Involvement of peripheral opioid receptors in electroacupuncture analgesia for carrageenan-induced hyperalgesia

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www.elsevier.com/locate/brainres

Research Report

Involvement of peripheral opioid receptors in electroacupuncture analgesia for carrageenan-induced hyperalgesia Reina Taguchi a,⁎, Tatsuki Taguchi b , Hiroshi Kitakoji a a

Department of Clinical Acupuncture and Moxibustion, Meiji University of Integrative Medicine, Nantan-shi, Kyoto, Japan Meiji School of Oriental Medicine, Suita-shi, Osaka, Japan

b

A R T I C LE I N FO

AB S T R A C T

Article history:

Acupuncture is widely used to relieve pain; however, the mechanism underlying

Accepted 5 August 2010

electroacupuncture analgesia (EAA) during inflammatory pain is unclear. We investigated

Available online 11 August 2010

whether endogenous peripheral opioid receptors participated in EAA during hyperalgesia elicited by carrageenan-induced inflammation. Moreover, we investigated which subtype of

Keywords:

opioid receptor was involved in EAA. Carrageenan was subcutaneously administered by

Hyperalgesia

intraplanter (i.pl.) injection into the left hind paw. Nociceptive thresholds were measured

Inflammation

using the paw pressure threshold (PPT). Rats received 3 Hz electroacupuncture (EA) for 1 h

Carrageenan

after carrageenan injection. The nonselective peripheral opioid receptor antagonist,

Acupuncture

naloxone methiodide, was administered by i.pl. injection of the inflamed paw 5 min

Peripheral opioid Receptors

before EA. Also, animals received i.pl. or intravenous (i.v.) injection of selective antagonists

Analgesia

against μ(D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-ThrNH2, CTOP), δ(naltrindole, NTI), or κ (norBinaltorphimine, nor-BNI) opioid receptors 1 h before EA. PPT decreased significantly 3 h after carrageenan injection. EA resulted in significant increases of PPT, moreover, PPT elevations persisted for 9 h after carrageenan injection. PPT elevations produced by EA were antagonized by local i.pl. injection of naloxone methiodide at 3 and 5 h after cessation of EA. NTI, nor-BNI and CTOP blocked EAA from immediately, 1 h, and 3 h after EA cessation, respectively. The EAA in the inflamed paw could not be blocked by i.v. injection of NTI, norBNI and CTOP. These findings suggest that peripheral μ, δ and κ receptors on peripheral nerve terminals are activated by EA, although there is a time difference among these activations. © 2010 Elsevier B.V. All rights reserved.

1.

Introduction

EA has been widely used to relieve various kinds of pain. Numerous investigations into the mechanism underlying EAA have been performed in humans and animals. The results

showed that EAA was reversed by naloxone, an opioid receptor antagonist (Chen et al., 1996; He, 1987; Mayer et al., 1977), and that the quantity of β-endorphin or enkephalin in the cerebrospinal fluid was increased after EA (Clement-Jones et al., 1979, 1980; He, 1987). It is well known that EAA is at least

⁎ Corresponding author. Department of Clinical Acupuncture and Moxibustion, Meiji University of Integrative Medicine, Hiyoshi-cho, Nantan-shi, Kyoto, 629-0392, Japan. Fax: + 81 771 72 0326. E-mail address: [email protected] (R. Taguchi). 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.08.014

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partially mediated by endogenous opioids and other neurotransmitters in the central nervous system (CNS) (Han and Trenius, 1982; He, 1987.) Recently, most studies have reported the effect of EA on inflammatory pain (Baek et al., 2005; Choi et al., 2005; Fu et al., 2006; Lee et al., 2006; Sekido et al., 2000, 2002, 2003, 2004; Zhang et al., 2004, 2005). Although the mechanisms of EAA have been investigated using various inflammatory models, most studies have resolved only the central mechanism of EAA, and the peripheral mechanism underlying EAA during inflammatory pain remains unclear. In a previous study, we showed that EAA during inflammatory hyperalgesia lasted longer and was blocked by i.pl. injection of naloxone to inflamed paw (Sekido et al., 2000, 2003), indicating that peripheral opioid receptors on peripheral nerve terminals might be involved in EAA during inflammatory hyperalgesia. In the present study, we investigated whether endogenous peripheral opioid receptors participated in EAA during hyperalgesia using a nonselective peripheral opioid receptor antagonist. Moreover, we investigated which subtype of opioid receptors is involved in EAA.

