Anesth Analg 2003;97:85-90
© 2003 International Anesthesia Research Society
ANESTHETIC PHARMACOLOGY
Procaine and Mepivacaine Have Less Toxicity In Vitro Than Other Clinically Used Local Anesthetics
Toshiharu Kasaba, MD,
Shin Onizuka, MD, and
Mayumi Takasaki, MD
Department of Anesthesiology, Miyazaki Medical College, Kiyotake-Cho, Miyazaki, Japan
Address correspondence and reprint requests to Toshiharu Kasaba, MD, Department of Anesthesiology, Miyazaki Medical College, Kiyotake-Cho, Miyazaki, 889-1692, Japan. Address e-mail to binjik{at}post1.miyazaki-med.ac.jp
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Abstract
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The neurotoxicity of local anesthetics can be demonstrated in vitro by the collapse of growth cones and neurites in cultured neurons. We compared the neurotoxicity of procaine, mepivacaine, ropivacaine, bupivacaine, lidocaine, tetracaine, and dibucaine by using cultured neurons from the freshwater snail Lymnaea stagnalis. A solution of local anesthetics was added to the culture dish to make final concentrations ranging from 1 x 10-6 to 2 x 10-2 M. Morphological changes in the growth cones and neurites were observed and graded 1 (moderate) or 2 (severe). The median concentrations yielding a score of 1 were 5 x 10-4 M for procaine, 5 x 10-4 M for mepivacaine, 2 x 10-4 M for ropivacaine, 2 x 10-4 M for bupivacaine, 1 x 10-4 M for lidocaine, 5 x 10-5 M for tetracaine, and 2 x 10-5 M for dibucaine. Statistically significant differences (P < 0.05) were observed between mepivacaine and ropivacaine, bupivacaine and lidocaine, lidocaine and tetracaine, and tetracaine and dibucaine. The order of neurotoxicity was procaine = mepivacaine < ropivacaine = bupivacaine < lidocaine < tetracaine < dibucaine. Although lidocaine is more toxic than bupivacaine and ropivacaine, mepivacaine, which has a similar pharmacological effect to lidocaine, has the least-adverse effects on cone growth among clinically used local anesthetics.
IMPLICATIONS: Systematic comparison was assessed morphologically in growth cones and neurites exposed to seven local anesthetics. The order of neurotoxicity was procaine = mepivacaine < ropivacaine = bupivacaine < lidocaine < tetracaine < dibucaine. Although lidocaine is more toxic than bupivacaine and ropivacaine, mepivacaine, which has a similar pharmacological effect to lidocaine, is the safest among clinically used local anesthetics.
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Introduction
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Covino and Wildsmith (1) have rated the relative systemic toxicity of commonly used local anesthetics, claiming that bupivacaine is more toxic than lidocaine or procaine. However, the toxicity of local anesthetics is different in sites of organs or tissues. Although bupivacaine is more cardiotoxic than lidocaine (2,3), bupivacaine is less neurotoxic than lidocaine or tetracaine (4,5). Electrophysiologic (6), histopathologic (7), and behavioral (8) animal studies have shown that bupivacaine is safer than lidocaine. The order and potency of neurotoxicity for clinically used local anesthetics are not known, and the neurotoxicity among local anesthetics is difficult to compare clinically. Clinical profiles of neurotoxicity have been based on many reports of cauda equina syndrome and transient neurologic symptoms after spinal anesthesia (5,9) and subarachnoid or epidural anesthesia (10).
Assaying for the collapse of growth cones isolated from chick embryos is an established method for quantifying neurotoxic effects of different substances. Some local anesthetics have been assessed with this technique (11,12). Cultured neurons from the freshwater snail Lymnaea stagnalis have been used to determine the toxicity of drugs (13). Previously, we reported that bupivacaine was less toxic than dibucaine in the growth cones and neurites of L. stagnalis under direct microscopic observation (14). In addition to the change in the growth cones, changes in the neurites also show damage caused by local anesthetics. Knowing the degree and order of neurotoxicity of local anesthetics would be useful for clinical practice.
