Remifentanil is a μ-opioid agonist extensively used in daily clinical anesthesia. It is characterized by a rapid onset of action and a metabolism independent of patient condition. However, according to some clinical studies, remifentanil may induce hyperalgesia after surgery or worsen postoperative pain. Pain threshold may decrease after remifentanil use in postoperative patients1–3 and volunteers,4,5 and recent animal studies suggest that administration of remifentanil may induce hyperalgesia.6,7 Generally, μ-opioid agonists have the potential to induce hyperalgesia, termed opioid-induced hyperalgesia (OIH). Elucidating the mechanism and clarifying the characteristics of remifentanil-induced hyperalgesia may improve management of perioperative pain.
The molecular mechanisms of remifentanil-induced hyperalgesia are poorly understood. Many different signaling pathways are involved in central sensitization. Furthermore, the primary molecules affecting the hyperalgesic state vary at different times of sensitization.8 We focused on the mitogen-activated protein kinase (MAPK) in this study, because recent studies have shown that the rapid central sensitization caused by neuropathic pain, inflammatory pain, and opioid administration are strongly associated with activation of the MAPK, especially extracellular signal-regulated protein kinase 1/2 (ERK1/2), in spinal dorsal horn neurons or glial cells.6,8–14 We hypothesized that remifentanil-induced hyperalgesia is related to ERK1/2, as observed in other pain models.
Thus, we performed animal experiments to determine whether IV remifentanil infusion induces hyperalgesia, the circumstances under which hyperalgesia occurs, and whether remifentanil-induced hyperalgesia is associated with ERK1/2 phosphorylation.
All experiments were approved by the Animal Care and Use Committee of Shimane University (approval nos. IZ20-167 and IZ21-80) and conform to the Guide for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats weighing 200 to 300 g were used (CLEA Japan, Tokyo, Japan). Rats were housed at 22°C ± 1°C with a 12-hour light-dark schedule and were fed food and water freely. Rats were habituated to the cage and experimental environment in the daytime for 3 days before experiments. Experiments were performed by the same experimenter in a blinded manner in the same daytime environment.
Remifentanil IV Infusion Model and Behavioral Testing
Each rat's tail vein was cannulated with a 24-gauge catheter at the root of the tail and flushed with heparinized saline. Remifentanil (Ultiva®; Janssen Pharmaceuticals, Tokyo, Japan) was dissolved in saline and systemically administered with an infusion syringe pump (KD Scientific, Holliston, MA). Rats were divided into several groups of 6 rats each according to drug administration dose and duration. In our preliminary study, we confirmed that these doses not only exerted antinociceptive effects, but also allowed survival without cardiorespiratory support. Experiment 1, designed to examine whether short-term infusion caused hyperalgesia, consisted of remifentanil 30 μg · kg−1 · min−1 for 10 minutes (remi30-10), remifentanil 10 μg · kg−1 · min−1 for 30 minutes (remi10-30), remifentanil 1 μg · kg−1 · min−1 for 30 minutes (remi1-30), remifentanil 0.1 μg · kg−1 · min−1 for 30 minutes (remi0.1-30), and saline for 30 minutes (saline30; control). Experiment 2, designed to examine whether long-term infusion caused hyperalgesia, consisted of remifentanil 10 μg · kg−1 · min−1 for 120 minutes (remi10-120), remifentanil 3 μg · kg−1 · min−1 for 120 minutes (remi3-120), remifentanil 1 μg · kg−1 · min−1 for 120 minutes (remi1-120), remifentanil 0.1 μg · kg−1 · min−1 for 120 minutes (remi0.1-120), and saline for 120 minutes (saline120; control).
Mechanical nociceptive thresholds were determined using a modification of the “up and down” method15 with calibrated von Frey monofilaments (North Coast Medical, Morgan Hill, CA). Rats were placed on a wire mesh platform consisting of 1-mm-diameter wire with a 9 × 9 mm grid pattern. The starting filament was 4.31 (2 g), and the upper limit cutoff was 5.18 (15 g). Measurements were performed every 10 minutes for 60 minutes after infusion termination in experiments 1 and 2.
Thermal nociceptive threshold was measured by tail withdrawal latency to focused radiant light using a tail-flick unit (Ugo Basile, Comerio, Italy) with a cutoff value of 10 seconds. The apparatus was calibrated to an average baseline latency of approximately 4 seconds. Tail-flick latency was measured at 0, 5, 10, 20, and 30 minutes during infusion and every 10 minutes for 60 minutes after infusion termination in experiment 1. It was also measured every 30 minutes during infusion and every 10 minutes for 60 minutes after infusion termination in experiment 2.
