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Department of Anesthesiology, Graduate School of Medicine, Chiba University, Japan
Address correspondence and reprint requests to Tatsuo Yamamoto, MD, Department of Anesthesiology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba 260-8670, Japan. Address e-mail to yamamotot{at}faculty.chiba-u.jp.
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Introduction |
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Sensitization of central neurons can be caused by a number of changes, including increased release of excitatory neurotransmitters from primary afferent terminals and changes in the responsiveness of postsynaptic neurons (3). Capsaicin stimulates C-fibers, and repetitive input from C-fibers can evoke a powerful, spinally mediated sensitization (wind-up) of the activity of dorsal horn wide dynamic range neurons (8). The development of wind-up is selectively prevented by N-methyl-d-aspartate (NMDA) receptor antagonist (9) and an opioid receptor like1 (ORL1) receptor agonist (10).
The level of mechanical hyperalgesia or allodynia induced by intradermal capsaicin is reported to be concentration dependent (11). However, a very large concentration or dose of capsaicin produces intense pain and, in turn, is followed by desensitization or hypoalgesia. In this study, the dose-response relationship for intradermal capsaicin in rats was first assessed to determine the smallest possible dose to induce the most intense secondary mechanical allodynia. After the determination of the above dose, we studied the role of the NMDA receptor and the ORL1 receptor in the development and maintenance of secondary mechanical allodynia induced by intradermal capsaicin; we examined the effect of an intrathecal injection of nociceptin, an ORL1 receptor agonist, and MK801, an NMDA receptor antagonist.
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Methods |
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For the capsaicin dose-response study, naïve male Sprague-Dawley rats (250–300 g) were used (n = 28). For the studies involving intrathecal drug administration, male Sprague-Dawley rats (220–270 g) were prepared with intrathecal catheters using a modification of the method described by Yaksh and Rudy (12) (n = 73). Briefly, a chronic intrathecal polyethylene catheter (PE-10) was inserted, under halothane anesthesia, through an incision in the atlantooccipital membrane to a position 8 cm caudal to the cisterna at the level of the lumbar enlargement. Rats with discernible neurologic deficits after surgery were discarded. Rats were given at least a week to recover from surgery before any testing was performed.
Capsaicin 50 µL (0.01%, 0.03%, 0.1%, 0.3%, and 1.0%) was injected intradermally into the plantar surface of the heel portion of the foot with a 30-gauge needle. Rats were briefly anesthetized with halothane during the injection. Capsaicin was dissolved in Tween 80 (10%), alcohol (10%), and saline. A vehicle (50 µL) containing Tween 80 (10%), alcohol (10%), and saline was injected as a control.
Rats were tested for responses to von Frey filaments applied to the plantar surface of the foot in an area within the tori and away from the capsaicin injection site as a measure of secondary mechanical allodynia. Von Frey filaments with logarithmic incremental stiffness (0.41, 0.70, 1.20, 2.00, 3.63, 5.50, 8.50, and 15.10 g; Stoelting, Wood Dale, IL) were used, and the 50% probability threshold of paw withdrawal to mechanical stimulus was determined using a previously described method (13). Rats were placed in plastic cages with a wire-meshed bottom. Beginning with a 2.0 g probe, von Frey filaments were applied to the planter surface of a hind paw for 6–8 s, in ascending or descending order, after a negative or positive response, respectively. Interpolation of 50% probability threshold was conducted according to the method of Dixon (14). In cases where continuous positive or negative responses were observed to the exhaustion of the stimulus set, values of 15.00 g and 0.25 g were assigned, respectively.
