Graduate School of Acupuncture and Moxibustion, Meiji University of Oriental Medicine , Japan

The American Journal of Chinese Medicine, Vol. 32, No. 2, 269 279 2004 World Scientific Publishing Company Institute for Advanced Research in Asian Science and Medicine Corticotropin-Releasing Factor and Interleukin-1β are Involved in the Electroacupuncture-Induced Analgesic Effect on Inflammatory Pain Elicited by Carrageenan Reina Sekido, * Keisou Ishimaru and Masakazu Sakita * * Departments of Surgery, and Clinical Acupuncture and Moxibustion Graduate School of Acupuncture and Moxibustion, Meiji University of Oriental Medicine 629-0392, Japan Abstract: Electroacupuncture (EA) is used to relieve various kinds of pain. However, the mechanistic basis of electroacupuncture analgesia (EAA) in inflammatory pain remains unclear. In the present study, we investigated whether endogenous peripheral corticotropin-releasing factor (CRF) or interleukin-1β (IL-1) participated in EAA during hyperalgesia elicited by carrageenan-induced inflammation. Carrageenan was subcutaneously administered by intraplantar (i.pl.) injection of the left hind paw to induce inflammation. Nociceptive thresholds were measured using the paw pressure threshold (PPT) (Randall Sellito Test). Rats received 3 Hz EA in the left anterior tibial muscles for 1 hour after carrageenan injection. The selective CRF antagonist, α-helical CRF, or the recombinant IL-1 receptor antagonist, IL-1ra, was administered by i.pl. injection of the inflamed paw or by intravenous (i.v.) injection 1 hour before EA. PPT decreased significantly 3 hours after carrageenan injection. This decrease persisted at least 24 hours after carrageenan injection. EA resulted in significant increases of PPT, moreover, PPT elevations lasted 24 hours after carrageenan injection. By contrast, PPT elevations produced by EA were dose-dependently antagonized by local i.pl. injection of α- helical CRF or IL-1ra. This PPT elevation was not influenced by i.v. injection of α-helical CRF or IL-1ra. These findings suggest that peripheral CRF or IL-1 participate in EAA during hyperalgesia. The release of CRF or IL-1 elicited by EA may trigger the release of opioid peptides within inflamed tissue which may activate peripheral opioid receptors and inhibit the pain. Keywords: Electroacupuncture; Analgesia; Corticotropin-releasing Factor; IL-1β; Carrageenan; Hyperalgesia; Inflammation. Correspondence to: Dr. Reina Sekido, Department of Surgery, Graduate School of Acupuncture and Moxibustion, Meiji University of Oriental Medicine, Hiyoshi, Funai, Kyoto, 629-0392, Japan. Tel: (+81) 771-72-1181, Fax: (+81) 771-72-0326, E-mail: r_sekido@muom.meiji-u.ac.jp. 269

270 R. SEKIDO et al. Introduction Pain, evoked by local injury and inflammation, can be controlled by various endogenous mechanisms. Under inflammatory conditions, opioid-containing immune cells migrate preferentially to inflamed sites (Przewlocki et al., 1992; Stein, 1995; Cabot et al., 1997). Experimental and clinical studies demonstrate that locally administered opioid receptor agonists can produce pronounced analgesic effects by interacting with peripheral opioid receptors in inflamed tissue (Antonijevic et al., 1995; Stein et al., 1989, 1990a, 1993 and 1995). Stein et al. (1990a and b) have also shown that environmental stimuli, such as in the cold water swim (CWS) in rats with unilateral hind paw inflammation elicit peripheral opioid receptor-mediated anti-nociception in the inflamed paw. These opioids are released locally from immunocytes during environmental stimuli and inhibit pain in animals (Stein et al., 1990a and b) and in humans (Stein et al., 1993). Moreover, these opioid peptides can be released by stimulating immunocytes with corticotropin-releasing factor (CRF) or interleukin- 1β (IL-1) in vivo and in vitro (Schafer et al., 1994; Cabot et al., 2001). It has also been shown that the anti-nociception produced by CWS is blocked by intraplantar (i.pl.) injection of CRF antagonist, suggesting a peripheral receptor-specific action of endogenous CRF (Schafer et al., 1996). Thus, it is believed that local immune system and peripheral opioid receptors are involved in inflammatory pain control (Stein et al., 1993; Stein, 1995). At present, electroacupuncture (EA) is used to relieve various kinds of pain. However, little is known about the effects of electroacupuncture analgesia (EAA) on inflammatory pain. In a previous study, we observed that peripheral opioid receptors might be involved in the EAA in inflamed tissue (Sekido et al., 2000). In this study, we investigated whether peripheral CRF or IL-1 might be the endogenous trigger for local opioid release and was involved in EAA in rats with carrageenan-induced inflammation. Materials and Methods Animals Male Sprague-Dawley rats weighing 280 380 g were purchased from Japan SLC, Inc. The animals were kept at 24 ± 1ºC and a 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 12/12-hour (7 am/7 pm) 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. Induction of Inflammation Carrageenan (2%, 0.1 ml; Sigma, St. Louis, MO) was subcutaneously administered by i.pl. injection of the left hind paw of rats under ether anesthesia using a 26-gauge needle to induce inflammation.

