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Reversible conduction block in isolated frog sciatic nerve by high concentration of bupivacaine
Pharmacological Research 47 (2003) 235–241
Reversible conduction block in isolated frog sciatic nerve by high concentration of bupivacaine Belgin Buyukakilli a,∗ , Ulku Comelekoglu a , Cengiz Tataroglu b , Arzu Kanik c a
Department of Biophysics, Medical Faculty, Mersin University Campus Yeni¸s ehir, 33160 Mersin, Turkey Department of Neurology, Medical Faculty, Mersin University Campus Yeni¸s ehir, 33160 Mersin, Turkey Department of Biostatistics, Medical Faculty, Mersin University Campus Yeni¸s ehir, 33160 Mersin, Turkey
b c
Accepted 6 December 2002
Abstract We evaluated the effects of a high concentration of bupivacaine commonly used for spinal anaesthesia on the reversibility of conduction block and compound nerve action potential (CNAP) parameters in isolated frog sciatic nerve measured by extracellular recording technique. Isolated frog sciatic nerves were bathed in 1.3% bupivacaine solution for 20 min. In each nerve, action potentials were recorded before exposure to bupivacaine solution, which served as the control data. The extracellular action potentials were recorded after 20 min in the drug by using a BIOPAC MP 100 Acquisition System Version 3.5.7 (Santa Barbara, USA). The nerves were washed for 3 h continuously with Ringer’s solution and action potentials were recorded. The nerve was then soaked overnight in Ringer’s solution at room temperature and tested for impulse recovery. There were significant differences among the experiments regarding CNAP peak-to-peak amplitude, area and duration but conduction velocities among the experiments did not show any statistical difference. In the presence of bupivacaine the extracellular action potential amplitude decreased by 46.99 ± 29.31% relative to the control amplitude (P < 0.05), recovered to 47.10 ± 26.90% after 3 h of wash, and reached 123.20 ± 39.70% after the overnight soak process. This study showed that exposing nerve to high concentration of bupivacaine causes reversible impulse blockade and that bupivacaine does not cause neurotoxic effect on isolated frog sciatic nerve. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Action potential; Local anaesthetics; Bupivacaine; Cauda equina syndrome
1. Introduction Nerve signals are transmitted by action potentials, which are rapid changes in cell membrane potential from the “resting” or depolarized state. In the depolarization stage, voltage-dependent Na+ channels are activated, leading to a rapid flux of Na+ ions into the nerve cell and action potential reaching its peak. A very short time after peak action potential, voltage-dependent K+ channels open and K+ ions rapidly exit to the extracellular space. As K+ flows outward, the Na+ channels gradually become deactivated. Na+ flux drops off and membrane repolarization occurs [1]. Measurements of action potential amplitude, area, duration and conduction velocity may provide information about membrane Na+ and K+ transport. Compound nerve action potential (CNAP) amplitude, area and conduction velocity are ∗ Corresponding author. Tel.: +90-324-341-2815; fax: +90-324-341-2400. E-mail address: [email protected] (B. Buyukakilli).
positively correlated with sodium transport. In addition, the action potential amplitude and area recorded from nerve can be used to estimate the number of activated nerve fibrils [2]. Local anaesthetics block the propagation of nerve impulses by binding to receptors on the sodium channel and preventing normal function [3,4]. This binding appears to involve a single local anaesthetic molecule [5] and is based on the concentration of local anaesthetic required to affect 50% inhibition of Na+ current. Ferguson and Watkins [6] were first to describe patients with cauda equina syndrome associated neurological sequela of spinal anaesthesia in 1937. Direct exposure of the cauda equina to local anaesthetics during continuous spinal and epidural anaesthesia may have caused the reported cauda equina syndrome [7–9]. In these cases, the use of large volumes of highly concentrated local anaesthetic was a common factor [7,8]. The main agent involved in cauda equina syndrome was lidocaine [7]. The toxicity of lidocaine is well known; therefore its use is now avoided in spinal anaesthesia. Thus, lidocaine is widely
1043-6618/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1043-6618(02)00337-7
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B. Buyukakilli et al. / Pharmacological Research 47 (2003) 235–241
replaced by bupivacaine. Bupivacaine hydrochloride (amide local anaesthetic) is the most frequently used local anaesthetic for preoperative and postoperative pain relief in many countries. It is a potent agent capable of producing prolonged anaesthesia. Its long duration of action plus its tendency to provide more sensory than motor block has made it a popular drug for providing prolonged anaesthesia during labour or the postoperative period [10]. Because anaesthesiologists cannot predict how cerebrospinal fluid will dilute administered drugs, the appropriate modification to ensure safe practice would be to give concentrations of drugs that in themselves never exceed a safe concentration. So it is important to identify a concentration below which the nerve was not irreversibly affected. Thus, we sought the effects of a high concentration of the bupivacaine commonly used for spinal anaesthesia on the reversibility of conduction block and CNAP parameters in isolated frog myelinated sciatic nerve.
