Asunción Blanco Romero

Pronociceptive effects of remifentanil in a mouse model of postsurgical pain: effect of a second surgery.

Background:Remifentanil anesthesia enhances postoperative pain in animals and humans. The authors evaluated the impact of the dose (&mgr;g · kg−1 · min−1) and duration of remifentanil infusion, and the effects of a second surgery on postoperative pain sensitization. Methods:Mice received different doses of remifentanil over 30 or 60 min. The authors assessed thermal (Hargreaves) and mechanical hyperalgesia (von Frey) at 2, 4, 7, and 10 days. In other experiments, mice had a plantar incision during sevoflurane with or without remifentanil anesthesia that was repeated 27 days later, when nociceptive thresholds returned to baseline. Linear mixed models were used for statistical analysis. Results:Remifentanil induced dose-dependent pronociceptive effects with calculated ED50s of 1.7 (95% confidence interval, 1.3–2.1) and 1.26 (1.0–1.6) &mgr;g · kg−1 · min−1 for thermal and mechanical hyperalgesia, respectively, which lasted longer with higher doses (P < 0.001). The duration of infusion did not alter the pronociceptive effects of remifentanil when administered at a constant dose of infusion. When given during surgery, high (2.66 &mgr;g · kg−1 · min−1) or low (0.66 &mgr;g · kg−1 · min−1) remifentanil increased the extent (P < 0.05) and duration (P < 0.01) of thermal and mechanical hyperalgesia. The latter was further enhanced after a second surgery performed in the same experimental conditions (P < 0.05). Surgery or remifentanil infusion, each one individually, induced significant mechanical hyperalgesia, which was greater when repeated (P < 0.05). Conclusions:In this model of incisional pain, remifentanil induces pronociceptive effects, which are dose dependent but unaltered by the duration of administration. A second surgery performed on the same site and experimental conditions induces greater postoperative hyperalgesia that is enhanced when remifentanil is used as an anesthetic.

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

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 (80μg/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 (1mg/kg), (+)-naloxone (1mg/kg), naloxone-methiodide (3mg/kg), or nor-binaltorphimine (5mg/kg). Another group received subcutaneous MK-801 (0.15mg/kg) before nor-binaltorphimine 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 5months 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.

Increased Spinal Dynorphin Levels and Phospho-Extracellular Signal-Regulated Kinases 1 and 2 and c-Fos Immunoreactivity after Surgery under Remifentanil Anesthesia in Mice

In humans, remifentanil anesthesia enhances nociceptive sensitization in the postoperative period. We hypothesized that activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the expression of c-Fos, prodynorphin (mRNA), and dynorphin in the spinal cord could participate in the molecular mechanisms underlying postoperative opioid-induced sensitization. In a mouse model of incisional pain, we evaluated thermal (Hargreaves test) and mechanical (von Frey) hyperalgesia during the first 21 postoperative days. Moreover, prodynorphin (mRNA, real-time polymerase chain reaction), dynorphin (enzymatic immunoassay), c-Fos expression, and ERK1/2 phosphorylation (both by immunohistochemistry) in the lumbar spinal cord were assessed. Surgery performed under remifentanil anesthesia induced a maximal decrease in nociceptive thresholds between 4 h and 2 days postoperatively (p < 0.001) that lasted 10 to 14 days compared with noninjured animals. In the same experimental conditions, a significant increase in prodynorphin mRNA expression (at 2 and 4 days) followed by a sustained increase of dynorphin (days 2 to 10) in the spinal cord was observed. We also identified an early expression of c-Fos immunoreactivity in the superficial laminae of the dorsal horn of the spinal cord (peak at 4 h; p < 0.001), together with a partial activation of ERK1/2 (4 h; p < 0.001). These findings suggest that activated ERK1/2 could induce c-Fos expression and trigger the transcription of prodynorphin in the spinal cord. This in turn would result in long-lasting increased levels of dynorphin that, in our model, could participate in the persistence of pain but not in the manifestation of first pain.

