Acute administration of opioids results in analgesia and side effects, whereas tolerance and dependence only occur after chronic administration (1). However, it has become more recently recognized that tolerance can also develop rapidly after an acute opioid exposure in animals (2–4) and humans (5–7). Tolerance to opioids is classically defined as a progressive reduction of their analgesic effect (1), thus explaining the need for a larger dose to achieve the same pharmacological effect. However, this definition now has to be revised because a growing body of evidence suggests that opioid analgesics can elicit delayed hyperalgesia (exaggerated nociceptive response to noxious stimulation) in experimental models after repeated opioid administrations (8,9) or continuous delivering (3,10,11). Such a pain sensitivity enhancement could explain a reduction of the opioid analgesic effect. Interestingly, we reported that a single administration of an opioid also induces a long-lasting increase of basal pain sensitivity, leading to delayed hyperalgesia (9), which, if not taken into account, gives the impression of less analgesia (i.e., apparent tolerance) when a new opioid administration is performed (4,12). A series of clinical reports also indicate that opioids can produce abnormal pain including allodynia (nociceptive responses to innocuous stimulation) or hyperalgesia (8,13–15). If tolerance and pain facilitation share some common pathways (8,9), then animals (or humans) with acute tolerance to opioid analgesia should be hyperalgesic after the opioid administration. In line with the current discussion on acute tolerance in humans (16), two recent clinical studies reported that perioperative fentanyl (6) or remifentanil (7) increase both postoperative pain and morphine requirement.
There is now a substantial amount of evidence that glutamate via N-methyl-d-aspartate (NMDA) receptors play a pivotal role in the development and maintenance of central hyperactive states underlying the behavioral manifestations of pain facilitation such as hyperalgesia, allodynia, and spontaneous pain (8,13). Moreover, tolerance is prevented by NMDA-receptor antagonists (4,8,9,14,17). In an animal model, we reported that an acute administration of fentanyl, an opioid widely used for surgery, induces in rats a dose-dependent long-lasting hyperalgesia (days) that can be prevented by ketamine pretreatment (18). It is in agreement with an alfentanil study in rats showing that ketamine suppressed rebound hyperalgesia observed the day after the alfentanil infusion (19). Moreover, NMDA-receptor antagonists also potentiate opioid-induced analgesia (4,12,18).
All experimental data related to acute tolerance and hyperalgesia would indicate that a decrease of morphine potency would be observed when injected after fentanyl. Because each year several millions of surgical patients benefit from fentanyl and related compounds during surgery and morphine during the postoperative period (20), it is crucial to develop an experimental model. We thus investigated, in rats, both a possible immediate fentanyl-induced hyperalgesia and a related acute tolerance to the analgesic effects of morphine and the potency of the clinically available NMDA-receptor antagonist, ketamine, to prevent this dual phenomenon.
Materials and Methods
Experiments were performed on male Sprague-Dawley rats (IFFA-CREDO, France) weighing 250–350 g, housed 5 per cage, maintained under a light/dark ratio of 12:12 h cycle (lights on at 8:00 am) and at a constant room temperature of 22°C ± 2°C. The rats had free access to food and water. Pharmacological tests and care of the animals were performed in accordance with the Guide for Animal Care and Use (National Institutes of Health, 1999).
Fentanyl citrate, naloxone hydrochloride, and ketamine hydrochloride were obtained from Sigma-Aldrich (Saint Quentin Fallavier, France). All drugs were dissolved in physiological saline 0.9% and injected subcutaneously in the dorsal surface of the neck (100 μL per 100 g of body weight). Control rats were injected with an equal volume of saline.
Measurement of Nociceptive Threshold
Nociceptive thresholds in rats were determined by a modification of the Randall-Selitto method (21), the paw-pressure vocalization test, in which a constantly increasing pressure is applied to the hind paw until the rat squeaks. The Basile analgesimeter (stylus tip diameter: 1 mm; Apelex, Massy, France) was used. A 600-g cutoff value was used for preventing tissue damage.
