Anesth Analg 2008; 106:1450-1455
© 2008 International Anesthesia Research Society
doi: 10.1213/ane.0b013e31816ba49d
ANESTHETIC PHARMACOLOGY
Propranolol Increases the Threshold for Lidocaine-Induced Convulsions in Awake Rats: A Direct Effect on the Brain
Taketo Nakamura, MD,
Yutaka Oda, MD, PhD,
Ryota Takahashi, MD, PhD,
Katsuaki Tanaka, MD, PhD,
Ichiro Hase, MD, PhD, and
Akira Asada, MD, PhD
From the Department of Anesthesiology, Osaka City University Graduate School of Medicine, Osaka, Japan.
Address correspondence and reprint requests to Yutaka Oda, MD, PhD, Department of Anesthesiology, Osaka City University Graduate School of Medicine, 1-5-7 Asahimachi, Abeno-ku, Osaka 545-8586, Japan. Address e-mail to odayou{at}msic.med.osaka-cu.ac.jp.
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Abstract
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BACKGROUND: Propranolol is a β-adrenoceptor antagonist used clinically. Local anesthetics are used for controlling pain, whereas propranolol is concomitantly given to treat hypertension and tachycardia. However, there are few studies examining the effects of propranolol on the toxicity of local anesthetics. We investigated the effect of propranolol on lidocaine-induced convulsions in awake, spontaneously breathing rats.
METHODS: Male Sprague-Dawley rats were randomly divided into six groups (n = 8, each group). Rats were pretreated with intracerebroventricular saline (cerebroventricle- control: CV-C group), 10 or 30 µg/kg of (S)-(–)-propranolol (propranolol) (cerebroventricle-small dose: CV-S and cerebroventricle-large dose: CV-L groups, respectively) or IV saline (IV-control: IV-C group), 1 or 3 mg/kg of propranolol (IV-small dose: IV-S and IV-large dose: IV-L groups, respectively). Three minutes later, lidocaine was administered IV at 4 mg · kg–1 · min–1 until tonic-clonic convulsions occurred.
RESULTS: The convulsive dose of lidocaine in the CV-L group was significantly larger than that in the CV-C group (30.6 ± 5.1 vs 23.5 ± 2.2 mg/kg, respectively, P = 0.008). Plasma concentrations of total and protein-unbound lidocaine, concentrations of lidocaine in the brain at the onset of convulsions were also significantly higher in the CV-L group than those in the CV-C group (36.1 ± 4.8 vs 26.0 ± 3.8 µg/mL, 22.5 ± 3.5 vs 13.7 ± 2.6 µg/mL, 82.7 ± 7.1 vs 57.3 ± 5.7 µg/g, P < 0.001 for all). The convulsive dose, plasma concentrations of total and protein-unbound lidocaine, and brain lidocaine in the IV-L group were also significantly larger than those in IV-C group and comparable with those in the CV-L group. The plasma concentration of propranolol before starting an infusion of lidocaine in the IV-L group was approximately 60-fold higher than that in the CV-L group (554.7 ± 104.6 and 9.3 ± 6.7 ng/mL, respectively).
CONCLUSIONS: Propranolol increased the threshold for lidocaine-induced convulsions by directly acting on the brain.
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Introduction
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β-Adrenoceptor antagonists exhibiting negative inotropic and chronotropic effects have been extensively used for treating hypertension and tachycardia. Besides their cardiovascular effects, β-adrenoceptor antagonists act directly on the central nervous system (CNS) to exert a sedative effect; therefore, they are used for treating anxiety disorders and acute stress reactions.1–3 Propranolol, a β-adrenoceptor antagonist, has anticonvulsant effects equipotent to phenytoin in addition to sympatholytic and cardiac effects.4 There have been several studies describing the anticonvulsant effects of propranolol; however, those studies were predominantly performed in animal models of electrically or chemically induced convulsions.4–6 Only a few studies have examined such effects on local anesthetic-induced convulsions and, furthermore, their results are still controversial.7–9 β-Adrenoceptor antagonists are commonly used for blunting adrenergic responses to noxious stimuli in combination with epidural and local infiltration anesthesia, and CNS toxicity is one of the common and life-threatening side effects of local anesthetics. In the present study, we examined the hypothesis that propranolol might increase the threshold for lidocaine-induced convulsions. For precise examination of the direct effect of propranolol on CNS, we administered propranolol IV and intracerebroventricularly to compare the effects of different administration routes.
