http://www.paper.edu.cn Pain 99 (2002) 525–535 www.elsevier.com/locate/pain Excitatory amino acid receptor antagonists and electroacupuncture synergetically inhibit carrageenan-induced behavioral hyperalgesia and spinal fos expression in rats Yu-Qiu Zhanga, Guang-Chen Jib, Gen-Cheng Wub, Zhi-Qi Zhaoa,* aInstitute of Neurobiology, Fudan University, 220 Han Dan Road, Shanghai, 200433, China bState Key Laboratory of Medical Neurobiology, Medical Center of Fudan University, 138 Yi Xue Yuan Road, Shanghai, 200032, China Received 30 January 2002; accepted 27 June 2002 Abstract The interaction between electroacupuncture and an N-methyl-d-aspartic acid (NMDA) receptor antagonist, (DL-2-amino-5-phosphono- pentanoic acid; AP5), or an ( ^ )-a-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainite (AMPA/KA) receptor antagonist, (6,7- dinitroquinoxaline-2,3 (1H,4H); DNQX) administered intrathecally on carrageenan-induced thermal hyperalgesia and spinal c-Fos expres- sion was investigated. The latency of paw withdrawal (PWL) from a thermal stimulus was used as a measure of hyperalgesia in awake rats. Intrathecal (i.t.) injection of 1 and 10 nmol AP5, but not DNQX, markedly increased the PWL of the carrageenan-injected paw. At a dose of 100 nmol, either AP5 or DNQX significantly increased the PWL of carrageenan-injected paw, with AP5 being more potent. The PWLs of the non-injected and normal saline (NS)-injected paws were not detectably affected by the administration of NMDA or AMPA/KA receptor antagonists at the doses tested. Unilateral electroacupuncture stimulation of the ‘Zu-San-Li’ (St 36) and ‘Kun-Lun’ (UB 60) acupuncture points (60 and 2 Hz alternately, 1–2–3 mA) contralateral to the carrageenan-injected paw significantly elevated the PWLs of carrageenan- and NS-injected paws. Although neither i.t. injection of 0.1 nmol AP5 nor 1 nmol DNQX alone had an effect on the PWL of the carrageenan- and NS-injected paws, both significantly potentiated electroacupuncture-induced analgesia in carrageenan-injected rats, especially 0.1 nmol AP5. Fos expression evoked by intraplantar (i.pl.) injection of carrageenan was examined in the spinal cord with immunohistochemical methods. Three hours after i.pl. injection of carrageenan, the number of Fos-like immunoreactive (Fos-LI) neurons was significantly increased in all the layers of the ipsilateral spinal cord at L4–5, with the highest density in laminae I–II and V–VI. Intrathecally pre- administered AP5 (10 nmol) or DNQX (100 nmol) significantly reduced the total number of carrageenan-induced Fos-LI neurons. The reduction was most apparent in laminae I-II and IV-V. Similarly, following bilateral electroacupuncture stimulation of the ‘Zu-San-Li’ and ‘Kun-Lun’ acupuncture points, the numbers of carrageenan-induced Fos-LI neurons in laminae I–II and V–VI were also markedly reduced. When a combination of electroacupuncture with 10 nmol AP5 or 100 nmol DNQX was used, the level of Fos expression in the spinal cord induced by carrageenan was significantly lower than electroacupuncture or i.t. injection of AP5 or DNQX alone. These results demonstrate that electroacupuncture and NMDA or AMPA/KA receptor antagonists have a synergetic anti-nociceptive action against inflammatory pain. Furthermore, this study supports the idea that both NMDA and AMPA/KA receptors are involved in spinal nociceptive transmission in carrageenan-inflamed rats, with the former more preferentially mediating transmission of nociceptive information from cutaneous tissue. q 2002 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. Keywords: N-methyl-d-aspartic acid receptor; AMPA/KA receptor; Electroacupuncture; Carrageenan; Fos; Spinal cord 1. Introduction (Zenz and Willweber-Strumpt, 1993). Unfortunately, the use of opiates is invariably limited and, ultimately, ineffec- Chronic pain remains a major health care problem afflict- tive in chronic pain patients due to problems with tolerance ing an estimated 70–94% of patients with advanced cancer, and life-threatening side effects. inflammatory disorders and nerve injuries. Currently, the Excitatory amino acids, such as glutamate and aspartate, mainstay of clinical pain management depends on opiates are richly contained in nociceptive primary afferent fiber terminals and play an important role in spinal nociceptive * Corresponding author. Tel.: 186-21-55612877; fax: 186-21- transmission (De Biasi and Rustioni, 1988). NMDA AMPA/ 55612876. KA and metabotropic receptors are distributed densely in E-mail addresses: [email protected] (Y.-Q. Zhang), zqzhao@- the superficial dorsal horn of the spinal cord where primary fudan.edu.cn (Z.-Q. Zhao). 0304-3959/02/$20.00 q 2002 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. PII: S0304-3959(02)00268-3 转载 中国科技论文在线 http://www.paper.edu.cn 526 Y.