Free
Education  |   September 2003
Pharmacologic Interaction between Cannabinoid and either Clonidine or Neostigmine in the Rat Formalin Test
Author Affiliations & Notes
  • Myung Ha Yoon, M.D.
    *
  • Jeong Il Choi, M.D.
  • *Associate Professor, †Fellow, Department of Anesthesiology and Pain Medicine.
  • Received from the Chonnam National University Medical School, Gwangju, Korea.
Article Information
Education
Education   |   September 2003
Pharmacologic Interaction between Cannabinoid and either Clonidine or Neostigmine in the Rat Formalin Test
Anesthesiology 9 2003, Vol.99, 701-707. doi:
Anesthesiology 9 2003, Vol.99, 701-707. doi:
CANNABINOID receptor agonists produce antinociception in animal models of acute pain and inflammatory pain by a direct spinal action, which is mediated cannabinoid receptors, mainly cannabinoid 1 (CB1) receptor. 1–5 Clonidine and neostigmine reverse not only acute nociception but also tissue injury hyperalgesia 6–9 through the action on spinal α2 adrenoceptor and cholinergic receptor, respectively. 10,11 These findings suggest that cannabinoid receptor agonists, clonidine, and neostigmine may have a comparable profile of antinociceptive actions at the spinal level regardless of their different binding sites. However, there is little information or data about the pattern of their interaction with the other drugs. Although the CB1 receptor, which is located in the central nervous system, has been identified in the spinal cord using autoradiography 1,12 and immunohistochemistry, 13–15 the presence of cannabinoid 2 (CB2) receptor in the spinal cord and its role for the nociceptive transmission at the spinal level have not been evaluated.
Therefore, the aim of the present study was to determine the characteristics of the drug interaction between intrathecal cannabinoid receptor agonist (WIN 55,212-2) and clonidine, and between WIN 55,212-2 and neostigmine, using the formalin test, which shows tissue-injury pain leading to the facilitated state and acute pain. In addition, we sought to further clarify the role of cannabinoid receptor subtypes on the antinociceptive effects of intrathecal cannabinoid receptor agonist using the selective cannabinoid receptor antagonists.
Materials and Methods
Animal Preparation
The studies were reviewed and approved by the Institutional Animal Care Committee, Research Institute of Medical Science, Chonnam National University. Male Sprague-Dawley rats (250–300 g) were used for all experiments. The rats were maintained on a 12-h night/day cycle and allowed free access to food and water at all times. For drug administration, an intrathecal catheter was implanted during enflurane anesthesia, as previously described. 16 The catheter was advanced caudally by 8.5 cm through an incision in the atlantooccipital membrane to the lumbar enlargement. The external end of the catheter was tunneled subcutaneously and exited at the top of head and plugged with a piece of steel wire. The skin was closed with 3-0 silk sutures. After surgery, rats were kept in individual cages. Only rats that displayed no postsurgical motor or sensory deficits were used. Animals showing neurologic dysfunction postoperatively were killed immediately. Studies were performed at least 4 or 5 days after intrathecal catheterization.
Drugs
The following drugs were used in this study: WIN 55,212-2 mesylate (Tocris Cookson Ltd., Bristol, UK), clonidine hydrochloride (Sigma Chemical Co., St., Louis, MO), neostigmine bromide (Research Biochemical Internationals, Natick, MA), AM 251 (Tocris), AM 630 (Tocris), and JWH 133 (Tocris). WIN 55,212-2, AM 251, AM 630 and JWH 133 were dissolved in 100% dimethylsulfoxide, and clonidine and neostigmine were dissolved in normal saline. Intrathecal administration of these agents was performed using a hand-driven, gear-operated syringe pump. All drugs were delivered in a volume of 10 μl solution.
Nociceptive Test
For the formalin test, 50 μl of 5% formalin solution was injected subcutaneously into the plantar surface of the hind paw using a 30-gauge needle. The formalin-injection produces characteristic pain behavior: biphasic flinching or shaking of the injected paw. Such pain behavior was therefore quantified by periodically counting the incident of spontaneous flinching or shaking of the injected paw. The number of flinches was counted for 1-min periods at 1 and 5 min and at 5-min intervals from 10 to 60 min. Two phases of spontaneous flinching were observed after the formalin injection. Phase 1 and 2 were defined as 0–9 and 10–60 min after formalin injection, respectively. After the observation period of 1 h, the animals were immediately killed.
Experimental Paradigm
Four to 5 days after surgery, rats were placed in a restraint cylinder for the experiment. After a 15- to 20-min adaptation, rats were then assigned to one of the drug treatment groups. The control study was done using intrathecal saline or dimethylsulfoxide depending on the solvent for experimental drug. Each animal was used in one experiment only. The total number of rats used was 298, and each group comprised 6–9 rats. The investigator was unaware of which drug was administered into each animal.
Effects of Intrathecal WIN 55,212-2, Clonidine, and Neostigmine
For evaluation of the dose-response of the antinociceptive action of cannabinoid receptor agonist (WIN 55,212-2 0.3, 1, 3, 10 μg), α2 receptor agonist (clonidine 1, 3, 10, 30 μg), and cholinesterase inhibitor (neostigmine 0.1, 0.3, 1, 3 μg), each of three agents was intrathecally administered. Intrathecal drugs were injected 10 min before formalin injection. Each ED50value (effective dose producing a 50% reduction of control formalin response) of three agents was separately determined in two phases.
