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Anesth Analg 2004;98:1056-1061
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000105877.07071.EF

PAIN MEDICINE

Intrathecal Clonidine and Bupivacaine Have Synergistic Analgesia for Acute Thermally or Inflammatory-Induced Pain in Rats

Tomoki Nishiyama, MD, PhD, and Kazuo Hanaoka, MD, PhD

From the Department of Anesthesiology, The University of Tokyo, Faculty of Medicine, Tokyo, Japan

Address correspondence and reprint requests to Tomoki Nishiyama, MD, PhD, Department of Anesthesiology, The University of Tokyo, Faculty of Medicine, 7–3-1, Hongo, Bunkyo-ku, Tokyo, 113–8655, Japan. Address email to nishit-tky{at}umin.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
We investigated the interaction between spinally administered bupivacaine and clonidine using an animal model of acute and inflammatory pain. Rats implanted with lumbar intrathecal catheters were injected intrathecally with saline (control), bupivacaine (1 to 100 µg), or clonidine (0.1 to 3 µg) and tested for their responses to thermal stimulation to the tail (tail flick test) and subcutaneous formalin injection into the hindpaw (formalin test). The effects of the combination of bupivacaine and clonidine on both stimuli were tested by isobolographic analysis. General behavior and motor function were examined as side effects. The 50% effective doses of bupivacaine and clonidine were significantly smaller when combined compared with each single drug in both the tail flick test (2.82 and 0.11 µg versus 7.1 and 0.29 µg, respectively) and phase 1 (0.24 and 0.009 µg versus 5.7 and 0.15 µg) and phase 2 (0.31 and 0.012 µg versus 3.2 and 0.16 µg) of the formalin test. Side effects were decreased by the combination. These results suggest a favorable combination of intrathecal bupivacaine and clonidine in the management of acute and inflammatory pain.

IMPLICATIONS: The analgesic interaction between intrathecally administered bupivacaine and clonidine was examined during acute thermal and inflammatory-induced pain in rats. The analgesia produced by the combination of these two drugs was synergistic in both acute thermal and inflammatory induced pain, with a decrease in behavioral side effects.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Local anesthetics are widely used in spinal anesthesia and in epidural analgesia to relieve postoperative pain. Clonidine, a {alpha}2-adrenoceptor agonist, is also analgesic when given by epidural (1) or intrathecal (2) administration. Clinically, epidural clonidine infusion improved analgesia when combined with epidural bupivacaine-fentanyl in patients after lower abdominal surgery (1). Intrathecal coadministration of clonidine and local anesthetics prolonged analgesic duration compared with local anesthetic alone (2). However, whether these interactions between clonidine and local anesthetics are synergistic or additive is not known. In rats, the interaction between clonidine and lidocaine for antinociception was synergistic (3). Bupivacaine is used more often than lidocaine for pain treatment by the epidural or intrathecal routes because of its longer duration of action. However, the interaction between clonidine and bupivacaine has not been investigated. The purpose of this study was to investigate the relative potency of the spinally-administered combination of bupivacaine and clonidine for the treatment of thermal-induced pain as a model of acute pain and of formalin-induced pain as a model of inflammatory pain.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
The protocol was approved by the IRB of the University of Tokyo. Male Sprague-Dawley rats (280–300 g for the tail flick test and behavioral test, 330–350 g for the formalin test; Nippon Bio-Supply, Tokyo, Japan) were implanted with chronic lumbar intrathecal catheters under halothane anesthesia. An 8.5-cm polyethylene catheter (PE-10; Clay Adams, Parsippany, NJ) was advanced caudally through an incision in the atlanto-occipital membrane to the thoracolumbar level of the spinal cord. The external part of the catheter was tunneled subcutaneously to exit on the top of the skull and plugged with a 28-gauge stainless steel wire. After surgery, all rats were housed individually in a temperature- and light-controlled environment with free access to food and water. Only rats with normal motor function and behavior 7 days after surgery were used. The position of the catheter in the intrathecal space at the lumbar enlargement was confirmed by exposing the lumbar spinal cord after killing the animals at the end of the study. Data from rats with incorrectly placed catheters were excluded from the study. In each dose group, eight randomly selected rats were used. In total 224 rats were used, and each rat was used only once.

