Delayed postoperative latent pain sensitization revealed by the systemic administration of opioid antagonists in mice

European Journal of Pharmacology 657 (2011) 89–96

Contents lists available at ScienceDirect

European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Neuropharmacology and Analgesia

Delayed postoperative latent pain sensitization revealed by the systemic administration of opioid antagonists in mice Ana Campillo, David Cabañero, Asunción Romero, Paula García-Nogales, Margarita María Puig ⁎ Department of Anesthesiology, Pain Research Unit, Institut Municipal d'Investigació Mèdica, Hospital del Mar, Universitat Autònoma de Barcelona, Barcelona, Spain, Passeig Marítim 25–29, E-08003 Barcelona, Spain

a r t i c l e

i n f o

Article history: Received 12 November 2010 Received in revised form 7 January 2011 Accepted 25 January 2011 Available online 4 February 2011 Keywords: Dynorphin Kappa opioid receptor Latent pain sensitization Opioid Postoperative pain Remifentanil

a b s t r a c t The long-lasting post-surgical changes in nociceptive thresholds in mice, indicative of latent pain sensitization, were studied. The contribution of kappa opioid and N-methyl-D-aspartate (NMDA) receptors was assessed by the administration of nor-binaltorphimine or MK-801; dynorphin levels in the spinal cord were also determined. Animals underwent a plantar incision and/or a subcutaneous infusion of remifentanil (80 μg/kg), and mechanical thresholds (von Frey) were evaluated at different times. On day 21, after complete recovery of mechanical thresholds and healing of the wound, one of the following drugs was administered subcutaneously: (−)-naloxone (1 mg/kg), (+)-naloxone (1 mg/kg), naloxone-methiodide (3 mg/kg), or norbinaltorphimine (5 mg/kg). Another group received subcutaneous MK-801 (0.15 mg/kg) before norbinaltorphimine administration. Dynorphin on day 21 was determined in the spinal cord by immunoassay. In mice receiving remifentanil during surgery, the administration of (−)-naloxone or nor-binaltorphimine induced significant hyperalgesia even 5 months after manipulation. Nociceptive thresholds remained unaltered after (+)-naloxone or naloxone-methiodide. On day 21 after manipulation, the administration of MK-801 prevented nor-binaltorphimine-induced hyperalgesia. No changes in dynorphin levels were observed before or after opioid antagonist administration. In conclusion, surgery produced latent pain sensitization evidenced by opioid antagonist-precipitated hyperalgesia. The effect was stereospecific, centrally originated, and mediated by kappa opioid receptors. The blockade of nor-binaltorphimine-induced hyperalgesia by MK-801, suggests that NMDA receptors are also involved. Our results show for the first time that surgery induces latent, long-lasting changes in the processing of nociceptive information that can be induced by non-nociceptive stimuli such as the administration of opioid antagonists. © 2011 Elsevier B.V. All rights reserved.

1. Introduction In a mouse model of post-surgical pain we have previously shown that the intraoperative administration of remifentanil enhances and extends postoperative pain sensitization (Cabañero et al., 2009a,b; Campillo et al., 2010; Célérier et al., 2006). It has also been reported that animals previously injured or exposed to opioids develop longlasting pain vulnerability shown by increased susceptibility to develop hyperalgesia in response to new stimuli or opioid administration (Cabañero et al., 2009a; Rivat et al., 2002, 2007). This phenomenon is known as latent pain sensitization (Rivat et al., 2007) and may reflect the transition from acute to chronic pain.

⁎ Corresponding author at: Department of Anesthesiology, Hospital del Mar, Passeig Marítim 25–29, E-08003 Barcelona, Spain. Tel.: +34 93 2483527; fax: + 34 93 2483254. E-mail addresses: [email protected] (A. Campillo), davidmail@graffiti.net (D. Cabañero), [email protected] (A. Romero), [email protected] (P. García-Nogales), [email protected] (M.M. Puig). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.01.059

In animals exposed to pain or opioids, latent pain sensitization can be evidenced by the naloxone test (Célérier et al., 2001; Kim et al., 1990; Laulin et al., 2002; Li et al., 2001; Richebe et al., 2005), in which the abrupt blockage of the opioid receptors precipitates hyperalgesia. This response has been tentatively explained by an increase in endogenous opioid peptides and/or increased signaling activity at the opioid receptors (Célérier et al., 2001). However, the specific type of endogenous opioids and/or the opioid receptors implicated remain unclear. In addition, N-methyl-D-aspartate (NMDA) antagonists prevent naloxone-precipitated hyperalgesia in rats previously exposed to opioids (Laulin et al., 2002; Richebe et al., 2005). Although the NMDA receptor system seems to have an important role in latent pain sensitization, the series of events leading to its activation after exposure to opioids is unknown. It has been proposed that exposure to opioids up-regulates spinal dynorphin, that in turn would induce the release of excitatory transmitters from primary afferents (Gardell et al., 2002a,b; Vanderah et al., 2001). Dynorphin is an endogenous opioid with antinociceptive (binding to kappa opioid receptors), and pronociceptive effects, possibly acting on NMDA and/or bradykinin