2.

Results

2.1. Effect of i.pl. injection of naloxone methiodide on EAA during hyperalgesia As shown in Fig. 1, PPT just before carrageenan injection was 79.5 ± 8.7 g in the control group. Three hours after carrageenan injection, a marked ipsilateral inflammatory response (swelling and redness) appeared and PPT decreased significantly (50.0 ± 11.0 g). Moreover, this decrease continued for 9 h after carrageenan injection. In the naloxone methiodide (125, 250 and 500 μg) + EA or vehicle + EA group, PPT decreased to almost the same level 3 h after carrageenan injection as the control group, respectively. In the vehicle + EA group, PPT increased significantly (88.0 ± 15.8 g) immediately after EA (P < 0.001). The control Vehicle+EA 125 µ g+EA 250 µ g+EA 500 µ g+EA

paw pressure threshold (g)

140 120 100 80

0

2.2. Effect of i.pl. injection of CTOP on EAA during hyperalgesia PPT decreased to almost the same level 3 h after carrageenan injection as the control group in the CTOP (2.5, 5.0 and 10.0 μg) + EA or vehicle + EA groups, respectively (Fig. 2A). In the vehicle + EA group, PPT significantly increased immediately after termination of EA (87.2 ± 14.3 g) compared to the control group (P < 0.001). In the 2.5 μg CTOP + EA group, PPT increased to the same level as the vehicle + EA group after EA cessation and no differences were observed between these two groups. In the 5.0 μg CTOP + EA group, PPT tended to increase immediately and 1 h after EA cessation compared to the control group; however, PPT elevations were significantly antagonized at 7 and 9 h after carrageenan injection (3 and 5 h after EA cessation, 46.2 ± 4.0 g and 49.7 ± 6.1 g, respectively) compared to the vehicle + EA group (P < 0.001). Also, in the 10.0 μg CTOP + EA group, PPT increased immediately and 1 h after EA cessation; however, PPT elevations were significantly antagonized at 7 and 9 h after carrageenan injection (3 and 5 h after EA cessation, 60.0 ± 15.9 g and 52.0 ± 10.5 g, respectively) compared to the vehicle + EA group (P < 0.01, P < 0.05) (Fig. 2A). CTOP per se did not have analgesic or hyperalgesic effect (Table 1). PPT elevations produced by EA were not influenced by i.v. injection of 10.0 μg CTOP, and PPT significantly increased during 9 h after the carrageenan injection compared to the control group (Fig. 2B).

2.3. Effect of i.pl. injection of NTI on EAA during hyperalgesia

60 40

PPT elevations produced by EA persisted for at least 5 h after EA compared to the control group (9 h carrageenan injection). In the 125 μg naloxone methiodide + EA group, PPT increased to the same level as the vehicle + EA group after the cessation of EA and no differences were observed between groups. In the 250 μg naloxone methiodide + EA group, PPT tended to increase immediately after EA cessation; however, PPT significantly decreased 3 and 5 h after the cessation of EA (46.7 ± 4.7 g and 46.2 ± 8.3 g, respectively) compared to the vehicle + EA group (P < 0.01). In the 500 μg naloxone methiodide + EA group, PPT did not increase 3 and 5 h after EA cessation (50.0 ± 8.0 g and 45.7 ± 10.5 g, respectively) and no differences were observed between this group and the control group, but significant differences were observed with the vehicle + EA group (P < 0.01). Naloxone methiodide per se did not have analgesic or hyperalgesic effect (Table 1).

carrageenan EA

-15min

0 3 4 Naloxone methiodide

5

7

9 h

Fig. 1 – Effects of i.pl. injection of naloxone methiodide on EAA during hyperalgesia elicited by carrageenan-induced inflammation. The results are expressed as the mean ± SD. N = 5 per group. *P < 0.001; #P < 0.01; †P < 0.05 (control vs. vehicle + EA, control vs. 125 μg + EA, control vs. 250 μg + EA, vehicle + EA vs. 250 μg + EA, vehicle + EA vs. 500 μg + EA).

