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0022-3565/99/2892-0840$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics JPET 289:840 –846, 1999 Vol. 289, No. 2 Printed in U.S.A. Spinal Blockade of Opioid Receptors Prevents the Analgesia Produced by TENS in Arthritic Rats1 KATHLEEN A. SLUKA, MEREK DEACON, ANDREA STIBAL, SHANNON STRISSEL, and AMY TERPSTRA Physical Therapy Graduate Program (M.D., A.S., S.S., A.T.) and Neuroscience Graduate Program (K.A.S.), The University of Iowa, Iowa City, Iowa Accepted for publication December 2, 1998 This paper is available online at http://www.jpet.org One noninvasive treatment commonly used to manage arthritic pain is transcutaneous electrical nerve stimulation (TENS). Studies have shown that TENS reduces pain in people with rheumatoid and osteoarthritis (Manheimer et al., 1978; Manheimer and Carlsson, 1979; Kumar and Redford, 1982). Although studies have demonstrated the effectiveness of TENS for reducing pain in people with arthritis, the physiological mechanism by which TENS produces analgesia is unknown. Two different theories have been proposed. The most popular theory for the mechanism of action of TENS is the gate control theory of pain (Melzack and Wall, 1965; Kumar and Redford, 1982; Garrison and Foreman, 1994; Hollman and Morgan, 1997). This theory proposes that stimulation of large-diameter afferent fibers inhibits secondorder neurons in the dorsal horn and prevents pain impulses carried by small-diameter fibers from reaching higher brain centers. Received for publication October 20, 1998. 1 This study was supported by grants from the Central Investment Fund for Research Enhancement from the University of Iowa and the Arthritis Foundation. as a control) administration, and after drug (or artificial cerebral spinal fluid) administration 1 TENS. Either high- (100 Hz) or low- frequency (4 Hz) TENS produced approximately 100% inhibition of hyperalgesia. Low doses of naloxone, selective for m opioid receptors, blocked the antihyperalgesia produced by low-frequency TENS. High doses of naloxone, which also block d and k opioid receptors, prevented the antihyperalgesia produced by high-frequency TENS. Spinal blockade of d opioid receptors dose-dependently prevented the antihyperalgesia produced by high-frequency TENS. In contrast, blockade of k opioid receptors had no effect on the antihyperalgesia produced by either low- or high-frequency TENS. Thus, low-frequency TENS produces antihyperalgesia through m opioid receptors and high-frequency TENS produces antihyperalgesia through d opioid receptors in the spinal cord. The second explanation for the mechanism of action of TENS is that it stimulates the release of endogenous opioids. Naloxone, an opioid receptor antagonist, blocks the analgesia produced by low-frequency electroacupuncture (,10 Hz), suggesting it works through the release of endorphins (Mayer et al., 1977; Woolf et al., 1977; Cheng and Pomeranz, 1979; Ha et al., 1981). Fox and Melzack (1976) compared the use of TENS and acupuncture in the treatment of lower back pain and concluded they have the same underlying mechanism of action. Others have demonstrated an increased content of opioid peptides in the cerebrospinal fluid in humans after administration of TENS (Salar et al., 1981; Hughes et al., 1984; Almay et al., 1985; Han et al., 1991). Several studies indicate that high- (.10 Hz) and low- (,10 Hz) frequency TENS work through different mechanisms. Abram et al. (1981) investigated the role of opioids in analgesia produced by high-frequency TENS. Specifically, no reversal of analgesia was seen after administration of naloxone, suggesting to the authors that high-frequency TENS does not work through the release of opioids. High-frequency TENS is, therefore, believed to work through mechanisms ABBREVIATIONS: TENS, transcutaneous electrical nerve stimulation; PWL, paw withdrawal latency; ACSF, artificial cerebral spinal fluid; MEAP, Met-enkephalin-Arg-Phe; NSAID: non-steroidal anti-inflammatory. 840 Downloaded from jpet.aspetjournals.org at ASPET Journals on May 17, 2016 ABSTRACT Transcutaneous electrical nerve stimulation (TENS) is commonly used for relief of pain. The literature on the clinical application of TENS is extensive. However, surprisingly few reports have addressed the neurophysiological basis for the actions of TENS. The gate control theory of pain is typically used to explain the actions of high-frequency TENS, whereas, low-frequency TENS is typically explained by release of endogenous opioids. The current study investigated the role of m, d, and k opioid receptors in antihyperalgesia produced by lowand high-frequency TENS by using an animal model of inflammation. Antagonists to m (naloxone), d (naltrinodole), or k (norbinaltorphimine) opioid receptors were delivered to the spinal cord by microdialysis. Joint inflammation was induced by injection of kaolin and carrageenan into the knee-joint cavity. Withdrawal latency to heat was assessed before inflammation, during inflammation, after drug (or artificial cerebral spinal fluid 1999 TENS and Opioids Materials and Methods Placement of the Microdialysis Fiber All experiments were approved by the animal care and use committee at our institution and are in accordance with National Institutes of Health guidelines. Male Sprague-Dawley rats (250 –350 g; n 5 122) were implanted with a microdialysis fiber in the dorsal horn (L4–L6 spinal level) for delivery of drugs to the spinal cord (Sluka and Westlund, 1992). A microdialysis fiber (Hospal AN69 with a cutoff of 45 