Spinal Blockade of Opioid Receptors Prevents the Analgesia Produced by TENS in Arthritic Rats 1

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1 /99/ $03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 289, No. 2 Copyright 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 289: , 1999 Spinal Blockade of Opioid Receptors Prevents the Analgesia Produced by in Arthritic Rats 1 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 ABSTRACT Transcutaneous electrical nerve stimulation () is commonly used for relief of pain. The literature on the clinical application of is extensive. However, surprisingly few reports have addressed the neurophysiological basis for the actions of. The gate control theory of pain is typically used to explain the actions of high-frequency, whereas, low-frequency is typically explained by release of endogenous opioids. The current study investigated the role of,, and opioid receptors in antihyperalgesia produced by lowand high-frequency by using an animal model of inflammation. Antagonists to (naloxone), (naltrinodole), or (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 One noninvasive treatment commonly used to manage arthritic pain is transcutaneous electrical nerve stimulation (). Studies have shown that 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 for reducing pain in people with arthritis, the physiological mechanism by which produces analgesia is unknown. Two different theories have been proposed. The most popular theory for the mechanism of action of 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, 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. Either high- (100 Hz) or low- frequency (4 Hz) produced approximately 100% inhibition of hyperalgesia. Low doses of naloxone, selective for opioid receptors, blocked the antihyperalgesia produced by low-frequency. High doses of naloxone, which also block and opioid receptors, prevented the antihyperalgesia produced by high-frequency. Spinal blockade of opioid receptors dose-dependently prevented the antihyperalgesia produced by high-frequency. In contrast, blockade of opioid receptors had no effect on the antihyperalgesia produced by either low- or high-frequency. Thus, low-frequency produces antihyperalgesia through opioid receptors and high-frequency produces antihyperalgesia through opioid receptors in the spinal cord. The second explanation for the mechanism of action of 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 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 (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 work through different mechanisms. Abram et al. (1981) investigated the role of opioids in analgesia produced by high-frequency. Specifically, no reversal of analgesia was seen after administration of naloxone, suggesting to the authors that high-frequency does not work through the release of opioids. High-frequency is, therefore, believed to work through mechanisms ABBREVIATIONS:, transcutaneous electrical nerve stimulation; PWL, paw withdrawal latency; ACSF, artificial cerebral spinal fluid; MEAP, Met-enkephalin-Arg-Phe; NSAID: non-steroidal anti-inflammatory. 840

2 1999 and Opioids 841 proposed by the gate control theory, producing only shortterm analgesia (Garrison and Foreman, 1994; Hollman and Morgan, 1997). Conversely, low-frequency 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 has a longer lasting effect than lowfrequency (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 in rats. The different mechanisms by which high- and low-frequency works still remain unclear. In response to the conflicting results of previous studies and the lack of research on the mechanisms through which works, this study investigated the spinal mechanisms through which low- and high-frequency exert their antihyperalgesic effects. We hypothesized that low-frequency activates endogenous opioid receptors in the spinal cord. 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 ( g; n 122) were implanted with a microdialysis fiber in the dorsal horn (L 4 L 6 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 T 13 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 treatment (n 6) control; 2) ACSF low- (n 8) or high-frequency (n 6) ; 3) Naloxone hydrochloride (Sigma Chemical Co., St. Louis, MO; mm) low- (1 mm, n 3; 5 mm, n 5; 10 mm, n 7) or high-frequency (1 mm, n 3; 5 mm, n 6; 10 mm, n 6) ; 4) Naltrinodole hydrochloride (Sigma Chemical Co.; mm) low- (1 mm; n 6) or high-frequency (0.01 mm, n 5; 0.1mM, n 3; 1 mm, n 6) ; or 5) nor-binaltorphimine (nor-bni; Research Biochemicals International, Natick, MA; 0.01 mm) low- (n 6) or high-frequency (n 7). All drugs were dissolved in ACSF and ph-corrected ( ). 