Drug Interaction
An isobolographic analysis 17 was used to determine the nature of pharmacologic interaction between spinal WIN 55,212-2 and clonidine, and spinal WIN 55,212-2 and neostigmine. This method is based on comparisons of doses that are determined to be equieffective. First, each ED50value was determined from the dose-response curves of three agents alone. Next, WIN 55,212-2 and either clonidine or neostigmine were intrathecally coadministered at a dose of the ED50values and fractions (1/2, 1/4, 1/8) of ED50of each drug. From the dose-response curves of the combined drugs, the ED50values of the mixture were calculated and these dose combinations were used for plotting the isobologram. In this experiment, the isobolograms were undertaken to characterize the effect of WIN 55,212-2-clonidine and WIN 55,212-2-neostigmine combinations. The isobologram was constructed by plotting the ED50values of the single agents on the x and y axes, respectively. The theoretical additive dose combination was calculated. From the variance of the total dose, individual variances for the agents in the combination were obtained. Furthermore, to describe the magnitude of the interaction, a total fraction value 16 was calculated:MATH
The fractional values indicate what portion of the single ED50value was accounted for by the corresponding ED50value for the combination. Values near 1 indicate additive interaction, values greater than 1 imply an antagonistic interaction, and values less than 1 indicate a synergistic interaction. The mixture was delivered intrathecally 10 min before the formalin test.
Role of Cannabinoid Receptor Subtypes
To determine whether the effect of intrathecal WIN 55,212-2 was mediated through certain subtypes of cannabinoid receptors, CB1 receptors antagonist (AM 251, 30 μg) and CB2 receptors antagonist (AM 630, 100 μg) were intrathecally administered 10 min before the delivery of WIN 55,212-2 (ED50). The maximal doses of cannabinoid receptor antagonists without affecting the control formalin response were determined from the pilot experiments. The formalin test was done 10 min after administration of WIN 55,212-2. These experiments were conducted in phase 1 and 2, respectively. Furthermore, we examined the effect of the CB2 receptor agonist (JWH 133). Because rats showed motor dysfunction at more than 100 μg of JWH 133, we evaluated its effect at 100 μg.
General Behavior
For evaluation of behavioral change of WIN 55,212-2, clonidine, and neostigmine, additional rats were received the highest doses of three agents used in this study, and examined at 5, 10, 20, 30, 40, 50, and 60 min after intrathecal administration. Motor function was assessed by the righting reflex and placing-stepping reflex. The former was evaluated 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. Drawing the dorsum of either hind paw across the edge of the table evoked the latter. Normally, rats try to put the paw ahead into a position to walk. Changes in motor function were scored as follows: 0, normal; 1, slight deficit; 2, moderate deficit; 3, severe deficit. Pinna reflex and corneal reflex were also evaluated and judged as present or absent.
Statistical Analysis
Data are expressed as means ± SD. The time-response data are presented as the number of flinches, whereas the dose-response data are presented as percentage of control in each phase. To calculate the ED50values of each drug, the number of flinches was converted to percentage of control as follows:MATH
Dose-response data were analyzed by one-way ANOVA with Scheffé for post hoc  . The dose-response lines were fitted using least-squares linear regression and ED50and its 95% confidence intervals were calculated according to the method described by Tallarida and Murray. 18 The difference between theoretical and experimental ED50, the antagonism for the effect of WIN 55,212-2, and the effect of CB2 receptor agonist were analyzed by t  test. P  < 0.05 was considered statistically significant.
Results
No change in pinna reflex, corneal reflex, and motor function was seen after intrathecal administration of WIN 55,212-2, clonidine, and neostigmine. The sum of the number of flinches in the saline or dimethylsulfoxide control group was not statistically different from each other in phase 1 (21 ± 4 vs.  19 ± 3) or phase 2 (166 ± 44 vs.  158 ± 38). Figure 1displays the time course of intrathecal WIN 55,212-2, clonidine, and neostigmine, administered 10 min before formalin injection.
Fig. 1. Time-effect curve of intrathecal WIN 55,212-2 (A  ), clonidine (B  ), and neostigmine (C  ) for flinching in the formalin test. Each drug was administered 10 min before formalin injection (arrow  ). Data are presented as the number of flinches. Each line  represents the mean ± SD of six to eight rats.
Fig. 1. Time-effect curve of intrathecal WIN 55,212-2 (A 
	), clonidine (B 
	), and neostigmine (C 
	) for flinching in the formalin test. Each drug was administered 10 min before formalin injection (arrow 
	). Data are presented as the number of flinches. Each line 
	represents the mean ± SD of six to eight rats.
Fig. 1. Time-effect curve of intrathecal WIN 55,212-2 (A  ), clonidine (B  ), and neostigmine (C  ) for flinching in the formalin test. Each drug was administered 10 min before formalin injection (arrow  ). Data are presented as the number of flinches. Each line  represents the mean ± SD of six to eight rats.
×
Intrathecal WIN 55,212-2, clonidine, and neostigmine produced a dose-dependent suppression of the flinching response during phase 1 and 2 in the formalin test (fig. 2). The ED50values (95% confidence intervals) of WIN 55,212-2, clonidine, and neostigmine in phase 1 were 1.4 (0.7–2.8), 10.3 (4.4–23.8), and 0.6 μg (0.4–0.8 μg), respectively. The phase 2 ED50values (95% confidence intervals) of WIN 55,212-2, clonidine, and neostigmine were 2.8 (1.3–6.1), 4.7 (3.1–7.2), and 0.3 μg (0.2–0.4 μg), respectively.