kDa) was prepared by marking a 2-mm gap and then applying an epoxy coating to the remaining length of the fiber. This allowed diffusion of the drug to occur only in the 2-mm gap to be positioned in the dorsal horn of the spinal cord. Rats were initially anesthetized with sodium pentobarbital (50 mg/kg i.p.) for placement of the microdialysis fiber. A hole was drilled just under the lip of the pedicle on both sides of the T13 spinal segment with a manual drill. The prepared microdialysis fiber was then threaded through the holes. Polyethylene (PE 20) tubing was secured to both ends of the fiber with super glue gel and epoxy. The fiber was positioned so that the 2-mm section was in the dorsal horn of the spinal cord and then secured in place with dental cement. The PE 20 tubing was then sutured to the fascia to prevent any unnecessary movement. Staples were used to close the incision and the rat was then placed in its cage for recovery overnight. The next day, rats were divided into the following treatment groups: 1) Artificial cerebrospinal fluid (ACSF) and no TENS treatment (n 5 6) control; 2) ACSF 1 low- (n 5 8) or high-frequency (n 5 6) TENS; 3) Naloxone hydrochloride (Sigma Chemical Co., St. Louis, MO; 1.0 –10.0 mM) 1 low- (1 mM, n 5 3; 5 mM, n 5 5; 10 mM, n 5 7) or high-frequency (1 mM, n 5 3; 5 mM, n 5 6; 10 mM, n 5 6) TENS; 4) Naltrinodole hydrochloride (Sigma Chemical Co.; 0.01–1.0 mM) 1 low- (1 mM; n 5 6) or high-frequency (0.01 mM, n 5 5; 0.1mM, n 5 3; 1 mM, n 5 6) TENS; or 5) nor-binaltorphimine (nor-BNI; Research Biochemicals International, Natick, MA; 0.01 mM) 1 low- (n 5 6) or high-frequency (n 5 7) TENS. All drugs were dissolved in ACSF and pH-corrected (7.2–7.4). Behavioral Testing and Treatment Protocol The day after implantation of the microdialysis fiber, withdrawal latencies of both hindpaws were determined according to the protocol described by Hargreaves et al. (1988). Rats were placed in clear plastic cages on an elevated glass plate and allowed to acclimate for 10 to 20 min. A radiant heat source was applied to the posterior plantar surface of the hindpaw and the time for the rat to withdraw its paw was measured. The light box had an on/off switch connected to a timer, which measured the duration of the paw withdrawal latency (PWL). If the PWL exceeded 20 s, the heat source was turned off to avoid tissue damage. The average of five trials for each paw was determined. The examiner was kept blinded to the treatment groups, both drug treatment and TENS treatment. The knee-joint circumferences were measured bilaterally with a flexible tape measure around the center of the fully extended knee. Injection of the Knee Joint. After baseline behavioral measurements, rats were anesthetized with 2 to 4% halothane via a face mask for approximately 5 min and a solution of 3% kaolin and 3% carrageenan (0.1 ml; pH 7.4) in sterile saline was injected into the left knee joint to induce inflammation (Sluka and Westlund, 1993). Four hours after the injection, the paw withdrawal responses to heat were tested as before. Spontaneous pain-related behaviors were rated on a scale from zero to five (0 5 normal, 1 5 curled toes, 2 5 everted foot, 3 5 partial weight bearing, 4 5 nonweight bearing, and 5 5 complete avoidance of limb by lying on side) (Sluka and Westlund, 1993). The rats were then given either a drug or ACSF for 1 h through the microdialysis fiber. After the 1-h infusion, PWL and spontaneous pain-related behaviors were assessed. TENS Treatment. Rats were then lightly anesthetized with halothane (1–2%, 20 min), their knee-joint circumferences were measured, and TENS was applied to the knee joint. Rats received either 1) low-frequency TENS at sensory intensity to the inflamed knee joint (4 Hz; 20 min.; EMPI Eclipse 1; EMPI, Inc., Minneapolis, MN), 2) high-frequency TENS at sensory intensity to the inflamed knee joint (100 Hz; 20 min.; EMPI Eclipse 1), or 3) halothane without TENS. One-inch round pregelled electrodes were placed on the medial and lateral aspects of the shaved knee joint. Sensory-intensity TENS was determined by increasing the intensity until a palpable muscle contraction was elicited and then reducing the intensity to just below that point. The study minimized variability of stimuli by maintaining a pulse duration constant at 100 ms and an intensity constant at sensory-level intensity (see Sluka et al., 1998). Thus, only the frequency of stimulation was varied. These parameters are based on those used clinically (see Robinson and Snyder-Mackler, 1995) and those previously published (Sluka et al., 1998). Immediately after TENS treatment, PWLs were determined and spontaneous pain-related behaviors were recorded. Finally, rats were anesthetized again to measure knee-joint circumferences. After the final measurements were taken, the rats were euthanized with an overdose of sodium pentobarbitol and the spinal cords were removed and dissected to verify the correct placement of the microdialysis fiber at the L4–L6 level of the spinal cord. Selectivity of Drugs. To test selectivity of naloxone to opioid receptors, 13 rats were implanted with microdialysis fibers. The m opioid receptor agonist DAMGO ([D-Ala2,N-Me-Phe4,Glyy-ol5]-enkephalin) (Research Biochemicals International; 1 mM; n 5 4), the d opioid receptor agonist SNC8O ((1)-4-[(aR)-a-((2S,5R)-4-allyl-2,5dimethyl-1-piperazinyl)-3-methoxybenzyl]-N, N-diethylbenzamide) (Tocris Cookson; 1 mM; n 5 4), or the k opioid receptor agonist U50,488 (trans-(1)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexy]benzenecetamide) (Tocris