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REGIONAL ANESTHESIA AND PAIN MEDICINE

The Effect of Diclofenac on the Expression of Spinal Cord c-fos-Like Immunoreactivity After Ischemia-Reperfusion-Induced Acute Hyperalgesia in the Rat Tail

Department of Anaesthesia and Pain Management, The University of Sydney at the Royal North Shore Hospital, St. Leonards, Australia

Address correspondence to Laurence E. Mather, PhD, Department of Anaesthesia and Pain Management, The University of Sydney at the Royal North Shore Hospital, St. Leonards NSW 2065, Australia. Address e-mail to lmather{at}med.usyd.edu.au

	Abstract

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Ischemia-reperfusion of the rat tail for 20 min induces local acute hyperalgesia of approximately 1-h duration. We studied how this stimulus affected the expression of c-fos-like immunoreactivity (c-fos-LI) labeling of neurons of the sacral spinal cord, and how diclofenac pretreatment influenced the outcome. After ischemia, the number of c-fos-LI-labeled neurons was significantly increased when assessed at 60, 90, and 120 min after reperfusion (to 183%, 283%, and 164% of control, respectively; all P < 0.01). At 90 min, the number of regional c-fos-LI-labeled neurons was increased to 585% in laminae I-II, 183% in laminae III-IV, 270% in laminae V-X, and 286% in total, compared with respective control values (all P < 0.01). After diclofenac pretreatment (subcutaneous 40 mg/Kg, 30 min before insult) the number of c-fos-LI-labeled neurons at 90 min was increased to 424% in laminae I-II, 150% in laminae III-IV, 142% in laminae V-X, and 183% in total (all P < 0.01). Thus diclofenac pretreatment partially prevented the insult-induced increase in total and regional neuronal c-fos-LI. This acute nociceptive model involves only natural algogens. However, the results were similar to acute chemically induced or chronic adjuvant induced arthritic inflammatory pain models in which increases in c-Fos were partially inhibited by nonsteroidal antiinflammatory drugs.

Implications: This study demonstrates a spinal action of the nonsteroidal antiinflammatory drug, diclofenac, in response to a peripheral insult. Whether the action is caused by reduced peripheral neural activity cannot be ascertained. The action was consistent with a ceiling effect of diclofenac as often found clinically with this class of analgesic drug.

	Introduction

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Increased expression of c-fos, an immediate-early gene, or of Fos-like protein, in the spinal cord of laboratory rodents has been widely used as an indicator of acute and chronic nociceptive spinal transmission (1–3). Many studies (4–10) have demonstrated that c-fos expression in spinal cord neurons increases shortly after the onset of a noxious peripheral stimulus, that the expression is anatomically well localized in superficial and deep laminae, and that the expression can be inhibited, however, not necessarily abolished, by treating the animal with a variety of known analgetic drugs. In most studies in laboratory rodents, nociception has been provoked by an exogenous irritant, typically formalin or carrageenan injected into a paw, or by an arthritic condition, typically induced by an adjuvant injected into the base of the tail. In this study, we wished to determine whether nociception stimulated peripherally by only endogenous mediators would exert a central effect and whether that effect could be influenced by treatment with a nonsteroidal antiinflammatory drug (NSAID).

Acute hyperalgesia induced by ischemia-reperfusion of the rat’s tail was first reported as a potential investigational model for acute pain studies in 1986 (11). Since then, a variety of physiological and pharmacological studies (12–15) have been performed to characterize this model. These studies have found that the hyperalgesia can be abolished by treating the animal with a variety of drugs, including opioids and NSAIDs. As part of a wider program to study the in vivo effects of NSAIDs in relation to their systemic and regional pharmacokinetics, we assessed the biochemical sequelae of the ischemia-reperfusion-hyperalgesia model and of the influence of diclofenac, an achiral NSAID with mixed COX-1 and COX-2 activity (16). We report the effects of rat tail ischemia-reperfusion-hyperalgesia on the expression of c-fos-like immunoreactivity (c-fos-LI) in neurons of the sacral spinal cord and its modification by pretreating with diclofenac.

	Methods

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Experimental Animals
Male Sprague-Dawley rats weighing 250–300 g were used. The procedures related to animal use were approved by our institutional animal care and ethics committee.