Fig. 2. Dose-response curve of intrathecal WIN 55,212-2, clonidine, and neostigmine for flinching during phase 1 (A  ) and phase 2 (B  ) in the formalin test. Data are presented as the percentage of control. WIN 55,212-2, clonidine, and neostigmine produced a dose-dependent inhibition of flinches in both phases. Each line  represents the mean ± SD of six to eight rats. C = control. *P  < 0.05, †P < 0.01, ‡P < 0.001 versus  control.
Fig. 2. Dose-response curve of intrathecal WIN 55,212-2, clonidine, and neostigmine for flinching during phase 1 (A 
	) and phase 2 (B 
	) in the formalin test. Data are presented as the percentage of control. WIN 55,212-2, clonidine, and neostigmine produced a dose-dependent inhibition of flinches in both phases. Each line 
	represents the mean ± SD of six to eight rats. C = control. *P 
	< 0.05, †P < 0.01, ‡P < 0.001 versus 
	control.
Fig. 2. Dose-response curve of intrathecal WIN 55,212-2, clonidine, and neostigmine for flinching during phase 1 (A  ) and phase 2 (B  ) in the formalin test. Data are presented as the percentage of control. WIN 55,212-2, clonidine, and neostigmine produced a dose-dependent inhibition of flinches in both phases. Each line  represents the mean ± SD of six to eight rats. C = control. *P  < 0.05, †P < 0.01, ‡P < 0.001 versus  control.
×
Isobolographic analysis revealed a synergistic interaction between intrathecal WIN 55,212-2 and clonidine, as well as intrathecal WIN 55,212-2 and neostigmine during phase 1 and 2 in the formalin test (figs. 3 and 4).
Fig. 3. Isobologram for the interaction between WIN 55,212-2 and clonidine during phase 1 (A  ) and phase 2 (B  ) in the formalin test. The ED50values for each agent are plotted on the x  and y  axes, respectively. Horizontal  and vertical bars  indicate confidence intervals. The straight line  connecting each ED50value is the theoretical additive line and the point  on this line is the theoretical additive ED50. The experimental ED50point was significantly different from the theoretical ED50point, indicating a synergistic interaction.
Fig. 3. Isobologram for the interaction between WIN 55,212-2 and clonidine during phase 1 (A 
	) and phase 2 (B 
	) in the formalin test. The ED50values for each agent are plotted on the x 
	and y 
	axes, respectively. Horizontal 
	and vertical bars 
	indicate confidence intervals. The straight line 
	connecting each ED50value is the theoretical additive line and the point 
	on this line is the theoretical additive ED50. The experimental ED50point was significantly different from the theoretical ED50point, indicating a synergistic interaction.
Fig. 3. Isobologram for the interaction between WIN 55,212-2 and clonidine during phase 1 (A  ) and phase 2 (B  ) in the formalin test. The ED50values for each agent are plotted on the x  and y  axes, respectively. Horizontal  and vertical bars  indicate confidence intervals. The straight line  connecting each ED50value is the theoretical additive line and the point  on this line is the theoretical additive ED50. The experimental ED50point was significantly different from the theoretical ED50point, indicating a synergistic interaction.
×
Fig. 4. Isobologram for the interaction between WIN 55,212-2 and neostigmine during phase 1 (A  ) and phase 2 (B  ) in the formalin test. The ED50values for each agent are plotted on the x  and y  axes, respectively. Horizontal  and vertical bars  indicate confidence intervals. The straight line  connecting each ED50value is the theoretical additive line and the point  on this line is the theoretical additive ED50.The experimental ED50point was significantly different from the theoretical ED50point, indicating a synergistic interaction.
Fig. 4. Isobologram for the interaction between WIN 55,212-2 and neostigmine during phase 1 (A 
	) and phase 2 (B 
	) in the formalin test. The ED50values for each agent are plotted on the x 
	and y 
	axes, respectively. Horizontal 
	and vertical bars 
	indicate confidence intervals. The straight line 
	connecting each ED50value is the theoretical additive line and the point 
	on this line is the theoretical additive ED50.The experimental ED50point was significantly different from the theoretical ED50point, indicating a synergistic interaction.
Fig. 4. Isobologram for the interaction between WIN 55,212-2 and neostigmine during phase 1 (A  ) and phase 2 (B  ) in the formalin test. The ED50values for each agent are plotted on the x  and y  axes, respectively. Horizontal  and vertical bars  indicate confidence intervals. The straight line  connecting each ED50value is the theoretical additive line and the point  on this line is the theoretical additive ED50.The experimental ED50point was significantly different from the theoretical ED50point, indicating a synergistic interaction.
×
The experimental ED50values were significantly lower than the calculated ED50values. Accordingly, the phase 1 ED50values (95% confidence intervals) of the WIN 55,212-2 in the mixture of WIN 55,212-2-clonidine and WIN 55,212-2-neostigmine were 0.02 (0–52.2), 0.08 μg (0.01–1.0 μg), respectively. The phase 2 ED50values (95% confidence intervals) of the WIN 55,212-2 in the mixture of WIN 55,212-2-clonidine and WIN 55,212-2-neostigmine were 0.3 (0.03–1.7), 0.6 μg (0.2–1.8 μg), respectively. The total fraction values of the mixture of WIN 55,212-2-clonidine were 0.03 in phase 1 and 0.19 in phase 2, and those of WIN 55,212-2-neostigmine were 0.12 in phase 1 and 0.41 in phase 2, indicating synergistic interactions in both phases.