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METHODS
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After approval from the Institutional Animal Care and Use Committee, 48 male Sprague-Dawley rats aged 8–9 wk and weighing 245–350 g (Clea Japan, Inc., Tokyo, Japan) were included in this study. Two or three days before experiments, rats were anesthetized with intraperitoneal ketamine, and the guide cannulas (12 mm length and 0.5 mm OD, GI-12; Eicom, Kyoto, Japan) were installed using a stereotaxic instrument (Narishige, Tokyo, Japan) to position the tip of the cannulas in the lateral cerebroventricle (posterior 0.8 mm, lateral 1.5 mm, vertically 5.0 mm from the Bregma suture) as reported previously.10 The position of the cannulas was checked by staining with Luxol fast blue and Saranin-O during our preliminary study.
On the day of experiments, the carotid artery and the cervical vein were cannulated with polyethylene catheters for monitoring mean arterial blood pressure (MAP) and heart rate (HR), as well as for blood sampling and infusion of drugs under general anesthesia with sevoflurane. These catheters were tunneled subcutaneously to the posterior cervical region so that the animals could move freely. Before emergence from anesthesia, the animals were placed in a plastic container to recover for at least 4 h before the experiment. During that time, the arterial catheter was connected to a pressure transducer, and MAP and HR were recorded continuously on a polygraph (RM-6000; Nihon Kohden, Tokyo, Japan) connected to a computer. HR was monitored with a cardiotachometer triggered by arterial pressure.
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EXPERIMENTAL PROCEDURES
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The animals were divided into six groups (n = 8, each group). After baseline measurement and blood sampling, three groups were assigned to receive infusion of saline as the cerebroventricle-control (CV-C) group, 10 or 30 µg/kg of (S)-(–)-propranolol (propranolol) as the cerebroventricle-small (CV-S) or the cerebroventricle-large (CV-L) dose groups, respectively, into the lateral cerebroventricle using a microsyringe pump (ESP-32: Eicom, Kyoto, Japan). Concentrations of intracerebroventricular propranolol were 1 and 3 mg/mL for CV-S and CV-L groups, respectively, and 10 µL/kg solution was administered in each group at a rate of 10 µL/min. The remaining three groups were assigned to receive IV infusion of saline, 1 or 3 mg/kg of propranolol (total volume of 1 mL/kg) as the IV-control (IV-C) group, the IV-small (IV-S), or the IV-large (IV-L) dose groups, respectively. Three minutes after the end of propranolol infusion, continuous IV infusion of lidocaine (4 mg · kg–1 · min–1) was started and continued until the onset of convulsions, which were defined as the occurrence of repeated backward falling accompanied by generalized tonic/clonic movements, as reported previously.11 Observation of rats was performed by one of the authors (Y.O.), who was unaware of group allocation.
Arterial blood (0.3 mL) was drawn before infusion of propranolol (baseline), before commencement of lidocaine infusion, and at the onset of convulsions to determine blood gas tensions and serum electrolyte levels. Before starting lidocaine infusion and at the onset of convulsions, an additional 0.5 mL of blood was obtained to measure the plasma concentrations of propranolol and lidocaine, respectively. Blood was replaced with the same volume of saline and blood sampling did not affect MAP or HR. At the onset of convulsions, blood sampling was immediately followed by IV infusion of thiopental (100 mg/kg) to euthanize the animals. After thoracotomy, the brain was perfused with 40 mL of ice-cold saline via a catheter inserted in the thoracic aorta and removed to determine the concentrations of lidocaine. Blood gas and electrolyte levels were measured immediately after sampling with a blood gas analyzer (ABL4; Radiometer, Copenhagen, Denmark). The remaining blood samples were centrifuged and plasma and brain samples were frozen and kept at –80°C until analysis.
