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PAIN MEDICINE

Nonsteroidal Antiinflammatory Drugs Suppress Pain-Related Behaviors, but Not Referred Hyperalgesia of Visceral Pain in Mice

Department of Anesthesiology and Pain Medicine, Ulsan University College of Medicine, Seoul, Korea

Address correspondence and reprint requests to Jeong-Gill Leem, MD, PhD, Department of Anesthesiology and Pain Medicine, Asan Medical Center, 388-1, Pungnap-dong, Songpa-Gu, Seoul, Korea. Address e-mail to jgleem{at}amc.seoul.kr.

	Abstract

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Visceral pain is characterized by spontaneous pain and referred hyperalgesia. After inducing visceral pain in mice using intracolonic mustard oil administration, we examined the effects of various nonsteroidal antiinflammatory drugs (NSAIDs) on pain-related behavior and on Evans blue dye extravasation. Animals were given one of the following: saline, ethanol, dimethylsulfoxide (DMSO), morphine, ketoprofen, ketorolac, or DFU (a cyclooxygenase-2 inhibitor). After drug treatment, mice underwent intracolonic administration of 50 µL 1.5% mustard oil. Spontaneous pain-related responses were assessed for the next 20 min. The frequency of withdrawal responses to the application of von Frey hairs to the abdomen, foot, and tail was determined. After completion of the behavioral tests, Evans blue was injected into the animals via the tail vein. Two hours later, the colon was removed postmortem and Evans blue content was measured. Spontaneous pain behaviors were significantly less in animals administered 3 and 10 mg/kg morphine, 50 mg/kg ketorolac, 100 mg/kg ketoprofen, and 20 mg/kg DFU (P < 0.05). Response frequencies to the application of von Frey hairs were lower in mice administered 3 and 10 mg/kg morphine (P < 0.05) but were not affected by ketorolac, ketoprofen, or DFU treatment. Colonic Evans blue content was smaller in mice given 100 mg/kg ketoprofen and 20 mg/kg DFU (P < 0.05). We concluded that NSAIDs reduced pain behavior and inflammation but had little effect on referred hyperalgesia.

	Introduction

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Visceral pain is associated with many diseases of the internal organs and, like somatic pain, signal amplification occurs through sensitization of peripheral nociceptors and subsequent changes in the excitability of spinal dorsal horn neurons (1). Inflammatory processes involving internal organs, or repeated and/or prolonged local application of non-noxious stimuli, can result in visceral nociceptors becoming frankly hypersensitive. Visceral and somatic afferent fibers converge on the same neurons in the relevant spinal cord segment (2) and an algogenic process in a viscus determines somatic, referred hyperalgesia in the area of referred pain (3,4).

The paucity of experimental studies of visceral pain is a result of the difficulty in accessing visceral tissue, with most animal models of visceral pain being technically demanding, mixing visceral and somatic mechanisms of peritoneal pain, or requiring surgery or intubation (5). However, one animal model involves intracolonic administration of capsaicin or mustard oil and provides a simple behavioral model that allows examination of visceral pain and hyperalgesia referred to the body wall and an indirect assessment of visceral inflammatory reactions by measuring the content of plasma extravasation (6).

Nonsteroidal antiinflammatory drugs (NSAIDs) exert their analgesic effects by inhibiting prostaglandin E2 formation generated from arachidonic acid. This is achieved via inhibiting cyclooxygenase-1 and -2 (COX-1 and COX-2) and, consequently, by blocking sensitization of peripheral nociceptors (7). COX-1 is expressed constitutively in virtually all tissues, most notably in the gastrointestinal tract, platelets, and kidney, and inhibition of COX-1 may cause serious complications from effects on physiological homeostasis. COX-2 is not detectable in most normal tissues but is induced at the site of injury and its expression is augmented in the central nervous system. Recently, Ghilardi et al. (8) have demonstrated that a significant amount of COX-2 is expressed in the spinal cord and inhibition of constitutively expressed spinal COX-2 prevents initial hyperalgesia after tissue injury. Thus, compared with COX-1 inhibitors, COX-2 inhibitors have less risk of side effects (9,10).

In the present study, we hypothesized that prostaglandin is involved in the inflammatory reaction as well as referred hyperalgesia of visceral pain. To test this hypothesis, we investigated the effects of morphine and NSAIDs on spontaneous pain reactions, referred hyperalgesia, and Evans blue dye extravasation in the mouse model of visceral pain induced by administration of intracolonic mustard oil. We were also interested in comparing the analgesic effects of NSAIDs with their antiinflammatory effects.