Tolerance to the antinociceptive effects of peripherally administered opioids: Expression of β-arrestins

Tolerance to peripheral antinociception after chronic exposure to systemic morphine was assessed in mice with chronic CFA-inflammation; cross-tolerance to locally administered mu, delta and kappa-opioid agonists and levels of beta-arrestins in the injured paw, were also evaluated. Tolerance was induced by the subcutaneous implantation of a 75 mg morphine-pellet, and antinociception evaluated with the Randall-Selitto test, 5 min after the subplantar injection of morphine, fentanyl, buprenorphine, DPDPE, U-50488H or CRF. Experiments were performed in the absence and presence of CFA-inflammation, in animals implanted with a morphine or placebo pellet. Beta-arrestin protein levels were determined by western blot. In mice without inflammation, subplantar opioids did not induce antinociception, while during CFA-inflammation, all drugs generated dose-response curves with an order of potency of: U-50488H < DPDPE < morphine < buprenorphine < fentanyl << CRF. During CFA-inflammation plus morphine-pellet, the potency of fentanyl decreased 1.25 times, while that of DPDPE, U-50488H and CRF diminished approximately 2.5-4.3 times. For each drug, the ratio between the ED(50)s in tolerant and naive animals, was significantly higher than 1 (except for buprenorphine and fentanyl), demonstrating partial cross-tolerance to systemic morphine. Inflammation induced a twofold increase in beta-arrestin expression (p<0.01), and the levels decreased after acute morphine exposure (p<0.05). Tolerance did not alter beta-arrestins, but partially prevented the increase induced by inflammation. The results suggest that peripheral beta-arrestins could facilitate peripheral OR-desensitization and tolerance development. Clinically, the experiments could be useful to establish the effectiveness of local opioid administration in patients with musculoskeletal pain, chronically receiving morphine analgesia.

Glial cell activation in the spinal cord and dorsal root ganglia induced by surgery in mice

In rodents, surgery and/or remifentanil induce postoperative pain hypersensitivity together with glial cell activation. The same stimulus also produces long-lasting adaptative changes resulting in latent pain sensitization, substantiated after naloxone administration. Glial contribution to postoperative latent sensitization is unknown. In the incisional pain model in mice, surgery was performed under sevoflurane+remifentanil anesthesia and 21 days later, 1 mg/kg of (-) or (+) naloxone was administered subcutaneously. Mechanical thresholds (Von Frey) and glial activation were repeatedly assessed from 30 min to 21 days. We used ionized calcium binding adaptor molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP) to identify glial cells in the spinal cord and dorsal root ganglia by immunohistochemistry. Postoperative hypersensitivity was present up to 10 days, but the administration of (-) but not (+) naloxone at 21 days, induced again hyperalgesia. A transient microglia/macrophage and astrocyte activation was present between 30 min and 2 days postoperatively, while increased immunoreactivity in satellite glial cells lasted 21 days. At this time point, (-) naloxone, but not (+) naloxone, increased GFAP in satellite glial cells; conversely, both naloxone steroisomers similarly increased GFAP in the spinal cord. The report shows for the first time that surgery induces long-lasting morphological changes in astrocytes and satellite cells, involving opioid and toll-like receptors, that could contribute to the development of latent pain sensitization in mice.

A 18F-fluorodeoxyglucose MicroPET Imaging Study to Assess Changes in Brain Glucose Metabolism in a Rat Model of Surgery-induced Latent Pain Sensitization

Background: Neuroplastic changes involved in latent pain sensitization after surgery are poorly defined. We assessed temporal changes in glucose brain metabolism in a postoperative rat model using positron emission tomography. We also investigated brain metabolism after naloxone administration. Methods: Rats were given remifentanil anesthetic and underwent a plantar incision, with 1 mg/kg of (−)-naloxone subcutaneously administered on postoperative days 20 and 21. Using the von Frey test, mechanical thresholds were measured pre- and postoperatively at different time points in awake animals during 18F-fluorodeoxyglucose (18F-FDG) uptake. Brain images were also obtained the day before mechanical testing, using a positron emission tomography R4 scanner (Concorde Microsystems, Siemens, Knoxville, TN). Differences in brain activity were assessed utilizing a statistical parametric mapping. Results: Surgery induced minor changes in 18F-FDG uptake in the cerebellum, hippocampus, and posterior cortex, which extended to the thalamus, hypothalamus, and brainstem on days 6 and 7. Changes were still present on day 21. Maximal postoperative hypersensitivity was observed on day 2. The administration of (−)-naloxone on day 21 induced significant hypersensitivity, greatly enhancing the effect on 18F-FDG uptake. In sham-operated rats, naloxone induced changes limited to the striatum and the cerebellum. Nonnociceptive stimulation with von Frey filaments had no effect on 18F-FDG uptake. Conclusions: Surgery, remifentanil, and their combination induced long-lasting and significant metabolic changes in the pain brain matrix, with a positive correlation with hypersensitivity after naloxone. Changes in brain 18F-FDG precipitated by naloxone suggest that surgery under remifentanil anesthetic induces the greatest neuroplastic brain adaptations in opioid-related pathways involved in nociceptive processing and long-lasting pain sensitization.