2.3. Experimental protocols After CNAP had stabilized in Ringer’s solution, CNAP were recorded using a BIOPAC MP100 Acquisition System Version 3.5.7 (Santa Barbara, USA) from each nerve before exposure to 1.3% bupivacaine solution and these data were accepted as control (experiment I). Then each nerve was bathed for 20 min in the 1.3% bupivacaine solution (40 mM). After the 20-min drug exposure, CNAP were recorded (experiment II). Then the nerves were washed (exposed to drug) continuously with Ringer’s solution for 3 h and then CNAP were again recorded (experiment III). The nerves were then removed from the chamber and soaked overnight at room temperature in 100 ml Ringer’s solution. The next morning (24 h after the drug exposure), the nerves were replaced in the nerve chamber and were tested for impulse recovery (experiment IV). 2.4. Statistical analysis
2. Material and methods The study design was approved by the ethic committee of Faculty of Medicine, University of Mersin. Animals were used throughout the experiments according to the proposals of the US National Institutes of Health Guide for the Care and Use of Laboratory Animals. 2.1. Tissue preparation Twenty-four Rana cameroni frogs weighing 30–40 g were used in the experiments. The sciatic nerves were excised from rapidly decapitated and pithed frogs and maintained in Ringer’s solution. This solution was composed of 111.87 mM NaCl, 2.47 mM KCl, 1.08 mM CaCl2 and 2.38 mM NaHCO3 . The pH of the Ringer’s solution was adjusted to 7.2 and all measurements were recorded with the preparations equilibrated at room temperature (21–23 ◦ C), conditions that are physiologic for the frog [11]. The bupivacaine hydrochloride powder used in this experiment was purchased from Sigma Chemical Co., St. Louis, USA (B-5274). 1.3% bupivacaine solution was prepared by dissolving 0.13 g bupivacaine hydrochloride powder to a total volume of 10 ml frog Ringer’s solution. 2.2. Electrophysiological techniques The experiments were carried out in vitro using extracellular recording techniques [12,13]. After 30 min of stabilization in Ringer’s solution, segments of nerve measuring 4–5 cm were placed in a 5 cm × 15 cm Plexiglas nerve chamber containing Ag/AgCl electrodes. The space between the electrodes was 0.5 cm. The nerves were stimulated with these electrodes. The stimulating voltage was set to produce a maximal CNAP using single square pulses of supra maximal strength and 0.5 ms in duration.
The same subject groups were observed four times using repeated-measure design. In this design, each subject serves as its own control. After testing normal distribution with Kolmogorov–Smirnov, the data were analysed with the repeated-measures analysis of variance by using SPSS 9.05 for windows. Least significant difference (LSD) was used for post hoc tests. The significance was set at P < 0.05. In this study, statistical power of our experiments was planned as minimum 80% for pathological differences of amplitude, area, duration and conduction velocity with MINITAB statistical program.
3. Results Table 1 shows the overall findings that were combined to derive the means for each parameter in four experimental groups. Table 2 shows significant values of post hoc tests with LSD. As seen in tables, there were significant differences among the experiments regarding CNAP peak-to-peak amplitude; area and duration but conduction velocity among the experiments did not show statistical difference. Table 1 shows that the amplitude and the area of action potential depressed in this concentration but both of these parameters were partially recovered to control values after 3- and 24-h washes, except for the values obtained after 24-h wash of area. As seen in Fig. 3, there was an important difference (P < 0.05) between CNAP areas in experiments I and IV. Although this difference was significant, there were no significant differences between CNAP areas in control and after 3-h wash, and no differences between CNAP areas were observed after 3- and 24-h washes (see Table 2). The amplitude of a given CNAP was defined as the height in milivolts from the peak of positive phase to peak of the negative phase. The CNAP amplitude of nerves exposed to 1.3% bupivacaine decreased by 46.99 ± 29.31% relative to
B. Buyukakilli et al. / Pharmacological Research 47 (2003) 235–241
237
Table 1 Descriptive statistics for action potential parameters studied Variables
Experiments
Mean
S.D.
Minimum
Maximum
Peak-to-peak amplitude (mV)
Control (I) Bupivacaine (II) After 3-h wash (III) After 24-h wash (IV)
2.21 0.97 0.98 2.54
0.54 0.50 0.54 0.53
1.48 0.21 0.21 0.98
3.11 1.92 2.35 3.36
Area (mV ms)
Control Bupivacaine After 3-h wash After 24-h wash
0.00353 0.00267 0.00278 0.00260
0.00076 0.00156 0.00204 0.00051
0.00230 0.00050 0.00014 0.00170
0.00500 0.00710 0.00910 0.00370
Total duration (ms)
Control Bupivacaine After 3-h wash After 24-h wash
0.28 0.66 0.65 0.24
0.12 0.24 0.37 0.11
0.17 0.34 0.08 0.17
0.56 1.51 2.13 0.62
Conduction velocity (m s−1 )
Control Bupivacaine After 3-h wash After 24-h wash
39.04 37.95 43.76 26.99
25.87 15.25 23.62 21.83
11.16 22.32 17.86 9.92
89.29 89.29 89.29 89.29
Table 2 Results of significant values of post hoc tests with least significant difference (LSD) Pair wise comparisons of experiments
Peak-to-peak amplitude (mV)
Area (mV ms)
Total duration (ms)
Conduction velocity (m s−1 )
I vs. II I vs. III I vs. IV II vs. III II vs. IV III vs. IV
0.001 0.001 0.034 0.968 0.001 0.001
0.035 0.170 0.001 0.761 0.622 0.520
0.000 0.000 0.265 0.969 0.000 0.000
0.634 0.603 0.181 0.237 0.126 0.061
the control amplitude during the 20-min exposure, recovered to 47.10 ± 26.90% after 3 h of wash, and reached 123.20 ± 39.70% after the overnight soak process. The effects of 40 mM concentration of bupivacaine applied extracellulary on sciatic nerve action potentials are shown in Fig. 1. As seen in figure, the amplitude of action potential depressed in this concentration but after 3- and 24-h washes the nerves recovered to control values. Figs. 2–5 show the calculated means (with confidence intervals) for the parameters. Each plot depicts the control data (experiment I) and the other three experimental group (experiments II–IV) findings for one of the four parameters.