Intrathecal AM 251 and AM 630 alone did not affect the control flinching response evoked by formalin injection (fig. 5, A). Intrathecal AM 251 reversed the antinociceptive effect of intrathecal WIN 55,212-2 during phase 1 and 2 in the formalin test (fig. 5, B and C). However, the antinociceptive effect of intrathecal WIN 55,212-2 was not antagonized by intrathecal AM 630 in both phases. Intrathecal JWH 133 did not alter the formalin-evoked flinching response in both phases (fig. 6).
Fig. 5. Effects of intrathecal AM 251 (30 μg) and AM 630 (100 μg) for the antinociceptive action of intrathecal ED50(WIN 55,212-2) during phase 1 (B  ) and phase 2 (C  ) in the formalin test. AM 251 and AM 630 were given 10 min before ED50administration, and the formalin test was done 10 min after ED50delivery. Data are presented as the percentage of control. Neither AM 251 nor AM 630 alone (A  ) affected the control response with formalin. The antinociceptive effect of ED50was reversed by AM 251 but not by AM 630. Each bar  represents the mean ± SD of six or eight rats. *P  < 0.05 versus  ED50.
Fig. 5. Effects of intrathecal AM 251 (30 μg) and AM 630 (100 μg) for the antinociceptive action of intrathecal ED50(WIN 55,212-2) during phase 1 (B 
	) and phase 2 (C 
	) in the formalin test. AM 251 and AM 630 were given 10 min before ED50administration, and the formalin test was done 10 min after ED50delivery. Data are presented as the percentage of control. Neither AM 251 nor AM 630 alone (A 
	) affected the control response with formalin. The antinociceptive effect of ED50was reversed by AM 251 but not by AM 630. Each bar 
	represents the mean ± SD of six or eight rats. *P 
	< 0.05 versus 
	ED50.
Fig. 5. Effects of intrathecal AM 251 (30 μg) and AM 630 (100 μg) for the antinociceptive action of intrathecal ED50(WIN 55,212-2) during phase 1 (B  ) and phase 2 (C  ) in the formalin test. AM 251 and AM 630 were given 10 min before ED50administration, and the formalin test was done 10 min after ED50delivery. Data are presented as the percentage of control. Neither AM 251 nor AM 630 alone (A  ) affected the control response with formalin. The antinociceptive effect of ED50was reversed by AM 251 but not by AM 630. Each bar  represents the mean ± SD of six or eight rats. *P  < 0.05 versus  ED50.
×
Fig. 6. Effect of intrathecal JWH 133 for flinching during phase 1 (A  ) and phase 2 (B  ) in the formalin test. The drug was administered 10 min before formalin injection. Data are presented as the sum of the number of flinches. Each bar  represents the mean ± SD of six or nine rats.
Fig. 6. Effect of intrathecal JWH 133 for flinching during phase 1 (A 
	) and phase 2 (B 
	) in the formalin test. The drug was administered 10 min before formalin injection. Data are presented as the sum of the number of flinches. Each bar 
	represents the mean ± SD of six or nine rats.
Fig. 6. Effect of intrathecal JWH 133 for flinching during phase 1 (A  ) and phase 2 (B  ) in the formalin test. The drug was administered 10 min before formalin injection. Data are presented as the sum of the number of flinches. Each bar  represents the mean ± SD of six or nine rats.
×
Discussion
In the current study, intrathecal WIN 55,212-2 attenuated the flinching response during phase 1 and 2 in the formalin test. In the formalin test, phase 1 response seems to result from the immediate and intense increase of primary afferent activity. However, phase 2 response mirrors the activation of wide dynamic range of dorsal horn neurons with very low level of ongoing activity of primary afferent. Therefore, phase 2 reflects a facilitated state that seems to be prominent, considering the decreased level of afferent input. These observations suggest that WIN 55,212-2 may alter the facilitated state and the acute nociception at the spinal level. An interesting aspect of the results was the relative effectiveness of WIN 55,212-2 on phase 1 and 2 of the formalin responses. A twofold-higher ED50for phase 2 was observed compared to that for phase 1 with WIN 55,212-2. This effect was further evident in the combination studies when WIN 55,212-2 was combined with clonidine or neostigmine. The phase 2 to phase 1 ratio was 8–15 under the combined administrations. However, for both clonidine and neostigmine, the phase 2 to phase 1 ratio was 0.5. These data suggest that WIN 55,212-2 seems to be much more effective on the acute pain than on the facilitated state. However, previous studies 19,20 showed different effects on formalin-induced response, in which injection of a cannabinoid receptor agonist attenuated phase 2 response, but not phase 1 or vice versa  . Such discrepancy may be caused by the use of a different drug, the route of drugs given, the kind of animal, the concentration of formalin solution, and the type of measures made.
