Introduction

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Chronic musculoskeletal pain is associated with significant disability and costs approximately 150 billion dollars per year in medical expenses within the United States alone (McCain, 1994; Yelin and Callahan, 1995). Musculoskeletal pain syndromes such as fibromyalgia and myofascial pain syndrome can be difficult to treat (McCain, 1994). For this reason, a new animal model of chronic muscle-induced pain was developed to examine mechanisms of chronic pain development and maintenance (Sluka et al., 2001b). Specifically, two injections of acidic saline into one gastrocnemius muscle 2 to 5 days apart produce a long-lasting bilateral hyperalgesia without associated tissue damage and without continued primary afferent input (Sluka et al., 2001b). This bilateral mechanical hyperalgesia is reversed by blockade of spinal NMDA or non-NMDA glutamate receptors (Skyba et al., 2002). Thus, this model produces a secondary mechanical hyperalgesia dependent on changes in the central nervous system.

Opioids are commonly used for pain relief clinically (Miyoshi and Leckband, 2001) and reduce hyperalgesia in most animal models (Millan, 1986; Sabbe and Yaksh, 1990). There are three opioid receptors located in the spinal cord, µ, , and , that when activated result in analgesia and a reduction in hyperalgesia (Millan, 1986). Following peripheral inflammation, there is an increased sensitivity to opioids in the spinal cord (Hylden et al., 1991b; Przewlocka et al., 1991; Stanfa et al., 1992). In contrast, in peripheral neuropathic pain, the sensitivity to opioids is greatly reduced (Ossipov et al., 1995). The hyperalgesia associated with carrageenan-induced inflammation of a muscle or joint is similarly responsive to morphine delivered intrathecally or systemically (Nagasaka and Yaksh, 1996; Kehl et al., 2000). Similarly, in musculoskeletal pain syndromes such as fibromyalgia, the pain is reduced by intrathecal or systemic administration of morphine (Bengtsson et al., 1989; Sorensen et al., 1997; Biasi et al., 1998). There is a wealth of evidence to show that morphine and [D-Ala²,N-Me-Phe⁴,Gly-ol⁵]-enkephalin (DAMGO), both µ-opioid agonists, are analgesic and reduce hyperalgesia in most animal models and that -opioid agonists similarly reduce hyperalgesia in inflammatory pain models. However, the effects of opioid agonists on chronic pain induced by stimulation of a muscle are not known.

The current study was undertaken to assess the effects of opioid agonists on mechanical hyperalgesia induced by repeated intramuscular injections of acid. Validation of this new model by showing sensitivity to opioids is critical. Portions of this data were presented in abstract form (Sluka et al., 2001a).

	Materials and Methods

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Induction of Hyperalgesia. Animals were injected with pH 4 saline (100 µl) into one lateral gastrocnemius muscle on day 0 and again on day 5 (Sluka et al., 2001b). pH was adjusted to within 3.9 to 4.1 with HCl.

Behavioral Testing. Animals were tested for withdrawal thresholds to mechanical stimuli (von Frey filaments) applied to the plantar aspect of the hindpaw (Gopalkrishnan and Sluka, 2000; Sluka et al., 2001b). von Frey filaments, with bending forces from 7 to 162 mN, were applied in a progressively increasing manner until the hindpaw was withdrawn or 162 mN was reached. Each filament was applied twice. The filament of lowest bending force from which the animal withdrew was considered the mechanical withdrawal threshold of the hindpaw. After a response, the filaments above and below were tested to confirm the withdrawal threshold. The test-retest reliability of this method was previously established (r² = 0.7; p = 0.007) (Gopalkrishnan and Sluka, 2000).

Intrathecal Catheterization. Intrathecal catheters (32 gauge, polyurethane; Recathco, Allison Park, PA) were placed 5 to 6 days before the first intramuscular injection of saline (Storkson et al., 1996). In brief, animals were anesthetized with halothane (2-5%), and a 23-gauge hypodermic needle was inserted into the intervertebral space between L5-L6. A 32-gauge polyurethane catheter was inserted through the needle and advanced cranially until 3.5 to 4.0 cm was under the skin. The external portion of the catheter was secured to the muscle and fascia. The free end was then inserted into PE-10 tubing and tunneled to the cervical region.