Cookson; 0.1 mM, n 5 5) was infused into the dorsal horn and PWL to radiant heat was tested. Naloxone (1, 5, or 10 mM) was tested for its ability to antagonize the analgesia produced by the opioid receptor agonists. Similarly, the selectivity of the d opioid receptor antagonist naltrinodole (n 5 14) and the k opioid receptor antagonist nor-BNI (n 5 13) was tested against DAMGO (1 mM), SNC8O (1 mM), or U50,488 (0.1 mM). The effects of 0.01, 0.1, and 1 mM naltrinodole and 0.1, 1, and 10 mM nor-BNI were tested against the opioid agonists. Statistical Analysis To minimize variability between groups, data were assessed for the percentage of inhibition by TENS for PWL with the following formula: (TENS or drug 2 arthritis)/(base 2 arthritis) 3 100. Thus, 100% inhibition is a full reversal of hyperalgesia and 0% inhibition is no change from the hyperalgesia measured 4 h after induction of Downloaded from jpet.aspetjournals.org at ASPET Journals on May 17, 2016 proposed by the gate control theory, producing only shortterm analgesia (Garrison and Foreman, 1994; Hollman and Morgan, 1997). Conversely, low-frequency TENS is proposed to work through release of endogenous opioids, which causes a more systemic and long-term response (Sjound and Eriksson, 1979). Some studies, however, have demonstrated that high-frequency TENS has a longer lasting effect than lowfrequency TENS (Manheimer and Carlsson, 1979; Walsh et al., 1995; Gopalkrishnan and Sluka, 1998; Sluka et al., 1998). Furthermore, Woolf et al. (1977) demonstrated that high doses of naloxone block the analgesia produced by highfrequency TENS in rats. The different mechanisms by which high- and low-frequency TENS works still remain unclear. In response to the conflicting results of previous studies and the lack of research on the mechanisms through which TENS works, this study investigated the spinal mechanisms through which low- and high-frequency TENS exert their antihyperalgesic effects. We hypothesized that low-frequency TENS activates endogenous opioid receptors in the spinal cord. 841 842 Sluka et al. Vol. 298 inflammation. The group effect of TENS and the group effect of drug on the percentage of inhibition of hyperalgesia were assessed by an ANOVA (p , .05). Post hoc tests were done with independent t tests for assessing differences between groups. TABLE 2 Spontaneous pain-related behavior ratings for control arthritic rats and those receiving TENS treatment. Ratings were based on a scale from zero to five, with zero being normal and five representing total avoidance of the inflammed limb. Values are the median and range. Results TABLE 1 Joint circumference measurements (cm) for control arthritic animals and those receiving either high- or low-frequency sensory TENS treatment. Values represent mean 6 S.E.M. Inflamed knee ACSF, no TENS ACSF 1 low frequency TENS ACSF 1 high frequency TENS 10 mM naloxone 1 low TENS 5 mM naloxone 1 low TENS 1 mM naloxone 1 low TENS 10 mM naloxone 1 high TENS 5 mM naloxone 1 high TENS 1 mM naloxone 1 high TENS 1 mM naltrinodole 1 low TENS 1 mM naltrinodole 1 high TENS 0.1 mM naltrinodole 1 high TENS 0.01 mM naltrinodole 1 high TENS 10 mM nor BNI 1 low TENS 10 mM nor BNI 1 high TENS Contralateral knee ACSF, no TENS ACSF 1 low frequency TENS ACSF 1 high frequency TENS 10 mM naloxone 1 low TENS 5 mM naloxone 1 low TENS 1 mM naloxone 1 low TENS 10 mM naloxone 1 high TENS 5 mM naloxone 1 high TENS 1 mM naloxone 1 high TENS 1 mM naltrinodole 1 low TENS 1 mM naltrinodole 1 high TENS 0.1 mM naltrinodole 1 high TENS 0.01 mM naltrinodole 1 high TENS 10 mM nor BNI 1 low TENS 10 mM nor BNI 1 high Baseline 4 h after Arthritis After TENS cm cm cm 5.5 6 0.9 5.6 6 .07 5.5 6 .08 5.8 6 .18 5.7 6 .29 5.9 6 .22 6.0 6 .26 5.8 6 .08 5.6 6 0.1 5.9 6 .03 5.9 6 .07 5.7 6 .03 6.9 6 .21 6.7 6 .12 6.6 6 .16 7.0 6 .26 6.7 6 .14 7.2 6 .15 7.3 6 .21 7.2 6 .15 7.3 6 .33 7.2 6 .11 7.4 6 .23 6.8 6 .15 6.9 6 .23 6.7 6 .16 6.7 6 .16 7.0 6 .22 7.1 6 .22 7.6 6 .13 7.4 6 .25 7.1 6 .16 7.2 6 .20 7.5 6 .08 7.4 6 .19 7.0 6 .21 5.6 6 .07 6.9 6 .16 6.9 6 .15 6.0 6 .06 5.7 6 .13 7.5 6 .08 7.1 6 .10 7.5 6 .09 7.1 6 .07 5.6 6 .09 5.6 6 .10 6.0 6 .22 5.6 6 .08 5.7 6 .19 5.7 6 .29 5.9 6 .25 5.8 6 .09 5.6 6 .07 5.9 6 .08 6.0 6 .08 5.7 6 .07 5.6 6 .10 5.6 6 .09 6.0 6 .23 5.6 6 .08 5.7 6 .19 5.8 6 .33 6.1 6 .23 5.8 6 .13 5.6 6 .09 5.8 6 .03 5.9 6 .06 5.6 6 .03 5.6 6 .11 5.6 6 .07 6.0 6 .22 5.5 6 .07 5.8 6 .19 5.8 6 .32 6.0 6 .28 5.8 6 .12 5.5 6 .06 5.8 6 .04 5.9 6 .06 5.7 6 .03 5.6 6 .08 5.6 6 .09 5.6 6 .06 6.1 6 .04 5.6 6 .06 6.0 6 .02 5.6 6 .08 6.1 6 .03 5.6 6 .04 ACSF, no TENS ACSF 1 low frequency TENS ACSF 1 high frequency TENS 10 mM naloxone 1 low TENS 5 mM naloxone 1 low TENS 1 mM naloxone 1 low TENS 10 mM naloxone 1 high TENS 5 mM naloxone 1 high TENS 1 mM naloxone 1 high TENS 1 mM naltrinodole 1 low TENS 1 mM naltrinodole 1 high TENS 0.1 mM naltrinodole 1 high TENS 0.01 mM naltrinodole 1 high TENS 10 mM nor BNI 1 low TENS 10 mM nor BNI 1 high TENS 1 h after Drug After TENS 4 (3–5) 4 (3–4) 4.5 (4–5) 4 (3–5) 4 (3–5) 3 (3–4) 4 (3–4) 4 (3–5) 4 (3–4) 4 (3–5) 4 (3–5) 4 (4) 4 (3–4) 4 (4) 4 (2–4) 4.5 (3–5) 4 (2–5) 4.5 (4–5) 4 (3–4) 5 (3–5) 4 (3–4) 4 (4–5) 4 (4–5) 4 (3–4) 4 (3–5) 4 (3–5) 4 (4) 4 (2–4) 4 (4–5) 4 (3–4) 4 (3–4) 3 (2–4) 4 (3–5) 4 (3–4) 4 (3–5) 4 (3–4) 4 (3–4) 4 (3–4) 4 (3–5) 4 (3–4) 4 (2–5) 4 (3–4) 4 (3–4) 4 (3–5) 4 (3–4) after induction of inflammation, to 8.3 6 0.26 s after treatment with TENS (baseline 5 9.0 6 0.75 s). Similarly, in the group of animals treated with high-frequency TENS, the PWL increased from 7.2 6 0.32 s, 4 h after induction