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PAIN MECHANISMS

The Effects of Intrathecal Cyclooxygenase-1, Cyclooxygenase-2, or Nonselective Inhibitors on Pain Behavior and Spinal Fos-Like Immunoreactivity

From the Departments of Anesthesiology and Pain Medicine, College of Medicine, Korea University, Seoul, Korea.

Address correspondence and reprint requests to Il-Ok Lee, MD, PhD, Departments of Anesthesiology and Pain Medicine, 97 Guro-dong gil, Guro-gu, Korea University Guro Hospital, College of Medicine, Korea University, Seoul, Korea 152–703. Address e-mail to iloklee{at}korea.ac.kr.

Abstract

BACKGROUND: Prostaglandins are synthesized by cyclooxygenase (COX) and are thought to play an important role in nociceptive transmission in the spinal cord. Fos expression is an indicator of spinal neuron activation. We examined the role of intrathecal selective and nonspecific COX inhibitors on spinal C-Fos expression.

METHODS: To evaluate the relative contribution of COX-1 and COX-2 in nociceptive transmission in the spinal cord, we assessed the effects of the selective COX-1 inhibitor SC 560, the selective COX-2 inhibitor celecoxib, and the nonselective COX inhibitor ketorolac on formalin-evoked behavior and spinal c-Fos-like immunoreactivity (FLI). Rats received each of the drugs (30, 60, or 90 µg) intrathecally before the subcutaneous administration of formalin (5%, 50 µL) to the plantar surface of a hindpaw. The control group received vehicle intrathecally before the administration of formalin.

RESULTS: Phase 1 flinching behavior decreased in rats given celecoxib or ketorolac 90 µg. Phase 2 flinching behavior decreased in rats given all doses of ketorolac or celecoxib 90 µg (P < 0.05). The FLI was significantly reduced in rats given celecoxib or ketorolac 90 µg for laminae I–II (P < 0.05). By contrast, for laminae V–VI, only the ketorolac 60 or 90 µg treatment group demonstrated a larger decrease in FLI (P < 0.05). The FLI expression in laminae V–VI had a significant correlation with phase 2 flinching behavior (P < 0.05).

CONCLUSIONS: A dual inhibitor of COX-1 and COX-2 suppressed both responses of formalin-evoked behaviors and FLI expression of whole laminae in the lumbar spinal cord. FLI expression of laminae I–II alone may not be a good indicator of the ability to produce anti-hypersensitivity; however, the FLI of laminae V–VI correlates with phase 2 responses.

Local injury causes the release of a variety of factors that alter the sensitivity of the primary afferent fibers that innervate damaged tissue. Prostaglandins (PG) are among those factors that are important components of this peripheral sensitization.1 PG are lipid acids that are locally synthesized by the cyclooxygenase (COX) enzymes after focal tissue injury. They sensitize peripheral nerve endings and enhance pain behavior in both animals and humans.2,3 It is now known that there is an equally important role in the spinal cord. Two different forms of COX have been characterized: COX-1 is constitutively expressed, whereas, COX-2 is expressed constitutively in the spinal cord but will show an increased expression in the spinal cord and in the periphery in response to cytokines, growth factors, or other inflammatory stimuli.4

The formalin test is a widely used model of peripheral inflammation and acute central sensitization. The intrathecal administration of nonsteroidal antiinflammatory drugs (NSAIDs) has been reported to produce an analgesic effect during the rat formalin test, emphasizing the role of spinal COX in this facilitated state.5 Spinal expression of the c-fos immediate early gene has been used as a marker of neuronal activation.6

In this study, we sought to define the roles of spinal COX-1 and/or COX-2 in the facilitatory effect on behavior otherwise induced by intraplantar injection of formalin and the activation of superficial and deep dorsal horn neurons c-fos-like immunoreactivity (FLI) expression by examining the effects of intrathecal administration of the inhibitors SC-560, celecoxib, or ketorolac.

METHODS

Intrathecal Catheterization
Experiments were performed on male Sprague-Dawley rats (250–300 g) with free access to food and water. The laboratory was controlled to a 12-h light-dark cycle. The experimental protocol was approved by our Institutional Animal Care and Use Committee and the ethical guidelines for the treatment of animals of the International Association for the Study of Pain were followed. After each rat was anesthetized with halothane (3% induction; 1.5%–2% maintenance in 100% O₂), each animal received lumbar intrathecal catheters after an incision through the atlantooccipital membrane, and a polyethene tube (PE 10, Becton Dickinson) was inserted into the intrathecal space and passed for 8.5 cm, so the caudal end of the tube was localized to the lumbar enlargement.7 The catheter was externalized on the top of the skull and sealed. The wound was closed with 3–0 silk sutures. Rats showing neurological deficits postoperatively were promptly killed by a barbiturate overdose. After the operation, each experimental rat was acclimated to the environment for at least 4 days before the formalin test.