In the NTI (25, 50 and 100 μg) + EA group, PPT decreased to almost the same level 3 h after carrageenan injection as the control group (Fig. 3A). In the 25 μg NTI + EA group, PPT increased to the same level as the vehicle + EA group after EA cessation and no differences were observed between these two groups; however, in the 50 μg NTI + EA group, PPT elevations were significantly antagonized at 4 and 7 h after carrageenan injection (immediately and 3 h after EA cessation, 58.1 ± 17.4 g and 56.1 ± 17.6 g, respectively) compared to the vehicle + EA group (P < 0.001, P < 0.05). Also, in the 100 μg NTI + EA group, PPT tended to be antagonized after EA cessation and significant differences were observed at 7 and 9 h after

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Table 1 – Effects of i.pl. injection of naloxone methiodide, CTOP, NTI and nor-BNI during hyperalgesia elicited by carrageenan-induced inflammation. The results are expressed as the mean ± SD (g). n = 6; control, n = 3 per group. Group Control (n = 6) Naloxone methiodide (n = 3)

125 μg 250 μg 500 μg 2.5 μg 5.0 μg 10.0 μg 25 μg 50 μg 100 μg 50 μg 100 μg 200 μg

CTOP (n = 3)

NTI (n = 3)

Nor-BNI (n = 3)

−15 min

0h

3h

4h

5h

7h

9h

86.2 ± 15.3 76.2 ± 11.2 80.4 ± 7.6 94.1 ± 5.0 70 6 ± 6.1 76.0 ± 3.8 78.1 ± 20.3 81.2 ± 18.8 89.5 ± 14.5 74.5 ± 3.6 70.4 ± 5.0 83.1 ± 11.4 86.6 ± 14.2

83.9 ± 13.4 76.2 ± 11.8 72.9 ± 6.4 81.2 ± 11.1 70.6 ± 0.8 74.3 ± 2.6 74.3 ± 15.0 80.3 ± 21.2 83.3 ± 18.0 72.5 ± 5.4 74.5 ± 3.1 76.2 ± 10.6 85.4 ± 11.2

54.1 ± 14.2 55.0 ± 8.7 47.9 ± 1.9 60.4 ± 7.6 56.2 ± 3.5 58.1 ± 6.1 56.8 ± 11.4 57.0 ± 8.3 55.8 ± 5.7 53.7 ± 3.3 52.0 ± 11.2 58.1 ± 9.7 53.3 ± 12.5

54.1 ± 9.2 51.6 ± 7.1 51.6 ± 6.4 58.7 ± 6.6 51.8 ± 0.8 57.5 ± 3.5 48.7 ± 8.8 47.9 ± 4.7 55.0 ± 4.3 44.5 ± 5.0 48.3 ± 5.0 53.1 ± 0.8 50.0 ± 2.1

60.0 ± 16.6 51.2 ± 9.4 43.7 ± 2.5 48.3 ± 4.3 54.3 ± 2.6 51.8 ± 4.4 51.2 ± 5.3 50.4 ± 12.6 56.2 ± 9.0 45.0 ± 3.3 47.5 ± 4.5 51.8 ± 6.1 49.1 ± 4.3

58.7 ± 20.3 48.3 ± 5.0 40.8 ± 1.4 45.8 ± 5.7 51.2 ± 3.5 51.8 ± 2.6 48.7 ± 5.3 53.7 ± 8.1 50.0 ± 7.5 48.7 ± 6.9 48.7 ± 3.3 61.8 ± 4.4 51.2 ± 2.1