Acute hyperalgesia of the rat tail was induced by ischemia-reperfusion by using the method of Gelgor et al. (11) with slight modification. The animals were placed in clear acrylic restrainers (62 mm x 62 mm, adjusted to the length of the animal), which allowed free movement of the tail and slightly restricted movement of the rest of the body. The animals were placed in these restrainers for 2–3 h/day on two consecutive days before any experimentation to allow them to adjust to these conditions. On experimental days, animals were placed in the restrainers for 15 min before any testing. Ischemia was induced by application of an inflatable cuff to the base of the restrained rat’s tail. The cuff was connected to a sphygmomanometer and inflated to a pressure of 240 mm Hg for 20 min. After the cuff was deflated, hyperalgesia from the insult was assessed by measuring the reduction in the time to tail flick after immersion of the tail in 49°C water, as previously described (11).

Three groups of animals were used in the study. Group 1 (n = 6) was a control group. These animals were placed in the restrainer and the cuff was placed on the tail but was not inflated; 90 min later, the animals were killed and c-fos expression in the sacral spinal cord was determined. Group 2 (n = 11) underwent ischemia-reperfusion of the tail. Subgroups of these animals were killed at 60, 90, and 120 min after the insult and c-fos expression in the sacral spinal cord was determined. Group 3 (n = 5) underwent ischemia-reperfusion of the tail after pretreating with diclofenac (40 mg/kg, subcutaneous) 30 min before the cuff was inflated. This dose chosen had been shown in our previous behavioral and biochemical experiments to inhibit the hyperalgesia and local prostaglandin synthesis (unpublished observations). At 90 min after insult, the animals were killed and the sacral spinal cord was assessed for c-fos expression.

Immunohistochemistry
After ischemic insult, each animal was deeply anesthetized with sodium pentobarbital (35 mg/kg IP) and perfused transcardially for 20 min with saline (500 mL 0.9%) followed by paraformaldehyde (4% wt/vol) and sucrose (500 mL 10%) in phosphate buffer (0.1 M, pH 7.4). The sacral spinal cord was removed and cryoprotected in a solution of sucrose 30% phosphate buffer (pH 7.4) for 24 h at 40°C. Coronal (bilateral) sections (50-µm thick) of the S1, S2, and S3 segments were cut on a freezing microtome and placed in phosphate buffer in sterile plastic jars. Sections were processed immunohistochemically for the presence of c-fos. Sections were washed in ethanol (50% in distilled water) for 30 min, followed by hydrogen peroxide (1% in 50% ethanol), then washed (90 min) in phosphate buffer followed by normal horse serum (20%) and placed in clean glass vials in phosphate-buffered horse serum in the presence of c-fos antibody (SC-52 rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:1000 (2–3 days at 4°C on a rotating table). After 3 days, sections were washed (30 min) in phosphate buffer and then incubated in the presence of biotinylated antirabbit IgG (RPN 1004; Amersham Pharmacia Biotech, Inc, Piscataway, NJ) at 1:200 dilution (room temperature, 2 h). Sections were washed (30 min) in phosphate buffer and then, incubated with Extravidin (Sigma Chemical Co, St. Louis, MO) at a dilution of 1:1000 (2.5 h). After another three washes in phosphate buffer, the sections were washed in a diaminobenzidine mix containing nickel and incubated in the presence of glucose and glucose-oxidase to visualize the reaction product. Sections were finally washed in phosphate buffer, mounted onto gelatinized slides, air dried, and cover slipped for visual counting of c-fos-LI-labeled neurons.

Counting of c-Fos-LI Cells
Bilateral sections from the spinal cord were examined microscopically to determine the location of c-fos-LI positive cells. The locations of positive cells in five sections, randomly selected from the relevant region of each spinal cord were plotted and counted by using a camera lucida technique by a technician not involved with the treatment or interpretation of results. Each of these regions was analyzed according to Rexed laminae of the spinal cord to determine differences in c-fos-LI positive neuron somatotopic distribution.

Statistical analysis was performed group-wise by using analysis of variance; multiple comparisons of the group mean values were made by the method of least significant differences.