After their arrival in the laboratory, the rats were left to become accustomed to the colony room for 2 days. To avoid stress resulting from the experimental conditions that might affect measurement of the nociceptive threshold, the experiments were performed by the same experimenter in quiet conditions in a test room close to the colony room. For 2 wk before the experiments, the rats were weighed daily, handled gently for 5 min, and placed in the test room for 2 h (from 11:00 am to 1:00 pm) to become accustomed to the nociceptive apparatus. All experiments began at 1:00 pm and were performed on groups of 8–10 rats. The rats were randomly assigned to the different experimental groups, and the experimenter was unaware of the treatment used.
To ensure nociceptive threshold stability, basal nociceptive threshold was measured twice (with an interval of 30 min) on the 2 days before the planned experimental day (data not shown). On the experimental day (D0), basal nociceptive threshold was also determined twice before drug injections. The first measurement of the nociceptive threshold performed on D0 was used as the reference value for the basal nociceptive threshold.
In the first set of experiments, we studied the early effect of various doses of fentanyl on nociceptive threshold using a procedure designed to partly mimic its use in surgery. Fentanyl (or saline) was injected four times at 15-min intervals (20, 60, or 100 μg/kg per injection, resulting in total doses of 80, 240, or 400 μg/kg, respectively). The nociceptive threshold was measured every 30 min. When the pharmacological effect of fentanyl had terminated, an injection of morphine (5 mg/kg) was performed. Subsequent changes in the nociceptive threshold were measured every 30 min until the end of the pharmacological effect of morphine. Using this protocol, we also investigated the effects of the noncompetitive NMDA-receptor antagonist, ketamine (10 mg/kg), on pharmacological effects of both fentanyl (4 × 60 μg/kg) and morphine (5 mg/kg). Ketamine (or saline) was injected 30 min before the first injection of fentanyl.
In the second set of experiments, we studied the long-lasting effects (5–10 days) of fentanyl (4 × 60 μg/kg), morphine (5 mg/kg), or both according to the aforementioned protocol. We also investigated the effects of the noncompetitive NMDA-receptor antagonist, ketamine (10 mg/kg), on pharmacological effects of both fentanyl (4 × 60 μg/kg) and fentanyl (4 × 60 μg/kg) plus morphine (5 mg/kg). Ketamine (or saline) was either injected once 30 min before the first injection of fentanyl or three times 30 min before the first injection of fentanyl, before and after the injection of morphine. At the end of these experiments, all rats received a naloxone injection (1 mg/kg), and the nociceptive threshold was measured 5 min later and every 30 min until recovery of the basal nociceptive threshold.
Data were expressed as the mean nociceptive threshold ± sem. Areas under the curves defining analgesic indexes were calculated by the trapezoidal method and expressed as arbitrary units (AU). Algesic indexes (i.e., hyperalgesia) were also calculated when the nociceptive threshold decreased less than its basal value. Student’s paired t-test was used to assess paired comparisons of nociceptive threshold values to evaluate changes of the nociceptive threshold before the experiments. To evaluate the time-course effects of treatments on nociception, analyses of variance (ANOVAs) were performed on the nociceptive threshold value, and post hoc analysis using Dunnett’s test came afterward. A difference was considered significant at P < 0.05.
No statistically significant difference was found among the basal nociceptive threshold of each experimental group (one-way ANOVA, P > 0.05; mean baseline threshold 286 ± 5 g, n = 86).
Biphasic Time Effects of Fentanyl on the Nociceptive Threshold of Saline or Ketamine-Pretreated Rats
The short-lasting changes (hours) in the nociceptive threshold induced by various doses of fentanyl or saline are shown in Figure 1A and C–E. Saline injection did not alter the nociceptive threshold (one-way ANOVA, P > 0.05;Fig. 1A). Four consecutive injections of fentanyl elicited increases of the nociceptive threshold followed by the largest fentanyl doses by sustained decreases of the nociceptive threshold for several hours (Fig. 1C–E). The fentanyl-induced analgesic effect increased with the dose of fentanyl used (one-way ANOVA, P < 0.05). As shown by evaluation of the algesic index (see Methods), hyperalgesia observed with fentanyl 4 × 100 μg/kg was more pronounced than hyperalgesia observed with fentanyl 4 × 60 μg/kg (one-way ANOVA, P < 0.05, Fig. 1, D and E). No decrease of the nociceptive threshold was observed with the smallest fentanyl dose (4 × 20 μg/kg). As shown in Figure 1F, a single pretreatment by ketamine (10 mg/kg) totally prevented the decrease of the nociceptive threshold induced by fentanyl (4 × 60 μg/kg) (one-way ANOVA, P > 0.05).