Cannabinoids are involved in the control of nociception at almost all relays of pain transmission, especially at the spinal level where they act through cannabinoid receptors. 21 Cannabinoids activate two receptor subtypes, the CB1 and CB2 receptors. Expression of the CB1 receptor is essentially restricted to neurons and is coupled with G-proteins, 22 whereas CB2 receptor expression is essentially restricted to immune cell lines. 23 Moreover, CB1 receptor has been identified in the dorsal horn of the spinal cord intimately concerned with the processing of nociceptive information and the modulation. 1,12–15 However, no reports have been made to date regarding the expression of the CB2 receptor at the spinal cord. The antinociceptive actions of peripheral CB1 and CB2 receptor agonists have been reported, 19,24 which implicates that peripheral CB1 and CB2 receptors participate in the control of nociception. However, the role of each subtype of CB receptors was not examined at the spinal level. In the current experiments, the CB1 antagonist, not the CB2 antagonist, antagonized the antinociceptive effect of WIN 55,212-2, and further administration of intrathecal CB2 agonist did not produce antinociception. These findings suggest that the antinociceptive action of cannabinoids is mediated through just the CB1 receptor at the spinal level.
There are at least three possible mechanisms by which cannabinoid may act spinally to reduce the nociceptive transmission. 1 First, it may prevent the afferent barrage by acting presynaptically to inhibit neurotransmitter release. Activation of CB1 receptor leads to reduction of calcium channel currents through N-type 25,26 and P/Q-type 27 channels. Activation of the cannabinoid receptor has been reported to inhibit adenylyl cyclase activity. 28 In addition, CB1 receptor activation has been shown to enhance potassium channel “A” currents 29 and activate potassium currents 30 through G-protein-coupled inwardly rectifying K+channels. Initiating these currents or second messenger systems aids in decreasing the cellular excitability and reducing subsequent neurosecretion. Indeed, activation of the CB1 receptor inhibits the release of transmitter from primary afferent fibers in the dorsal horn of the spinal cord. 1,3 Electrophysiologic studies have shown that cannabinoids suppress noxious stimulus-evoked activity of nociceptive neurons in the spinal cord, 31 and that activity-dependent facilitation of nociceptive dorsal horn neurons is decreased after the application of cannabinoids to the spinal cord. 32 Second, cannabinoids may act postsynaptically to stabilize membrane potentials at subthreshold levels via  the enhancement of K+currents and thus prevent the transduction of the nociceptive message. Third, cannabinoids may lead to disinhibition of an inhibitory circuit. Inhibition of terminal A removes the inhibition of terminal B resulting in the release of an antihyperalgesic substance.
Isobolographic analysis of this study revealed the synergistic interaction between intrathecal WIN 55,212-2 and clonidine, and between intrathecal WIN 55,212-2 and neostigmine during phase 1 and 2 in the formalin test. These results indicate that spinal combination of WIN 55,212-2 with clonidine or neostigmine can augment the antinociceptive effect of each drug alone, in both an acute nociceptive state and a tissue-injury state evoked by formalin stimulus. Although a pharmacologic interaction between two kinds of drugs is most likely complicated to characterize, several explanations could be possible for this synergy. First, drugs may interact by altering the kinetics of each other. One agent may alter the actions of the other agent at the receptor or channel. Second, such interaction may occur when both drugs affect different critical points along a common pathway. 33 Cannabinoids, clonidine, and neostigmine act on receptors that are G-protein coupled. Hence, the action of these three agents may independently alter intracellular second messenger systems coupled with G-protein activation and mediate a synergistic interaction. 34 Third, functional interaction may result from distinct drug effects at separate anatomic sites that may act independently and together to inhibit spinal nociceptive processing. 35 As mentioned previously, cannabinoids, clonidine, and neostigmine possesses both presynaptic and postsynaptic actions. Therefore, simultaneous engagement of presynatptic and postsynaptic mechanisms may augment the antinociceptive action produced by either drug acting at one site independently. 36 
Clinically, spinal cannabinoids are not yet available. However, in the future they can be used with clonidine or neostigmine to manage pain because their combination with clonidine or neostigmine may provide a decreased dose of either drug or an increased maximum achievable. Taken together, intrathecal WIN 55,212-2, clonidine, and neostigmine reduce the pain behavior evoked by formalin stimulus, and WIN 55,212-2 interacts with clonidine or neostigmine in a synergistic fashion. Spinal CB1 receptor, but not CB2 receptor, is involved in the antinociception of intrathecal WIN 55,212-2.
