Anesth Analg 2002;95:1708-1712
© 2002 International Anesthesia Research Society
PAIN MEDICINE
The Effects of Intrathecal Administration of an Antagonist for Prostaglandin E Receptor Subtype EP1 on Mechanical and Thermal Hyperalgesia in a Rat Model of Postoperative Pain
Keiichi Omote, MD,
Hiroki Yamamoto, MD,
Tomoyuki Kawamata, MD,
Yoshito Nakayama, MD, and
Akiyoshi Namiki, MD
Department of Anesthesiology, Sapporo Medical University School of Medicine, Sapporo, Japan
Address correspondence and reprint requests to Keiichi Omote, MD, Associate Professor, Department of Anesthesiology, Sapporo Medical University School of Medicine, South-1, West-16, Chuoke, Sapporo 060-8543, Japan. Address e-mail to komote{at}sapmed.ac.jp
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Abstract
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Despite substantial advances in understanding acute pain mechanisms and in the treatment of pain, postoperative pain, especially mechanically evoked pain (incident pain), is generally not effectively treated. Tissue injury and inflammation increase the release of prostaglandin E2 in the spinal cord, contributing to the development of hyperalgesia. We designed the present study to determine whether the intrathecal administration of an antagonist for prostaglandin E2 receptor subtype EP1, ONO-8711, has an analgesic effect on incision-induced mechanical and thermal hyperalgesia. A 1-cm longitudinal skin incision was made in the plantar aspect of the rat foot. The withdrawal threshold to mechanical stimulation and the withdrawal latency to thermal stimulation applied adjacent to the wound of the hindpaw were investigated. Both mechanical and thermal hyperalgesia were observed at 2 h and 24 h after the incision had been made. ONO-8711 (50, 80, 100 µg) or saline was administered intrathecally. ONO-8711 significantly increased the withdrawal thresholds to mechanical stimulation, but not to thermal stimulation, in a dose- and time-dependent manner. We conclude that EP1 receptor-mediated sensitization of the spinal dorsal horn may contribute to the generation of mechanical, but not thermal, hyperalgesia and that an EP1 receptor antagonist administered intrathecally is a potential analgesic for postoperative pain, especially mechanically evoked pain (incident pain).
IMPLICATIONS: We examined the effects of an intrathecally administered selective EP1 receptor antagonist on mechanical and thermal hyperalgesia in a postoperative pain model. The intrathecal EP1 receptor antagonist inhibited the mechanical, but not thermal, hyperalgesia, indicating the potential for an EP1 receptor antagonist to be used as an analgesic for postoperative pain, especially incident pain.
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Introduction
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Despite substantial advances in understanding acute pain mechanisms and in the treatment of pain, postoperative pain still causes significant suffering and continues to challenge physicians. Postoperative pain not only causes considerable discomfort for the patient but also prolongs the recovery period, causes various complications, and is a burden on scarce health care resources (1). The ability to provide adequate postoperative pain re-lief is one of the major challenges for health care providers.
In peripheral tissue damage and inflammation after surgery, peripherally released prostaglandin E2 (PGE2) acts on nociceptive neurons (2). PGE2 has important intra- and intercellular roles in nociception (3). In our previous study (4), we demonstrated that a peripherally administered antagonist for the PGE2 receptor subtype EP1 effectively inhibited the mechanical hyperalgesia induced by an incision. Thus, EP1 receptor-mediated sensitization of sensory nerve fibers might contribute to the generation of mechanical hyperalgesia produced by an incisional injury. Recent evidence has indicated that PGE2 is also produced in the spinal cord after tissue injury (5,6). Moreover, behavioral and electrophysiologic studies have suggested that PGE2 facilitates nociceptive transmission in the spinal cord (7,8), contributing to central sensitization. Because continuing inputs from damaged peripheral sites persist and affect central regions, spinal EP1 receptors may be repeatedly activated. The repetitive activation of EP1 receptors may contribute to activation of the intracellular cascade in the dorsal horn neurons, resulting in induction of central sensitization and hyperalgesia after surgery.
Mechanical hyperalgesia, which is an exaggerated response to noxious stimuli, occurs directly on, or immediately adjacent to, surgical incisions in patients and is correlated with pain during coughing or movement (9–11). Although pain during rest is generally easy to treat, the evoked pain during a body movement (incident pain) is not.
