Anesth Analg 2001;92:733-738
© 2001 International Anesthesia Research Society
REGIONAL ANESTHESIA AND PAIN MEDICINE
Antinociceptive Interaction Between Spinal Clonidine and Lidocaine in the Rat Formalin Test: An Isobolographic Analysis
Shuanglin Hao, MD, PhD,
Osamu Takahata, MD, PhD, and
Hiroshi Iwasaki, MD, PhD
Department of Anesthesiology & Critical Care Medicine, Asahikawa Medical College, Asahikawa, Japan
Address correspondence to Shuanglin Hao, MD, PhD, Department of Neurology, University of Pittsburgh, S-522, BST, Pittsburgh, PA 15213. Address reprint requests to Dr. Hiroshi Iwasaki, Department of Anesthesiology & Critical Care Medicine, Asahikawa Medical College, Midorigaoka-Higashi, 2-1-1-1, Asahikawa, 078-8510 Japan.
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Abstract
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Clinical and basic science studies suggest that spinal 
-2-adrenergic receptor agonists and local anesthetics produce analgesia, but interaction between -2-adrenergic receptor agonists and local anesthetics in the persistent pain model has not been examined. In the present study, using isobolographic analysis, we investigated the antinociceptive interaction of intrathecal clonidine and lidocaine in the rat formalin test. Sprague-Dawley rats were implanted with chronic lumbar intrathecal catheters, and were tested for paw flinch by formalin injection. Biphasic painful behavior was counted. Intrathecal clonidine (3–12 nmol) was administered 15 min before formalin, and intrathecal lidocaine (375–1850 nmol) was administered 5 min before formalin. To examine the interaction of intrathecal clonidine and lidocaine, an isobolographic design was used. Spinal administration of clonidine produced dose-dependent suppression of the biphasic responses in the formalin test. Spinal lidocaine resulted in dose-dependent transient motor dysfunction and the motor dysfunction recovered to normal at 10–15 min after administration. Spinal lidocaine produced dose-dependent suppression of phase-2 activity in the formalin test. Isobolographic analysis showed that the combination of intrathecal clonidine and lidocaine synergistically reduced Phase-2 activity. We conclude that intrathecal clonidine synergistically interacts with lidocaine in reducing the nociceptive response in the formalin test.
Implications: Preformalin administration of intrathecal clonidine and lidocaine dose-dependently produced antinociception in the formalin test. The combination of clonidine and lidocaine, synergistically produced suppression of nociceptive response in the persistent pain model.
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Introduction
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Injection of formalin into the hindpaw of the rat leads to a hyperalgesic state involving protracted afferent input and produces a biphasic nociceptive response; Phase 1 reflects the acute pain response and Phase 2 reflects the increased input from primary afferent fiber after tissue injury and local inflammatory response (1). Similarly, some of the clinical features of pain in humans are considered a consequence of injury-induced sensitization of dorsal horn neuron (2).
The spinal cord is considered to be an important site for the action of drugs in mediation of pain relief. Several classes of drugs act spinally to alter nociceptive processing. There has been increasing research in recent years on drug combinations that produce analgesia in experimental animals that may be useful in clinical populations. The general purpose of the combinations is to enhance analgesia by either synergism or additive effects, and to reduce the side effects of the drugs by either reducing the dose of each drug or allowing the drugs to interact, so that one drug reduces the side effects of the other. In cancer pain management, World Health Organization guidelines emphasize the combination of nonopioid drugs. The combination of spinal clonidine and lidocaine possesses antinociceptive synergy in the rat tail-flick test (3). In the neuronal mechanism, the tail-flick test is different from the formalin test. The tail-flick reflex is organized at the level of the spinal cord and can be elicited in spinally transected rats, whereas behavior responses (e.g., flinching or licking the paw injected) in the formalin test are mediated by both spinal and supraspinal structures (4). A body of evidence shows that spinal clonidine and lidocaine produce a significant suppression of the formalin-induced nociceptive response (1,5). However, the character of the antinociceptive interaction between intrathecal clonidine and lidocaine in the rat formalin test is unclear.
