Anesth Analg 2004;98:677-682
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000101984.50597.E9
ANESTHETIC PHARMACOLOGY
Vasodilation Increases the Threshold for Bupivacaine-Induced Convulsions in Rats
Yutaka Oda, MD PhD,
Tomoharu Funao, MD,
Katsuaki Tanaka, MD, and
Akira Asada, MD PhD
From the Department of Anesthesiology and Intensive Care Medicine, Graduate School of Medicine, Osaka City University, Osaka, Japan
Address correspondence and reprint requests to Yutaka Oda, Department of Anesthesiology and Intensive Care Medicine, Graduate School of Medicine, Osaka City University, 1-5-7 Asahimachi, Abeno-ku, Osaka 545-8586, Japan. Address email to odayou{at}msic med.osaka-cu.ac.jp.
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Abstract
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Bupivacaine affects the vascular resistance by peripheral and central nervous system (CNS) mechanisms. As vasoconstrictors increase the CNS toxicity of IV bupivacaine, vasodilators may decrease its CNS toxicity. We examined the hypothesis that vasodilators decrease the CNS toxicity of bupivacaine in awake, spontaneously breathing rats. Male Sprague-Dawley rats were randomly divided into control (C), nicardipine (N), and phentolamine (P) groups (n = 12 in each group). Racemic bupivacaine was administered IV at 1 mg/kg/min until tonic/clonic convulsions occurred. Saline, nicardipine (0.4 µg/min), and phentolamine (10 µg/min within 5 min, 50 µg/min thereafter) were simultaneously administered with bupivacaine in groups C, N, and P, respectively. Mean arterial blood pressure was significantly increased by infusion of bupivacaine in group C and was maintained at baseline levels before the onset of convulsions in groups N and P. The convulsive dose of bupivacaine in group C was 5.8 ± 1.5 mg/kg, but was significantly larger in groups N and P (7.6 ± 1.5 and 8.1 ± 1.1 mg/kg, P = 0.02 and 0.001, respectively). However, there were no differences in total or protein-unbound plasma concentration of bupivacaine or in concentration of bupivacaine in the brain at the onset of convulsions among the 3 groups. We conclude that nicardipine and phentolamine increase the cumulative dose but do not affect the threshold plasma or brain concentrations required for bupivacaine-induced convulsions.
IMPLICATIONS: Bupivacaine, a long-acting local anesthetic, induces central nervous system toxicity when its plasma concentration is increased. Nicardipine and phentolamine increased the cumulative dose but did not affect the threshold plasma concentrations, required for bupivacaine-induced convulsions, suggesting that both nicardipine and phentolamine inhibited the increase in the plasma concentration of bupivacaine by inducing peripheral vasodilation.
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Introduction
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Adding vasoconstrictors to local anesthetics reduces central nervous system (CNS) and cardiovascular toxicity by preventing their absorption from the site of injection into the systemic circulation. However, adding vasoconstrictors increases the CNS toxicity of IV administered local anesthetics (1–4), although the reason for this is unclear. Yokoyama et al. (1) showed that adding epinephrine decreases the plasma concentration as well as the dose of lidocaine required to induce convulsions and suggested that increased intracerebral catecholamine levels or local cerebral ischemia induced by epinephrine-induced hypertension contribute to the increased CNS toxicity of lidocaine. Notably, reversal of epinephrine-induced hypertension by vasodilators increases the convulsive dose and plasma concentration of lidocaine at the onset of convulsions to baseline levels present without epinephrine (1). Bernards et al. (4) showed that adding epinephrine reduces the convulsive dose of bupivacaine; however, the plasma concentration of bupivacaine at the onset of convulsions was not affected by epinephrine, suggesting that the increase in plasma concentration of bupivacaine resulting from the decreased volume of distribution induced by peripheral vasoconstriction accounted for the differential effects of epinephrine on the convulsive dose and the threshold of plasma concentration for convulsions.
Bupivacaine affects the peripheral vascular resistance, and its effects are controversial, with both vasodilation and vasoconstriction having been reported (5–8). Of those reports, IV bupivacaine reduced the diameter of muscle arterioles and increased the systemic vascular resistance with reduced cardiac output at concentrations less than 100 µg/mL (7,8), suggesting the vasoconstrictive effect of bupivacaine. As vasoconstrictors increase CNS toxicity and an increase in the plasma concentration of bupivacaine may be the reason for this, vasodilators may decrease its CNS toxicity by counteracting the vasoconstrictive effect and inhibiting the increase in plasma concentration of bupivacaine. Although several studies have examined the effect of vasodilators on the CNS toxicity of bupivacaine (9,10), the convulsive dose and plasma concentrations of bupivacaine required to induce convulsions have not been measured, and the mechanism by which vasodilators decrease the CNS toxicity of bupivacaine has not been examined.