A rat model of postoperative pain has been described (12). It has been suggested that the model displays some similarities to the human postoperative pain state. The present study was designed to determine the effects of an intrathecally administered selective EP1 receptor antagonist, ONO-8711, on mechanical and thermal hyperalgesia in a rat postoperative pain model.
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Methods
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The protocol for this study was approved by the Sapporo Medical University Animal Care and Use Committee. Experiments were conducted on male Sprague-Dawley rats weighing 250–300 g (Japan SLC, Hamamatsu, Japan), which were housed individually in a temperature-controlled (21 ± 1°C) room with a 12-h light-dark cycle and given free access to food and water.
Under general anesthesia (3% isoflurane in oxygen), a polyethylene intrathecal catheter (PE-10; Becton Dickinson, Sparks, MD) was inserted 15 mm cephalad into the lumbar subarachnoid space at the L4-5 intervertebrae with the tip of the catheter located near the lumbar enlargement of the spinal cord using a method described previously (13). The catheter was tunneled subcutaneously and externalized through the skin in the neck region. The animals were allowed at least 6 days of postsurgical recovery before being used in experiments. In the experiments, we used only animals that showed normal behavior and motor function and had shown complete paralysis of bilateral hind legs and tail after the administration of 2% lidocaine 10 µL through the intrathecal catheter. After the experimentation, each rat was killed by an overdose of halothane. The spread of injected materials was determined by a postmortem intrathecal injection of 1% methylene blue (10 µL) followed by a flush of saline, and the position of the catheter tip was verified.
The incisional surgery was based on the procedure recently described by Brennan et al. (12). Under general anesthesia (3% isoflurane in oxygen), the plantar surface of the right hindpaw of each rat was disinfected with povidone iodine, and 30,000 IU of penicillin-G (benzylpenicillin; Sigma Chemical Co., St. Louis, MO) was injected into the triceps muscle. A 1-cm longitudinal incision was made through the skin and fascia of the plantar aspect of the foot, starting 0.5 cm from the edge of the heel and extending toward the toes. The plantaris muscle was elevated by using forceps and incised longitudinally. The skin was apposed tightly with 3 simple sutures of 5-0 nylon using a CV-6 needle. After surgery, the animals were allowed to recover in cages.
To evaluate mechanical hyperalgesia, withdrawal thresholds to mechanical stimulation were determined by using calibrated von Frey filaments (0.0045–75.8580 g bending force; Stoelting Co., Wood Dale, IL), which were applied from underneath the cage through openings (12 x 12 mm) in the wire mesh floor to the area adjacent to the wound and to the same area on the noninjured foot. Each filament was applied once starting with 0.0045 g and continuing until a withdrawal response occurred. The test was repeated three times in each time point. A withdrawal response was considered to be complete lifting of the hindpaw off the surface of the cage or flinching. The lowest force producing a response was considered to be the withdrawal threshold.
To evaluate thermal hyperalgesia, withdrawal latency to thermal stimulation was measured by using testing equipment (Plantar Test 7370; Ugo Basile, Comerio, Italy). A rat was placed in a clear plastic chamber (17 x 22 x 14 cm) with a glass floor (2 mm thick). A radiant heat source was positioned under the glass floor so that it focused on the plantar surface of one hindpaw. Thermal stimulation was applied to the area adjacent to the wound and to the same area on the contralateral side. The radiant heat source consisted of a high-intensity projector lamp bulb (Halogen "Bellaphot," model 64607, 8 V-50 W; Osram, München, Germany) located 30 mm below the glass floor and projecting through a 5 x 10 mm aperture in the top of a movable case. The withdrawal latency to the thermal stimulation was defined as the time from onset of radiant heat to withdrawal of the rat hindpaw.
Before the incisional surgery, the control value of withdrawal thresholds to mechanical stimulation or withdrawal latencies to thermal stimulation on both hindpaws was measured. After 2 h of recovery time after the incision, the baseline value of the threshold or latency was determined. ONO-8711 (50, 80, or 100 µg) or saline was administered intrathecally. After the administration, withdrawal threshold or the latency was measured at 15-min intervals for 120 min. On postoperative day 1 (24 h after the incision), the withdrawal threshold or the latency was again determined. ONO-8711 at doses of 50, 80, or 100 µg or saline was administered intrathecally, and the withdrawal threshold or latency was measured. This experiment was also performed on nonsurgical rats.
