- Email: [email protected]
Disruption of glial function enhances electroacupuncture analgesia in arthritic rats
Experimental Neurology 198 (2006) 294 – 302 www.elsevier.com/locate/yexnr
Disruption of glial function enhances electroacupuncture analgesia in arthritic rats Shan Sun, Wen-Ling Chen, Pei-Fen Wang, Zhi-Qi Zhao, Yu-Qiu Zhang ⁎ Institute of Neurobiology, Fudan University, 220 Handan Road, Shanghai 200433, China Shanghai Research Center of Acupuncture and Meridian, Shanghai, China Received 6 August 2005; revised 6 October 2005; accepted 18 November 2005 Available online 20 February 2006
Abstract Activated glia play a major role in mediating behavioral hypersensitive state following peripheral inflammation. Electroacupuncture is well known to relieve persistent inflammatory pain. The present study was undertaken to examine whether fluorocitrate, a glial metabolic inhibitor, could synergize electroacupuncture antagonizing thermal hyperalgesia and mechanical allodynia evoked by ankle joint inflammation. Monoarthritis of rat ankle joint was induced by an intra-articular injection of Complete Freund's Adjuvant (CFA). The paw withdrawal latency (PWL) from a thermal stimulus and paw withdrawal threshold (PWT) from von Frey hairs were measured in awake rats. Intrathecal (i.t.) injection of 1 nmol fluorocitrate markedly suppressed monoarthritis-induced thermal hyperalgesia and mechanical allodynia. Unilateral electroacupuncture stimulation of “Huantiao” (GB30) and “Yanglingquan” (GB34) acupuncture points (100/2 Hz alternation, 1–2–3 mA) significantly elevated the PWLs and PWTs for 45 min after cessation of electroacupuncture in monoarthritic rats. Co-application of 0.1 or 1 nmol fluorocitrate with electroacupuncture significantly potentiated electroacupuncture analgesia, although 0.1 nmol fluorocitrate alone had no effect on PWLs and PWTs in monoarthritic rats. These results suggested that electroacupuncture and disrupting glial function could synergistically antagonize inflammatory pain, which might provide a potential strategy for the treatment of arthritic pain. © 2005 Elsevier Inc. All rights reserved. Keywords: Glial cells; Fluorocitrate; Electroacupuncture; Monoarthritis; Hyperalgesia; Allodynia; Intrathecal injection; Rat
Introduction Arthritis remains a major health care problem in our society. One in seven people of all age is affected by arthritis or joint inflammation, resulting in pain and impaired joint function. Currently, non-steroidal anti-inflammatory drugs remain the major recommended strategy for its treatment, while long-term treatment with non-steroidal anti-inflammatory drugs may result in serious side effects, such as gastrointestinal ulcer and renal morbidity (Pincus et al., 1992; Campbell, 1988). Therefore, a safe and effective therapeutic strategy is desired for treatment of arthritis. As a traditional complementary and alternative medicine approach, electroacupuncture, an acupuncture therapy, has been shown to treat human rheumatoid arthritis and osteoar-
⁎ Corresponding author. Fax: +86 21 55522876. E-mail address: [email protected] (Y.-Q. Zhang). 0014-4886/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2005.11.011
thritis with satisfactory results (Guan and Zhang, 1995; Berman et al., 2000; Ezzo et al., 2001). In spite that electroacupuncture has been used successfully in the treatment of acute and/or chronic pain in clinic and investigated extensively with normal (uninjured) animal model for several decades, the precise mechanisms of electroacupuncture analgesia, especially under pathological conditions, are not fully understood. Recent published data have shown that healthy and pathological conditions respond differently to electroacupuncture (Zhang et al., 2002; Han, 2003; Lao et al., 2004). Our previous study revealed that intrathecal injection of a widespectrum excitatory amino acid receptor(s) antagonist (kynurenic acid), an N-methyl-D-aspartic acid (NMDA) receptor(s) antagonist (AP-5), and an (±)-α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainite (AMPA/KA) receptor(s) antagonist (DNQX) significantly potentiated electroacupuncture analgesia in carrageenan inflammatory rats rather than normal ones, suggesting that glutamate/glutamate receptors play an important role in electroacupuncture antihyperalgesia
S. Sun et al. / Experimental Neurology 198 (2006) 294–302
under conditions of inflammatory pain (Zhang et al., 2002, 2003). Spinal dorsal horn glial cells are now known to be major regulators of synaptic glutamate concentration (Meller et al., 1992; Paini et al., 1993; Parpura et al., 1994; McBean et al., 1995). It is well known that glutamate/glutamate receptors (especially NMDA receptors) in the spinal cord play a key role in the development and maintenance of central sensitization, which is an important component of inflammatory pain such as arthritic pain (Sluka and Westund, 1993a,b; Zhang et al., 2003). Mounting evidence has demonstrated that spinal glia might be important for hyperalgesia mechanisms (Watkins et al., 2001; Watkins and Maier, 2003). Following inflammation and damage of peripheral tissues, spinal glia become activated with changes in morphology and pro-inflammatory cytokines (Colburn et al., 1999; Hashizume et al., 2000; Bao et al., 2001; Watkins et al., 2001; Tanga et al., 2004; Twining et al., 2005). Spinal glial activation and up-regulation of cytokines appear to parallel the development and maintenance of mechanical allodynia induced by nerve injury, formalin, and zymosan (Colburn et al., 1999; Sweitzer et al., 1999). Disruption of glial function (using glial metabolic inhibitor, fluorocitrate) or the action of glial products (using a human recombinant interleukin-1 receptor antagonist) markedly reduces formalininduced hyperalgesia and sciatic inflammatory neuropathyinduced mechanical allodynia without influence on baseline (Watkins et al., 1999; Milligan et al., 2003). Along this line, we considered whether electroacupuncture might alter spinal chemistry by modulating glial function, thereby producing a higher efficient antinociceptive under pathological pain conditions. Complete Freund's adjuvant (CFA) has been utilized to induce an arthritic immunopathological disease that displays many of the pathological features of human rheumatoid arthritis (Colpaert, 1987). In the present study, CFA-induced ankle joint monoarthritis model was used to evaluate the synergetic antinociception effects of electroacupuncture and inhibiting spinal glial function. Both thermal hyperalgesia and mechanical allodynia were measured following different treatments.
295
Intrathecal surgery and injection An intrathecal catheter (PE-10 tube) was inserted through the gap between the L4 and L5 vertebrae and extended to the subarachniod space of the lumbar enlargement (L4 and L5 segments) under Chloral Hydrate (300 mg/kg, intraperitoneal (i.p.)) anesthesia. The catheter was filled with sterile normal saline (NS, approximately 4 μl), and the outer end was plugged. The cannulated rats were allowed to recover for 3–4 days and were housed individually. Rats that showed any neurological deficits resulting from the surgical procedure were excluded from the experiments. Drug or vehicle was injected over a period of 1 min via the catheter at a volume of 10 μl, followed by 5 μl NS for flushing. The location of the distal end of the i.t. catheter was verified at the end of each experiment by the injection of Pontamine Sky Blue via the i.t. catheter. Induction of monoarthritis Monoarthritis was induced by an intra-articular injection of complete Freund's adjuvant (CFA). The rat was briefly anesthetized with isoflurane. The skin around the site of injection was sterilized with 75% alcohol. The left foot of the rat was held and the fossa of the lateral malleolus of the fibula was located. A 28-gauge needle was inserted vertically to penetrate the skin, and turned distally to insert into the articular cavity from the gap between the tibiofibular and tarsus bone until a distinct loss of resistance was felt. A volume of 50 μl CFA was then injected. Sham monoarthritis control animals were similarly injected with sterile NS. Drugs Fluorocitrate (0.1 or 1 nmol in 10 μl; Sigma), a reversible glial metabolic inhibitor, acts by inhibiting aconitase, an enzyme of the Krebs energy cycle of glia but not neurons (Paulsen et al., 1987; Hassel et al., 1992). Fluorocitrate was dissolved initially in 2 M HCl and then diluted in sterile 10 mM phosphate buffered saline (PBS) to attain a final concentration, pH 6.0 at the time of testing.
Materials and methods Electroacupuncture treatment Animals Experiments were performed on adult male Sprague– Dawley rats (Experimental Animal Center, Shanghai Medical College of Fudan University, China) weighing 200–250 g. Prior to experimental manipulation, rats were allowed to acclimate to the housing facilities for 1 week and maintained on a 12:12 h light–dark cycle and a constant room temperature of 21°C with free access to food and water. All experimental protocols and animal handling procedures were approved by Animal Care and Use Committee of Fudan University, and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize the number of animals used and their suffering.
Rats were loosely immobilized in a specially made restrainer with the head, hind legs, and tail protruding. A pair of stainless steel pins of 0.34 mm in diameter were inserted with a depth of 5 mm into the left (ipsilateral to CFA-injected joint) “Huantiao” (GB30, located the lateral 1/3 and medial 2/3 of the distance between the sacral hiatus and the greater trochanter of femur), and “Yanglingquan” (GB34, located in the depression anterior and inferior to the fibula capitulum) acupuncture points (Fig. 1). The two pins were connected with the output terminals of the H.A.N.S. Acupuncture point Nerve Stimulator (LH-202H, Huawei Co., Ltd., Beijing, China). The electroacupuncture parameters were set as follows: square wave current output (pulse width: 0.2 ms); intensities ranging from 1–2–3 mA (each intensity for 10 min, totaling 30 min); at a 2 Hz and
296
S. Sun et al. / Experimental Neurology 198 (2006) 294–302
intensity, which produced a stable withdrawal latency of approximately 10–12 s in the absence of arthritis. A 20 s cutoff was used to prevent tissue damage in the absence of a response. Both hind paws were tested independently with 10min interval between trials. von Frey test for mechanical allodynia
Fig. 1. Illustration of the acupuncture points in human and rats. Panels a and a′ present Huantiao (GB 30), and panels b and b′ present Yanglingquan (GB 34) points in human and rats, respectively.
100 Hz alternating frequencies (automatically shifting between 100 Hz and 2 Hz stimulation for 3 s each) (Tang et al., 1997). Sham electroacupuncture control animals received needle insertion into GB30 and GB34 without manipulation.
The hind paw withdrawal threshold (PWT) was determined using a calibrated series of von Frey hairs (Stoelting, IL, USA) ranging from 1 to 26 g. Animals were placed individually into Plexiglass chamber with customized platform that contains 1.5 mm diameter holes in a 5 mm grid of perpendicular rows throughout the entire area of the platform (Pitcher et al., 1999). The protocol used in this study was a variation of that described by Takaishi et al. (1996). After acclimation to the test chamber, a series of 9 calibrated von Frey hairs were applied to the central region of the plantar surface of one hind paw in ascending order (1, 1.4, 2, 4, 6, 8, 10, 15, and 26 g) with the lowest hair at 1 g. A particular hair was applied until buckling of the hair occurred. This was maintained for approximately 2 s. The hair was applied only when the rat was stationary and standing on all four paws. A withdrawal response was considered valid only if the hind paw was completely removed from the customized platform. A trial consisted of application of a von Frey hair to the hind paw five times at 15-s intervals. If withdrawal did not occur during five applications of a particular hair, the next larger hair in the series was applied in a similar manner. When the hind paw was withdrawn from a particular hair in three out of the five consecutive applications, the value of that hair in grams was considered to be the withdrawal threshold. Once the threshold was determined for one hind paw, the same testing procedure was repeated on the other hind paw after 5 min. All behavioral tests were performed in a temperaturecontrolled room (22 ± 2°C). The experimenter was blind with respect to treatments.