The general behavior of each rat was carefully observed and tested. Motor functions were evaluated by the performance of two specific behavioral tasks: (a) The placing/stepping reflex: this response was evoked by drawing the dorsum of either hindpaw over the edge of a tabletop. In normal rats, this stimulus elicits an upward lifting of the paw onto the surface of the table, called stepping. Rats with any degree of hindlimb flaccidity will demonstrate an altered or absent reflex. (b) The righting reflex: an animal placed horizontally with its back on the table will normally show an immediate coordinated twisting of the body around its longitudinal axis to regain its normal position on its feet. Rats displaying ataxic behavior will show a decreased ability to right themselves. To quantify the evaluation of motor functions, both tasks were scored on a scale of 0 to 2, in which 0 = absence of function and 2 = normal motor functions. Rats that were able to perform the motor tasks but did so more slowly than normal rats were assigned a score of 1.
All drugs were dissolved in 0.9% saline such that the final dose was delivered in 10 µL followed by 10 µL of saline to flush the catheter. The drugs used in this study were nociceptin (molecular weight [MW] = 1809; Peptide Institute, Osaka, Japan), CompB ((1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3- dihydro-2H-benzimidazol-2-one, MW = 436; Banyu Pharmaceutical, Tsukuba, Japan), and MK801 (MW = 337; Research Biochemicals, Natick, MA). CompB is a nonpeptidyl ORL1 receptor selective antagonist (15).
After the measurement of the baseline mechanical withdrawal threshold, capsaicin was injected as described. Mechanical withdrawal thresholds were determined every 30 min for 180 min for the capsaicin dose-response study. To test the effect of intrathecal drugs, drugs were administered through the intrathecal catheter either 10 min before (pretreatment) or 30 min after (posttreatment) the capsaicin injection. Mechanical withdrawal thresholds were tested every 30 min for 120 min.
In the present study, we estimated the level of the secondary mechanical allodynia by the 50% probability threshold (g) for mechanical paw withdrawal of the capsaicin-injected hindpaw. To analyze the effect of drugs on the mechanical threshold, the area under the curve (AUC) of the time-effect curve was calculated by use of the trapezoidal rule over the curve. In the capsaicin dose-response study, AUC was calculated from the time of capsaicin injection to 180 min after the capsaicin injection. In the pretreatment group, AUC was calculated from the time of capsaicin injection to 120 min after the capsaicin injection, and in the posttreatment group, AUC was calculated from 30 min after capsaicin injection to 120 min after capsaicin injection (AUC30–120). Capsaicin dose response and nociceptin dose response were calculated by AUC and analyzed by using one-way analysis of variance (ANOVA) followed by the Tukey test. The effect of MK801 was analyzed with a t-test. Results are presented as mean ± sem, or otherwise stated. A P value of <0.05 was considered significant.
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Results |
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Based on the preceding study, the capsaicin concentration of 0.03% was chosen as the maximum effective dose and was used for further testing.
Preliminary dose range studies demonstrated that when 30 µg of MK801 was administered intrathecally, all rats scored 1 (impairment of motor function) in the placing/stepping reflex and righting reflex tests and that this motor impairment lasted longer than 30 min and was considered unacceptable for behavioral testing. When 10 µg of MK801 was administered intrathecally, all rats scored 2 (normal motor function) in the placing/stepping reflex and righting reflex tests. Thus, 10 µg of MK801 was the largest dose applied in the present study. Intrathecal injection of 10 µg of MK801, both pre- and posttreatment, had no effect on the level of AUC and AUC30–120 as compared with the saline-treated rats, respectively (Fig. 3; pretreatment, P > 0.7; posttreatment, P > 0.6 by t-test; n = 5–7).
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After the intrathecal injection of nociceptin or CompB, all rats scored 2 (normal motor function) in the placing/stepping reflex and righting reflex tests.
Intrathecal injection of nociceptin (3, 10, and 30 µg) pretreatment showed no effect on the level of AUC as compared with saline-treated rats (Fig. 3 and 4; pretreatment, P > 0.4 by ANOVA; n = 5–6). However, posttreatment with intrathecal injection of nociceptin (3, 10, and 30 µg) dose-dependently reversed the threshold decrease observed after the intradermal injection of capsaicin (Fig. 3 and 4; posttreatment. P < 0.001 by ANOVA; n = 5–7), with a significant effect with 30 µg of nociceptin as compared with saline-treated rats (P < 0.001 by Tukey test).