Bupivacaine (Sigma, St. Louis, MO) 1, 10, 30, and 100 µg, and clonidine (Sigma) 0.1, 0.3, 1, and 3 µg were dissolved in 10 µL saline. After intrathecal drug injection, the catheter was flushed with 10 µL of normal saline to clear the dead space of the catheter (8 ± 0.6 µL, mean ± SE). Microinjector syringes were used for all injections. Normal saline 10 µL was injected in the control group.

Nociceptive Tests
Tail Flick Test.
The rats were placed in a clear plastic cylindrical cage with their tails extending through a slot in the rear of the cylinder. Noxious stimulation was provided by a beam of high intensity light (Tail-flick Analgesia Meter MK-330A; Muromachi Kikai Co. Ltd., Tokyo, Japan) focused 2 to 3 cm from the tip of the tail. The response time, defined as the interval between the onset of the thermal stimulation and an abrupt flick of the tail, was measured. The cut-off time in the absence of a response was set at 14 s, to prevent tissue injury. Data are reported as the percentage of maximum possible effect (%MPE) as follows: %MPE = (postdrug latency - predrug latency) x 100/(cut off time - predrug latency).

Formalin Test
Ten minutes after the intrathecal administration of the drug or saline, 50 µL of 5% formalin was injected subcutaneously into the dorsal surface of the right hindpaw with a 30-gauge needle. Immediately after injection, the rat was placed in an open Plexiglas chamber and observed for 60 min. Quantification of pain behavior was made by counting the incidence of spontaneous flinches/shaking of the injected paw for 1 min at 1–2 min, 5–6 min, and then at 5-min intervals until 60 min after formalin injection. Two distinct phases were observed after formalin injection: phase 1, during 0–6 min after injection, and phase 2, beginning approximately 10 min after injection. Phase 1 is an acute response to formalin injection and the phase 2 shows a facilitated state induced by inflammation.

Behavioral and Motor Function Test (Side Effects)
The general behavior (including agitation and allodynia-like behavior), motor function, flaccidity, pinna reflex, and corneal reflex were examined. They were judged as present or absent. Agitation was judged as spontaneous irritable movement and/or vocalization. The presence of allodynia-like behavior was examined by looking for agitation (escape and/or vocalization) evoked by lightly stroking the flank of the rat with a small probe. Motor function was evaluated by the placing/stepping reflex and the righting reflex. The former was evoked by drawing the dorsum of either hindpaw across the edge of the table. Normally rats try to put the paw ahead into a position to walk. The latter was assessed by placing the rat horizontally with its back on the table, which normally gives rise to an immediate, coordinated twisting of the body to an upright position. The disturbance of the righting reflex also shows impairment of central nervous system function. Flaccidity was judged as muscle weakness by putting the forepaw 3 to 5 cm higher than the hindpaw. Normally the rat will walk up. We judged the rat flaccid when it did not move after positioning. Pinna and corneal reflexes were examined with a paper string. When a paper string is put into the ear canal or touches the cornea, rats normally shake their heads or blink.

Experimental Paradigm
The first series of experiments was performed to determine the dose-dependency of the antinociceptive effects of intrathecally-administered bupivacaine or clonidine on both the tail flick test and the formalin test.

To investigate the interaction between bupivacaine and clonidine, an isobolographic analysis was used (4). First, the respective 50% effective dose (ED50) values were determined from the dose response curves of each drug alone using 1, 10, 30, and 100 µg of bupivacaine or 0.1, 0.3, 1, and 3 µg of clonidine. Subsequently, a dose-response curve was obtained by coadministration of the two drugs of 1/2 ED50, 1/4 ED50, 1/8 ED50, or 1/16 ED50 doses in 10 µL. From the dose-response curve of the combined drugs, the ED50 values of clonidine and bupivacaine in combination were calculated.

Data are expressed as mean ± SD. The ED50 values were calculated using the %MPE in the tail flick test and the area under the curve of the number of the flinches versus time in the formalin test, using a computer programs developed in the Anesthesiology Laboratory of University of California, San Diego.

To describe the magnitude of interaction between drugs, a total fractional dose value was also calculated as follows: [(ED50 of bupivacaine in combination)/(ED50 of bupivacaine alone)] + [(ED50 of clonidine in combination)/(ED50 of clonidine alone)]. The values were normalized by assigning the ED50 value of each drug given alone as 1. Values near 1 show an additive interaction, values more than 1 imply an antagonistic interaction, whereas values <1 indicate a synergistic interaction.