Measurement of ankle perimeter Statistical analysis The perimeters of ankles were measured around the lower edge of lateral and medial malleoli with a scaled soft ruler without elasticity. Hargreave's test for thermal hyperalgesia After acclimation to the test chamber, thermal hyperalgesia was assessed by measuring the latency of paw withdrawal in response to a radiant heat source. Rats were placed individually into Plexiglas chamber on an elevated glass platform, under which a radiant heat source (model 336 combination unit, IITC/life Science Instruments, Woodland Hill, CA, USA) was applied to the glabrous surface of the paw through the glass plate. The heat source was turned off when the rat lifted the foot, allowing the measurement of time from onset of radiant heat application to withdrawal of the rat's hind paw. This time was defined as the hind paw withdrawal latency (PWL). The heat was maintained at a constant
Data were expressed as mean ± SEM. Prearthritis baseline measures were analyzed by one-way analysis of variance (ANOVA). Pre- and postdrug time course measures for both hyperalgesia and allodynia were analyzed by two-way ANOVA (treatment × time) followed by Newman–Keuls post hoc test. The differences in joint circumference between before and after intra-articular injection were determined with paired t test. P b 0.05 was considered statistically significant. Experimental procedures Experiment 1: effects of intrathecal fluorocitrate on monoarthritis-induced thermal hyperalgesia and mechanical allodynia After baseline behavioral assessments, rats received an intra-articular injection of either CFA (50 μl) or equivolume sterile NS. Behavior was assessed 48 h later to confirm
S. Sun et al. / Experimental Neurology 198 (2006) 294–302
297
development of thermal hyperalgesia and mechanical allodynia in the CFA-injected rats. Directly after testing at 48 h, rats received an intrathecal injection of either fluorocitrate (0.1 or 1 nmol) or vehicle (0.3% 2 M HCl in PBS, pH 6.0). Hargreave's and von Frey tests were then performed 15, 30, 45, 60, 90 min, and 2–20 h later. Experiment 2: inhibition of monoarthritis-induced thermal hyperalgesia and mechanical allodynia by electroacupuncture After baseline behavioral assessments, intra-articular CFA was performed as above. Following testing at 48 h, electroacupuncture or sham electroacupuncture stimulation (needle insertion without manipulation) was applied to ipsilateral (arthritis) hind limb at “GB30” and “GB34” acupuncture point for 30 min. Behavior was then assessed 15, 30, 45, 60, 90 min, and 2–20 h after cessation of electroacupuncture stimulation. Experiment 3: enhancement of electroacupuncture analgesia by intrathecal fluorocitrate in monoarthritis rats After baseline behavioral assessments, intra-articular CFA was performed as above. Following testing at 48 h, rats received an intrathecal injection of fluorocitrate (0.1 or 1 nmol). Two hours and thirty minutes after intrathecal fluorocitrate, rats received electroacupuncture stimulation for 30 min. Behavior was then assessed at the same time-point as experiment 2. Results General results Baseline measures of the paw withdrawal latency (PWL) to radiant heat stimulation and the paw withdrawal threshold (PWT) to von Frey hairs did not differ across groups (one-way ANOVA, PWL: F9,60 = 1.245, P N 0.05; PWT: F9,60 = 0.541, P N 0.05). Following unilateral intra-articular injection of CFA, joint inflammation (edema and erythema), thermal hyperalgesia, and mechanical allodynia developed at 4 h and persisted for more than 3 days in the ipsilateral hind paw (Figs. 2A, B). The PWLs to thermal stimuli and PWTs to von Frey hairs in contralateral hind paw to monoarthritis remained stable within 3 days after CFA injection. The joint circumference for the ipsilateral ankle joint increased significantly on day 2 after CFA compared to before CFA injection (paired t test, P b 0.01). There were no significant differences in contralateral intact and NSinjected ankles between before and after intra-articular injection (Fig. 2C). Inhibition of fluorocitrate on thermal hyperalgesia and mechanical allodynia in CFA-induced monoarthritic rats The effects of i.t. injection of fluorocitrate (0.1 or 1 nmol), a glia metabolic inhibitor, on the PWLs to thermal stimuli and PWTs to von Frey hairs were examined on day 2 after intraarticular injection of CFA when the arthritic features stably occurred. I.t. injection of vehicle and low dose fluorocitrate
Fig. 2. Three variables measured before and after induction of the CFA model of rat monoarthritis. (A, B) Unilateral intra-articular injection of CFA induced significant thermal hyperalgesia (A) and mechanical allodynia (B). (C) Injection of CFA into the unilateral articular cavity produced a significant increase in circumference of the ipsilateral ankle joint that is measured 2 days after injection. *P b 0.05 and **P b 0.01 vs. before intra-articular injection. MA, monoarthritis; ipsi, ipsilateral; contra, contralateral; CFA, complete Freund's adjuvant.
(0.1 nmol) had no effect on PWLs and PWTs in monoarthritic rats. At the dose of 1 nmol, fluorocitrate significantly reduced the CFA-induced thermal hyperalgesia and mechanical allodynia in ipsilateral paw from 3 to 12 h after i.t. injection (Fig. 3). Two-way ANOVA analysis revealed significant effect of i.t. fluorocitrate (1 nmol) treatment (F1,192 = 516.56, P b 0.01) and significant interaction between fluorocitrate treatment and time (F15,192 = 43.67, P b 0.01). I.t. injection of 1 nmol fluorocitrate had no effect on the PWLs and PWTs of contralateral intact hind paw to monoarthritis (data not shown) and sham monoarthritic hind paw (Fig. 3).
298
S. Sun et al. / Experimental Neurology 198 (2006) 294–302
electrode into the acupuncture points (sham electroacupuncture) in monoarthritic rats (Fig. 4). Fluorocitrate and electroacupuncture synergistically inhibited monoarthritis-induced thermal hyperalgesia and mechanical allodynia The preceding results showed that neither PWLs nor PWTs were influenced by i.t. injection of 0.1 nmol fluorocitrate in monoarthritic rats. When a combination of 0.1 nmol fluorocitrate with electroacupuncture was given, both PWLs and PWTs of ipsilateral hind paw to monoarthritis had a robust increase in magnitude and duration, compared with the effects obtained in either the electroacupuncture or 0.1 nmol fluorocitrate alone group (PWL: F2,288 = 1485.39, P b 0.01; PWT: F2,288 = 1475.17, P b 0.01) (Figs. 4A, B). Notably, the PWLs to thermal stimuli even exceeded baseline before CFA injection within 1 h after electroacupuncture when co-applied with fluorocitrate (Fig. 4A). The effects of co-applied 1 nmol fluorocitrate and electroacupuncture on monoarthritis-induced thermal hyperalgesia and mechanical allodynia were similar to those of 0.1 nmol fluorocitrate combined with electroacupuncture group (PWL: F1,192 = 75.87, P b 0.01; PWT: F1,192 = 0.708, P N 0.05) (Fig. 4). A significant interaction between treatments and time was also found (PWL: F30,288 = 46.84, P b 0.01; PWT: F30,288 = 16.11, P b 0.01). In contralateral intact hind paw, PWLs and PWTs were also significantly increased following coapplication of 1 nmol fluorocitrate and electroacupuncture (Figs. 4C, D). Fig. 3. Intrathecal fluorocitrate inhibited monoarthritis-induced thermal hyperalgesia (A) and mechanical allodynia (B) in ipsilateral hind paw to monoarthritis, but not altered the PWLs and PWTs in sham monoarthritic rats. Fluorocitrate or vehicle was given 2 days after intra-articular injection of CFA. **P b 0.01 vs. vehicle control. Fc, fluorocitrate; MA, monoarthritis.
Inhibition of electroacupuncture on monoarthritis-induced thermal hyperalgesia and mechanical allodynia On day 2 after unilateral intra-articular injection of CFA, electroacupuncture stimulation applied to the unilateral hind limb (ipsilateral to CFA injection) at the “GB 30” and “GB 34” acupuncture points markedly inhibited pain hypersensitivity of the rats. The PWLs and PWTs of ipsilateral hind paw to monoarthritis significantly increased from 15 to 60 min after cessation of electroacupuncture stimulation (Figs. 4A, B). Twoway ANOVA analysis revealed significant effect of electroacupuncture treatment (PWL: F1,160 = 97.27, P b 0.01; PWT: F1,160 = 230.48, P b 0.01) and significant interaction between electroacupuncture treatment and time (PWL: F15,160 = 51.33, P b 0.01; PWT: F15,160 = 89.99, P b 0.01). At the time of the maximal electroacupuncture analgesic effect, 15 min, PWL and PWT of ipsilateral hind paw to monoarthritis were increased by 144.85% and 280.95%, respectively. Also, electroacupuncture stimulation produced a transient increase in the PWLs of contralateral intact hind paw to monoarthritis, whereas PWTs were not significantly influenced (Figs. 4C, D). Neither PWLs nor PWTs were affected by just inserting the stimulation
Discussion An important finding in the present study is that coapplication of electroacupuncture with intrathecal injection of a glial metabolic inhibitor fluorocitrate synergistically antagonized rat monoarthritic pain. Consistent with previous reports (Sluka et al., 1998; Zhang et al., 2002; Lao et al., 2004), electroacupuncture produced a robust but brief (60 min) inhibitory effect on CFA-induced thermal hyperalgesia and mechanical allodynia. Low dose (0.1 nmol) of fluorocitrate alone did not alter PWLs to thermal stimuli and PWTs to von Frey hairs in CFA-induced monoarthritic rats. However, a combination of low dose fluorocitrate and electroacupuncture completely reversed intra-articular injection of CFA-induced thermal hyperalgesia and mechanical allodynia for 12 h, suggesting a significant inter-reinforcing of electroacupuncture and disrupting spinal glial function. Involvement of spinal glia in inflammatory pain Glial cells were first considered as contributing to exaggerated “pain” by Garrison et al. (1994). They found that manipulation that created exaggerated “pain” also activated astrocytes, and the drug that blocked exaggerated “pain” also blocked astrocytic activation. Since then, glial (both microglia and astrocytes) activation has been observed at the lumbar spinal cord in various animal models of pathological pain,
S. Sun et al. / Experimental Neurology 198 (2006) 294–302
299
Fig. 4. Co-application of electroacupuncture with an intrathecal injection of fluorocitrate completely reversed monoarthritis-induced thermal hyperalgesia (A) and mechanical allodynia (B) in ipsilateral hind paw, and briefly increased the PWLs and PWTs of contralateral hind paw (C, D). Electroacupuncture was given 2.5 h after intrathecal fluorocitrate, when they are co-applied. *P b 0.05, **P b 0.01 vs. sham electroacupuncture; ++P b 0.01 vs. electroacupuncture alone; ##P b 0.01 vs. 1 nmol fluorocitrate plus electroacupuncture. Fc, fluorocitrate; EA, electroacupuncture.