References
Richardson JD, Aanonsen L, Hargreaves KM: Antihyperalgesic effects of spinal cannabinoids. Eur J Pharmacol 1998; 345: 145–53Richardson, JD Aanonsen, L Hargreaves, KM
Martin WJ, Loo CM, Basbaum AI: Spinal cannabinoids are anti-allodynic in rats with persistent inflammation. Pain 1999; 82: 199–205Martin, WJ Loo, CM Basbaum, AI
Drew LJ, Harris J, Millns PJ, Kendall DA, Chapman V: Activation of spinal cannabinoid 1 receptors inhibits C-fibre driven hyperexcitable neuronal responses and increases [35S]GTPgammaS binding in the dorsal horn of the spinal cord of noninflamed and inflamed rats. Eur J Neurosci 2000; 12: 2079–86Drew, LJ Harris, J Millns, PJ Kendall, DA Chapman, V
Johanek LM, Heitmiller DR, Turner M, Nader N, Hodges J, Simone DA: Cannabinoids attenuate capsaicin-evoked hyperalgesia through spinal and peripheral mechanisms. Pain 2001; 93: 303–15Johanek, LM Heitmiller, DR Turner, M Nader, N Hodges, J Simone, DA
Gühring H, Schuster J, Hamza M, Ates M, Kotalla CE, Brune K: HU-210 shows higher efficacy and potency than morphine after intrathecal administration in the mouse formalin test. Eur J Pharmacol 2001; 429: 127–34Gühring, H Schuster, J Hamza, M Ates, M Kotalla, CE Brune, K
Tejwani GA, Rattan AK: Antagonism of antinociception produced by intrathecal clonidine by ketorolac in the rat: The role of the opioid system. Anesth Analg 2000; 90: 1152–6Tejwani, GA Rattan, AK
Buerkle H, Schapsmeier M, Bantel C, Marcus MA, Wusten R, Van Aken H: Thermal and mechanical antinociceptive action of spinal vs peripherally administered clonidine in the rat inflamed knee joint model. Br J Anaesth 1999; 83: 436–41Buerkle, H Schapsmeier, M Bantel, C Marcus, MA Wusten, R Van Aken, H
Muerkle H, Boschin M, Marcus MAE, Brodner G, Wusten R, Van Aken H: Central and peripheral analgesia mediated by the acetylcholinesterase-inhibitor nesostigmine in the rat inflamed knee joint model. Anesth Analg 1998; 86: 1027–32Muerkle, H Boschin, M Marcus, MAE Brodner, G Wusten, R Van Aken, H
Prado WA, Goncalves AS: Antinociceptive effect of intrathecal neostigmine evaluated in rats by two different pain models. Braz J Med Biol Res 1997; 30: 1225–31Prado, WA Goncalves, AS
Bouchenafa O, Livingston A: Autoradiographic localisation of α 2 adrenoceptor binding sites in the spinal cord of the sheep. Res Vet Sci 1987; 42: 382–5Bouchenafa, O Livingston, A
Villiger JW, Faull RLM: Muscarinic cholinergic receptors in the human spinal cord: Differential localization of [3H] pirenzepine and [3H] quinuclidinylbenzilate binding sites. Brain Res 1985; 345: 196–9Villiger, JW Faull, RLM
Hohmann AG, Briley EM, Herkenham M: Pre- and postsynaptic distribution of cannabinoid and mu opioid receptors in rat spinal cord. Brain Res 1999; 822: 17–25Hohmann, AG Briley, EM Herkenham, M
Tsou K, Brown S, Sanudo-Pena MC, Mackie K, Walker JM: Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central nervous system. Neuroscience 1998; 83: 393–411Tsou, K Brown, S Sanudo-Pena, MC Mackie, K Walker, JM
Ong WY, Mackie K: A light and electron microscopic study of the CB1 cannabinoid receptor in the primate spinal cord. J Neurocytol 1999; 28: 39–45Ong, WY Mackie, K
Farquhar-Smith WP, Egertova M, Bradbury EJ, McMahon SB, Rice AS, Elphick MR: Cannabinoid CB(1) receptor expression in rat spinal cord. Mol Cell Neurosci 2000; 15: 510–21Farquhar-Smith, WP Egertova, M Bradbury, EJ McMahon, SB Rice, AS Elphick, MR
Yaksh TL, Rudy TA: Chronic catheterization of the spinal subarachnoid space. Physiol Behav 1976; 17: 1031–6Yaksh, TL Rudy, TA
Yoon MH, Yaksh TL: Evaluation of interaction between gabapentin and ibuprofen on the formalin test in rats. A nesthesiology 1999; 91: 1006–13Yoon, MH Yaksh, TL
Tallarida RJ, Murray RB: Manual of pharmacologic calculations with computer programs, 2nd edition. New York, Springer-Verlag, 1987, pp 1–95
Calignano A, La Rana G, Giuffrida A, Piomelli D: Control of pain initiation by endogenous cannabinoids. Nature 1998; 394: 277–81Calignano, A La Rana, G Giuffrida, A Piomelli, D
Jaggar SI, Hasnie FS, Sellaturay S, Rice AS: The anti-hyperalgesic actions of the cannabinoid anandamide and the putative CB2 receptor agonist palmitoylethanolamide in visceral and somatic inflammatory pain. Pain 1998; 76: 189–99Jaggar, SI Hasnie, FS Sellaturay, S Rice, AS
Rice AS, Farquhar-Smith WP, Nagy I: Endocannabinoids and pain: Spinal and peripheral analgesia in inflammation and neuropathy. Prostaglandins Leukot Essent Fatty Acids 2002; 66: 243–56Rice, AS Farquhar-Smith, WP Nagy, I
Munro S, Thomas KL, Abu-Shaar M: Molecular characterization of a peripheral receptor for cannabinoids. Nature 1993; 365: 61–5Munro, S Thomas, KL Abu-Shaar, M
Schatz AR, Lee M, Condie RB, Pulaski JT, Kaminski NE: Cannabinoid receptors CB1 and CB2: A characterization of expression and adenylate cyclase modulation within the immune system. Toxicol Appl Pharmacol 1997; 142: 278–87Schatz, AR Lee, M Condie, RB Pulaski, JT Kaminski, NE
Malan TP Jr, Ibrahim MM, Deng H, Liu Q, Mata HP, Vanderah T, Porreca F, Makriyannis A: CB2 cannabinoid receptor-mediated peripheral antinociception. Pain 2001; 93: 239–45Malan, TP Ibrahim, MM Deng, H Liu, Q Mata, HP Vanderah, T Porreca, F Makriyannis, A