90

A. Campillo et al. / European Journal of Pharmacology 657 (2011) 89–96

receptors (Lai et al., 2006; Tan-No et al., 2002). Thus, dynorphin could play a role maintaining a balance between opioid-dependent antinociception and the pronociceptive systems. In a mouse model of post-incisional pain, we reported that the intraoperative administration of remifentanil increases spinal dynorphin levels between days 2 and 10 after surgery (Campillo et al., 2010). The increased dynorphin may disrupt the initial equilibrium between the two pain modulating systems, inducing postoperative hyperalgesia lasting up to 10–14 days. The recovery of nociceptive thresholds could be related to the return of dynorphin levels to basal values, and/or to a new highregulating equilibrium reached between the two pain modulating systems (Célérier et al., 2001). In the present study, we used a mouse model of post-incisional pain that closely mimics the surgical procedure in humans and explored whether latent pain sensitization may occur after a surgical injury (incision) and/or remifentanil administration; we also investigated the role of the dynorphin/kappa opioid receptor system and NMDA receptors in this phenomenon. Our results could contribute to a better understanding of the underlying mechanisms involved in persistent pain after surgery in humans. 2. Material and methods 2.1. Animals Swiss CD1 male mice weighing 25–30 g obtained from CharlesRiver (CRIFFA, France) were used in all experiments. Animals were housed four per cage and maintained in a room under a 12 h light/ dark cycle (lights on at 8 AM), at controlled temperature (21 ± 1 °C) and relative humidity (55 ± 10%). Food and water were available ad libitum except during behavioral evaluation. All procedures and animal handling met the guidelines of the European Communities directive 86/609/EEC regulating animal research. The protocol was approved by the institutional review board of our institution (CEEAPRBB, Barcelona, Spain).

subcutaneously (s.c.) over a period of 30 min (rate 0.8 ml/h) using a pump (KD Scientific Inc., Holliston, MA). (−)-Naloxone (1 mg/kg, a non receptor-specific dose) (Célérier et al., 1999, 2000, 2001; Richebe et al., 2005), (+)-naloxone hydrochloride (1 mg/kg) (at the same doses of the active isomer), and naloxone methiodide (3 mg/kg) (at a ratio 3:1) were used with reference to (−)-naloxone, as previously used in our laboratory (Pol et al., 1995). In vivo doses of Nor-BNI (5 mg/kg) (Endoh et al., 1992), and MK-801 (0.15 mg/kg) (Bilsky et al., 1996; Célérier et al., 1999, 2001) were selected according to previous reports, and administrated as a s.c. injection of 250 μl. All drugs were dissolved in saline (NaCl 0.9%), and were injected at the same dose and route to sham-treated animals that served as control. 2.4. Behavioral testing Hyperalgesia to punctate mechanical stimulus (referred as mechanical hyperalgesia throughout the text) served as a measure of nociception. Before the experiments, animals were habituated to the equipment (without nociceptive stimulation) for 2–3 days. All behavioral experiments were performed between 9:00 AM and 4:00 PM. Mechanical hyperalgesia was measured by the hind paw withdraw response to von Frey filament stimulation. Animals were placed in methacrylate cylinders (30 cm high, 9 cm diameter; acquired from Servei Estació, Barcelona, Spain) with a wire grid bottom through which the von Frey filaments were applied (bending force range from 0.008 to 2 g; North Coast Medical, Inc., San Jose, CA). Animals were allowed to habituate for 2 h before testing, to achieve immobility. The filament force was increased or decreased according to the response. The upper limit value (2 g) was assigned when there was no response, and the threshold of response was calculated using the up–down method (Chaplan et al., 1994). Paw shaking or licking were considered nociceptive-like responses. Both hind paws were alternately tested. 2.5. Dynorphin immunoassay

We used the incisional postoperative pain model adapted from rats (Brennan et al., 1996) and validated in mice in our laboratory (Cabañero et al., 2009a,b; Campillo et al., 2010; Célérier et al., 2006). Animals were anesthetized with sevoflurane delivered for 30 min via a nose mask (induction, 3.5% v/v; surgery, 3.0% v/v) in a sterile operating room. A 0.7 cm longitudinal incision was made with a number 20 blade through the skin and fascia of the plantar surface of the right hind paw, starting 0.3 cm from the proximal edge of the heel extending toward the toes. The underlying plantaris muscle was exposed and incised longitudinally, keeping the muscle origin and insertion intact. After homeostasis with slight pressure, the skin was closed with two 6–0 silk sutures and the wound covered with povidone-iodine antiseptic ointment. After surgery, the animals were allowed to recover under a heat source in cages with sterile bedding.

In the control group, we used the lumbar spinal cord (from L4–L6) of 2 animals per sample. In the experimental group remifentanil + incision (Section 2.6), we used the ipsilateral and contralateral spinal cord from the same spinal section (L4–L6) of 4 animals per sample. After sacrifice, the spinal cord was removed and frozen in liquid nitrogen. To perform the assay, tissue samples were placed in 1 N acetic acid, disrupted with a homogenizer (Ultra-Turf, T8; Ika Werke, Staufen, Germany), and incubated for 30 min at 95 °C. After centrifugation at 12,000 rpm for 20 min (4 °C) the supernatant was lyophilized and stored at −80 °C. Protein concentrations were determined using the bichinchoninic acid method (BCATM Protein Assay Kit, Thermo Scientific, Rockford, IL, USA) with bovine serum albumin as standard. Immunoassay was performed with a commercial enzyme immunoassay kit using a specific antibody for dynorphin A (1–17) (Peninsula Laboratories, Belmont, CA). Each experiment was repeated at least four times. Standard curves were constructed and the dynorphin content determined with the Prism program (GraphPad, San Diego, CA).

2.3. Drugs

2.6. Experiments performed

Sevoflurane (Sevorane®; Abbot Laboratories S.A., Madrid, Spain), remifentanil (Ultiva®, GlaxoSmithKline, Madrid, Spain), and (−)naloxone hydrochloride (Naloxona KERN PHARMA®, Kern Pharma, Barcelona, Spain) were supplied by the Department of Anesthesiology of the Hospital del Mar (Barcelona, Spain). (+)-Naloxone hydrochloride was obtained from the National Institute on Drug Abuse (Bethesda, MD, USA). Nor-binaltorphimine dihydrocloride (nor-BNI), naloxone-methiodide and dizocilpine hydrogen maleate (MK-801) were purchased from Sigma (St. Louis, MO, USA). Drug doses were selected on the basis of previous studies (Cabañero et al., 2009a; Campillo et al., 2010): remifentanil (80 μg/kg), was infused

2.6.1. Behavioral studies Mechanical thresholds were evaluated using von Frey filaments. Special care was taken to reduce interindividual variability and to use the smallest number of animals per group. After the habituation period, the average of the measures of two to three consecutive days was obtained and the baseline response calculated. Experiments were performed in mice receiving one of the following treatments:

2.2. Surgery

– Sevoflurane + s.c. saline, a treatment that does not alter nociceptive thresholds (Célérier et al., 2006) (the group will be referred as control or sham-treated group).

