Involvement of Peripheral Opioid Mechanisms in Electroacupuncture Analgesia

ORIGINAL RESEARCH

INVOLVEMENT OF PERIPHERAL OPIOID MECHANISMS IN ELECTROACUPUNCTURE ANALGESIA Grant G. Zhang, PhD,1,# Chengsi Yu, PhD,1 Wenlin Lee, PhD,1 Lixing Lao, PhD,1 Ke Ren, PhD,2 and Brian M. Berman, MD1

The involvement of the peripheral opioid system in modulating inflammatory pain has been well documented. This study aimed to investigate the possibility of electroacupuncture (EA)mediated peripheral opioid release. Rats were injected with complete Freund’s adjuvant in one of the hind paws to induce localized inflammatory pain. The pain behavioral changes were measured by paw withdrawal latency (PWL) to a noxious thermal stimulus. At day 5 of inflammation, rats received a second injection of saline or opioid antagonists into the inflamed paw, followed by EA at 30 Hz, 2 mA, and 0.1 ms for 30 minutes. The EA was conducted at acupuncture point GB30. A control was used in which needles were inserted at GB30 but no electrical stimulation was applied. Rats receiving EA showed a signifi-

cantly longer PWL as compared with the control from 30 minutes to three hours after EA treatment. Intraplantar but not intraperitoneal injection of naloxone methiodide, a peripherally acting opioid receptor antagonist, eliminated the analgesic effect at 30 minutes after EA treatment. Intraplantar injection of an antibody against ␤-endorphin and a corticotropin-releasing factor antagonist also produced a reduction in PWL in rats receiving EA. These data strongly suggest that peripheral opioids are released by EA at the inflammatory site.

Studies have shown the presence of peripheral mechanisms in the inhibition of pain. Such mechanisms involve hormones, cytokines, and neurotransmitters that interact at the peripheral terminals of afferent nerves.1,2 Opioid receptors are present on the peripheral sensory nerve fibers and their terminals.3–5 Exogenous opioid receptor agonists, when administered locally, bind to these receptors to produce analgesia. The sources of endogenous opioid peptides in the peripheral tissues have been identified to be immune cells, including macrophages, mast cells, lymphocytes, and plasma cells.3,6,7 Cells containing high levels of opioid peptides have been identified at inflammatory sites. The local release of opioids from these cells can be triggered by a stressful event such as a cold water swim3,8 and is mediated by a variety of hormones and cytokines, such as corticotropin-releasing factor (CRF), tumor necrosis factor, and interleukins 1␤ and6,9 –13 as well as sympathetic nerve activation.14 This neuroimmune network appears to be activated during an acute inflammatory pain condition. For example, the number of opioid receptors in normal tissues is barely detectable but increases substantially after the initiation of an inflammatory reaction.15 Several opioid peptides, ␤-endorphin, met-enkephalin,

and dynorphin, have been found in increased levels in the inflamed tissue, ␤-endorphin being the most abundant.6,16,17 It appears that, at the early stage of inflammation (six hours), both peripheral and central opioid systems are activated to inhibit nociception; at the later stages (four days), antinociception is exclusively produced by leukocyte-derived ␤-endorphin acting at peripheral ␮ and ␦ receptors.18 Acupuncture has been used in China for thousands of years to treat a variety of diseases. Its clinical application has grown steadily in the West in recent years, with chronic pain as the most common indications.19-21 Studies have demonstrated that acupuncture-mediated analgesia is modulated through a central mechanism involving neurohumoral pathways.22,23 The current working model proposes that, when an acupuncture point is needled, the large (A␤) and small (A␦) myelinated nerve fibers are selectively activated. This subsequently results in activation of certain neurons in the spinal cord and supraspinal regions, including neurons in the deep layers of spinal dorsal horn,24-26 the nucleus raphe magnus in the brain stem,27 and the hypothalamus and thalamus.28,29 These neurons modulate pain by inhibiting neurons located at the superficial layer of the dorsal horn and small unmyelinated fibers24,26,30 and by releasing neurotransmitters, such as opioids.31-38 The expression of all four subsets of opioid peptides, ␤-endorphin, met-enkaphelin, dynorphin, and endomorphins, have been found to be up-regulated following acupuncture treatment.39-41 Opioid peptides bind to their receptors on central neurons and produce analgesic effects.42 However, whether, and to what extent, the peripheral release of opioids plays a role in acupuncture analgesia has not been investigated. Two lines of evidence suggest that acupuncture-induced peripheral opioid release is possible. First, acupuncture has been shown to modulate immunological activi-

