Opioids are considered excellent analgesics for the treatment of mild to severe pain, but their adverse effects, such as opioid-induced hyperalgesia (OIH) and tolerance, may limit their use.1 OIH is defined as an increased pain sensitivity caused by opioid exposure. Remifentanil administration has been associated with an increase in the degree and duration of postoperative pain in animal models,2 which in patients may lead to higher postoperative morphine consumption and pain scores.3 Tolerance is defined as a decrease in the efficacy of a drug over time that requires step increases in dosing to maintain the same level of effect. Tolerance may develop in chronic opioid treatments, but it has also been observed following acute opioid administration.4 In healthy volunteers given remifentanil, a decrease in the analgesic effect produced by this opioid was observed after only 90 min.4
The development of acute opioid tolerance (AOT) may modify anaesthetic requirements during surgery, and a decreased efficacy of remifentanil in its ability to reduce the sevoflurane minimum alveolar concentration (MAC) over the short term has been observed in rats.5 Then, when the remifentanil dose was increased, the initial MAC was restored,5 suggesting an AOT effect rather than OIH effect. Low doses of ketamine have been successfully employed to prevent or treat OIH and delayed opioid tolerance,6 but have failed to prevent or treat remifentanil AOT during sevoflurane anaesthesia in rats.7 Gabapentin, an antiepileptic drug used to treat hyperalgesia, and paracetamol, may blunt or prevent AOT to remifentanil under volatile anaesthesia, probably because of their additive sevoflurane-sparing action.8,9 In contrast, other cyclooxygenase-inhibitors, such as dipyrone, ketoprofen or parecoxib, blunted AOT once it was established without affecting MAC.8
Antidepressants such as amitriptyline,10 neurokinin-1 (NK-1) antagonists such as maropitant11,12 and inhibitors of microglia activation such as minocycline13 have shown their efficacy in the treatment of morphine tolerance, but they have not yet been assessed in AOT or OIH during volatile anaesthesia. We hypothesised that, apart from a potential reduction in anaesthetic requirements, these drugs, alone or combined with an opioid such as remifentanil,14 may prevent the observed increase on the sevoflurane requirements during remifentanil infusion related to AOT and the development of OIH. The aim was to determine the interactions of amitriptyline, minocycline and maropitant on sevoflurane MAC in rats and whether they blunt or block the OIH and AOT produced by remifentanil.
Materials and methods
After obtaining ethical approval for this study (Ethics Animal Experimentation Committee (CEA) of the University Complutense, Madrid, Spain, CEA approval reference date: 29 May 2008), the sevoflurane MAC and mechanical nociceptive thresholds (MNTs) in response to amitriptyline, minocycline, maropitant and remifentanil were evaluated in rats (Microsurgery Laboratory, Experimental Surgery Department). The care of animal and licensing guidelines under which the study was performed were in accordance with the ARRIVE (Animals in Research: Reporting In Vivo Experiments) statement. Sevoflurane was obtained from Abbott (Sevorane; Abbott Laboratories, Madrid, Spain), amitriptyline and minocycline from Sigma-Aldrich (St. Quentin Fallavier, France), maropitant from Pfizer (Cerenia; Pfizer PGM, Pocé sur Ciss, France) and remifentanil from Glaxo-Wellcome (Ultiva; Glaxo-Wellcome Laboratories, Madrid, Spain).
Adult male Wistar rats (Charles River Laboratories, Barcelona, Spain), weighing 321 (34) g, were housed in groups of four to six animals per cage (macrolon Type IV, Souralit 3000, Souralit S.L., Girona, Spain) with a 12 h light-12 h dark cycle, relative humidity of 40 to 70% and room temperature of 20 (2)°C. Food (SAFE A04, Panlab, Barcelona, Spain) and water were provided ad libitum. The animals were allowed to acclimatise for at least 1 week. All of the experiments were performed during the morning (starting at 08.30h). Rats were randomly allocated to two experiments: Experiment I aimed to determine the interactions of amitriptyline, minocycline and maropitant on sevoflurane MAC and whether they blunt or block the AOT produced by remifentanil; and, Experiment II aimed to determine whether these drugs could reduce or block OIH to remifentanil.