In the current study, we sought to define the antinociceptive effects of intrathecal clonidine and lidocaine in the formalin test, and characterize the spinal interaction between the two drugs by using isobolographic analysis. The formalin test was chosen because it is a model of tonic inflammatory pain and resembles clinically relevant pain in humans.
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Methods
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The experimental protocol was approved by the Animal Experiment Committee of Asahikawa Medical College. Male Sprague-Dawley rats (250–350 g) were housed in individual cages with free access to food and water, and maintained on a 12-h light-dark cycle at an ambient temperature (21° ± 1°C). On the day of testing, rats were removed from the animal-care facility to the testing area at least 1 h before testing.
Chronic intrathecal catheters were implanted using isoflurane anesthesia. Briefly, through an incision in the atlanto-occipital membrane, a polyethylene (PE-10) catheter, filled with 0.9% saline, was advanced 8.5 cm caudally to position its tip at the level of the lumbar enlargement. The rostral tip of the catheter was passed subcutaneously, externalized on top of the skull, and sealed with a stainless-steel plug. Animals showing neurological deficits after implantation were excluded. Rats were neither used sooner than 4 days nor later than 10 days after implantation.
For formalin injection, 50 µL of 5% formalin was injected subcutaneously into the dorsal surface of the right hindpaw using a 27-gauge needle. Animals were then placed in a clear Plexiglas cylinder (20 x 30 cm) for observation. A mirror was placed below the floor (Plexiglas) at a 45-degree angle for unencumbered observation during the test. Within 1 min after the injection, the rat displayed the typical behavior of this model, i.e., holding the injected paw just off the floor. During this period, spontaneous flinching of the injected paw could also be observed. Flinching is readily discriminated and is characterized as a rapid and brief withdrawing or flexing of the injected paw. Pain-related behavior was quantified by counting the number of flinches for 1-min periods at 1–2 min and 5–6 min, and then at 5-min intervals during the period from 10 to 60 min after the formalin injection. Two phases of spontaneous flinching behavior were observed. An initial acute pain response (Phase 1, during the 0- to 6-min interval after the formalin injection) was followed by a relatively short quiescent period and then by a prolonged tonic response (Phase 2, beginning approximately 10 min after the formalin injection). Criteria for exclusion from the study included incomplete formalin injection, or excessive bleeding from the injection site.
Motor blockade was graded according to the scale proposed by Langerman et al. (6), for rabbits, which we applied to the rat model as follows: 0 = free movement of hindlimbs without limitation, 1 = limited or asymmetrical movement of the hindlimbs to support the body and walk, 2 = inability to support the back of the body on the hindlimbs, with detectable movement of the limbs and response to a pain stimulus, and 3 = total paralysis of the hindlimbs.
Time-response data were presented as the mean ± SEM per minute for the period of 1 to 2 min, 5 to 6 min, and then for 1-min periods at 5-min intervals up to 60 min. For the dose-response analysis, data from Phase 1 and Phase 2 observations were considered separately. In each case, the mean of the responses per minute obtained for each counting period in the respective observation intervals was calculated for each rat. The cumulative flinching response was calculated for each animal, and the dose-response curve represents the mean ± SEM.
To determine the dose dependency and time course of the antinociceptive action of an intrathecal injection of clonidine, animals were randomly assigned to four groups receiving intrathecal injection of different doses of clonidine 15 min before formalin: 3, 6, 9, and 12 nmol (n = 8–12). In the control group, intrathecal saline was given. These doses were chosen based on preliminary experiments. To determine the dose dependency and time course of the antinociceptive action of intrathecal lidocaine, animals were randomly organized to four groups receiving intrathecal injection of different doses of lidocaine 5 min before formalin: 375, 750, 1500, and 1850 nmol (n = 8–12). In the control group, intrathecal saline was given. These doses were chosen based on preliminary experiments and a previous study (3). The dose-response lines for the Phase 1 and Phase 2 effects then were fitted by using least squares linear regression analysis, and the doses that resulted in 50% of the saline response (ED50) and their 95% confidence intervals (CI95) were calculated.