Analysis of the opioid–opioid combinations according to the nociceptive stimulus in mice

The purpose of the present study was to characterize the antinociceptive effects of tramadol, fentanyl and morphine, when two of them were systemically combined in a 1:1 potency ratio, in the hot plate, the acetic acid writhing, and the formalin tests in mice. Interaction indexes and isobolographic analysis were used to assess the type of interaction. Fentanyl was the most potent drug, followed by morphine and tramadol, with the exception in the phase I of formalin test. Synergistic interactions were obtained when tramadol was combined with fentanyl or with morphine in the writhing and formalin tests. But, in the hot plate only additive interactions were obtained. Changes were induced on the type of interaction depending on the level of effect of opioid-opioid combinations. Moreover, co-administration of fentanyl with morphine showed additivity, regardless of the type of stimulus. Standard rotarod test analysis confirmed intact motor coordination. The present findings suggest that the type of interaction between opioids is not only related to the nature of nociceptive stimulus but also to non-opioid analgesic pathways.

Deletion of the inducible nitric oxide synthase gene reduces peripheral morphine tolerance in a mouse model of chronic inflammation

The implication of inducible nitric‐oxide synthase (iNOS) on peripheral tolerance to morphine was evaluated in wild‐type (WT) and iNOS knockout mice. Chronic inflammation was induced by subplantar (s.p.) injection of Complete Freund’s Adjuvant (CFA), and morphine tolerance by subcutaneous implantation of a 75 mg morphine‐pellet. Withdrawal was assessed after the intraperitoneal injection of 2 mg/kg naloxone. Antinociception was assessed (Randall‐Selitto test) 5 min after a fixed dose of s.p. morphine (16 μg). In the absence of inflammation, s.p. morphine did not induce antinociception, while during CFA‐inflammation produced 47.4 ± 0.8 and 38.8 ± 2.7% inhibitions respectively, in each genotype (P < 0.05). In morphine‐tolerant mice with CFA‐inflammation, no antinociception could be elicited in WT mice (2.4 ± 0.3% inhibition); however, iNOS knockout mice showed significant antinociception (33.1 ± 0.9%) (P < 0.001). Thus, iNOS gene deletion partially prevented tolerance to the peripheral effects of morphine, and significantly attenuated withdrawal‐induced hyperactivity.

Antinociceptive effects of morphine, fentanyl, tramadol and their combination, in morphine-tolerant mice

The development of morphine-tolerance after chronic administration, reduces analgesic efficacy and is a significant clinical problem in some patients; may be managed clinically by increasing the doses of morphine and/or the administration of a second mu-opioid agonist. In morphine-tolerant mice, we investigated the presence of an interaction when two opioids are administered simultaneously. We determined the antinociceptive effects of morphine (M), fentanyl (FEN), and tramadol (TRM) individually and combined in a 1:1 proportion, based on their potency. Nociceptive thresholds were evaluated in CD1 mice using the hot plate test. Morphine tolerance was induced by the subcutaneous implantation of a 75mg morphine pellet, whereas control animals received a placebo pellet; the experiments were performed three days later. In both (placebo and morphine pellets), dose-response curves for M, FEN and TRM, individually and combined were obtained, and the doses that produced 50% inhibition (ED(50)) were determined. Sustained exposure to morphine induced a significant decrease in antinociceptive potency to acute M or FEN administration (tolerance), which was of a lesser magnitude after acute TRM; in these experiments the analysis of the interaction between chronic morphine and each opioid, demonstrated functional antagonism. The simultaneous administration of two opioids in morphine-tolerant mice, demonstrated antagonism for the M:FEN combination, whereas the effects of TRM combined with M or FEN, remained additive. The results suggest that during morphine-tolerance, TRM could be a useful drug to induce effective analgesia when combined with FEN or M.

Antihyperalgesic effects of dexketoprofen and tramadol in a model of postoperative pain in mice - effects on glial cell activation.

To define likely targets (i.e. glia) and protocols (analgesic combinations) to improve postoperative pain outcomes and reduce chronic pain after surgery. Specifically, to assess the antihyperalgesic effects of the dexketoprofen : tramadol (DEX : TRM) combination, exploring the implication of glial activation.