Tissue damage associated with a surgical lesion often produces peripheral and central sensitization that may outlast the stimuli, leading to exaggerated postoperative pain (known as hyperalgesia), allodynia, and persistent pain.1 Paradoxically, clinical and experimental studies2,3 report that opioid therapy designed to alleviate surgical pain may render patients more sensitive to nociception, can aggravate early postoperative pain, and increase the risk of developing both acute postoperative opioid tolerance and persistent postoperative pain.3–5 Indeed, preclinical studies in animals indicate that prior pain experiences, especially those that have been treated using an opioid, produce long-lasting pain vulnerability, as demonstrated by hyperalgesia, in response to subsequent painful stimuli,6 even after postoperative pain has disappeared. This phenomenon is referred to as latent pain sensitization.7 Excitatory amino acids play a major role in pain sensitization, mainly by activating the N-methyl-d-aspartate receptor (NMDA-R).1,2,6,8–10
Based on these data, an interesting therapeutic strategy for surgery would be to use not only analgesics but also drugs with antihyperalgesic properties to improve postoperative pain management and reduce the development of pain sensitization that may lead to persistent pain. NMDA-R antagonists, such as ketamine, have been shown to reduce postoperative pain in animals6 and humans.11–13 The use of ketamine also reduces postoperative morphine consumption in humans.14 However, the use of such treatments is limited in clinical practice by the associated side effects.
It has been reported that nefopam, an analgesic used in humans since 1976 to control postoperative pain,15–17 may also have antihyperalgesic properties.18 Nefopam hydrochloride (Acupan®) is a cyclic analog of orphenadrine with antinociceptive properties demonstrated in rodents.19 Administering nefopam in patients either 30 min before the anticipated end of surgery or postoperatively improves postoperative analgesia and reduces morphine consumption.18,20 These observations from clinical use at the end of surgery do not allow us to determine the relative analgesic and antihyperalgesic contributions of this drug, prompting us to undertake this study.
In this work, we used an experimental rat model that allows evaluation of the magnitude of postoperative pain induced by a surgical incision in the rat hindpaw21 and its enhancement by fentanyl, as previously reported.10 The model also allows determination of the level of long-term pain vulnerability in animals subjected to surgical injury and fentanyl, based on the hyperalgesia induced by inflammation in the unlesioned contralateral hindpaw 3 wk later. This experimental pain model may be clinically relevant by mimicking not only postoperative pain but also the long-term changes in pain sensitivity observed in surgical patients. Acute postoperative pain is followed by persistent pain in 10%–50% of individuals after common operations.5
Using this pain model, we first examined the ability of a single preoperative dose of nefopam to reduce postoperative pain and long-term pain vulnerability. Second, we examined the ability of a single dose of nefopam to prevent hyperalgesia induced by fentanyl administration. Third, because fentanyl administration enhances both postoperative pain and long-lasting pain vulnerability,6 we evaluated the preventive effects of a single dose of nefopam in rats treated with fentanyl before surgical incision. Fourth, the ability of preoperative nefopam to improve postoperative morphine analgesic efficacy was evaluated in the incisional pain model in fentanyl-treated rats.
Experiments were performed on adult male Sprague-Dawley rats (Charles River Laboratories, L’Arbresle, France) that weighed 300–400 g and were housed in groups of five per cage on a 12-h/12-h light/dark cycle (lights on at 7:00 am) and at a constant room temperature of 23°C ± 2°C. The animals had access to food and water ad libitum. Pharmacologic tests and care of the animals were conducted in accordance with the guidelines laid out in the Guide for the Care and Use of Laboratory Animals (U.S. National Academy of Science, 1996) and were approved by the Ethics Committee for Animal Experimentation of Aquitaine and Poitou-Charentes (France). At the end of the experiment, the rats were killed with pentobarbital sodium (120 mg/kg). These experiments were conducted in an authorized laboratory under the supervision of an authorized researcher, E. Laboureyras.