In Experiment I, rats were placed in an induction chamber into which 8%vol. sevoflurane in a continuous oxygen flow of 3 l min−1 was directed (Sevoflurane Vaporizer; Sevorane Dragër Vapor 2000, Lubeck, Germany). Tracheal intubation was performed using a 14-G polyethylene catheter (Terumo Surflo IV Catheter; Terumo Europe NV, Leuven, Belgium) with the animal positioned in sternal recumbency. An otoscope was used to insert a flexible, blunt-tipped wire guide into the trachea, which was used to direct the catheter. After the catheter was properly positioned, it was connected to a small T piece breathing system with minimum dead space. Fresh gas flow to the T piece was adjusted to 1 l min−1 of oxygen (100%), and the sevoflurane concentration was adjusted to 1.5 × MAC (3.5 to 4%vol.). The rats were kept under spontaneous ventilation during the entire experiment because remifentanil at the doses administered did not produce significant hypercapnia,15 which might have modified the MAC. However, when signs of hypoventilation occurred, spontaneous ventilation was stimulated by softly touching the rat's thorax. Remifentanil was administered with an infusion pump (syringe pump, model Sep11S; Ascor S.A., Medical Equipment, Warsaw, Poland) by a 22-G polyethylene catheter inserted in a lateral tail vein.
Arterial oxygen haemoglobin saturation (via pulse oximetry) and heart and respiratory rates were monitored continuously (SA5; Datex Ohmeda, Helsinki, Finland). Rectal temperature was also monitored and was maintained between 37.0°C and 38.5°C by a water-circulating warming blanket (Heat Therapy Pump, Model TP-220; Gaymar, Orchard Park, New York, USA) and a heating light.
At the end of the experiment, animals were euthanised with an overdose of potassium chloride given intravenously while they were still deeply anaesthetised.
Determination of the minimum alveolar concentration
Gas samples were obtained from the tracheal tube through a needle connected to a side-stream infrared analyser (Capnomac Ultima; Datex-Ohmeda, Hertfordshire, UK) inserted into the small T piece breathing system. They were used to measure the anaesthetic gas concentration to determine the MAC and end-tidal carbon dioxide. After every step change in anaesthetic concentration delivered by the anaesthetic circuit, a period of at least 10 min was allowed for equilibration before the noxious stimulus was applied.
The MAC of sevoflurane was established according to a method described elsewhere.16 A supramaximal noxious stimulus was applied with a long haemostat (8-inch Rochester Dean Haemostatic Forceps) clamped on the tail for 60 s or until a positive response was obtained. A positive response was considered to be a gross purposeful movement of the head, extremities or body. Lack of movement, swallowing, chewing, tail flicking and grimacing indicated a negative response. Following a negative response, the sevoflurane concentration was reduced in decrements of 0.2% until the response became positive. Similarly, when a positive response was observed, the sevoflurane concentration was increased until the response became negative. The MAC was considered to be the concentration mid-way between the highest concentration that permitted movement in response to the stimulus and the lowest concentration that prevented such movement. The MAC values were corrected for barometric pressure at sea level.
Nociceptive behaviour test
To determine whether the studied drugs could reduce or block OIH to remifentanil in Experiment II, MNTs were evaluated by measuring the hind paw withdrawal response to the application of electronically calibrated von Frey filaments (Electronic von Frey Anesthesiometer, Model 2393 IITC Inc., California, USA). To minimise the influence of stress during the experimental procedure, the animals were acclimatised over 3 days to the testing procedure before baseline values were obtained. The animals were placed in a methacrylate cylinder (18 × 30 cm) with a wire grid bottom (1-cm2 perforations).
Four filaments provided with the electronic von Frey device were used. These filaments were chosen to cover a pressure range between 10 and 50 g. The estimated pressure produced by every filament when applied was previously calculated as the mean of 25 tests. The thinnest filament (approximately 10 g) was first used, followed by an increase in pressure, after which pressure was increased or decreased according to the previous response. When a negative response was observed, the filament with the next greater thickness was used; similarly, when a positive response was observed, the filament with the next lesser thickness was used. The maximum pressure applied, as determined by the device, was recorded. The MNT of each animal was calculated as the mean of the six applied pressures from the first crossover.
Experimental design and drug groups
The MAC was determined three times in each animal (Fig. 1). Once the rats were anaesthetised and instrumented, a baseline MAC (MAC-1) was determined, and each animal acted as its own control. Then, the drugs (amitriptyline, or minocycline, or maropitant) were administered intraperitoneally, and remifentanil (240 μg kg−1 h−1) or isotonic saline was continuously infused into the tail vein 30 min later with no loading dose. The MAC was then re-determined (MAC-2) and again approximately 90 min later (MAC-3). Periods of 30 min were allotted between the MAC determinations, and periods of 40 to 60 min were usually necessary to determine the MAC value. Overall, every experiment lasted more than 6 h.