Isobolographic analysis for drug-drug interaction was conducted according to the procedure of Tallarida et al. (7). The method is based on comparisons of doses that are determined to be equieffective. To perform the isobolographic analysis, clonidine and lidocaine were administered in combination as fixed ratios of equieffective ED50 dose for each drug (clonidine/lidocaine = 1:200 nmol). As determined in preliminary studies, clonidine and lidocaine were administered intrathecally 15 min and 5 min, respectively, before the formalin test so that the peak effect of each drug coincided. Rats randomly received one of the following combinations of clonidine and lidocaine: 3.5:700 nmol, 1.5:300 nmol, 0.5:100 nmol, and 0.15:30 nmol (n = 8–12). The dose-response curve of the combined drugs was used to calculate the actual (experimental) ED50 value and CI95.
The isobolos were drawn by plotting the experimental determined ED50 value of lidocaine on the x axis and that of clonidine on the y axis, delivered alone and in combination. The theoretically additive ED50 value, assuming simple additivity and CI95, was calculated according to Tallarida (8). For statistical comparison of the difference between the theoretical additive point and the experimentally derived ED50 value, student’s t-test was used. To describe the magnitude of the interaction, a total dose fraction value (see formula below) was calculated. The value of total dose fraction = ([ED50 dose in combination of drug 1]/[ED50 value for Drug 1 given alone]) + ([ED50 dose in combination of Drug 2]/[ED50 value for Drug 2 given alone]). The fractional value describes the experimental ED50 as a fraction of the additive ED50. A value near 1 indicates additive interaction, a value >1 implies an antagonistic interaction, and a value <1 indicates a synergistic, multiplicative interaction.
Drugs used in the study included clonidine hydrochloride (Nacalai Tesque, Kyoto, Japan) and lidocaine hydrochloride (Research Biochemicals International, Natick, MA). The drugs were delivered with a microsyringe in a total volume of 10 µL followed immediately by 10 µL of saline to flush the catheter. All drugs were dissolved in saline.
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Results
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Preformalin administration of intrathecal clonidine at the doses used in the study did not affect motor function during the observation period (75 min). Intrathecal lidocaine dose- and time-dependently resulted in motor dysfunction ( Table 1). The motor dysfunction was reliably localized and forelimb function was unaffected. During the recovery phase of the block, animals retained the ability to withdraw the paw evoked by applied pressure. Fifteen minutes after the injection of lidocaine, motor function recovered to normal. Thus, considering that formalin was injected at 5 min after administration of intrathecal lidocaine and that Phase 2 began 10 min after injection of formalin, we think that the motor dysfunction was not sufficient to affect observation of the Phase 2 response of the formalin test. In the group consisting of the combination of 3.5 nmol of clonidine and 700 nmol of lidocaine, some rats showed limited movement of the hindlimbs to support the body (scale = 1) (Table 1). There was not significant motor dysfunction observed in the other combinations of clonidine and lidocaine.
Injection of formalin into the hindpaw of the rat resulted in a reliable flinching response with two distinct phases. The biphasic behavioral activities are indicated in the typical results presented in Figure 1. The timing or magnitude of the behavioral response did not differ in the control groups (intrathecal saline 15 min and/or 5 min before formalin; one-way analysis of variance, P > 0.05). The control experiments were, therefore, pooled and used as a common control group.
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Figure 1. Time-effect curves of intrathecal clonidine (Clo) (A), lidocaine (Lido) (B), and the combination of clonidine and lidocaine (C) administered before formalin. The number of flinches per minute is plotted versus the time after the formalin injection into the hindpaw. Each line on the graph represents the mean ± SEM from 8–12 rats.