including inflammation and damage of peripheral tissues, peripheral nerves, spinal nerves, and spinal cord, as well as chronic opioid treatment (Sweitzer et al., 1999, 2001; Raghavendra et al., 2002; Milligan et al., 2003; Ledeboer et al., 2005). Activated microglia and astrocytes release a variety of the neuron- and glial-excitatory substances, including reactive oxygen species, nitric oxide, prostaglandin, and excitatory amino acids. As resident immune cells in the CNS, activated glia also produce and release pro-inflammatory cytokines interleukin-1, interleukin-6, and tumor necrosis factor (Liu et al., 2004; Watkins and Maier, 2002; WieselerFrank et al., 2004). The substances from activated glia importantly contribute to the amplification of pain (Raghavendra et al., 2003; Sommer, 2003; Watkins and Maier, 2003). Blocking the activation of spinal glia markedly inhibits inflammation- or injury-induced release of glial- and neuroexcitatory substances, as well as the resultant exaggerated “pain” state (Milligan et al., 2000, 2001, 2003; Tikka et al., 2001; Ledeboer et al., 2005). In agreement with the previous studies on different models (Watkins et al., 1997, 1999; Raghavendra et al., 2003; Milligan et al., 2001, 2004), the present study showed that i.t. injection of 1 nmol fluorocitrate
reversibly suppressed thermal hyperalgesia and mechanical allodynia evoked by intra-articular CFA, whereas there was no significant increase in the PWLs and PWTs of contralateral intact hind paw to monoarthritis and sham monoarthritic hind paw. This result further supports the conclusion that glia is critically involved in the mediation of “exaggerated pain” evoked by inflammation but not acute pain. Synergetic inhibition of electroacupuncture and glial dysfunction on inflammatory pain Acupuncture is an important component of Chinese traditional medicine. Numerous studies have shown that acupuncture stimulation increases experimental pain threshold in various pain models and animal species (Sluka et al., 1998, 1999; Ma and Sluka, 2001). The commonly held theory for the mechanisms of electroacupuncture analgesia is activation of endogenous opioid system. The studies from Han's group demonstrated that low frequency (2 Hz) electroacupuncture accelerated the release of endomorphine, enkephalin, and βendorphin, and could be blocked by pretreatment with μ- and δopioid receptor antagonists, whereas high frequency (100 Hz)
300
S. Sun et al. / Experimental Neurology 198 (2006) 294–302
electroacupuncture produced the release of dynorphin, and could be blocked by κ-opioid receptor antagonists (Han et al., 1991; Han, 2003; Ulett et al., 1998; Wang et al., 2005). Given a dense-spare frequency (2 Hz/100 Hz alternation) of electroacupuncture stimulation used in the present study, all four kinds of opioid peptides release together and their receptors might be activated to a greater extent. It has been demonstrated that all three classes of opioid receptors are located on primary nociceptive afferent terminals and on the dendrites of pain sensitive neurons in the dorsal horn of the spinal cord. Presynaptic activation of opioid receptors on the primary afferent terminals results in a direct reduction of Ca2+ conductance or indirect increase in K+ conductance, and in turn decreases release of transmitters such as excitatory amino acids and substance P (Dickenson and Sullivan, 1986; Malmberg and Yaksh, 1995; Ulett et al., 1998; Cheunsuang et al., 2002). Several studies have shown that glial cells in culture express the μ-, κ-, and δ-opioid receptors, and respond to opioid stimulation by changing their morphology and proliferation rate in vitro (Ruzica et al., 1996; Ruzica and Akil, 1997; Tryoen et al., 2000). If this case occurred in the spinal glia in vivo, it is possible that electroacupuncture acts directly or indirectly on glial cells, triggering alterations in their metabolism, morphology, and functions via opioid peptides and opioid receptors. On the other hand, electroacupuncture has been found to modulate the function of the immune system and alleviated inflammatory pain (Sun et al., 2000; Ceccherelli et al., 2002; Hahm et al., 2004; Zhang et al., 2004). In the CNS, one cellular target of immune system activation is the glia. Evidence is accumulating that acupuncture inhibits activation of glia and decreases the levels of pro-inflammatory cytokine. Son et al. (2002) reported that electroacupuncture efficiently suppressed lipopolysaccharide-induced up-regulation of interleukin-6 and interleukin-1β mRNA levels in hypothalamus. Also, the middle cerebral artery occlusion-induced increase in interleukin-1β mRNA in ischemic cortex, the medial forebrain bundle axotomy-induced activation of microglia in substantia nigra, and resultant increase in the levels of tumor necrosis factor and interleukin-1β mRNA were significantly blocked by electroacupuncture (Xu et al., 2002; Liu et al., 2004). Thus, fluorocitrate synergized electroacupuncture, blocking activation of spinal glia, reducing release of pain enhancing substances in the spinal dorsal horn, and disrupting neuron-to-glia-to-neuron excitatory circuit, thereby leading to a robust inhibition on monoarthritis-induced thermal hyperalgesia and mechanical allodynia. In addition, the release of supraspinal opioid, supraspinal and spinal serotonin, and noradrenalin as well as the activation of brainstem endogenous descending inhibitory system induced by electroacupuncture (Kalra et al., 2001) may also contribute to the synergetic antinociceptive effect of electroacupuncture with glial metabolic inhibitor. It is worthy of mention that PWLs of ipsilateral paw to monoarthritis even went beyond that of normal rats by almost 40% when fluorocitrate and electroacupuncture were given together; suggesting that combination of electroacupuncture and fluorocitrate might interfere with normal sensory physiology. This presume was further supported by the fact that
co-application of the two treatments prolonged PWLs of contralateral intact hind paw or sham monoarthritic hind paw, although fluorocitrate alone could not affect them. Clinically, electroacupuncture induces only a moderate degree of analgesia on arthritis, and high dose fluorocitrate should not be administered because of neurotoxicity. The present results that co-application of electroacupuncture with low dose glial metabolic inhibitor synergistically antagonized monoarthritic pain may provide a potential strategy for the treatment of arthritis.
Acknowledgments The authors wish to thank Dr. Judith Strong for her critical reading of the manuscript and helpful criticism. We also thank Mr. Zhou Dong for his drawing assistance. This work was supported by the National Natural Science Fund of China (NSFC) (30330230, 30570594), NSFC for Distinguished Young Scholars (30425022), National Basic Research Program of China (2006CB500807), and Science Foundation of Shanghai Municipal Commission of Science and Technology (02DZ19150-4).
References Bao, L., Zhu, Y., Elhassan, A.M., Wu, Q., Xiao, B., Zhu, J., Undgren, J.U., 2001. Adjuvant-induced arthritis: IL-1β, IL-6 and TNF-α are up-regulated in the spinal cord. NeuroReport 12, 3905–3908. Berman, B.M., Swyers, J.P., Ezzo, J., 2000. The evidence for acupuncture as a treatment for rheumatologic conditions. Rheum. Dis. Clin. North Am. 26, 103–115. Campbell, S.M., 1988. Rheumatiod arthritis: current strategies. Hosp. Med. 34, 29–32. Ceccherelli, F., Gagliardi, G., Ruzzante, L., Giron, G., 2002. Acupuncture modulation of capsaicin-induced inflammation: effect of intraperitoneal and local administration of naloxone in rats. A blinded controlled study. J. Altern. Complement. Med. 8, 341–349. Cheunsuang, O., Maxwell, D., Morris, R., 2002. Spinal lamina I neurons that express neurokinin 1 receptors: II. Electrophysiological characteristics, responses to primary afferent stimulation and effects of a selective muopioid receptor agonist. Neuroscience 111, 423–434. Colburn, R.W., Rickman, A.J., Deleo, J.A., 1999. The effect of site and type of nerve injury on spinal glial activation and neuropathic pain behavior. Exp. Neurol. 157, 289–304. Colpaert, F.C., 1987. Evidence that adjuvant arthritis in the rat is associated with chronic pain. Pain 28, 201–222. Dickenson, A.H., Sullivan, A.F., 1986. Electrophysiological studies on the effects of intrathecal morphine on nociceptive neurons in the rat dorsal horn. Pain 24, 211–222. Ezzo, J., Hadhazy, V., Birch, S., 2001. Acupuncture for osteoarthritis of the knee: a systematic review. Arthritis Rheum. 44, 819–825. Garrison, C.J., Dougherty, P.M., Carlton, S.M., 1994. GFAP expression in lumbar spinal cord of naive and neuropathic rats treated with MK-801. Exp. Neurol. 129, 237–243. Guan, Z., Zhang, L., 1995. Effects of acupuncture on immunoglobulins in patients with asthma and rheumatoid arthritis. J. Tradit. Chin. Med. 15, 102–105. Hahm, E.T., Lee, J.J., Lee, W.K., Bae, H.S., Min, B.I., Cho, Y.W., 2004. Electroacupuncture enhancement of natural killer cell activity suppressed by anterior hypothalamic lesions in rats. Neuroimmunomodulation 11, 268–272.