A. Campillo et al. / European Journal of Pharmacology 657 (2011) 89–96

– Sevoflurane + s.c. remifentanil 80 μg/kg (remifentanil group). – Surgery performed under sevoflurane + s.c. saline (incision group). – Surgery performed under sevoflurane + s.c. remifentanil 80 μg/kg (remifentanil + incision group). According to previous work performed in our laboratory, the highest degree of mechanical hyperalgesia was observed in the remifentanil + incision group 2 days after manipulation; in those experiments, mechanical nociceptive thresholds returned to baseline between days 10 and 14 (Cabañero et al., 2009a; Campillo et al., 2010). In the present investigation, mechanical hyperalgesia was assessed in all experimental groups on days 2, 20, and 21 after manipulation. Testing on day 20 was performed in order to confirm that mechanical thresholds were similar to baseline values. On day 21, animals received one of the drugs of study, and were tested again for mechanical hyperalgesia. Thus, animals were evaluated at two consecutive time points (days 20 and 21), to facilitate the experimental process. A pilot study was performed to determine the time of maximal effect of (−)-naloxone (1 mg/kg) or nor-BNI (5 mg/kg) in the remifentanil + incision group. Animals were tested with von Frey filaments 5, 60, and 120 min after a s.c. injection of each antagonist, until nociceptive thresholds returned to baseline values. (−)-Naloxone showed a maximal peak effect between 5 and 30 min, disappearing 1 h later. However, the maximal effect of nor-BNI was observed between 120 and 150 min, completely disappearing 2 weeks later (data not shown). After systemic administration in vivo, kappa opioid receptor blockade by norBNI has been reported to have a slow-onset and long-lasting effect (Endoh et al., 1992), a fact that cannot be demonstrated after intrathecal (i.t.) administration (Hao et al., 1998). To explore the presence of latent pain sensitization after surgery and/or remifentanil administration, and the possible involvement of the dynorphin/kappa opioid receptor/NMDA systems, we performed the following experiments to assess. 2.6.1.1. The effect of (−)-naloxone or nor-BNI on mechanical thresholds after complete recovery of nociceptive thresholds. On day 21, mice from the four groups of study (see Section 2.6) received a s.c. injection of (−)-naloxone (1 mg/kg) or nor-BNI (5 mg/kg). For each group of treatment, 6–12 animals were used. 2.6.1.2. The effect of (+)-naloxone and naloxone methiodide on mechanical thresholds after complete recovery of surgery performed under remifentanil anesthesia (remifentanil + incision group). On postoperative day 21, mice received a s.c. injection of (+)-naloxone (1 mg/kg) or naloxone methiodide (3 mg/kg). We used for comparison a group of mice receiving (−)-naloxone (1 mg/kg); 6–12 animals per group were tested. 2.6.1.3. The duration of the effect of (−)-naloxone or nor-BNI on mechanical thresholds, after complete recovery of surgery performed under remifentanil anesthesia (remifentanil+ incision group). (−)-Naloxone (1 mg/kg) or nor-BNI (5 mg/kg) was administered on days 21, 51, 111 or 151 after surgery. All mice were also tested for mechanical hyperalgesia on the previous days (20, 50, 110, and 150) to confirm that mechanical thresholds had been restored to baseline values. Between 6 and 11 mice were used for each time point. 2.6.1.4. The effect of MK-801 administered prior to nor-BNI in the remifentanil + incision group. On day 21 after surgery, mice received a s.c. injection of MK-801 (0.15 mg/kg) or saline and were tested for mechanical hyperalgesia between the next 5 and 30 min. Immediately afterwards [on min 30 (Bilsky et al., 1996)], the same mice received a s.c. injection of nor-BNI (5 mg/kg) and were tested again 120 min later (i.e. 150 min from MK-801 injection). A control sham-treated

91

group received the same treatments. For each group of treatment, 6–9 animals were used. 2.6.2. Dynorphin determination 2.6.2.1. Dynorphin levels were determined in the spinal cord after the administration of s.c. (−)-naloxone, in the remifentanil + incision group. On day 21 after surgery, animals were sacrificed 30 min after a s.c. injection of saline or (−)-naloxone (1 mg/kg). Each determination was repeated at least five times. 2.7. Statistical analysis In the behavioral studies for each mouse and time point, the responses in grams (von Frey) are expressed as percent changes (%)±S.D., with respect to baseline values (represented in the figures by a horizontal broken line). Negative values indicate net pronociceptive effects. In the biochemical study, the data obtained from the dynorphin A (1–17) immunoassay (expressed in pg/mg of total protein±S.D.) was normalized with respect to the mean control values, set to a value of 1. In the text, dynorphin levels are expressed as the mean relative value±S.D. For the statistical analysis, we used the Kruskal–Wallis ANOVA test with the GraphPad Prism 4 software (San Diego, CA). Paired comparisons were performed by the non-parametric Mann Whitney test for independent samples (GraphPad Prism 4). A P value less than 0.05 was considered statistically significant. 3. Results The results are reported according to the groups of experiments described in Material and methods. 3.1. The effect of (−)-naloxone or nor-BNI on mechanical thresholds after complete recovery of nociceptive thresholds The pronociceptive effects of s.c. (−)-naloxone or nor-BNI were evaluated in all groups (Fig. 1). In the experiments with (−)naloxone, baseline thresholds (day 0) were similar in all groups, with pooled mean values of 1.28 ± 0.11 and 1.23 ± 0.11 g for the right and left paws, respectively. Baseline thresholds in mice receiving nor-BNI were also comparable in all groups with mean values of 1.27 ± 0.10 g (right paw) and 1.26 ± 0.12 g (left paw). In the control group (sham-treated) non significant changes in nociceptive thresholds were observed when animals were tested on days 2, 20 and 21 after manipulation. In the other groups, significant hyperalgesia was observed on day 2, but was absent on day 20 after treatment. On day 21, the s.c. administration of (−)-naloxone (1 mg/kg) or nor-BNI (5 mg/kg) induced significant hyperalgesia in all treatment groups, that in all instances was of a similar magnitude than the observed on day 2 (Fig. 1). In animals receiving s.c. (−)-naloxone, the decrease in nociceptive thresholds on day 21 in the operated paw (Fig. 1A) was: −23.6±23.0% in the remifentanil group (Pb 0.05 vs. control), −26.7±15.9% (Pb 0.05) in the incision group, and −58.3±19.3% (Pb 0.001) in the remifentanil + incision group. The hyperalgesia observed in the remifentanil+incision group was significantly greater than that of each treatment individually (remifentanil group Pb 0.05; incision Pb 0.01), suggesting a summation of effects when both treatments were combined. When evaluating the left non-injured paw (Fig. 1B), significant hyperalgesia was observed in all experimental conditions and times of evaluation, except in the incision group. Nor-BNI administration on day 21 also decreased nociceptive thresholds in the right paw in all groups of study (when compared to controls), although the reduction in the remifentanil group was not statistically significant (N.S.) (Fig.1C). Percent decreases were: −23.2±30.6% (N.S.) in the remifentanil group, −39.4±14.6% (Pb 0.01) in the incision group,