Student’s t-test was used to compare the ED50 values in combination with the theoretical additive values. A value of P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Tail Flick Test
Both intrathecal bupivacaine and clonidine induced dose-dependent increases of the tail flick latency (Fig. 1). The combination of bupivacaine and clonidine also showed a dose-dependent increase of the tail flick latency (Fig. 1), and the ED50 values obtained by the combination were significantly lower than the theoretical additive values, which indicates synergistic interaction (Fig. 2, Table 1). Total fractional dose value was 0.75 ± 0.04.



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  		Figure 1. Time course of the effects of intrathecal bupivacaine (upper), clonidine (middle), and bupivacaine + clonidine (lower) on the tail flick test. Each point represents mean ± SD of eight animals. Bupv = bupivacaine; Cl = clonidine. The unit in the legend on the lower figure is µg.

 


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  		Figure 2. Isobolograph for the intrathecal interaction of bupivacaine and clonidine in the tail flick test. The X and Y axes show the dose (µg) of bupivacaine and clonidine, respectively. Horizontal and vertical bars indicate SD. The oblique lines between the X-axis and Y-axis are the theoretical additive lines. The points in the middle of this line are the theoretical additive points calculated from separate ED50 values. The experimental points lie far below the additive line, indicating significant synergism (P < 0.05).

 

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  		Table 1. 50% Effective Doses
 
Formalin Test
The number of flinches in both phase 1 and 2 were decreased by increasing doses of bupivacaine, clonidine, or their combination (Fig. 3). The isobolographic analysis and ED50 values showed significant synergistic effects of bupivacaine and clonidine in both phases of the formalin test (Fig. 4, Table 1). Total fractional dose value of phase 1 was 0.10 ± 0.05 and that of phase 2 was 0.17 ± 0.08.



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  		Figure 3. Time course of the effects of intrathecal bupivacaine (upper), clonidine (middle), and bupivacaine + clonidine (lower) on the formalin test. Each point represents mean ± SD of eight animals. The unit in the legend on the lower figure is µg.

 


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  		Figure 4. Isobolograph for the intrathecal interaction of bupivacaine and clonidine in the phase 1 (upper) and phase 2 (lower) of the formalin test. The X and Y axes show the dose (µg) of bupivacaine and clonidine, respectively. Horizontal and vertical bars indicate SD. The oblique lines between the X-axis and Y-axis are the theoretical additive lines. The points in the middle of this line are the theoretical additive points calculated from separate ED50 values. The experimental points lie far below the additive line, indicating very marked significant synergism in both phase 1 and 2 (P < 0.01).

 
Behavioral and Motor Function Test
Pinna and corneal reflexes were not disturbed in the rats tested in this study. Motor disturbances were observed in rats that received large doses of both bupivacaine and clonidine. The combination of bupivacaine and clonidine did not induce any observable behavioral and motor function abnormalities (Table 2).


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  		Table 2. Side Effects
 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The results of this study show that intrathecally administered combinations of bupivacaine and clonidine produce synergistic analgesic effects on both acute thermal and inflammation-induced pain with decreased side effects. The synergistic potency was higher for inflammatory-induced pain than for thermal-induced pain.

The synergistic analgesic effects between clonidine and bupivacaine are consistent with the results using clonidine and lidocaine in the tail flick test on rats (3) and clinical evidence showing that epidural clonidine dose-dependently improved analgesia by bupivacaine-fentanyl (1).

The analgesic effects of local anesthetics are mediated by blockade of neuronal sodium channels (5), potassium current (6), presynaptic muscarinic receptors (7), presynaptic calcium channels (8), and dopamine receptors (9).

Clonidine inhibits nociceptive transmission by inhibitory effects on C-fiber terminals in the spinal cord (10), by decreasing the release of glutamate or substance P from primary afferent nerve terminals (11), and by mimicking the action of spinally released norepinephrine from descending noradrenergic inhibitory pathways (12). In addition, clonidine can act postsynaptically to hyperpolarize dorsal horn wide dynamic range neurons (13) and increase acetylcholine in the dorsal horn of the spinal cord (14).