S. Sun et al. / Experimental Neurology 198 (2006) 294–302 Han, J.S., 2003. Acupuncture: neuropeptide release produced by electrical stimulation of different frequencies. Trends Neurosci. 26, 17–22. Han, J.S., Chen, X.H., Sun, S.L., Xu, X.J., Yuan, Y., Yan, S.C., Hao, J.X., Terenius, L., 1991. Effect of low- and high-frequency TENS on Metenkephalin-Arg-Phe and dynorphin immunoreactivity in human lumbar CSF. Pain 47, 295–298. Hashizume, H., Deleo, J.A., Colburn, R.W., Weinstein, J.N., 2000. Spinal glial activation and cytokine expression after lumbar root injury in the rat. Spine 25, 1206–1217. Hassel, B., Paulsen, R.E., Johnson, A., Fonnum, F., 1992. Selective inhibition of glial cell metabolism by fluorocitrate. Brain Res. 249, 120–124. Kalra, A., Urban, M.O., Sluka, K.A., 2001. Blockade of opioid receptors in rostral ventral medulla prevents antihyperalgesia produced by transcutaneous electrical nerve stimulation (TENS). J. Pharmacol. Exp. Ther. 298, 257–263. Lao, L., Zhang, R.X., Zhang, G., Wang, X., Berman, B.M., Ren, K., 2004. A parametric study of electroacupuncture on persistent hyperalgesia and Fos protein expression in rats. Brain Res. 1020, 18–29. Ledeboer, A., Sloane, E.M., Milligan, E.D., Frank, M.G., Mahony, J.H., Maier, S.F., Watkins, L.R., 2005. Minocycline attenuates mechanical allodynia and proinflammatory cytokine expression in rat models of pain facilitation. Pain 115, 71–83. Liu, X.Y., Zhou, H.F., Pan, Y.L., Liang, X.B., Niu, D.B., Xue, B., Li, F.Q., He, Q.H., Wang, X.H., Wang, X.M., 2004. Electro-acupuncture stimulation protects dopaminergic neurons from inflammation-mediated damage in medial forebrain bundle-transected rats. Exp. Neurol. 189, 189–196. Ma, Y.T., Sluka, K.A., 2001. Reduction in inflammation-induced sensitization of dorsal horn neurons by transcutaneous electrical nerve stimulation in anesthetized rats. Exp. Brain Res. 137, 94–102. Malmberg, A.B., Yaksh, T.L., 1995. The effect of morphine on formalin-evoked behavior and spinal release of excitatory amino acids and prostaglandin E2 using microdialysis in conscious rats. Br. J. Pharmacol. 114, 1069–1075. McBean, G.J., Doorty, K.B., Tipton, K.F., Kollegger, H., 1995. Alternation in the glial cell metabolism of glutamate by kainite and N-methyl-D-aspartate. Toxicon 33, 569–576. Meller, S.T., Dykstra, C., Gebhart, G.F., 1992. Production of endogenous nitric oxide and activation of soluble guanylate cyclase are required for N-methylD-aspartate-produced facilitation of the nociceptive tail-flick reflex. Eur. J. Pharmacol. 214, 93–96. Milligan, E.D., Mehmert, K.K., Hinde, J.L., Harvey, L.O.J., Martin, D., Tracey, K.J., Maier, S.F., Watkins, L.R., 2000. Thermal hyperalgesia and mechanical allodynia produced by intrathecal administration of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein, gp120. Brain Res. 861, 105–116. Milligan, E.D., O'Connor, K.A., Nguyen, K.T., Armstrong, C.B., Twining, C., Gaykema, R.P., Holguin, A., Martin, D., Maier, S.F., Watkins, L.R., 2001. Intrathecal HIV-1 envelope glycoprotein gp120 induces enhanced pain states mediated by spinal cord proinflammatory cytokines. J. Neurosci. 21, 2808–2819. Milligan, E.D., Twining, C., Chacur, M., Biedenkapp, J., O'Connor, K., Poole, S., Tracey, K., Martin, D., Maier, S.F., Watkins, L.R., 2003. Spinal glia and proinflammatory cytokines mediate mirror-image neuropathic pain in rats. J. Neurosci. 23, 1026–1040. Milligan, E.D., Zapata, V., Chacur, M., Schoeniger, D., Biedenkapp, J., O'Connor, K.A., Verge, G.M., Chapman, G., Green, P., Foster, A.C., Naeve, G.S., Maier, S.F., Watkins, L.R., 2004. Evidence that exogenous and endogenous fractalkine can induce spinal nociceptive facilitation in rats. Eur. J. Neurosci. 20, 2294–2302. Paini, D., Frei, K., Pfister, H.W., Fontana, A., 1993. Glutamate uptake by astrocytes is inhibited by reactive oxygen intermediates but not by other macrophage-derived molecules including cytokines, leukotrienes or plateletactivating factor. J. Neuroimmunol. 48, 153–156. Parpura, V., Basarsky, T.A., Liu, F., Jeftinija, K., Jeftinija, S., Haydon, P.G., 1994. Glutamate-mediated astrocytes-neuron signaling. Nature 369, 744–747. Paulsen, R.E., Contestabile, A., Villani, L., Fonnum, F., 1987. An in vivo model for studying function of brain tissue temporarily devoid of glial cell metabolism: the use of fluorocitrate. J. Neurochem. 48, 1377–1385.
301
Pincus, T., Marcum, S.B., Callahan, L.F., 1992. Long-term drug therapy for rheumatoid arthritis in seven rheumatology private practices: II. Second line drugs and prednisone. J. Rheumatol. 19, 1885–1894. Pitcher, G.M., Ritchie, J., Henry, J.L., 1999. Paw withdrawal threshold in the von Frey hair test is influenced by the surface on which the rat stands. J. Neurosci. Method 87, 185–193. Raghavendra, V., Rutkowski, M.D., Deleo, J.A., 2002. The role of spinal neuroimmune activation in morphine tolerance/hyperalgesia in neuropathic and sham-operated rats. J. Neurosci. 22, 9980–9989. Raghavendra, V., Tanga, F., Deleo, J.A., 2003. Inhibition of microglial activation attenuates the development but not existing hypersensitivity in a rat model of neuropathy. J. Pharmacol. Exp. Ther. 306, 624–630. Ruzica, B.B., Akil, H., 1997. The interleukin-1-beta-mediated regulation of proenkephalin and opioid receptor messenger RNA in primary astrocytes enriched cultures. Neuroscience 19, 517–524. Ruzica, B.B., Thompson, R.C., Watson, S.J., Akil, H., 1996. Interleukin-1-betamediated regulation of mu-opioid receptor messenger RNA in primary astrocytes enriched cultures. Neurochemistry 66, 425–428. Sluka, K.A., Westund, K.N., 1993a. Behavioral and immunohistochemical changes in an experimental arthritis model in rats. Pain 55, 367–377. Sluka, K.A., Westund, K.N., 1993b. Centrally administered non-NMDA but not NMDA receptor antagonists block peripheral knee joint inflammation. Pain 55, 217–225. Sluka, K.A., Bailey, K., Bogush, J., Olson, R., Ricketts, A., 1998. Treatment with either high or low frequency TENS reduces the secondary hyperalgesia observed after injection of kaolin and Carrageenan into the knee joint. Pain 77, 97–102. Sluka, K.A., Deacon, M., Stibal, A., Strissel, S., Terpstra, A., 1999. Spinal blockade of opioid receptors the analgesia produced by TENS in arthritic rats. J. Pharmacol. Exp. Ther. 289, 840–846. Sommer, C., 2003. Painful neuropathies. Curr. Opin. Neurol. 16, 623–628. Son, Y.S., Park, H.J., Kwon, O.B., Jung, S.C., Shin, H.C., Lim, S., 2002. Antipyretic effects of acupuncture on the lipopolysaccharide-induced fever and expression of interleukin-6 and interleukin-1 beta mRNAs in the hypothalamus of rats. Neurosci. Lett. 319, 45–48. Sun, T., Du, L.N., Wu, G.C., Cao, X.D., 2000. Effect of intrathecal morphine and electroacupuncture on cellular immune function of rats and increment of mu-opioid receptor mRNA expression in PAG following intrathecal morphine. Acupunct. Electro-ther. Res. 25, 1–8. Sweitzer, S.M., Colburn, R.W., Rutkowski, M., Deleo, J.A., 1999. Acute peripheral inflammation induces moderate glial activation and spinal IL-1β expression that correlates with pain behavior in the rat. Brain Res. Int. 829, 209–221. Sweitzer, S.M., Schubert, P., Deleo, J.A., 2001. Propentofyline, a glial modulating agent, exhibits antiallodynia properties in a rat model of neuropathic pain. J. Pharmacol. Exp. Ther. 297, 1210–1217. Takaishi, K., Eisele Jr., J.H., Carstens, E., 1996. Behavioral and electrophysiological assessment of hyperalgesia and changes in dorsal horn responses following partial sciatic nerve ligation in rats. Pain 66, 297–306. Tang, N.M., Dong, H.W., Wang, X.M., Tsui, Z.C., Han, J.S., 1997. Cholecystokinin antisense RNA increases the analgesic effect induced by electroacupuncture or low dose morphine: conversion of low responder rats into high responders. Pain 71, 71–80. Tanga, F.Y., Raghavendra, V., Deleo, J.A., 2004. Quantitative real-time RT-PCR assessment of spinal microglial and astrocytic activation markers in a rat model of neuropathic pain. Neurochem. Int. 45, 397–407. Tikka, T., Fiebich, B.L., Goldsteins, G., Keinanen, R., Koistinaho, J., 2001. Minocycline, a tetracycline derivative, a neuroprotective against excitotoxicity by inhibiting activation and proliferation of microglia. J. Neurosci. 21, 2580–2588. Tryoen, P., Gaveraux, C., Labourdette, G., 2000. Down-regulation of mu-opioid receptor expression in rat oligodendrocytes during their development in vitro. J. Neurosci. Res. 60, 10–20. Twining, C.M., Sloane, E.M., Schoeniger, D.K., Milligan, E.D., Martin, D., Marsh, H., Maier, S.F., Watkins, L.R., 2005. Activation of the spinal cord complement cascade might contribute to mechanical allodynia induced by three animal models of spinal sensitization. J. Pain 6, 174–183.
302
S. Sun et al. / Experimental Neurology 198 (2006) 294–302
Ulett, G.A., Han, S., Han, J.S., 1998. Electroacupuncture: mechanisms and clinical application. Biol. Psychiatry 44, 129–138. Wang, Y., Zhang, Y., Wang, W., Cao, Y., Han, J.S., 2005. Effects of synchronous or asynchronous electroacupuncture stimulation with low versus high frequency on spinal opioid release and tail flick nociception. Exp. Neurol. 192, 156–162. Watkins, L.R., Maier, S.F., 2002. Beyond neurons: evidence that immune and glial cells contribute to pathological pain states. Physiol. Rev. 82, 981–1011. Watkins, L.R., Maier, S.F., 2003. Glia: a novel drug discovery target for clinical pain. Nat. Rev., Drug Discov. 2, 973–985. Watkins, L.R., Martin, D., Ulrich, P., Tracey, K.J., Maier, S.F., 1997. Evidence for the involvement of spinal cord glia in subcutaneous formalin induced hyperalgesia in the rat. Pain 71, 225–235. Watkins, L.R., Hansen, M.K., Nguyen, K.T., Lee, J.E., Maier, S.F., 1999. Dynamic regulation of the proinflammatory cytokine, interleukin-1β: molecular biology for non-molecular biologists. Life Sic. 65, 449–481.
Watkins, L.R., Milligan, E.D., Maier, S.F., 2001. Glial activation: a driving force for pathological pain. Trends Neurosci. 24, 450–455. Wieseler-Frank, J., Maier, S.F., Watkins, L.R., 2004. Glial activation and pathological pain. Neurochem. Int. 45, 389–395. Xu, Z.F., Wu, G.C., Cao, X.D., 2002. Effect of electroacupuncture on the expression of interleukin-1 beta mRNA after transient focal cerebral ischemia. Acupunct. Electro-ther. Res. 27, 29–35. Zhang, Y.Q., Ji, G.C., Wu, G.C., Zhao, Z.Q., 2002. Excitatory amino acid receptor antagonists and electroacupuncture synergistically inhibit Carrageenan-induced behavioral hyperalgesia and spinal fos expression in rats. Pain 99, 525–535. Zhang, Y.Q., Ji, G.C., Wu, G.C., Zhao, Z.Q., 2003. Kynurenic acid enhances electroacupuncture analgesia in normal and Carrageenan-injected rats. Brain Res. 966, 300–307. Zhang, S.P., Zhang, J.S., Yung, K.K., Zhang, H.Q., 2004. Non-opioiddependent anti-inflammatory effects of low frequency electroacupuncture. Brain Res. Bull. 62, 327–334.
