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REGIONAL ANESTHESIA AND PAIN MEDICINE

The Effects of Epidural Bupivacaine, Morphine, and Their Combination on Thermal Nociception with Different Stimulus Intensity in Rats

Department of Anesthesiology, The University of Tokyo, Tokyo, Japan

Address correspondence and reprint requests to Tomoki Nishiyama, MD, PhD, 3-2-6-603, Kawaguchi, Kawaguchi-shi, Saitama, 332-0015, Japan. Address e-mail to nishiyamat-ane{at}h.u-tokyo.ac.jp

	Abstract

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The analgesic effect of drugs depends on the stimulus intensity as well as the potency of the drugs. We investigated the effects of stimulus intensity on antinociceptive potencies of epidural bupivacaine + morphine. Sprague-Dawley rats implanted with chronic lumbar epidural catheters were tested for paw withdrawal response to thermal stimulation after the epidural injection of bupivacaine, morphine, or bupivacaine + morphine. Two stimulation currents were used, 5.1 and 4.6 A, to provide baseline response latency of approximately 5.0 s (high intensity) and 10.0 s (low intensity), respectively. Increasing the dose of epidural morphine in a dose range that had a maximum effect on low-intensity stimulation was not effective for high-intensity stimulation. Bupivacaine, which alone had no effect, potentiated the antinociceptive effect of epidural morphine at both high- and low-intensity stimuli similarly. We concluded that bupivacaine potentiated the analgesic effect of epidural morphine at both weak and strong nociceptive stimuli similarly, whereas increasing the dose of epidural morphine was not as effective for strong nociceptive stimulation. Therefore, adding bupivacaine might be more effective than increasing the dose of epidural morphine for intense nociception.

Implications: When patients have severe pain even when receiving epidural morphine, adding bupivacaine might be more effective than increasing the dose of epidural morphine.

	Introduction

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The analgesic effect of an analgesic depends on the stimulus intensity as well as the potency of the drug. The more intense the nociceptive stimulus, the more intense is the evoked activity of nociceptive neurons, and the larger the dose of the analgesic drug required to block the nociceptive responses (1). Saeki and Yaksh (2) reported that, in rats, intrathecally administered µ-opioids with high intrinsic efficacy, such as sufentanil and [D-Ala2-N-Phe4, Gly-ol]-enkephalin, showed less shift in the dose-response curves with an increase in stimulus intensity than morphine, an agonist with low intrinsic efficacy. Further, at the high-intensity stimulation, intrathecal morphine, but not sufentanil, was suggested to be a partial agonist (3).

Epidural drug administration is frequently used for pain management and drugs acting on different receptors (e.g., local anesthetics and opioids) are often coadministered epidurally. Clinically, small-dose epidural bupivacaine potentiates postoperative small-dose epidural morphine analgesia during movement and cough but not at rest (4). This result suggests that there are differences in the interaction of epidural bupivacaine and morphine at different stimulus intensities. However, in experimental studies of the interaction of epidural or spinal opioids and local anesthetics, the nociceptive intensity had not been considered (5–8). Large doses of bupivacaine and lidocaine potentiate spinal morphine antinociception in a synergistic manner in thermal stimulation studies (5–7). Epidurally coadministered morphine and lidocaine produce synergistic analgesia and prolong the duration of analgesia in somatic and visceral nociception (8). We investigated the effects of stimulus intensity on antinociceptive potencies of epidural bupivacaine, morphine, and the combination of these two drugs using a new rat model of epidural catheterization (9,10). The new model requires no hole, groove, or dissection in the bone and is less invasive than the previous models (11,12) in which the bone is injured. In addition, the efficacy ratio between epidural and intrathecal drug administration by using this new model is the same as that in clinical practice (9,10). Therefore, we expect the results of the present study to be clinically relevant.