1 Complementary Medicine Program, University of Maryland School of Medicine, Baltimore, MD 2 Department of OCBS, Dental School, University of Maryland, Baltimore, MD Supported by a grant award (1R21RT00279) from the National Center for Complementary and Alternative Medicine, NIH. # Corresponding author. Address: Kernan Mansion, 2200 Kernan Dr, Baltimore, MD 21207 e-mail [email protected]

© 2005 by Elsevier Inc. Printed in the United States. All Rights Reserved ISSN 1550-8307/05/$30.00

Key words: electroacupuncture, peripheral opioids, rat, hyperalgesia, naloxone (Explore 2005; 1:365-371. © Elsevier Inc. 2005)

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ties,43-51 and immune cells are a key component of the peripheral opioid system. Second, central opioid mechanisms do not explain why needling an acupuncture point adjacent to a painful area, a common practice in clinic, is generally more effective in relieving pain than needling distal areas, a phenomenon that is consonant with the presence of a localized analgesic mechanism. The aim of the present study was to investigate the involvement of the peripheral opioid system in the acupuncture treatment of a painful condition. Using a rat model of unilateral inflammatory hyperalgesia, we examined the effect of electroacupuncture on hyperalgesia and the effect of locally administered opioid receptor antagonists on acupuncture analgesia.

MATERIALS AND METHODS Animal Preparation Male Sprague-Dawley rats weighing 250 to 350 g were kept under controlled environmental conditions (22°C ⫾ 0.5°C, relative humidity 40% to 60%, 7 AM to 7 PM alternate light-dark cycles, food and water ad libitum). The animals were housed in cages in which the floor is covered with paper pellets to minimize potentially painful contacts with a hard surface. Rats were randomly assigned to each experimental group. The University of Maryland School of Medicine Animal Care and Use Committee approved the animal model. The International Association for the Study of Pain ethical guidelines for the treatment of animals was adhered to in these experiments.52

Induction of inflammation. Inflammation and hyperalgesia were induced by injecting 0.05 mL complete Freund’s adjuvant (CFA; suspended in an 1:1 oil/saline emulsion, containing 0.05% heat-killed and dried Mycobacterium butyricum, CalBiochem) subcutaneously into the plantar surface of one hind paw of the rat using a 25-gauge hypodermal needle. The inflammation, manifested as redness, edema, and hyperresponsiveness to noxious stimuli, was limited to the injected paw. Hyperalgesia was determined by a reduction of paw withdrawal latency (PWL) to a noxious thermal stimulus. On day 5 after the CFA injection, 0.05 mL saline or reagents dissolved in saline was injected into the inflamed hind paw. CFA-inflamed rats show normal grooming behavior and levels of activity, and the effect of hyperalgesia on the normal behavior of the animal is minimal.53,54 Measurement of thermal hyperalgesia. Rats were tested for hind paw thermal hyperalgesia by a method described in detail by Hargreaves et al.54 The rats were placed under a clear plastic chamber on an elevated glass surface and allowed to acclimatize for 30 minutes. A radiant heat stimulus was applied from underneath the glass floor with a high-intensity projector lamp bulb (Osram 58-8007, USHIO, Tokyo, Japan; 8 V, 50 W). The heat stimulus was directed onto the plantar surface of each hind paw, and the PWL to the nearest 0.1 second is determined using an electronic clock circuit and a microcomputer (Model 336, IITC Life Science, Woodland Hills, CA). Bulb voltage was adjusted to derive an average baseline PWL of approximately 10.0 seconds

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in naive animals. A 20-second cut-off was used to prevent tissue damage. The PWL of the inflamed paw is usually 4 to 6 seconds faster than that of the normal paw, indicative of a state of thermal hyperalgesia. PWL was established by averaging the latency of 3 tests with a 5-minute interval between each test. The PWL were measured pre-CFA and at designated intervals post-CFA injection. To avoid possible conditioning, two groups of rats were used in experiments involving hourly measure of PWL. One group was measured at 30 minutes and two and five hours, and the other group was measured at one and three hours after EA treatment. The investigator who performed PWL testing and all other measurements was unaware of the treatment assignments.