4. Discussion
Fig. 1. Effects of 1.3% bupivacaine on the compound nerve action potential (CNAP) and recovery of the CNAP after 3-h and 24-h washes. CNAP records before bupivacaine (A), after bupivacaine (B), after 3-h wash (C), after 24-h wash (D). Calibrations for all traces are shown in A; vertical bar = 0.8 mV; horizontal bar = 0.07 ms.
In this study, we examined the effects of a high concentration of the bupivacaine commonly used for spinal anaesthesia on the reversibility of conduction block and CNAP parameters in isolated frog sciatic nerve. Our results confirm that the peak-to-peak amplitude and the area of CNAP recorded in frog sciatic nerve are abolished reversibly by 20-min exposure to bupivacaine. This reversible inhibition was due to local anaesthetics that block the propagation of nerve impulses by binding to receptors on the
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Fig. 2. Peak-to-peak amplitude values with 95% confidence intervals of the means for experiments I–IV in the study. I: controls, II: after bupivacaine exposure, III: after 3-h wash, IV: after 24-h wash.
Fig. 3. The effect of bupivacaine on compound nerve action potential (CNAP) area in isolated frog sciatic nerve. I: controls, II: after bupivacaine exposure, III: after 3-h wash, IV: after 24-h wash.
sodium channel and preventing normal function [3]. In this study, the decrease in CNAP area accompanying with the decrease in CNAP amplitude and increase in CNAP duration suggests that not all Na+ channels were open and the opening–closing kinetics of Na+ channels slowed down. Bupivacaine is the most frequently used drug for spinal anaesthesia. In reported cases of cauda equina syndrome
after continuous spinal anaesthesia, it was suggested that volumes of highly concentrated local anaesthetics were responsible [7,8]. Studies of spinal canal models show that slow injection through misdirected intrathecal catheters may cause nonhomogenous mixing of local anaesthetic with the cerebrospinal fluid [14–16]. As a result, the relatively unprotected nerve fibres of the cauda equina may be
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Fig. 4. The effect of bupivacaine on compound nerve action potential (CNAP) duration in isolated frog sciatic nerve. I: controls, II: after bupivacaine exposure, III: after 3-h wash, IV: after 24-h wash.
Fig. 5. The effect of bupivacaine on conduction velocity in isolated frog sciatic nerve. I: controls, II: after bupivacaine exposure, III: after 3-h wash, IV: after 24-h wash.
unintentionally and directly exposed to a high concentration of local anaesthetic. Although the frog myelinated sciatic nerve is not a mammalian nerve, it has physiologic and morphologic properties much like those of mammalian peripheral nerves [17,18] and responds to blocking doses
of local anaesthetics in a way that is comparable to its mammalian counterpart [19,20]. Bupivacaine is commonly used in 0.5–0.75% concentrations for spinal anaesthesia [11]. It has been reported that bupivacaine solution at 0.75% concentration causes partially
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reversible conduction block [11]. Based on this finding, Lambert et al. suggested that bupivacaine could not cause cauda equina syndrome, which is caused by irreversible conduction block. It is not known whether the effect of bupivacaine on neural substrates at higher concentrations is reversible. To answer this question, we have chosen a high dose, which is double the amount of the dose that is used in clinics, in our study. The present study did not evaluate the potential neurotoxicity of such high concentration of bupivacaine. Lambert et al. [11] found that nerves exposed to 0.75% bupivacaine recovered to nearly 76 ± 3% after 3 h of wash and 44 ± 8% after soaking overnight. Similar findings were reported for 1.5% lidocaine and Ringer’s solution alone after soaking overnight [11]. In our study, we used higher bupivacaine dose and prolonged duration of exposure than Lambert et al. However, our results suggest that bupivacaine does not produce any toxic effect on frog sciatic nerves when used at the concentration of 1.3%. Nerves exposed to 1.3% bupivacaine for 20 min, recovered their impulse activity approximately 50% during the 3-h wash and almost completely during the 24-h wash. Different methods that were used in these two studies may have caused the differences in recovery observed after 3- and 24-h washes. The nerves were exposed to bupivacaine for 20 min instead of 15 min—the exposure time was used by Lambert et al. The duration of exposure to the local anaesthetic is an important factor that affects reversibility of the conduction block [11]. In this respect, the recovery after 3-h wash in this study was less than the recovery in Lambert et al.’s study, which may be due to the result of 5 min plus in our exposure time; causing more accumulation of the local anaesthetic within the individual nerve fibres. Also, the recording procedure was different than that was used by Lambert et al. They used the sucrose-gap method for recording CNAP and studied the reversibility of conduction blockade in desheathed frog sciatic nerves. However, we used intact (not desheathed) nerves and used extracellular recording technique instead of sucrose-gap chamber method. Both desheathing procedure and sucrose-gap chamber method were shown to be traumatic to the nerve fibres [21]. It is suggested that the low values of CNAPs measured 24 h after the initial mounting in the sucrose-gap chamber, removal and remounting result from both mechanical trauma (handling the nerve and threading it through orifices in the sucrose-gap chamber) and from the intrinsic “run-down” that occurs after the nerve is desheathed [21]. As seen in Fig. 3, there was an important difference (P < 0.05) between CNAP areas in experiments I and IV. Although this difference was significant, there were no significant differences between CNAP areas in control and after 3-h wash, and also between CNAP areas observed during 3and 24-h washes (see Table 2). These results indicate that this high concentration of bupivacaine, used in this study, causes partially reversible impulse blockade, measured by CNAP
areas, just after 3-h wash. Thus, we concluded that bupivacaine at this concentration did not cause any axonal damage. In the present study, the nerves exposed to 1.3% bupivacaine showed an apparently greater recovery after 24-h wash than the control nerves. Also Lambert et al [11] found that the nerves exposed to 0.06% tetracaine showed an apparently greater recovery than the control nerves. The observed greater recovery than the control nerves remain unexplained. In conclusion, this study shows that exposing frog myelinated sciatic nerves to the high concentrations of bupivacaine causes reversible impulse blockade and that bupivacaine does not cause any neurotoxic effect on isolated frog sciatic nerve. Whether these in vitro actions on amphibian nerves also occur in vivo on mammalian nerves will be examined in further studies in our laboratory.