-Q. Zhang et al. / Pain 99 (2002) 525–535 nociceptive afferents terminate (Aanonsen et al., 1990). It acupuncture stimulation and has been widely used in clin- has been considered that excitatory amino acid receptors are ical and basic research. The release of various neurotrans- important targets for the treatment of pain, especially those mitters in the central nervous system (CNS) is produced by resulting from inflammation or nerve injury. The involve- electroacupuncture (Han and Terenius, 1982; Tang et al., ment of NMDA receptors in tonic pain processing has been 1997; Zhu et al., 1997). Extensive work has shown that widely reported. Administration of NMDA receptor low-frequency and high-frequency electroacupuncture agonists elicits hyperalgesia, while administration of stimulation selectively induces the release of enkephalins NMDA receptor antagonists, on the other hand, produces and dynorphins in both experimental animals and humans antinociception. Electrophysiological studies have demon- (Han et al., 1991). Also, electroacupuncture has been strated that NMDA receptor agonists activate nociceptive reported to inhibit the release of some neurotransmitters neurons in the spinal dorsal horn (Dougherty and Willis, such as substance P (SP) (Shen et al., 1996). Recent studies 1991). In contrast, NMDA receptor antagonists attenuate in our laboratory revealed that electroacupuncture stimula- inflammation-induced responses in the spinal dorsal horn tion markedly reduced the release of glutamate and aspar- (Haley et al., 1990; Ren et al., 1992a). Behavioral studies tate in the spinal dorsal horn, suggesting an interaction have also indicated that intrathecal (i.t.) injection of NMDA between electroacupuncture analgesia and excitatory receptor agonists induces biting and scratching behavior and amino acid receptors in the spinal cord. Therefore, the hyperalgesia in the tail-flick and hot-plate tests in mice second aim of the present study is to determine whether (Aanonsen and Wilcox, 1987) and rats (Raigorodsky and electroacupuncture has synergetic antinociceptive effects Urca, 1987). I.t. injection of NMDA receptor antagonists with NMDA and AMPA/KA receptor antagonists in normal suppresses a variety of nociceptive responses produced by and carrageenan-injected rats. formalin (Nishiyama, 2000; Hamada et al., 2001), nerve injury (Yamamoto and Yaksh, 1992; Chaplan et al., 1997), peripheral inflammation (Ren et al., 1992a,b; Sakur- 2. Methods ada et al., 1998), C-fiber stimulation or cutaneous mustard oil application (Woolf and Thompson, 1991) and surgical 2.1. Animal preparation wounds (Zahn et al., 1998; Zahn and Brennan, 1998). By contrast, studies of the function of AMPA/KA receptors in Experiments were performed on adult male Sprague– spinal nociceptive processing have produced conflicting Dawley rats weighing 230–300 g. Rats were on a 12:12 h results. I.t. injection of AMPA/KA receptor antagonists light–dark cycle and received food and water. Prior to produced dose-dependent antinociception in the tail flick experimental manipulation, the animals were given a period (Advokat and Rutherford, 1995) and hot plate tests of 2 weeks to adjust to the new surroundings. All the experi- (Nishiyama et al., 1998) in rats, but did not affect forma- ments were carried out at the same time of the day between lin-induced pain or the hyperalgesia induced by intraplantar 8:00 and 4:00 to avoid diurnal variation in behavioral tests (i.pl.) injection of prostaglandin E2 or carrageenan (Coderre and c-fos expression. Rats were supplied by the Experimen- and Melzack, 1992; Coderre and Van Empel, 1994; Ferreira tal Animal Center of Fudan University. The treatment of the and Lorenzetti, 1994). Similarly, our previous studies animals conformed to the guidelines of the International showed that AMPA/KA antagonists markedly reduced Association for the Study of Pain (Zimmermann, 1983). muscular but not cutaneous nociceptive responses in spinal Peripheral inflammation was induced by i.pl. of carragee- dorsal horn neurons (Song and Zhao, 1993; Hu and Zhao, nan (2 mg/100 ml of 0.9% normal saline (NS); l-Carragee- 2001). However, the opposite results which showed that nan, Sigma) into one hindpaw in rats. AMPA/KA and NMDA receptors were similarly involved An intrathecal catheter (PE-10 tube) was inserted through in cutaneous and deep tissue and in acute, prolonged acute the gap between the L4 and L5 vertebrae and extended to the and chronic inflammatory processing (Chaplan et al., 1997; subarachnoid space of the lumbar enlargement (L4 and L5 Zahn et al., 1998; Nishiyama, 2000) were reported as well. segments) under sodium pentobarbital (40 mg/kg, intraper- To explore further the role of NMDA and AMPA/KA recep- itoneal (i.p.)) anesthesia. The catheter was filled with sterile tors in prolonged acute inflammatory processing, the first normal saline (approximately 4 ml), and the outer end was aim of the present study was to compare the effects of the plugged. The cannulated rats were allowed to recover for 3 NMDA receptor antagonist, AP5, and the AMPA/KA recep- days and were housed individually. Rats that showed any tor antagonist, DNQX, on carrageenan-induced behavioral neurological deficits resulting from the surgical procedure hyperalgesia and spinal Fos expression in rats. were excluded from the experiments.