	Methods

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Adult male ICR (Institute of Cancer Research) mice with body weights of 25–35 g (Bio-Zenomics, Kapyoung, Kyunggi-do, Korea) were used in this study, which was approved by the Asan Lifescience Research Center, where the animals were housed 7 days before testing. Experiments observing pain were performed during a 4 h test period from 1:00 pm to 5:00 pm. Mice were placed on raised wire mesh under a clear plastic ventilated box and the frequency of withdrawal responses to mechanical stimulation after application of von Frey hairs (Monofilaments, Stoelting, IL) to the abdomen, foot, and tail was examined. On application of a hair for 1–2 s, the following responses were scored as positive: sharp retraction of abdomen, immediate licking or scratching of hair application site, and sharp withdrawal response of foot and tail. Five hairs with forces of 1, 4, 8, 16, and 32 mN were applied 5 times each in ascending order of force with an inter-stimulus interval of 5–10 s. The number of responses was noted and the "response rate" was obtained by dividing the number of positive responses by five.

Immediately after the von Frey hair procedure, 12 randomized groups of mice (n = 10 per group) were subcutaneously administered agents (50 µL) in the subscapular region. Vehicles were saline for morphine, 10% ethanol for ketorolac and ketoprofen or 60% dimethylsulfoxide (DMSO) for 3-(3-fluorophenyl)-4-(4-[methylsulfonylphenyl]-5,5-dimethyl-5H-furan-2-one (DFU), whereas animals of experimental groups were given one of the following; 1, 3, or 10 mg/kg morphine (morphine sulfate; RBI, Natick, MA); 5 or 50 mg/kg ketorolac (Tarasyn®, Dongkuk Pharm., Seoul, Korea); 10 or 100 mg/kg ketoprofen (Ketofen®, Korea United Pharm., Seoul, Korea); 2 or 20 mg/kg DFU. DFU, a selective COX-2 inhibitor, was supplied by the MSD Korea Ltd. as an experimental reagent and was dissolved in 60% DMSO. The initial doses of NSAIDs were chosen to be 100 times the human administration dose following the result of the previous study (11,12), showing that analgesic doses of intraperitoneally injected ketoprofen and ketorolac on the mouse writhing test were 30 mg/kg and 7.5 mg/kg, respectively, which are 30 times more than the human administration doses. DFU is a selective COX-2 inhibitor; because it resembles the chemical structure and pharmacological properties of the clinically used rofecoxib, the maximal dose used was 20 mg/kg. When initial doses were effective, one tenth of the initial doses were tested on new groups of mice. For the groups without effective results, no more smaller doses were tested.

Thirty minutes after drug administration, petroleum jelly was applied in the perianal area and 50 µL mustard oil (Sigma, St. Louis, MO; 1.5% vol/vol dissolved in 50% ethanol) was administered into the colon by inserting a fine catheter (Minipack; Portex, Hythe, UK) through the anus to a 4-cm length. After administration of mustard oil, spontaneous pain responses were directly observed for the next 20 min by experimenters blinded as to the identity of the administered drugs. Behaviors defined as pain-related were licking of the abdomen, stretching the abdomen, squashing the lower abdomen against the floor, and abdominal retractions. After these observations, response rates to von Frey hairs were again determined as described before drug administration. The percentage change in response rate was based on the response rate 20 min after intracolonic administration minus that before administration. Values of percent changes smaller than those of the corresponding vehicle-treated animals at the same forces represent that the administered drug had an inhibitory effect on the referred hyperalgesia.

Plasma extravasation caused by tissue inflammation can be quantified indirectly by measuring the concentration of Evans blue dye (13,14). After completion of behavioral tests, animals were administered 50 mg/kg (<100 µL volume) Evans blue dye (Sigma) dissolved in saline via a tail vein using a 30-gauge needle. Two hours later, animals were humanely killed by cervical dislocation and the terminal colon was removed for determination of Evans blue content. The tissue was dried at 60°C for 24 h, weighed, and then incubated in formamide (Sigma) at 60°C for 24 h to extract the Evans blue. Tissue was removed and the absorbance of the solution measured using a spectrophotometer (8453E UV visible spectrophotoscopy system; Agilent, Taufkirchen, Germany) ( = 620 nm). The Evans blue concentration was calculated using a standard absorbance curve generated by linear regression from measurements of five samples of known concentrations.

The number of spontaneous pain behaviors, the percentage change in response rates to application of von Frey hairs before and after intracolonic mustard oil administration, and Evans blue content of the distal colon were analyzed using Mann-Whitney U-tests and the level of statistical significance was set at P < 0.05.