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Figure 1 shows the time course of clonidine and lidocaine on the formalin test. Figure 2 shows that clonidine and lidocaine alone produced a dose-dependent suppression of the behavioral response induced by formalin. ED50 (CI95) values of clonidine in Phases 1 and 2 were 7.6 nmol (5.6–10.3 nmol) and 6.9 nmol (4.8–9.8 nmol), respectively. The ED50 (CI95) value of lidocaine in Phase 1 was not calculated because the rats showed motor dysfunction during Phase 1. The ED50 (CI95) value of lidocaine in Phase 2 was 1349 nmol (910–1992 nmol). The isobologram of the combination of clonidine and lidocaine showed that the experimentally derived ED50 (CI95) value decreased below the theoretical dose-additive line, and CIs of the theoretical additive point and those of the experimental point did not overlap ( Fig. 3). This result indicated a significant difference between the experimental ED50 point and the theoretical additive ED50 point (P < 0.05) and a synergistic interaction between clonidine and lidocaine in the rat formalin test. The total fraction value in Phase 2 was 0.28, which was <1, indicating a synergistic interaction ( Table 2). A combination of intrathecal clonidine and lidocaine did not enhance motor dysfunction.
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Figure 2. Dose-response curves for intrathecal clonidine (A) and lidocaine (B). Mean values for behavioral activities expressed as a percent of control for clonidine and lidocaine.
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Table 2. ED50 Values and CI95 for Intrathecally Administered Clonidine and Lidocaine Alone and in Combination in a Fixed-Dose Ratio on Phase-2 Response
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Discussion
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In the current study, both the Phase-1 and Phase-2 responses in the formalin test were attenuated in a dose-dependent manner by preformalin administration of clonidine. Intrathecal lidocaine given before formalin resulted in a powerful suppression of the Phase-2 formalin response. These results support previous investigations indicating that clonidine has antinociceptive effects in the formalin test in rats (5), and that spinal local anesthetics suppress the spinal sensitization induced by formalin (1). The potency of intrathecal clonidine, in suppressing Phases 1 and 2 is very similar, which is consistent with a previous study (5). A previous report showed that the combination of intrathecal clonidine and lidocaine synergistically produced antinociception in the rat tail-flick test (3). The present study extended the previous work, indicating that a combination of intrathecal clonidine and lidocaine in the rat formalin test also produced antinociception in a synergistic manner.
Several investigations showed that an intrathecal combination of clonidine and local anesthetics improved analgesia compared with local anesthetics alone in humans (9) and animals (3). Although investigators have postulated a synergistic analgesic interaction between epidural or intrathecal clonidine and local anesthetics, there is no clinical evidence to prove such an effect (additive or synergetic effect). This interaction would be difficult to elucidate clinically because the administration of ineffective analgesia doses of each drug has practical and ethical problems.
The formalin test reflects a surprisingly complex series of events. It is well known that wide-dynamic range (WDR) neurons of the dorsal horn of the spinal cord play an important role in the transmission and integration of nociceptive information. Electrophysiologically, Dickenson and Sullivan (10) observed that injection of formalin resulted in a profound augmentation in the discharge of dorsal horn WDR neurons in rats, and that formalin results in a biphasic paw-flinching behavior with a time course and amplitude that correspond to activity in spinal WDR neurons. In the formalin test, the first phase is representative of an acute effect mediated by the activation of nociceptive afferent C-fiber; the second phase is attributed to the development of the increased input from primary afferent fiber and local inflammatory response at the injection site (11). Nociceptive sensitization, an increase in dorsal horn neuron response to noxious stimulation, is thought to be responsible for the second phase of the formalin test (10). The wind-up phenomenon is evoked by repetitive C-fiber stimulation in dorsal horn WDR neurons and is mediated partly by the glutamate receptor of N-methyl D-aspartate type (12).
Intrathecal -2-adrenergic agonists mimic the effects of endogenous norepinephrine and produce antinociception resulting from an agonist interaction with presynaptic -2-adrenergic receptors located on small primary afferent, which decreases the release of C-fiber transmitters (substance P, calcitonin gene-related peptide, etc.). The -2-adrenergic agonists also inhibited voltage-dependent Ca2+ channels (13). The release of the C-fiber peptide neurotransmitter was inhibited by the blockade of the activation of voltage-sensitive Ca2+ channels (14). Thus, -2-adrenergic agonists can inhibit the release of the C-fiber peptide neurotransmitter through blocking the voltage-sensitive Ca2+ channels. Direct evidence showed that intrathecal clonidine reduced electrically evoked C-fiber activity in the rat dorsal horn in a dose-dependent manner (15). Moreover, the -2-adrenergic receptor may increase potassium conductance and hyperpolarize the membrane of neurons (16). The patch-clamp technique indicated that -2-adrenergic receptor drugs had a hyperpolarizing property (17). Thus, a hyperpolarization caused by stimulation of the postsynaptic -2-adrenergic receptor is a possible model for the observed antinociception induced by clonidine.