Nefopam hydrochloride (Biocodex, Gentilly, France), fentanyl citrate, morphine, and carrageenan (Sigma-Aldrich, Saint-Quentin Fallavier, France) were dissolved in physiologic saline (0.9%). Nefopam (10 mg/kg), morphine (2 mg/kg), and fentanyl (100 μg/kg) were administered subcutaneously at doses of 3 mL/kg body weight for nefopam and 1 mL/kg body weight for morphine and fentanyl. Control animals received an equal volume of saline injections. We decided to limit the nefopam administration to a single, moderate dose immediately before surgery in rats. The dose of 10 mg/kg was preferred to higher doses because it only induced a moderate analgesic effect. Therefore, by evaluating pharmacological effects of nefopam on several postoperative days, our experimental design allows us to distinguish putative antihyperalgesic effects from analgesic effects.
Carrageenan (0.2 mL of a 1% carrageenan solution in saline) was prepared 24 h before each experiment. In the model of incisional pain, an ointment with 2% Fucidine (Léo, St. Quentin-en-Yvelines, France) and Primyxine (oxytetracycline hydrochloride and polymyxin B sulfate; Chemineau, Vouvray, France) was placed on the wound after the surgery.
Two pain tests were used to assess pain behavior bilaterally. Nociceptive thresholds (NT) were determined in hand-held rats by a modification of the Randall-Selitto method,22 which is a paw-pressure vocalization test in which a constantly increasing pressure is applied to the hindpaw until the rat vocalizes. The Basile analgesimeter (Apelex, Massy, France; stylus tip diameter, 1 mm) was used for this test. A 600-g cutoff value was imposed to prevent tissue damage.
Pain score was based on changes in weight bearing (WB) using an incapacitance apparatus that detects changes in postural equilibrium after a unilateral hindpaw injury (Bioseb, Chaville, France). As previously described,10 the rats were trained to stand on their hindpaws in a box with an inclined plane (65° from horizontal). This box was placed above the incapacitance apparatus. This allowed us to independently measure the weight that the animal applied on each hindpaw. The value considered for each animal was the mean of 10 consecutive measurements. To avoid changes related to increases in the rat’s body weight, the results were expressed as a percentage of total body weight. In the absence of hindpaw injury, rats applied equal weight (approximately 45% of total body weight) on each hindpaw, indicating postural equilibrium.
Carrageenan Pain Model
Rats were placed in a plastic cage and then anesthetized with 3% halothane for 3 min. Carrageenan (0.2 mL of a 1% carrageenan solution in saline) was then injected into one of the rat’s hindpaws subcutaneously. Injections were performed with a 25-gauge needle.
Surgical Pain Model
Immediately before surgery, rats were anesthetized with 1%–3% halothane vaporized via a nose cone. The plantar incision was performed as previously described by Brennan et al.21 The wound site was covered with an antibiotic mixture of polymyxin B, oxytetracycline, and fusidate. At the end of the surgery, halothane administration was stopped, the rats awakened, and they were allowed to recover in their home cage.
Upon arrival in the laboratory, animals were randomly assigned to the different experimental groups and left to acclimate in the animal care unit for 4 days. To avoid stress that might result from the experimental conditions and affect pain measures, the experiments were performed by the same experimenter in quiet conditions, in a test room close to the animal care unit. On each day of the 2 wk before the experiments, the animals were weighed, handled gently for 5 min, and placed in the test room for 2 h (from 9:00 am to 11:00 am), to become accustomed to the various apparatus. All experiments began at 9:00 am and were performed on groups of 10 animals during the light part of the cycle.
Behavioral tests were performed 2 days preceding the scheduled experimental day (i.e., on D−2 and D−1) and were repeated on the experimental day (D0), immediately before hindpaw injury. Experiments were initiated only when no statistical change in basal pain variables was observed for 3 successive days (D−2, D−1, and D0, one-way analysis of variance [ANOVA], P > 0.05). The value on D0 was used as the baseline value in subsequent data analyses. The experimenter performing pain evaluations was unaware of the treatment used.
Experiment 1: Effect of Nefopam on Development of Pain Sensitization by Surgical Incision
Rats were allocated into one of the following groups: control group, incision group, or nefopam-incision group. On D0, a plantar incision was performed in the left hindpaw during halothane anesthesia. No incision was performed in the control group. Nefopam (10 mg/kg) or saline injection was performed 30 min before injury. Pain variables were assessed at 2, 4, and 6 h after tissue injury (D0), and once daily on subsequent postoperative days for 3 wk. On D21, a carrageenan injection was performed in the contralateral hindpaw (right hindpaw). Pain variables were assessed at 2, 4, and 6 h after this second injury and once daily during the subsequent postoperative days until NT returned to basal values.