92

A. Campillo et al. / European Journal of Pharmacology 657 (2011) 89–96

Fig. 1. Mechanical hyperalgesia after (−)-naloxone or nor-BNI administration. Nociceptive thresholds on days 2, 20, and 21, in the following groups of study: sham-treated (white columns, n = 6), remifentanil (dotted columns, n = 6), incision (lined columns, n = 7), and remifentanil + incision (gray columns, n = 12). On day 21 all animals received a s.c. injection (arrows) of (−)-naloxone (1 mg/kg) (A, B right and left hind paw, respectively) or nor-BNI (5 mg/kg) (C, D right and left hind paw). Values are expressed as mean percent change ± S.D. with respect to baseline (horizontal broken lines). *P b 0.05; **P b 0.01; ***P b 0.001 vs. control group; &P b 0.05; &&P b 0.01 vs. remifentanil group; #P b 0.05; ##P b 0.01; ### P b 0.001 vs. incision group. Comparison performed with Kruskal–Wallis ANOVA test followed by Mann Whitney test.

and −57.4±20.4% (Pb 0.01) in the remifentanil+incision group. In the left paw (Fig. 1D), significant hyperalgesia was only observed in the remifentanil+incision group. In the remifentanil + incision group, the hyperalgesia precipitated by (−)-naloxone or nor-BNI was of a similar magnitude (Fig. 1A, B, C, D). These experiments show that after complete recovery of surgery and/or remifentanil administration (day 20), the subcutaneous administration of (−)-naloxone or nor-BNI induces significant mechanical hyperalgesia.

controls (Fig. 2). As active control, we also tested another group of mice receiving (−)-naloxone (1 mg/kg) on day 21, that showed significant mechanical hyperalgesia with mean threshold reductions of −52.9 ± 26.7% (P b 0.01, compared to controls) and −32.9 ± 23.3% (P b 0.01) in the operated and non-operated paw, respectively. Thus, neither (+)-naloxone or naloxone-methiodide modified nociceptive thresholds in animals that had previous surgery under remifentanil anesthesia, suggesting that the effect is stereospecific and is mediated by opioid receptors located in the central nervous system.

3.2. Effect of (+)-naloxone and naloxone methiodide on mechanical thresholds after complete recovery of surgery performed under remifentanil anesthesia

3.3. Duration of the effect of (−)-naloxone or nor-BNI on mechanical thresholds, after complete recovery of surgery performed under remifentanil anesthesia (remifentanil + incision group)

Baseline thresholds in both paws were similar in the control and remifentanil + incision groups, with mean pooled values of 1.20±0.17 g in the right operated paw, and 1.23 ± 0.12 g in the left paw (nonoperated). (+)-Naloxone (inactive isomer of naloxone) or naloxonemethiodide (peripherally acting naloxone) administered on day 21, did not significantly change nociceptive thresholds when compared to the

We assessed the time-course of mechanical hyperalgesia precipitated by (−)-naloxone (1 mg/kg) or nor-BNI (5 mg/kg), in the remifentanil + incision group. Mechanical thresholds were tested on day 2 and on days 20/21, 50/51, 110/111 and 150/151 after manipulation; the antagonists were administered on days 21, 51, 111 and 151. Different sets of animals were used for each time of evaluation (see Section 2.6.1.3).

A. Campillo et al. / European Journal of Pharmacology 657 (2011) 89–96

93

postoperative latent pain sensitization substantiated by the administration of opioid antagonists. 3.4. Effect of MK-801 administered prior to nor-BNI in the remifentanil+incision group We performed these experiments to assess if nor-BNI-precipitated hyperalgesia could be prevented by the s.c. administration of MK-810 (0.15 mg/kg). Baseline thresholds in both paws were similar in the control and remifentanil + incision groups. Pre-drug mean baseline values were 1.16 ± 0.17 g and 1.21 ± 0.16 g in the MK-801 group. On day 21, the s.c. administration of MK-801 individually, did not alter nociceptive thresholds in any of the groups during the first 5–30 min after injection (data not shown). However, the subsequent administration of nor-BNI induced significant hyperalgesia in the remifentanil + incision group (Fig. 4), corroborating the results shown in Figs. 1 and 2. The s.c. administration of MK-801 completely prevented nor-BNI precipitated hyperalgesia in the operated (−8.34 ± 26.6%; P b 0.01) and non-operated paw (−4.79 ± 26.4%; P b 0.05), as shown in Fig. 4A, B. 3.5. Dynorphin levels in the spinal cord after the administration of s.c. (−)-naloxone, in the remifentanil + incision group

Fig. 2. Mechanical hyperalgesia after (+)-naloxone or naloxone-methiodide administration. Nociceptive thresholds on days 2, 20, and 21, in control sham-treated (white columns, n = 6) and remifentanil + incision group (gray columns, n = 12). On day 21 all animals received a s.c. injection (arrows) of (+)-naloxone (1 mg/kg) (A, B right and left hind paw, respectively) or naloxone-methiodide (naloxone-met, 3 mg/kg) (C, D right and left hind paw). Values are expressed as mean percent change ± S.D. with respect to the baseline (horizontal broken lines). **P b 0.01 vs. control group, using Kruskal–Wallis ANOVA test followed by Mann Whitney test.