The mechanism of interaction between two different types of the drugs might be that the different receptors localized on individual primary afferent neurons are coupled to a single class of ion channels. Thus, activation of a common second messenger pathway within individual neurons simultaneously by two different receptors may facilitate the effector mechanisms. The {alpha}2 adrenoceptor is metabotropic and the predominant action of bupivacaine is on ion channels. Interactions of clonidine with other analgesic drugs are reported to involve spinal muscarinic receptors, serotonergic mechanisms, and local nitric oxide synthesis (15,16). Therefore, the synergistic effects of clonidine and bupivacaine might be mediated, at least in part, by muscarinic receptors. In addition, clonidine may complement the action of local anesthetics on sodium channels by opening potassium channels, resulting in membrane hyperpolarization, a state unresponsive to excitatory input (17).

Clonidine may decrease spinal cord blood flow with vasoconstriction, thereby affecting the pharmacokinetics of local anesthetics (18,19). Intrathecal clonidine prolongs analgesic duration of local anesthetics in humans (2). In the rat study, the combination of clonidine and lidocaine seemed to prolong the analgesic effect of lidocaine but not of clonidine (3). In the present study, we did not measure the duration of the analgesic effects. However, for both the tail flick test and the formalin test, the analgesic effects did not appear to be prolonged by the combination of the two drugs. The duration of the effect of bupivacaine might be long enough to be uninfluenced by clonidine but that of lidocaine might be too short to be affected by clonidine.

The tail flick response is organized at the level of the spinal cord, whereas behavior responses in the formalin test are mediated by both spinal and supraspinal structures. The more potent synergism in the formalin test than the tail flick test between clonidine and bupivacaine in the present study might be attributable to the supraspinal action of clonidine. Clonidine is rapidly absorbed into the blood after intrathecal administration (15). The binding affinity for clonidine to {alpha}2 adrenoceptors was not different between spinal cord and brain (20). Therefore, intrathecally administered clonidine might exert some supraspinal effects.

In the study by Kawamata et al. (3), the intrathecal combination of clonidine and lidocaine decreased blood pressure significantly. Therefore, bupivacaine and clonidine might also decrease blood pressure although we did not measure it. Clonidine induces hypnosis (21), hypotension, and motor blockade (2), and altered thermoregulation (22). Bupivacaine also induces hypotension and motor blockade. All of these effects might have some influence on the analgesic results. Although motor disturbance was evident in the large dose of both drugs, especially bupivacaine, the rats still produced a vigorous tail flick response and paw flinches, indicating that the motor disturbance did not interfere with the animals’ ability to respond to pain in the present study. We did not measure hypnosis, blood pressure, or body temperature. However, neither apparent sedation nor hemodynamic shock were observed. Therefore, hypnosis and hypotension, if any, would have little effect on the results. Body temperature, especially tail temperature in the tail flick test, would have some effect on the results. This should be further clarified in future studies, but in the meantime we should assume that temperature changes should be included in the analgesic effects on thermally-induced pain.

Motor disturbance and other behavioral side effects seen with each drug were not observed in combination. Decreasing the dose of each drug could decrease behavioral side effects.

In conclusion, the intrathecally administered combination of bupivacaine and clonidine produced synergistic analgesic effects on both acute thermal and inflammation-induced pain. The synergistic potency was higher in inflammatory-induced pain than thermal induced pain. Motor disturbance and other behavioral side effects were decreased by the combination.