Experiment 2: Effect of Nefopam on Fentanyl-Induced Hyperalgesia
Rats were allocated into the fentanyl group or the nefopam-fentanyl group. On D0, one fentanyl (100 μg/kg) injection was performed every 15 min for 1 h, resulting in a total dose of 400 μg/kg. Nefopam (10 mg/kg) injection (or saline in the fentanyl group) was performed 30 min before the first administration of fentanyl. NT was assessed at 2, 4, and 6 h after fentanyl administration and once daily on subsequent postoperative days until the values returned to baseline.
Experiment 3: Effect of Nefopam on Development of Pain Sensitization by Surgical Incision Accompanied by Fentanyl
Rats were allocated into one of the following groups: control group, fentanyl group, or nefopam-fentanyl group. One fentanyl (100 μg/kg) injection (or saline in the control group) was performed every 15 min for 1 h, resulting in a total dose of 400 μg/kg. Five minutes after the first fentanyl or saline injection, a plantar incision was made on the left hindpaw during halothane anesthesia. No incision was performed in the control group. Nefopam (10 mg/kg) or saline injection was performed 30 min before injury. Pain variables were assessed at 2, 4, and 6 h after tissue injury (D0) and once daily on subsequent postoperative days for 3 wk. On D21, a carrageenan injection in the absence of fentanyl or nefopam administration was performed in the contralateral hindpaw (right hindpaw). Pain variables were assessed at 2, 4, and 6 h after this second injury and once daily during the subsequent postoperative days until the NT returned to baseline values.
Experiment 4: Effect of Nefopam on Morphine Analgesia 1 Day After Surgical Incision Accompanied by Fentanyl
Rats were allocated into the control group or the nefopam group. Analgesic effectiveness of morphine (2 mg/kg) was estimated on D1, 24 h after one plantar incision (D0) in fentanyl-treated rats (4 × 100 μg/kg). Nefopam (10 mg/kg) or saline administration was performed 30 min before injury on D0. Pain variables were estimated at 2, 4, and 6 h after tissue injury on D0 and every 30 min for 3 h after morphine injection on D1. Pain variables were also estimated daily for the subsequent 12 days.
Calculation and Statistical Analysis
Hyperalgesic indexes (HI), represented by the area above the curve for the postoperative days, were calculated for each rat by the trapezoidal method23 and were expressed as a mean percentage (±sd) of the reference index (100%: HI associated with hyperalgesia observed in the control group). Analgesic indexes (AI) for morphine analgesia, represented by the area under the curve, were also calculated by the trapezoidal method.
To evaluate the time course effects of treatments on pain parameters, an ANOVA followed by post hoc analyses using the Dunnett test or the Newman-Keuls test was performed on measures from D0. The basal value in all experiments was defined as the preinjury value on D0. Another ANOVA was performed for the days after treatment in each group. The Mann–Whitney test was used to compare the AI or HI when the experiments involved only two groups. An ANOVA followed by post hoc analyses using the Dunnett test or the Newman-Keuls test was performed when the experiments involved more than two groups. The criterion of statistical significance was P < 0.05.
Enhancement of Inflammatory Hyperalgesia in Rats Subjected to Prior Surgical Pain: Prevention by Nefopam
As expected, the unilateral left hindpaw plantar incision produced a significant decrease in NT for 9 days (Dunnett test, P = 0.005; Fig. 1A), as measured using the paw-pressure vocalization test. At the contralateral hindpaw, the unilateral left hindpaw incision induced a significant decrease in the NT for 3 days (Dunnett test, P = 0.001; Fig. 1B). Moreover, incision of the hindpaw produced a significant decrease in WB for 5 days (Dunnett test, P = 0.008; Fig. 1C).