In sham-treated mice and in animals in the remifentanil +incision group, we determined dynorphin levels on day 21 after manipulation. The groups received either s.c. (−)-naloxone (1 mg/kg) or the same volume of saline. In the spinal cord, mean dynorphin levels in shamtreated mice (control) receiving saline were 358.7±151.0 pg/mg of total protein in the ipsilateral, and 291.7±165.7 pg/mg in the contralateral spinal cord. Similar values were observed in sham-treated animals receiving (−)-naloxone (456.6±39.1 and 372.8±108.8 pg/mg for the ipsilateral and contralateral side). Thus, (−)-naloxone did not alter dynorphin levels in control animals. Similarly, no changes in dynorphin were observed in the remifentanil+incision group after saline or (−)naloxone administration. Compared to the control group (values set to 1), the relative expression of dynorphin in the ipsilateral side were 1.28 ± 0.12 (N.S.) and 0.93 ± 0.23 (N.S.) for saline and (−)-naloxone treated animals, respectively, while in the contralateral side were of 1.04 ± 0.35 and 0.87 ± 0.32. When a challenge with s.c. nor-BNI (instead of naloxone) was administered to another group of animals, we did not observe any changes in dynorphin levels in the spinal cord in two consecutive experiments (results not shown), and thus the group of experiments was not completed to avoid unnecessary killing of mice. Consequently we can conclude that, in mice that had surgery under remifentanil anesthesia, the administration of opioid antagonists did not modify dynorphin levels in the spinal cord. 4. Discussion

In animals receiving a challenge with (−)-naloxone, pooled mean baseline values were 1.22 ± 0.16 g and 1.25 ± 0.11 g for the right and left paws respectively, while in those receiving nor-BNI they were 1.24 ± 0.15 g and 1.25 ± 0.13 g. In sham-treated mice (control) nociceptive thresholds remained unaltered at all times of evaluation. Similarly, in the remifentanil + incision group, thresholds in the previous days to the antagonist administration were similar to baseline (Fig. 3). The results show that in the operated paw (Fig. 3A, C) antagonist-precipitated hyperalgesia was still present on day 151 (i.e. 5 months after manipulation), although at this time point it was of a lesser magnitude when compared to day 2. In the non-operated paw (Fig. 3B, D), antagonist-induced mechanical hyperalgesia disappeared between days 111 and 151. The results show bilateral, long lasting,

The present study shows that surgery performed under remifentanil anesthesia induces long lasting neuroplastic changes in the central nervous system that could be involved in latent pain sensitization in mice. This long-lasting pain vulnerability was demonstrated by the precipitated hyperalgesia induced after (−)-naloxone or nor-BNI administration, that was present even 5 months after the surgical procedure. This effect suggests long-term changes in the activity of the endogenous opioid system at the level of the kappa opioid receptors, and/or changes in other systems resulting in altered effects of kappa opioid receptor activation. Nociceptive thresholds remained unaltered after (+)-naloxone or naloxone-methiodide administration, showing the stereospecificity and the central origin of the hyperalgesic response, respectively. In this group, the NMDA antagonist MK-801 prevented nor-BNI precipitated hyperalgesia. Although dynorphin levels remained

94

A. Campillo et al. / European Journal of Pharmacology 657 (2011) 89–96

Fig. 3. Time course of the mechanical hyperalgesia after (−)-naloxone or nor-BNI administration. Nociceptive thresholds on days 2, 20, 21, 50, 51, 110, 111, 150, and 151, in control sham-treated (white columns, n = 6–10 for each time point) and remifentanil+ incision group (gray columns, n = 7–11). On day 21, 51, 111, and 151 all animals received a s.c. injection of (−)-naloxone (1 mg/kg) (A, B right and left hind paw, respectively) or nor-BNI (5 mg/kg) (C, D right and left hind paw). Values are expressed as mean percent change± S.D. with respect to the baseline (horizontal broken lines). *Pb 0.05; **P b 0.01; ***P b 0.001 vs. control group; +P b 0.05; ++P b 0.01 vs. remifentanil+ incision group on day 2, using Kruskal–Wallis ANOVA test followed by Mann Whitney test.

unchanged during antagonist-induced hyperalgesia, our results suggest that an increased signaling activity at kappa opioid receptor could be implicated in postoperative latent pain sensitization. In sham-treated mice, the administration of (−)-naloxone or norBNI did not modify mechanical thresholds in our model, a fact that has been also reported by others (Crain and Shen, 2007; Li et al., 2001; Schepers et al., 2008); consequently, the development of latent pain sensitization requires either a surgical nociceptive input (Li et al., 2001) or exposure to an opioid (Célérier et al., 2001; Richebe et al., 2005; Rivat et al., 2007). In our model, (−)-naloxone or norBNI-precipitated hyperalgesia was observed after each treatment individually (remifentanil or surgical incision) and their combination (remifentanil + incision), being of a greater magnitude in the later group. This finding shows that the administration of remifentanil during surgery, enhances and extends postoperative pain (Cabañero et al., 2009a; Campillo et al., 2010), and in addition is able to induce long-lasting latent pain sensitization (up to 151 days after surgery). Given that in our study the remifentanil + incision group roughly reproduces the events taking place during surgery in humans, the present results suggest that the intraoperative administration of remifentanil in clinical practice could make patients more susceptible to develop latent pain sensitization and possibly chronic postsurgical pain. As the greatest antagonist-induced hyperalgesia was observed in the remifentanil + incision group, we investigated postoperative antagonist precipitated hyperalgesia and its underlying mechanisms in this group. By using (+)-naloxone, we could demonstrate the stereospecificity of the antagonist-precipitated hyperalgesia when binding to neuronal opioid receptors. Thus, in contrast to (−)-naloxone, the dextro-isomer did not significantly alter nociceptive thresholds in the control or remifentanil + incision groups. It has been hypothesized that opioid