Acupuncture treatment. Acupuncture treatment was given immediately following the second injection at day five after the CFA injection. The equivalent human acupuncture point GB30 (Huantiao) was selected ipsilateral to the animal’s inflamed hind limb. In humans, GB30 is located at the junction of the lateral one third and medial two thirds of the distance between the great trochanter and the hiatus of the sacrum.55,56 In the rat, the equivalent anatomical landmarks were used to locate this point. A nonacupuncture point site 10 mm below GB30 at the same side of the inflamed paw was also used. Two disposable, stainless-steel acupuncture needles (gauge No. 32-30, one inch in length) were slanted inserted into the two points with needle tip toward the ankle, and the needles were fixed at the points by adhesive paper tape. A pair of electrodes from an electrical stimulator (A300; WPI, Sarasota, FL) was attached to the ends of the needles. The EA was applied at 2.0 mA, 30 Hz, and 0.1 ms pulse for 30 minutes. Our previous study on EA parameters using a similar rat pain model found that EA at a moderate frequency (10-100 Hz) and intensity (2-3 mA) produced a significantly greater antihyperalgesic effect.24,26 In our pilot study, EA with moderate frequency and intensity for 10 or 20 minutes did not produce significant effect on thermal hyperalgesia (data not shown). No anesthesia was used during the EA treatment. Animals were placed under a plastic chamber (5” ⫻ 8” ⫻ 11”) with no physical restraint. No sign of stress, such as increased urination or defecation, was observed. For EA control, rats received acupuncture needles inserted at the same points as for EA treatment but no electrical stimulation was supplied. For vehicle controls, rats received the identical procedure as the EA control except that no acupuncture needles were inserted. Administration of antagonists. The following reagents were used: rat CRF (1 ng, 3 ng, Sigma, St Louis, MO); ␣-helical CRF (2 ng, Sigma); naloxone methiodide (5 ␮g, 50 ␮g, Sigma); rabbit anti-␤-endorphin, (0.2 ␮g, Peninsula Laboratories). All reagents were dissolved in saline. Routes and volumes of administration were intraplantar (i.pl., 0.05 mL) or intraperitoneal (i.p., 0.05 mL). Data Analysis The results are presented as means ⫾ SEM. For initial experiments establishing the model, the percentage change of PWL was calculated based on the formula: [(PWLpost-CFA ⫺ PWLpre-CFA)/

Local Opioid Receptor Antagonists Block EA Analgesia

3 hours (EA: 20.0% ⫾ 3.9%, EA control: 9.9% ⫾ 3.0%, vehicle control: 4.6% ⫾ 1.7%) after EA treatment. No significant differences were found among the three groups at five hours after the EA treatment. No significant changes in PWL were recorded on the contralateral paws at any of the time points (Figure 2, contralateral).

Figure 1. Time course of inflammatory pain behavior. Paw withdrawal latency (PWL) to a noxious thermal stimulus was determined at the indicated time points. The percentage changes in PWL were defined by the formula: [(PWLpost-CFA ⫺ PWLpre-CFA)/PWLpre-CFA] ⫻ 100%. The data are expressed as means (the closed or open symbols) ⫾ SEM. N ⫽ 8 for both groups.

The Effect of Local CRF on Unilateral Hyperalgesia Intraplantar injection of CRF (3 ng) into the inflamed hind paw also produced a significant increase in PWL at one hour following the injection (9.7% ⫾ 8.5%) as compared with the rats receiving saline only (⫺16.5% ⫾ 5.3%, P ⬍ .01; Figure 3, ipsilateral). The increased PWL continued to be detectable at two hours and five hours after the administration of CRF, although no significant difference was found at these time points because of the large standard variation within the CRF group. The injection of a reduced dose of CRF (1 ng) also produced an increase in PWL, but the increase did not reach statistical significance when compared with the saline control.

PWLpre-CFA] ⫻ 100%. For all other experiments, the percentage change of PWL was calculated as follows: [(PWLpostsecond injection ⫺ PWLpresecond injection)/PWLpresecond injection ] ⫻ 100%. Separate analyses of variance (ANOVAs) were performed to examine the difference between groups on the percentage change of PWL as the dependent variable. To identify further the locus of any significant effects, post hoc Tukey’s tests of significance were employed to make pairwise comparisons. The significant criterion for all analyses was set at P ⬍ .05.