References [1] Koester J, Siegelbaum SA. Propagated signalling: the action potentials. In: Kandel ER, Schwartz JH, Jassel TM, editors. Principles of neural sciences. New York: McGraw-Hill; 2000. p. 151–70. [2] Daube JR. Clinical Neurophysiology. Philadelphia: FA Davis; 1996. p. 60–8. [3] Taylor RE. Effect on procaine on electrical properties of squid axon membrane. Am J Physiol 1959;196:1071–8. [4] Guo X, Castle NA, Chernoff DM, Strichart GR. Comparative inhibition of voltage-gated cation channels by local anaesthetics. Ann NY Acad Sci 1991;625:181–99. [5] Khodorov B, Shishkova L, Peganov E, Revenko S. Inhibition of sodium currents in frog Ranvier node treated with local anaesthetics. Role of slow sodium inactivation. Biochem Biophys Acta 1976;433:433–5. [6] Ferguson FH, Watkins KH. Paralysis of the bladder and associated neurological sequelae of spinal anaesthesia (cauda equina syndrome). Br J Surg 1937;25:735–52. [7] Rigler ML, Drasner K, Krejcie TC, et al. Cauda equina syndrome after continuous spinal anaesthesia. Anesth Analg 1991;72:275–81. [8] Schell RM, Brauer FS, Cole DJ, Applegate RL. Persistent sacral nerve root deficits after continuous spinal anaesthesia. Can J Anaesth 1991;38:908–11. [9] Drasner K, Rigler ML, Sessler DI, Stoller ML. Cauda equina syndrome following intended epidural anaesthesia. Anesthesiology 1992;77:582–5. [10] Catterall W, Mackie K. Local anaesthetics. In: Hardman JG, Gilman AG, Limbird LE, editors. Goodman & Gilman’s the pharmacological basis of therapeutics. New York: McGraw-Hill; 1996. p. 339. [11] Lambert LA, Lambert DH, Strichartz GR. Irreversible conduction block in isolated nerve by high concentrations of local anaesthetics. Anesthesiology 1994;80:1082–93. [12] Andrew BI. Experimental Physiology. London: Churchill Livingstone; 1972. p. 17–34. [13] Katz B. Nerve Muscle and Synapse. New York: McGraw-Hill; 1966. p. 9–25. [14] Rigler ML, Drasner K. Distribution of catheter injected local anaesthetic in a model of the subarachnoid space. Anesthesiology 1991;75:684–92. [15] Lambert DH, Hurley RJ. Cauda equina syndrome and continuous spinal anaesthesia. Anesth Analg 1991;72:817–9. [16] Ross BK, Coda B, Heath CH. Local anaesthetic distribution in a spinal model. A possible mechanism of neurological injury after continuous spinal anaesthesia. Reg Anesth 1992;17:69–77.
B. Buyukakilli et al. / Pharmacological Research 47 (2003) 235–241 [17] Landon DN, Hall SM. The myelinated nerve fibre, the peripheral nerve. In: Landon DN, editor. London: Chapman and Hall; 1976. p. 1–105. [18] Chiu SY, Ritchie JM, Rogart RB, Stagg D. A quantitative description of membrane currents in rabbit myelinated nerve. J Physiol (Lond) 1979;292:149–66. [19] Lee-Son S, Wang GK, Concus A, Crill E, Strichartz G. Stereo selective inhibition of neuronal sodium channels by local anaesthet-
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ics: Evidence for two sites of action? Anesthesiology 1992;77:324– 35. [20] Bader AM, Datta S, Moller RA, Covino BG. Acute progesterone treatment has no effect on bupivacaine-induced conduction blockade in the isolated in the isolated rabbit vagus nerve. Anesth Analg 1990;71:545–8. [21] Wang GK. The long-term excitability of myelinated nerve fibres in the transacted frog sciatic nerve. J Physiol (Lond) 1985;368:309–21.