agonists activate glial cells interacting with toll-like receptors subtype 4, and that opioid-induced hyperalgesia may have a non-neuronal component (Hutchinson et al., 2007); these effects would be blocked by both naloxone steroisomers. In this group of experiments the administration of (−)-naloxone precipitated hyperalgesia 21 days after surgery, while (+)-naloxone did not induce a significant effect. Thus on the basis of the present results, glial cells did not appear to be involved in latent pain sensitization in this experimental model. However, von Frey filaments might have been inadequate to detect a slight or minor antinociceptive effect induced by (+)-naloxone. In a different group of experiments we also tested the effects of the peripherally acting opioid antagonist naloxone-methiodide, a drug that apparently does not cross the blood-brain barrier (Labuz et al., 2007). Since this antagonist did not induce any changes in nociceptive thresholds, we could conclude that antagonist-precipitated hyperalgesia (and probably latent pain sensitization in our model) involves neuroplastic changes in the central nervous system; however, with the present experiments we could not establish if changes occurred at the level of the brain or the spinal cord. In our study, long-term pain sensitization was observed over a long period of time and also in the contralateral paw, supporting the central origin of this phenomenon. Long-lasting (up to 4 months) naloxone-precipitated hyperalgesia has been reported in rats after exposure to opioids (Célérier et al., 2001); however, we are showing for the first time that when using remifentanil during surgery, a challenge with nor-BNI, a kappa opioid receptor antagonist, can also precipitate significant hyperalgesia up to 5 months after surgery, thus clearly implicating kappa opioid receptors in latent pain sensitization. Nevertheless, 5 months after manipulation, hyperalgesia decreased in the operated paw, while completely disappeared in the non-operated one. Probably, surgery induces higher and more durable neuroplastic changes in the

A. Campillo et al. / European Journal of Pharmacology 657 (2011) 89–96

Fig. 4. Mechanical hyperalgesia after MK-801 administration prior to nor-BNI injection. Nociceptive thresholds on days 2, 20, and 21. On day 21, sham-treated mice (control) received a s.c. injection of saline (SSF) (white columns, n = 6), or s.c. MK-801 (0.15 mg/kg) (dotted columns, n = 6), followed 30 min later by s.c. nor-BNI (5 mg/kg). Animals of the remifentanil + incision group received s.c. SSF (lined columns, n = 6) or s.c. MK-801 at the same dose (gray columns, n = 9), followed 30 min later by s.c. nor-BNI (A, B right and left hind paw, respectively). Values are expressed as mean percent change± S.D. with respect to the baseline (broken lines). *P b 0.05 vs. control group; #P b 0.05; ##P b 0.01 vs. remifentanil + incision group (SSF+ nor-BNI) on day 21, using Kruskal–Wallis ANOVA test followed by Mann Whitney test.

ipsilateral than in the contralateral spinal cord, as suggested by previous results from our group, when demonstrating a lesser increase in c-Fos and the extracellular signal-regulated kinases 1 and 2 (ERK1/2) in the contralateral spinal cord at early times after manipulation (Campillo et al., 2010). All these findings suggest that the long-lasting imprint of acute pain in the central nervous system would contribute to the transition from acute to chronic pain. In all the experiments, the selective kappa opioid receptor antagonist nor-BNI induced a similar degree of hyperalgesia than high doses of (−)-naloxone that is an antagonist at mu, delta and kappa receptors (Figs. 1 and 3). Thus, the kappa opioid receptor seems to be the main opioid-receptor involved in long-term pain sensitization in our model. These results are in agreement with other studies in rodents, in which nor-BNI administration enhanced nociceptive behavior after sciatic nerve ligation, implicating kappa opioid receptors in the hyperalgesic response (Obara et al., 2003; Xu et al., 2004). To the best of our knowledge, this is the first time that nor-BNI-precipitated hyperalgesia has been demonstrated after the complete recovery of nociceptive behavior in injured and/or opioid-exposed animals. It has been reported that nor-BNI itself may produce long-lasting effects in the kappa opioid receptor signaling complex, probably