Drugs
Hamilton syringes (Hamilton microliter syringe, Hamilton, NV), which contained vehicle or experimental drugs, respectively, were prepared and blinded. All rats were randomly divided into five groups: saline, vehicle, SC560, celecoxib, or ketorolac group. Each group consisted of 6 to 8 rats. The intrathecally administered drugs were delivered in a total volume of 10 µL, followed by 10 µL of saline to flush the catheter. For intrathecal injection, the drugs were dissolved in 70% dimethyl sulfoxide (DMSO, Sigma; St. Louis, MO) and 30% saline so that the final dose was delivered in a 10 µL volume. The vehicle group received the same volume of vehicle (70% DMSO and 30% saline) intrathecally. Ketorolac was diluted in saline so that the final dose was also delivered in a 10 µL volume. The drugs used in this study were ketorolac (ketorolac tromethamine; Roche, Seoul, Korea), SC 560 (5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethyl pyrazole; Sigma, St. Louis, MO), and celecoxib (4-[5-(4-methylphenyl)-3-trifluoromethyl]-(1H-pyrazol-1-yl)-benzenesulfonamide; Pfizer; New York, NY). During the pilot tests for intrathecal COX inhibitors, we administered 10 µL of saline, 15, 30, 60, and 90µg of each COX inhibitor for the formalin-induced phase 1 behavior. Each dose of the COX inhibitors was administered to three rats. Using saline or a dose of 15 µg of the COX inhibitors, we were unable to observe an obvious consistent inhibition of the formalin-induced phase 1 behavioral response with all three of the COX inhibitors. Using a dose of 30 µg of COX inhibitors, we observed some inhibition of the phase 1 behavioral response in all inhibitors. Using a dose of 60 µg or 90 µg of COX inhibitors, we also observed an obvious inhibition of the phase 1 behavioral response in all COX inhibitors. With the doses administered in these pilot tests, no motor disturbance or behavioral side effects were observed. Because we wished to apply the effective dose of COX inhibitors, a dose of 30, 60, or 90 µg was chosen.

Behavioral Assessment
Rats in all groups were tested for possible side effects, such as reduction of righting, stepping, and irritability in the form of touch-evoked agitation or vocalization, before and after drug administration.

Formalin Test
Antinociception was assessed using the formalin test.8,9 To habituate the rats to the formalin test environment, the animals were placed in test chambers in groups of 3 for 15 min for 4 days and then placed individually on the fifth day. For the generation of inflammatory pain, we diluted a 37% formaldehyde solution (formalin, Sigma Chemical CO) to a final concentration of 5% formalin by mixing with saline. The formalin test was performed with a 5% formalin 50 µL injection into the right plantar surface of the hindpaw with a 26G needle. The formalin test was delivered 10 min after intrathecal injection of COX inhibitors or vehicle. Intraplantar injection of dilute formalin first stimulates nociceptors, resulting in a barrage of primary afferent fiber activity (phase 1: 0–10 min after formalin injection) and the continuing stimulation of nociceptors increases primary afferent fiber activity (phase 2: 10–60 min after formalin injection). After the formalin was injected, each rat was placed in a transparent plexiglas box and observed for behavioral reactions for 60 min. The number of flinches defined as lifting the paw injected with formalin was counted during 5 min-intervals for 60 min after the injection. Lifting the paw from the ground or shaking the lifted paw once was regarded as one flinching.

Tissue Preparation and Immunohistochemical Analysis
The rats were killed 2 h after formalin injections in the following manner. After anesthetizing each rat with 75 mg/kg pentobarbital intraperitoneally, surgery proceeded with a sternotomy, transcardiac aortic needle cannulation, and perfusion with 200 mL of phosphate buffered saline (PBS), followed by the addition of 500 mL of 4% paraformaldehyde/PBS. The lumbar spinal cord was extracted, postfixed for 8 h in 4% paraformaldehyde/PBS, then cryoprotected for 48 h in 30% sucrose/PBS at 4°C. The spinal cords were frozen on dry ice and 50-µm transverse sections were cut with a refrigerated Leica cryostat and collected at intervals of 100 µm. These free-floating sections were collected and immunohistochemistry for the Fos-like protein (polyclonal antibody 1:20,000 dilution, Oncogene Research Products, Cambridge, MA) was performed by the avidin-biotin peroxidase method of Hsu et al.10 After air-drying for 24 h, the sections were dehydrated in an ascending alcohol series and defatted in xylene. Negative control experiments were conducted with tissue sections from the formalin-injected controls by omitting the primary antibody from the above protocol and by the addition of 2 µg/mL of N-terminal Fos peptide to the primary antibody incubation solution. Neither of these controls showed any expression of FLI.