Reversible conduction block in isolated frog sciatic nerve by high concentration of bupivacaine Belgin Buyukakilli a,∗ , Ulku Comelekoglu a , Cengiz Tataroglu b , Arzu Kanik c a
Department of Biophysics, Medical Faculty, Mersin University Campus Yeni¸s ehir, 33160 Mersin, Turkey Department of Neurology, Medical Faculty, Mersin University Campus Yeni¸s ehir, 33160 Mersin, Turkey Department of Biostatistics, Medical Faculty, Mersin University Campus Yeni¸s ehir, 33160 Mersin, Turkey
b c
Accepted 6 December 2002
Abstract We evaluated the effects of a high concentration of bupivacaine commonly used for spinal anaesthesia on the reversibility of conduction block and compound nerve action potential (CNAP) parameters in isolated frog sciatic nerve measured by extracellular recording technique. Isolated frog sciatic nerves were bathed in 1.3% bupivacaine solution for 20 min. In each nerve, action potentials were recorded before exposure to bupivacaine solution, which served as the control data. The extracellular action potentials were recorded after 20 min in the drug by using a BIOPAC MP 100 Acquisition System Version 3.5.7 (Santa Barbara, USA). The nerves were washed for 3 h continuously with Ringer’s solution and action potentials were recorded. The nerve was then soaked overnight in Ringer’s solution at room temperature and tested for impulse recovery. There were significant differences among the experiments regarding CNAP peak-to-peak amplitude, area and duration but conduction velocities among the experiments did not show any statistical difference. In the presence of bupivacaine the extracellular action potential amplitude decreased by 46.99 ± 29.31% relative to the control amplitude (P < 0.05), recovered to 47.10 ± 26.90% after 3 h of wash, and reached 123.20 ± 39.70% after the overnight soak process. This study showed that exposing nerve to high concentration of bupivacaine causes reversible impulse blockade and that bupivacaine does not cause neurotoxic effect on isolated frog sciatic nerve. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Action potential; Local anaesthetics; Bupivacaine; Cauda equina syndrome
1. Introduction Nerve signals are transmitted by action potentials, which are rapid changes in cell membrane potential from the “resting” or depolarized state. In the depolarization stage, voltage-dependent Na+ channels are activated, leading to a rapid flux of Na+ ions into the nerve cell and action potential reaching its peak. A very short time after peak action potential, voltage-dependent K+ channels open and K+ ions rapidly exit to the extracellular space. As K+ flows outward, the Na+ channels gradually become deactivated. Na+ flux drops off and membrane repolarization occurs [1]. Measurements of action potential amplitude, area, duration and conduction velocity may provide information about membrane Na+ and K+ transport. Compound nerve action potential (CNAP) amplitude, area and conduction velocity are ∗ Corresponding author. Tel.: +90-324-341-2815; fax: +90-324-341-2400. E-mail address: [email protected] (B. Buyukakilli).
positively correlated with sodium transport. In addition, the action potential amplitude and area recorded from nerve can be used to estimate the number of activated nerve fibrils [2]. Local anaesthetics block the propagation of nerve impulses by binding to receptors on the sodium channel and preventing normal function [3,4]. This binding appears to involve a single local anaesthetic molecule [5] and is based on the concentration of local anaesthetic required to affect 50% inhibition of Na+ current. Ferguson and Watkins [6] were first to describe patients with cauda equina syndrome associated neurological sequela of spinal anaesthesia in 1937. Direct exposure of the cauda equina to local anaesthetics during continuous spinal and epidural anaesthesia may have caused the reported cauda equina syndrome [7–9]. In these cases, the use of large volumes of highly concentrated local anaesthetic was a common factor [7,8]. The main agent involved in cauda equina syndrome was lidocaine [7]. The toxicity of lidocaine is well known; therefore its use is now avoided in spinal anaesthesia. Thus, lidocaine is widely
1043-6618/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1043-6618(02)00337-7
236
B. Buyukakilli et al. / Pharmacological Research 47 (2003) 235–241
replaced by bupivacaine. Bupivacaine hydrochloride (amide local anaesthetic) is the most frequently used local anaesthetic for preoperative and postoperative pain relief in many countries. It is a potent agent capable of producing prolonged anaesthesia. Its long duration of action plus its tendency to provide more sensory than motor block has made it a popular drug for providing prolonged anaesthesia during labour or the postoperative period [10]. Because anaesthesiologists cannot predict how cerebrospinal fluid will dilute administered drugs, the appropriate modification to ensure safe practice would be to give concentrations of drugs that in themselves never exceed a safe concentration. So it is important to identify a concentration below which the nerve was not irreversibly affected. Thus, we sought the effects of a high concentration of the bupivacaine commonly used for spinal anaesthesia on the reversibility of conduction block and CNAP parameters in isolated frog myelinated sciatic nerve.