The novel selective EP1 antagonist ONO-8711 was supplied by Ono Pharmaceutical Co., Ltd. (Osaka, Japan). This compound was freshly dissolved in physiologic saline in concentrations that allowed intrathecal injections in 10-µL volumes.
The results of withdrawal threshold to mechanical stimulation and withdrawal latency to thermal stimulation were expressed as mean ± SEM, and were analyzed by using two-way analysis of variance for repeated measurements, followed by Scheffé F-test A P value < 0.05 was considered statistically significance.
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Results
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Table 1 shows the control and baseline (2 h after the incision) values of withdrawal thresholds to mechanical stimulation and withdrawal latencies to thermal stimulation on the ipsi- and contralateral sides. Both mechanical and thermal hyperalgesia were observed on the ipsilateral side after the incisional surgery.
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Table 1. Control and Baseline Values Showing Mechanical and Thermal Hyperalgesia on the Ipsi- and Contralateral Sides
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Withdrawal thresholds to mechanical stimulation were remarkably decreased at 2 h after the surgery (Fig. 1, top). The intrathecally administered ONO-8711, but not saline, significantly (P < 0.01) increased the threshold in a dose-dependent manner (n = 7–8 in each group) (Fig. 1, top). At 24 h after the surgery, the baseline withdrawal thresholds (3.76 ± 0.56 g) were comparable to those on the day of surgery; thus, mechanical hyperalgesia was persistent. No effect from the previous treatment was evident. ONO-8711, but not saline, significantly (P < 0.01) increased the withdrawal threshold in a dose-dependent manner (n = 7–8 in each group) (Fig. 1, bottom). Withdrawal thresholds to mechanical stimulation on the contralateral hindpaw did not show any changes at 2 and 24 h after the surgery, and none of the doses of ONO-8711 caused any significant changes (data not shown).
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Figure 1. Effects of intrathecally administered ONO-8711 (50, 80, and 100 µg) and saline on mechanical hyperalgesia caused by incision at 2 h (top) and 24 h (bottom) after the incision (n = 7–8 in each group). The data are expressed as means ± SEM. *P < 0.05 versus baseline, **P < 0.01 versus baseline,  P < 0.01 among groups. Note that the decreased withdrawal thresholds to mechanical stimulation increased after the intrathecal administration of ONO-8711.
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Table 2 shows the 50% effective dose (ED50) for intrathecally administered ONO-8711 and that for peripherally administered ONO-8711 that had been described in the previous report (4). ED50 was calculated from a simple area under the curve. The ED50 for intrathecal ONO-8711 was about twice that for peripheral ONO-8711.
Figure 2 shows the thermal latencies after the incisional surgery and the effects of intrathecally administered ONO-8711. At 2 h after the surgery, the mean latency was significantly (P < 0.05) shortened compared with that before the surgery. Intrathecally administered ONO-8711 did not have any significant effects on the latency for up to 120 min after administration (Fig. 2, top). At 24 h after the surgery, the baseline withdrawal latencies (4.58 ± 0.45 s) were similar to those on the day of surgery; thus, thermal hyperalgesia was persistent. As seen from the case at 2 h after the surgery, no significant effects of intrathecally administered ONO-8711 on the shortened latencies were observed (Fig. 2, bottom). No significant changes in withdrawal latencies to thermal stimulation on the contralateral hindpaw were observed at 2 h and 24 h after the surgery, and none of the dose of ONO-8711 caused any changes (data not shown).
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Figure 2. Effects of intrathecally administered ONO-8711 (50, 80, and 100 µg) and saline on thermal hyperalgesia caused by incision at 2 h (top) and 24 h (bottom) after the incision (n = 7–8 in each group). The data are expressed as means ± SEM. Note that the withdrawal latency to thermal stimulation did not show any changes after the intrathecal administration of ONO-8711 at any doses used.
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In nonsurgical rats, intrathecally administered ONO- 8711 did not cause any significant changes in the withdrawal thresholds to mechanical stimulation and the withdrawal latencies to thermal stimulation (data not shown).
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Discussion
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The present study demonstrated that withdrawal thresholds to mechanical stimulation decreased and withdrawal latencies to thermal stimulation were shortened on the ipsilateral side to the incision at 2 hours and 24 hours after surgery. The intrathecal administration of a selective EP1 antagonist, ONO-8711, inhibited the mechanical hyperalgesia in a dose-dependent manner. In contrast, thermal hyperalgesia was not attenuated by intrathecally administered ONO-8711.