   Acknowledgments
 
Supported, in part, by Grant-in-Aid #12671453 from the Ministry of Education, Science and Culture, Japan.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Peach MJ, Pavy TJG, Orlikowski CEP, et al. Postoperative epidural infusion: a randomized, double-blind, dose-finding trial of clonidine in combination with bupivacaine and fentanyl. Anesth Analg 1997; 84: 1323–8.[Abstract]
  2. Klimscha W, Chiari A, Krafft P, et al. Hemodynamic and analgesic effects of clonidine added repetitively to continuous epidural and spinal blocks. Anesth Analg 1995; 80: 322–7.[Abstract]
  3. Kawamata T, Omote K, Kawamata M, et al. Antinociceptive interaction of intrathecal {alpha}2-adrenergic agonists, tizanidine and clonidine, with lidocaine in rats. Anesthesiology 1997; 87: 436–48.[Web of Science][Medline]
  4. Tallarida RJ, Porreca F, Cowan A. Statistical analysis of drug-drug and site-site interactions with isobolograms. Life Sci 1989; 45: 947–61.[Web of Science][Medline]
  5. Butterworth JF, Strichartz GR. Molecular mechanisms of local anesthesia: a review. Anesthesiology 1990; 72: 711–34.[Web of Science][Medline]
  6. Olschewski A, Hempelmann G, Vogel W, Safronov BV. Blockade of Na+ and K+ currents by local anesthetics in the dorsal horn neurons of the spinal cord. Anesthesiology 1998; 88: 172–9.[Web of Science][Medline]
  7. Aguilar JS, Criado M, De Roberts E. Inhibition by local anesthetics, phentol-amine and propranolol of [3H] quinuclidinyl benzylate binding to central muscarinic receptors. Eur J Pharmacol 1980; 68: 317–26.[Web of Science][Medline]
  8. Frelin C, Vigne P, Lazdunski M. Biochemical evidence for pharmacological similarities between {alpha}2-adrenoceptors and voltage-dependent Na+ and Ca++ channels. Biochem Biophys Res Commun 1982; 106: 967–73.[Medline]
  9. Bittencourt AL, Takahashi RN. Mazindol and lidocaine are antinociceptives in the mouse formalin model: involvement of dopamine receptor. Eur J Pharmacol 1997; 330: 109–13.[Web of Science][Medline]
  10. Calvillo O, Ghignone M. Presynaptic effect of clonidine on unmyelinated afferent fibers in the spinal cord of the cat. Neurosci Lett 1986; 64: 335–9.[Web of Science][Medline]
  11. Pang IH, Vasko MR. Morphine and norepinephrine but not 5-hydroxy-tryptamine and {alpha}-aminobutyric acid inhibit the potassium-stimulated release of substance P from rat spinal cord slices. Brain Res 1986; 376: 268–79.[Web of Science][Medline]
  12. Stamford JA. Descending control of pain. Br J Anaesth 1995; 75: 217–27.[Abstract/Free Full Text]
  13. Fleetwood-Walker SM, Mitchell R, Hope PJ, et al. An {alpha}2 receptor mediates the selective inhibition by noradrenaline of nociceptive responses of identified dorsal horn neurones. Brain Res 1985; 334: 243–54.[Web of Science][Medline]
  14. Klimscha W, Tong C, Eisenach JC. Intrathecal {alpha}2-adrenergic agonists stimulate acetylcholine and norepinephrine release from the spinal cord dorsal horn in sheep. Anesthesiology 1997; 87: 110–6.[Web of Science][Medline]
  15. Eisenach JC, Detweiler D, Hood D. Hemodynamic and analgesic actions of epidurally administered clonidine. Anesthesiology 1993; 78: 277–87.[Web of Science][Medline]
  16. Eisenach JC, De Kock M, Klimscha W. Alpha2-adrenergic agonists for regional anesthesia. Anesthesiology 1996; 85: 655–74.[Web of Science][Medline]
  17. Butterworth JF, Strichartz GR. The {alpha}2-adrenergic agonists clonidine and guanfacine produce tonic and phasis block of conduction in rat sciatic nerve fibers. Anesth Analg 1993; 76: 295–301.[Web of Science][Medline]
  18. Crosby G, Russo MA, Szabo MD, Davies KR. Subarachnoid clonidine reduces spinal cord blood flow and glucose utilization in conscious rats. Anesthesiology 1990; 73: 1179–85.[Web of Science][Medline]
  19. Gordh T Jr, Fenk U, Morten K. Effect of epidural clonidine on spinal cord blood flow and regional and central hemodynamics in pigs. Anesth Analg 1986; 65: 1312–8.[Abstract/Free Full Text]
  20. Asano T, Dohi S, Ohta S, et al. Antinociception by epidural and systemic {alpha}2-adrenoceptor agonists and their binding affinity in rat spinal cord and brain. Anesth Analg 2000; 90: 400–7.[Abstract/Free Full Text]
  21. Mizobe T, Maghsoudi K, Sitwala K, et al. Antisense technology reveals the alpha2A adrenoceptor to be the subtype mediating the hypnotic response to the highly selective agonist, dexmedetomidine, in the locus coeruleus of the rat. J Clin Invest 1996; 98: 1076–80.[Web of Science][Medline]
  22. LoPachin RM, Rudy TA. The effects of intrathecal sympathomimetic agents on neural activity in the lumbar sympathetic chain of rats. Brain Res 1981; 204: 195–8.
Accepted for publication October 23, 2003.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 2004 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press