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REGIONAL ANESTHESIA AND PAIN MEDICINE

Lidocaine Disrupts Axonal Membrane of Rat Sciatic Nerve In Vitro

Department of Anesthesiology, Miyazaki Medical College, Miyazaki, Japan

Address correspondence and reprint requests to Yuko Kanai, MD, Department of Anesthesiology, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan. Address e-mail to yukanai{at}post1.miyazaki-med.ac.jp

	Abstract

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Highly concentrated lidocaine has been reported to induce irreversible loss of membrane potential in crayfish nerve, which implies membrane disruption as one of the direct mechanisms of lidocaine-induced neurotoxicity. To confirm lidocaine-induced membrane disruption in mammalian nerve, a lactate dehydrogenase (LDH) leakage from rat sciatic nerve was measured in vitro. Before applying lidocaine, the desheathed nerve was incubated for 60 min in Krebs-Ringer solution at 37°C to examine basal LDH activity. It was then incubated in 80 mM lidocaine solution at pH 7.3 for 15, 30, 60, or 120 min. Other nerves were immersed in 800 mM choline solution for 120 min. Total LDH activity per wet weight of nerve tissue was assayed using spectrophotometry. It was also determined using nerves cut into 10 segments and incubated in distilled water for 60 min. The LDH activity in the lidocaine group showed a time-dependent increase. After the 60- and 120-min incubation with lidocaine, the amount of LDH activity was significantly increased compared with the choline group and was similar to that of the group incubated in distilled water. We conclude that 80 mM lidocaine may be sufficient to cause membrane damage and facilitate the leakage of enzymes from cytoplasm.

Implications: This study demonstrates that exposing the rat myelinated nerve to lidocaine at a clinically used concentration for more than 30 min causes enough membrane damage to allow enzyme leakage. In clinical practice, the smallest effective dose should be used.

	Introduction

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Local anesthetics such as lidocaine have been shown to be neurotoxic (1,2). Studies of clinical adverse effects of intrathecal local anesthetics suggest that cauda equina syndrome results from local anesthetic neurotoxicity (1–3).

The neurological manifestations appearing after spinal anesthesia are divided into two types: reversible or irreversible, i.e., transient neurologic symptoms (4) or cauda equina syndrome (2), respectively. The former has been hotly debated in recent years, but whether they result from local anesthetic neurotoxicity is still controversial (5,6). Permanent neurological damage, however, is a very acute phenomenon occurring before or during recovery of spinal anesthesia, and it closely correlates with the dosage of local anesthetic applied to the intrathecal space (2). Although many modifying factors contribute to the extent of neurological effects, permanent complications could be induced by severe axonal degeneration (7).

Morphological observations have shown that degeneration of the axon and Schwann cell appears 2–4 days after the injection of local anesthetics in the rat (8,9). However, the relationship of acute neurological deficit immediately after the recovery period of spinal anesthesia to the degenerative changes over 2 days after the injection cannot be explained. In addition to the acute appearance of neurological deficits, concurrent persistent or irreversible expression cannot be explained by the injury of the Schwann cell alone (10), because Schwann cell injury will most likely recover by myelin regeneration. Therefore, this long-lasting neurological deficit and the permanent, irreversible damage induced by local anesthetics are possibly related to the infliction of an acute injury on the axon or neuronal cell body.

Our previous study, using intracellular recording of the crayfish giant axon in vitro, demonstrated that a 30-min perfusion of 80 mM lidocaine induces irreversible loss of action and membrane potentials (11). Gold et al. (12) have demonstrated that depolarization of resting membrane potential is proportional to nerve cell death. This was shown by evaluating cell survival using trypan blue exclusion in the dorsal root ganglion neuron of the rat. We had postulated that irreversible loss of membrane potential in the crayfish axon was a result of membrane disruption, which may induce severe nerve cell injury and clinically permanent neurological sequelae. Although studies of local anesthetic-lipid membrane interaction have shown that the disruption of liposomes by tetracaine is related to the formation of mixed micelles of tetracaine and phosphatidylcholine bilayers in vitro (13,14), an abrupt destruction of isolated axonal membrane has not been demonstrated. We believe that the irreversible loss of membrane potential reflects not only the severe membrane damage but also membrane disruption.