Effects of Fentanyl on Morphine-Induced Changes of the Nociceptive Threshold of Saline or Ketamine-Pretreated Rats
Morphine (5 mg/kg) injection induced a significant increase of the nociceptive threshold (Fig. 1B; one-way ANOVA, P < 0.05). When morphine was injected after the biphasic fentanyl effects, we observed a decrease of morphine-induced analgesia (Fig. 1C–E; one-way ANOVA, P > 0.05). The analgesic index of morphine decreased by 15.8% (Student’s t-test, P > 0.05), 46.6% (Student’s t-test, P < 0.01), and 85.1% (Student’s t-test, P < 0.001) after 4 × 20, 4 × 60, and 4 × 100 μg/kg fentanyl injections, respectively. As shown in Figure 1F, a single pretreatment by ketamine (10 mg/kg) restored the morphine analgesic effect (83.2% of the control areas under the curves; Student’s t- test, P > 0.05 compared with the control analgesic index of morphine).
Long-Lasting Effects of Fentanyl and Morphine on the Nociceptive Threshold of Saline or Ketamine-Pretreated Rats
Fentanyl (4 × 60 μg/kg) induced a delayed decrease of the nociceptive threshold for several days (D+1 to D+4;Fig. 2A; one-way ANOVA, P < 0.05). No delayed hyperalgesia was observed after a single morphine injection (5 mg/kg;Fig. 2B; one-way ANOVA, P > 0.05). When morphine (5 mg/kg) was injected after the biphasic fentanyl effects, the decrease of the nociceptive threshold was lengthened (D+1 to D+6;Fig. 2C; one-way ANOVA, P < 0.05). Further analysis indicated that the algesic index obtained after the fentanyl/morphine administrations (−495.0 ± 125.3 AU) was statistically different from the algesic index calculated when fentanyl was injected alone (−70.8 ± 10.9 AU; Student’s t- test, P < 0.01).
Naloxone (1 mg/kg) administered after the delayed fentanyl-induced hyperalgesia (Fig. 2A) induced a significant decrease of the nociceptive threshold (Fig. 2A; one-way ANOVA, P < 0.001). No change was observed when naloxone was injected in morphine-treated rats (Fig. 2B; one-way ANOVA, P > 0.05). The naloxone-precipitated hyperalgesia was also present in the Fentanyl/Morphine-Treated group (Fig. 2C; one-way ANOVA, P < 0.001).
The significant decrease of the nociceptive threshold observed in the Fentanyl-Treated group was prevented by a single administration of ketamine (10 mg/kg;Fig. 3A). A similar ketamine pretreatment failed to suppress the decrease of the nociceptive threshold in the Fentanyl/Morphine-Treated group (Fig. 3B; one-way ANOVA, P < 0.05). Ketamine administrations repeated three times before fentanyl and before and after morphine injections totally prevented delayed hyperalgesia (Fig. 3C; one-way ANOVA, P > 0.05).
A single ketamine injection before fentanyl (with no subsequent administration of morphine) was able to totally prevent naloxone-precipitated hyperalgesia (Fig. 3A; one-way ANOVA, P > 0.05). Conversely, it was unable to prevent naloxone-precipitated hyperalgesia in Fentanyl/Morphine-Treated rats (Fig. 3B; one-way ANOVA, P < 0.001). Three injections of ketamine did not completely block the naloxone-precipitated hyperalgesia (Fig. 3C; one-way ANOVA, P < 0.05).