The number of animals per group was determined based on our preliminary experiments. In that study, the convulsive dose of lidocaine was 25 ± 3 mg/kg. We assumed that a 20% increase of the convulsive dose of lidocaine by propranolol would be needed. On the basis of analysis of variance, sample size calculations, and assuming a Type I error protection of 0.05 and a power of 0.80, eight animals were required in each of the groups. The findings of our preliminary study—that more than 30 µg/kg of intracerebroventricular, or more than 3 mg/kg of IV propranolol induced significant respiratory depression during IV infusion of lidocaine—guided our decision regarding the dose of propranolol used in this study.
Measurement of Lidocaine and Propranolol Concentrations
Concentrations of lidocaine and propranolol were measured by high-performance liquid chromatography-mass spectrometry. The plasma concentration of protein-unbound lidocaine was determined after ultrafiltration using a membrane (Centricon YM-30; Amicon, Inc., Beverly, MA). Lidocaine in plasma and in the whole brain homogenate was extracted by the method reported previously.12 In the present study, racemic bupivacaine was used as an internal standard. Propranolol in plasma was extracted using a solid-phase extraction column (Oasis MCX, Waters, Milford, MA), followed by an addition of midazolam as an internal standard. All samples were reconstructed with liquid chromatography-mass spectrometry mobile phase and were injected onto high-performance liquid chromatography (Agilent 1100 series, Palo Alto, USA) fitted with a C18 column (ODS-100Z, 20 x 50 mm, particle size 5 µm, Tosoh, Tokyo, Japan) with use of an isocratic mobile phase consisting of 10 mM ammonium acetate (pH 5.0) and acetonitrile (51/49, v/v) at a flow rate of 0.2 mL/min. Analysis was performed by 4000 Qtrap tandem mass spectrometer (Applied Biosystems, Foster City, CA) equipped with an electrospray ionization interface and was operated in the positive ionization mode. The interface was maintained at 600°C and detection was performed at m/z 235.2 and 289.3 for measuring lidocaine and bupivacaine, respectively. It was maintained at 700°C and detection was performed at m/z 261.3 and 327.2 for propranolol and midazolam, respectively. The peak area ratios of lidocaine to bupivacaine, propranolol to midazolam were used to calculate the concentrations based on least-squares regression of calibrators (0.5–100 µg/mL for lidocaine, 1–1000 ng/mL for propranolol) included in each run. The lower limit of quantitation was 0.5 µg/mL and 1 ng/mL for lidocaine and propranolol, respectively. The coefficients of within-day and between-day variance of lidocaine were 4.8% and 5.5%, respectively, at 10 µg/mL. The coefficients of within-day variance of propranolol were 3.8% and 4.8% and between-day variance were 5.0% and 5.0% at concentrations of 10 and 200 ng/mL, respectively.
Statistics
All values are expressed as mean ± sd. Statistical analysis was performed using Stat View 5.0 (SAS Institute Inc., Cary, NC) and Sigma Stat 3.0 (Systat Software Inc., Richmond, CA). Differences in convulsive doses and in concentrations of lidocaine in plasma and brain among groups CV-C, CV-S, and CV-L and among groups IV-C, IV-S, IV-L groups were tested using one-way analysis of variance (ANOVA) followed by Scheffé testing. Differences in blood gas and plasma electrolyte levels were examined using two-factor functional ANOVA. Differences in MAP and HR during the all experiments were examined using ANOVA for repeated measures; subsequently, we examined the differences of MAP and HR within the same study groups at baseline, before infusion of lidocaine, and at the onset of convulsions, and between different groups at the same time points using Scheffé test, considering the number of measurements. Values were considered significant when P < 0.05.