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 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 normal, 1 curled toes, 2 everted foot, 3 partial weight bearing, 4 nonweight bearing, and 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. Treatment. Rats were then lightly anesthetized with halothane (1 2%, 20 min), their knee-joint circumferences were measured, and was applied to the knee joint. Rats received either 1) low-frequency at sensory intensity to the inflamed knee joint (4 Hz; 20 min.; EMPI Eclipse ; EMPI, Inc., Minneapolis, MN), 2) high-frequency at sensory intensity to the inflamed knee joint (100 Hz; 20 min.; EMPI Eclipse ), or 3) halothane without. One-inch round pregelled electrodes were placed on the medial and lateral aspects of the shaved knee joint. Sensory-intensity 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 s 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 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 L 4 L 6 level of the spinal cord. Selectivity of Drugs. To test selectivity of naloxone to opioid receptors, 13 rats were implanted with microdialysis fibers. The opioid receptor agonist DAMGO ([D-Ala 2,N-Me-Phe 4,Glyy-ol 5 ]-enkephalin) (Research Biochemicals International; 1 mm; n 4), the opioid receptor agonist SNC8O (( )-4-[( R)- -((2S,5R)-4-allyl-2,5- dimethyl-1-piperazinyl)-3-methoxybenzyl]-n, N-diethylbenzamide) (Tocris Cookson; 1 mm; n 4), or the opioid receptor agonist U50,488 (trans-( )-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexy]benzenecetamide) (Tocris Cookson; 0.1 mm, n 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 opioid receptor antagonist naltrinodole (n 14) and the opioid receptor antagonist nor-bni (n 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 M nor-bni were tested against the opioid agonists. Statistical Analysis To minimize variability between groups, data were assessed for the percentage of inhibition by for PWL with the following formula: ( or drug arthritis)/(base arthritis) 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

3 842 Sluka et al. Vol. 298 inflammation. The group effect of 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. Results Control Arthritic Animals and Effect of. 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 ). 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 s to s, 4 h after induction of arthritis. In contrast, treatment with either high- or low-frequency produced approximately 100% reversal of the hyperalgesia (Figs. 1 and 2). In the group of animals treated with lowfrequency, the PWL increased from s, 4 h TABLE 1 Joint circumference measurements (cm) for control arthritic animals and those receiving either high- or low-frequency sensory treatment. Values represent mean S.E.M. Baseline 4 h after Arthritis After cm cm cm Inflamed knee ACSF, no ACSF low frequency ACSF high frequency mm naloxone low mm naloxone low mm naloxone low mm naloxone high mm naloxone high mm naloxone high mm naltrinodole low mm naltrinodole high mm naltrinodole high mm naltrinodole high M nor BNI low M nor BNI high Contralateral knee ACSF, no ACSF low frequency ACSF high frequency mm naloxone low mm naloxone low mm naloxone low mm naloxone high mm naloxone high mm naloxone high mm naltrinodole low mm naltrinodole high mm naltrinodole high mm naltrinodole high M nor BNI low M nor BNI high TABLE 2 Spontaneous pain-related behavior ratings for control arthritic rats and those receiving 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. 4h Arthritis 1 h after Drug After ACSF, no 4 (3 5) 4.5 (3 5) 4 (3 4) ACSF low frequency 4 (3 4) 4 (2 5) 3 (2 4) ACSF high frequency 4.5 (4 5) 4.5 (4 5) 4 (3 5) 10 mm naloxone low 4 (3 5) 4 (3 4) 4 (3 4) 5 mm naloxone low 4 (3 5) 5 (3 5) 4 (3 5) 1 mm naloxone low 3 (3 4) 4 (3 4) 4 (3 4) 10 mm naloxone high 4 (3 4) 4 (4 5) 4 (3 4) 5 mm naloxone high 4 (3 5) 4 (4 5) 4 (3 4) 1 mm naloxone high 4 (3 4) 4 (3 4) 4 (3 5) 1 mm naltrinodole low 4 (3 5) 4 (3 5) 4 (3 4) 1 mm naltrinodole high 4 (3 5) 4 (3 5) 4 (2 5) 0.1 mm naltrinodole high 4 (4) 4 (4) 4 (3 4) 0.01 mm naltrinodole high 4 (3 4) 4 (2 4) 4 (3 4) 10 M nor BNI low 4 (4) 4 (4 5) 4 (3 5) 10 M nor BNI high 4 (2 4) 4 (3 4) 4 (3 4) after induction of inflammation, to s after treatment with (baseline s). Similarly, in the group of animals treated with high-frequency, the PWL increased from s, 4 h after induction of inflammation, to s after treatment with (baseline s). There was an overall significant effect across time (F 1, ; p.