We randomly selected 4 sections at the L4/5 segmental level from each rat. Quantification of the FLI neurons was performed in the gray matter of both sides of the cord. Tissue sections were first examined by light-field microscopy to find the L4–5 segmental level according to the procedure of Molander et al.11 To study the laminar distribution of the spinal dorsal horn regions, they were defined as follows: the superficial dorsal horn (laminae I–II) and the neck of the dorsal horn (laminae V–VI). Cells were considered positive only if they demonstrated the appropriate size and shape, had dark brown to black nuclei, and were distinct from the background. For each rat, four randomly selected sections were counted. The investigator responsible for plotting and counting the FLI neurons was unaware of the left and right side of the section, control or drug-treated animals, and experimental procedures performed on the rats.

Data Analysis
To evaluate the drug effects, one-way analysis of variance was used. For all pair-wise multiple comparisons SigmaStat (SigmaStat, v. 3.01; Systat Software; Chicago, IL) was used. Pearson's correlation was used to determine the relationship between the flinching behavior and FLI. Wherever appropriate, the results are expressed as the mean ± sd. P values <0.05 were considered statistically significant.

RESULTS

Effects of Intrathecal COX Inhibition of Flinching Behavior
After intrathecal delivery of 70% DMSO/saline or saline, intraplantar injection of formalin evoked a biphasic incidence of flinching behavior (Table 1, Fig. 1).

View larger version (16K): [in this window] [in a new window]   Figure 1. Time-course curves of flinching behavior for 30µg of ketorolac, SC 560, celecoxib, vehicle, or saline given intrathecally 10 min before the formalin test. The total number of flinches per 5 min is plotted. Each line represents the group mean and sd.

Intrathecal injection of SC-560 did not demonstrate an inhibitory effect on flinching behavior (P > 0.05 vs vehicle) (Table 1). In contrast, celecoxib 90 µg was effective in producing inhibition of the flinching behavior during phase 1 and phase 2 (P < 0.05 vs vehicle). Ketorolac 90 µg reduced phase 1 flinching behavior (P < 0.05 vs saline). All doses of ketorolac reduced phase 2 flinching behavior (P < 0.05 vs saline) (Table 1).

No detectable effects on motor function and general behavior were observed in any of the drug-treatment groups, vehicle, or saline group.

Effects of Intrathecal COX Inhibition on Intraplantar Formalin-Evoked C-fos Expression
After unilateral intraplantar injection of formalin into the hind paw, there was a marked increase in the expression of c Fos (+) neurons in laminae I–II and laminae V–VI of L4–5 lumbar segments (Fig. 2). Ketorolac or celecoxib 90 µg groups demonstrated a significant decrease in FLI only in laminae I–II when compared with their each control (ketorolac versus saline, celecoxib versus vehicle) (P < 0.05) (Table 1).

View larger version (56K): [in this window] [in a new window]   Figure 2. Photomicrographs of 50 µm thick lumbar spinal cord sections show Fos-like immunoreactivity in L4–5 spinal segments (ipsilateral side to the formalin injection). (A) 70% DMSO/saline, (B) saline, (C–E) SC 560, celecoxib and ketorolac pretreatment 10 min before the formalin test.

By contrast, in laminae V–VI, only the ketorolac 60 or 90 µg groups produced a significant decrease in FLI when compared with the saline group (Table 1).

There was a significant correlation between flinching behavior and FLI at phase 2 and FLI of laminae V–VI (Table 2). In laminae V–VI, there was a significant inhibition of FLI by drug that showed a significant inhibition of phase 2 flinching behavior. There were no significant relationships between any pair of variables in the correlation (P > 0.05).

DISCUSSION

It is noteworthy that the administration of intrathecal ketorolac prevented formalin-related enhancement of nociceptive behavior and suppressed FLI. Celecoxib also prevented formalin-related behavior when 90 µg was administered intrathecally, but did not inhibit FLI expression in deep lamina.

Because ketorolac is a COX-1 preferred nonselective inhibitor (COX-2/COX-1 IC₅₀ ratio of 7 to several hundreds),12,13 the antihyperalgesic effect of ketorolac is mediated through COX-1 inhibition initially. But SC560 in this study was not able to reduce formalin– induced behavior. Therefore, we cannot conclude that the antinociceptive efficacy of ketorolac in this model was due to COX-1 inhibition mainly, even if ketorolac is a COX-1 preferred nonselective inhibitor. But we still have some questions about the role of COX-1. The role of COX-1 in spinal pain processing has been supported by other investigators in models of incisional model,14,15 formalin model,16 or neuropathic model.17