Immunohistochemistry of ERK1/2
We used remi0.1-120, remi0.1-30, remi10-120, remi10-30, saline120, and sham (no pretreatment) rats to evaluate ERK1/2 phosphorylation. Agents were administered as above to groups of 6 rats each. Rats underwent fixation 30 minutes after infusion termination.
Rats were anesthetized with 100 mg/kg intraperitoneal pentobarbital and transcardially perfused with 200 mL saline, followed by 250 mL of 4% paraformaldehyde. The spinal cord (L4-5) was extracted and postfixed with the same fixative for 3 hours at room temperature and cryoprotected overnight at 4°C. Transverse frozen spinal cord sections (40 μm) were prepared using a sliding microtome (SM-2000R; Leica Microsystems, Wetzlar, Germany). Sections were blocked with 3% normal goat serum and incubated for 48 hours at 4°C with primary antibodies: phospho (p)-ERK1/2 (1:100; Cell Signaling, Danvers, MA), neuronal nuclei (1:1000; Chemicon, Temecula, CA), OX-42 (1:100; Serotec, Oxford, UK), or glial fibrillary acidic protein (1:400; Chemicon). Sections were incubated for 3 hours at room temperature in Alexa Fluor® 594- or 488-conjugated secondary antibodies (1:1000; Molecular Probes, Eugene, OR). Sections were analyzed using a confocal laser microscope (CLSM FV-300; Olympus, Tokyo, Japan). The number of p-ERK1/2–immunoreactive cells in laminae I and II was counted. Counted values of 5 random sections were averaged.
MEK Inhibitor and Hyperalgesia
Under 1.5% halothane anesthesia, an intrathecal catheter was implanted in each of 14 rats in a caudal direction at the L4-5 level.16 The correct placement of each catheter was confirmed by injection of 15 μL of 2% lidocaine after the experiment. Data from rats without paralysis of the tail and bilateral hindpaw after lidocaine injection were excluded. After catheterization, 3 days were allowed for recovery. Rats with motor deficits or health problems after catheter placement were excluded.
1,4-Diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene (U0126; Wako Pure Chemical Industries, Osaka, Japan) was dissolved in 20% dimethyl sulfoxide to achieve a concentration of 2 mM. Rats were intrathecally injected with U0126 (2 mM/10 μL) or vehicle (20% dimethyl sulfoxide/10 μL), followed by 10 μL saline.17 Two hours later, rats were infused IV with remifentanil 10 μg · kg−1 · min−1 for 120 minutes as described above. Tail-flick latency was measured 10, 20, and 30 minutes after infusion. Immediately after each measurement, p-ERK1/2 protein expression in the spinal cord was assessed. Rats were anesthetized with 100 mg/kg intraperitoneal pentobarbital. Lumbosacral spinal cords were extracted and immediately homogenized in lysis buffer containing protease and phosphatase inhibitors on ice and centrifuged at 12,000 rpm for 5 minutes at 4°C. Supernatants were dissolved in sample buffer (ATTO Corporation, Tokyo, Japan) and denatured at 100°C for 5 minutes; protein aliquots were separated using 5% to 20% sodium dodecylsulfate–polyacrylamide gel electrophoresis. After transfer, membranes were blocked with 1% w/v bovine serum albumin in Tris-buffered saline with Tween-20 for 1 hour at room temperature. Membranes were incubated overnight at 4°C with the primary antibodies anti–p-ERK1/2 (1:2000; Cell Signaling) and anti–β-actin (1:2000; Sigma-Aldrich, St. Louis, MO). Membranes were washed with Tris-buffered saline with Tween-20 and incubated with secondary antibodies conjugated with horseradish peroxidase for 1 hour at room temperature. Results were visualized using enhanced chemiluminescence (GE Healthcare, Buckinghamshire, UK).
Results of behavioral experiments were evaluated using 2-way repeated-measures analysis of variance or 1-way repeated-measures analysis of variance followed by the Dunnett post hoc test. Immunohistochemistry results were evaluated using the Kruskal-Wallis test, followed by the Steel-Dwass18,19 post hoc test.