2.3. Experimental protocols After CNAP had stabilized in Ringer’s solution, CNAP were recorded using a BIOPAC MP100 Acquisition System Version 3.5.7 (Santa Barbara, USA) from each nerve before exposure to 1.3% bupivacaine solution and these data were accepted as control (experiment I). Then each nerve was bathed for 20 min in the 1.3% bupivacaine solution (40 mM). After the 20-min drug exposure, CNAP were recorded (experiment II). Then the nerves were washed (exposed to drug) continuously with Ringer’s solution for 3 h and then CNAP were again recorded (experiment III). The nerves were then removed from the chamber and soaked overnight at room temperature in 100 ml Ringer’s solution. The next morning (24 h after the drug exposure), the nerves were replaced in the nerve chamber and were tested for impulse recovery (experiment IV). 2.4. Statistical analysis
2. Material and methods The study design was approved by the ethic committee of Faculty of Medicine, University of Mersin. Animals were used throughout the experiments according to the proposals of the US National Institutes of Health Guide for the Care and Use of Laboratory Animals. 2.1. Tissue preparation Twenty-four Rana cameroni frogs weighing 30–40 g were used in the experiments. The sciatic nerves were excised from rapidly decapitated and pithed frogs and maintained in Ringer’s solution. This solution was composed of 111.87 mM NaCl, 2.47 mM KCl, 1.08 mM CaCl2 and 2.38 mM NaHCO3 . The pH of the Ringer’s solution was adjusted to 7.2 and all measurements were recorded with the preparations equilibrated at room temperature (21–23 ◦ C), conditions that are physiologic for the frog [11]. The bupivacaine hydrochloride powder used in this experiment was purchased from Sigma Chemical Co., St. Louis, USA (B-5274). 1.3% bupivacaine solution was prepared by dissolving 0.13 g bupivacaine hydrochloride powder to a total volume of 10 ml frog Ringer’s solution. 2.2. Electrophysiological techniques The experiments were carried out in vitro using extracellular recording techniques [12,13]. After 30 min of stabilization in Ringer’s solution, segments of nerve measuring 4–5 cm were placed in a 5 cm × 15 cm Plexiglas nerve chamber containing Ag/AgCl electrodes. The space between the electrodes was 0.5 cm. The nerves were stimulated with these electrodes. The stimulating voltage was set to produce a maximal CNAP using single square pulses of supra maximal strength and 0.5 ms in duration.
The same subject groups were observed four times using repeated-measure design. In this design, each subject serves as its own control. After testing normal distribution with Kolmogorov–Smirnov, the data were analysed with the repeated-measures analysis of variance by using SPSS 9.05 for windows. Least significant difference (LSD) was used for post hoc tests. The significance was set at P < 0.05. In this study, statistical power of our experiments was planned as minimum 80% for pathological differences of amplitude, area, duration and conduction velocity with MINITAB statistical program.
3. Results Table 1 shows the overall findings that were combined to derive the means for each parameter in four experimental groups. Table 2 shows significant values of post hoc tests with LSD. As seen in tables, there were significant differences among the experiments regarding CNAP peak-to-peak amplitude; area and duration but conduction velocity among the experiments did not show statistical difference. Table 1 shows that the amplitude and the area of action potential depressed in this concentration but both of these parameters were partially recovered to control values after 3- and 24-h washes, except for the values obtained after 24-h wash of area. As seen in Fig. 3, there was an important difference (P < 0.05) between CNAP areas in experiments I and IV. Although this difference was significant, there were no significant differences between CNAP areas in control and after 3-h wash, and no differences between CNAP areas were observed after 3- and 24-h washes (see Table 2). The amplitude of a given CNAP was defined as the height in milivolts from the peak of positive phase to peak of the negative phase. The CNAP amplitude of nerves exposed to 1.3% bupivacaine decreased by 46.99 ± 29.31% relative to
B. Buyukakilli et al. / Pharmacological Research 47 (2003) 235–241
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Table 1 Descriptive statistics for action potential parameters studied Variables
Experiments
Mean
S.D.
Minimum
Maximum
Peak-to-peak amplitude (mV)
Control (I) Bupivacaine (II) After 3-h wash (III) After 24-h wash (IV)
2.21 0.97 0.98 2.54
0.54 0.50 0.54 0.53
1.48 0.21 0.21 0.98
3.11 1.92 2.35 3.36
Area (mV ms)
Control Bupivacaine After 3-h wash After 24-h wash
0.00353 0.00267 0.00278 0.00260
0.00076 0.00156 0.00204 0.00051
0.00230 0.00050 0.00014 0.00170
0.00500 0.00710 0.00910 0.00370
Total duration (ms)
Control Bupivacaine After 3-h wash After 24-h wash
0.28 0.66 0.65 0.24
0.12 0.24 0.37 0.11
0.17 0.34 0.08 0.17
0.56 1.51 2.13 0.62
Conduction velocity (m s−1 )
Control Bupivacaine After 3-h wash After 24-h wash
39.04 37.95 43.76 26.99
25.87 15.25 23.62 21.83
11.16 22.32 17.86 9.92
89.29 89.29 89.29 89.29
Table 2 Results of significant values of post hoc tests with least significant difference (LSD) Pair wise comparisons of experiments
Peak-to-peak amplitude (mV)
Area (mV ms)
Total duration (ms)
Conduction velocity (m s−1 )
I vs. II I vs. III I vs. IV II vs. III II vs. IV III vs. IV
0.001 0.001 0.034 0.968 0.001 0.001
0.035 0.170 0.001 0.761 0.622 0.520
0.000 0.000 0.265 0.969 0.000 0.000
0.634 0.603 0.181 0.237 0.126 0.061
the control amplitude during the 20-min exposure, recovered to 47.10 ± 26.90% after 3 h of wash, and reached 123.20 ± 39.70% after the overnight soak process. The effects of 40 mM concentration of bupivacaine applied extracellulary on sciatic nerve action potentials are shown in Fig. 1. As seen in figure, the amplitude of action potential depressed in this concentration but after 3- and 24-h washes the nerves recovered to control values. Figs. 2–5 show the calculated means (with confidence intervals) for the parameters. Each plot depicts the control data (experiment I) and the other three experimental group (experiments II–IV) findings for one of the four parameters.