	Methods

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After approval of our institutional animal care committee, Sprague-Dawley rats (300–350 g; Nippon Bio-Supply, Tokyo, Japan) were implanted with chronic lumbar epidural catheters under halothane (2%) anesthesia according to the method described previously (9). Briefly, an epidural catheter was made with a 2-cm polyethylene tube (PE-5; Clay Adams, Parsippany, NJ) heat-melted to a distal side of 12-cm polyethylene tube (PE-10; Clay Adams). The catheter had two knots; the first knot was at the connection between PE-5 and PE-10, and the second knot was 5 mm distal to the first knot. Tying the tube and covering the knot with acrylic dental cement made the second knot. A midline skin incision was made at the T13 area. Muscles were bluntly dissected from the vertebrae and the intervertebral space was exposed. The intervertebral ligament was cut and a catheter was inserted into the epidural space 2 cm caudally. The catheter tip was located at the L3-4 level, which was anatomically confirmed after the study by exposing the spinal column and injecting a dye infusion into the catheter. The first knot was placed in the space between the two vertebrae, and the second knot was covered by muscle. The PE-10 portion was tunneled subcutaneously to the neck to exit. The catheter tip was plugged with a 28-gauge steel wire. Only rats with no motor disturbance were used on the second postoperative day according to our previous result (10).

In each dose group, eight randomly selected rats were used. Morphine sulfate (Merck, Sharpe, and Dohme, West Point, PA) 1, 10, 30, 100, and 300 µg and bupivacaine (Sigma Chemical Company, St. Louis, MO) 100, 200, and 400 µg (maximal soluble dose in saline) were dissolved in 20 µL of saline. For the combination study, bupivacaine 100 µg or 200 µg + morphine 1, 10, or 30 µg were dissolved in 20 µL of saline. Naloxone (Sigma) 90 µg was dissolved in 300 µL of saline and injected intraperitoneally 10 min before the epidural administration of bupivacaine 100 µg + morphine 30 µg. After the epidural drug injection, the catheter was flushed with a subsequent injection of 10 µL of normal saline. Micro injector syringes were used for all injections.

Electrical thermal stimulation was used to assess nociceptive responses. The rats were placed in a clear plastic cage on a clear glass. The floor of the cage was kept at 30°C. A movable radiant heat source (halogen projector lamp CXL/CXP 50 W 8 V; Ushio, Tokyo, Japan) was positioned to focus on the plantar surface of one hind paw where it was in contact with the glass (13). The latency was defined as the time required for the paw to withdraw from thermal stimulation. Cutoff time was 20 s to avoid injury to the paw. Two stimulation currents were used, 5.1 and 4.6 A, to provide baseline response latency (control without drug injection) of approximately 5.0 s (high intensity) and 10.0 s (low intensity), respectively. The temperature was monitored on the movable heat source.

All rats were allowed 10 to 15 min to acclimate to the device. Baseline latency was measured. At 15, 30, 60, 90, 120, 180, 240, and 300 min after the drug administration, the latency was measured. The latency was indicated as the average of the left and right paw of each rat as shown in the previous study (3).

The general behavior (including agitation and allodynia), motor function, flaccidity, pinna reflex, and corneal reflex were also examined at 15, 30, 60, 90, 120, 180, 240, and 300 min after drug administration. Agitation was judged as spontaneous irritable movement and/or vocalization. The presence of allodynia was examined by looking for agitation (escape and/or vocalization) evoked by lightly stroking the flank of the rat with a small probe. Motor function was evaluated by the placing/stepping reflex and the righting reflex. The former was evoked by drawing the dorsum of either hind paw across the edge of the table. Normally, rats try to put the paw ahead to walk. The latter was assessed by placing the rat horizontally with its back on the table, which normally gives an immediate, coordinated twisting of the body to an upright position. The disturbance of the righting reflex also shows damage to the central nervous system. Flaccidity was judged as muscle weakness. Pinna and corneal reflexes were examined with a paper string. When a paper string is put into the ear canal or touched to the cornea, normally, rats shake their head or blink, respectively, to avoid the stimulation.