Remifentanil Induces Hyperalgesia After Long-Term Infusion
In experiment 1, mechanical and thermal hyperalgesia were not observed in any10- or 30-minute infusion groups, regardless of dose (Fig. 1). However, in experiment 2, 120-minute remifentanil infusion led to mechanical hyperalgesia in all groups (Fig. 2A) and thermal hyperalgesia in all groups except remi0.1-120 (Fig. 2B). In the von Frey test, nociception returned to baseline values 60 minutes after infusion termination in all groups except remi0.1-120 and remi3-120. Remi0.1-120 and remi1-120 groups showed hyperalgesia despite that the total drug dose was lower than that of remi10-30 and remi30-10 groups.
ERK1/2 Phosphorylation Was Observed After Remifentanil Infusion
Most p-ERK1/2–immunoreactive cells showed neuronal nuclei immunoreactivity (Fig. 3) and were located in laminae I and II of the spinal dorsal horn (Fig. 4). These cells expressed no immunoreactivity for OX-42 or glial fibrillary acidic protein (data not shown). Significantly more p-ERK1/ 2–immunoreactive cells were observed in the remifentanil 120-minute groups than the other groups (Fig. 5).
Intrathecal MEK Inhibitor Could Not Suppress Hyperalgesia
None of the 14 rats used in this study showed any motor deficits or health problems. Two rats were excluded after ineffective placement of the catheter was confirmed by the lidocaine test, resulting in 6 rats per treatment group. Behavioral tests showed that remifentanil induced hyperalgesia even after administration of the MAPK inhibitor U0126, despite the fact that Western blotting showed that U0126 inhibited ERK1/2 phosphorylation in the spinal cord (data not shown).
We report 2 significant findings. First, remifentanil-induced hyperalgesia was dependent on infusion duration, with minimal, if any, dose effect. Second, remifentanil administration leads to rapid ERK1/2 phosphorylation in spinal dorsal horn neurons.
OIH is a well-known phenomenon, especially in basic research.20 Various pathways may be involved in OIH, including those related to N-methyl-D-aspartate receptors,21,22 MAPK,6 the nitric oxide system,12,23 μ-opioid receptors,21 and neurokinin-1 receptors.24 Although morphine is one of the most prototypical opioids that induces OIH, several reports showed that other μ-opioids such as fentanyl, frequently used in clinical practice, also induce OIH.23 Remifentanil is widely used as an analgesic during surgery. However, some studies have indicated that remifentanil decreases pain thresholds and worsens postoperative pain,1–5 suggesting that remifentanil induces hyperalgesia. Conversely, some studies have reported that remifentanil does not induce hyperalgesia.25 Based on its definition, OIH is a phenomenon that occurs during opioid administration; thus, the remifentanil “withdrawal” hyperalgesia we observed may be a discrete phenomenon. Cèlérier et al.26 suggested that opioid withdrawal hyperalgesia results from disequilibrium between pronociceptive and antinociceptive systems. Opioid administration sensitizes pronociceptive systems, leading to hyperalgesia, and opioid-dependent antinociceptive systems counteradapt after discontinuation of opioids to offset the hyperalgesia. Remifentanil may have resulted in more-pronounced pronociceptive system sensitization because of its rapid offset (which may be masked in other opioids).
Some in vivo research of remifentanil-induced hyperalgesia has been published.6,23 In those studies, remifentanil was administered intraperitoneally or subcutaneously using incisional pain models. Remifentanil is frequently administered through IV infusion in clinical anesthesia. Therefore, we established an IV infusion model, which may lead to clinically relevant outcomes. We chose intact rats as subjects for our study, because we first wanted to determine whether remifentanil itself affects pain behaviors in intact animals. We selected remifentanil infusion doses based on the dose response to continuous infusion of remifentanil measured preliminarily, which ranged up to the tail-flick cutoff value. Our results show that IV remifentanil induced rapid withdrawal hyperalgesia dependent on infusion duration. Additionally, the hyperalgesia was transient and observed in the absence of any painful pretreatment, such as incision or inflammation. Previous studies have suggested that occurrence of remifentanil-induced hyperalgesia correlates with remifentanil dose.3,27 However, the nature and time course of the hyperalgesia we observed completely differ from those previously reported. We therefore hypothesize that a sufficient duration of exposure is necessary to trigger the molecular cascades that lead to hyperalgesia. We observed a discrepancy between the tail-flick and von Frey tests only in the remi0.1-120 group. One possible reason for this finding is the different modality of nociception between these tests.