The measurement of lactate dehydrogenase (LDH) activity leaked into the incubation medium from nerve cells was performed as an index of the quantitative analysis of nerve cell death in the experiments (for example, an evaluation of the neurotoxic or apoptotic effects of some drugs) (15–17). Therefore, using such an index, we examined the membrane disruption associated with the topical administration of lidocaine at clinically used concentrations by measuring LDH activity leaked from the mammalian myelinated nerve in vitro.

	Methods

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After approval by the Animal Research and Use Committee of Miyazaki Medical College, Sprague-Dawley rats weighing 200–350 g were studied. The rats were individually housed in a temperature and humidity controlled environment with a 12-h light-dark cycle and free access to food and water.

The rats were anesthetized by the intraperitoneal administration of pentobarbital sodium (50 mg/kg) (Nembutal^® sodium; Dainabot, Osaka, Japan). After skin incision, the sciatic nerves were separated by reflection of the superficial muscle tissue and then carefully removed. The branches of the sciatic nerve were tied with cotton threads. The nerves were quickly immersed in Krebs-Ringer solution and bubbled with a 95% O₂/5% CO₂ gas mixture to maintain a pH of 7.25–7.35. The composition of Krebs-Ringer solution, in mmol/L, was as follows: NaCl, 118; KCl, 4.7; KH₂PO₄, 1.2; MgSO₄ 7H₂O, 1.2; NaHCO₃, 24; CaCl₂, 1.9; and glucose, 11. The nerves were desheathed by inverting and stripping the epineurium with the aid of an operating microscope. The blood vessels were removed, and the nerve was measured for wet weight.

The nerves were placed into each test group at random. To detect an incomplete ligation of the nerves and/or any mechanical damage on the desheathed nerves, the nerves were immersed for 60 min in 0.2 mL plain Krebs-Ringer solution bubbled with 95% O₂/5% CO₂. Lidocaine hydrochloride 80 mM (Sigma Chemical, St. Louis, MO) or choline chloride 800 mM (Nacalai Tesque Inc, Kyoto, Japan) was dissolved in Krebs-Ringer solution and adjusted to pH 7.3 by adding 2 M NaOH. After preincubation, the nerves were transferred to the 0.2 mL 80 mM lidocaine (80L) or 800 mM choline (800C) solution. The nerve was incubated for 15, 30, 60, or 120 min in 80L solution (80L-15, 80L-30, 80L-60, or 80L-120 groups) or for 120 min in 800C solution (800C-120 group) bubbled with 95% O₂/5% CO₂. To measure total LDH activity, the other desheathed nerves were cut into 10 pieces with scissors, placed into 0.2 mL of distilled water, and likewise incubated for 60 min (hypotonically shocked group). A volume of 0.1 mL incubation medium was sampled and used for spectrophotometric assay. All incubations were performed at 37°C.

The LDH activity that leaked from the nerve was measured spectrophotometrically (18). The amount of L-lactate oxidized at 30°C, measured by the continuous increase in absorbance at 334 nm caused by the reduction of nicotinamide-adenine dinucleotide (NAD), is a measure of the catalytic activity of LDH. A sample of 0.1 mL of incubated medium was added to the 2.4 mL of 112 mM Tris-buffered 56 mM L-lactate (pH 9.3) and 0.1 mL of 172 mM NAD. The last concentration in the assay mixture was 6.5 mM NAD, 52 mM L-lactate, 155 mM KCl, and 100 mM Tris. After an ordinary stable reaction for 30 s, the change in absorbance at 334 nm was measured for 2 min by using a spectrophotometer (UV-210A, Shimadzu, Kyoto, Japan). Although absorbance at 334 nm was mostly independent of the temperature change, the temperature during the reaction was kept at 30°C with a temperature controller (SPR-5, Shimadzu). The catalytic activity of LDH was calculated from the measured change in absorbance by an equation (18). The LDH activity was standardized by the wet weight of each nerve.