	Results

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There was no significant difference in the spontaneous pain-related behaviors or response rates to von Frey hairs and Evans blue content of the DMSO, ethanol, and saline groups. Compared with mice administered saline, we found that administration of 3 or 10 mg/kg morphine resulted in less pain-related behavior after intracolonic administration of mustard oil (P < 0.05). There was no significant effect of 1 mg/kg morphine administration. In regard to the effect of NSAIDs, pain behavior was significantly less in mice given 50 mg/kg ketorolac, 100 mg/kg ketoprofen, and 20 mg/kg DFU than their corresponding vehicle groups (P < 0.05). There was no significant effect of 5 mg/kg ketorolac, 10 mg/kg ketoprofen, or 2 mg/kg DFU (Fig. 1).

View larger version (27K): [in this window] [in a new window]   Figure 1. Effects of morphine and nonsteroidal anti-inflammatory drugs on the pain-related behaviors induced by intracolonic administration of mustard oil in mice. The boxes are interquartile ranges; error bars are 10th and 90th percentiles. S, saline; M1, 1 mg/kg morphine; M2, 3 mg/kg morphine; M3, 10 mg/kg morphine; ET, ethanol; E1, 5 mg/kg ketorolac; E2, 50 mg/kg ketorolac; P1, 10 mg/kg ketoprofen; P2, 100 mg/kg ketoprofen; DM, dimethylsulfoxide; D1, 2 mg/kg DFU; D2, 20 mg/kg DFU. *Significantly (P < 0.05) different from group S; significantly (P < 0.01) different from group S; significantly (P < 0.05) different from group ET; $significantly (P < 0.05) different from group DM.

In the vehicle groups, compared with von Frey hair response rates before mustard oil administration, response rates 20 min after administration were more frequent at 1, 4, 8, 16, and 32 mN on the abdomen, and at 8, 16, and 32 mN on the foot and tail. Compared with the percentage changes of response rates in saline controls, 3 mg/kg morphine significantly decreased those values at 1, 4, and 16 mN on the abdomen, whereas there were no differences at 8 and 32 mN. At 10 mg/kg morphine, the percent changes of response rates were smaller at all intensities of abdomen compared with the saline group (P < 0.01), whereas for foot and tail stimulation the percent changes of response rates were smaller at 4, 8, 16, and 32 mN. In contrast, administration of ketorolac, ketoprofen, and DFU had no effect on the percent changes of response rates compared with the corresponding vehicle groups (Fig. 2).

View larger version (32K): [in this window] [in a new window]   Figure 2. Effects of morphine and nonsteroidal anti-inflammatory drugs on the referred hyperalgesia of visceral pain. The percentage change in response rate was based on the response rate 20 min after intracolonic administration of mustard oil minus that before administration. The boxes are interquartile ranges; error bars are 10th and 90th percentiles. S, saline; M1, 1 mg/kg morphine; M2, 3 mg/kg morphine; M3, 10 mg/kg morphine; ET, ethanol; E1, 5 mg/kg ketorolac; E2, 50 mg/kg ketorolac; P1, 10 mg/kg ketoprofen; P2, 100 mg/kg ketoprofen; DM, dimethylsulfoxide; D1, 2 mg/kg DFU; D2, 20 mg/kg DFU. *Significantly (P < 0.05) different from group S; significantly (P < 0.01) different from group S.

The amount of Evans blue released from colon tissue isolated from control vehicle treated mice was 0.62 ± 0.17 µg/mg, and a similar amount was released from colons of mice administered morphine and ketorolac. Less dye was released from colons of mice treated with 100 mg/kg ketoprofen and 20 mg/kg DFU (Fig. 3).

View larger version (16K): [in this window] [in a new window]   Figure 3. Effects of morphine and nonsteroidal anti-inflammatory drugs on the plasma extravasation represented by tissue contents of Evans blue. The boxes are interquartile ranges; error bars are 10th and 90th percentiles. S, saline; M1, 1 mg/kg morphine; M2, 3 mg/kg morphine; M3, 10 mg/kg morphine; ET, ethanol; E1, 5 mg/kg ketorolac; E2, 50 mg/kg ketorolac; P1, 10 mg/kg ketoprofen; P2, 100 mg/kg ketoprofen; DM, dimethylsulfoxide; D1, 2 mg/kg DFU; D2, 20 mg/kg DFU. *Significantly (P < 0.05) different from group S; significantly (P < 0.05) different from group DM.

	Discussion

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In the present study we demonstrated that NSAIDs inhibit peripheral inflammation and reduce spontaneous pain-related behaviors but that they are not effective on the hyperalgesia referred to the superficial somatic areas in the mouse model of visceral pain induced by intracolonic administration of mustard oil.