Disruption of glial function enhances electroacupuncture analgesia in arthritic rats Shan Sun, Wen-Ling Chen, Pei-Fen Wang, Zhi-Qi Zhao, Yu-Qiu Zhang ⁎ Institute of Neurobiology, Fudan University, 220 Handan Road, Shanghai 200433, China Shanghai Research Center of Acupuncture and Meridian, Shanghai, China Received 6 August 2005; revised 6 October 2005; accepted 18 November 2005 Available online 20 February 2006
Abstract Activated glia play a major role in mediating behavioral hypersensitive state following peripheral inflammation. Electroacupuncture is well known to relieve persistent inflammatory pain. The present study was undertaken to examine whether fluorocitrate, a glial metabolic inhibitor, could synergize electroacupuncture antagonizing thermal hyperalgesia and mechanical allodynia evoked by ankle joint inflammation. Monoarthritis of rat ankle joint was induced by an intra-articular injection of Complete Freund's Adjuvant (CFA). The paw withdrawal latency (PWL) from a thermal stimulus and paw withdrawal threshold (PWT) from von Frey hairs were measured in awake rats. Intrathecal (i.t.) injection of 1 nmol fluorocitrate markedly suppressed monoarthritis-induced thermal hyperalgesia and mechanical allodynia. Unilateral electroacupuncture stimulation of “Huantiao” (GB30) and “Yanglingquan” (GB34) acupuncture points (100/2 Hz alternation, 1–2–3 mA) significantly elevated the PWLs and PWTs for 45 min after cessation of electroacupuncture in monoarthritic rats. Co-application of 0.1 or 1 nmol fluorocitrate with electroacupuncture significantly potentiated electroacupuncture analgesia, although 0.1 nmol fluorocitrate alone had no effect on PWLs and PWTs in monoarthritic rats. These results suggested that electroacupuncture and disrupting glial function could synergistically antagonize inflammatory pain, which might provide a potential strategy for the treatment of arthritic pain. © 2005 Elsevier Inc. All rights reserved. Keywords: Glial cells; Fluorocitrate; Electroacupuncture; Monoarthritis; Hyperalgesia; Allodynia; Intrathecal injection; Rat
Introduction Arthritis remains a major health care problem in our society. One in seven people of all age is affected by arthritis or joint inflammation, resulting in pain and impaired joint function. Currently, non-steroidal anti-inflammatory drugs remain the major recommended strategy for its treatment, while long-term treatment with non-steroidal anti-inflammatory drugs may result in serious side effects, such as gastrointestinal ulcer and renal morbidity (Pincus et al., 1992; Campbell, 1988). Therefore, a safe and effective therapeutic strategy is desired for treatment of arthritis. As a traditional complementary and alternative medicine approach, electroacupuncture, an acupuncture therapy, has been shown to treat human rheumatoid arthritis and osteoar-
⁎ Corresponding author. Fax: +86 21 55522876. E-mail address: [email protected] (Y.-Q. Zhang). 0014-4886/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2005.11.011
thritis with satisfactory results (Guan and Zhang, 1995; Berman et al., 2000; Ezzo et al., 2001). In spite that electroacupuncture has been used successfully in the treatment of acute and/or chronic pain in clinic and investigated extensively with normal (uninjured) animal model for several decades, the precise mechanisms of electroacupuncture analgesia, especially under pathological conditions, are not fully understood. Recent published data have shown that healthy and pathological conditions respond differently to electroacupuncture (Zhang et al., 2002; Han, 2003; Lao et al., 2004). Our previous study revealed that intrathecal injection of a widespectrum excitatory amino acid receptor(s) antagonist (kynurenic acid), an N-methyl-D-aspartic acid (NMDA) receptor(s) antagonist (AP-5), and an (±)-α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainite (AMPA/KA) receptor(s) antagonist (DNQX) significantly potentiated electroacupuncture analgesia in carrageenan inflammatory rats rather than normal ones, suggesting that glutamate/glutamate receptors play an important role in electroacupuncture antihyperalgesia
S. Sun et al. / Experimental Neurology 198 (2006) 294–302
under conditions of inflammatory pain (Zhang et al., 2002, 2003). Spinal dorsal horn glial cells are now known to be major regulators of synaptic glutamate concentration (Meller et al., 1992; Paini et al., 1993; Parpura et al., 1994; McBean et al., 1995). It is well known that glutamate/glutamate receptors (especially NMDA receptors) in the spinal cord play a key role in the development and maintenance of central sensitization, which is an important component of inflammatory pain such as arthritic pain (Sluka and Westund, 1993a,b; Zhang et al., 2003). Mounting evidence has demonstrated that spinal glia might be important for hyperalgesia mechanisms (Watkins et al., 2001; Watkins and Maier, 2003). Following inflammation and damage of peripheral tissues, spinal glia become activated with changes in morphology and pro-inflammatory cytokines (Colburn et al., 1999; Hashizume et al., 2000; Bao et al., 2001; Watkins et al., 2001; Tanga et al., 2004; Twining et al., 2005). Spinal glial activation and up-regulation of cytokines appear to parallel the development and maintenance of mechanical allodynia induced by nerve injury, formalin, and zymosan (Colburn et al., 1999; Sweitzer et al., 1999). Disruption of glial function (using glial metabolic inhibitor, fluorocitrate) or the action of glial products (using a human recombinant interleukin-1 receptor antagonist) markedly reduces formalininduced hyperalgesia and sciatic inflammatory neuropathyinduced mechanical allodynia without influence on baseline (Watkins et al., 1999; Milligan et al., 2003). Along this line, we considered whether electroacupuncture might alter spinal chemistry by modulating glial function, thereby producing a higher efficient antinociceptive under pathological pain conditions. Complete Freund's adjuvant (CFA) has been utilized to induce an arthritic immunopathological disease that displays many of the pathological features of human rheumatoid arthritis (Colpaert, 1987). In the present study, CFA-induced ankle joint monoarthritis model was used to evaluate the synergetic antinociception effects of electroacupuncture and inhibiting spinal glial function. Both thermal hyperalgesia and mechanical allodynia were measured following different treatments.
295
Intrathecal surgery and injection An intrathecal catheter (PE-10 tube) was inserted through the gap between the L4 and L5 vertebrae and extended to the subarachniod space of the lumbar enlargement (L4 and L5 segments) under Chloral Hydrate (300 mg/kg, intraperitoneal (i.p.)) anesthesia. The catheter was filled with sterile normal saline (NS, approximately 4 μl), and the outer end was plugged. The cannulated rats were allowed to recover for 3–4 days and were housed individually. Rats that showed any neurological deficits resulting from the surgical procedure were excluded from the experiments. Drug or vehicle was injected over a period of 1 min via the catheter at a volume of 10 μl, followed by 5 μl NS for flushing. The location of the distal end of the i.t. catheter was verified at the end of each experiment by the injection of Pontamine Sky Blue via the i.t. catheter. Induction of monoarthritis Monoarthritis was induced by an intra-articular injection of complete Freund's adjuvant (CFA). The rat was briefly anesthetized with isoflurane. The skin around the site of injection was sterilized with 75% alcohol. The left foot of the rat was held and the fossa of the lateral malleolus of the fibula was located. A 28-gauge needle was inserted vertically to penetrate the skin, and turned distally to insert into the articular cavity from the gap between the tibiofibular and tarsus bone until a distinct loss of resistance was felt. A volume of 50 μl CFA was then injected. Sham monoarthritis control animals were similarly injected with sterile NS. Drugs Fluorocitrate (0.1 or 1 nmol in 10 μl; Sigma), a reversible glial metabolic inhibitor, acts by inhibiting aconitase, an enzyme of the Krebs energy cycle of glia but not neurons (Paulsen et al., 1987; Hassel et al., 1992). Fluorocitrate was dissolved initially in 2 M HCl and then diluted in sterile 10 mM phosphate buffered saline (PBS) to attain a final concentration, pH 6.0 at the time of testing.
Materials and methods Electroacupuncture treatment Animals Experiments were performed on adult male Sprague– Dawley rats (Experimental Animal Center, Shanghai Medical College of Fudan University, China) weighing 200–250 g. Prior to experimental manipulation, rats were allowed to acclimate to the housing facilities for 1 week and maintained on a 12:12 h light–dark cycle and a constant room temperature of 21°C with free access to food and water. All experimental protocols and animal handling procedures were approved by Animal Care and Use Committee of Fudan University, and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize the number of animals used and their suffering.
Rats were loosely immobilized in a specially made restrainer with the head, hind legs, and tail protruding. A pair of stainless steel pins of 0.34 mm in diameter were inserted with a depth of 5 mm into the left (ipsilateral to CFA-injected joint) “Huantiao” (GB30, located the lateral 1/3 and medial 2/3 of the distance between the sacral hiatus and the greater trochanter of femur), and “Yanglingquan” (GB34, located in the depression anterior and inferior to the fibula capitulum) acupuncture points (Fig. 1). The two pins were connected with the output terminals of the H.A.N.S. Acupuncture point Nerve Stimulator (LH-202H, Huawei Co., Ltd., Beijing, China). The electroacupuncture parameters were set as follows: square wave current output (pulse width: 0.2 ms); intensities ranging from 1–2–3 mA (each intensity for 10 min, totaling 30 min); at a 2 Hz and
296
S. Sun et al. / Experimental Neurology 198 (2006) 294–302
intensity, which produced a stable withdrawal latency of approximately 10–12 s in the absence of arthritis. A 20 s cutoff was used to prevent tissue damage in the absence of a response. Both hind paws were tested independently with 10min interval between trials. von Frey test for mechanical allodynia
Fig. 1. Illustration of the acupuncture points in human and rats. Panels a and a′ present Huantiao (GB 30), and panels b and b′ present Yanglingquan (GB 34) points in human and rats, respectively.
100 Hz alternating frequencies (automatically shifting between 100 Hz and 2 Hz stimulation for 3 s each) (Tang et al., 1997). Sham electroacupuncture control animals received needle insertion into GB30 and GB34 without manipulation.
The hind paw withdrawal threshold (PWT) was determined using a calibrated series of von Frey hairs (Stoelting, IL, USA) ranging from 1 to 26 g. Animals were placed individually into Plexiglass chamber with customized platform that contains 1.5 mm diameter holes in a 5 mm grid of perpendicular rows throughout the entire area of the platform (Pitcher et al., 1999). The protocol used in this study was a variation of that described by Takaishi et al. (1996). After acclimation to the test chamber, a series of 9 calibrated von Frey hairs were applied to the central region of the plantar surface of one hind paw in ascending order (1, 1.4, 2, 4, 6, 8, 10, 15, and 26 g) with the lowest hair at 1 g. A particular hair was applied until buckling of the hair occurred. This was maintained for approximately 2 s. The hair was applied only when the rat was stationary and standing on all four paws. A withdrawal response was considered valid only if the hind paw was completely removed from the customized platform. A trial consisted of application of a von Frey hair to the hind paw five times at 15-s intervals. If withdrawal did not occur during five applications of a particular hair, the next larger hair in the series was applied in a similar manner. When the hind paw was withdrawn from a particular hair in three out of the five consecutive applications, the value of that hair in grams was considered to be the withdrawal threshold. Once the threshold was determined for one hind paw, the same testing procedure was repeated on the other hind paw after 5 min. All behavioral tests were performed in a temperaturecontrolled room (22 ± 2°C). The experimenter was blind with respect to treatments.