This study shows that, in rats, four injections of fentanyl within one hour induced a classic antinociceptive effect followed by an immediate hyperalgesia that may have lasted several hours. When morphine was injected at the termination of these two opposite effects, an acute tolerance to its analgesic effects was observed: the larger the fentanyl dose, the less potent was the morphine. A single ketamine administration was effective in preventing acute morphine tolerance. Nevertheless, the morphine administration reinforced the fentanyl-induced delayed hyperalgesia observed for several days. A single ketamine administration before fentanyl treatment was not effective in preventing this enhanced delayed hyperalgesia. A main finding of this study is that repeated ketamine administration (before and after the fentanyl and morphine administrations) was required for totally preventing the development of both immediate and delayed hyperalgesia and acute tolerance to the analgesic effect of morphine. Indeed, this suggests that both of these phenomena have common mechanisms.
Three main points of this study have to be considered: the first is that the analgesic effect of fentanyl was immediately followed by a hyperalgesia revealed by a dose-dependent decrease of the nociceptive threshold less than the basal value (the larger the fentanyl dose, the larger was the hyperalgesia). Of interest is the recent observation that the alfentanil-induced hyperalgesia is similar in both naive and inflamed rats (19). It is likely that we spontaneously observed what is classically observed during opioid-receptor antagonist challenge with naloxone when it is administered during morphine analgesia (22,23), heroin analgesia (4), or fentanyl analgesia (23). This leads us and others (22–24) to suggest that opioids activate not only antinociceptive systems, but also pronociceptive systems with a longer activity. Although multiple mechanisms may account for pain hypersensitivity (8,13), a growing body of evidence indicates that pronociceptive systems associated with central pain sensitization are mainly underlain by excitatory amino acid activity at the NMDA-receptor level (8,14,25). In agreement with this hypothesis, our results showed that the NMDA-receptor antagonist, ketamine, devoid of analgesic effect on its own and at the dose used in this study (18), prevented fentanyl-induced early hyperalgesia. μ-Opioid receptors trigger the activation of NMDA receptors by reducing Mg2+ block via a protein kinase Cγ activation (26). It has been suggested that the subsequent increase in intracellular Ca2+ concentrations further stimulates protein kinase Cγ, leading to an enduring reinforcement of glutamate synaptic efficiency associated with pronociceptive systems in a positive feedback loop (8,26). The activation of these pronociceptive systems associated with NMDA-receptor synaptic activity thus goes beyond the activation of nociceptive inhibitory systems, which are not usually coupled with such a reinforcement system.
In this context, we previously suggested that opioids produce two opposite effects on nociception concomitantly that mask one another: analgesia prevails at first and is replaced by hyperalgesia at later times (12,23). This could explain why the NMDA-receptor antagonist, ketamine, which prevented early hyperalgesia, also enhanced fentanyl-induced analgesia. Using an IV alfentanil infusion model in rats, ketamine and MK-801 attenuated the gradual analgesia decline observed during a four-hour infusion period (27). The enhancing effect of the NMDA-receptor antagonist on the fentanyl-induced analgesia observed in this study is then indicative of the reversal of an acute tolerance. This is in agreement with our previous suggestion (4,23) that the enhancement of opioid-induced analgesia by the block of NMDA-receptor antagonists (4,12,18,27) results neither from a true potentiating effect nor from an additive effect of NMDA-receptor antagonists. Rather it is from the block of NMDA-dependent counteracting systems, which oppose the full expression of analgesia induced by opioid receptor stimulation. These data confirm that opioid-induced acute tolerance may be observed during the opioid-induced analgesia or as soon as analgesia has ended. They also support the fact that acute tolerance and early pain hypersensitivity stem from a common NMDA-receptor-dependent mechanism.