However, with three different doses of experiment groups we also observed the inhibitory effect of celecoxib 90 µg. COX-2 is also important in hyperalgesia. Others have emphasized the role of COX-2 alone in a formalin model18,19 or thermal hyperalgesia model.20 But we are still uncertain whether large doses of intrathecal celecoxib can act locally, systemically or supraspinally and whether 30 or 60 µg of celecoxib can act as an antihyperalgesia in this model. We think that a dual inhibition of COX-1 and COX-2 is more effective than COX-2 alone to inhibit formalin pain. The role of nonselective COX inhibitors in spinal pain processing has also been supported by other investigators of the formalin model.21

Nishiyama has also reported that intrathecal celecoxib decreased the flinch response with a dose of less than a 5 µg dose.22 The results of this study are not consistent with our intrathecal 30 or 60 µg studies. The reason for this difference may be in the application of the formalin test and the volume used for intrathecal administration. In the Nishiyama study, 20 µL was injected, and 10 µL was injected in the present study. To obtain a localized action of intrathecal drug, we had to control the total volume of intrathecal injection because the volume is the main controlling factor of the level of drug spread while the animals are freely moving. Therefore, a positive effect of celecoxib was possibly due to the capability of a wide delivery of drug along the intrathecal space, even though quite a small dose of celecoxib was administered in Nishiyama study. We think that the effects of COX-2 inhibitors on hyperalgesia are still controversial, as are the effects on formalin-induced pain.

According to previous investigations, NSAIDs have essentially no effect on Fos expression after acute noxious stimuli or on basal Fos expression after chronic inflammation.23 By contrast, other investigations have shown that NSAIDS reduce both Fos expression and behavioral response after inflammatory stimuli.24 Thus, NSAIDS reduce acute inflammatory pain and Fos in concert, but do not reduce Fos expression after noninflammatory stimuli or long-term inflammation. In this model, we demonstrated that intraplantar formalin-evoked pain activated c-Fos in the ipsilateral superficial and deep dorsal horn.

Our findings also demonstrated that FLI expression of laminae I–II was reduced by 90 µg of celecoxib or ketorolac treatment, regardless of behavioral responses. Nonselective COX inhibitors may have advantages in the suppression of FLI expression of whole laminae. We failed to obtain a significant inhibition of FLI expression of laminae V–VI in COX-2 inhibitors; even COX-2 inhibitors showed inhibition of phase 2 response efficiently. Both COX isoforms are constitutively expressed in the spinal cord, COX-1 mainly in dorsal horn glial cells and COX-2 in motoneurones of the ventral horns.25,26 As in the periphery, COX-2 behaves as an immediate early gene which is rapidly up-regulated in dorsal horn neurons in response to local or peripheral noxious stimulation.26 Thus, we assumed that the initial nociceptive behavior in the formalin test was probably basically independent of COX-2 up-regulation and, therefore, less responsive to celecoxib. However we observed reduced flinching behaviors in the celecoxib 90 µg group in this model. We cannot explain why celecoxib 90µg is less effective for inhibiting FLI expression in deep laminae than ketorolac, even if it can significantly inhibit phase 2 flinching. There are two possible explanations. First, this study of FLI was for a 2-h period after formalin administration, whereas behavioral studies only investigated during the first hour after administering formalin. Because nociceptive processing might continue 1 h after formalin, residual FLI expression could occur even in the absence of measurable pain behaviors. Therefore, we think that given the time frame of the inhibition (e.g., <2 h) is still not relevant to spinal inhibition of PG synthesis within this time frame. Second, we assume that COX-2 inhibitors do not completely suppress PG production in inflamed tissue, which is in part COX-1 dependent.

FLI expression of laminae V–VI correlates with the phase 2 response.

In summary, nonselective COX inhibitors result in consistent antihyperalgesic activity of behavioral responses and FLI expression of spinal dorsal horns. Nonselective or COX-2 inhibitor suppressed the phase 2 response of the formalin test. COX-2 inhibitors may be weaker antihyperalgesic drugs than the nonselective COX inhibitors. FLI expression of laminae V–VI correlates with the phase 2 response of the formalin test. These results reflect the role of PGs in the spinal dorsal horn in initiating facilitated pain states, findings which may relate to a presynaptic facilitation of transmitter release and a postsynaptic mechanism that serves to reduce inhibitory glycinergic neurotransmission. Inhibition of these processes is probably the major mechanism of the analgesic action of COX inhibitors.

Footnotes

Accepted for publication November 20, 2007.

Supported by KUIACF grant funded by the Korea University (K0517781).

Abstract was presented partially at the 11th World Congress on Pain on August 22, 2005 in Sydney, Australia.

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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 Google Scholar Google Scholar Articles by Lee, I. O. Articles by Seo, Y. Search for Related Content PubMed PubMed Citation Articles by Lee, I. O. Articles by Seo, Y. Related Collections Mechanisms Pain Mechanisms Preclinical Pharmacology Pain Pharmacology