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 (F 14, ; P.001) but not after infusion of drug (or ACSF) alone (F ; p.08). The PWL of the contralateral hindpaw remained unchanged after induction of inflammation in animals with ACSF or those treated with or drug. For example, baseline PWL for the contralateral paw in control arthritic animals treated only with ACSF was s, and 4 h after inflammation the PWL remained at s. After treatment with either high- or low-frequency, the contralateral PWL was s and s, respectively, compared with baseline values of s and s. Effects of Naloxone on Analgesia. Spinal infusion of 1 mm naloxone had no effect on the inhibition of hyperalgesia produced by either high- or low-frequency ; the percentage of inhibition of hyperalgesia remained at approximately 100%. However, 5 and 10 mm naloxone prevented the inhibition of hyperalgesia by low- frequency (Fig. 1). Thus, there was still a decrease in the PWL to radiant heat after treatment similar to that observed 4 h after induction of inflammation. Only 10 mm naloxone blocked the inhibition of hyperalgesia produced by high-frequency (Fig. 2). To test selectivity of naloxone for different opioid receptors, naloxone was tested against agonists selective for (DAMGO), (SNC80), or (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 ( agonist) returned to the baseline.

4 1999 and Opioids 843 Therefore, at a dose of 5 mm, naloxone selectively blocks receptors but not or opioid receptors. After increasing the concentration of naloxone to 10 mm, all of the groups PWL returned to the baseline, indicating all opioid receptors (,, and ) were blocked at this dose. Effects of Naltrinodole on Analgesia. Blockade of opioid receptors with 1 mm naltrinodole prevented the inhibition of hyperalgesia produced by high-frequency but not low-frequency (Figs. 1 and 2). The effects of naltrinodole on preventing the inhibition of hyperalgesia by high-frequency were dose-dependent (Fig. 2, inset). The selectivity of naltrinodole was tested against agonists to (DAMGO), (SNC80), and (U50,488) opioid receptors. Figure 3B demonstrates that 1 mm naltrinodole selectively blocks opioid receptors. The analgesia produced by spinal infusion of DAMGO or U50,488 was unaffected by naltrinodole. Effects of nor-bni on Analgesia. Blockade of opioid receptors with nor-bni had no effect on the analgesia produced by either high- or low-frequency (Figs. 1 and Fig. 1. The percentage of inhibition of hyperalgesia is represented after administration of drug or ACSF (E) or treatment with drug or ACSF (F) in the group of animals treated with low-frequency. A, inhibition of hyperalgesia after treatment with ACSF (n 6), low-frequency (, n 8), or naloxone at 1 (n 3),5(n 5), or 10 mm (n 7). The percentage of analgesia was significantly increased after treatment with (p.009) when compared with animals treated with ACSF and no. Treatment with 5 (p.01) or 10mM (p.02) naloxone significantly prevented the analgesia produced by low-frequency. B, inhibition of hyperalgesia after treatment with 10 M nor-bni (n 6) or 1mM naltrinodole (n 6). No significant difference was observed between the group treated with ACSF low-frequency and those treated with nor-bni or naltrinodole. *, significantly different from group. Values are mean S.E.M. Fig. 2. The percentage of inhibition of hyperalgesia is represented After administration of drug or ACSF (E) or treatment with drug or ACSF (F) in the group of animals treated with high-frequency. A, inhibition of hyperalgesia after treatment with ACSF (n 6), highfrequency (, n 6), or naloxone at 1 (n 3),5(n 6), or 10 mm (n 6). The percentage of analgesia was significantly increased after treatment with (p.001) when compared with animals treated with ACSF and no. Treatment with 10mM (p.0001) naloxone significantly prevented the analgesia produced by high-frequency. B, inhibition of hyperalgesia after treatment with 10 M nor-bni (n 7) or 1mM naltrinodole (n 6). A significant blockade of the inhibition of hyperalgesia was observed After treatment with 1 mm naltrinodole (p.002) when compared with treatment with high-frequency ACSF. Inset, dose response effect after administration of.01 (n 5),.1 (n 3), or 1 naltrinodole (n 6). A significant inhibition of hyperalgesia occurred in the group treated with.1 (p.03) and 1 mm (p.002). *, significantly different from group. Values are mean S.E.M. 2). The percentage of inhibition of hyperalgesia was similar to that observed in animals treated with ACSF and