CRF AND IL-1β ON ACUPUNCTURE ANALGESIA 271 Experimental Protocols To determine the involvement of peripheral CRF or IL-1 on local opioid release, we examined whether α-helical CRF or recombinant IL-1 receptor antagonist (IL-1ra) given by i.pl. injection to the inflamed paw blocked the paw pressure threshold (PPT) elevations produced by EA. Animals received i.pl. injection (0.1 ml) of α-helical CRF (2.5, 3.7 and 5.0 ng) or IL-1ra (25, 50 and 100 ng) 1 hour 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 intravenous (i.v.) injection (0.1 ml) of 5.0 ng α-helical CRF or 100 ng IL-1ra 1 hour before EA (n = 6 per group). Algesiometry Nociceptive thresholds were evaluated using an Analgesy-meter (Ugo Basile). Rats were gently restrained under a soft cloth jacket and incremental pressure was applied onto the dorsal surface of the left hind paw. The pressure required to elicit paw withdrawal, the PPT, was determined. 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 minutes, was determined after a rest period of 15 minutes. PPT was determined 15 minutes before, just before, and 3, 4, 5, 7, 9 and 24 hours after the carrageenan injection. Electroacupuncture A pair of stainless steel needles, 0.20 mm in diameter and 30 mm in length, were inserted into an acupoint of Zusanli (ST36) and the left anterior tibial muscles (5 mm from the Zusanli). A 3 Hz biphasic square wave pulse of 0.1 ms pulse width was delivered via the needles for a period of 60 minutes using a constant current programmed pulse generator. EA was started from 3 hours after carrageenan injection. The intensity of EA was increased according to a schedule of 1-2-3 ma, for 20 minutes at each intensity. The intensity was sufficient to produce a rhythmic muscle contraction of the hind legs. 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. Drugs We used the selective CRF antagonist α-helical CRF (Sigma) and recombinant IL-1 receptor antagonist IL-1ra (R&D Systems). Both agents were dissolved in sterile distilled water and administered by i.pl. injection (0.1 ml) of the inflamed paw or i.v. injection via a tail vein 1 hour before EA. Control animals received 0.1 ml of sterile distilled water.

272 R. SEKIDO et al. Data Analysis The experimental data were expressed as means ± SD. Repeated measures ANOVA were performed to determine the overall effect. Tukey s post-hoc test was then used to determine probability values when repeated measure ANOVAs indicated a significant effect. p < 0.05 was considered as statistically significant. Results Effect of EA during Hyperalgesia Elicited by Carrageenan-induced Inflammation Figure 1 shows the time course of analgesia produced by EA during inflammatory hyperalgesia. In the control group, PPT just before carrageenan injection was 83.9 ± 13.4 g. Three hours after the carrageenan injection, a marked ipsilateral inflammatory response (swelling and redness) appeared and PPT decreased significantly (54.1 ± 14.2 g). Moreover, this decrease continued for 24 hours after carrageenan injection. In the EA group, PPT decreased to almost the same level as the control group 3 hours after the carrageenan injection. However, PPT increased significantly (86.4 ± 12.3 g) immediately after the EA (p < 0.001). The PPT elevations produced by EA lasted at least 20 hours after the EA (24 hour carrageenan injection) (Fig. 1). paw pressure threshold (g) 120 * # 100 80 60 40 20 ~ carrageenan EA -15min 0 3 4 5 7 9 24 * h cont EA Figure 1. Effects of EA during hyperalgesia elicited by carrageenan-induced inflammation. The results are expressed as mean ± SD. n = 6 per group, * p < 0.001, # p < 0.01, p < 0.05 (control versus EA).

