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Preoperative Inhibition of Cyclooxygenase-1 in the Spinal Cord Reduces Postoperative Pain

Zhu, Xiaoying MD; Conklin, Dawn R. BS; Eisenach, James C. MD

doi: 10.1213/01.ANE.0000148127.53832.8E
Pain Medicine: Research Report

Intrathecal administration of cyclooxygenase (COX)-1, but not COX-2, specific inhibitors given on postoperative day 1 has analgesic effects in an incisional model of postoperative pain. We investigated the effects of preoperative administration of intrathecal COX inhibitors in this model. Fifteen minutes before surgery, rats received intrathecally the COX-1 preferring inhibitor, ketorolac, the specific COX-1 inhibitor, SC-560, the COX-2 inhibitor, NS-398, or vehicle. A 1-cm longitudinal incision was then made through skin, fascia, and muscles of the plantar aspect of a left paw in male rats. Withdrawal threshold to von Frey filaments was measured at 2 h, 4 h, and at intervals up to 5 days later. Ketorolac and SC-560 increased withdrawal threshold to mechanical stimulation, but NS-398 had no significant effect. These results suggest that COX-1 plays an important role in spinal cord pain processing and sensitization after surgery and that preoperative intrathecal administration of specific COX-1 inhibitors may be useful to treat postoperative pain.

IMPLICATIONS: Spinal administration of ketorolac in humans is under investigation, and these data in animals suggest that spinal ketorolac injection before surgery could reduce postoperative pain.

Program of Neuroscience, Department of Anesthesiology and Center for the Study of Pharmacologic Plasticity in the Presence of Pain, Wake Forest University School of Medicine, Winston-Salem, North Carolina

Supported, in part, by the National Institutes of Health grants GM48085 and NS41386.

Accepted for publication September 29, 2004.

Address correspondence and reprint requests to Xiaoying Zhu, MD, Department of Anesthesiology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157. Address e-mail to xzhu@wfubmc.edu.

Prostaglandins (PGs) are important mediators of pain and inflammation (1). Cyclooxygenase (COX) is the rate-limiting enzyme that catalyzes the conversion of arachidonic acid to PGs. Two isoforms of COX, COX-1 and COX-2, are expressed constitutively in the spinal cord (2–4). Hindpaw injection of complete Freund’s adjuvant produces mechanical allodynia and thermal hyperalgesia in the hindpaws and joints, accompanied by a significant increase in COX-2 mRNA (5,6) and protein (6) in the lumbar spinal cord. However, COX-1 mRNA (5) and protein (7) remain unchanged by these manipulations. Intrathecal COX-2 inhibitors attenuate inflammation-induced mechanical allodynia and thermal hyperalgesia (6,8–10), but a COX-1 inhibitor has no such effect (9). These studies demonstrate that COX-2 plays a prominent role in inflammatory pain.

In contrast to these results with inflammation, in an incisional model of postoperative pain, COX-1 expression increases in glia in the ipsilateral lumbar spinal dorsal horn. In addition, postoperative intrathecal administration of COX-1, but not COX-2, specific inhibitors exerts analgesic effects (4,11). Because intrathecal injections are usually administered before surgery and preoperative treatment with some drugs reduces postoperative pain (12,13), we investigated whether intrathecal preoperative treatment with COX-1 inhibitors attenuates postoperative pain in this paw incision model by examining the effects of intrathecal COX-1 and COX-2 inhibitors given 15 min before surgery on postoperative hypersensitivity to mechanical stimulation. Lack of selective COX-3 inhibitors precluded us from examining the role of this isoenzyme.

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Methods

With approval by the Animal Care and Use Committee in Wake Forest University, male Sprague-Dawley rats (220–260 g) were used in this study. Rats were anesthetized with 2%–3% halothane and lumbar intrathecal catheters were implanted by a slightly modified procedure to that previously described (14). Briefly, an incision was made through the atlanto-occipital membrane and a 7.5- to 8.0-cm 32-gauge catheter was inserted into the intrathecal space such that the caudal end of the catheter was near to the lumbar enlargement. One week later, after paw withdrawal threshold was measured and 15 min before paw incision surgery, rats were randomized to receive intrathecal ketorolac, 50 μg, NS-398, 50 μg, SC-560, 100 μg, or either saline or 100% dimethyl sulfoxide vehicle control in a volume of 10 μL. Then paw incision surgery was conducted on these rats as described by Brennan et al. (15). Rats were anesthetized with 2%–3% halothane and a 1.0-cm longitudinal incision was made through skin, fascia, and muscles of the plantar aspect of a left paw in these rats starting 0.5 cm from the proximal edge of the heel and extending toward the toes. The muscle origin and insertion remained intact. After manipulation of the muscles and hemostasis, the skin was sutured and animals were allowed to recover in their cages. Paw withdrawal threshold to mechanical stimulus was then measured at 2 h and 4 h after drug treatment and at 1, 2, 3, and 5 days after paw incision. For measurement of paw withdrawal threshold, rats were placed in clear plastic cages on an elevated mesh floor and allow to accommodate for 20 min. Paw withdrawal threshold in response to mechanical stimulation was measured using the up-and-down method (16) by applying calibrated von Frey filaments (Stoelting, Wood Dale, IL) from underneath the cage through openings in the mesh floor to the medial area adjacent to the incision and to the same area before paw incision (15). Fiber 5.88 was used as the cutoff fiber because fiber stronger than 5.88 lifted the paw and may also have caused tissue damage.