of inflammation, to 10.1 6 0.42 s after treatment with TENS (baseline 5 9.8 6 0.31 s). There was an overall significant effect across time (F1,66 5 131.95; p 5 .001) for changes in PWL in all of the groups of animals. A significant effect for group by time occurred for the percentage of inhibition of hyperalgesia after treatment with TENS (F14,66 5 4.05; P 5 .001) but not after infusion of drug (or ACSF) alone (F14.66 5 1.75; p 5 .08). The PWL of the contralateral hindpaw remained unchanged after induction of inflammation in animals with ACSF or those treated with TENS or drug. For example, baseline PWL for the contralateral paw in control arthritic animals treated only with ACSF was 9.3 6 0.655 s, and 4 h after inflammation the PWL remained at 8.7 6 0.655 s. After treatment with either high- or low-frequency TENS, the contralateral PWL was 10.5 6 0.53 s and 9.3 6 0.55 s, respectively, compared with baseline values of 9.8 6 0.53 s and 9.6 6 0.56 s. Effects of Naloxone on TENS Analgesia. Spinal infusion of 1 mM naloxone had no effect on the inhibition of hyperalgesia produced by either high- or low-frequency TENS; the percentage of inhibition of hyperalgesia remained at approximately 100%. However, 5 and 10 mM naloxone prevented the inhibition of hyperalgesia by low- frequency TENS (Fig. 1). Thus, there was still a decrease in the PWL to radiant heat after TENS treatment similar to that observed 4 h after induction of inflammation. Only 10 mM naloxone blocked the inhibition of hyperalgesia produced by high-frequency TENS (Fig. 2). To test selectivity of naloxone for different opioid receptors, naloxone was tested against agonists selective for m (DAMGO), d (SNC80), or k (U50,488) opioid receptors. As Fig. 3A shows, all three agonists produced analgesia as indicated by the significant increase in PWL (p , .05). After administration of 1 mM naloxone, the PWL remained significantly increased. After administration of 5 mM naloxone, only the group receiving DAMGO (m agonist) returned to the baseline. Downloaded from jpet.aspetjournals.org at ASPET Journals on May 17, 2016 Control Arthritic Animals and Effect of TENS. Four hours after induction of arthritis, there was a significant decrease in PWL to radiant heat that was maintained throughout the testing period. There was also an increase in joint circumference and an increase in spontaneous painrelated behavior ratings 4 h after inflammation. Changes in joint circumference and spontaneous pain behavior ratings are given in Tables 1 and 2, respectively. There were no significant differences between groups for joint circumference or spontaneous pain-related behaviors at any time period (baseline, 4 h after inflammation, after administration of a drug or ACSF, or after TENS). ACSF had no effect on the decreased withdrawal latency normally observed after joint inflammation (Fig. 1). In the group of animals treated only with ACSF, the withdrawal latency decreased from 9.0 6 0.22 s to 7.2 6 0.24 s, 4 h after induction of arthritis. In contrast, treatment with either high- or low-frequency TENS produced approximately 100% reversal of the hyperalgesia (Figs. 1 and 2). In the group of animals treated with lowfrequency TENS, the PWL increased from 6.7 6 0.32 s, 4 h 4h Arthritis 1999 TENS and Opioids 843 Fig. 2. The percentage of inhibition of hyperalgesia is represented After administration of drug or ACSF (E) or treatment with TENS 1 drug or ACSF (F) in the group of animals treated with high-frequency TENS. A, inhibition of hyperalgesia after treatment with ACSF (n 5 6), highfrequency TENS (TENS, n 5 6), or naloxone at 1 (n 5 3), 5 (n 5 6), or 10 mM (n 5 6). The percentage of analgesia was significantly increased after treatment with TENS (p 5 .001) when compared with animals treated with ACSF and no TENS. Treatment with 10mM (p 5 .0001) naloxone significantly prevented the analgesia produced by high-frequency TENS. B, inhibition of hyperalgesia after treatment with 10 mM nor-BNI (n 5 7) or 1mM naltrinodole (n 5 6). A significant blockade of the inhibition of hyperalgesia was observed After treatment with 1 mM naltrinodole (p 5 .002) when compared with treatment with high-frequency TENS 1ACSF. Inset, dose response effect after administration of .01 (n 5 5), .1 (n 5 3), or 1 naltrinodole (n 5 6). A significant inhibition of hyperalgesia occurred in the group treated with .1 (p 5 .03) and 1 mM (p 5 .002). *, significantly different from TENS group. Values are mean 6 S.E.M. 2). The percentage of inhibition of hyperalgesia was similar to that observed in animals treated with ACSF and TENS and was not significantly different from that group. The selectivity of nor-BNI was tested against agonists to m (DAMGO), d (SNC80), and k (U50,488) opioid receptors. As Fig. 3C shows, spinal infusion of 10 mM nor-BNI selectively blocks k opioid receptors. The analgesia produced by spinal infusion of DAMGO or SNC80 was unaffected by nor-BNI. Fig. 1. The percentage of inhibition of hyperalgesia is represented after administration of drug or ACSF (E) or treatment with TENS 1 drug or ACSF (F) in the group of animals treated with low-frequency TENS. A, inhibition of hyperalgesia after treatment with ACSF (n 5 6), low-frequency TENS (TENS, n 5 8), or naloxone at 1 (n 5 3), 5 (n 5 5), or 10 mM (n 5 7). The percentage of analgesia was significantly increased after treatment with TENS (p 5 .009) when compared with animals treated with ACSF and no TENS. Treatment with 5 (p 5 .01) or 10mM (p 5 .02) naloxone significantly prevented the analgesia produced by low-frequency