When administered 30 min before incision on D0, the subcutaneous injection of nefopam (10 mg/kg) induced an analgesic effect, because it prevented the NT decrease for 4 h (Newman-Keuls test, P < 0.001; Figs. 1A and B). Moreover, nefopam also reduced the WB decrease at 2 and 6 h after incision (Dunnett test, P = 0.002 for 2 h and P = 0.029 for 6 h; Fig. 1C). Once the analgesic effect of nefopam subsided, it had no further effect on long-lasting (10 day) hyperalgesia induced by incision (one-way ANOVA, P > 0.05; Figs. 1A and B). A limited but significant reduction in WB change was observed on several days between D4 and D15 (Dunnett test, P < 0.05; Fig. 1C). No significant decrease was observed in the HI (Mann–Whitney test, P > 0.05; Fig. 1C, inset).
When injected on D21 into the right hindpaw (nonincised hindpaw), the proinflammatory drug carrageenan induced more sustained hyperalgesia of the previously incised hindpaw (left hindpaw) compared with rats that had no incision on D0 (Fig. 1A). HI was sixfold higher in the incision group than in the control group without incision on D0 (Newman-Keuls test, P < 0.001; Fig. 1A, inset). At the nonincised hindpaw (right hindpaw), injection of the proinflammatory drug carrageenan induced more sustained hyperalgesia in rats that had received surgical injury compared with rats that received no incision on D0 (Fig. 1B). HI was 2.8-fold higher than in those without an incision on D0 (Newman-Keuls test, P < 0.001; Fig. 1B, inset).
The enhancement of long-lasting inflammatory hyperalgesia was reduced in rats that received nefopam immediately before the incision 21 days earlier on D0. For the left hindpaw, HI was 68.8% lower in the nefopam-incision group than in the incision group (Newman-Keuls test, P < 0.001; Fig. 1A, inset). No significant difference was observed between the HI of the nefopam-incision group and that of the control group (Newman-Keuls test, P = 0.702; Fig. 1A, inset). For the right hindpaw, HI was 46.8% lower in the nefopam-incision group than in the incision group (Newman-Keuls test, P < 0.001; Fig. 1B, inset). No significant difference between the nefopam-incision group and the control group was observed (Newman-Keuls test, P = 0.516; Fig. 1B, inset).
Preventive Effect of Nefopam on Fentanyl- Induced Hyperalgesia
As previously described, fentanyl administration induced analgesia that was followed by hyperalgesia for several days (Fig. 2). When administered 30 min before fentanyl on D0, nefopam (10 mg/kg) injection induced an increase in analgesia (Dunnett test, P = 0.016) and strongly reduced hyperalgesia observed for several days after fentanyl administration. Indeed, this hyperalgesia was smaller in amplitude in the nefopam-fentanyl group compared with the fentanyl group, and only lasted 1 day in the nefopam-fentanyl group (Dunnett test, P < 0.001), compared with 6 days in the fentanyl group (Dunnett test, P = 0.004). HI was 93% lower in the nefopam-fentanyl group than in the fentanyl group (Mann–Whitney test, P = 0.004; Fig. 2, inset).
Enhancement of Inflammatory Hyperalgesia in Rats Subjected to Prior Surgical Pain Combined with Fentanyl Administration: Prevention by Nefopam
As expected, fentanyl administration (four boluses of 100 μg/kg) initially induced an analgesic effect on D0 for 4 h after the incision of the left hindpaw (Dunnett test, P < 0.001; Fig. 3A). Subsequent daily measurements of NT and WB showed long-lasting hyperalgesia and postural disequilibrium that lasted 12 and 8 days, respectively (Dunnett test, P = 0.023; Fig. 3A and Dunnett test, P = 0.044; Fig. 3C). At the contralateral hindpaw level (nonincised hindpaw), hyperalgesia was observed for 10 days after analgesia (Dunnett test, P = 0.020; Fig. 3B).
Subcutaneous injection of one 10 mg/kg nefopam dose 30 min before the incision on D0 strongly reduced long-lasting hyperalgesia and postural disequilibrium observed after incision in fentanyl-treated rats (Figs. 3A–C). HI was lower in rats treated with nefopam and fentanyl than in rats treated with saline and fentanyl, with reductions of 60.3% and 59.7%, respectively, for the ipsilateral and contralateral hindpaws (Newman-Keuls test, P < 0.001; Figs. 3A and B, insets). In tests of postural disequilibrium, HI was lower by 69.2% in rats treated with nefopam and fentanyl than in rats treated with saline and fentanyl (Fig. 3C, inset).