Hyperalgesia can be produced by peripheral or central sensitization. Many molecules may be involved in central sensitization. We hypothesized that the modality of sensitization in remifentanil-induced hyperalgesia is similar to that involved in the early phase of other pain models and that the sensitization is induced by the MAPK phosphorylation cascade, considered to be the quickest change in the central nervous system induced by nociception or nerve injury.9,11 ERK1/2, well known as the classic MAPK family, is quickly activated by various extracellular stimuli and is involved in short-term pain hypersensitivity, has been shown to be phosphorylated in various pain models, and has been proved to be a sensitive marker of central sensitization.9
We found significantly more p-ERK1/2–immunoreactive neurons in remi10-120 and remi0.1-120 groups with behavioral hyperalgesia than groups without hyperalgesia. This result indicates some excitatory effect of remifentanil on neurons. Because ERK1/2 phosphorylation was localized in laminae I and II, where primary afferent fibers terminate, and was not observed in short-term or control groups, we considered that this change was related to the hyperalgesia. Although administration of opioids such as morphine induces ERK1/2 phosphorylation via μ-opioid receptors in the central nervous system,28 the significance of this phenomenon remains controversial.13,28,29 Campillo et al.6 recently reported a possible relationship between ERK1/2 and remifentanil-induced hyperalgesia. They concluded that ERK1/2 phosphorylation modulates dynorphin or c-Fos and then induces hyperalgesia, but that other signaling pathways may also be implicated. However, their hypothesis cannot explain the early-phase hyperalgesia we observed. In fact, Campillo et al. suggested that dynorphin could be involved in the persistence of hyperalgesia, but not its initiation. We speculate that the mechanism of acute hyperalgesia during remifentanil withdrawal and that of persistent hyperalgesia after remifentanil administration, which has been reported in many previous studies,27 are inherently different. The combination of remifentanil and incisional stimulation induces persistent hyperalgesia that may last for several days,6,23 whereas remifentanil administration alone induced rapid, short-duration hyperalgesia in our study. We found that ERK1/2 phosphorylation was consistent with behavioral hyperalgesia. However, U0126 could not suppress this hyperalgesia. Thus, we cannot conclude that ERK1/2 phosphorylation is the essential factor that induces hyperalgesia. We suggest the following mechanisms. Generally, ERK1/2 phosphorylation induces rapid central sensitization by posttranslational regulation, such as by increasing the activity of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) or N-methyl-D-aspartate receptors and suppressing the activity of potassium channels (Kv4.2).9 These mechanisms are likely related to the initiation of remifentanil-induced hyperalgesia, although signaling pathways other than ERK1/2, such as protein kinase C or phospholipase C, may concurrently be involved in this early-phase sensitization. Simultaneous inhibition of all pathways may be necessary to prevent remifentanil-induced hyperalgesia. We suspect that ERK and other signaling pathways together sensitize neurons and trigger subsequent transcriptional changes when combined with surgical incision, thus leading to transition from withdrawal hyperalgesia to persistent hyperalgesia.
It is important to recognize that remifentanil administration may result in hyperalgesia. We may avoid this hyperalgesia by controlling the duration of remifentanil administration, although the results of this study do not indicate specific limitations to infusion duration in human clinical practice.
In conclusion, remifentanil induced hyperalgesia after long-term (120 minutes), but not short-term (30 minutes), IV infusion, suggesting that the duration of exposure to remifentanil is the primary factor influencing hyperalgesia induction. Contrary to our hypothesis, ERK1/2 alone was not the essential factor involved in hyperalgesia induction. Further investigation is needed to clarify the mechanisms of remifentanil-induced hyperalgesia to prevent it, and thus overall improve the quality of anesthesia.
Name: Ryosuke Ishida, MD.
Contribution: This author helped design the study, analyze the data, write the manuscript, and collect data.
Attestation: Ryosuke Ishida has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Tetsuro Nikai, MD.
Contribution: This author helped design the study, conduct the study, analyze the data, write the manuscript, and collect data.
Attestation: Tetsuro Nikai has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Tatsuya Hashimoto, MD, PhD.
Contribution: This author helped design the study and analyze the data.
Attestation: Tatsuya Hashimoto has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Toshiko Tsumori, PhD.
Contribution: This author helped analyze the data and collect data.
Attestation: Toshiko Tsumori has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Yoji Saito, MD, PhD.
Contribution: This author helped design the study, conduct the study, and write the manuscript.
Attestation: Yoji Saito has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
This manuscript was handled by: Tony L. Yaksh, PhD.
The authors are very grateful to Mai Takahashi, MSc, and Yukiko Katsube for their excellent technical assistance in Western blotting and behavioral experiments.
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