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Discussion |
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Capsaicin-induced secondary mechanical allodynia is a well established experimental human pain model. Simone et al. (11) have shown concentration-dependent pain and hyperalgesia from 0.01 to 100 µg of capsaicin in humans. In most studies, 1% capsaicin solution was injected intradermally (1,7,16–18). The dose-response curve in humans has not been shown to be bell-shaped in this dose range. In our experiments, intradermal injection of capsaicin 1% solution was observed with a slightly hypoalgesic state in the secondary area. In human studies, only the injected site—within the small bleb produced by the capsaicin injection—has been shown to be hypoalgesic or desensitized, and this is only seen at large concentrations (by injection) but not at small concentrations (by topical application) (18). Our data indicated that 0.3% and 1.0% of capsaicin solutions did not produce significant secondary mechanical allodynia and that these concentrations are not suitable for studying secondary mechanical allodynia. In rats, a 0.1% solution (5,6,19) is often used as an identical stimulus in human experiments. Our dose-response study demonstrated that 0.03% capsaicin solution was the smallest possible dose to produce the most intense secondary mechanical allodynia, and we used a 0.03% capsaicin solution in the following study.
Ten micrograms of MK801 failed to alter the development and maintenance of the capsaicin-induced secondary mechanical allodynia. Larger doses of MK801 could not be used because of the evolving motor dysfunction, and an intrathecal injection of 10 µg of MK801 has been reported to alleviate the central sensitization induced by peripheral nerve injury or peripheral tissue injury (20,21). These reports suggested that 10 µg of MK801 is the suitable dose for the study of NMDA-dependent central sensitization. The sensitization of spinothalamic tract neurons by intradermal injection of capsaicin is alleviated by the administration of antagonists of NMDA receptors (4), and capsaicin-induced hyperalgesia is thought to be caused by NMDA-dependent central sensitization. In that experiment, 3% capsaicin was used for the stimulus in anesthetized primates, which would likely be a stimulus intense enough to produce desensitization. The authors’ result, showing NMDA independence in the small-dose (0.03%) capsaicin-induced secondary mechanical allodynia, is in accordance with the results shown in a skin incision model (22) and in a mild thermal injury model (23), in which the stimulus or the tissue injury is mild. Accordingly, our results suggest involvement of different central mechanisms in small-dose capsaicin-induced secondary mechanical allodynia in which the NMDA receptor is a significant factor, including hyperalgesia induced by large-dose capsaicin.
In the human model of capsaicin-induced pain, the NMDA antagonist, ketamine, given IV, is effective in reducing continuing pain and hyperalgesia (17). However, drugs given systemically have several sites of action. Indeed, peripherally applied ketamine has been reported to effectively inhibit the development of burn-induced secondary hyperalgesia to a level equivalent to systemic infusion (24,25).
The ORL1 receptor agonist nociceptin effectively attenuated the level of secondary mechanical allodynia induced by intradermal capsaicin, and this effect was antagonized by the ORL1 receptor selective antagonist CompB. However, this effect was only observed with posttreatment and not pretreatment. These data suggested that the maintenance of mechanical allodynia, but not the development of mechanical allodynia, induced by intradermal injection of 0.03% capsaicin depends on the spinal ORL1 receptor system.