The low dose used for each drug and the time of administration were selected according to previous studies: amitriptyline 10 mg kg−1, 1 h before remifentanil or saline administration;17–19 minocycline 30 mg kg−1, twice at 20 h and 1 h before remifentanil or saline administration13,20 and maropitant 10 mg kg−1, 30 min before remifentanil or saline administration.21 Then, a high dose of amitriptyline (50 mg kg−1), minocycline (100 mg kg−1) or maropitant (30 mg kg−1) was selected by a three to five-fold increase in the low dose to achieve similar MAC reductions and ensure the use of a sufficient dose in the rat. To accommodate two different doses of the three drugs, remifentanil alone and saline control, 14 groups were needed.
OIH was defined as a decrease in MNT as determined by the von Frey method. Following acclimatisation, baseline MNT was determined at day 0. Rats then received the (more effective) high dose of the drugs amitriptyline, minocycline, maropitant or saline, followed by a remifentanil infusion or saline under sevoflurane anaesthesia. This required five groups. On day 2, animals were evaluated for MNT determination.
On the basis of previous MAC studies in our laboratory, the sample size calculations indicated an n value of 6 per group as being necessary to determine differences with a power of 80% and a P value of 0.05. With 14 groups required for Experiment l and 5 for Experiment II, 114 rats were required in all. The mean and standard deviation were obtained from a previous study,5 and the statistical package used was N Query Advisor (v 2.0; Statistical Solutions. Saugus, Massachusetts, USA).
The results are presented as the mean (standard deviation). The rats in every experiment were randomly allocated using a random number generator (function RAND; Excel 2007, Microsoft Office). The data were tested for normality with the Kolmogorov–Smirnov test. Assessment of the interactions between remifentanil and each of the studied drugs on the MAC and AOT prevention was explored with repeated measures analysis of variance (ANOVA) according to the drug administered: three drugs (amitriptyline, minocycline or maropitant) at two doses (high and low) and saline, combined with remifentanil or saline. The Bonferroni test was used to compare groups. A paired Student's t test was employed to assess the OIH by comparing MNT between days 0 and 2. A P value of less than 0.05 was considered to indicate statistical significance. The data analyses were performed using the SPSS statistical package (v. 15 for Windows; SPSS Inc., Chicago, Illinois, USA).
Effects of amitriptyline, minocycline and maropitant on minimum alveolar concentration
The baseline MAC (MAC-1) determined in the control group (saline) was 2.4 (0.2)%vol. (n = 6) and did not differ from the subsequent MAC determinations (MAC-2 and MAC-3; P = 1.000) (Fig. 2).
Only the high doses of the studied drugs reduced the MAC to a significant level: amitriptyline reduced the sevoflurane MAC by 24 (8)% and 24 (10)% at MAC-2 and MAC-3, respectively (P <0.001); minocycline reduced MAC by 23 (6)% and 17 (10)% (MAC-2 and MAC-3; P < 0.001 and P = 0.005, respectively), and maropitant reduced MAC by 15 (5)% and 15 (8)% (MAC-2 and MAC-3; P < 0.001 and P = 0.003, respectively). The low doses of the drugs did not reduce MAC at any time point (P
Effects of the combined action of amitriptyline, minocycline or maropitant with remifentanil on minimum alveolar concentration and acute opioid tolerance
Remifentanil administered alone reduced the MAC by 36 (6)% (MAC-2; P < 0.001) (Fig. 2). When the high doses of amitriptyline, minocycline and maropitant were administered with the remifentanil infusion, the MAC reduction was further increased to 76 (9)%, 75 (16)% and 59 (5)%, respectively (MAC-2; P < 0.001 vs. remifentanil group).
When remifentanil was administered alone, an AOT effect was observed approximately 1.5 h later, as determined by a lower MAC reduction of 16 (12)% (MAC-2 vs. MAC-3; P = 0.001), even though a constant rate of remifentanil infusion was maintained. When amitriptyline, minocycline and maropitant were administered before the remifentanil infusion was started, an AOT effect to remifentanil was also observed, with the MAC reduction (MAC-3) being significantly lower (MAC-2 vs. MAC-3, P < 0.01).
Effects of the combined action of amitriptyline, minocycline or maropitant with remifentanil opioid-induced hyperalgesia
The baseline MNT at day 0 determined in all rats was 37.1 (6.9) g (n = 30) and was similar between treatment groups (P = 1.000) (Fig. 3). Remifentanil produced a significant decrease in MNT values (35% at day 2 compared to baseline; P = 0.009). Amitriptyline, minocycline and maropitant, combined with remifentanil, produced a similar decrease in MNT (34, 28 and 35%, respectively) at day 2 compared with day 0 (P = 0.012, P = 0.080 and P = 0.007, respectively).