The analytic system was calibrated by an application of exogenous pure LDH (Wako Pure Chemical, Osaka, Japan). The LDH was added to 0.1 mM phosphate buffer and prepared by various concentrations. The pH of the solution was adjusted to 7.3. The volume of the 0.1 mL LDH solution was used for spectrophotometric assay. The linear correlation between the LDH dose and measured absorbance is shown in Figure 1A. The pure LDH solution was likewise prepared at a concentration of 150 µg/mL, and 0.1 mL of the LDH solution was added to 0.9 mL of each test solution (Ringer’s solution, 800C, 80L, or distilled water). After the incubation at 37°C for 60 min, the 0.1-mL incubation medium was assayed. The LDH activity was not significantly different in each test solution (Fig. 1B). When leaked LDH activity was detected in pretreatment in Krebs solution, the specimen was excluded from the experiment because there may have been an incomplete ligation of nerve branching or mechanical damage to the desheathed nerve.

View larger version (15K): [in this window] [in a new window]   Figure 1. Preparations of study. A, The analytic system was calibrated by an application with exogenous pure lactate dehydrogenase (LDH) to the assay medium. Change in absorbance at 334 nm is plotted to the dose of LDH. The change in absorbance at 334 nm reflects a catalytic activity of LDH (n = 3 in each LDH dose). B, Change in absorbance at 334 nm when a same measured dose of LDH was incubated in each test solution for 60 min. There is no significant difference (n = 4, each). Ringer = plain Krebs-Ringer solution, 800C = 800 mM choline chloride solution, 80L = 80 mM lidocaine solution, DW = distilled water.

	Results

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The wet weight of the desheathed rat sciatic nerve was 25 ± 4 mg. The LDH activity of the pretreatment samples, which were incubated for 60 min in plain Krebs-Ringer solution, showed no significant difference among the groups studied.

Figure 2 shows typical recordings of changes in absorbance at 334 nm, which reflect the catalytic activity of LDH in each assay medium (Fig. 2A), and recordings of absorbance versus wavelength (Fig. 2B). The LDH activity in the 80 mM lidocaine group increased in a time-dependent manner. The nerves exposed to 80L for 30, 60, and 120 min showed a significant increase in LDH activity compared with that of the pretreatment. Those exposed to 800C for 120 min or 80L for 15 min showed no significant increase in LDH activity (Fig. 3). The LDH activity in the posttreatment of the 80L-30 group was significantly increased over that of the 80L-15 group. The LDH activity of the 80L-60, 80L-120, and hypotonically shocked nerve groups was significantly increased over that of the 800C-120, 80L-15, and 80L-30 groups in the posttreatment.

View larger version (21K): [in this window] [in a new window]   Figure 2. Typical tracings of absorbance versus time (A) or wavelength (B). The increase in absorbance at 334 nm reflects a catalytic activity of lactate dehydrogenase (LDH) leaked from the desheathed rat sciatic nerve. A, Increase in absorbance at 334 nm in each group. B, Absorbance against wavelength 3 min after starting the enzyme reaction. The absorbance at 334 nm in 80 mM lidocaine group showed a time-dependent increase as a result of enzyme products and reactions. 800C-120 = 800 mM choline chloride group applied for 120 min; 80L-15, -30, -60, or -120 = 80 mM lidocaine groups applied for 15, 30, 60, or 120 min; HS = hypotonically shocked group, the nerves cut into 10 pieces and immersed in distilled water for 60 min.