CRF AND IL-1β ON ACUPUNCTURE ANALGESIA 273 Effect of i.pl. Injection of α-helical CRF on EAA during Hyperalgesia Elicited by Carrageenan-induced Inflammation Figure 2A shows the effect of i.pl. injection of α-helical CRF on EAA during inflammatory hyperalgesia. To observe the effect of α-helical CRF on the PPT elevations induced by EA, rats were given an i.pl. injection of α-helical CRF (2.5, 3.7 and 5.0 ng) or vehicle 1 hour before EA. In the α-helical CRF (2.5, 3.7 and 5.0 ng) + EA or vehicle + EA groups, PPT decreased to almost the same level as the control group 3 hours after the carrageenan injection. In the vehicle + EA group, PPT significantly increased immediately, 5 hours and 20 hours after termination of EA (89.1 ± 10.5 g, 77.7 ± 10.4 g and 78.5 ± 6.4 g, respectively), compared to that of the control group. In the 2.5 ng α-helical CRF + EA group, PPT increased to the same level as the vehicle + EA group after the termination of EA, and no differences were observed between these two groups. In the 3.7 ng α-helical CRF + EA group, PPT tended to increase immediately after the termination of EA and thereafter compared to that of the control group. In the 5.0 ng α-helical CRF + EA group, however, PPT did not increase immediately, 5 hours and 20 hours after the termination of EA (55 ± 6.1 g, 53.7 ± 2.6 g and 48.7 ± 4.1 g, respectively) and no differences were observed between this group and the control group, but significant differences were observed compared to that of the vehicle + EA group. PPT elevations produced by EA were not influenced by i.v. injection of 5.0 ng α-helical CRF and PPT significantly increased during 24 hours after the carrageenan injection compared to that of the control group (Fig. 2B). Effect of i.pl. Injection of IL-1ra on EAA during Hyperalgesia Elicited by Carrageenan-induced Inflammation Figure 3A shows the effect of i.pl. injection of IL-1ra on EAA during inflammatory hyperalgesia. Rats were given an i.pl. injection of 25, 50 and 100 ng IL-1ra 1 hour before EA. In the IL-1ra (25, 50 and 100 ng) + EA groups, PPT decreased to the same level as the control group 3 hours after carrageenan injection. In the 25 ng IL-1ra + EA group, PPT increased to the same level as the vehicle + EA group immediately, 1 hour and 5 hours after the termination of EA (86.8 ± 13.3 g, 81.0 ± 7.9 g and 80.4 ± 9.6 g, respectively). In the 50 ng IL-1ra + EA group, PPT tended to increase after the termination of EA compared to that of the control group. In the 100 ng IL-1ra + EA group, however, PPT did not increase after the termination of EA. Thus, PPT elevations elicited by EA were dose-dependently antagonized by i.pl., but not by i.v. injection of α-helical CRF or IL-1ra (Figs. 2 and 3). In addition, PPT elevations elicited by EA were not antagonized by i.v. injection of 100 ng IL-1ra (Fig. 3B). Moreover, neither i.pl. injection of α-helical CRF nor IL-1ra per se did not have analgesic or hyperalgesic effects during carrageenan-induced inflammation (Table 1).