56.4 ± 9.4 52.9 ± 14.5 51.2 ± 7.6 55.8 ± 7.5 47.5 ± 7.0 53.7 ± 8.8 48.1 ± 0.8 53.7 ± 2.1 52.5 ± 4.5 50.0 ± 4.3 52.0 ± 3.8 51.2 ± 7.0 46.6 ± 2.8

carrageenan injection (3 and 5 h after EA cessation, 54.3 ± 7.6 g and 54.5 ± 5.8 g, respectively) compared to the vehicle + EA group (P < 0.05) (Fig. 3A). NTI per se did not have analgesic or hyperalgesic effect (Table 1).

140

A

control Vehicle+EA 2.5 µg+EA 5.0 µg+EA 10.0 µg+EA

100 80 60 carrageenan EA

0

140

*

*

*

*

60 40

80 60

carrageenan EA -15min

0

140

paw pressure threshold (g)

paw pressure threshold (g)

100

0

* 80

carrageenan EA

NTI control 10µg I.v.+EA

120

40

100

0

B

control Vehicle+EA 25µg+EA 50µg+EA 100µg+EA

120

CTOP 140

A *

paw pressure threshold (g)

paw pressure threshold (g)

120

40

PPT elevations produced by EA were not influenced by i.v. injection of 100 μg NTI and PPT continuously increased during 9 h after carrageenan injection compared to the control group (Fig. 3B).

3

B

control 100µg I.v.+EA

120

*

100

#

*

80 60 40 carrageenan

4

5

7

9 h

CTOP

0

EA

-15min

0

3

4

5

7

9 h

NTI Fig. 2 – Effects of i.pl. (A) or i.v. (B) injection of CTOP on EAA during hyperalgesia elicited by carrageenan-induced inflammation. The results are expressed as the mean ± SD. N = 6 per group. *P < 0.001; #P < 0.01; †P < 0.05 (control vs. vehicle+ EA, control vs. 2.5 μg + EA, control vs. 10.0 μg + EA, vehicle+ EA vs. 5.0 μg + EA, vehicle+ EA vs. 10.0 μg + EA, control vs. 10.0 μg i.v. + EA).

Fig. 3 – Effects of i.pl. (A) or i.v. (B) injection of NTI on EAA during hyperalgesia elicited by carrageenan-induced inflammation. The results are expressed as the mean ± SD. N = 6 per group. *P < 0.001; #P < 0.01; †P < 0.05 (control vs. vehicle + EA, control vs. 25 μg + EA, vehicle + EA vs. 50 μg + EA, vehicle + EA vs. 100 μg + EA, control vs. 100 μg i.v. + EA).

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3.

Discussion

The results of the present study suggested that EAA during inflammation-induced hyperalgesia could be dose-dependently blocked by local i.pl. injection of naloxone methiodide, CTOP, NTI, and nor-BNI. In addition, we found that naloxone methiodide blocked EAA from 1 h after EA cessation, and NTI, nor-BNI and CTOP blocked EAA from immediately, 1 h, and 3 h after EA cessation, respectively; however, EAA could not be blocked by i.v. injection of NTI, nor-BNI and CTOP. These results indicated that peripheral μ, δ and κ receptors on peripheral nerve terminals were activated by EA, although there was a time difference among these activations. Recently, most studies have reported the effect of EA on inflammatory pain. Fu et al. suggested that the spinal nociceptin/orphanin FQ system might be involved in EAA, which may be one of the mechanisms underlying the antinociceptive effect of EA in the complete Freund's adjuvant (CFA) model (Fu et al., 2006). Also, Choi et al. (2005) concluded that EA could attenuate inflammatory edema and hyperalgesia in CFA-induced rats by modulating the expression of ionotropic glutamate receptors, and especially N-methyl-Daspartate receptors, in the dorsal horn of the spinal cord. Similarly, using CFA-induced rats, spinal μ and δ, but not κ receptors were involved in EA-produced antinociception (Zhang et al., 2004). In collagen-induced arthritis, analgesic effect of low-frequency EA were blocked by intraperitoneal injection of 5-HT1a (spiroxatrine) and 5-HT3 (ondansetron) selective serotonergic receptor antagonist, suggesting that its analgesic effect could be mediated by 5-HT1a and 5-HT3 receptors (Baek et al., 2005). In carrageenan-induced inflammation, Lee et al. (2006) showed that low-frequency EA at low intensity might be a useful therapy for mitigation of edema and hyperalgesia through the regulation of COX-2 expression and PGE2 production in paws and lumbar spinal cords. Thus, although the mechanisms of EAA have been investigated using various inflammatory models, most studies have