View larger version (19K): [in this window] [in a new window]   Figure 3. The lactate dehydrogenase (LDH) activity of pre- and posttreatments (n = 7, each group). The values were calculated from the measured change in absorbance at 334 nm and standardized by wet weight (mg) of the nerve. Data are mean ± SD. 800C-120 = 800 mM choline chloride group applied for 120 min, 80L-15, -30, -60, or -120 = 80 mM lidocaine groups applied for 15, 30, 60, or 120 min; HS = hypotonically shocked group, the nerves cut into 10 pieces and immersed in distilled water for 60 min. *P < 0.05 compared with the values of predrug application in each group.

	Discussion

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This concentration of lidocaine (80 mM = 2.17%) is commonly used in clinical practice. In this study, we used 80 mM lidocaine, which was also used in a previous study describing the irreversible toxic effect of lidocaine in crayfish axon (11). There was a graded irreversibility of axonal function. The rapid irreversible conduction block of measured compound action potential has been observed in 2% lidocaine with an application of 15 min in frog myelinated nerve (19,20). We believe that the results of the present study are markedly different from those of previous reports (19,20) regarding the irreversibility of membrane function. Our observations may relate to the irreversible depolarization of membrane potential (11,12) and an irreversible breakdown in the integrity of axonal membrane, which is sufficient to facilitate a LDH leakage. The differences in these studies may also have been caused by the differences of the species used, the temperature of the experiment, and desheathed but myelinated fibers or nonmyelinated axons.

Although the mechanism of axonal membrane destruction is unclear, local anesthetics do produce a toxic reaction in neurological tissues. Highly concentrated tetracaine can solubilize lipid when directly applied to the membrane lipid bilayer (13,14). We hypothesized that lidocaine also had a similar effect on the membrane lipid and had a direct interaction with membranes. A previous study indicated the possibility that an increase in intracellular Ca²⁺ could contribute to both neuronal death and long-lasting conduction loss (12). Although an increase in intracellular Ca²⁺ can disrupt the cytoskeleton (21,22), it is unknown whether intracellular Ca²⁺ increase has had an effect on our conclusion that lidocaine disrupts the myelinated nerve membrane. Further studies are necessary to investigate the mechanism of membrane disruption.

The LDH measurement of the incubation medium is used as an index of neuronal cell death. However, even if myelinated axons are not completely destroyed, a LDH leakage could be observed when the nerve membrane was injured by a sufficient leakage of enzymes. Thus, our observations may also apply to mild neurotoxic effects of local anesthetics on axonal membrane. Because hyperosmolar 800 mM choline chloride produced little LDH leakage, we believe that high osmolarity per se did not affect our results. Although lidocaine has an absorbance in the ultraviolet range (23), we confirmed that the absorbance of lidocaine was different from that of NADH⁺. Lidocaine therefore did not affect the LDH measurements of our study. We obtained a small amount of LDH leakage in preincubation in plain Krebs-Ringer, which may have been caused by the remaining unligated small branches and unremoved small interneuronal vessels. We set up the hypotonically shocked nerve group, in which the nerves were cut and immersed in distilled water for 60 min. The Schwann cell consists almost entirely of myelin. Some of the LDH leakage, however, may come from the Schwann cell. Therefore, these conditions can contribute to the membrane damage of both the axon and the Schwann cell.

In summary, 80 mM lidocaine caused enough membrane damage to allow LDH leakage from the desheathed rat sciatic nerve in vitro. The nerve membrane was disrupted by lidocaine in a time-dependent manner. Even if 2% lidocaine is injected into the intrathecal space as in current clinical practice, it can be assumed that the concentration around the spinal cord or nerve roots will decrease rapidly. Although nerves are not consistently exposed to this concentration, the membrane disruption could happen when more concentrated lidocaine is used and/or when an intrathecal maldistribution of local anesthetic occurs.

	Acknowledgments

 
Support was provided solely from institutional and departmental sources.

The authors thank Mr. Richard L. White for his help.

	References

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