Mackie K, Hille B: Cannabinoids inhibit N-type calcium channels in neuroblastoma-glioma cells. Proc Natl Acad Sci U S A 1992; 89: 3825–9Mackie, K Hille, B
Pan X, Ikeda SR, Lewis DL: Rat brain cannabinoid receptor modulates N-type Ca2+channels in a neuronal expression system. Mol Pharmacol 1996; 49: 707–14Pan, X Ikeda, SR Lewis, DL
Twitchell W, Brown S, Mackie K: Cannabinoids inhibit N- and P/Q-type calcium channels in cultured rat hippocampal neurons. J Neurophysiol 1997; 78: 43–50Twitchell, W Brown, S Mackie, K
Howlett AC, Fleming RM: Cannabinoid inhibition of adenylate cyclase: Pharmacology of the response in neuroblastoma cell membranes. Mol Pharmacol 1984; 26: 532–8Howlett, AC Fleming, RM
Deadwyler SA, Hampson RE, Bennett BA, Edwards TA, Mu J, Pacheco MA, Ward SJ, Childers SR: Cannabinoids modulate potassium current in cultured hippocampal neurons. Recept Channels 1993; 1: 121–34Deadwyler, SA Hampson, RE Bennett, BA Edwards, TA Mu, J Pacheco, MA Ward, SJ Childers, SR
Henry DJ, Chavkin C: Activation of inwardly rectifying potassium channels (GIRK1) by co-expressed rat brain cannabinoid receptors in Xenopus oocytes. Neurosci Lett 1995; 186: 91–4Henry, DJ Chavkin, C
Hohmann AG, Martin WJ, Tsou K, Walker JM: Inhibition of noxious stimulus-evoked activity of spinal cord dorsal horn neurons by the cannabinoid WIN 55,212-2. Life Sci 1995; 56: 2111–8Hohmann, AG Martin, WJ Tsou, K Walker, JM
Strangman NM, Walker JM: Cannabinoid WIN 55,212-2 inhibits the activity-dependent facilitation of spinal nociceptive responses. J Neurophysiol 1999; 82: 472–7Strangman, NM Walker, JM
Berenbaum MC: What is synergy? Pharmacol Rev 1989; 41: 93–141Berenbaum, MC
Malmberg AB, Yaksh TL: Pharmacology of the spinal action of ketorolac, morphine, ST-91, U50488H, and L-PIA on the formalin test and an isobolographic analysis of the NSAID interaction. A nesthesiology 1993; 79: 270–81Malmberg, AB Yaksh, TL
Roerig SC, Fujimoto JM: Multiplicative interaction between intrathecally and intracerebroventricularly administered mu opioid agonists but limited interactions between delta and kappa agonists for antinociception in mice. J Pharmacol Exp Ther 1989; 249: 762–8Roerig, SC Fujimoto, JM
Solomon RE, Gebhart GF: Synergistic antinociceptive interactions among drugs administered to the spinal cord. Anesth Analg 1994; 78: 1164–72Solomon, RE Gebhart, GF
Fig. 1. Time-effect curve of intrathecal WIN 55,212-2 (A  ), clonidine (B  ), and neostigmine (C  ) for flinching in the formalin test. Each drug was administered 10 min before formalin injection (arrow  ). Data are presented as the number of flinches. Each line  represents the mean ± SD of six to eight rats.
Fig. 1. Time-effect curve of intrathecal WIN 55,212-2 (A 
	), clonidine (B 
	), and neostigmine (C 
	) for flinching in the formalin test. Each drug was administered 10 min before formalin injection (arrow 
	). Data are presented as the number of flinches. Each line 
	represents the mean ± SD of six to eight rats.
Fig. 1. Time-effect curve of intrathecal WIN 55,212-2 (A  ), clonidine (B  ), and neostigmine (C  ) for flinching in the formalin test. Each drug was administered 10 min before formalin injection (arrow  ). Data are presented as the number of flinches. Each line  represents the mean ± SD of six to eight rats.
×
Fig. 2. Dose-response curve of intrathecal WIN 55,212-2, clonidine, and neostigmine for flinching during phase 1 (A  ) and phase 2 (B  ) in the formalin test. Data are presented as the percentage of control. WIN 55,212-2, clonidine, and neostigmine produced a dose-dependent inhibition of flinches in both phases. Each line  represents the mean ± SD of six to eight rats. C = control. *P  < 0.05, †P < 0.01, ‡P < 0.001 versus  control.
Fig. 2. Dose-response curve of intrathecal WIN 55,212-2, clonidine, and neostigmine for flinching during phase 1 (A 
	) and phase 2 (B 
	) in the formalin test. Data are presented as the percentage of control. WIN 55,212-2, clonidine, and neostigmine produced a dose-dependent inhibition of flinches in both phases. Each line 
	represents the mean ± SD of six to eight rats. C = control. *P 
	< 0.05, †P < 0.01, ‡P < 0.001 versus 
	control.
Fig. 2. Dose-response curve of intrathecal WIN 55,212-2, clonidine, and neostigmine for flinching during phase 1 (A  ) and phase 2 (B  ) in the formalin test. Data are presented as the percentage of control. WIN 55,212-2, clonidine, and neostigmine produced a dose-dependent inhibition of flinches in both phases. Each line  represents the mean ± SD of six to eight rats. C = control. *P  < 0.05, †P < 0.01, ‡P < 0.001 versus  control.
×
Fig. 3. Isobologram for the interaction between WIN 55,212-2 and clonidine during phase 1 (A  ) and phase 2 (B  ) in the formalin test. The ED50values for each agent are plotted on the x  and y  axes, respectively. Horizontal  and vertical bars  indicate confidence intervals. The straight line  connecting each ED50value is the theoretical additive line and the point  on this line is the theoretical additive ED50. The experimental ED50point was significantly different from the theoretical ED50point, indicating a synergistic interaction.