PGE2 is thought to be involved in central sensitization because it has been shown that PGE2 is released in the spinal cord in some models of persistent nociception, that PGE2 induces neuronal hyperexcitability and release of neuroactive compounds, that application of PGE2 to the spinal cord induces hyperalgesia and allodynia, which are typical of central sensitization, and that spinal administration of cyclooxygenase inhibitors reduces manifestations of central sensitization in various experimental models and in humans (3). PGE2 activates different second messenger pathways. The receptors for PGE2 are divided into four subtypes (EP1, EP2, EP3, and EP4) on the basis of the distinct genes and signal transduction pathways (14). The activation of EP1 receptors initiates an influx of calcium ions. The EP2 and EP4 receptors are essentially coupled to stimulation of adenylate cyclase, which leads to an increase of intracellular adenosine 3',5'-cyclic monophosphate. The EP1 receptor mediates increases in intracellular calcium ions (14), probably by opening a novel calcium channel that allows inflow from the extracellular milieu and thus facilitates neurotransmitter release. EP1 receptors probably have some role in neurotransmitter release from primary afferents (3), although the type of such afferents is not known. It has been reported that EP1 receptor antagonists (SC-51089 and SC-51234A) inhibited only the late phase of a formalin test, which is thought to arise mainly from sensitization of spinal neurons, but the early phase, which is thought to be attributed mainly to afferent activity, was unaffected (15). ONO-8711 used in this study is the most selective antagonist for EP1 receptors currently available (16). Thus, it seems that spinal EP1 receptors are activated in the postoperative pain model and that this antagonist for EP1 receptors is able to inhibit the development of incision-induced hyperalgesia.
Intrathecally administered ONO-8711 inhibited mechanical, but not thermal, hyperalgesia in the postoperative pain model used in the present study. However, it has been shown that the oral administration of ONO-8711 reduced both mechanical and thermal hyperalgesia in a chronic constriction injury model (17). The different degree of effectiveness of the EP1 antagonist on mechanical and thermal hyperalgesia may reflect the different mechanisms that are thought to underlie these two phenomena in a postoperative pain model. Heat-sensitive and mechanosensitive channels or receptors have been identified and cloned (18,19). It is possible that the patterns and modes of the transmission of nociceptive information from thermal and mechanical nociceptions to the spinal cord are different. This might reflect the results showing that intrathecally administered ONO-8711 has antihyperalgesic effects on mechanical, but not thermal, hyperalgesia in postoperative pain.
In a clinical situation, pain from a surgical incision is exacerbated by mechanical stimulation such as coughing and deep breathing. This increased mechanical sensitivity of the surgical incision is crucial because mechanically evoked pain during body function (incident pain) is difficult to manage. Although the clinical significance of thermal hyperalgesia in postoperative pain is not yet clear, thermal hyperalgesia is very rarely a clinical problem in humans. However, it has been proposed that fever, local inflammation, and warmth from vasodilatation might enhance nociceptor activity in surgical wounds (20).
We analyzed the ED50 for intrathecal and peripheral ONO-8711 in the postoperative pain model. The ED50 for intrathecal ONO-8711 was about twice that for peripheral ONO-8711. This might mean that, although the EP1 receptors at the spinal cord as well as those at the incisional site were activated, peripheral EP1 receptors would greatly contribute to producing a mechanical hyperalgesic state than the spinal EP1 receptors after the surgery.
In present clinical practice, nonsteroidal antiinflammatory drugs have been widely used for management of postoperative pain. In the present study, we demonstrated the analgesic effect of EP1 antagonist ONO-8711; however, a further study needs to determine whether EP1 antagonists offer any benefit over nonsteroidal antiinflammatory drugs, including potency and side effects.
In summary, the EP1 receptor antagonist ONO-8711 administered intrathecally inhibited the mechanical, but not thermal, hyperalgesia induced by incisional surgery. EP1 receptor-mediated sensitization of the spinal dorsal horn might contribute to the generation of mechanical, but not thermal, hyperalgesia. Consequently, an EP1 receptor antagonist administered intrathecally is a potential analgesic for postoperative pain, especially mechanically evoked pain (incident pain).
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Acknowledgments
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Financial support for this work was provided by a Grant-in Aid for Scientific Research (No. 0930737) from the Ministry of Education, Tokyo, Japan.
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Accepted for publication August 13, 2002.
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Abstract
Introduction