95

mediated by JNK activation (Bruchas et al., 2007); however it is unlikely that potential signaling effects of the antagonists by themselves (nor-BNI or naloxone) contributed to the present results. In our report, a single dose of the antagonist was administered on day 21 after surgery, and hyperalgesia was tested at the peak of maximal effect (see Material and methods section) both in control and postoperated mice. The antagonists had no effect in control mice, while they produced significant hyperalgesia in mice that had surgery. Thus our results indicate that the antagonists produced their acute hyperalgesic effect by blocking the different types of opioid receptors. We hypothesize that after the clinical recovery of hyperalgesia (in the incision+remifentanil group), naloxone would induce the release of dynorphin that in the presence of nor-BNI, could have a direct or indirect effect on NMDA receptors, mediating hyperalgesia. Dynorphin is an endogenous opioid peptide that shows preference for kappa receptors, and has been reported to have anti- and pro-nociceptive effects (Gardell et al., 2002a,b; Vanderah et al., 2001), although it is unclear at present if both effects may occur as a consequence of binding of dynorphin to kappa receptors. We used the systemic administration of nor-BNI in order to block kappa receptors at both spinal and supraspinal sites, including those that could be involved in the descending pain modulatory pathways in the brainstem rostral ventromedial medulla. Thus, the precise site of action of the antagonists (spinal vs. supraspinal) cannot be established from the present investigation. The fact that we were unable to demonstrate changes in dynorphin levels in the spinal cord after (−)-naloxone administration does not exclude that increased dynorphin levels at supraspinal sites could be present, since reversal of nor-BNI precipitated hyperalgesia by MK-801 could be mediated by mechanisms involving transmitter systems other than dynorphin. Although MK-801 administration prevented antagonist-precipitated hyperalgesia, we did not observe antinociception when administered individually, a finding that is in agreement with other studies with NMDA receptor antagonists in rodents (Célérier et al., 2001; Richebe et al., 2005). In addition, clinical studies report that the NMDA antagonist ketamine prevents postoperative hyperalgesia, particularly when surgery is performed under remifentanil anesthesia (Angst and Clark, 2006; Joly et al., 2005). Thus, another possibility to explain our results would be that, in the presence of unchanged normal levels of dynorphin in the spinal cord, antagonistprecipitated hyperalgesia could be related to an increased signaling activity of kappa opioid receptors and/or NMDA receptors (Célérier et al., 2001), or by the sensitization of these receptors (McLaughlin et al., 2004), a hypothesis that has not been explored in the present investigation. 5. Conclusion In summary, surgical injury (incision) and/or remifentanil administration induce long term pain sensitization as shown by opioidantagonist precipitated hyperalgesia. The hyperalgesic effect was stereospecific, centrally originated, and seems to be mediated by kappa opioid receptors. The reversal of the hyperalgesia by the administration of MK-801, also shows the direct or indirect involvement of the NMDA receptors. To the best of our knowledge, this is the first time that nor-BNI-precipitated hyperalgesia has been demonstrated after the complete recovery of nociceptive behavior in injured and/or opioid-exposed animals. The results would suggest that the use of intraoperative opioids in humans may account for a higher susceptibility to suffer pain after new stimulus, and that the long-lasting imprint of acute pain in the central nervous system could contribute to the transition of acute to chronic pain. Acknowledgements The authors thank Ms. Carolina Zamora for her excellent technical assistance, Klaus Langohr, PhD, for his contribution in the statistical analysis. (+)-Naloxone hydrochloride was kindly provided from the National Institute on Drug Abuse (Bethesda, MD, USA). This work was

96

A. Campillo et al. / European Journal of Pharmacology 657 (2011) 89–96

supported by grants from Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Madrid, Spain (Grants PS09/01270); Marató de Televisió de Catalunya, TV3 (Grant 071110); the Endowed Chair in Pain Management Universitat Autònoma de Barcelona-Institut Municipal d'Assitència Sanitària-MENARINI; and a Predoctoral Fellowship from the Spanish Ministry of Education (Grant AP2006-4718) held by Ana Campillo. References Angst, M.S., Clark, J.D., 2006. Opioid-induced hyperalgesia: a qualitative systematic review. Anesthesiology 104, 570–587. Bilsky, E.J., Inturrisi, C.E., Sadee, W., Hruby, V.J., Porreca, F., 1996. Competitive and noncompetitive NMDA antagonists block the development of antinociceptive tolerance to morphine, but not to selective mu or delta opioid agonists in mice. Pain 68, 229–237. Brennan, T.J., Vandermeulen, E.P., Gebhart, G.F., 1996. Characterization of a rat model of incisional pain. Pain 64, 493–501. Bruchas, M.R., Yang, T., Schreiber, S., DeFino, M., Kwan, S.C., Li, S., Chavkin, C., 2007. Longacting κ opioid antagonists disrupt receptor signaling and produce noncompetitive effects by activating c-Jun N-terminal kinase. J. Biol. Chem. 282, 29803–29811. Cabañero, D., Campillo, A., Célérier, E., Romero, A., Puig, M.M., 2009a. Pronociceptive effects of remifentanil in a mouse model of postsurgical pain: effect of a second surgery. Anesthesiology 111, 1334–1345. Cabañero, D., Célérier, E., Garcia-Nogales, P., Mata, M., Roques, B.P., Maldonado, R., Puig, M.M., 2009b. The pro-nociceptive effects of remifentanil or surgical injury in mice are associated with a decrease in delta-opioid receptor mRNA levels: prevention of the nociceptive response by on-site delivery of enkephalins. Pain 141, 88–96. Campillo, A., Gonzalez-Cuello, A., Cabañero, D., Garcia-Nogales, P., Romero, A., Milanes, M.V., Laorden, M.L., Puig, M.M., 2010. Increased spinal dynorphin levels and phosphoextracellular signal-regulated kinases 1 and 2 and c-Fos immunoreactivity after surgery under remifentanil anesthesia in mice. Mol. Pharmacol. 77, 185–194. Célérier, E., Laulin, J., Larcher, A., Le Moal, M., Simonnet, G., 1999. Evidence for opiateactivated NMDA processes masking opiate analgesia in rats. Brain Res. 847, 18–25. Célérier, E., Rivat, C., Jun, Y., Laulin, J.P., Larcher, A., Reynier, P., Simonnet, G., 2000. Longlasting hyperalgesia induced by fentanyl in rats: preventive effect of ketamine. Anesthesiology 92, 465–472. Célérier, E., Laulin, J.P., Corcuff, J.B., Le Moal, M., Simonnet, G., 2001. Progressive enhancement of delayed hyperalgesia induced by repeated heroin administration: a sensitization process. J. Neurosci. 21, 4074–4080. Célérier, E., Gonzalez, J.R., Maldonado, R., Cabañero, D., Puig, M.M., 2006. Opioidinduced hyperalgesia in a murine model of postoperative pain: role of nitric oxide generated from the inducible nitric oxide synthase. Anesthesiology 104, 546–555. Chaplan, S.R., Bach, F.W., Pogrel, J.W., Chung, J.M., Yaksh, T.L., 1994. Quantitative assessment of tactile allodynia in the rat paw. J. Neurosci. Methods 53, 55–63. Crain, S.M., Shen, K.F., 2007. Naloxone rapidly evokes endogenous kappa opioid receptor-mediated hyperalgesia in naive mice pretreated briefly with GM1 ganglioside or in chronic morphine-dependent mice. Brain Res. 1167, 31–41. Endoh, T., Matsuura, H., Tanaka, C., Nagase, H., 1992. Nor-binaltorphimine: a potent and selective kappa-opioid receptor antagonist with long-lasting activity in vivo. Arch. Int. Pharmacodyn. Thér. 316, 30–42. Gardell, L.R., Burgess, S.E., Dogrul, A., Ossipov, M.H., Malan, T.P., Lai, J., Porreca, F., 2002a. Pronociceptive effects of spinal dynorphin promote cannabinoid-induced pain and antinociceptive tolerance. Pain 98, 79–88. Gardell, L.R., Wang, R., Burgess, S.E., Ossipov, M.H., Vanderah, T.W., Malan Jr., T.P., Lai, J., Porreca, F., 2002b. Sustained morphine exposure induces a spinal dynorphin-dependent