An electrophysiological study showed that intrathecal lidocaine produced a dose-dependent inhibition of the C-fiber-evoked response and wind-up (18). Moreover, evidence indicated that lidocaine selectively reduced the neuronal activity evoked by C-fiber in rat spinal cord through decreasing N-methyl D-aspartate receptor-mediated postsynaptic depolarization (19). Lidocaine inhibited action potential propagation via binding to membrane sodium channels, leading to a reduction in inward sodium currents and also caused the hyperpolarization of the resting membrane potential by the blockade of sodium channels that opened spontaneously under resting conditions (20). Although the principal effect of lidocaine remains on voltage-sensitive sodium channels, it may interact with voltage-sensitive K+ and Ca2+ channels (18,21).
The mechanism of the antinociceptive interaction between clonidine and lidocaine is unclear. A common cellular mechanism might provide the basis for the pharmacological interaction between an -2-adrenergic agonist and lidocaine. Local anesthetics have extensive effects on the sodium channels but also membrane-associated enzymes, second messenger system, and voltage-sensitive K+ and Ca2+ channels (21,22). Thus, local anesthetics have effects on synaptic transmission in addition to their effects on nerve conduction. However, spinal -2-adrenergic agonists can alter pain transmission by acting presynaptically on C-fibers to reduce neurotransmitters release, as well as acting postsynaptically to hyperpolarize dorsal horn WDR neurons. The -2-adrenergic agonists also activate K+ channels and inhibit Ca2+ channels (13). Importantly, Gaumann et al. (23) showed that a small dose of clonidine enhanced lidocaine-induced inhibition of C-fiber action potential, which provided electrophysiological evidence of interaction between clonidine and lidocaine.
It is unclear whether the pharmacokinetic variables of one drug may be altered by the combination of a second drug. Although we did not study the redistribution of the intrathecal drugs, Monasky et al. (24) showed that spinal -2-adrenoceptor agonists did not alter the spinal clearance of other spinal drugs. Thus, it seems unlikely that the synergistic interaction between clonidine and lidocaine depends on altered clearance of either drug.
To produce a powerful analgesic effect and to reduce the doses of each drug for the management of severe pain, the combination of drugs may be used. However, there are some combinations that may not display a synergistic or additive interaction. For example, a recent study demonstrated that antinociception of intrathecal clonidine was antagonized by intrathecal ketorolac (25), which highlights the need to consider the effect of interaction of drugs before using a combination. Our current work emphasizes a powerful interaction between clonidine and lidocaine. The potent synergistic interaction of clonidine with lidocaine supports the use of these drugs in managing some pain states generated secondary to afferent fiber input.
In conclusion, the intrathecal combination of clonidine and lidocaine synergistically produced antinociception in the persistent (inflammatory) pain model. Such a synergistic combination may provide an important theory foundation to control clinical pain.
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Acknowledgments
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We thank Drs. K. Omote and K. Kawamata (Department of Anesthesiology, Sapporo Medical University, Sapporo, Japan) for statistical assistance. We also thank Profs. D. Fink and M. Mata (Department of Neurology, University of Pittsburgh, Pittsburgh, PA) for their useful comments.
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Footnotes
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This work was presented as a poster discussion at the 12th World Congress of Anesthesiologists, Montreal, Canada, June 4–9, 2000.
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Accepted for publication October 31, 2000.
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Abstract
Introduction
) for the drug combination is shown on the additive line. The experimental ED50 value (
) of the combination of the two drugs was significantly lower than the theoretical additive value (P < 0.05), and 95% confidence intervals did not overlap, indicating a synergistic interaction.