Data were expressed as mean ± SE (SEM). Time-course studies were indicated as latencies in seconds and were analyzed with two-way repeated measures analysis of variance followed by Newman-Keuls test if appropriate. Latencies were also converted to percent of maximal possible effect (%MPE) as follows: %MPE = (postdrug latency - predrug latency) x 100/(cut off latency - predrug latency). Dose-response curves associated with low- and high-intensity stimuli were compared for parallelism. The 50% effective dose (ED₅₀), dose ratio (ED₅₀ at high-intensity stimulation/ED₅₀ at low-intensity stimulation), and slope of the regression line were calculated with 95% confidential interval. The slopes of regression line of morphine were not linear; therefore, the slopes were calculated by dividing them into two parts (initial and late parts). A P value < 0.05 was considered statistically significant.

	Results

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The basal paw withdrawal latency (control value without drug injection, time = 0) and the temperature at the heating point were 5.1 ± 0.3 s and 53.1 ± 2.6°C for the high-intensity stimulation and 10.0 ± 0.4 s and 47.8 ± 3.2°C for the low-intensity stimulation.

Epidural administration of morphine and bupivacaine + morphine resulted in a dose-dependent increase in the thermally evoked response latency at both high- and low-intensity stimuli whereas bupivacaine alone did not induce dose-dependent effects (Figs. 1 and 2). Intraperitoneal naloxone inhibited the increase in the response latencies by bupivacaine + morphine (Fig. 3).

View larger version (24K): [in this window] [in a new window]   Figure 1. Time course of the effects of morphine (upper), bupivacaine (middle), and morphine + bupivacaine (lower) at high- and low-intensity stimulation. High = high-intensity stimulation, Low = low-intensity stimulation, Mor = morphine, Bup = bupivacaine, 10, 30, 100, 200, 300, 400 = µg. Bars indicate SEM. *P < 0.05 versus the control value (Time 0). **P < 0.01 versus the control value (Time 0).

View larger version (26K): [in this window] [in a new window]   Figure 3. Effects of intraperitoneal injection of naloxone 90 µg/300 µL of saline 10 min before intrathecal drug administration. High = high-intensity stimulation, low = low-intensity stimulation, mor = morphine 30 µg, bup = bupivacaine 100 µg, nal = naloxone 90 µg/300 µL of saline (intraperitoneal injection). *P < 0.05 versus the control value (Time 0). **P < 0.01 versus the control value (Time 0). P < 0.05 versus the group without naloxone. P < 0.01 versus the group without naloxone.

View larger version (15K): [in this window] [in a new window]   Figure 2. Dose response of morphine (upper), bupivacaine (middle), and morphine + bupivacaine 200 µg (lower) at high- and low-intensity stimulation. High = high-intensity stimulation, Low = low-intensity stimulation, %MPE = percent of maximal possible effect: = (postdrug latency - predrug latency) x 100/(cutoff latency - predrug latency). *P < 0.05 versus high-intensity stimulation. **P < 0.01 versus high-intensity stimulation.

	Discussion

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The present results showed that increasing the dose of epidural morphine in a dose range, which had a maximum effect on low-intensity stimulation, was not effective for high-intensity stimulation. Epidurally administered bupivacaine had no effect on thermal nociception, but potentiated the effects of epidural morphine at both high- and low-intensity stimuli similarly. The effect of the combination of epidural bupivacaine and morphine might be mediated by µ-opioid receptors because the effect was inhibited by intraperitoneal administration of naloxone.

We used 4.6 A (low intensity) and 5.1 A (high intensity) of current to give the basal response latencies of 10 and 5 seconds, respectively. Dirig and Yaksh (3) used three different currents: 5.0 A (low intensity), 5.25 A (medium intensity), and 6.0 A (high intensity) to give the basal response latencies of 15, 9, and 6 seconds, respectively. There is no definition of low- and high-intensity stimuli in the literature. However, 15 seconds might be too long to detect an antinociceptive effect when 20 seconds is used as the stimulus cutoff time. We considered that 5 and 10 seconds are different enough to separate the stimulus intensity into high and low. The temperature at paw withdrawal is not a determinant factor of stimulus intensity, but the changes of the temperature that might be indicated by the area under the curve of time times temperature are important to determine the stimulus intensity. Therefore, we showed current, not temperature, to show the stimulus intensity because the current controls the increasing rate of the temperature.