The purpose of this study was to determine whether lidocaine is more neurotoxic than other clinically used local anesthetics, including procaine, mepivacaine, ropivacaine, bupivacaine, tetracaine, and dibucaine, and to determine which of these is the safest.
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Methods
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Laboratory-raised stocks of the freshwater snail L. stagnalis were maintained at room temperature. For cell culture studies, 3- to 6-mo-old snails with a shell length of 10–25 mm were selected.
Lymnaea normal saline contained 51.3 mM NaCl, 1.7 mM KCl, 4.1 mM CaCl2, 1.5 mM MgCl2, and 5.0 mM HEPES and had the pH adjusted to 7.9 with 1 M NaOH. Antibiotic normal saline contained gentamicin at 150 µg/mL (G-3632; Sigma, St. Louis, MO) as the sole antibiotic. The defined growth medium consisted of serum-free 50% Liebowitz L-15 medium (Life Technologies, Grand Island, NY) with added inorganic salts and 20 µg/mL of gentamicin. Inorganic salts in the medium contained 40 mM NaCl, 1.7 mM KCl, 4.1 mM CaCl2, 1.5 mM MgCl2, and 10 mM HEPES, with the pH adjusted to 7.9.
After de-shelling, snails were transferred to antibiotic normal saline in a sterile dissection dish. The central ganglionic rings were isolated with standard dissection procedures and then pinned to the silicone rubber base of a tissue culture plate. Ganglia were treated with a trypsin (Sigma) at 2 mg/mL in defined medium for 25 min. Subsequently, ganglia were treated with a soybean trypsin inhibitor (Sigma) at 2 mg/mL in defined medium for 15 min. Ganglia were maintained in a high-osmolarity medium that contained 30 mM glucose rather than normal saline. Before removal of identified cultured neurons (A cluster), the inner connective tissue sheath was dissected from the ganglia with fine forceps. Neurons were removed by gentle suction with a siliconized (Sigmacoate; Sigma) microforge fine-polished pipette that had an outside diameter of 1.5 mm (IB-150F; WPI, Sarasota, FL). After removal, neurons were transferred to poly-L-lysine-coated culture dishes (3001; Becton Dickinson, Franklin Lakes, NJ) with 3 mL of conditioned medium. Conditioned medium was made to incubate the ganglion into the defined medium (one ganglion per milliliter) for a few days (15). After 16–20 h of cell pulling, evidence of nerve growth was observed under an optical microscope. The changes in cultured neurons were observed by using a color videocamera mounted directly on an inverted microscope, and images were recorded with a digital videotape recorder. We examined the changes in the growth cones and the neurites before and 30 min after exposure of one of local anesthetics.
After baseline morphological features of the growth cones and neurites were recorded, large concentrations (1 x 10-3, 1 x 10-2, 1 x 10-1, and 1 M) of local anesthetics were gently added to the culture medium to give final concentrations ranging from 5 x 10-5 to 2 x 10-2 M in procaine and mepivacaine, 1 x 10-5 to 1 x 10-3 M in ropivacaine, 1 x 10-5 to 2 x 10-3 M in bupivacaine, 5 x 10-5 to 1 x 10-2 M in lidocaine, 1 x 10-5 to 2 x 10-3 M in tetracaine, and 1 x 10-6 to 1 x 10-3 M in dibucaine. Procaine (Sigma), mepivacaine (AstraZeneca, London, UK), ropivacaine (AstraZeneca), bupivacaine (Sigma), lidocaine (Sigma), tetracaine (Kyorin, Tokyo, Japan), and dibucaine (Sigma) were prepared in highly purified distilled water. Insolubility prevented concentrations of ropivacaine and bupivacaine larger than 2 x 10-3 M in the conditioned medium. The volume of added local anesthetic was less than one twentieth of the volume of culture media. Cultured neurons were examined 30 min after exposure to each local anesthetic. The values for pH and osmolarity of the medium containing local anesthetic were measured before and after the experiment.
Morphological changes of the growth cones and the neurites were scored as 0 (no change), 1 (moderate change), or 2 (severe change) compared with baseline features. Scores were defined by the following. For a score of 1, growth cones with lamellipodia and filopodia were retracted and shrunken while still retaining their shape. Neurites were narrowed irregularly while still remaining connected. For a score of 2, growth cones with lamellipodia and filopodia were shrunken and diminished, and neurites were shrunken and cut (Fig. 1). The investigator scoring the morphological changes was blinded to the different local anesthetics.