4. Discussion
Fig. 1. Effects of 1.3% bupivacaine on the compound nerve action potential (CNAP) and recovery of the CNAP after 3-h and 24-h washes. CNAP records before bupivacaine (A), after bupivacaine (B), after 3-h wash (C), after 24-h wash (D). Calibrations for all traces are shown in A; vertical bar = 0.8 mV; horizontal bar = 0.07 ms.
In this study, we examined the effects of a high concentration of the bupivacaine commonly used for spinal anaesthesia on the reversibility of conduction block and CNAP parameters in isolated frog sciatic nerve. Our results confirm that the peak-to-peak amplitude and the area of CNAP recorded in frog sciatic nerve are abolished reversibly by 20-min exposure to bupivacaine. This reversible inhibition was due to local anaesthetics that block the propagation of nerve impulses by binding to receptors on the
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Fig. 2. Peak-to-peak amplitude values with 95% confidence intervals of the means for experiments I–IV in the study. I: controls, II: after bupivacaine exposure, III: after 3-h wash, IV: after 24-h wash.
Fig. 3. The effect of bupivacaine on compound nerve action potential (CNAP) area in isolated frog sciatic nerve. I: controls, II: after bupivacaine exposure, III: after 3-h wash, IV: after 24-h wash.
sodium channel and preventing normal function [3]. In this study, the decrease in CNAP area accompanying with the decrease in CNAP amplitude and increase in CNAP duration suggests that not all Na+ channels were open and the opening–closing kinetics of Na+ channels slowed down. Bupivacaine is the most frequently used drug for spinal anaesthesia. In reported cases of cauda equina syndrome
after continuous spinal anaesthesia, it was suggested that volumes of highly concentrated local anaesthetics were responsible [7,8]. Studies of spinal canal models show that slow injection through misdirected intrathecal catheters may cause nonhomogenous mixing of local anaesthetic with the cerebrospinal fluid [14–16]. As a result, the relatively unprotected nerve fibres of the cauda equina may be
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Fig. 4. The effect of bupivacaine on compound nerve action potential (CNAP) duration in isolated frog sciatic nerve. I: controls, II: after bupivacaine exposure, III: after 3-h wash, IV: after 24-h wash.
Fig. 5. The effect of bupivacaine on conduction velocity in isolated frog sciatic nerve. I: controls, II: after bupivacaine exposure, III: after 3-h wash, IV: after 24-h wash.
unintentionally and directly exposed to a high concentration of local anaesthetic. Although the frog myelinated sciatic nerve is not a mammalian nerve, it has physiologic and morphologic properties much like those of mammalian peripheral nerves [17,18] and responds to blocking doses
of local anaesthetics in a way that is comparable to its mammalian counterpart [19,20]. Bupivacaine is commonly used in 0.5–0.75% concentrations for spinal anaesthesia [11]. It has been reported that bupivacaine solution at 0.75% concentration causes partially
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reversible conduction block [11]. Based on this finding, Lambert et al. suggested that bupivacaine could not cause cauda equina syndrome, which is caused by irreversible conduction block. It is not known whether the effect of bupivacaine on neural substrates at higher concentrations is reversible. To answer this question, we have chosen a high dose, which is double the amount of the dose that is used in clinics, in our study. The present study did not evaluate the potential neurotoxicity of such high concentration of bupivacaine. Lambert et al. [11] found that nerves exposed to 0.75% bupivacaine recovered to nearly 76 ± 3% after 3 h of wash and 44 ± 8% after soaking overnight. Similar findings were reported for 1.5% lidocaine and Ringer’s solution alone after soaking overnight [11]. In our study, we used higher bupivacaine dose and prolonged duration of exposure than Lambert et al. However, our results suggest that bupivacaine does not produce any toxic effect on frog sciatic nerves when used at the concentration of 1.3%. Nerves exposed to 1.3% bupivacaine for 20 min, recovered their impulse activity approximately 50% during the 3-h wash and almost completely during the 24-h wash. Different methods that were used in these two studies may have caused the differences in recovery observed after 3- and 24-h washes. The nerves were exposed to bupivacaine for 20 min instead of 15 min—the exposure time was used by Lambert et al. The duration of exposure to the local anaesthetic is an important factor that affects reversibility of the conduction block [11]. In this respect, the recovery after 3-h wash in this study was less than the recovery in Lambert et al.’s study, which may be due to the result of 5 min plus in our exposure time; causing more accumulation of the local anaesthetic within the individual nerve fibres. Also, the recording procedure was different than that was used by Lambert et al. They used the sucrose-gap method for recording CNAP and studied the reversibility of conduction blockade in desheathed frog sciatic nerves. However, we used intact (not desheathed) nerves and used extracellular recording technique instead of sucrose-gap chamber method. Both desheathing procedure and sucrose-gap chamber method were shown to be traumatic to the nerve fibres [21]. It is suggested that the low values of CNAPs measured 24 h after the initial mounting in the sucrose-gap chamber, removal and remounting result from both mechanical trauma (handling the nerve and threading it through orifices in the sucrose-gap chamber) and from the intrinsic “run-down” that occurs after the nerve is desheathed [21]. As seen in Fig. 3, there was an important difference (P < 0.05) between CNAP areas in experiments I and IV. Although this difference was significant, there were no significant differences between CNAP areas in control and after 3-h wash, and also between CNAP areas observed during 3and 24-h washes (see Table 2). These results indicate that this high concentration of bupivacaine, used in this study, causes partially reversible impulse blockade, measured by CNAP
areas, just after 3-h wash. Thus, we concluded that bupivacaine at this concentration did not cause any axonal damage. In the present study, the nerves exposed to 1.3% bupivacaine showed an apparently greater recovery after 24-h wash than the control nerves. Also Lambert et al [11] found that the nerves exposed to 0.06% tetracaine showed an apparently greater recovery than the control nerves. The observed greater recovery than the control nerves remain unexplained. In conclusion, this study shows that exposing frog myelinated sciatic nerves to the high concentrations of bupivacaine causes reversible impulse blockade and that bupivacaine does not cause any neurotoxic effect on isolated frog sciatic nerve. Whether these in vitro actions on amphibian nerves also occur in vivo on mammalian nerves will be examined in further studies in our laboratory.