TENS. B, inhibition of hyperalgesia after treatment with 10 mM nor-BNI (n 5 6) or 1mM naltrinodole (n 5 6). No significant difference was observed between the group treated with ACSF 1 low-frequency TENS and those treated with nor-BNI or naltrinodole. *, significantly different from TENS group. Values are mean 6 S.E.M. Discussion The current study supports the theory that TENS works through release of endogenous opioids at the spinal cord level. Spinal administration of 5 mM naloxone, which selectively blocks m opioid receptors, significantly reduced the antihyperalgesic effects of low-frequency TENS. A greater, nonselective dose of naloxone (10 mM) reduced the antihyperalgesia produced by high-frequency TENS, suggesting the involvement of endogenous opioids acting at d or k opioid Downloaded from jpet.aspetjournals.org at ASPET Journals on May 17, 2016 Therefore, at a dose of 5 mM, naloxone selectively blocks m receptors but not k or d opioid receptors. After increasing the concentration of naloxone to 10 mM, all of the groups’ PWL returned to the baseline, indicating all opioid receptors (m, d, and k) were blocked at this dose. Effects of Naltrinodole on TENS Analgesia. Blockade of d opioid receptors with 1 mM naltrinodole prevented the inhibition of hyperalgesia produced by high-frequency but not low-frequency TENS (Figs. 1 and 2). The effects of naltrinodole on preventing the inhibition of hyperalgesia by high-frequency TENS were dose-dependent (Fig. 2, inset). The selectivity of naltrinodole was tested against agonists to m (DAMGO), d (SNC80), and k (U50,488) opioid receptors. Figure 3B demonstrates that 1 mM naltrinodole selectively blocks d opioid receptors. The analgesia produced by spinal infusion of DAMGO or U50,488 was unaffected by naltrinodole. Effects of nor-BNI on TENS Analgesia. Blockade of k opioid receptors with nor-BNI had no effect on the analgesia produced by either high- or low-frequency TENS (Figs. 1 and 844 Sluka et al. receptors, or both. The antihyperalgesic effect of high-frequency TENS was reduced by blockade of d opioid receptors but not k opioid receptors. Thus, the current study demonstrated that the analgesia produced by high-frequency sensory TENS is mediated by d opioid receptors spinally, and that produced by low-frequency sensory TENS is mediated by m opioid receptors spinally. Previous studies support the conclusion that both highand low-frequency TENS result in the release of endogenous opioids. Increased b-endorphin concentrations in the cerebral spinal fluid were observed after administration of either high- or low-frequency TENS (Salar et al., 1981; Hughes et al., 1984; Almay et al., 1985). Han et al. (1991) analyzed the opioid peptides Met-enkephalin-Arg-Phe (MEAP) and dynorphin A in the cerebral spinal fluid of human subjects after application of either high- or low-frequency TENS. They found that high-frequency stimulation produced an increase in dynorphin A but not in MEAP, whereas low-frequency TENS increased MEAP but not dynorphin A. Although similar frequencies of stimulation were used by Han et al. (1991; 2 and 100 Hz), the intensity of stimulation was greater, eliciting a motor contraction in the subjects. Previously, we demonstrated that increasing intensity of stimulation resulted in an increase in inhibition of hyperalgesia in carrageenan-inflamed rats (Gopalkrishnan and Sluka, 1998). Similarly, Garrison and Foreman (1996) showed an increased inhibition of responses to noxious stimuli with increased intensity of stimulation when recording from unsensitized dorsal horn neurons. Differences between the studies, thus, could be explained by differences in intensity of stimulation. Several studies have demonstrated that acupuncture-induced analgesia is reduced by naloxone in normal subjects and a variety of patient populations (Mayer et al., 1977; Ha et al., 1981; Homma et al., 1985; Eriksson et al., 1991). Similarly, low-frequency, high-intensity electroacupuncture suppresses responses of dorsal horn neurons to noxious stimuli and this suppression is reversed by naloxone (Pomeranz and Cheng, 1979). Sjolund and Eriksson (1979) demonstrated that analgesia produced by low-frequency, high-intensity TENS but not high-frequency, low-intensity TENS is reversible by administration of naloxone systemically. The dose used by Sjolund and Eriksson (1979) was at a concentration expected to block m opioid receptors. In contrast, high-frequency TENS was unaffected by systemic naloxone in patient populations (Abram et al., 1981; Freeman et al., 1983). However, analgesia induced in rats by high-frequency TENS was reversed by high doses of systemic naloxone expected to block m, d, and k opioid receptors (Woolf et al., 1977; Han et al., 1984). The release of endogenous opioids in the spinal cord in response to TENS stimulation could result from activation of local circuits within the spinal cord or from activation of descending inhibitory pathways. Opioid peptides, enkephalin, and dynorphin are contained in spinal dorsal horn neurons (Hokfelt et al., 1977; Glazer and Basbaum, 1981). Likewise, m and d opioid receptors have been localized to the dorsal horn, both presynaptically on primary afferent fibers and postsynaptically on dorsal horn neurons (LaMotte et al., 1976; Atweh and Kuhar, 1983; Cheng et al., 1997). By using immunohistochemistry, Zhang et al. (1998) demonstrated that small dorsal root ganglia neurons labeled for the d opioid receptor also contain Substance P and calcitonin gene-related peptide. Further spinal localization of the d receptor was reduced