The second point is that previous fentanyl administration drastically reduced the amplitude of morphine-induced analgesia. This is also indicative of an acute tolerance. Our results showed a dose-dependent tolerance to the analgesic effect of morphine: the larger the fentanyl dose, the greater was the decrease of morphine’s analgesic effect. A dramatic decrease of the morphine-induced analgesic index (−85%) was observed when the largest dose of fentanyl was administered. Interestingly, this paralleled the magnitude of the early fentanyl-induced hyperalgesia. A main finding is that the morphine analgesic effect was totally restored in rats receiving a single dose of ketamine before the series of fentanyl boluses. This suggests that the secondary morphine administration has reactivated a vicious cycle initiated by the primary opioid injection (8,18). Studies are in progress in our laboratory to evaluate the level of opioid-induced acute tolerance to morphine analgesic effects in suffering rats.
The third point is that injecting morphine just after fentanyl enhanced the delayed hyperalgesia observed for several days after a series of fentanyl boluses. Because morphine did not induce delayed hyperalgesia on its own, this indicates that the secondary morphine administration has reactivated pronociceptive systems for a long period. Our study shows that a single ketamine administration before the fentanyl administration, which was effective in preventing long-lasting hyperalgesia in rats receiving fentanyl alone or alfentanil infusion for four hours (19), was not able to prevent the delayed hyperalgesia in rats receiving fentanyl and morphine. It is noteworthy that ketamine has to be reinjected both before and after morphine to totally prevent long-lasting hyperalgesia from occurring. The bioavailability of ketamine and its rapid redistribution from the brain to other tissues (28) may explain this limited effect. Nonetheless, this indicates that by using an opioid scenario mimicking a clinical opioid use in human surgery, the NMDA-dependent pain sensitization process activated by opioid receptor stimulation is effective for a long period (18). This is further supported by our observation that naloxone induced hyperalgesia in rats, and the nociceptive threshold was returned to basal 10 days after the fentanyl/morphine administrations. A similar naloxone hyperresponsiveness was observed for a long time (two months) after the cessation of a two-week daily heroin administration (17). This indicates that the rats were not back to their initial biological state (homeostasis) but were in a new biological state (allostasis) associated with a high level balance between opioid-dependent analgesic systems and pronociceptive systems. Because ketamine reduced the magnitude of such a naloxone-triggered hyperalgesia, this indicates that pronociceptive systems involved in this new biological equilibrium were NMDA-dependent.
Could our experimental results be relevant to the clinical experiment with intraoperative and postoperative acute opioid exposure in surgical patients? Although this study was performed in rats without tissue damage, our data support the hypothesis that opioids may lead to pain enhancement and acute tolerance via pronociceptive systems and through to a common pathophysiological continuum. Recently, several clinical studies report that intraoperative opioids such as fentanyl (6,15) or remifentanil (7) increase both postoperative pain and opioid analgesic consumption, suggesting acute tolerance. This should not dissuade us from using moderate or even large doses of opioids to alleviate pain, especially surgical pain. Indeed, although many clinical studies examined the use of ketamine, most are difficult to interpret because of fundamental problems with design, methods, or statistical treatment of data; however, it is becoming increasingly clear that ketamine has beneficial effects on both postoperative pain and opioid consumption [for review, see Ref. (29)]. IV intraoperative ketamine diminishes the incidence of residual pain as expressed by analgesic requirements until the sixth postoperative month (30). Recently, Kissin (31) suggested the possibility that the effect of ketamine on opioid-induced acute tolerance could explain reduction of opioid consumption. Our results provide experimental data supporting such a proposal and clearly indicate that acute tolerance to the analgesic effects of morphine is the result of a pain sensitization process triggered by fentanyl. Our study shows that the pharmacological treatment of such a phenomenon requires not only a ketamine treatment only before the opioid (fentanyl), but also a sustained block of NMDA receptors should be subsequently administered with a long-lasting opioid such as morphine. This could explain why beneficial effects were not reported when the ketamine administration was limited to the postoperative period (32). Because different mechanisms may account for hyperalgesia after surgery, the consequences of ketamine for preventing fentanyl-induced hyperalgesia and related acute morphine tolerance should be evaluated in the rat incisional pain model (33) —an interesting model of postoperative pain.
The authors thank Patrick Reynier, MD, for providing helpful advice and discussion about the manuscript.
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