Measurement of ankle perimeter Statistical analysis The perimeters of ankles were measured around the lower edge of lateral and medial malleoli with a scaled soft ruler without elasticity. Hargreave's test for thermal hyperalgesia After acclimation to the test chamber, thermal hyperalgesia was assessed by measuring the latency of paw withdrawal in response to a radiant heat source. Rats were placed individually into Plexiglas chamber on an elevated glass platform, under which a radiant heat source (model 336 combination unit, IITC/life Science Instruments, Woodland Hill, CA, USA) was applied to the glabrous surface of the paw through the glass plate. The heat source was turned off when the rat lifted the foot, allowing the measurement of time from onset of radiant heat application to withdrawal of the rat's hind paw. This time was defined as the hind paw withdrawal latency (PWL). The heat was maintained at a constant
Data were expressed as mean ± SEM. Prearthritis baseline measures were analyzed by one-way analysis of variance (ANOVA). Pre- and postdrug time course measures for both hyperalgesia and allodynia were analyzed by two-way ANOVA (treatment × time) followed by Newman–Keuls post hoc test. The differences in joint circumference between before and after intra-articular injection were determined with paired t test. P b 0.05 was considered statistically significant. Experimental procedures Experiment 1: effects of intrathecal fluorocitrate on monoarthritis-induced thermal hyperalgesia and mechanical allodynia After baseline behavioral assessments, rats received an intra-articular injection of either CFA (50 μl) or equivolume sterile NS. Behavior was assessed 48 h later to confirm
S. Sun et al. / Experimental Neurology 198 (2006) 294–302
297
development of thermal hyperalgesia and mechanical allodynia in the CFA-injected rats. Directly after testing at 48 h, rats received an intrathecal injection of either fluorocitrate (0.1 or 1 nmol) or vehicle (0.3% 2 M HCl in PBS, pH 6.0). Hargreave's and von Frey tests were then performed 15, 30, 45, 60, 90 min, and 2–20 h later. Experiment 2: inhibition of monoarthritis-induced thermal hyperalgesia and mechanical allodynia by electroacupuncture After baseline behavioral assessments, intra-articular CFA was performed as above. Following testing at 48 h, electroacupuncture or sham electroacupuncture stimulation (needle insertion without manipulation) was applied to ipsilateral (arthritis) hind limb at “GB30” and “GB34” acupuncture point for 30 min. Behavior was then assessed 15, 30, 45, 60, 90 min, and 2–20 h after cessation of electroacupuncture stimulation. Experiment 3: enhancement of electroacupuncture analgesia by intrathecal fluorocitrate in monoarthritis rats After baseline behavioral assessments, intra-articular CFA was performed as above. Following testing at 48 h, rats received an intrathecal injection of fluorocitrate (0.1 or 1 nmol). Two hours and thirty minutes after intrathecal fluorocitrate, rats received electroacupuncture stimulation for 30 min. Behavior was then assessed at the same time-point as experiment 2. Results General results Baseline measures of the paw withdrawal latency (PWL) to radiant heat stimulation and the paw withdrawal threshold (PWT) to von Frey hairs did not differ across groups (one-way ANOVA, PWL: F9,60 = 1.245, P N 0.05; PWT: F9,60 = 0.541, P N 0.05). Following unilateral intra-articular injection of CFA, joint inflammation (edema and erythema), thermal hyperalgesia, and mechanical allodynia developed at 4 h and persisted for more than 3 days in the ipsilateral hind paw (Figs. 2A, B). The PWLs to thermal stimuli and PWTs to von Frey hairs in contralateral hind paw to monoarthritis remained stable within 3 days after CFA injection. The joint circumference for the ipsilateral ankle joint increased significantly on day 2 after CFA compared to before CFA injection (paired t test, P b 0.01). There were no significant differences in contralateral intact and NSinjected ankles between before and after intra-articular injection (Fig. 2C). Inhibition of fluorocitrate on thermal hyperalgesia and mechanical allodynia in CFA-induced monoarthritic rats The effects of i.t. injection of fluorocitrate (0.1 or 1 nmol), a glia metabolic inhibitor, on the PWLs to thermal stimuli and PWTs to von Frey hairs were examined on day 2 after intraarticular injection of CFA when the arthritic features stably occurred. I.t. injection of vehicle and low dose fluorocitrate
Fig. 2. Three variables measured before and after induction of the CFA model of rat monoarthritis. (A, B) Unilateral intra-articular injection of CFA induced significant thermal hyperalgesia (A) and mechanical allodynia (B). (C) Injection of CFA into the unilateral articular cavity produced a significant increase in circumference of the ipsilateral ankle joint that is measured 2 days after injection. *P b 0.05 and **P b 0.01 vs. before intra-articular injection. MA, monoarthritis; ipsi, ipsilateral; contra, contralateral; CFA, complete Freund's adjuvant.
(0.1 nmol) had no effect on PWLs and PWTs in monoarthritic rats. At the dose of 1 nmol, fluorocitrate significantly reduced the CFA-induced thermal hyperalgesia and mechanical allodynia in ipsilateral paw from 3 to 12 h after i.t. injection (Fig. 3). Two-way ANOVA analysis revealed significant effect of i.t. fluorocitrate (1 nmol) treatment (F1,192 = 516.56, P b 0.01) and significant interaction between fluorocitrate treatment and time (F15,192 = 43.67, P b 0.01). I.t. injection of 1 nmol fluorocitrate had no effect on the PWLs and PWTs of contralateral intact hind paw to monoarthritis (data not shown) and sham monoarthritic hind paw (Fig. 3).
298
S. Sun et al. / Experimental Neurology 198 (2006) 294–302
electrode into the acupuncture points (sham electroacupuncture) in monoarthritic rats (Fig. 4). Fluorocitrate and electroacupuncture synergistically inhibited monoarthritis-induced thermal hyperalgesia and mechanical allodynia The preceding results showed that neither PWLs nor PWTs were influenced by i.t. injection of 0.1 nmol fluorocitrate in monoarthritic rats. When a combination of 0.1 nmol fluorocitrate with electroacupuncture was given, both PWLs and PWTs of ipsilateral hind paw to monoarthritis had a robust increase in magnitude and duration, compared with the effects obtained in either the electroacupuncture or 0.1 nmol fluorocitrate alone group (PWL: F2,288 = 1485.39, P b 0.01; PWT: F2,288 = 1475.17, P b 0.01) (Figs. 4A, B). Notably, the PWLs to thermal stimuli even exceeded baseline before CFA injection within 1 h after electroacupuncture when co-applied with fluorocitrate (Fig. 4A). The effects of co-applied 1 nmol fluorocitrate and electroacupuncture on monoarthritis-induced thermal hyperalgesia and mechanical allodynia were similar to those of 0.1 nmol fluorocitrate combined with electroacupuncture group (PWL: F1,192 = 75.87, P b 0.01; PWT: F1,192 = 0.708, P N 0.05) (Fig. 4). A significant interaction between treatments and time was also found (PWL: F30,288 = 46.84, P b 0.01; PWT: F30,288 = 16.11, P b 0.01). In contralateral intact hind paw, PWLs and PWTs were also significantly increased following coapplication of 1 nmol fluorocitrate and electroacupuncture (Figs. 4C, D). Fig. 3. Intrathecal fluorocitrate inhibited monoarthritis-induced thermal hyperalgesia (A) and mechanical allodynia (B) in ipsilateral hind paw to monoarthritis, but not altered the PWLs and PWTs in sham monoarthritic rats. Fluorocitrate or vehicle was given 2 days after intra-articular injection of CFA. **P b 0.01 vs. vehicle control. Fc, fluorocitrate; MA, monoarthritis.
Inhibition of electroacupuncture on monoarthritis-induced thermal hyperalgesia and mechanical allodynia On day 2 after unilateral intra-articular injection of CFA, electroacupuncture stimulation applied to the unilateral hind limb (ipsilateral to CFA injection) at the “GB 30” and “GB 34” acupuncture points markedly inhibited pain hypersensitivity of the rats. The PWLs and PWTs of ipsilateral hind paw to monoarthritis significantly increased from 15 to 60 min after cessation of electroacupuncture stimulation (Figs. 4A, B). Twoway ANOVA analysis revealed significant effect of electroacupuncture treatment (PWL: F1,160 = 97.27, P b 0.01; PWT: F1,160 = 230.48, P b 0.01) and significant interaction between electroacupuncture treatment and time (PWL: F15,160 = 51.33, P b 0.01; PWT: F15,160 = 89.99, P b 0.01). At the time of the maximal electroacupuncture analgesic effect, 15 min, PWL and PWT of ipsilateral hind paw to monoarthritis were increased by 144.85% and 280.95%, respectively. Also, electroacupuncture stimulation produced a transient increase in the PWLs of contralateral intact hind paw to monoarthritis, whereas PWTs were not significantly influenced (Figs. 4C, D). Neither PWLs nor PWTs were affected by just inserting the stimulation
Discussion An important finding in the present study is that coapplication of electroacupuncture with intrathecal injection of a glial metabolic inhibitor fluorocitrate synergistically antagonized rat monoarthritic pain. Consistent with previous reports (Sluka et al., 1998; Zhang et al., 2002; Lao et al., 2004), electroacupuncture produced a robust but brief (60 min) inhibitory effect on CFA-induced thermal hyperalgesia and mechanical allodynia. Low dose (0.1 nmol) of fluorocitrate alone did not alter PWLs to thermal stimuli and PWTs to von Frey hairs in CFA-induced monoarthritic rats. However, a combination of low dose fluorocitrate and electroacupuncture completely reversed intra-articular injection of CFA-induced thermal hyperalgesia and mechanical allodynia for 12 h, suggesting a significant inter-reinforcing of electroacupuncture and disrupting spinal glial function. Involvement of spinal glia in inflammatory pain Glial cells were first considered as contributing to exaggerated “pain” by Garrison et al. (1994). They found that manipulation that created exaggerated “pain” also activated astrocytes, and the drug that blocked exaggerated “pain” also blocked astrocytic activation. Since then, glial (both microglia and astrocytes) activation has been observed at the lumbar spinal cord in various animal models of pathological pain,
S. Sun et al. / Experimental Neurology 198 (2006) 294–302
299
Fig. 4. Co-application of electroacupuncture with an intrathecal injection of fluorocitrate completely reversed monoarthritis-induced thermal hyperalgesia (A) and mechanical allodynia (B) in ipsilateral hind paw, and briefly increased the PWLs and PWTs of contralateral hind paw (C, D). Electroacupuncture was given 2.5 h after intrathecal fluorocitrate, when they are co-applied. *P b 0.05, **P b 0.01 vs. sham electroacupuncture; ++P b 0.01 vs. electroacupuncture alone; ##P b 0.01 vs. 1 nmol fluorocitrate plus electroacupuncture. Fc, fluorocitrate; EA, electroacupuncture.