by dorsal rhizotomy, suggesting presynaptic localization on primary afferents. Release of the primary afferent peptides, Substance P and calcitonin gene-related peptide, is blocked by opioid agonists (Yaksh et al., 1980; Downloaded from jpet.aspetjournals.org at ASPET Journals on May 17, 2016 Fig. 3. Bar graphs representing the selectivity of opioid receptor antagonists to the agonists. The paw withdrawal latency was measured before (base) and after spinal infusion of opioid agonists (drug), and then agonist plus increasing doses of the antagonists. A, effects of naloxone at reducing the increase in PWL induced by DAMGO (n 5 4) to activate m receptors; SNC80 (n 5 4) to activate d receptors, and U50,488 (n 5 5) to activate k receptors. A significant reversal of the analgesia produced by DAMGO occurred after spinal infusion of 5 and 10 mM naloxone. 10mM naloxone also reversed the analgesia produced by SNC80 and U50,488. B, effects of increasing doses of naltrinodole on the increased paw withdrawal latency produced by DAMGO (n 5 4), SNC80 (n 5 5), and U50,488 (n 5 5). A significant reversal of the analgesia by SNC80 was produced with spinal infusion of 1mM naltrinodole. C, effects of increasing doses of nor-BNI on the increased paw withdrawal latency produced by DAMGO (n 5 4), SNC80 (n 5 4), and U50,488 (n 5 4). A significant reversal of the analgesia produced by U50,488 occurred with spinal infusion of 10 mM nor-BNI. *p , .05, significantly decreased from infusion of agonist. Values are mean 6 S.E.M. Vol. 298 1999 845 ation of mood, mental clouding, and increased tolerance to the drug with continued use. Thus, the use of TENS in conjunction with opioids could lower the intake of drugs and limit side effects. Herrero and Headley (1996) found that naloxone blocks the antinociceptive effects of the nonsteroidal anti-inflammatory drug (NSAID) flunixin in rats with an inflamed paw. They concluded that naloxone acts as a noncompetitive antagonist to flunixin and that spinal antinociception caused by the NSAID was mediated via release of endogenous opioid peptides. This implies that TENS could be an effective alternative to NSAIDs as an analgesic when used in conditions of acute inflammation. Furthermore, if a patient is taking NSAIDs, TENS may be less effective. Conclusions. Both high- and low-frequency TENS at sensory intensity reverse the hyperalgesia produced by kneejoint inflammation. The antihyperalgesic effects of low-frequency TENS are reversed by spinal administration of low doses of naloxone that are selective for m opioid receptors. However, the antihyperalgesia produced by high-frequency TENS is prevented by blockade of d opioid receptors in the spinal cord. Thus, low-frequency TENS works through activation of m opioid receptors and high-frequency TENS works through activation of d opioid receptors. Acknowledgments We thank Dr. G. F. Gebhart for critically reading the manuscript and EMPI for providing the TENS units. References Abram S, Reynolds A and Cusick J (1981) Failure of naloxone to reverse analgesia from transcutaneous electrical stimulation in patients with chronic pain. Anesth Analg 60:81– 84. Almay BGL, Johansson F, Knorring L, Sakurada T and Terenius L (1985) Long-term high frequency transcutaneous electrical nerve stimulation (hi-TNS) in chronic pain. Clinical response and effects on CSF-endorphins, monoamine metabolites, substance P-like immunoreactivity (SPLI) and pain measures. J Psychosom Res 29:247–257. Atweh SF and Kuhar MJ (1983) Distribution and physiological significance of opioid receptors in the brain. Br Med Bull 39:47–52. Basbaum AI and Fields HL (1984) Endogenous pain control systems: Brainstem spinal pathways and endorphin circuitry. Ann Rev Neurosci 7:309 –338. Besson JM and Chaouch A (1987) Peripheral and spinal mechanisms of nociception. Physiol Rev 67:67–186. Bourgoin S, Benoliel JJ, Collin E, Mauborgne A, Pohl M, Hamon M and Cesselin F (1994) Opioidergic control of the spinal release of neuropeptides. Possible significance for the analgesic effects of opioids. Fundam Clin Pharmacol 8:307–321. Chang HM, Berde CB, Holz GG 4th, Steward GF and Kream RM (1989) Sufentanil, morphine, met-enkephalin, and kappa-agonist (U-50,488H) inhibit substance P release from primary sensory neurons: A model for presynaptic spinal opioid actions. Anesthesiology 70:672– 677. Cheng PY, Moriwaki A, Wang JB, Uhl GR and Pickel VM (1996) Ultrastructural localization of d opioid receptors in the superficial layers of the rat cervical spinal cord: Extrasynaptic localization and proximity to Leu5-enkephalin. Brain Res 731:141–154. Collin E, Frechilla D, Pohl M, Bourgoin S, LeBars D, Hamon M and Cesselin F (1993) Opioid control of the release of calcitonin gene-related peptide-like material from the rat spinal cord in vivo. Brain Res 609:211–222. Collin E, Mauborgne A, Bourgoin S, Chantrel D, Hamon M and Cesselin F (1991) In vivo tonic inhibition of spinal substance P (-like material) release by endogenous opioid(s) acting at delta receptors. Neuroscience 44:725–731. Cross S (1994) Pathophysiology of pain. Mayo Clin Proc 69:375–383. Eriksson SV, Lundeberg T and Lundeberg S (1991) Interaction of diazepam and naloxone on acupuncture induced pain relief. Am J Chin Med 19:1–7. Fields HL, Barbaro NM and Heinricher MM (1988) Brain stem neuronal circuitry underlying the antinociceptive action of opiates. Prog Brain Res 77:245–257. Fox E and Melzack R (1976) Transcutaneous electrical stimulation and acupuncture: Comparison of treatment for low back pain. Pain 2:141–148. Freeman TB, Campbell