enhancement of excitatory transmitter release from primary afferent fibers. J. Neurosci. 22, 6747–6755. Hao, J.X., Yu, W., Xu, X.J., 1998. Evidence that spinal endogenous opioidergic systems control the expression of chronic pain-related behaviors in spinally injured rats. Exp. Brain Res. 118, 259–268. Hutchinson, M.R., Bland, S.T., Johnson, K.W., Rice, K.C., Maier, S.F., Watkins, L.R., 2007. Opioid-induced glial activation: mechanisms of activation and implications for opioid analgesia, dependence, and reward. Scientific World Journal 7, 98–111. Joly, V., Richebe, P., Guignard, B., Fletcher, D., Maurette, P., Sessler, D.I., Chauvin, M., 2005. Remifentanil-induced postoperative hyperalgesia and its prevention with small-dose ketamine. Anesthesiology 103, 147–155. Kim, D.H., Fields, H.L., Barbaro, N.M., 1990. Morphine analgesia and acute physical dependence: rapid onset of two opposing, dose-related processes. Brain Res. 516, 37–40. Labuz, D., Mousa, S.A., Schafer, M., Stein, C., Machelska, H., 2007. Relative contribution of peripheral versus central opioid receptors to antinociception. Brain Res. 1160, 30–38. Lai, J., Luo, M.C., Chen, Q., Ma, S., Gardell, L.R., Ossipov, M.H., Porreca, F., 2006. Dynorphin A activates bradykinin receptors to maintain neuropathic pain. Nat. Neurosci. 9, 1534–1540. Laulin, J.P., Maurette, P., Corcuff, J.B., Rivat, C., Chauvin, M., Simonnet, G., 2002. The role of ketamine in preventing fentanyl-induced hyperalgesia and subsequent acute morphine tolerance. Anesth. Analg. 94, 1263–1269 table of contents. Li, X., Angst, M.S., Clark, J.D., 2001. Opioid-induced hyperalgesia and incisional pain. Anesth. Analg. 93, 204–209. McLaughlin, J.P., Myers, L.C., Zarek, P.E., Caron, M.G., Lefkowitz, R.J., Czyzyk, T.A., Pintar, J.E., Chavkin, C., 2004. Prolonged kappa opioid receptor phosphorylation mediated by G-protein receptor kinase underlies sustained analgesic tolerance. J. Biol. Chem. 279, 1810–1818. Obara, I., Mika, J., Schafer, M.K., Przewlocka, B., 2003. Antagonists of the kappa-opioid receptor enhance allodynia in rats and mice after sciatic nerve ligation. Br. J. Pharmacol. 140, 538–546. Pol, O., Planas, E., Puig, M.M., 1995. Peripheral effects of naloxone in mice with acute diarrhea associated with intestinal inflammation. J. Pharmacol. Exp. Ther. 272, 1271–1276. Richebe, P., Rivat, C., Laulin, J.P., Maurette, P., Simonnet, G., 2005. Ketamine improves the management of exaggerated postoperative pain observed in perioperative fentanyl-treated rats. Anesthesiology 102, 421–428. Rivat, C., Laulin, J.P., Corcuff, J.B., Célérier, E., Pain, L., Simonnet, G., 2002. Fentanyl enhancement of carrageenan-induced long-lasting hyperalgesia in rats: prevention by the N-methyl-D-aspartate receptor antagonist ketamine. Anesthesiology 96, 381–391. Rivat, C., Laboureyras, E., Laulin, J.P., Le Roy, C., Richebe, P., Simonnet, G., 2007. Nonnociceptive environmental stress induces hyperalgesia, not analgesia, in pain and opioid-experienced rats. Neuropsychopharmacology 32, 2217–2228. Schepers, R.J., Mahoney, J.L., Gehrke, B.J., Shippenberg, T.S., 2008. Endogenous kappaopioid receptor systems inhibit hyperalgesia associated with localized peripheral inflammation. Pain 138, 423–439. Tan-No, K., Esashi, A., Nakagawasai, O., Niijima, F., Tadano, T., Sakurada, C., Sakurada, T., Bakalkin, G., Terenius, L., Kisara, K., 2002. Intrathecally administered big dynorphin, a prodynorphin-derived peptide, produces nociceptive behavior through an N-methyl-Daspartate receptor mechanism. Brain Res. 952, 7–14. Vanderah, T.W., Ossipov, M.H., Lai, J., Malan Jr., T.P., Porreca, F., 2001. Mechanisms of opioid-induced pain and antinociceptive tolerance: descending facilitation and spinal dynorphin. Pain 92, 5–9. Xu, M., Petraschka, M., McLaughlin, J.P., Westenbroek, R.E., Caron, M.G., Lefkowitz, R.J., Czyzyk, T.A., Pintar, J.E., Terman, G.W., Chavkin, C., 2004. Neuropathic pain activates the endogenous kappa opioid system in mouse spinal cord and induces opioid receptor tolerance. J. Neurosci. 24, 4576–4584.