In this study, we tested the hypothesis that vasodilators decrease the CNS toxicity of bupivacaine by inhibiting an increase in its plasma concentration. We administered vasodilators with different mechanisms of action, nicardipine and phentolamine, to awake, spontaneously breathing rats to maintain mean arterial blood pressure (MAP) at baseline levels before administration of bupivacaine and measured concentrations of bupivacaine in both plasma and brain to elucidate the relationships between convulsive dose and concentration of bupivacaine at the onset of convulsions.
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Methods
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After approval from the Institutional Animal Care and Use Committee, 36 male Sprague-Dawley rats aged 9 wk and weighing 320–360 g (CLEA Japan, Inc., Tokyo, Japan) were included in the study. Under general anesthesia with sevoflurane, the carotid artery and the cervical and femoral veins were cannulated with polyethylene catheters to monitor MAP, heart rate (HR), and blood sampling and infusion of drugs. 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. Rectal temperature was maintained at 37°C with an infrared heat lamp. 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. The animals were divided into control (C), nicardipine (N), and phentolamine (P) groups (n = 12 in each group). After baseline measurement and blood sampling, racemic bupivacaine was infused at 1 mg · kg-1 · min-1 by an infusion pump (Model 1235; Atom, Tokyo, Japan) until tonic/clonic convulsions occurred. Infusion of nicardipine (8 µg/mL) at 0.4 µg/min or phentolamine (200 µg/mL) at 10 µg/min was started at the same time as bupivacaine in groups N and P, respectively. The rate of infusion of phentolamine was increased to 50 µg/min at 5 min after starting infusion. The infusion rates of nicardipine and phentolamine were determined based on our preliminary study to prevent the increase of MAP induced by bupivacaine and to maintain the MAP at baseline. An increase of the rate of infusion of phentolamine was required to prevent the increase of MAP by bupivacaine. In our pilot study, infusion of nicardipine or phentolamine alone did not induce convulsions. Infusion of saline at 50 µL/min was started with bupivacaine in group C. Observation of rats was performed by one of the authors (TF) who was unaware of which groups rats belonged to and judged fine skeletal muscle twitching in the head and digits to be evidence of convulsions. The number of animals in each group was determined based on our previous findings for 10 animals. In that study, the convulsive dose of bupivacaine was 6.2 ± 1.5 mg/kg. Based on the formula for normal theory and assuming type I error protection of 0.05 and a power of 0.80 to detect 30% change of the convulsive dose, 12 animals were required for each of the 3 groups.
Arterial blood (0.3 mL) was drawn before infusion of bupivacaine (baseline) and at the onset of convulsions to determine blood gas tensions and serum potassium levels. At the onset of convulsions, an additional 0.5 mL of blood was obtained to measure the plasma concentrations of bupivacaine. This was immediately followed by IV infusion of pentobarbital (100 mg/kg) to euthanize the animal and perfusion of the brain with 40 mL of ice-cold saline via a catheter inserted in the thoracic aorta and removal of the brain to determine the concentrations of bupivacaine. Blood gas and electrolyte levels were measured immediately after sampling with a blood gas analyzer (ABL4; Radiometer, Copenhagen, Denmark). Remaining blood samples were centrifuged and plasma and brain samples were frozen and kept at -80°C until analysis.
Plasma concentrations of total (protein-bound and unbound) bupivacaine were determined by high-performance liquid chromatography as reported previously (11). Plasma concentrations of unbound bupivacaine were measured after treatment using an ultrafiltration system (Centricon YM-30; Amicon, Inc., Beverly, MA). Briefly, 0.2 mL of plasma was injected into a centrifugation tube with ultrafiltration membrane, centrifuged at 2000g for 40 min, and unbound bupivacaine in the filtrate was measured. Concentrations of bupivacaine in the whole brain were measured after a method described previously (11). Regression coefficients of the calibration curves for bupivacaine in plasma and brain were above 0.99. The lower limits of quantitation were 0.1 µg/mL for bupivacaine in plasma and 0.3 µg/g tissue for bupivacaine in the brain. Intra-assay and inter-assay coefficient of variations were <7% throughout the range of testing. Concentrations of bupivacaine in plasma were the mean of duplicate measurements; concentrations in the brain were the mean of triplicate measurements.