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RESULTS
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Blood Gas and Hemodynamic Data
Rats were not sedated during experiments. Plasma concentrations of propranolol before starting infusion of lidocaine were 3.2 ± 3.0, 9.3 ± 6.7, 164.1 ± 50.9, and 554.7 ± 104.6 ng/mL in groups CV-S, CV-L, IV-S, and IV-L, respectively. There were no differences in blood gas data within groups, among groups CV-C, CV-S, and CV-L or among groups IV-C, IV-S, and IV-L at baseline, before infusion of lidocaine, or at the onset of convulsions (Tables 1 and 2). There were no within-group differences in MAP in any groups, no between-group differences among groups CV-C, CV-S, and CV-L or among groups IV-C, IV-S, and IV-L, either (Figs. 1 and 2). HR was decreased during infusion of lidocaine, significantly lower in groups CV-S and CV-L than CV-C group (P = 0.04 and 0.02, respectively, Fig. 1), and significantly lower in groups IV-S and IV-L than IV-C group during the whole experiment (P = 0.02 and 0.01, respectively, Fig. 2); however, there were no differences in HR among groups CV-C, CV-S, and CV-L or among groups IV-C, IV-S, and IV-L at any time points.
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Figure 1. Mean arterial blood pressure (MAP) and heart rate (HR) at baseline, before infusion of lidocaine, 3 min after starting infusion of lidocaine (3 min), immediately before convulsions, and at the onset of convulsions after cerebroventricular infusion of 0, 10, or 30 µg/kg of (S)-(–)-propranolol (cerebroventricle-control: CV-C, cerebroventricle-small dose: CV-S and cerebroventricle-large dose: CV-L groups, respectively). Overall changes of HR in CV-S and CV-L groups were significantly different from those in CV-C group (P = 0.04 and 0.02, respectively). **P < 0.01 compared with baseline within the group.
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Figure 2. Mean arterial blood pressure (MAP) and heart rate (HR) at baseline, before infusion of lidocaine, 3 min after starting infusion of lidocaine (3 min), immediately before convulsions and at the onset of convulsions after IV infusion of 0, 1, or 3 mg/kg of (S)-(–)-propranolol (IV-control: IV-C, IV-small dose: IV-S and IV-large dose: IV-L groups, respectively). Overall changes of HR in IV-S and IV-L groups were significantly different from those in IV-C group (P = 0.02 and 0.01, respectively). **P < 0.01 compared with baseline within the group.
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Convulsive Dose, Plasma, and Brain Concentration of Lidocaine
The convulsive dose of lidocaine in the CV-L group was significantly larger than that in group CV-C (P = 0.008, Table 3). Plasma concentrations of total and protein-unbound lidocaine and brain lidocaine at the onset of convulsions in group CV-L were significantly higher than those in the CV-C group (P < 0.001 for all), although there were no differences in these values between groups CV-C and CV-S.
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Table 3. Convulsive Dose and Concentration of Lidocaine in Plasma and Brain at the Onset of Convulsions After Cerebroventricular Infusion of Propranolol
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The convulsive dose of lidocaine in group IV-L was significantly larger than that in group IV-C (P = 0.03, Table 4). Plasma concentrations of total and protein-unbound lidocaine in group IV-L were significantly higher than those in group IV-C (P < 0.001 and P = 0.01, respectively). Concentrations of lidocaine in the brain in the IV-S and IV-L groups were significantly higher than that in the IV-C group (P = 0.04 and P < 0.001, respectively).
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Table 4. Convulsive Dose and Concentration of Lidocaine in Plasma and Brain at the Onset of Convulsions After Intravenous Infusion of Propranolol
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DISCUSSION
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In the present study, intracerebroventricular propranolol significantly increased the convulsive dose and threshold plasma concentration of lidocaine for inducing convulsions. IV propranolol also exerted an anticonvulsant effect at plasma concentrations of the same order of magnitude as those required for controlling HR in humans13; however, the plasma concentration of propranolol required to produce a comparable anticonvulsant effect was approximately 60-fold higher than that by intracerebroventricular injection. These results suggest that IV propranolol acts directly on the brain to exert an anticonvulsant effect.