When injected on D21 into the right hindpaw (nonincised hindpaw), carrageenan induced more prolonged hyperalgesia of the previously incised hindpaw (left hindpaw) in fentanyl-treated rats compared with rats that had neither incision nor fentanyl administration on D0 (Fig. 3A). On the nonincised hindpaw (right hindpaw), injection of carrageenan induced more prolonged hyperalgesia in rats with prior fentanyl treatment and tissue injury compared with rats that had neither incision nor fentanyl administration on D0. HI was 4.5-fold higher in rats given fentanyl before incision than in rats that did not have any incision and fentanyl treatment on D0 (Newman-Keuls test, P < 0.001; Fig. 3B, inset). In this last experiment, the enhancement of long-lasting hyperalgesia was completely prevented in fentanyl-treated rats that had received nefopam 21 days earlier on D0 (Newman-Keuls test, P < 0.001; Fig. 3B, inset). On the incised hindpaw, nefopam administration on D0 reduced the enhancement of long-lasting hyperalgesia induced by carrageenan injection on D21 in rats treated with saline and fentanyl. HI was lower by 79.8% in rats treated with nefopam and fentanyl than in rats treated with saline and fentanyl (Newman-Keuls test, P < 0.001; Fig. 3A, inset).
Improvement of Postoperative Morphine-Induced Analgesia by Nefopam in Fentanyl-Treated Rats
When administered 1 day after incision in rats treated with fentanyl on D0, morphine induced an analgesic effect, measured by the increase in NT observed in saline- or nefopam-treated rats using the paw-pressure vocalization test (Dunnett test, P < 0.001 by comparison with the D1 basal value; Fig. 4A). Although the two experimental groups showed a similar AI of morphine (Mann–Whitney test, P > 0.05; Fig. 4A, inset), morphine was more effective in nefopam-treated rats, because of a higher basal NT on D1 than in untreated rats. Morphine induced a partial reversal of WB decrease in the non-nefopam-treated group (Dunnett test, P < 0.001; Fig. 4B) when compared with the D1 basal value. In nefopam-treated rats, morphine completely reversed the WB decrease for 1 h (Dunnett test, P > 0.05 when compared with the basal value on D0).
From a pathophysiological viewpoint, we report that a surgical lesion in rats induced a latent and long-term pain hypersensitivity, which manifested when additional tissue injury was created at a significant distance from the preliminary injury. From a pharmacological viewpoint, we show that a single preoperative administration of nefopam reduced the exaggerated postoperative pain induced by preoperative fentanyl treatment and diminished the development of long-term pain hypersensitivity. Preoperative nefopam administration also improved morphine analgesic efficacy in the context of fentanyl-induced postoperative hyperalgesia.
From a mechanistic viewpoint, abnormal persistence of excitatory neuroplasticity induced by tissue injury is considered to be a major candidate mechanism for the development of postoperative hyperalgesia and related chronic pain.5 Here, we show that the initial surgical incision induced long-lasting and latent changes of pain sensitivity. In other words, despite the disappearance of postoperative pain, its imprint in the central nervous system remained because pain hypersensitivity was observed at both ipsilateral and contralateral levels. This finding is in agreement with previous observations on repeated paw inflammation in animals.6,7,24,25 Although our experimental animal study was limited to several weeks, these results suggest that a pain experience can affect pain sensitivity even several days after postoperative pain has disappeared. Independent of genetic differences, this may partly explain some individual differences in pain sensitivity in humans,26 and this topic requires further study. Both animal27–29 and human studies30 reported that pain experienced by neonates induced long-term pain hypersensitivity and altered nociceptive neuronal circuits in the infants and adults.27 Taken together, these findings suggest that evaluation of individual pain histories in adults before surgery may help identify those at risk of pain vulnerability.
Interestingly, our study shows that a single administration of nefopam immediately before the incision, which had limited analgesic effects, strongly reduced this long-term pain hypersensitivity. In fact, this beneficial effect was observed in both hindpaws. This clearly indicates that nefopam not only has analgesic effects as previously reported15,17 but also inhibits central neuroplasticity mechanisms leading to the pain hypersensitivity triggered by surgical injury.