paw pressure threshold (g)

In the nor-BNI (50, 100 and 200 μg) + EA group, PPT decreased to almost the same level 3 h after carrageenan injection as the control group (Fig. 4A). In the 50 μg nor-BNI + EA group, PPT increased to the same level as the vehicle + EA group after EA cessation and no differences were observed between these two groups. In the 100 μg nor-BNI + EA group, PPT elevations tended to be antagonized from 5 h after carrageenan injection (1 h after EA cessation) compared to the vehicle + EA group. Also, in the 200 μg nor-BNI + EA group, PPT elevations were significantly antagonized at 5 and 7 h after carrageenan injection (1 and 3 h after EA cessation, 49.1 ± 13.0 g and 46.2 ± 7.7 g, respectively) compared to the vehicle + EA group (P < 0.001, P < 0.05) (Fig. 4A). Nor-BNI per se did not have analgesic or hyperalgesic effect (Table 1). PPT elevations produced by EA were not influenced by i.v. injection of 200 μg nor-BNI, and PPT continuously increased during 9 h after carrageenan injection compared to the control group (Fig. 4B).

140

A

control Vehicle+EA 50µg+EA 100µg+EA 200µg+EA

120 100

*

*

80

# 60 40

*

carrageenan EA

0 nor-BNI 140

paw pressure threshold (g)

2.4. Effect of i.pl. injection of nor-BNI on EAA during hyperalgesia

B

control 200µg I.v.+EA

120 100

*

#

4

5

80 60 40 0

carrageenan

EA

-15min 0

3

7

9 h

nor-BNI Fig. 4 – Effects of i.pl. (A) or i.v. (B) injection of nor-BNI on EAA during hyperalgesia elicited by carrageenan-induced inflammation. The results are expressed as the mean ± SD. N = 6 per group. *P < 0.001; #P < 0.01; †P < 0.05 (control vs. vehicle + EA, control vs. 50 μg + EA, control vs. 100 μg + EA, control vs. 200 μg + EA, vehicle + EA vs. 200 μg + EA, control vs. 200 μg i.v. + EA).

resolved only the central mechanism of EAA, and only a few reports have investigated the peripheral mechanism of EAA in inflammatory pain. Zhang et al. (2005) showed that peripheral opioid receptors were activated by EA at the inflammatory site on the CFA model. This is consistent with our previous reports (Sekido et al., 2000, 2002, 2003, 2004) and this study that peripheral opioid receptors might be involved in EAA during inflammatory pain. In painful inflammation, POMC mRNA, β-endorphin, metenkephalin, and dynorphin are found in circulating cells and lymph nodes (Cabot et al., 1997, 2001). Furthermore, localized inflammation of the rat's hindpaw elicits the accumulation of immune cells containing opioid peptides, such as β-endorphin, met-enkephalin, dynorphin, and endomorphins. These peptides are produced by T- and B-lymphocytes, monocytes/ macrophages, and granulocytes (Cabot et al., 1997; Przewlocki et al., 1992; Rittner et al., 2001; Stein et al., 1993), and released on environmental stimuli into inflamed tissue (Stein et al., 1990). The released peptides can also bind to opioid receptors on peripheral nerve endings, the receptors of which are synthesized in dorsal root ganglia and transported intra-