Synergistic interactions can occur when drugs affect different critical points along a common pathway (14). Small doses of intrathecal lidocaine and bupivacaine, which alone have no effect on antinociception or motor function, significantly augment the antinociceptive activity of intrathecal morphine on the hot plate and paw pressure tests (15). Intrathecal coadministration of morphine and bupivacaine does not alter the morphine concentration either in spinal cord tissue or in plasma (15). Therefore, the prominent and selective potentiation of the morphine effect by bupivacaine was thought to be caused by a nonpharmacokinetic mechanism and probably reflects the interaction of small concentrations of local anesthetics with systems in the spinal dorsal horn that process acute high-threshold afferent input. This, in combination with the postsynaptic actions of the opioids, may serve as an effective therapeutic approach to pain management (16). In the study conducted by Tejwani et al. (7), intrathecal administration of bupivacaine 5 to 50 µg significantly potentiated the antinociception produced by intrathecal administration of morphine 10 µg, but was not so effective at larger doses of morphine (20 µg). This facilitation of morphine-induced antinociception by bupivacaine was thought to be associated with a conformational change in the spinal opioid receptors induced by bupivacaine (7). This change allows morphine to bind more easily to spinal opioid receptors. In the present study, epidural bupivacaine also potentiated antinociception of epidural morphine similar to the effect with intrathecal administration in other studies (15,16). Thus, the interaction between epidural bupivacaine and morphine might be attributed to the same mechanism as intrathecal administration. Complete block by naloxone of the interaction between epidural bupivacaine and morphine suggests that the blockade by naloxone could inhibit conformational change of the spinal opioid receptors. However, we cannot deny the possibility that naloxone antagonized a potentiating effect of bupivacaine in other mechanisms.

Dirig and Yaksh (3) reported that the slope of the dose-response curve of intrathecal morphine tended to show a prominent reduction at the highest stimulus intensity. Their results were consistent with only the small-dose range (10 to 100 µg) of the epidural morphine portion of the data in the present study. The large doses of epidural morphine (late phase of the slope) did not show any changes of the slope of dose-response regression line according to the changes in stimulus intensity. That might be because the large doses we used were already enough to block high-intensity stimulation. Adding bupivacaine to epidural morphine decreased the slope of the dose-response regression line at low-intensity stimulation and at large doses of morphine with high-intensity stimulation. At small doses of morphine with high-intensity stimulation, bupivacaine increased the slope of the regression line. These results suggest that, for severe pain, in a small-dose range of epidural morphine, adding bupivacaine is more effective than increasing the dose of morphine.

In an electrophysiological experiment, high rates of noxious radiant skin heating induced A- nociceptor activation whereas a low rate of skin heating activated C fiber nociceptors (17). This was confirmed by a behavioral study in which capsaicin was used as a sensitizer or desensitizer of C fiber nociceptors and morphine was administered to attenuate nociception produced by the activation of C fiber nociceptors (18). We do not know whether the high- and low-intensity stimuli in the present study are equal to the high and low rates of noxious radiant heating in their studies. However, considering the effects of morphine, low-intensity stimulation might be mediated by C fiber nociceptors whereas high-intensity stimulation might be mainly mediated by non-C fiber nociceptors, e.g., A- nociceptors.

As stimulus intensity rises, the fractional receptor occupancy, the portion of the total receptor population that must be occupied to generate a given effect, also increases (19), and the release of primary afferent transmitters increases (20). For a given change in stimulus intensity, anesthetics with a high intrinsic efficacy show less shift in their dose-response curves with a given increase in stimulus intensity than an agonist with a low intrinsic efficacy (2). Therefore, epidural bupivacaine with morphine could have higher intrinsic efficacy than morphine alone.

We conclude that bupivacaine potentiated the analgesic effect of epidural morphine at both weak and strong nociceptive stimuli similarly, whereas increasing the dose of epidural morphine was not as effective for strong nociceptive stimulation. Therefore, adding bupivacaine might be more effective than increasing the dose of epidural morphine for strong nociception.

	Acknowledgments

 
We thank Professor Chingmuh Lee, MD, Department of Anesthesiology, University of California, Los Angeles, School of Medicine, for his helpful comments.

	References

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