RESULTS The Effect of EA on Local Hyperalgesia Injection of CFA into one hind paw induced a unilateral hyperalgesia that lasted approximately 10 days, with significant reductions of PWL at 2.5 hours, continuing at 24 hours, and gradually recovering around day five after the injection of CFA (Figure 1). A second i.pl. injection of saline into the inflamed paw on day five did not significantly change PWL between day five and day 9 (Figure 1). The PWLs of the contralateral paws in either group did not show any significant changes during the observation period (day zero to day nine, Figure 1). All subsequent experiments were conducted on day five after CFA injection. Thirty minutes after completion of the EA treatment (60 minutes after the second injection), rats receiving EA treatment showed a significant increase of PWL (1.7% ⫾ 2.5%, Figure 2 ipsilateral) as compared with the EA control group (⫺19.4% ⫾ 4.3%, P ⬍ .05) and the vehicle control group (⫺10.7% ⫾ 4.0%, P ⬍ .05). The EA-treated rats continued to show a significantly longer PWL (P ⬍ .05) at 1 hour (EA: 10.5% ⫾ 5.5%, EA control: ⫺7.9% ⫾ 3.65%, vehicle control: ⫺2.9% ⫾ 2 %), 2 hours (EA: 18.2% ⫾ 2.1%, EA control: 1% ⫾ 4.5%, vehicle control: 5% ⫾ 3%), and

Local Opioid Receptor Antagonists Block EA Analgesia

Figure 2. The effect of EA on PWL. Rats were randomized into either EA group or control groups at day 5 after CFA injection. PWL was tested at the indicated time points. Top panel: PWL of ipsilateral paw (inflamed paw). Lower panel: PWL of contralateral paw (normal paw). The percentage changes of PWL were defined as [(PWLpostsecond injection ⫺ PWLbefore second injection)/PWLbefore second injection ] ⫻ 100%. The data was expressed as mean (the bars) ⫾ SEM. *P ⬍ .05 EA group (n ⫽ 11) compared with EA control group and vehicle group (n ⫽ 8 for each group).

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PWL was still present (⫺9.9% ⫾ 5.0%, P ⬍ .05) but with lesser magnitude (Figure 4A). Intraperitoneal injection of the higher dose of naloxone (50 ␮g) did not cause a significant change of PWL in EA-treated rats in either paw at any time points as compared with the saline-EA group. Intraplantar administration of an antibody against ␤-endorphin or ␣-helical CRF in EAtreated rats resulted in a significant reduction of PWL at 30 minutes after the EA treatment as compared with the saline-EAtreated rats (P ⬍ .05; Figure 4B, ipsilateral). No significant difference was found among these groups at one hour and two hours after the EA treatment. No significant difference of PWL was found in the contralateral paws of the three groups (Figure 4B, contralateral).

Figure 3. The effect of local CRF on PWL. Rats were randomized into either CRF group or saline group at day 5 after CFA injection. PWL was tested at the indicated time points. Top panel: PWL of ipsilateral paw (inflamed paw). Lower panel: PWL of contralateral paw (normal paw). The percentage changes of PWL were defined as [(PWLpostsecond injection ⫺ PWLbefore second injection)/PWLbefore second injection ] ⫻ 100%. The data are expressed as mean (the bars) ⫾ SEM. *P ⬍ .05 CRF group compared with saline group. N ⫽ 8 for saline and CRF 3-ng groups. N ⫽ 7 for CRF 1-ng group.

The Effect of Locally Administered Nalaxone, Anti-␤-Endorphin Antibody, and CRF Antagonist on EA-Mediated Analgesia Rats receiving EA immediately following the i.pl. injection of naloxone methiodide (50 ␮g) showed a reduction of PWL (⫺16.6% ⫾ 4.5%; Figure 4A) to the level of EA control (⫺19.4% ⫾ 4.3%, Figure 2) at 30 minutes after the completion of EA treatment (60 minutes after naloxone injection), suggesting attenuation of EA-produced antihyperalgesia. A significant difference was found between the naloxone-EA-treated rats and salineEA-treated rats (P ⬍ .05; Figure 4A, ipsilateral). The naloxoneproduced suppression of EA-induced PWL modulation was not observed at one hour and two hours after EA treatment. Neither EA nor i.pl. injection of naloxone showed any significant effects on PWL of the contralateral paws at any time points (Figure 4A, contralateral). A dose-response trend was observed when 10-fold less naloxone (5 ␮g) was used, whereas a significant reduction of