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axonally to peripheral nerve endings. Opioid receptors are involved in three receptors; μ, δ and κ opioid receptors. CRF and IL-1 receptors also have been demonstrated to be expressed in splenocytes, macrophages, and T- and B-lymphocytes (Crofford et al., 1992; Dinarello and Thompson, 1991; Karalis et al., 1991; Mousa et al., 1996) and are upregulated in inflamed tissue (Mousa et al., 1996). CRF or IL-1 triggers the release of opioid peptides within inflamed tissue and these peptides activate peripheral opioid receptors, reducing neuronal excitability or the release of proinflammatory neuropeptides (e.g. substance P), thereby inhibiting pain (Schafer et al., 1994, 1996). We previously suggested that EA might be able to release CRF and IL-1 from immunocytes within inflamed tissue (Sekido et al., 2004). Our previous study also showed that systemic pretreatment with cyclosporin A, an immunosuppressant, blocked EAA in inflammatory pain (Sekido et al., 2002), suggesting that immunocytes might be involved in EAA. In the present study, we found that EAA could be blocked by i.pl. injection of a peripheral opioid receptor antagonist. Accordingly these results suggested that peripheral opioid receptors on peripheral nerve terminals and immunocytes might be involved in EAA. Immunocyte types are different in the inflammation stage. In early inflammation, granulocytes are the major source of opioid peptide production (Rittner et al., 2001). Later in the inflammatory course, monocytes and macrophages are the predominant supply of opioid peptides (Brack et al., 2004). Thus, opioid peptides are processed and present in the circulation and in cells infiltrating injured tissue. In the present study, EA started from 3 h after carrageenan injection, in the early inflammation stage. Accordingly, granulocytes may be the major source of opioid peptide. In rats with CFA inflammation, stress induced by cold water swimming (CWS) elicits potent analgesia only in inflamed paws. In early inflammatory stages (6 h), all three families of opioid peptides, peripheral and central opioid receptors are involved, whereas in later stages (4–6 days), endogenous analgesia is mediated exclusively by peripheral opioid receptors β-endorphin, acting at μ and δopioid receptors. (Machelska et al., 2003; Stein et al., 1990). Peripheral opioid mechanisms of pain control seem to become more prevalent with the duration and severity of inflammation. Differences between CWS-induced antinociception and EAA are the inflammatory agent used and the stage of inflammatory reaction (Sekido et al., 2004). Accordingly, the mechanism of CWS-induced antinociception and EAA may be distinguished. It is also known that the analgesic effect of EA is mediated by endogenous opioids and other neurotransmitters, including serotonin and norepinephrine in the CNS (Han and Trenius L., 1982). Also, most studies have resolved the central mechanism of EAA using various inflammatory models; therefore, we could not completely exclude the involvement of the CNS in the mechanism of EAA, although peripheral opioid receptors on peripheral nerve endings are strongly involved in EAA. Indeed, naloxone methiodide could not block EAA from immediately after EA cessation. Thus, in addition to the endogenous opioid mechanism in the CNS, the local immune system may be involved in EAA; however, the relationship between the CNS and peripheral mechanisms in EAA is unclear. Moreover, there might be interactions with the peripheral opioid receptor

101

subtype in the EAA, because there was a time difference among these activations. Further study is needed to clarify the mechanism of EAA during peripheral inflammation. In conclusion, we demonstrated that peripheral μ, δ and κ receptors on peripheral nerve terminals were activated by EA during peripheral inflammation, although there was a time interval among these activations.

4.

Experimental procedures

4.1.

Animals

Male Sprague–Dawley rats weighing 280–380 g (n = 111) were purchased from Japan SLC, Inc. The animals were kept at 24± 1 °C and relative humidity of 50% to 60%. Standard laboratory rodent food and tap water were available ad libitum. All experiments were conducted in the light phase of a 12/12h (7 a.m./7 p.m.) light–dark cycle. This study followed NIH regulations for humane experimentation on animals, and the guidelines of the International Association for the Study of Pain. Animals were treated in accordance with the guidelines of our Institutional Committee on the Treatment of Experimental Animals.

