-activated protein kinase (MAPK) activation in adult rat dorsal root ganglion (DRG) neurons as assayed by ELISA. Results showed that specific activation of p38 MAPK and c-jun terminal N-kinase (JNK) is induced in adult rat DRG neurons by both bupivacaine and ropivacaine. Neurons were incubated with 10 mM bupivacaine or 10 mM ropivacaine for 4 h, and phosphorylation states of the three major MAPKs (p38, p42/44, JNK) were analyzed by ELISA. p38 MAPK and JNK were activated after drug treatment, as shown by a significant increase of their respective ratios (active/total). The same treatment had no effect on the activation of p42/44 MAPK extracellular signal-regulated kinase (ERK) 1,2. Data are presented as mean ± sd of ≥three cultures, *P < 0.05 when compared with controls.

 

Pharmacologic Inhibition of p38 or JNK Is Neuroprotective In Bupivacaine-Induced Neurotoxicity, But Only Inhibition of p38 Is Protective in Ropivacaine Neurotoxicity
Pharmacologic inhibition of JNK by coincubation of DRG neurons with 10 mM bupivacaine and the JNK inhibitor SP600125 at 10 µM highly significantly attenuated bupivacaine-induced neurotoxicity, as shown by a significant increase of relative neuron numbers from 44 ± 8% (n = 15) in cultures treated with bupivacaine alone to 75 ± 25% (n = 12). Average neuron numbers of respective control cultures were defined as 100% (n = 15). Addition of the p38 MAPK inhibitor SB203580 (10 µM) showed a highly significant neuroprotective effect, resulting in neuron numbers of 68 ± 9% (n = 10). In contrast, coincubation with the ERK inhibitor U0126 (10 µM) did not attenuate neurotoxicity, as represented in decreased neuron numbers of 48 ± 5% (n = 10) relative to control cultures (Fig. 6A).


Figure 622
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  		Figure 6. Pharmacologic inhibition of mitogen-activated protein kinase (MAPK) and its effect on bupivacaine- and ropivacaine-induced neurotoxicity. A, Inhibition of c-jun terminal N-kinase (JNK) by coincubation with the JNK inhibitor SP600125 at 10 µM was neuroprotective in bupivacaine-treated cultures, as was inhibition of p38 MAPK by the p38 MAPK inhibitor SB203580 (10 µM). Both inhibitors caused a highly significant increase of neuron survival. In contrast, inhibition of p42/44 MAPK by the inhibitor U0126 (10 µM) had no protective effect. B, Addition of the same inhibitors to ropivacaine-treated cultures showed similar effects on neuronal survival regarding the inhibition of p38 MAPK. However, inhibition of both JNK and p42/44 MAPK showed no protective effect on neuron survival in these cultures. Data are presented as mean ± sd of ≥10 cultures. ***P < 0.001.

 

Addition of the same inhibitors to ropivacaine-treated cultures affected neuronal survival as follows. Incubation with 10 mM ropivacaine significantly reduced neuron numbers to 36 ± 11% (n = 10) compared with controls (100%, n = 10), and coincubation with the p38 MAPK inhibitor SB203580 (10 µM) highly significantly increased neuron numbers to 62 ± 9% (n = 10) compared with cultures treated with ropivacaine alone (Fig. 6B). Neither addition of the JNK inhibitor SP600125 at 10 µM nor coincubation with the ERK inhibitor U0126 at 10 µM significantly increased neuron survival (neuron numbers 43 ± 16% and 47 ± 12%, respectively, n = 10).

Incubation of cell cultures with MAPK inhibitors alone demonstrated no discernable effect on cell survival (data not shown).


   DISCUSSION
Top
 Abstract
 Introduction
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The main findings of the present study are that, in an in vitro model of dissociated primary sensory neurons, bupivacaine and ropivacaine (A) have neurotoxic properties that do not seem significantly different from that of lidocaine (B) affect all primary sensory neuron cell types uniformly and (C) activate p38 MAPK and JNK, but not ERK.

There is both clinical and laboratory evidence that all LAs are neurotoxic to some degree. This is the case for the classical LA lidocaine, as well as newer LAs such as bupivacaine and its n-propyl derivative, ropivacaine. The literature seems to agree that lidocaine elicits neuronal injury more frequently than, e.g., bupivacaine.18 Auroy et al. demonstrated that the incidence of persistent lumbosacral neuropathy was between 0.8 and 1.4 per 1000 patients for lidocaine and roughly 0.1 per 1000 for bupivacaine.2,3 Nevertheless, cases of persistent cauda equina syndrome after administration of bupivacaine have been reported in the presence of potential risk factors such as spinal stenosis,19 and in healthy individuals.20 Similarly, ropivacaine has been shown to elicit toxic effects after neuraxial application,8 though less frequently than lidocaine.