The anticonvulsant effects of β-adrenoceptor antagonists have been well documented in animal models, where electric stimulation or chemicals such as penthylenetetrazol have predominantly been used for inducing convulsions.4–6 However, the mechanisms of convulsions induced by local anesthetics are different from those by electroshock or chemicals, in that sedation frequently precedes local anesthetics-induced convulsions, and the threshold for lidocaine-induced, but not for penthylenetetrazol-induced, convulsions increases by manipulating intracerebral monoamines.14 Although several studies have examined the effect of propranolol on local anesthetic-induced toxicity,7–9 plasma concentrations of propranolol or local anesthetics have not been measured and, moreover, whether they induce or inhibit convulsions remains to be clarified. In the present study, it was found that intracerebroventricular propranolol increased the threshold for lidocaine-induced convulsions in an awake rat model. Since the intracerebral noradrenergic system mediated by the β-adrenoceptor is involved in controlling convulsions,15,16 our results might suggest that propranolol exerted an anticonvulsant effect via the noradrenergic system.
Unlike previous studies,1–3 the sedative effects of propranolol were not observed in the present study before infusion of lidocaine. This difference might be attributed to the necessity of a longer observation time or larger dose of propranolol than in our study to exert its sedative effects. In awake animals, physiological changes, such as acidosis and a decrease of arterial carbon dioxide tension, are thought to affect the CNS toxicity of local anesthetics.17 In the present study, there were no differences in blood pH or carbon dioxide tensions within or between groups at any time point, suggesting that blood gas data did not influence the convulsive potency of lidocaine in any group. A major metabolite of lidocaine, monoethylglycinexylidide, also has convulsive potency at the ratio of approximately 80% of that of lidocaine.18 However, the plasma concentration of monoethylglycinexylidide was <10% of that of lidocaine in our previous study following a similar protocol,12 suggesting that monoethylglycinexylidide would have made a small contribution to the results of the present study, whether its plasma concentration was influenced by propranolol or not.
We used S(–)-propranolol as the β-adrenoceptor antagonist, since the anticonvulsant potency of this stereoisomer is higher than R(+)-isomer while the lack of effects of R(+)- and racemic isomer on chemically induced convulsions has been reported previously.5 The IV dose of propranolol used in the present study is comparable to those for suppressing convulsions after systemic administration.5,6 Since clearance of propranolol from the blood is predominantly dependent on hepatic blood flow,19 and since there were no changes in MAP during the experiments in any groups, there would still be large differences in the plasma concentrations of propranolol after intracerebroventricular and IV administration observed before infusion of lidocaine at the onset of convulsions.
One of the major limitations of the present study is that the mechanisms of the anticonvulsant effect of propranolol on lidocaine-induced convulsions are not clear. Measurement of intracerebral monoamines, such as dopamine and noradrenaline, would be required for elucidating the mechanism. Second, we only examined the effect of propranolol on the convulsive potency of lidocaine, not other local anesthetics. Further studies are required for extrapolating the results of the present study to other local anesthetics.
In summary, we have examined the effect of intracerebroventricular and IV propranolol on lidocaine-induced convulsions. Intracerebroventricular propranolol increased the convulsive dose of lidocaine to an approximately 60-fold lower concentration compared with IV administration. Most likely, systemically administered propranolol exerts its anticonvulsant potency through direct actions on the brain.
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Footnotes
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Accepted for publication January 22, 2008.
Supported, in part, by Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology in Japan, no. 17591648 and 18791098.
Yutaka Oda, Department of Anesthesiology, Osaka City University Graduate School of Medicine, 1-5-7 Asahimachi, Abeno-ku, Osaka 545-8586, Japan.
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Abstract
Introduction