	Results

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After ischemia-reperfusion-hyperalgesia, c-fos-LI-labeled neurons in the sacral spinal cord increased significantly from control at 60, 90, and 120 min (Figure 1). As 90 min was the time of greatest change, it was chosen for evaluation of the effects of diclofenac.

View larger version (26K): [in this window] [in a new window]   Figure 1. Summed c-fos neurones in control rats killed at different times after the ischemia-reperfusion-hyperalgesia insult. Results are expressed as mean (± SEM) of the number of c-fos neurones per section in laminae I-II, III-IV, and V-X.

View larger version (43K): [in this window] [in a new window]   Figure 2. Number of c-fos neurones in control, ischemic, and diclofenac treated rats. Results are expressed as mean (± SEM) of the number of c-fos neurones per section (total) and per laminar region (laminae I-II, III-IV, and V-X). Treatments were control, ischemia-reperfusion-hyperalgesia, and diclofenac treatment before ischemia-reperfusion-hyperalgesia.

	Discussion

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This study clearly demonstrates that an acute ischemia-reperfusion-hyperalgesia insult to the rat tail can produce a marked increase in the number of c-fos-LI positive neurons in the sacral spinal cord within 60 min. These data add to a long list of noxious stimuli that can cause increased c-fos-LI in the spinal cord (1–3). However, an important difference to most previous reports is that the algogens of this study were entirely endogenous. Most previous acute studies have used exogenous noxious stimuli, such as subdermal injection into the rat’s paw of formalin or carrageenan, or injection of an adjuvant into the base of the tail. Nevertheless, as with those studies, the somatotopic distribution of the positive activity was found to be greatest in the regions processing nociceptive information: the superficial laminae I-II that contain A and C fiber primary afferent terminations and the deep laminae V-X that contain secondary interneurons of superficial primary afferents, as well as cranio-caudal projections (reference 1, for more detailed background). Central neural tissue c-fos activity, like that of a number of immediate early genes, increases in response to a variety of types of acute and chronic noxious stimulation-induced peripheral neuronal activity. However, the induction of c-fos-LI should not be necessarily equated with nociception and with pain per se, although this is tempting because the effect is ameliorated by known analgesic drugs (17–21).

It has been shown that, even when nociception is completely inhibited with neural block, the induction of c-fos may only be attenuated rather than abolished (22). Our study also indicates that the spinal c-Fos-LI response was only partially inhibited by pretreating with diclofenac. This is consistent with the results of other studies (6–8,10,17) with a variety of NSAIDs in other nociceptive models involving overt inflammatory sequelae. Although a diclofenac dose-response analysis was not performed in this study, our other studies (23, and unpublished observations) with this model indicate that the dose used was required to suppress the hyperalgesia from the insult and is associated with suppression of prostanoid synthesis locally in the tail and in the brain, however, not necessarily in the spinal cord. Smaller doses of diclofenac do not suppress the hyperalgesia, and significantly larger doses are not feasible because of the risk of renal damage and other adverse effects (24). Our studies (23) indicate that peak diclofenac plasma concentrations occur at approximately 20 min after subcutaneous injection, a period before the insult with sufficient time for the development of the biochemical action of the drug. Thus, it appears that NSAID as a class of drug may only partially prevent the c-fos reaction to nociception within reasonable limits of dose. This would appear to be a corollary of the "analgesic ceiling" often found when NSAID "unimodal analgesia" is used for the treatment of severe "clinical pain" in humans (25). It also suggests that "multimodal analgesia" may be required to obtund reliably the central mechanisms associated with the development of acute hyperalgesia (25).

Because it appears that the NSAIDs insufficiently reduce the peripheral and presynaptic afferent neurotransmitter production and descending inhibitory control associated with the peripheral nociception, it is useful to question whether there is sufficient penetration of the NSAIDs to their sites of action (spinal). Our ongoing studies (23) also indicate that the distribution coefficient of diclofenac into the spinal cord, although low, is similar to that in the tail and the brain. Although this line of inquiry cannot discern between central and peripheral mechanisms of diclofenac action, the partial central neural inhibition of c-fos-LI gives one more piece of information that indirectly supports a possible central action of diclofenac (26–28).