274 R. SEKIDO et al. paw pressure threshold (g) 120 A 100 80 60 40 0 ~ carrageenan EA * * * * * * * Control Vehicle + EA 2.5 ng + EA 3.7 ng + EA 5.0 ng + EA paw pressure threshold (g) 120 B * 100 80 60 40 ~ carrageenan 0-15min EA # 0 3 4 5 7 9 24 h Control 5.0 ng i.v. + EA Figure 2. Effects of i.pl. (A) or i.v. (B) injection of α-helical CRF on EAA during hyperalgesia elicited by carrageenan-induced inflammation. The results are expressed as mean ± SD. n = 6 per group, * p < 0.001, # p < 0.01, p < 0.05 (control versus vehicle + EA, vehicle + EA versus 2.5 ng + EA, vehicle + EA versus 5.0 ng + EA, control versus 5.0 ng i.v.+ EA).

CRF AND IL-1β ON ACUPUNCTURE ANALGESIA 275 paw pressure threshold (g) paw pressure threshold (g) 120 100 80 60 40 0 120 100 80 60 40 A ~ carrageenan B ~ carrageenan 0-15min EA EA * * # # # * * * * * # 0 3 4 5 7 9 24 Figure 3. Effects of i.pl. (A) or i.v. (B) injection of IL-1ra on EAA during hyperalgesia elicited by carrageenaninduced inflammation. The results are expressed as mean ± SD. n = 6 per group, * p < 0.001, # p < 0.01, p < 0.05 (control versus vehicle + EA, vehicle + EA versus 25 ng + EA, vehicle + EA versus 100 ng + EA, vehicle + EA versus 50 ng + EA, control versus 100 ng i.v. + EA). * Control Vehicle + EA 25 ng + EA 50 ng + EA 100 ng + EA Control 100 ng i.v. + EA h

276 Table 1. Effects of i.pl. Injection of α-helical CRF or IL-1ra During Hyperalgesia Elicited by Carrageenan-induced Inflammation Group 15 Minutes 0 Hours 3 Hours 4 Hours 5 Hours 7 Hours 9 Hours 24 Hours Carrageenan control 86.2 ± 15.3 83.9 ± 13.4 54.1 ± 14.2 54.1 ± 9.2 60.0 ± 16.6 58.7 ± 20.3 56.4 ± 9.4 57.5 ± 8.2 (n = 6) (100) (97.3) (62.8) (62.8) (69.5) (68.1) (65.4) (66.6) Carrageenan + 73.3 ± 11.6 72.0 ± 10.1 58.7 ± 11.4 43.7 ± 9.7 43.7 ± 5.0 49.5 ± 6.8 47.9 ± 10.6 50.8 ± 7.3 α-helical CRF 2.5 ng (100) (98.2) (80.1) (59.6) (59.6) (67.6) (65.3) (69.3) (n = 3) Carrageenan + 74.5 ± 8.7 72.0 ± 10.0 55.0 ± 4.5 53.3 ± 4.3 53.3 ± 6.2 50.4 ± 5.7 50.8 ± 5.0 58.3 ± 8.5 α-helical CRF 3.7 ng (100) (96.6) (73.7) (71.5) (71.5) (67.5) (68.1) (78.2) (n = 3) Carrageenan + 77.5 ± 19.2 77.9 ± 22.5 53.7 ± 9.9 51.2 ± 8.7 50.8 ± 8.3 48.7 ± 3.3 55.0 ± 2.5 60.4 ± 4.7 α-helical CRF 5.0 ng (100) (100.5) (69.3) (66.1) (65.5) (62.9) (70.9) (77.9) (n = 3) Carrageenan + 65.4 ± 4.0 64.1 ± 1.9 46.6 ± 8.1 45.4 ± 0.7 42.5 ± 1.2 45.8 ± 8.0 48.3 ± 5.0 51.6 ± 5.2 IL-1ra 25 ng (100) (98.0) (71.3) (69.4) (64.9) (70.0) (73.8) (78.9) (n = 3) Carrageenan + 78.7 ± 9.1 75.9 ± 6.8 55.9 ± 2.5 52.1 ± 4.8 46.8 ± 5.9 53.7 ± 6.5 52.1 ± 4.6 55.6 ± 5.2 IL-1ra 50 ng (100) (96.4) (71.0) (66.2) (59.5) (68.2) (66.2) (70.6) (n = 4) Carrageenan + 75.9 ± 15.8 78.4 ± 18.7 57.8 ± 12.1 57.1 ± 11.1 50.0 ± 5.3 57.0 ± 16.7 55.3 ± 9.8 63.1 ± 10.2 IL-1ra 100 ng (100) (103.2) (76.1) (75.3) (65.8) (75.1) (72.8) (83.1) (n = 4) The results are expressed as mean ± SD (g) and as percentage change from PPT of 15 minutes before carrageenan injection (100%). n = 6; control, n = 3 4 per group. R. SEKIDO et al.