4.2.

Induction of hyperalgesia

Carrageenan (2%, 0.1 ml; Sigma, St. Louis, MO) was subcutaneously administered by i.pl. injection into the left hind paw of rats under ether anaesthesia using a 26-gauge needle to induce hyperalgesia.

4.3.

Experimental design

First, to determine the involvement of peripheral opioid receptors in EAA, we examined whether naloxone methiodide (a nonselective peripheral opioid receptor antagonist) given by i.pl. injection to the inflamed paw blocked the PPT elevation produced by EA. Animals received i.pl. injection (0.1 ml) of naloxone methiodide (125, 250 and 500 μg) 5 min before EA (n = 5 per group). Second, to determine the involvement of the subtype of opioid receptors in EAA, we examined whether D-Phe-Cys-TyrD-Trp-Orn-Thr-Pen-ThrNH2 (CTOP), naltrindole hydrochloride (NTI), and nor-Binaltorphimine dihydrochloride (nor-BNI) given by i.pl. injection to the inflamed paw blocked the PPT elevation produced by EA. Animals received i.pl. injection (0.1 ml) of CTOP (2.5, 5.0 and 10.0 μg), NTI (25, 50 and 100 μg), and nor-BNI (50, 100 and 200 μg) 1 h before EA (n = 6 per group). To confirm that these effects were not mediated through a central site of action, another group of animals received an i.v. injection (0.1 ml) of 10.0 μg CTOP, 100 μg NTI and 200 μg nor-BNI 1 h before EA (n = 6 per group).

4.4.

Algesiometry

Nociceptive thresholds were evaluated using an Analgesymeter (Ugo Basile). Rats were gently restrained under a soft cloth jacket and incremental pressure was applied to the dorsal surface of the left hindpaw. The pressure required to

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elicit paw withdrawal, the paw pressure threshold (PPT), was determined (Randall Sellito Test). We used a cut-off threshold of 250 g to avoid tissue damage to the paw. The mean of two consecutive measurements, separated by 2 min, was determined after a rest period of 15 min. PPT was determined 15 min before, just before, and 3, 4, 5, 7, and 9 h after the carrageenan injection.

4.5.

Electroacupuncture

A pair of stainless steel needles, 0.20 mm in diameter and 30 mm in length, was inserted into the acupoint of Zusanli (ST36) and 5 mm from the Zusanli (left anterior tibial muscles). A 3 Hz biphasic square wave pulse of 0.1 ms width was delivered via the needles for periods of 60 min using a constant current programmed pulse generator. EA was started 3 h after carrageenan injection. The intensity of EA was increased according to a schedule of 1–3 mA for 20 min at each intensity. This intensity was sufficient to produce rhythmic muscle contraction of the hind leg. The rats were gently restrained under a soft cloth jacket during the PPT measurement and EA procedure. Except at these times, rats were left in their cage to move freely.

4.6.

Drugs

We used naloxone methiodide (a nonselective peripheral opioid receptor antagonist) (Sigma), CTOP (a selective μ opioid receptor antagonist) (Sigma), NTI (a selective δ opioid receptor antagonist) (Sigma) and nor-BNI (a selective κ opioid receptor antagonist) (Sigma). All agents were dissolved in sterile distilled water. Naloxone methiodide was administered by i.pl. injection (0.1 ml) into the inflamed paw 5 min before EA. CTOP, NTI or nor-BNI was administered by i.pl. injection (0.1 ml) into the inflamed paw or i.v. injection via a tail vein 1 h before EA. Control animals received 0.1 ml sterile distilled water.

4.7.

Data analysis

The experimental data are presented as the means ± SD. Repeated-measures ANOVA was performed to determine the overall effect. Tukey's post-hoc test was then used to determine probability values when repeated measures ANOVAs indicated a significant effect. P < 0.05 was considered significant.

Acknowledgments This work was supported by a grant for Project Research from Meiji Univesity of Integrative Medicine in Japan. REFERENCES

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