and was not significantly different from that group. The selectivity of nor-bni was tested against agonists to (DAMGO), (SNC80), and (U50,488) opioid receptors. As Fig. 3C shows, spinal infusion of 10 M nor-bni selectively blocks opioid receptors. The analgesia produced by spinal infusion of DAMGO or SNC80 was unaffected by nor-bni. Discussion The current study supports the theory that works through release of endogenous opioids at the spinal cord level. Spinal administration of 5 mm naloxone, which selectively blocks opioid receptors, significantly reduced the antihyperalgesic effects of low-frequency. A greater, nonselective dose of naloxone (10 mm) reduced the antihyperalgesia produced by high-frequency, suggesting the involvement of endogenous opioids acting at or opioid

5 844 Sluka et al. Vol. 298 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 4) to activate receptors; SNC80 (n 4) to activate receptors, and U50,488 (n 5) to activate 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 4), SNC80 (n 5), and U50,488 (n 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 4), SNC80 (n 4), and U50,488 (n 4). A significant reversal of the analgesia produced by U50,488 occurred with spinal infusion of 10 M nor-bni. *p.05, significantly decreased from infusion of agonist. Values are mean S.E.M. receptors, or both. The antihyperalgesic effect of high-frequency was reduced by blockade of opioid receptors but not opioid receptors. Thus, the current study demonstrated that the analgesia produced by high-frequency sensory is mediated by opioid receptors spinally, and that produced by low-frequency sensory is mediated by opioid receptors spinally. Previous studies support the conclusion that both highand low-frequency result in the release of endogenous opioids. Increased -endorphin concentrations in the cerebral spinal fluid were observed after administration of either high- or low-frequency (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. They found that high-frequency stimulation produced an increase in dynorphin A but not in MEAP, whereas low-frequency 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 but not high-frequency, low-intensity is reversible by administration of naloxone systemically. The dose used by Sjolund and Eriksson (1979) was at a concentration expected to block opioid receptors. In contrast, high-frequency was unaffected by systemic naloxone in patient populations (Abram et al., 1981; Freeman et al., 1983). However, analgesia induced in rats by high-frequency was reversed by high doses of systemic naloxone expected to block,, and opioid receptors (Woolf et al., 1977; Han et al., 1984). The release of endogenous opioids in the spinal cord in response to 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, and 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 opioid receptor also contain Substance P and calcitonin gene-related peptide. Further spinal localization of the 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;

6 1999 and Opioids 845 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 and 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 or receptors reduces the behavioral response to formalin injection in rats (Hammond et al., 1998). Wang et al. (1996) demonstrated that, 1, and 2 receptor agonists inhibit activity of trigeminal dorsal horn neurons to A and C-fiber stimulation. Thus, the effects of 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 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 will better enable clinicians to determine which patients will benefit from 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. This finding implies that the mechanism of action of involves the same neural substrate as opioid-induced analgesia. If the patient is taking opioids or has taken opioids in the past, may not be the modality of choice to control pain. Solomon et al. (1980) also show that 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, alteration of mood, mental clouding, and increased tolerance to the drug with continued use. Thus, the use of 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 could be an effective alternative to NSAIDs as an analgesic when used in conditions of acute inflammation. Furthermore, if a patient is taking NSAIDs, may be less effective. Conclusions. Both high- and low-frequency at sensory intensity reverse the hyperalgesia produced by kneejoint inflammation. 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Brain Res 236: 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 kathleen-sluka@uiowa.edu