JN and Long DM (1983) Naloxone does not affect pain relief induced by electrical stimulation in man. Pain 17:189 –195. Garrison DW and Foreman RD (1994) Decreased activity of spontaneous and noxiously evoked dorsal horn cells during transcutaneous electrical nerve stimulation. Pain 58:309 –315. Garrison DW and Foreman RD (1996) Effects of prolonged transcutaneous electrical nerve stimulation (TENS) and variation of stimulation variables on dorsal horn cell activity in cats. Eur J Phys Med Rehab 6:87–94. Glazer EJ and Basbaum AI (1981) Immunohistochemical localization of leucine- Downloaded from jpet.aspetjournals.org at ASPET Journals on May 17, 2016 Collin et al., 1991; Collin et al., 1993; Bourgoin et al., 1994), suggesting a role for opioid receptors in presynaptic neurotransmitter release. Specifically, activation of m and d opioid receptors inhibits the release of Substance P and calcitonin gene-related peptide (Hirota et al., 1985; Chang et al., 1989; Collin et al., 1991; Ray et al., 1991; Yonehara et al., 1992). Furthermore, opioids applied directly to the spinal cord block behavioral responses in animals to a noxious stimulation and produce significant antinociception in humans (Lazorthes et al., 1988; Hammond et al., 1998). For example, intrathecal administration of agonists to either m or d receptors reduces the behavioral response to formalin injection in rats (Hammond et al., 1998). Wang et al. (1996) demonstrated that m, d1, and d2 receptor agonists inhibit activity of trigeminal dorsal horn neurons to A d and C-fiber stimulation. Thus, the effects of TENS may be to reduce the release of neurotransmitters from primary afferent terminals in the dorsal horn and/or to reduce activity of dorsal horn neurons through the release of endogenous opioids. Opioid release in the dorsal horn can also occur through activation of descending inhibitory pathways. The opioid peptides and their receptors are distributed throughout the central nervous system in areas involved in pain transmission including the spinal cord, medullary and pontine nuclei, midbrain, amygdala, hypothalmus, thalamus, and cortex (Mansour et al., 1988; Basbaum and Fields, 1984). Descending projections originate in the reticular formation in the brainstem, the ventral portion of the periaqueductal gray in the midbrain, and the rostral ventral medulla (Besson and Chaouch, 1987; Basbaum and Fields, 1996). All of these nuclei are involved in descending inhibition of the dorsal horn neurons either by direct descending fibers or by intermediary brainstem structures (Fields et al., 1988; Cross, 1994; Basbaum and Fields, 1984). Descending raphe spinal axons exert their antinociceptive effect through spinal opioid receptors because intrathecal injection of naloxone blocks the analgesia produced by stimulation of the raphe nucleus (Zorman et al., 1982). Support for a role of descending systems in TENS comes from work on acupuncture analgesia. Zhou et al. (1981) injected small amounts of naloxone into different brain areas to assess its effect on acupuncture analgesia. They concluded that the nuclei accumbens, amygdala, habenula, and periaqueductal gray are sites where acupuncture exerts its analgesic effect and that acupuncture involves the release of opioids at these sites. Clinical Implications. Knowledge of the mechanism of TENS will better enable clinicians to determine which patients will benefit from TENS treatment based on the type of medication the patient is currently taking for pain control. Solomon et al. (1980) demonstrated that patients who used opioids in amounts sufficient to produce tolerance also experienced tolerance to the effects of TENS. This finding implies that the mechanism of action of TENS involves the same neural substrate as opioid-induced analgesia. If the patient is taking opioids or has taken opioids in the past, TENS may not be the modality of choice to control pain. Solomon et al. (1980) also show that TENS could reduce the need for postoperative opioids in a group of patients who had not used opioid analgesics before the operation. This is significant because, in addition to providing analgesia, opioid drugs produce several unwanted side effects. These include respiratory depression, nausea and vomiting, constipation, alter- TENS and Opioids 846 Sluka et al. Pomeranz B and Cheng R (1979) Suppression of noxious responses in single neurons of cat spinal cord by electroacupuncture and its reversal by the opiate antagonist, naloxone. Exp Neurol 64:327–341. Ray NJ, Jones AJ and Keen P (1991) Morphine, but not sodium cromoglycate, modulates the release of substance P from capsaicin-sensitive neurones in rat trachea in vitro. Br J Pharmacol 102:797– 800. Robinson AJ and Snyder-Mackler L (1995) Clinical electrophysiology: Electrotherapy and electrophysiological testing. Williams & Wilkins, Baltimore. Salar G, Job I, Minigrino S, Bosio A and Trabucchi M (1981) Effect of transcutaneous electrotherapy on CSF beta-endorphin content in patients without pain problems. Pain 10:169 –172. Sjolund B and Eriksson M (1979) The influence of naloxone on analgesia produced by peripheral conditioning stimulation. Brain Res 173:295–301. Sluka KA, Bailey K, Bogush J, Olson R and 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 KA and Westlund KN (1992) An experimental arthritis in rats: Dorsal horn aspartate and glutamate increases. Neurosci Lett 145:141–144. Sluka K and Westlund K (1993) Behavioral and immunohistochemical changes in an experimental arthritis model in rats. Pain 55:367–377. Solomon RA, Viernstein