Fig. 3. Isobologram for the interaction between WIN 55,212-2 and clonidine during phase 1 (A 
	) and phase 2 (B 
	) in the formalin test. The ED50values for each agent are plotted on the x 
	and y 
	axes, respectively. Horizontal 
	and vertical bars 
	indicate confidence intervals. The straight line 
	connecting each ED50value is the theoretical additive line and the point 
	on this line is the theoretical additive ED50. The experimental ED50point was significantly different from the theoretical ED50point, indicating a synergistic interaction.
Fig. 3. Isobologram for the interaction between WIN 55,212-2 and clonidine during phase 1 (A  ) and phase 2 (B  ) in the formalin test. The ED50values for each agent are plotted on the x  and y  axes, respectively. Horizontal  and vertical bars  indicate confidence intervals. The straight line  connecting each ED50value is the theoretical additive line and the point  on this line is the theoretical additive ED50. The experimental ED50point was significantly different from the theoretical ED50point, indicating a synergistic interaction.
×
Fig. 4. Isobologram for the interaction between WIN 55,212-2 and neostigmine during phase 1 (A  ) and phase 2 (B  ) in the formalin test. The ED50values for each agent are plotted on the x  and y  axes, respectively. Horizontal  and vertical bars  indicate confidence intervals. The straight line  connecting each ED50value is the theoretical additive line and the point  on this line is the theoretical additive ED50.The experimental ED50point was significantly different from the theoretical ED50point, indicating a synergistic interaction.
Fig. 4. Isobologram for the interaction between WIN 55,212-2 and neostigmine during phase 1 (A 
	) and phase 2 (B 
	) in the formalin test. The ED50values for each agent are plotted on the x 
	and y 
	axes, respectively. Horizontal 
	and vertical bars 
	indicate confidence intervals. The straight line 
	connecting each ED50value is the theoretical additive line and the point 
	on this line is the theoretical additive ED50.The experimental ED50point was significantly different from the theoretical ED50point, indicating a synergistic interaction.
Fig. 4. Isobologram for the interaction between WIN 55,212-2 and neostigmine during phase 1 (A  ) and phase 2 (B  ) in the formalin test. The ED50values for each agent are plotted on the x  and y  axes, respectively. Horizontal  and vertical bars  indicate confidence intervals. The straight line  connecting each ED50value is the theoretical additive line and the point  on this line is the theoretical additive ED50.The experimental ED50point was significantly different from the theoretical ED50point, indicating a synergistic interaction.
×
Fig. 5. Effects of intrathecal AM 251 (30 μg) and AM 630 (100 μg) for the antinociceptive action of intrathecal ED50(WIN 55,212-2) during phase 1 (B  ) and phase 2 (C  ) in the formalin test. AM 251 and AM 630 were given 10 min before ED50administration, and the formalin test was done 10 min after ED50delivery. Data are presented as the percentage of control. Neither AM 251 nor AM 630 alone (A  ) affected the control response with formalin. The antinociceptive effect of ED50was reversed by AM 251 but not by AM 630. Each bar  represents the mean ± SD of six or eight rats. *P  < 0.05 versus  ED50.
Fig. 5. Effects of intrathecal AM 251 (30 μg) and AM 630 (100 μg) for the antinociceptive action of intrathecal ED50(WIN 55,212-2) during phase 1 (B 
	) and phase 2 (C 
	) in the formalin test. AM 251 and AM 630 were given 10 min before ED50administration, and the formalin test was done 10 min after ED50delivery. Data are presented as the percentage of control. Neither AM 251 nor AM 630 alone (A 
	) affected the control response with formalin. The antinociceptive effect of ED50was reversed by AM 251 but not by AM 630. Each bar 
	represents the mean ± SD of six or eight rats. *P 
	< 0.05 versus 
	ED50.
Fig. 5. Effects of intrathecal AM 251 (30 μg) and AM 630 (100 μg) for the antinociceptive action of intrathecal ED50(WIN 55,212-2) during phase 1 (B  ) and phase 2 (C  ) in the formalin test. AM 251 and AM 630 were given 10 min before ED50administration, and the formalin test was done 10 min after ED50delivery. Data are presented as the percentage of control. Neither AM 251 nor AM 630 alone (A  ) affected the control response with formalin. The antinociceptive effect of ED50was reversed by AM 251 but not by AM 630. Each bar  represents the mean ± SD of six or eight rats. *P  < 0.05 versus  ED50.
×
Fig. 6. Effect of intrathecal JWH 133 for flinching during phase 1 (A  ) and phase 2 (B  ) in the formalin test. The drug was administered 10 min before formalin injection. Data are presented as the sum of the number of flinches. Each bar  represents the mean ± SD of six or nine rats.
Fig. 6. Effect of intrathecal JWH 133 for flinching during phase 1 (A 
	) and phase 2 (B 
	) in the formalin test. The drug was administered 10 min before formalin injection. Data are presented as the sum of the number of flinches. Each bar 
	represents the mean ± SD of six or nine rats.
Fig. 6. Effect of intrathecal JWH 133 for flinching during phase 1 (A  ) and phase 2 (B  ) in the formalin test. The drug was administered 10 min before formalin injection. Data are presented as the sum of the number of flinches. Each bar  represents the mean ± SD of six or nine rats.
×