Specific pain behavior in the inflammation of the colon was not clear, but abdominal retraction was most frequently observed. All four types of pain behaviors observed were dose-dependently inhibited by morphine, suggesting that they are pain related. In regard to analgesic effects of various NSAIDs, maximal doses of ketoprofen, ketorolac and DFU led to significant reduction in spontaneous pain behaviors, but 1 of 10 maximum doses made no difference. At the smaller doses of NSAIDs, absence of analgesic effects with ketoprofen was consistent with a previous study (11), but the result with ketorolac was somewhat different from other studies (15) that 5 mg/kg or less doses of ketorolac were effective on the suppression of the writhing response to acetic acids. The difference with our study, especially with the smaller doses, might have originated from the difference in animal strains, route of administration, algogens used, and observed pain behaviors.

Although mechanisms of simple referred pain are now fully elucidated, the pathophysiological bases of referred pain with hyperalgesia are still somewhat unknown (16). Two mechanisms have traditionally been proposed to account for referred phenomena, which are not mutually exclusive (3). According to the first, the phenomenon can be sustained by central mechanisms, i.e., the abnormal visceral input produces an irritable focus in the relevant spinal cord segment, thus facilitating messages coming from somatic structures at the level of viscero-somatic convergent neurons. According to the second hypothesis, the visceral afferent barrage induces activation of a reflex arc whose afferent branch is represented by visceral afferent fibers: the efferent branch is represented by somatic efferents towards the muscle and sympathetic efferents towards subcutis and skin. The "central" hypothesis has received substantial support from animal experiments, but some evidence in favor of a sympathetic loop has also been found.

We found evidence of referred hyperalgesia in the abdomen, foot, and tail after intracolonic administration of mustard oil. Referred hyperalgesia was reduced by administration of morphine but was not affected by NSAIDs, even at maximal doses. Because both peripheral nociceptor and spinal dorsal horn neurons work together to provoke referred hyperalgesia in visceral pain, analgesic drugs should be able to act at both the peripheral and central levels to get an effective inhibition against referred hyperalgesia. Possible reasons why NSAIDs inhibited spontaneous pain behavior, but not referred hyperalgesia, are that these drugs did not affect the release of neurotransmitters or the activity of receptors in the synapses related to the reflex arc mediating referred hyperalgesia.

Using visceral pain model in rats induced by uterine cervical distension, Shin et al. (17) measured electrical activity of the abdominal muscle by electromyography and determined the electrical activity as the index of muscular hyperalgesia from visceral pain. They found that IV administration of 10 mg/kg ketorolac inhibited visceral pain-related muscular electrical activity. The data from the present study differed from those data, probably as a result of the discrepancy between electrical activity of muscle and pain behaviors. Because there are spontaneous electrical activities in the normal motor endplate (18), electromyographic activity may not always represent subjective pain perception and may not provoke pain behavior.

A maximum dose of NSAIDs reduced both plasma extravasation indirectly measured by Evans blue dye concentration and pain-related behaviors. These findings suggest that prostaglandin production and subsequent sensitization of nociceptors were inhibited by NSAIDs. At the site of inflammation, analgesic and antiinflammatory effects of ketoprofen and ketorolac could be the result of inhibition of constitutively expressed COX-1. As COX-2 is not normally expressed in most peripheral tissues, the analgesic effect of DFU could be the result of inhibition of the constitutively expressed COX-2 in. the spinal cord, whereas the antiinflammatory effect is achieved via blocking inducible COX-2 in migrating inflammatory cells at the site of inflammation. In the early events of inflammatory response, Funk (19) suggests that vasodilation and increased permeability of post-capillary venules reflect the effect of COX-2-derived prostaglandin and leukotriene, and prostaglandin synergize with other mediators to elicit enhanced vascular permeability and edema at the site of inflammation. In this study, it is unlikely that inducible COX-2 in the spinal cord was involved in the pain-related behaviors and referred hyperalgesia because 20 minutes after intracolonic administration of mustard oil does not allow enough time for induction of spinal COX-2 (8,20). The more intense analgesic than antiinflammatory effect indicates that analgesic effects of NSAIDs are not solely dependent on the prostaglandin synthesis and that NSAIDs also act at the central levels such as constitutively expressed spinal COX-2, opioid receptors (21,22), or N-methyl-d-aspartate receptors (23). Moreover, because a complex process involving the interaction of multiple mediators is responsible for peripheral inflammation as well as activation and sensitization of nociceptors, it is likely that certain drugs inhibiting only part of the inflammatory cascade may be not effective for inhibiting plasma extravasation (13).

In summary, this study provides evidence that in the visceral pain model induced by mustard oil in mice, NSAIDs reduce Evans blue plasma extravasation and spontaneous pain-related behaviors, but they are not effective for referred hyperalgesia. In addition, the data indicate NSAIDs inhibit pain behaviors more effectively than they inhibit peripheral inflammatory reactions.

	Footnotes

 
Supported, in part, by a grant (No 2001-190) from the Asan Institute for Life Science, Seoul, Korea.

	References

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