Despite their analgesic effects, opioids induce delayed hyperalgesia after analgesia. This paradoxical phenomenon has been observed after a single administration of a large analgesic dose of opioids similar to the doses typically used in surgery.3,23 This phenomenon is an important clinical problem because opioid-induced pain hypersensitivity may lead to exaggerated postoperative pain. For the first time, our study shows that a single nefopam administration performed immediately before fentanyl administration nearly eliminated fentanyl-induced hyperalgesia. On the basis of this positive result, we evaluated the ability of nefopam to prevent both the heightened postoperative pain induced by fentanyl and long-term pain vulnerability. Our study indicates that a single dose of nefopam reduced the fentanyl-mediated increase in postoperative pain, which is evident from the comparable levels of incision-induced lowering of the paw-pressure withdrawal threshold after nefopam-fentanyl (Fig. 3A) and after incision alone (Fig. 1A). Interestingly, nefopam-treated rats recover postural equilibrium more rapidly in the WB test, suggesting improved postoperative rehabilitation. Moreover, nefopam strongly reduced the inflammation-associated mechanical pain hypersensitivity in both hindpaws when inflammation was induced 3 wk later in the contralateral nonincised hindpaw. Taken together, these results confirm that nefopam has potent antihyperalgesic properties in addition to its analgesic effects.
From a pharmacological viewpoint, we have previously demonstrated that the acute tolerance to morphine’s analgesic effects induced by preoperative opioid use is not due primarily to a decrease in the potency of analgesic opioids, but rather results from the postoperative pain hypersensitivity induced by both surgical incision and preoperative opioid use.6,10,14,31 Experimental studies have shown that drugs that inhibit pain sensitization reduce acute tolerance to morphine’s analgesic effects.10,31–33 In agreement with such a mechanistic viewpoint, clinical studies report that the use of the NMDA-R antagonist ketamine reduces postoperative morphine consumption in humans.14 Our study shows that a single dose of nefopam, by reducing postoperative hyperalgesia in fentanyl-treated rats, also improved morphine efficacy.
Several aspects of the mechanism of pain hypersensitivity and nefopam effects require clarification. There is a compelling body of evidence that neural NMDA-R activation plays a critical role in the development and maintenance of pain hypersensitivity. Preventive effects on pain hypersensitivity have previously been reported in animals6,10 and humans3,14 treated with NMDA-R antagonists, such as ketamine. Moreover, there is mounting evidence that glia may play a key role in creating exaggerated pain states of diverse etiologies.34 Recently, electrophysiological studies using patch clamp recording of lamina II neurons in isolated spinal cord slices showed that centrally produced proinflammatory cytokines in injury conditions induce central sensitization and pain hypersensitivity.35 Unfortunately, the exact mechanism of action of nefopam is not fully understood. Nefopam shows no affinity for ionotropic glutamate receptors36 and its putative effect on glial function is unknown. However, it has been shown that nefopam blocks voltage-sensitive sodium channels and may modulate glutamatergic transmission in rodents.36–38 Its main analgesic mechanism of action seems to be one of inhibiting monoamine reuptake,39–43 leading to an increased concentration of these neuromediators in the synaptic cleft41,43–45 and thereby reinforcing descending serotoninergic inhibitory systems.41,46 Transcriptomic studies in the spinal cord are in progress to explore neural and nonneural mechanisms involved in both pain hypersensitivity and antihyperalgesic effects mediated by nefopam.
This study could have therapeutic implications. Preemptive analgesia is defined as treatment initiated before surgery to reduce pain sensitization47 and its outcomes. Nefopam given in this manner may be effective at improving postoperative management clinically and at reducing the risk of developing postoperative chronic pain. Indeed, nefopam may be beneficial because the drug has both analgesic and antihyperalgesic properties and induces only mild adverse events in humans.20 Clinical trials will be necessary to clarify the risk of developing pain vulnerability after surgery and to design preventive antihyperalgesic strategies. Because this study indicates that a moderate analgesic dose of nefopam has antihyperalgesic properties in rats, it would be interesting to evaluate whether analgesic doses of nefopam15–18,20 currently used for relieving postoperative pain (0.3 mg/kg) have antihyperalgesic effects in humans. The long-term effects (months) of nefopam on pain vulnerability also have to be documented.
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