Expeimental Design. Animals were tested for withdrawal to mechanical stimuli before injection 1 on day 0, before injection 2 on day 5, and 24 h after injection 2. Following the development of hyperalgesia, 24 h after injection 2, the opioid agonist or vehicle was injected. All experiments were performed with the tester blinded to the drug injected. The following drugs were injected intrathecally: 1) morphine, µ-opioid receptor agonist (n = 28; 0.07-7.0 nmol, dissolved in saline; Sigma-Aldrich, St. Louis, MO); 2) DAMGO (Sigma/RBI, Natick, MA), µ-opioid receptor agonist (n = 30; 0.03-10 nmol, dissolved in saline); 3) 4-[(R)--((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80; Tocris Cookson, Inc., Ballwin, MO), -opioid receptor agonists (n = 33, 6-60 nmol, dissolved in 20% DMSO and water); and 4) (1S-trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cylcohexyl]-benzeneacetamide hydrochloride (U50,488; Sigma/RBI), -opioid receptor agonist (n = 23, 1-300 nmol, dissolved in 20% DMSO and saline). Vehicle controls included saline (n = 8), 20% DMSO and saline (n = 12), and 20% DMSO and water (n = 6). Each animal received one intrathecal injection of the opioid agonist or vehicle. Animals were tested for mechanical withdrawal threshold in 15-min intervals for 1 h after injection of the opioid agonist or vehicle.

Data Analysis. A Kruskall-Wallis analysis of variance compared differences between vehicle controls and drug at each time period after injection. Post hoc testing was done using a sign rank test. The percentage of maximal possible effect (%MPE) was calculated using the following formula: (withdrawal threshold after drug  withdrawal threshold 24 h after the second injection)/(cutoff withdrawal threshold  withdrawal threshold 24 h after second injection). ED₅₀ values and confidence intervals were calculated on the %MPE for each drug at 30 min (Pharm Tools Pro; The McCary Group, Elkins Park, PA) to compare with published literature. A one-way analysis of variance compared differences for the percent inhibition and vehicle for all drug doses. A t test compared the percent inhibition of drug + antagonist to drug.

	Results

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Control. Twenty-four hours after the second injection of pH 4 saline into one gastrocnemius muscle, there was a significant decrease in mechanical withdrawal threshold of the paw bilaterally. This decrease in mechanical withdrawal threshold remained decreased throughout the 1-h testing period after intrathecal injection of saline, 20% DMSO in saline, or 20% DMSO in water (Fig. 1).

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Line graphs representing the withdrawal threshold across time of the effects of drug compared with vehicle controls (circles) for animals treated with morphine (7 nmol; squares) (A and D), DAMGO (10 nmol; triangles) (A and D), SNC80 (60 nmol; upside down triangles) (B and E), or U50,488 (300 nmol; diamonds) (C and F). The ipsilateral paw is represented by closed symbols (A, B, and C) and the contralateral paw with open symbols (D, E, and F). Data are represented as the median with the 25th and 75th percentile. *, significantly increased from vehicle.

Effects of Morphine. Intrathecal injection of morphine resulted in an increase in mechanical withdrawal threshold within 15 min and remained elevated for 60 min (Fig. 1). When compared with vehicle controls, the withdrawal threshold to mechanical stimuli was significantly increased bilaterally 15, 30, 45, and 60 min after intrathecal injection of 7 nmol of morphine. Dose response curves showed a reversal of the withdrawal threshold for the 7-nmol dose for the ipsilateral side and for the 2- and 7-nmol dose for the contralateral side. The ED₅₀ values for morphine were 2.2 ± 0.86 and 0.18 ± 0.09 nmol for the ipsilateral and contralateral paws, respectively. Percent inhibition following pretreatment with 2 nmol of CTAP was 6.2 ± 10.7 and 4.7 ± 8.7% ipsilaterally and contralaterally, respectively, and was significantly less that the percent inhibition by 7 nmol of morphine.

Effects of DAMGO. Intrathecal injection of the µ-opioid receptor agonist DAMGO similarly reversed the decrease in mechanical withdrawal threshold that occurs following the second injection of pH 4 saline (Fig. 1). The increase occurred bilaterally by 15 min and remained elevated for 1 h after intrathecal injection of 10 nmol of DAMGO when compared with vehicle controls. The ED₅₀ values for DAMGO were 0.10 ± 0.06 and 0.09 ± 0.03 nmol for the ipsilateral and contralateral paws, respectively. Significant increases from vehicle occurred following intrathecal injection of 0.1, 0.3, 1, 3, and 10 nmol of DAMGO ipsilaterally and following 0.3, 1, and 10 nmol contralaterally (Fig. 2). Percent inhibition following pretreatment with 2 nmol of CTAP was 3.1 ± 5.2 and 26 ± 16% ipsilaterally and contralaterally, respectively, and was significantly less that the percent inhibition by 10 nmol of DAMGO.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Dose response curves representing the %MPE for the mechanical withdrawal threshold 30 min after administration of the agonist for the ipsilateral (closed symbols) and contralateral (open symbols) paws for animals treated with morphine (square), DAMGO (circle), SNC80 (triangle), or U50,488 (diamond). Data are represented as the mean ± S.E.M. *, significantly different from vehicle.