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DISCUSSION The Animal Model The rat model of unilateral inflammatory pain has been used extensively in the study of peripheral opioid system.2 The localized persistent pain provides an ideal condition for examining the effect of peripheral opioids and its mechanism. Most previous studies have used the mechanical paw pressure threshold test for the behavioral study.3,8,10,11,53,57 In the present study, we used paw withdrawal response to a noxious thermal stimulus as a behavioral end point. This test is well established and is highly reliable.54 We have used this model previously to demonstrate acupuncture-produced antihyperalgesia in rats.24,25 The significant increase in PWL following local delivery of CRF (Figure 3) further supports the use of this model in present study. The features of PWL change after EA, eg, adjacent to the injected paw and short duration, are characteristic of the release of peripheral opioids. An important advantage of the thermal PWL test is that the test does not require physical restraint of the animal. The stress of the animal, which is one of the main confounding factors in acupuncture animal study, was therefore reduced to a minimum. After EA treatment, however, longer waiting time (10 to 15 minutes) was needed to allow the rat to acclimatize before the PWL test can be reliably conducted. Studies have shown that significant levels of local opioids were not detected until day four or five after the CFA challenge.16 We chose day five after CFA injection for our experiments, based on our observations that EA was less effective when the inflammatory pain was very severe, such as at day two or three after CFA injection (data not shown). The Effectiveness of EA on Thermal Hyperalgesia In this study, we found that EA at 2 mA, 30 Hz, 0.1 ms for 30 minutes produced a significant effect on thermal hyperalgesia. The effect was detected 30 minutes after EA treatment (our first time point for measuring) and lasted for at least three hours thereafter. The analgesic effect was localized to the affected paw, with no significant behavioral changes recorded on the contralateral paw (Figure 2). In our study on the specificity of acupuncture points, we have observed that EA at the point adjacent to the painful area (GB 30) was more effective than the points at the distal areas (on the forepaw or on the abdominal region26), suggesting a more localized action. It is known that peripheral oipoids were released when the animal is exposed to a stressful

Local Opioid Receptor Antagonists Block EA Analgesia

Figure 4. The effect of opioid receptor antagonists on PWL of rats receiving EA. Rats were injected with naloxone (panel A) or antibody against ␤-endorphin or antagonist to CRF (panel B) followed by EA treatment at day 5 after CFA injection. PWL was tested at the indicated time points. Top panel: PWL of ipsilateral paw (inflamed paw). Lower panel: PWL of contralateral paw (normal paw). The percentage changes in PWL were defined as [(PWLpostsecond injection ⫺ PWLbefore second injection)/PWLbefore second injection ] ⫻ 100%. The data were expressed as mean (the bars) ⫾ SEM. *P ⬍ .05 EA saline group compared with EA drug groups. N ⫽ 8 for each group. event, such as cold-water swimming.8 Was the antihyperalgesia effect of EA mediated by stress? Our results suggested this was unlikely. First, all of the animals in our experiments were under no physical restraint even during the acupuncture treatment. Animals were observed as quiet and relaxed. To achieve this, we used moderate electrical stimulation that was within the tolerance of the animals. We have not observed signs of increased stress, such as increased urination and defecations. Second, our EA control animals, which received needle insertion at the same place as EA-treated rats, showed no increase in PWL. Third, in contrast to the short duration (minutes) of the analgesic effect mediated by a stressful event,8 the EA-mediated analgesic effect characterized a long-lasting effect (at least three hours in this study and days in our other studies25). The Effect of Locally Administered Opioid Receptor Antagonists on EA Analgesia In this study, we demonstrated that intraplantar administration of naloxone effectively blocked the EA analgesia. The blockage

Local Opioid Receptor Antagonists Block EA Analgesia

of the analgesic effect by naloxone has the following features: (1) The duration of the blockage was brief. The EA analgesic effect was blocked totally only at the 30 minutes but not at the one hour after EA treatment. This differed from both the classical peripheral opioid blockage studies, in which a total blockage of the analgesic effect was observed within five minutes after the trigger event8 and the systemic blockage studies, which showed a total blockage of EA analgesia for at least 120 minutes.31 The short-lived blockage by local naloxone in our study suggested that factors other than peripheral opioid system were involved in EA analgesia. One of the factors is the central opioid system, which is likely to be activated following EA treatment, and local naloxone would be expected to have limited effect on its activity. (2) The effect of naloxone was local. The decreased PWL was only recorded at the paw receiving naloxone but not the contralateral paw, consistent with other reports.3,8,10,11 (3) The dosage of naloxone methiodide employed was low. The dosage at 50 ␮g (approximately 0.25 mg/kg) effectively reversed the EA analgesia to the EA control level, whereas the naloxone dosage