The basal level of c-fos-LI-labeled neurons in the lumbar spinal cord of our control animals was higher than has been reported by others (6,7). The difference could be because our control animals are not necessarily "unstimulated" or "naïve" animals and are trained for several days to being used to the restrainers for the experiment. During this process, the tail was positioned in a hole of the restrainer and, to maintain the tail in the correct position, the tail was held for a short time. This may constitute a stimulation and thus, emphasizes the need for relevant control animals (3). Nevertheless, the outcome in this hyperalgesia model was generally similar to that seen in chemical inflammogen (formalin and carrageenan) paw injection models, in which increased c-fos-LI has been found most extensively localized in laminae I-II and in the neck of the dorsal horn (laminae V-VI) with differences in distribution varying with the time after injection (20,29). Thus, the similarity in distribution of c-fos-LI-labeled neurons between the models with endogenous mediators and chemical inflammogens may imply a qualitatively similar processing of nociceptive input at the spinal cord level.

The duration of the increase in c-fos-LI-labeled neurons from ischemia-reperfusion-hyperalgesia appears to be brief. With the rat paw carrageenan inflammation model, Honore et al. (18) found increased c-fos-LI at two hours and maximal increases three to four hours after the injection of carrageenan. With the rat paw formalin inflammation model, Presley et al. (20) found that Fos labeling in the dorsal horn was present at one hour and was most extensive at two to four hours postinjection. Although Honore et al. (18) claimed that the time of peak c-fos expression is similar to that of peak behavioral hyperalgesia, we found that c-fos time-course lags behind the behavioral hyperalgesia, suggesting that the mechanisms underlying the various pain models may differ. Nevertheless, our results do support a linkage between the behavioral hyperalgesia and the expression of c-fos-labeled neurons. Hyperalgesia in this model was maximal immediately after cuff release, however, it was maintained only for approximately 30 minutes after the insult (unpublished observations, manuscript submitted); the peak c-fos expression was similarly brief. In the formalin injection model, hyperalgesia commences later, lasts longer, and with increased c-fos labeling for up to 24 hours after the insult (18,20).

In our study, diclofenac was more effective in reducing the number of the c-fos-LI neurons in the deep laminae (47% reduction) than in the superficial laminae (26% reduction). These effects with subcutaneous diclofenac are in accord with the ability of IV diclofenac (7), indomethacin (18), and oral piroxicam (6) to reduce c-fos-LI-labeled neurons in the superficial and deep laminae of the dorsal horn in the carrageenan injection model. Others (30) have shown that c-fos neuronal labeling is determined to some extent by the nature and duration of the stimulus. As the acute pain model of the current study appears to be without a primary inflammatory reaction, it may not evoke the antiinflammatory effect of diclofenac although this dose abolished the hyperalgesia. Once the cuff is released there may be no "peripheral generator" to drive central processes, allowing return to baseline levels.

This study, in an acute hyperalgesia model with only endogenous nociceptive mediators generated by nonprimary inflammatory stimulus, adds further indirect support for a central component of the analgesic effect of diclofenac. It is possible that the inhibitory effect of c-fos-LI expression is associated with the suppression of prostanoid formation in the spinal cord. Our study may also support the view that there are two possible components of diclofenac-induced analgesia—an early, indirect opioid-related effect (26,27) and a later effect mediated by cyclo-oxygenase inhibition (28). Our data demonstrated that the c-fos-LI expression was increased in the sacral spinal cord after the induction of acute hyperalgesia by ischemia-reperfusion of the tail, that the increased expression was partially inhibited by diclofenac pretreatment, and that the results were similar to other acute pain models, such as formalin and carrageenan paw injection models, although the stimuli differ greatly.

	Acknowledgments

 
Supported, in part, by the National Health & Medical Research Council of Australia.

We are pleased to acknowledge the technical expertise of Ms. Cao-Ling Xu and the helpful criticisms of Dr. Philip Siddall.

	Footnotes

 
Presented, in part, at the 33rd Annual Meeting of the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists, Sydney, Australia, December 1999.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Web of Science (5) Citing Articles via Google Scholar Google Scholar Articles by Lin, Y. Articles by Cousins, M. J. Search for Related Content PubMed PubMed Citation Articles by Lin, Y. Articles by Cousins, M. J.