References [1] Koester J, Siegelbaum SA. Propagated signalling: the action potentials. In: Kandel ER, Schwartz JH, Jassel TM, editors. Principles of neural sciences. New York: McGraw-Hill; 2000. p. 151–70. [2] Daube JR. Clinical Neurophysiology. Philadelphia: FA Davis; 1996. p. 60–8. [3] Taylor RE. Effect on procaine on electrical properties of squid axon membrane. Am J Physiol 1959;196:1071–8. [4] Guo X, Castle NA, Chernoff DM, Strichart GR. Comparative inhibition of voltage-gated cation channels by local anaesthetics. Ann NY Acad Sci 1991;625:181–99. [5] Khodorov B, Shishkova L, Peganov E, Revenko S. Inhibition of sodium currents in frog Ranvier node treated with local anaesthetics. Role of slow sodium inactivation. Biochem Biophys Acta 1976;433:433–5. [6] Ferguson FH, Watkins KH. Paralysis of the bladder and associated neurological sequelae of spinal anaesthesia (cauda equina syndrome). Br J Surg 1937;25:735–52. [7] Rigler ML, Drasner K, Krejcie TC, et al. Cauda equina syndrome after continuous spinal anaesthesia. Anesth Analg 1991;72:275–81. [8] Schell RM, Brauer FS, Cole DJ, Applegate RL. Persistent sacral nerve root deficits after continuous spinal anaesthesia. Can J Anaesth 1991;38:908–11. [9] Drasner K, Rigler ML, Sessler DI, Stoller ML. Cauda equina syndrome following intended epidural anaesthesia. Anesthesiology 1992;77:582–5. [10] Catterall W, Mackie K. Local anaesthetics. In: Hardman JG, Gilman AG, Limbird LE, editors. Goodman & Gilman’s the pharmacological basis of therapeutics. New York: McGraw-Hill; 1996. p. 339. [11] Lambert LA, Lambert DH, Strichartz GR. Irreversible conduction block in isolated nerve by high concentrations of local anaesthetics. Anesthesiology 1994;80:1082–93. [12] Andrew BI. Experimental Physiology. London: Churchill Livingstone; 1972. p. 17–34. [13] Katz B. Nerve Muscle and Synapse. New York: McGraw-Hill; 1966. p. 9–25. [14] Rigler ML, Drasner K. Distribution of catheter injected local anaesthetic in a model of the subarachnoid space. Anesthesiology 1991;75:684–92. [15] Lambert DH, Hurley RJ. Cauda equina syndrome and continuous spinal anaesthesia. Anesth Analg 1991;72:817–9. [16] Ross BK, Coda B, Heath CH. Local anaesthetic distribution in a spinal model. A possible mechanism of neurological injury after continuous spinal anaesthesia. Reg Anesth 1992;17:69–77.
B. Buyukakilli et al. / Pharmacological Research 47 (2003) 235–241 [17] Landon DN, Hall SM. The myelinated nerve fibre, the peripheral nerve. In: Landon DN, editor. London: Chapman and Hall; 1976. p. 1–105. [18] Chiu SY, Ritchie JM, Rogart RB, Stagg D. A quantitative description of membrane currents in rabbit myelinated nerve. J Physiol (Lond) 1979;292:149–66. [19] Lee-Son S, Wang GK, Concus A, Crill E, Strichartz G. Stereo selective inhibition of neuronal sodium channels by local anaesthet-
241
ics: Evidence for two sites of action? Anesthesiology 1992;77:324– 35. [20] Bader AM, Datta S, Moller RA, Covino BG. Acute progesterone treatment has no effect on bupivacaine-induced conduction blockade in the isolated in the isolated rabbit vagus nerve. Anesth Analg 1990;71:545–8. [21] Wang GK. The long-term excitability of myelinated nerve fibres in the transacted frog sciatic nerve. J Physiol (Lond) 1985;368:309–21.