All values are means ± SD. Statistical analysis was performed using StatView 5.0 (SAS Institute Inc. Cary, NC). Differences in convulsive doses and in concentrations of bupivacaine in plasma and brain among the groups were tested using one-way analysis of variance (ANOVA). Differences in blood gas and plasma potassium levels were examined using two-factor functional ANOVA followed by Scheffé testing. Differences in MAP and HR among the groups were examined using ANOVA for repeated measures. If a significant difference among the groups was observed, overall differences among the values of the 3 groups were analyzed by Scheffé tests. In addition, comparisons among the values at baseline, 3 min after administration of bupivacaine, immediately before convulsions, and at the onset of convulsions were performed using one-way ANOVA for repeated measures followed by Scheffé tests to determine which time point had values significantly different from those of baseline. Values were considered significant when P < 0.05.
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Results
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All the animals were included in the study. There were no differences in MAP at baseline among the 3 groups. Overall changes of MAP in groups N and P were significantly different from those in group C (P = 0.03 and 0.01, respectively). In group C, MAP continuously increased during infusion of bupivacaine and was significantly higher than baseline both immediately before convulsions and at the onset of convulsions (P = 0.02 and P < 0.001, respectively). However, MAP in groups N and P was stable and significantly higher than baseline only at the onset of convulsions (P < 0.001). In groups N and P, MAP was significantly lower than in group C both immediately before and at the onset of convulsions (Fig. 1). HR was significantly slower than baseline at 3 min after starting bupivacaine infusion, immediately before convulsions, and at the onset of convulsions in all three groups (P < 0.001), but there were no differences among groups at any time points (Fig. 2). There were no differences in blood gas data or plasma potassium levels at baseline or at the onset of convulsions among the groups, with the exception that base excess at baseline was significantly lower in group P than in group C (Table 1).
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Figure 2. Heart rate at baseline, 3 min after starting bupivacaine infusion (3 min), immediately before convulsions, and at the onset of convulsions in the control (C), nicardipine (N), and phentolamine (P) groups. There were no differences in overall changes of heart rate among the 3 groups. Heart rates at 3 min after starting bupivacaine infusion, immediately before convulsions, and at the onset of convulsions were significantly slower than that at baseline.
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The doses of bupivacaine required to induce convulsions in groups N and P were significantly larger than that in group C (P = 0.02 and 0.001, respectively). However, there were no differences in plasma concentration of total, unbound bupivacaine or concentration of bupivacaine in the brain among the three groups. There were no differences in the tissue-to-plasma concentration ratio of bupivacaine among groups, either (Table 2).
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Discussion
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Nicardipine and phentolamine each increased the convulsive dose of bupivacaine. However, plasma concentrations of bupivacaine at the onset of convulsions were identical in the 3 groups, suggesting that these drugs did not affect the threshold of plasma concentration for convulsions. A likely explanation is that addition of nicardipine or phentolamine to bupivacaine produced peripheral vasodilation, which resulted in increased volume of distribution. As a result, the CNS was exposed to a smaller plasma concentration of bupivacaine than would have been the case without nicardipine or phentolamine. This explanation is supported by the very similar concentrations of bupivacaine in the brain at the onset of convulsions among the groups. In our preliminary study, using the same animal model as in this study, intermittent administration of nicardipine or phentolamine to prevent an increase of MAP by bupivacaine significantly increased the convulsive dose of bupivacaine, further supporting this explanation.
As we have not measured the pharmacokinetic changes of bupivacaine induced by nicardipine or phentolamine, we cannot eliminate the possibility that increased clearance of bupivacaine from the plasma by these vasodilators accounts for the finding of comparable plasma concentrations of bupivacaine in the groups despite the larger cumulative doses in groups N and P than in C group. This is a major limitation of our study. However, elimination of bupivacaine from plasma depends principally on hepatic metabolic activity rather than hepatic blood flow because of its low hepatic extraction ratio (12,13), and neither nicardipine or phentolamine would have increased hepatic metabolic activity and thereby increased clearance of bupivacaine. There were no differences in plasma concentration of protein-unbound bupivacaine at the onset of convulsions among the groups, suggesting that neither nicardipine nor phentolamine affected the protein binding of bupivacaine.