including inflammation and damage of peripheral tissues, peripheral nerves, spinal nerves, and spinal cord, as well as chronic opioid treatment (Sweitzer et al., 1999, 2001; Raghavendra et al., 2002; Milligan et al., 2003; Ledeboer et al., 2005). Activated microglia and astrocytes release a variety of the neuron- and glial-excitatory substances, including reactive oxygen species, nitric oxide, prostaglandin, and excitatory amino acids. As resident immune cells in the CNS, activated glia also produce and release pro-inflammatory cytokines interleukin-1, interleukin-6, and tumor necrosis factor (Liu et al., 2004; Watkins and Maier, 2002; WieselerFrank et al., 2004). The substances from activated glia importantly contribute to the amplification of pain (Raghavendra et al., 2003; Sommer, 2003; Watkins and Maier, 2003). Blocking the activation of spinal glia markedly inhibits inflammation- or injury-induced release of glial- and neuroexcitatory substances, as well as the resultant exaggerated “pain” state (Milligan et al., 2000, 2001, 2003; Tikka et al., 2001; Ledeboer et al., 2005). In agreement with the previous studies on different models (Watkins et al., 1997, 1999; Raghavendra et al., 2003; Milligan et al., 2001, 2004), the present study showed that i.t. injection of 1 nmol fluorocitrate
reversibly suppressed thermal hyperalgesia and mechanical allodynia evoked by intra-articular CFA, whereas there was no significant increase in the PWLs and PWTs of contralateral intact hind paw to monoarthritis and sham monoarthritic hind paw. This result further supports the conclusion that glia is critically involved in the mediation of “exaggerated pain” evoked by inflammation but not acute pain. Synergetic inhibition of electroacupuncture and glial dysfunction on inflammatory pain Acupuncture is an important component of Chinese traditional medicine. Numerous studies have shown that acupuncture stimulation increases experimental pain threshold in various pain models and animal species (Sluka et al., 1998, 1999; Ma and Sluka, 2001). The commonly held theory for the mechanisms of electroacupuncture analgesia is activation of endogenous opioid system. The studies from Han's group demonstrated that low frequency (2 Hz) electroacupuncture accelerated the release of endomorphine, enkephalin, and βendorphin, and could be blocked by pretreatment with μ- and δopioid receptor antagonists, whereas high frequency (100 Hz)
300
S. Sun et al. / Experimental Neurology 198 (2006) 294–302
electroacupuncture produced the release of dynorphin, and could be blocked by κ-opioid receptor antagonists (Han et al., 1991; Han, 2003; Ulett et al., 1998; Wang et al., 2005). Given a dense-spare frequency (2 Hz/100 Hz alternation) of electroacupuncture stimulation used in the present study, all four kinds of opioid peptides release together and their receptors might be activated to a greater extent. It has been demonstrated that all three classes of opioid receptors are located on primary nociceptive afferent terminals and on the dendrites of pain sensitive neurons in the dorsal horn of the spinal cord. Presynaptic activation of opioid receptors on the primary afferent terminals results in a direct reduction of Ca2+ conductance or indirect increase in K+ conductance, and in turn decreases release of transmitters such as excitatory amino acids and substance P (Dickenson and Sullivan, 1986; Malmberg and Yaksh, 1995; Ulett et al., 1998; Cheunsuang et al., 2002). Several studies have shown that glial cells in culture express the μ-, κ-, and δ-opioid receptors, and respond to opioid stimulation by changing their morphology and proliferation rate in vitro (Ruzica et al., 1996; Ruzica and Akil, 1997; Tryoen et al., 2000). If this case occurred in the spinal glia in vivo, it is possible that electroacupuncture acts directly or indirectly on glial cells, triggering alterations in their metabolism, morphology, and functions via opioid peptides and opioid receptors. On the other hand, electroacupuncture has been found to modulate the function of the immune system and alleviated inflammatory pain (Sun et al., 2000; Ceccherelli et al., 2002; Hahm et al., 2004; Zhang et al., 2004). In the CNS, one cellular target of immune system activation is the glia. Evidence is accumulating that acupuncture inhibits activation of glia and decreases the levels of pro-inflammatory cytokine. Son et al. (2002) reported that electroacupuncture efficiently suppressed lipopolysaccharide-induced up-regulation of interleukin-6 and interleukin-1β mRNA levels in hypothalamus. Also, the middle cerebral artery occlusion-induced increase in interleukin-1β mRNA in ischemic cortex, the medial forebrain bundle axotomy-induced activation of microglia in substantia nigra, and resultant increase in the levels of tumor necrosis factor and interleukin-1β mRNA were significantly blocked by electroacupuncture (Xu et al., 2002; Liu et al., 2004). Thus, fluorocitrate synergized electroacupuncture, blocking activation of spinal glia, reducing release of pain enhancing substances in the spinal dorsal horn, and disrupting neuron-to-glia-to-neuron excitatory circuit, thereby leading to a robust inhibition on monoarthritis-induced thermal hyperalgesia and mechanical allodynia. In addition, the release of supraspinal opioid, supraspinal and spinal serotonin, and noradrenalin as well as the activation of brainstem endogenous descending inhibitory system induced by electroacupuncture (Kalra et al., 2001) may also contribute to the synergetic antinociceptive effect of electroacupuncture with glial metabolic inhibitor. It is worthy of mention that PWLs of ipsilateral paw to monoarthritis even went beyond that of normal rats by almost 40% when fluorocitrate and electroacupuncture were given together; suggesting that combination of electroacupuncture and fluorocitrate might interfere with normal sensory physiology. This presume was further supported by the fact that
co-application of the two treatments prolonged PWLs of contralateral intact hind paw or sham monoarthritic hind paw, although fluorocitrate alone could not affect them. Clinically, electroacupuncture induces only a moderate degree of analgesia on arthritis, and high dose fluorocitrate should not be administered because of neurotoxicity. The present results that co-application of electroacupuncture with low dose glial metabolic inhibitor synergistically antagonized monoarthritic pain may provide a potential strategy for the treatment of arthritis.
Acknowledgments The authors wish to thank Dr. Judith Strong for her critical reading of the manuscript and helpful criticism. We also thank Mr. Zhou Dong for his drawing assistance. This work was supported by the National Natural Science Fund of China (NSFC) (30330230, 30570594), NSFC for Distinguished Young Scholars (30425022), National Basic Research Program of China (2006CB500807), and Science Foundation of Shanghai Municipal Commission of Science and Technology (02DZ19150-4).
References Bao, L., Zhu, Y., Elhassan, A.M., Wu, Q., Xiao, B., Zhu, J., Undgren, J.U., 2001. Adjuvant-induced arthritis: IL-1β, IL-6 and TNF-α are up-regulated in the spinal cord. NeuroReport 12, 3905–3908. Berman, B.M., Swyers, J.P., Ezzo, J., 2000. The evidence for acupuncture as a treatment for rheumatologic conditions. Rheum. Dis. Clin. North Am. 26, 103–115. Campbell, S.M., 1988. Rheumatiod arthritis: current strategies. Hosp. Med. 34, 29–32. Ceccherelli, F., Gagliardi, G., Ruzzante, L., Giron, G., 2002. Acupuncture modulation of capsaicin-induced inflammation: effect of intraperitoneal and local administration of naloxone in rats. A blinded controlled study. J. Altern. Complement. Med. 8, 341–349. Cheunsuang, O., Maxwell, D., Morris, R., 2002. Spinal lamina I neurons that express neurokinin 1 receptors: II. Electrophysiological characteristics, responses to primary afferent stimulation and effects of a selective muopioid receptor agonist. Neuroscience 111, 423–434. Colburn, R.W., Rickman, A.J., Deleo, J.A., 1999. The effect of site and type of nerve injury on spinal glial activation and neuropathic pain behavior. Exp. Neurol. 157, 289–304. Colpaert, F.C., 1987. Evidence that adjuvant arthritis in the rat is associated with chronic pain. Pain 28, 201–222. Dickenson, A.H., Sullivan, A.F., 1986. Electrophysiological studies on the effects of intrathecal morphine on nociceptive neurons in the rat dorsal horn. Pain 24, 211–222. Ezzo, J., Hadhazy, V., Birch, S., 2001. Acupuncture for osteoarthritis of the knee: a systematic review. Arthritis Rheum. 44, 819–825. Garrison, C.J., Dougherty, P.M., Carlton, S.M., 1994. GFAP expression in lumbar spinal cord of naive and neuropathic rats treated with MK-801. Exp. Neurol. 129, 237–243. Guan, Z., Zhang, L., 1995. Effects of acupuncture on immunoglobulins in patients with asthma and rheumatoid arthritis. J. Tradit. Chin. Med. 15, 102–105. Hahm, E.T., Lee, J.J., Lee, W.K., Bae, H.S., Min, B.I., Cho, Y.W., 2004. Electroacupuncture enhancement of natural killer cell activity suppressed by anterior hypothalamic lesions in rats. Neuroimmunomodulation 11, 268–272.