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caused a systemic effect typically ranging from 2 mg to 3 mg/ kg.31 (4) Naloxone methiodide showed a dose-dependent trend with dosage at 5 ␮g showing a smaller blockage effect as compared with the 50-␮g dosage. (5) Abdominal delivery of naloxone methiodide showed no significant effect on the PWL of both paws. We want to point out that the naloxone methiodide does not pass the brain barrier58; hence, no central effect was expected. Taken together, our results strongly suggest that EA induces peripheral opioid release and activates peripheral opioid receptors, which were blocked by naloxone administrated at the inflammatory site. Further support of this notion comes from the observation that EA analgesia was blocked by an antibody against ␤-endorphin and a CRF antagonist that were delivered locally. This study is the first to demonstrate a relationship between the analgesic effect of acupuncture and peripheral opioid system. We are currently examining the levels of opioid peptides in the peripheral tissues and sources of peripheral opioids following EA treatment. This will further support the involvement of peripheral opioid system in acupuncture-produced attenuation of inflammatory hyperalgesia. Acknowledgments The authors thank Xiaoya Wang and Dr. Rui-xin Zhang for their support during the study and Dr. Barker Bussell for his critical review of the paper.

REFERENCES 1. Machelska H, Stein C. Pain control by immune-derived opioids. Clin Exp Pharmacol Physiol. 2000;27:533-536. 2. Janson W, Stein C. Peripheral opioid analgesia. Curr Pharmaceut Biotech. 2003;4:270-274. 3. Stein C, Hassan AHS, Przewlocki R, Gramsch C, Peter K, Herz A. Opioids from immunocytes interact with receptors on sensory nerves to inhibit nociception in inflammation. Proc Natl Acad Sci U S A. 1990;87:5935-5939. 4. Coggeshall RE, Zhou S, Carlton SM. Opiate receptors on peripheral sensory axons. Brain Res. 1997;764:126-132. 5. Wenk HN, Honda CN. Immunohistochemical localization of delta opioid receptors in peripheral tissues. J Comp Neurol. 1999;408:567579. 6. Przewlocki R, Hassan AHS, Lason W, Epplen C, Herz A, Stein C. Gene expression and localization of opioid peptides in immune cells of inflamed tissue: functional role in antinociception. Neuroscience 1992;48:491-500. 7. Brack A, Labuz D, Schiltz A, Rittner HL, Machelsa H, Schafer M, Reszka R, Stein C. Tissue monocytes/macrophages in inflammation: hyperalgesia versus opioid-mediated peripheral antinociception. Anesthesiology. 2004;101:204-211. 8. Stein C, Gramsch C, Herz A. Intrinsic mechanisms of antinociception in inflammation: local opioid receptors and b-endorphin. J Neurosci. 1990;10:1292-1298. 9. Mousa SA, Schafer M, Mitchell WM, Hassan AH and Stein C (1996) Local upregulation of corticotropin-releasing hormone and interleukin-1 receptors in rats with painful hindlimb inflammation. Eur J Pharmacol. 1996;311:221-231. 10. Czlonkowski A, Stein C, Herz A. Peripheral mechanism of opioid antinociception in inflammation: involvement of cytokines. Eur J Pharmacol. 1993;242:229-235.