The equipotency ratios between the classic LA lidocaine and bupivacaine or ropivacaine as calculated from electrophysiological experiments were 5.1:1 and 3.4:1, respectively. This finding corresponds to evidence from previous investigations in which clinical variables were used to estimate equipotency. For example, Sakura et al. compared efficacy of lidocaine and bupivacaine in sensory block in vivo and assumed lidocaine/bupivicaine ratios between 4.3:1 and 4.7:1.6,21 Notably, in the latter study, lidocaine at these concentrations elicited a greater degree of neurotoxicity in vivo than bupivacaine. Similarly, Yamashita et al.22 compared in vivo the neurotoxicity of lidocaine, bupivacaine, and ropivacaine, assuming an equipotency ratio of 5:1 for lidocaine versus both bupivacaine and ropivacaine. Under these conditions, neurotoxicity of lidocaine was greatest and that of ropivacaine smallest. Moreover, the degree of vacuolation in the dorsal funiculus was higher with lidocaine, corresponding to a higher degree of sensory function loss. Yamashita et al. speculated that axonal injuries of primary afferent fibers induce Wallerian degeneration and secondarily result in vacuolation in the dorsal funiculus.22 Electrophysiological investigations have found different equipotency ratios, probably most dependent upon cell type and species.23 For example, a study in Xenopus laevis nerve fibers found a potency ratio between lidocaine and bupivacaine of 7.5:124 while, in dissociated DRG cells, the same authors found a ratio of 5.7:1.25 In newborn rat dorsal horn neurons, 4.3 µM lidocaine was found to be equipotent to 1 µM bupivacaine,26 approximately corresponding to our results in GH3 cells.

Our in vitro results seem to suggest that the neurotoxicity of bupivacaine and ropivacaine is not significantly different from that of lidocaine. Several factors may account for this discrepancy between our findings, and results from epidemiological3 and in vivo22 investigations. First, our in vitro culture model does not consider the complex physiological environment of human nerves in vivo. In particular, the various neurotoxic and neuroprotective effects of cell types such as immune cells27 or glial cells28 or the protective function of the perineurium against noxious substances29 cannot be modeled adequately in vitro, even though they may profoundly influence toxic processes. Therefore, we understand that any in vitro model of neurotoxic effects can only be a first step towards better understanding this pathophysiological entity in its intricacy. However, we believe that among the cell culture models available, organotypic primary sensory neurons represent the model most closely resembling the physiologic in vivo state.

Although toxicity of both conventional12,30 and investigational17,31 LAs has been described before, these experiments lumped all types of neurons into a single category, although the clinical and preclinical profile of LAs (e.g.,23,32 suggests differences in the fiber selectivity of different compounds). For example, ropivacaine has been suggested to preferentially block sensory fibers over motor fibers. However, we found no evidence to suggest substantial fiber selectivity in the assessment of bupivacaine and ropivacaine neurotoxicity, which corresponds to previous findings that lidocaine does not affect different cell types selectively.10 Therefore, it appears that neither drug is a candidate substance for neurolytic blocks.

Finally, we sought to determine the relevance of the main MAPKs thought to participate in neurodegeneration: p38 MAPK, JNK, and p44/42 MAPK (ERK1,2). We found that bupivacaine triggers activation of both p38 MAPK and JNK, followed by neuronal death, and that p38 MAPK and JNK inhibition significantly attenuated this toxic effect, which is in agreement with studies of other LAs such as lidocaine9 and tetracaine.11 Ropivacaine, being closely related to bupivacaine in terms of structure and activity, induced JNK similarly to bupivacaine, while the extent of p38 MAPK activation was less pronounced. However, only inhibition of p38 MAPK significantly reduced ropivacaine-induced neurotoxicity, whereas inhibition of JNK did not significantly attenuate toxic effects. This last finding may indicate a subordinate role of JNK in ropivacaine-induced neural damage, further underlining the need for compound-specific neuroprotective strategies.

In general, the activation of different MAPKs by bupivacaine and ropivacaine was not as specific or clear-cut as for lidocaine.10 Since MAPK can trigger apoptosis,33 our findings are in concordance with reports that apoptosis is the main mechanism mediating the neurotoxicity of bupivacaine.13 Interestingly, this mechanism has not been described for ropivacaine.12 Specifically, Unami et al.13 demonstrated in promyelocytic leukemia cells (HL60) that bupivacaine induced DNA fragmentations and caspase activation, indicating apoptosis, whereas ropivacaine was not effective in inducing apoptosis in a t-cell lymphoma cell line.12 Further evidence to support the notion that bupivacaine and lidocaine share mechanisms of neurotoxicity comes from Arai et al., who demonstrated that calcium- and mitochondria-triggered pathways were involved in bupivacaine neurotoxicity,34 which would correspond with pathways for lidocaine described by us and others.9,30,35 It is difficult to predict the extent of neuroprotection of selective MAPK or JNK inhibition in the clinical situation in vivo. We hypothesize that there may be situations in which pharmacologic inhibition of MAPK could be beneficial. For example, lidocaine, bupivacaine, and ropivacaine activate the p38 MAPK as part of the neurotoxic cascade, and p38 MAPK activation occurs in relevant comorbidities such as diabetic neuropathy.36 Theoretically, these two harmful stimuli could coincide, explaining clinical37 and experimental38 evidence that diabetic nerves are more susceptible to LA-induced neurotoxicity and, conversely, that inhibition of the p38 MAPK could reduce the elevated risk of neurotoxicity in this growing patient collective.

We conclude that, in dissociated primary sensory neuron cultures, the neurotoxic potential of lidocaine, bupivacaine, and ropivacaine is comparable. Moreover, the neurotoxicity of bupivacaine and ropivacaine does not differentially affect specific subsets of DRG neurons. Finally, we show that bupivacaine and ropivacaine exert their neurotoxic effects by activating specific MAPKs, while specific pharmacologic inhibition of these kinases attenuates neurotoxicity in vitro.


   Footnotes
 
Accepted for publication December 21, 2007.

Supported by the National Institutes of Health, Bethesda, MD (Research Grant No. GM64051 to P.G.).


   REFERENCES
Top
 Abstract
 Introduction
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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