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Figure 1. Retraction and collapse of the growth cones and the neurites induced by local anesthetics. A: Each arrow indicates morphological changes of growth cones. Growth cones with lamellipodia and filopodia are retracted and shrunken (score of 1) or diminished (score of 2). Bars = 50 µm. B, Each arrow indicates the morphological changes of neurites. Neurites are narrowed irregularly (score of 1) or shrunken and cut (score of 2). Bars = 50 µm.
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Values of pH and osmolarity are presented as the mean ± SD. Data were analyzed with Student’s t-test before and after exposure of local anesthetics. Morphological grading scores were analyzed with the Kruskal-Wallis test, followed by the Mann-Whitney U-test. StatView (Abacus Concepts, Berkley, CA) was used for these analyses. P < 0.05 was considered statistically significant.
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Results
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The pH and osmolarity of the control conditioned medium were 7.60 ± 0.08 mOsm and 123 ± 3 mOsm, respectively. After addition of the local anesthetics used in this experiment, these values did not change significantly. Morphological changes in the growth cones and neurites were seen after addition of each of the local anesthetics. The relationship between the score and the concentration for each drug is shown in Figure 2. The median concentration resulting in a score of 1 in the growth cones was 5 x 10-4 M for the procaine solution, 5 x 10-4 M for mepivacaine, 2 x 10-4 M for ropivacaine, 2 x 10-4 M for bupivacaine, 1 x 10-4 M for lidocaine, 5 x 10-5 M for tetracaine, and 2 x 10-5 M for dibucaine (Fig. 3). The median concentrations of procaine and mepivacaine were the largest and that of dibucaine was the smallest among the seven drugs. The dose of lidocaine was significantly smaller than that of bupivacaine. Significant differences between the median concentrations causing damage to the growth cones gave a dosing scale as follows: procaine = mepivacaine > ropivacaine = bupivacaine > lidocaine > tetracaine > dibucaine (Fig. 3A). The results for the neurites were similar, although values for ropivacaine and bupivacaine were not obtained (Fig. 3B).
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Figure 2. Relationship among the doses of procaine, mepivacaine, ropivacaine, bupivacaine, lidocaine, tetracaine, and dibucaine and morphological changes for the growth cones (A) and neurites (B). Morphological changes of the growth cones and the neurites were scored as 1 (moderate) or 2 (severe).
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Figure 3. Comparison of the doses of local anesthetics giving a score of 1 for the growth cones (A) and the neurites (B). Boxes indicate median and range of 10% to 90%. Small circles indicate the cases that were outside of the range between 10% and 90%. *P < 0.05
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Discussion
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Our results demonstrated that mepivacaine and procaine had the least-adverse effects on cone growth among the seven local anesthetics used in clinical practice. Lidocaine was more toxic than ropivacaine and bupivacaine, although it was less toxic than tetracaine and dibucaine. The order of neurotoxicity was as follows: procaine = mepivacaine < ropivacaine = bupivacaine < lidocaine < tetracaine < dibucaine. Assuming a relative neurotoxicity of 1 for procaine and mepivacaine, then the toxicity would be 2.5 for ropivacaine and bupivacaine, 5 for lidocaine, 10 for tetracaine, and 25 for dibucaine.
The neurotoxicity of the seven local anesthetics was determined by the damage to the growth cones with small concentrations, because the growth cone is the most sensitive part of the neuron (13,14). The damage (score of 1) to the neurites was induced at concentrations 10–20 times larger than those affecting the growth cones. Concentrations of bupivacaine and ropivacaine causing damage to the neurites were not obtained because these local anesthetics were not soluble in the conditioned medium at the concentrations to be studied.