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11. Schäfer M, Carter L, Stein C. interleukin 1␤ and corticotropinreleasing factor inhibit pain by releasing opioids from immune cells in inflamed tissue. Proc Natl Acad Sci U S A. 1994;91:4219-4223. 12. Schäfer M, Mousa SA, Stein C. Corticotropin-releasing factor in antinociception and inflammation (review). Eur J Pharmacol. 1997; 323:1-10. 13. Mousa SA, Bopaiah CP, Stein C, Schafer M. Involvement of corticotropin-releasing hormone receptor subtypes 1 and 2 in peripheral opioid-mediated inhibition of inflammatory pain. Pain. 2003;16: 297-307. 14. Binder W, Mousa SA, Sitte N, Kaiser M, Stein C, Schafer M. Sympathetic activation triggers endogenous opioid release and analgesia within peripheral inflamed tissue. Eur J Neurosci. 2004;20:92-100. 15. Hassan AHS, Ableitner A, Stein C, Herz A. Inflammation of the rat paw enhances axnal transport of opioid receptors in the sciatic nerve and increases their density in the inflamed tissue. Neuroscience. 1993; 55:185-195. 16. Cabot PJ, Carter L, Gaiddon C, et al. Immune cell-derived ␤-endorphin: production, release and control of inflammatory pain in rats. J Clin Invest. 1997;100:142-148. 17. Cabot PJ, Carter L, Schäfer M, Stein C. Methionine-enkephalinand dynorphin A-release from immune cells and control of inflammatory pain. Pain. 2001;93:207-212. 18. Machelska H, Schopohl JK, Mousa SA, Labuz D, Schafer M, Stein C. Different mechanisms of intrinsic pain inhibition in early and late inflammation. J Neuroimmunenol. 2003;141:30-39. 19. Bullock ML, Pheley AM, Kiresuk TJ, Lenz SK, Culliton PD. Characteristics and complaints of patients seeking therapy at a hospitalbased alternative medicine clinic. J Altern Compl Med. 1997;3:31-37. 20. Patel M, Gutzwiller F, Paccaud F, Marazzi A. A meta-analysis of acupuncture for chronic pain. Int J Epidemiol. 1989;18:900-906. 21. Riet GT, Kleunen J, Knipschild P. Acupuncture and chronic pain: a criteria-based meta-analysis. J Clin Epidemiol. 1990;43:1191-1199. 22. Pomeranz B. Acupuncture analgesia-basic research. In: Stux G, Hammerschlag R, eds. Clinical Acupuncture Scientific Basis. Berlin, Heidelberg, New York: Springer-Verlag; 2001 23. Sim J. The mechanism of acupuncture analgesia: a review. Com Ther Med. 1997;5:102-111. 24. Lao L, Zhang G, Wei F, Berman BM, Ren K. Electroacupuncture attenuates hyperalgesia and modulates Fos protein expression in rats with unilateral persistent inflammation. J Pain. 2001;2:111-117. 25. Lao L, Zhang R-X, Zhang G, Wang X, Berman B, Ren K. A parametric study of electroacupuncture on persistent hyperglasia and Fos protein expression in rats. Brain Res. 2004;1020:18-29. 26. Zhang R-X, Lao L, Wang L, et al. Involvement of opioid receptors in electroacupuncture-produced antihyperalgesia in rats with peripheral inflammation. Brain Res. 2004:1020:12-17. 27. Toda K. Response of raphe neurons after acupuncture stimulation in rat. Brain Res. 1982;242:350-353. 28. Takeshige C, Sato T, Mera T, Hisamitru T, Fang JQ. Descending pain inhibitory system invovled in acupuncture analgesia. Brain Res Bull. 1992;29:617-634. 29. Takeshige C, Oka K, Mizuno T, et al. The acupuncture point and its connecting central pathway for producing acupuncture analgesia. Brain Res Bull. 1993;30:53-67. 30. Liu X, Zhu B, Zhang SX. Relationship between electroacupuncture analgesia and descending pain inhibitory mechanism of nucleus raphe magnus. Pain. 1986;24:383-396. 31. Pomeranz B, Chiu D. Naloxone blocks acupuncture analgesia and causes hyperalgesia: endorphin is implicated. Life Sci. 1976;19:17571762. 32. Cheng RSS, Pomeranz B, Yu G. Dexamethaxone partially reduces and 2% saline treatment abolished electroacupuncture analgesia:

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33.

34. 35.

36. 37.

38.

39.

40.

41.

42. 43.

44.