The antidepressant amitriptyline, minocycline the inhibitor of microglia activation, and the NK-1 antagonist maropitant, each decreased sevoflurane MAC in the rat and potentiated the remifentanil anaesthetic-sparing action. These drugs have been reported to share a potential analgesic action14,22,23 and an effect against opioid tolerance and OIH. Although no previous experimental evidence suggests that amitriptyline or minocycline has a volatile anaesthetic-sparing effect, maropitant has already been shown to decrease MAC.14 The mechanism may involve common targets for both volatile anaesthetics and the three studied drugs. Amitriptyline, minocycline and volatile agents have been shown to modulate sodium channels associated with immobility, potentially modifying the MAC.24–26
Amitriptyline is a tricyclic antidepressant that inhibits the re-uptake of transmitters norepinephrine and serotonin at nerve terminals, thus potentiating their action. In addition, amitriptyline may inhibit cellular adenosine uptake,27 antagonise the N-methyl-D-aspartate receptor28 and activate opioid receptors.29 Altogether, these mechanisms of action may explain the efficacy of this antidepressant drug as an analgesic for acute and persistent pain;22 nevertheless, its analgesic effect is controversial.18,22 Minocycline is an inhibitor of microglia proliferation and cytokine release [tumour necrosis factor-alpha (TNF-α), interleukin (IL)-1β and IL-6] known to prevent central neural sensitisation and persistent pain induced by nerve injury.30 Although minocycline does not appear to produce thermal analgesia,31 it may exert analgesia peripherally.26 Maropitant antagonises the NK-1 receptor expressed in spinal dorsal horn neurons that binds to substance P, an excitatory neurotransmitter released by primary afferent nociceptors. The NK-1 antagonists have been evaluated to produce somatic analgesia14 and visceral analgesia demonstrating a consistent effect.32–35 In addition, maropitant has been shown to decrease the sevoflurane MAC in dogs between 15 and 30% following tail-clamp and visceral nociceptive stimulation, respectively.14,35 A similar effect is found in rats in our study with a reduction in the MAC of 15%.36
Opioids such as remifentanil are generally administered during volatile anaesthesia both to provide analgesia and to reduce anaesthetic requirements. In addition to their effects on MAC when administered alone, amitriptyline, minocycline and maropitant also potentiated the remifentanil anaesthetic-sparing action. This effect has been brought about primarily by high drug doses, in an additive fashion, and with decreases in MAC that are clinically significant (> 50%). These results suggest that patients treated with any of these drugs may have lower anaesthetic requirements. However, this study only tested sevoflurane and further research is needed to determine whether the interactions observed here can be obtained with other anaesthetics, for example, when a target-controlled infusion of propofol-remifentanil anaesthesia is used.
Extrapolation of the results from rats to patients has limitations. Although doses of amitriptyline, minocycline and maropitant were adjusted to obtain the desired clinical effects,13,17–21 these doses are higher than the therapeutic range for humans according to allometric escalation between species.37 The drug doses employed in rats may be 1 to 2 orders of magnitude higher than the human doses. Consequently, variations in the effects of the opioid, which may explain the differences in the modulation of tolerance observed between humans and rodents, cannot be ruled out. Maropitant produced a lower MAC reduction than amitriptyline or minocycline, though this result could be attributed to a low dose of the former. A higher maropitant dose (50 mg kg−1) was employed in a pilot study but produced severe side effects in one rat and was discarded. Another potential limitation of the study is the method of MAC reduction used, which should not be considered as a measure of analgesia. Anaesthetic immobility and analgesia are not necessarily linked phenomena,38 and nonanalgesic drugs also decrease MAC. Accordingly, the aim of the study was not to determine their analgesic action, but rather the interaction between anaesthetics and the studied drugs. Therefore, MAC was considered because it is a reliable method that effectively simulates the perioperative clinical setting using volatile anaesthetics.
In conclusion, amitriptyline, minocycline and maropitant reduced the sevoflurane MAC and potentiated the remifentanil MAC reduction in rats. However, these drugs were unable to block the OIH and AOT produced by remifentanil during volatile anaesthesia.
Acknowledgements relating to this article
Assistance with the study: the authors wish to thank the personnel from the Department of Experimental Surgery, La Paz University Hospital, Madrid, Spain.
Financial support and sponsorship: this work was supported by a grant from the Fondo de Investigaciones Sanitarias. Spanish Health Ministry. Grant number FIS 08/0422 and PI11-01241.
Conflict of interests: none.
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