S. Sun et al. / Experimental Neurology 198 (2006) 294–302 Han, J.S., 2003. Acupuncture: neuropeptide release produced by electrical stimulation of different frequencies. Trends Neurosci. 26, 17–22. Han, J.S., Chen, X.H., Sun, S.L., Xu, X.J., Yuan, Y., Yan, S.C., Hao, J.X., Terenius, L., 1991. Effect of low- and high-frequency TENS on Metenkephalin-Arg-Phe and dynorphin immunoreactivity in human lumbar CSF. Pain 47, 295–298. Hashizume, H., Deleo, J.A., Colburn, R.W., Weinstein, J.N., 2000. Spinal glial activation and cytokine expression after lumbar root injury in the rat. Spine 25, 1206–1217. Hassel, B., Paulsen, R.E., Johnson, A., Fonnum, F., 1992. Selective inhibition of glial cell metabolism by fluorocitrate. Brain Res. 249, 120–124. Kalra, A., Urban, M.O., Sluka, K.A., 2001. Blockade of opioid receptors in rostral ventral medulla prevents antihyperalgesia produced by transcutaneous electrical nerve stimulation (TENS). J. Pharmacol. Exp. Ther. 298, 257–263. Lao, L., Zhang, R.X., Zhang, G., Wang, X., Berman, B.M., Ren, K., 2004. A parametric study of electroacupuncture on persistent hyperalgesia and Fos protein expression in rats. Brain Res. 1020, 18–29. Ledeboer, A., Sloane, E.M., Milligan, E.D., Frank, M.G., Mahony, J.H., Maier, S.F., Watkins, L.R., 2005. Minocycline attenuates mechanical allodynia and proinflammatory cytokine expression in rat models of pain facilitation. Pain 115, 71–83. Liu, X.Y., Zhou, H.F., Pan, Y.L., Liang, X.B., Niu, D.B., Xue, B., Li, F.Q., He, Q.H., Wang, X.H., Wang, X.M., 2004. Electro-acupuncture stimulation protects dopaminergic neurons from inflammation-mediated damage in medial forebrain bundle-transected rats. Exp. Neurol. 189, 189–196. Ma, Y.T., Sluka, K.A., 2001. Reduction in inflammation-induced sensitization of dorsal horn neurons by transcutaneous electrical nerve stimulation in anesthetized rats. Exp. Brain Res. 137, 94–102. Malmberg, A.B., Yaksh, T.L., 1995. The effect of morphine on formalin-evoked behavior and spinal release of excitatory amino acids and prostaglandin E2 using microdialysis in conscious rats. Br. J. Pharmacol. 114, 1069–1075. McBean, G.J., Doorty, K.B., Tipton, K.F., Kollegger, H., 1995. Alternation in the glial cell metabolism of glutamate by kainite and N-methyl-D-aspartate. Toxicon 33, 569–576. Meller, S.T., Dykstra, C., Gebhart, G.F., 1992. Production of endogenous nitric oxide and activation of soluble guanylate cyclase are required for N-methylD-aspartate-produced facilitation of the nociceptive tail-flick reflex. Eur. J. Pharmacol. 214, 93–96. Milligan, E.D., Mehmert, K.K., Hinde, J.L., Harvey, L.O.J., Martin, D., Tracey, K.J., Maier, S.F., Watkins, L.R., 2000. Thermal hyperalgesia and mechanical allodynia produced by intrathecal administration of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein, gp120. Brain Res. 861, 105–116. Milligan, E.D., O'Connor, K.A., Nguyen, K.T., Armstrong, C.B., Twining, C., Gaykema, R.P., Holguin, A., Martin, D., Maier, S.F., Watkins, L.R., 2001. Intrathecal HIV-1 envelope glycoprotein gp120 induces enhanced pain states mediated by spinal cord proinflammatory cytokines. J. Neurosci. 21, 2808–2819. Milligan, E.D., Twining, C., Chacur, M., Biedenkapp, J., O'Connor, K., Poole, S., Tracey, K., Martin, D., Maier, S.F., Watkins, L.R., 2003. Spinal glia and proinflammatory cytokines mediate mirror-image neuropathic pain in rats. J. Neurosci. 23, 1026–1040. Milligan, E.D., Zapata, V., Chacur, M., Schoeniger, D., Biedenkapp, J., O'Connor, K.A., Verge, G.M., Chapman, G., Green, P., Foster, A.C., Naeve, G.S., Maier, S.F., Watkins, L.R., 2004. Evidence that exogenous and endogenous fractalkine can induce spinal nociceptive facilitation in rats. Eur. J. Neurosci. 20, 2294–2302. Paini, D., Frei, K., Pfister, H.W., Fontana, A., 1993. Glutamate uptake by astrocytes is inhibited by reactive oxygen intermediates but not by other macrophage-derived molecules including cytokines, leukotrienes or plateletactivating factor. J. Neuroimmunol. 48, 153–156. Parpura, V., Basarsky, T.A., Liu, F., Jeftinija, K., Jeftinija, S., Haydon, P.G., 1994. Glutamate-mediated astrocytes-neuron signaling. Nature 369, 744–747. Paulsen, R.E., Contestabile, A., Villani, L., Fonnum, F., 1987. An in vivo model for studying function of brain tissue temporarily devoid of glial cell metabolism: the use of fluorocitrate. J. Neurochem. 48, 1377–1385.
301
Pincus, T., Marcum, S.B., Callahan, L.F., 1992. Long-term drug therapy for rheumatoid arthritis in seven rheumatology private practices: II. Second line drugs and prednisone. J. Rheumatol. 19, 1885–1894. Pitcher, G.M., Ritchie, J., Henry, J.L., 1999. Paw withdrawal threshold in the von Frey hair test is influenced by the surface on which the rat stands. J. Neurosci. Method 87, 185–193. Raghavendra, V., Rutkowski, M.D., Deleo, J.A., 2002. The role of spinal neuroimmune activation in morphine tolerance/hyperalgesia in neuropathic and sham-operated rats. J. Neurosci. 22, 9980–9989. Raghavendra, V., Tanga, F., Deleo, J.A., 2003. Inhibition of microglial activation attenuates the development but not existing hypersensitivity in a rat model of neuropathy. J. Pharmacol. Exp. Ther. 306, 624–630. Ruzica, B.B., Akil, H., 1997. The interleukin-1-beta-mediated regulation of proenkephalin and opioid receptor messenger RNA in primary astrocytes enriched cultures. Neuroscience 19, 517–524. Ruzica, B.B., Thompson, R.C., Watson, S.J., Akil, H., 1996. Interleukin-1-betamediated regulation of mu-opioid receptor messenger RNA in primary astrocytes enriched cultures. Neurochemistry 66, 425–428. Sluka, K.A., Westund, K.N., 1993a. Behavioral and immunohistochemical changes in an experimental arthritis model in rats. Pain 55, 367–377. Sluka, K.A., Westund, K.N., 1993b. Centrally administered non-NMDA but not NMDA receptor antagonists block peripheral knee joint inflammation. Pain 55, 217–225. Sluka, K.A., Bailey, K., Bogush, J., Olson, R., Ricketts, A., 1998. Treatment with either high or low frequency TENS reduces the secondary hyperalgesia observed after injection of kaolin and Carrageenan into the knee joint. Pain 77, 97–102. Sluka, K.A., Deacon, M., Stibal, A., Strissel, S., Terpstra, A., 1999. Spinal blockade of opioid receptors the analgesia produced by TENS in arthritic rats. J. Pharmacol. Exp. Ther. 289, 840–846. Sommer, C., 2003. Painful neuropathies. Curr. Opin. Neurol. 16, 623–628. Son, Y.S., Park, H.J., Kwon, O.B., Jung, S.C., Shin, H.C., Lim, S., 2002. Antipyretic effects of acupuncture on the lipopolysaccharide-induced fever and expression of interleukin-6 and interleukin-1 beta mRNAs in the hypothalamus of rats. Neurosci. Lett. 319, 45–48. Sun, T., Du, L.N., Wu, G.C., Cao, X.D., 2000. Effect of intrathecal morphine and electroacupuncture on cellular immune function of rats and increment of mu-opioid receptor mRNA expression in PAG following intrathecal morphine. Acupunct. Electro-ther. Res. 25, 1–8. Sweitzer, S.M., Colburn, R.W., Rutkowski, M., Deleo, J.A., 1999. Acute peripheral inflammation induces moderate glial activation and spinal IL-1β expression that correlates with pain behavior in the rat. Brain Res. Int. 829, 209–221. Sweitzer, S.M., Schubert, P., Deleo, J.A., 2001. Propentofyline, a glial modulating agent, exhibits antiallodynia properties in a rat model of neuropathic pain. J. Pharmacol. Exp. Ther. 297, 1210–1217. Takaishi, K., Eisele Jr., J.H., Carstens, E., 1996. Behavioral and electrophysiological assessment of hyperalgesia and changes in dorsal horn responses following partial sciatic nerve ligation in rats. Pain 66, 297–306. Tang, N.M., Dong, H.W., Wang, X.M., Tsui, Z.C., Han, J.S., 1997. Cholecystokinin antisense RNA increases the analgesic effect induced by electroacupuncture or low dose morphine: conversion of low responder rats into high responders. Pain 71, 71–80. Tanga, F.Y., Raghavendra, V., Deleo, J.A., 2004. Quantitative real-time RT-PCR assessment of spinal microglial and astrocytic activation markers in a rat model of neuropathic pain. Neurochem. Int. 45, 397–407. Tikka, T., Fiebich, B.L., Goldsteins, G., Keinanen, R., Koistinaho, J., 2001. Minocycline, a tetracycline derivative, a neuroprotective against excitotoxicity by inhibiting activation and proliferation of microglia. J. Neurosci. 21, 2580–2588. Tryoen, P., Gaveraux, C., Labourdette, G., 2000. Down-regulation of mu-opioid receptor expression in rat oligodendrocytes during their development in vitro. J. Neurosci. Res. 60, 10–20. Twining, C.M., Sloane, E.M., Schoeniger, D.K., Milligan, E.D., Martin, D., Marsh, H., Maier, S.F., Watkins, L.R., 2005. Activation of the spinal cord complement cascade might contribute to mechanical allodynia induced by three animal models of spinal sensitization. J. Pain 6, 174–183.
302
S. Sun et al. / Experimental Neurology 198 (2006) 294–302
Ulett, G.A., Han, S., Han, J.S., 1998. Electroacupuncture: mechanisms and clinical application. Biol. Psychiatry 44, 129–138. Wang, Y., Zhang, Y., Wang, W., Cao, Y., Han, J.S., 2005. Effects of synchronous or asynchronous electroacupuncture stimulation with low versus high frequency on spinal opioid release and tail flick nociception. Exp. Neurol. 192, 156–162. Watkins, L.R., Maier, S.F., 2002. Beyond neurons: evidence that immune and glial cells contribute to pathological pain states. Physiol. Rev. 82, 981–1011. Watkins, L.R., Maier, S.F., 2003. Glia: a novel drug discovery target for clinical pain. Nat. Rev., Drug Discov. 2, 973–985. Watkins, L.R., Martin, D., Ulrich, P., Tracey, K.J., Maier, S.F., 1997. Evidence for the involvement of spinal cord glia in subcutaneous formalin induced hyperalgesia in the rat. Pain 71, 225–235. Watkins, L.R., Hansen, M.K., Nguyen, K.T., Lee, J.E., Maier, S.F., 1999. Dynamic regulation of the proinflammatory cytokine, interleukin-1β: molecular biology for non-molecular biologists. Life Sic. 65, 449–481.
Watkins, L.R., Milligan, E.D., Maier, S.F., 2001. Glial activation: a driving force for pathological pain. Trends Neurosci. 24, 450–455. Wieseler-Frank, J., Maier, S.F., Watkins, L.R., 2004. Glial activation and pathological pain. Neurochem. Int. 45, 389–395. Xu, Z.F., Wu, G.C., Cao, X.D., 2002. Effect of electroacupuncture on the expression of interleukin-1 beta mRNA after transient focal cerebral ischemia. Acupunct. Electro-ther. Res. 27, 29–35. Zhang, Y.Q., Ji, G.C., Wu, G.C., Zhao, Z.Q., 2002. Excitatory amino acid receptor antagonists and electroacupuncture synergistically inhibit Carrageenan-induced behavioral hyperalgesia and spinal fos expression in rats. Pain 99, 525–535. Zhang, Y.Q., Ji, G.C., Wu, G.C., Zhao, Z.Q., 2003. Kynurenic acid enhances electroacupuncture analgesia in normal and Carrageenan-injected rats. Brain Res. 966, 300–307. Zhang, S.P., Zhang, J.S., Yung, K.K., Zhang, H.Q., 2004. Non-opioiddependent anti-inflammatory effects of low frequency electroacupuncture. Brain Res. Bull. 62, 327–334.