MC and Long DM (1980) Reduction of postoperative pain and narcotic use by transcutaneous electrical nerve stimulation. Surgery 87:142– 146. Walsh DM, Liggett C, Baxter D and Allen JM (1995) A double-blind investigation of the hypoalgesic effects of transcutaneous electrical nerve stimulation upon experimentally induced ischaemic pain. Pain 61:39 – 45. Wang XM, Yan JQ, Zhang KM and Mokha SS (1996) Role of opioid receptors (mu, delta 1, delta 2) in modulating responses of nociceptive neurons in the superficial and deeper dorsal horn of the medulla (trigeminal nucleus caudalis) in the rat. Brain Res 739:235–243. Woolf CJ, Barrett D, Mitchell D and Myers R (1977) Naloxone-reversible peripheral electroanalgesia in intact and spinal rats. Eur J Pharmacol 45:311–314. Yaksh TL, Jessell TM, Gamse R, Mudge AW and Leeman SE (1980) Intrathecal morphine inhibits substance P release from mammalian spinal cord in vivo. Nature (London) 286:155–157. Yonehara N, Imai Y, Chen JQ, Takiuchi S and Inoki R (1992) Influence of opioids on substance P release evoked by antidromic stimulation of primary afferent fibers in the hind instep of rats. Regul Peptides 38:13–22. Zhang X, Bao L, Arvidsson U, Elde R and Hokfelt T (1998) Localization and regulation of the delta-opioid receptor in dorsal root ganglia and spinal cord of the rat and monkey: Evidence for association with the membrane of large dense-core vesicles. Neurosci 82:1225–1242. Zhou ZF, Du MY, Wu WY, Jiang Y and Han JS (1981) Effect of intracerebral microinjection of naloxone on acupuncture and morphine-analgesia in the rabbit. Sci Sin 24:1166 –1178. Zorman G, Belcher G, Adams JE and Fields HL (1982) Lumbar intrathecal naloxone blocks analgesia produced by microstimulation of the ventromedial medulla in the rat. Brain Res 236:77– 84. Send reprint requests to: Kathleen A. Sluka, P.T., Ph.D., Physical Therapy Graduate Program, The University of Iowa, 2600 Steindeler Bldg., Iowa City, IA 52242. E-mail: [email protected] Downloaded from jpet.aspetjournals.org at ASPET Journals on May 17, 2016 enkephalin in the spinal cord of the cat: Enkephalin-containing marginal neurons and pain modulation. J Comp Neurol 196:377–389. Gopalkrishnan P and Sluka KA (1998) TENS reduces primary hyperalgesia and dorsal horn neuron sensitization in rats (Abstract). Soc Neurosci Abstr 24:893 Ha H, Tan EC, Fukunaga H and Aochi O (1981) Naloxone reversal of acupuncture analgesia in the monkey. Exp Neurol 73:298 –303. Hammond DL, Wang H, Nakashima N and Basbaum AI (1998) Differential effects of intrathecally administered delta and mu opioid receptor agonists on formalinevoked nociception and on the expression of Fos-like immunoreactivity in the spinal cord of the rat. J Pharmacol Exp Ther 284:378 –387. Han JS, Chen XH, Sun SL, Xu XJ, Yuan Y, Yan SC, Hao JX and Terenius L (1991) Effect of low- and high-frequency TENS on Met-enkephalin-Arg-Phe and dynorphin A immunoreactivity in human lumbar CSF. Pain 47:295–298. Han JS, Xie GX, Ding ZX and Fan SG (1984) High and low frequency electroacupuncture analgesia are mediated by different opioid peptides. Pain 2(Suppl):543 Hargreaves K, Dubner R, Brown F, Flores C and Joris J (1988) A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32: 77– 88. Herrero JF and Headley PM (1996) Reversal by naloxone of the spinal antinociceptive actions of a systemically-administered NSAID. Br J Pharmacol 118:968 –972. Hirota, N, Kuraishi Y, Hino Y, Sato Y, Satoh, M and Takagi H (1985) Metenkephalin and morphine but not dynorphin inhibit noxious stimuli-induced release of substance P from rabbit dorsal horn in situ. Neuropharmacology 24:567– 570. Hökfelt T, Ljungdahl A, Terenius L, Elde R and Nilsson G (1977) Immunohistochemical analysis of peptide pathways possibly related to pain and analgesia: Enkephalin and substance P. Proc Natl Acad Sci USA 74:3081–3085. Hollman JE and Morgan BJ (1997) Effect of transcutaneous electrical nerve stimulation on the pressor response to static handgrip exercise. Phys Ther 77:28 –36. Homma S, Hori Y and Yonezawa T (1985) The antagonistic effects of naloxone on acupuncture inhibition of the vibration-induced grasp reflex in man. Neurosci Lett 61:227–232. Hughes G, Lichstein P, Whitlock D and Harker C (1984) Response of plasma beta-endorphins to transcutaneous electrical nerve stimulation in healthy subjects. Phys Ther 64:1062–1066. Kumar VN and Redford JB (1982) Transcutaneous nerve stimulation in rheumatoid arthritis. Arch Phys Med Rehab 63:595–596. LaMotte C, Pert CB and Snyder SH (1976) Opiate receptor binding in primate spinal cord. Distribution and changes after dorsal root section. Brain Res 112:407– 412. Lazorthes Y, Verdie JC, Caute B, Maranhao R and Tafani M (1988) Intracerebroventricular morphinotherapy for control of chronic cancer pain. Prog Brain Res 77:395– 405. Manheimer C and Carlsson CA (1979) The analgesic effect of transcutaneous electrical nerve stimulation (TNS) in patients with rheumatoid arthritis. A comparative study of different pulse patterns. Pain 6:329 –334. Manheimer C, Lund S and Carlsson CA (1978) The effect of transcutaneous electrical nerve stimulation (TNS) on joint pain in patients with rheumatoid arthritis. Scand J Rheumatol 7:13–16. Mayer D, Price D and Rafii A (1977) Antagonism of acupuncture analgesia in man by the narcotic antagonist naloxone. Brain Res 121:368 –372. Melzack R and Wall PD (1965) Pain mechanisms: A new theory. Science (Wash DC) 150:971–979. Mansour A, Khachaturian H, Lewis ME, Akil H and Watson SJ (1988) Anatomy of CNS opioid receptors Trends Neurosci 11:308 –314. Vol. 298