these finding implicate pituitary endorphins. Life Sci. 1979;24:1481-1486. Cheng RSS, Pomeranz B. Electro-acupuncture analgesia could be mediated by at least two pain-relieving mechanisms: endorphin and non-endorphin systems. Life Sci. 1979;25:1957-1962. Han JS, Tang J, Ren MF, et al. Central neurontrasmitters and acupuncture analgesia. Am J Chin Med 1980;8:331-348. Han JS, Xie GX, Zhou ZF, Folkesson R, Terenius L. Enkephalin and B-endorphin as mediators of electroacupuncture analgesia in rabbits: an antiserum microinjection study. Adv Biochem Pharmacol. 1982;33:369-377. Han JS, ed. The Neurochemical Basis Of Pain Relief By Acupuncture. Beijing: Chinese Medical and Technology Press; 1987. Mayer JD, Price DD, Rafii A. Antagonism of acupuncture analgesia in man by the narcotic antagonist naloxone. Brain Res 1977;21:368372. Chen XH, Geller EB, Adler MW. Electrical stimulation at traditional acupuncture sites in periphery produces brain opioid-receptor-mediated antinociception in rats. J Pharm Exp Ther. 1996;277: 654-660. Chen XH, Han JS. All three types of opioid receptors in the spinal cord are important for 2/15 Hz electroacupuncture analgesia. Eur J Pharmacol. 1992;211:203-210. Guo HF, Tian J, Wang X, Fang Y, Hou Y, Han J. Brain substrates activated by electroacupuncture of different frequencies (I): comparative study on the expression of oncogene c-fos and genes coding for three opioid peptides. Mol Brain Res 1996;43:157-166. Han Z, Jiang YH, Wan Y, Wang Y, Chang JK, Han JS. Endomorphin-1 mediates 2 Hz but not 100 Hz electroacupuncture analgesia in the rat. Neurosci Lett. 1999;22:75-78. Pasternak GW. Pharmacological mechanisms of opioid analgesics. Clin Neuropharma. 1993;16:1-18. Bianchi M, Jotti E, Sacerdote P, Panerai AE. Traditional acupuncture increases the content of ␤-endorphin in immune cells and influences mitogen induced proliferation. Am J Chin Med. 1991;19: 101-104. Chen XD, Wu GC, He QZ, Cao XD. Effect of continued electroacupuncture on induction of interleukin-2 production of spleen lymphocytes from the injured rats. Acup Electro-Ther Res. 1997;22: 1-8.

Local Opioid Receptor Antagonists Block EA Analgesia

45. Kasahara T, Wu Y, Sakurai Y, Oguchi K. Suppressive effect of acupuncture on delayed type hypersensitivity to trinitrochlorobenzene and involvement of opiate receptors. Int J Immunophar. 1992;14:661665. 46. Liu X, Sun L, Xiao J, et al. Effect of acupuncture and point-injection treatment on immunologic function in rheumatoid arthritis. J Trad Chin Med. 1993;13:174-178. 47. Yu Y, Kasahara T, Sato T, et al. Enhancement of splenic interferongamma, interleukin-2, and NK cytotoxicity by S36 acupoint acupuncture in F344 rats. Jpn J Physiol. 1997 47:173-178. 48. Yuan J, Zhou R. Effect of acupuncture on T-lymphocyte and its subsets from the peripheral blood of patients with malignant neoplasm. Chen Tzu Yen Chiu Acupunct Res. 1993;18:174-177. 49. Zhao X. Effect of HC-3 on electroacupuncture-induced immunoregulation. Chen Tzu Yen Chiu Acupunct Res. 1995;20:59-62. 50. Zhao J, Zjao X, Liu W, et al. The role sympathetic mechanism in the effect of electroacupuncture on immunoregulation. Chen Tzu Yen Chiu Acupunct Res. 1995;20:40-43. 51. Zhao R, Ma C, Tan L, Zhao X, Zhuang D. The effect of acupuncture on the function of macrophages in rats of immunodepression. Chen Tzu Yen Chiu Acupunct Res. 1994;19:66-68. 52. Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain. 1983;16:109-110. 53. Stein C, Millan MJ, Herz A. Unilateral inflammation of the hind paws in rats as a modle of prolonged noxious stimulation: alterations in behavior and mociceptive thresholds. Pharm Biochem Behav. 1988;31:445-451. 54. Hargreaves K, Dubner R, Brown F, Flores C, Joris J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain. 1988;32:77-88. 55. Cheng X, ed. Chinese Acupuncture and Moxibustion. Beijing: Foreign Languages Press; 1987. 56. O’Connor J, Bensky D, eds. Acupuncture—A Comprehensive Text. Chicago: Eastland Press; 1981. 57. Schäfer M, Mousa SA, Zhang Q, Carter L, Stein C. Expression of corticotropin-releasing factor in inflamed tissue is required for intrinsic peripheral opioid analgesia. Proc Natl Acad Sci U S A. 1996; 93:6096-6100. 58. Iorio MA, Frigeni V. Narcotic agonist/antagonist properties of quatemary diastereoisomers derived from oxymorphone and naloxone. Eur J Med Chem Chin Ther. 1984;19:301.

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