CRF AND IL-1β ON ACUPUNCTURE ANALGESIA 277 Discussion EA has been widely used to relieve various kinds of pain. Numerous investigations of the mechanism underlying EAA have been performed in humans and animals. The results have shown that EAA was reversed by naloxone, an opioid receptor antagonist (Mayer et al., 1977; He, 1987; Chen et al., 1996), and that the quantity of β-endorphin or enkephalin in the cerebrospinal fluid was increased after EA (Clement-Jones et al., 1979 and 1980; He, 1987). Although it is well known that EAA is at least partially mediated by endogenous opioids and other neurotransmitters in the central nervous system (CNS) (Han and Trenius, 1982; He, 1987), the mechanism underlying EAA during inflammatory pain is unclear. In a previous study, we showed that the EAA during inflammatory hyperalgesia lasted longer and was blocked by i.pl. injection of naloxone (Sekido et al., 2000). This indicated that peripheral opioid receptors might be involved in the EAA during inflammatory hyperalgesia. We then investigated whether the endogenous peripheral CRF or IL-1 participated in EAA during hyperalgesia elicited by carrageenan-induced inflammation. In the present study, we found that the EAA in inflamed tissue could be dose-dependently blocked by local i.pl. injection of α-helical CRF or IL-1ra. This phenomenon is in accordance with the finding that anti-nociception elicited by CWS is blocked by i.pl. injection of α-helical CRF (Schafer et al., 1996). CRF and IL-1 receptors have been demonstrated to be expressed in splenocytes, macrophages, and T- and B-lymphocytes (Webster et al., 1990; Dinarello and Thompson, 1991; Karalis et al., 1991; Crofford et al., 1992; Mousa et al., 1996) and these receptors are upregulated in inflamed tissue (Mousa et al., 1996). CRF and IL-1 receptors are also detectable in the CNS (Heijnen et al., 1991). However, peripheral CRF and IL-1 receptors on immunocytes were probably involved in the EAA since the EAA in inflamed tissue could not be blocked by i.v. injection of α-helical CRF or IL-1ra. There are several differences between CWS-induced anti-nociception and EAA. First, EAA in inflamed tissue lasted 20 hours after termination of EA while the CWS-induced anti-nociception returned to baseline 10 17 minutes after termination of CWS (Schafer et al., 1996). Secondly, EAA was blocked by i.pl. injection of both α-helical CRF or IL-1ra. However, CWS-induced anti-nociception could not be blocked by i.pl. injection of IL-1ra (Schafer et al., 1996). These differences might be due to the difference in inflammatory agent used and stage of inflammatory reaction. Stein et al. used Freund s complete adjuvant to induce inflammation and CWS was conducted 4 days after inoculation. By contrast, we used carrageenan to induce inflammation and EA was conducted 3 hours after carrageenan injection. Antonijevic et al. (1995) showed that inflammation-induced disruption of the perineurial barrier and peripheral opioid analgesia coincides with earlier stages of an inflammatory reaction. In this study, disruption of the perineurial barrier might be involved in EAA. IL-1 gene expression has also been shown to peak very early, i.e. 1 2 hours after inflammatory stimuli (Ulich et al., 1992). Therefore, the EAA might be antagonized by i.pl. injection of IL-1ra. The other possible difference between EAA and CWS-induced anti-nociception is in the mechanism of action. It is well known that the analgesic effect of EA is mediated by

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