The drug doses of COX inhibitors were chosen based on studies by others, and by us, with intrathecal administration. SC-560, 100 μg intrathecally given, has no effect on intrathecal substance P-induced thermal hyperalgesia (9) but was shown to increase the paw withdrawal threshold (4) in this model of peripheral surgery. Intrathecal NS-398, 30 μg, prevents hypersensitivity induced by intrathecal N-methyl-d-aspartic acid (NMDA) or alpha amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) (17), attenuates thermal hyperalgesia induced by paw inflammation from carrageenan (10). However, this dose (11) or 50 μg (4) has no significant effect in the paw incision model. Therefore, in the current study 100 μg SC-560 and 50 μg NS-398 were chosen. This study was partially blinded because the drug NS-398 is yellow in color, and the same person did both the injection and the behavioral test.

Data are presented as mean ± sem, and were analyzed using two-way analysis of variance followed by Student-Newman-Keuls test. P < 0.05 was considered to be significant.

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Results

Paw incision reduced the paw withdrawal threshold to mechanical stimulation to a similar extent in animals with or without indwelling intrathecal catheters. A COX-1 preferring inhibitor ketorolac (18) given intrathecally 15 min before surgery increased the paw withdrawal threshold to mechanical stimulation compared with saline control (Fig. 1). Two other COX inhibitors were tested: SC-560 (COX-1 selective) and NS-398 (COX-2 selective). Because these two drugs had very limited solubility in water, 100% dimethyl sulfoxide was used to dissolve these drugs. Intrathecal SC-560, when administrated 15 min before surgery, increased the paw withdrawal threshold, whereas NS-398 had no effect compared to dimethyl sulfoxide control (Fig. 2). The analgesic effect of SC-560 lasted longer than that of ketorolac (Figs. 1 and 2).

Figure 1

Figure 1

Figure 2

Figure 2

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Discussion

The current study showed that, 15 minutes before surgery, intrathecal administration of a COX-1 preferring inhibitor, ketorolac, and a selective COX-1 inhibitor, SC-560, reduced the hypersensitivity induced by paw incision but a COX-2 inhibitor, NS-398, had no significant effect. These results support our previous observation that spinal COX-1 plays an important role in postoperative pain processes (4). In contrast, other studies have shown that inhibition of spinal COX-1 by SC-560 has no effect on intrathecal substance P-induced thermal hyperalgesia (9). In addition, intrathecal injection of the COX-2 inhibitor, NS-398, prevents hypersensitivity induced by intrathecal NMDA or AMPA (17) and attenuates thermal hyperalgesia induced by paw inflammation from carrageenan (10). These results underscore the unique pharmacology of postoperative pain.

Similar to postoperative treatment (4), preoperative COX-1 inhibitors are analgesic in the paw incision model. Studies have shown that there is a basal release of PGs in the spinal cord, and COX-1 contributes to this basal release of PGs because it is constitutively expressed in rat spinal cord (2–4). COX-1 inhibitors, injected 15 minutes before peripheral surgery, would decrease PGs in the spinal cord by inhibiting the constitutive COX-1. After peripheral surgery, COX-1 is up-regulated (4). The enzyme activity of the up-regulated COX-1 will also be reduced by the inhibitors, although it is not clear how long it takes for the inhibitors to be completely cleared from the cerebrospinal fluid. The final result is that the total PGs in the spinal cord are decreased, thus reducing the sensitizing effects of PGs.

We do not argue that COX-2 inhibitors are useless for treating postoperative pain. Actually, selective COX-2 inhibitors, systemically administrated, have been shown to relieve postoperative pain in humans (19) and to reduce paw incision-induced mechanical hypersensitivity in rats (20,21). Because surgical tissue injury results in the peripheral synthesis and release of many inflammatory mediators, including PGs, and because these mediators sensitize peripheral nociceptors by altering their firing threshold and sometimes causing direct stimulation (22), the COX-2 inhibitors are considered to achieve analgesic effects by inhibiting COX-2 enzyme in the peripheral tissue and thus reduce PG synthesis. Yet our results argue that the COX-1 enzyme is also important in the underlining mechanisms of postoperative pain.

In this study and our previous investigation (4), COX-1 inhibitors did not totally reverse but only reduced the hypersensitivity induced by paw incision. This could be because COX-1 is also induced in the gracile nucleus (4), a site distant from the lumbar intrathecal catheter tip and unlikely to be inhibited by these drugs given through the lumbar intrathecal catheter. Inhibition of both COX-1 and COX-2 is probably needed to achieve more effective analgesia in this pain model because it has been shown that nonselective COX inhibitors offer better analgesia than selective COX-2 inhibitors in the formalin test (23) and for arthritis (24). In addition, PGs synthesized by COX-3 (25), if existing in the spinal cord, could also contribute to sensitization. SC-560 has a longer-lasting analgesic effect than ketorolac. This is probably a result of the very low water solubility of SC-560 likely leading to slower clearance from cerebrospinal fluid.

In conclusion, COX-1 preferring and selective inhibitors, when administrated preoperatively, reduce paw incision-induced mechanical hypersensitivity, but the COX-2 inhibitors lack efficacy. These data support the notion that COX-1 may play an important role in pain processing and sensitization in spinal cord after peripheral surgery and that preoperative intrathecal COX-1 inhibitors may be useful for treating postoperative pain.

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