Effects of SNC80. Intrathecal injection of the nonselective -opioid receptor agonist SNC80 reversed the decrease in mechanical withdrawal threshold after the second injection of pH 4 saline (Fig. 1). SNC80 (60 nmol) reversed this hyperalgesia bilaterally 15, 30, 45, and 60 min after intrathecal injection when compared with vehicle controls (Fig. 1). Significant increases from vehicle-treated animals occurred for 30 and 60 nmol of SNC80 (Fig. 2). ED₅₀ values for SNC80 were 6.3 ± 2.9 and 35 ± 18 nmol for the ipsilateral and contralateral paws, respectively. Percent inhibition following pretreatment with 100 nmol of naltrindole was 7 ± 20 and 4 ± 2% ipsilaterally and contralaterally, respectively, and was significantly less than the percent inhibition by 60 nmol of SNC80.

Effects of U50,488. Intrathecal injection of the -opioid agonist U50,488 at doses from 1 to 300 nmol had no effect on the decreased withdrawal threshold to mechanical stimuli induced by repeated intramuscular injection of acidic saline (Fig. 1). The data for a 300-nmol dose are shown in Fig. 1, and all doses are shown in Fig. 2 for the 30-min time period.

	Discussion

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Intramuscular injection of acidic saline produces a bilateral mechanical hyperalgesia of the paw that lasts through 4 weeks and is sensitive to spinally administered NMDA and non-NMDA ionotropic glutamate receptor antagonists (Sluka et al., 2001b; Skyba et al., 2002). Further there is no observable damage to the muscle tissue, and the hyperalgesia does not depend on continued primary afferent input from the muscle (Sluka et al., 2001b). The hyperalgesia is interpreted as secondary mechanical hyperalgesia since it occurs outside the area of injection and spreads to the contralateral side. These data suggest that changes in the central nervous system maintain the bilateral, long-lasting hyperalgesia. The current data shows that this model of chronic muscle pain induced by repeated intramuscular injections of acidic saline is sensitive to agonists of µ- and -opioid receptors. The inhibition of the hyperalgesia by morphine and DAMGO was prevented by the selective µ-opioid antagonist CTAP, and that of SNC80 was prevented by the selective -opioid agonist naltrindole. Activation of -opioid receptors spinally has no effect on the decreased withdrawal threshold induced by repeated acidic saline.

Role of µ-Opioid Receptors in Pain. Intrathecal administration of µ-opioid agonists has been studied extensively and reliably reduces pain behaviors in most animal models of pain. Activation of spinal µ-opioid receptors with morphine or DAMGO causes analgesia in acute pain tests, including tail-flick, hot plate test, paw withdrawal to heat, paw pressure test, and colorectal distension (Miaskowski et al., 1991, 1992; Mjanger and Yaksh, 1991; Malmberg and Yaksh, 1992; Tiseo and Yaksh, 1993; Stewart and Hammond, 1993; Danzebrink et al., 1995; Hammond et al., 1995; Sluka et al., 1999). The ED₅₀ values range from 1.6 to 50 nmol for morphine, and that of DAMGO ranges from 0.04 to 0.72 nmol in acute pain tests.

Role of -Opioid Receptors in Pain. Activation of -opioid receptors spinally consistently produces analgesia in acute pain tests and reduces hyperalgesia in animal models of pain. Spinally administered -opioid agonists produce analgesia in the tail-flick test, hot plate test, paw pressure test, and colorectal distension (Malmberg and Yaksh, 1992; Stewart and Hammond, 1993; Tiseo and Yaksh, 1993; Bilsky et al., 1995; Danzebrink et al., 1995; Hammond et al., 1995; Hosohata et al., 2000). Intrathecal SNC80 is a nonselective -opioid agonist that produces analgesia in the tail-flick test, with an ED₅₀ of 69 nmol in mice; 60 nmol produced approximately 40% inhibition, whereas 300 nmol produced a maximal effect of approximately 95% inhibition (Bilsky et al., 1995). In the current study, the 30- and 60-nmol dose of SNC80 significantly reversed the mechanical hyperalgesia bilaterally, with a maximal inhibition of 86%, and the ED₅₀ values were 17 and 46 nmol for the ipsilateral and contralateral paws, respectively. These doses are lower than those found for acute pain models, suggesting an increased sensitivity to -opioid agonists.

Role of -Opioid Receptors in Pain. The lack of effect of a spinally administered -opioid agonist in the current study, agrees with several other studies. Spinally administered U50,488 has no effect on the tail-flick latency to heat (Stevens and Yaksh, 1986; Schmauss, 1987; Leighton et al., 1988; Przewlocka et al., 1991), hot plate test (Stevens and Yaksh, 1986), paw pressure test (Leighton et al., 1988), and visceromotor response to colorectal distension (Danzebrink et al., 1995). Following complete Freund's adjuvant inflammation or carrageenan paw inflammation, U50,488 was still without effect (Hylden et al., 1991a; Przewlocka et al., 1991).