The ORL1 receptor agonist, nociception, inhibits the development of wind-up (10), and this may be the main mechanism to attenuate the level of secondary mechanical allodynia. The wind-up–like sensitization is thought to play an important role in the development and maintenance of allodynia induced by inflammation (26). The wind-up phenomenon in the spinal dorsal horn is induced by the repetitive input from C-fibers (10–15 times; 0.5 Hz), and this phenomenon is not maintained when the nociceptive input to the spinal dorsal horn ends (8). In the capsaicin model, central sensitization is initially induced by the intradermal injection of 0.03% capsaicin. Once central sensitization is established, continuous C-fiber input to the spinal dorsal horn may be required to maintain central sensitization. In the capsaicin model, the central sensitization may be maintained by the nociceptive input from the site of the capsaicin injection where an inflammatory reaction is induced by the capsaicin injection. The level of nociceptive input induced by intradermal injection of 0.03% capsaicin may be much higher than that of the nociceptive input from an inflammatory reaction induced by capsaicin. We think this difference is the key mechanism to differentiate the development and the maintenance of central sensitization in the capsaicin model. In the capsaicin model, nociceptin is not able to prevent the development of central sensitization induced by the 0.03% capsaicin injection itself but can alleviate central sensitization maintained by nociceptive input from the site of capsaicin injection, where the inflammatory reactions are induced by response to the capsaicin.
In the present study, 30 µg of nociceptin applied 10 minutes before the capsaicin injection had no effect on the level of the secondary mechanical allodynia 30, 60, 90, and 120 minutes after the capsaicin injection. In contrast, 30 µg of nociceptin applied 30 minutes after the capsaicin injection attenuated the level of secondary mechanical allodynia 60, 90, and 120 minutes after the capsaicin injection (Fig. 3). If maintenance of mechanical allodynia induced by intradermal injection of 0.03% capsaicin depends on the spinal ORL1 receptor system, 30 µg of nociceptin applied 10 minutes before the capsaicin injection should have some effect 30 minutes after capsaicin injection. Although the level of nociceptive input from the inflammatory reaction induced by the capsaicin injection may have been weak, 30 µg of nociceptin was required to produce a significant antiallodynic effect in the posttreatment study. This suggested that a large concentration of nociceptin is required to alleviate the central sensitization maintained by nociceptive input from the inflammatory reaction induced by capsaicin injection. Thus, in the pretreatment study, 30 µg of nociceptin did not prevent the development of central sensitization, and after the development of the central sensitization, 30 µg of nociceptin did not alleviate central sensitization maintained by nociceptive input from the inflammatory reaction induced by the capsaicin injection. In the posttreatment study, the effect of 30 µg of nociceptin lasted more than 90 minutes. This suggests that once central sensitization disappeared from a large concentration of nociceptin, a relatively small concentration of nociceptin prevented the development of central sensitization. Probably, the nociceptive input from the inflammatory reaction induced by the capsaicin injection was weak, and a relatively small concentration of nociceptin was enough to prevent the development of central sensitization.
Spinal nociceptin has been shown to be analgesic in pain models, which produce inflammatory pain, such as in the formalin test (27) and carrageenan test (28), and is antihyperalgesic in neuropathic pain models, such as chronic constriction nerve injury (29) in rats. Pain and hyperalgesia observed in these models are also maintained by NMDA receptor-dependent central sensitization. Nociceptin has also been shown to attenuate mechanical hyperalgesia induced by skin incision, a model reported to be non-NMDA receptor-dependent (30). Moreover, thermal hyperalgesia induced by partial sciatic nerve injury was alleviated by an intrathecal injection of nociceptin but not by MK801 (31). Our results—the effective reversal of secondary mechanical allodynia induced by intradermal capsaicin and by intrathecal nociceptin but not by intrathecal NMDA receptor antagonist—adds to the idea that there is central sensitization that is maintained by the ORL1 receptor-dependent and NMDA receptor-independent mechanisms.
In conclusion, 0.03% of capsaicin solution is the smallest concentration of an intradermal injection that develops the most intense secondary mechanical allodynia. Mechanical allodynia induced by intradermal injection of 0.3% capsaicin is maintained by the spinal ORL-1 receptor-dependent mechanism but not by the spinal NMDA receptor-dependent mechanism.
We thank Professor Takashi Nishino, Department of Anesthesiology, Graduate School of Medicine, Chiba University, for his generous support of our study.
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Accepted for publication September 22, 2004.
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