The retraction of the growth cone may be due to the reversible inhibition of axonal transport and neurite growth (16). The changes that resulted in neurite damage in our experiments may be similar to those seen in previous reports that showed irreversible damage in rat nerve roots (17,18). The mechanism of the neurotoxicity by local anesthetics is not clearly understood, although it may involve increases in intracellular Ca2+ (19). Kim et al. (20) have suggested that the increase in intracellular Ca2+ levels induced by dibucaine is a factor in neural damage. They report that an increase in intracellular Ca2+ caused condensation of chromatin, resulting in cell apoptosis. We were unable to confirm these changes in our cells.
Many local anesthetics with different clinical profiles are commercially available. We found that procaine and mepivacaine had the least-adverse effects of seven local anesthetics tested; however, procaine was used at concentrations larger than those for other local anesthetics in clinical practice. The relative potency of these drugs has been calculated and reported by Covino and Wildsmith (1) and Berde and Strichartz (21). When used for spinal anesthesia, procaine is used at 10%, mepivacaine at 4%, ropivacaine at 1%, bupivacaine at 0.75%, lidocaine at 5%, tetracaine at 0.5%, and dibucaine at 0.5% (21–23). Considering the results obtained in our experiments, lidocaine carries the greatest risk of nerve damage after spinal anesthesia.
Anderson and Bamburg (24) used dorsal root ganglia from chick embryos and reported the retraction of microspikes in the growth cones and formation of large swellings along the neurites with 2.25 x 10-3 M lidocaine, 2 x 10-3 M procaine, and 1 x 10-4 M tetracaine. Similarly, 50% effective dose values of 10-3.8M for lidocaine, 10-2.9 M for ropivacaine, 10-2.6 M for bupivacaine, and 10-2 M for mepivacaine were shown to damage the growth cones in cultured dorsal root ganglia of chick embryos (12). These two reports describe neurotoxic doses that are larger than those we report with L. stagnalis. The differences are most likely due to differences in experimental materials. The relative neurotoxicity of different local anesthetics reported by Radwan et al. (12) is similar to our findings; however, they did not compare procaine, tetracaine, or dibucaine. Saito et al. (11) reported that the 50% effective dose of tetracaine causing damage to the growth cones was 1.53 mM in dorsal root ganglia, 0.15 mM in retinal ganglia, and 0.06 mM in sympathetic ganglion chain cultures from chick embryos, indicating that different ganglia display different sensitivities for local anesthetics. Because it is difficult to compare neurotoxicity levels of local anesthetics directly with results from other reports, our studies on L. stagnalis neurons, using consistent experimental conditions, provided us with a useful comparison of the relative neurotoxicity of clinically used local anesthetics.
In our experiments, the growth cones and neurites were able to regenerate in conditioned media after washout only when local anesthetics were administered at concentrations that resulted in morphological changes scoring 1 or less. When local anesthetics were administered at larger concentrations, these structures collapsed and were not able to regenerate. These observations were similar to a previous report (11) that reversibility was observed in retinal neurons and dorsal root ganglia neurons and was not observed in sympathetic ganglion neurons. Several studies (5,16,19) showed neuronal damage shortly after exposure. We compared the morphological change 30 minutes after local anesthetic administration. In addition to dose-response studies, previous histopathologic studies (25) have assessed neuronal damage at several hours or several days after exposure to local anesthetics. We need further study about the duration of exposure to local anesthetics and the reversibility of growth cone and neurites.
Although the results of this study cannot be directly applied to humans, our findings were similar to previous studies that showed that tetracaine and dibucaine are more toxic than lidocaine (4,5). Our results also agree with reports that procaine is much less likely than lidocaine to produce transient neurologic symptoms after spinal anesthesia (10). The pharmacological effect of mepivacaine is similar to lidocaine, but the use of mepivacaine is not popular in clinical practice. There are conflicting reports about the relative safety of mepivacaine and lidocaine (26,27). Further studies are needed to define the relationship between morphological changes induced in vitro and the occurrence of clinical symptoms.
In conclusion, we found that lidocaine is more toxic than bupivacaine and ropivacaine. Mepivacaine, which is pharmacologically similar to lidocaine, has the least-adverse effects on cone growth among the clinically used local anesthetics.
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Acknowledgments
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Supported by a Grant-in-Aid (11671510) from the Ministry of Education, Science, and Culture of Japan.
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Accepted for publication February 24, 2003.
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Abstract
Introduction