We performed experiments in awake, spontaneously breathing rats. This experimental condition is of clinical importance because the symptoms of bupivacaine-induced CNS toxicity such as agitation, sedation, and convulsions precede the manifestation of life-threatening cardiovascular toxicity. These symptoms are diminished and the convulsive dose of bupivacaine is increased by general anesthesia (14). In awake animals, physiological changes such as acidosis and decrease of carbon dioxide tension may affect the CNS toxicity of local anesthetics (15). In the present study, although overall arterial blood pH was higher because of the decrease in carbon dioxide tensions by hyperventilation at the onset of convulsions than at baseline, there were no differences in these values among the groups at baseline or at the onset of convulsions, suggesting that changes in blood gases did not differentially affect the convulsive potency of bupivacaine among the groups. The convulsive doses and concentrations of bupivacaine both in plasma and in brain in the control group were similar to those reported previously (16).
In the present study, continuous infusion of bupivacaine significantly increased MAP in group C, as noted in another report (4). Bupivacaine-induced hypertension has been previously studied and the possible mechanism is that bupivacaine inhibits the intracerebral 
-aminobutyric acid-ergic neurons, increasing the autonomic nervous system outflow from the brainstem (17,18). MAP was maintained at baseline levels by vasodilators and abruptly increased at the onset of convulsions in groups N and P. An increase of MAP increases the permeability of the blood-brain barrier and the diffusion of amines from peripheral blood vessels into the brain (19,20), which has been suggested to be one mechanism of increase in CNS toxicity of lidocaine by addition of epinephrine (1). In our study, MAP was significantly higher in group C than in groups N and P immediately before and at the onset of convulsions, but the concentration of bupivacaine in the brain was comparable among the three groups, suggesting that entry of bupivacaine into the brain would not be affected by the degree of increase in MAP.
We examined the effects of vasodilators with different mechanisms of action. Nicardipine is a calcium channel blocker commonly used clinically for treatment of hypertension. Although bupivacaine is used for epidural analgesia in patients receiving calcium channel blockers, few studies have examined their effects on the toxicity, particularly the CNS toxicity, of bupivacaine (9,10,21,22). Of calcium channel blockers, nicardipine increases the survival rate of rats receiving bupivacaine (9), a finding that is consistent with our results. Nimodipine, another calcium channel blocker with a peripheral vasodilating effect, also reduces the toxicity of bupivacaine (10). However, some calcium channel blockers, such as diltiazem and verapamil, enhance the toxicity of bupivacaine (21,22), although the reason for the differences in effects between drugs is not clear. Phentolamine is a nonselective 
-adrenoceptor antagonist and would have counteracted the vasoconstrictive effect of bupivacaine via 1-blocking effect and inhibited the increase in plasma concentration of bupivacaine observed in the present study. In contrast to bupivacaine, phentolamine has no protective effect against lidocaine-induced convulsions (23), suggesting that it does not affect the increase in plasma concentration of lidocaine. This resulted from differences in effect of phentolamine on the concentrations of bupivacaine and lidocaine, although the reasons for these differences are not known in detail.
Selective 2-adrenoceptor agonists such as clonidine, dexmedetomidine, and tizanidine reduce the convulsive potency of local anesthetics, a change that is reversed by 2-adrenoceptor antagonists (23–25). Importantly, dexmedetomidine increases the threshold of plasma concentration as well as the convulsive dose of bupivacaine (Tanaka K et al., unpublished data). Although the reason for this is not entirely clear, 2-adrenoceptor agonists may modulate levels of intracerebral catecholamines, which contribute to the onset of convulsions (26). Recent studies using a microdialysis technique showed that convulsions were suppressed by inhibiting the increase in intracerebral dopamine by dexmedetomidine (25). In our study, phentolamine did not affect the threshold of plasma concentration of bupivacaine for inducing convulsions despite its 2- as well as 1- blocking effect, suggesting that phentolamine affected only peripheral blood vessels rather than 2-adrenoceptors in the brain, as found in previous studies (27).
In summary, we have shown that nicardipine and phentolamine increased the convulsive dose of bupivacaine. Because these drugs did not affect the concentrations of total or protein-unbound bupivacaine in either plasma or brain, inhibition of the increase of plasma concentration of bupivacaine by vasodilation is the reason for this increase.
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Acknowledgments
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Supported, in part, by a Fund for Medical Research from the Osaka City University Medical Research Foundation (2001) and by Grants-in-Aid for Research from the Ministry of Education, Science and Culture of Japan, Nos. 11671517 and 14571460.
We thank Prof. Hisayo O. Morishima and Dr. Masataka Yokoyama for their critical comments.
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Accepted for publication October 1, 2003.
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Abstract
Introduction
P < 0.05, 
