Opioids produce excellent analgesia, but their antinociceptive effects may be limited by the development of tolerance and hyperalgesia.1,2 Tolerance may develop during months of chronic opioid treatment,3 but it has also been observed after acute administration over days and hours.4 In the rat, remifentanil is related to the development of hyperalgesia and opioid tolerance, which, in turn, may produce a decrease in the sevoflurane-sparing effect of opioids.5 The clinical consequences may include an increase in anesthetic and opioid requirements during surgery, potentially increasing their undesirable dose-dependent side effects.6 This is determined by measuring the reduction in the minimum alveolar concentration (MAC) produced by analgesics and is considered an indirect, although clinically valuable, method of determining analgesic potency during the intraoperative period.
Glial cell activation has been reported to have an important role in modulating the analgesia induced by chronic opioid administration or by high doses of opioids7–10 and may contribute to opioid tolerance and withdrawal, factors that reduce the clinical efficacy of opioids as pain therapeutics.11–13 Interestingly, the opioid antagonist and also toll-like receptor 4 (TLR4) antagonist, naloxone, may block remifentanil-induced hyperalgesia and the associated increase in anesthetic requirements (MAC)6 in the naive nonoperated rat. Ibudilast (AV-411) is a nonselective phosphodiesterase inhibitor and TLR4 receptor antagonist. It is also known to suppress glial cell activation14,15 and has potential for the treatment of multiple sclerosis or neuropathic pain. It may also improve the efficacy and safety of opioids by decreasing opioid tolerance, withdrawal, and reinforcement.16 Ibudilast may not only suppress the production of proinflammatory cytokines, such as tumor necrosis factor α and interleukin 1β, but also increase the production of the anti-inflammatory cytokine interleukin 10 and various neurotrophic factors.14,17–20 We hypothesized that ibudilast would significantly reduce the opioid tolerance effect associated with remifentanil in rats and, second, that it would have a significant MAC-sparing effect with sevoflurane. The aim of this study was to determine whether ibudilast could blunt or block the tolerance to remifentanil in terms of MAC reduction as well as the delayed increase in the MAC produced by this opioid in rats. In addition, the effects of ibudilast on the MAC were studied.
Thirty-five adult male Wistar rats (originally purchased from Charles River Laboratories, Barcelona, Spain) weighing 372 ± 65 g were used. The animals were housed in groups of 5 animals per cage (Macrolon type IV) with a 12:12-hour light–dark cycle at a relative humidity of 40% to 70% and 22°C ± 2°C ambient room temperature. Food (SAFE A03; Augy, France) and water were provided ad libitum. The animals were allowed to acclimatize to the animal facility for at least 1 week. All of the studies started at 10:00 AM and were performed during the light period. The study was approved by the Institutional Animal Care Committee, Madrid, Spain (Approval date, March 17, 2011).
Anesthetic Induction and Instrumentation Procedures
Rats were induced with 8% sevoflurane vaporized in a continuous oxygen flow of 2 L/min (Sevoflurane Vaporizer, Sevorane, Dragër Vapor 2000, Lubeck, Germany). All rats were intubated using an otoscope to introduce a flexible, blunt-tipped wire into the trachea and to direct a 14-gauge polyethylene IV catheter (Surflo, Terumo, Leuven, Belgium) with the animals positioned in sternal recumbency. To ensure that the catheter was well positioned, a capnograph was used to determine the presence of exhaled carbon dioxide. The catheter was then connected to a small T piece breathing system with minimum dead space. Fresh gas flow to the T piece was adjusted to 0.5 to 1.0 L/min of oxygen (100%), and the sevoflurane concentration was adjusted to 1× MAC: 2.4 to 2.6 %vol in the control group and close to the expected MAC value with the given treatments. Rats were kept under spontaneous ventilation throughout the experiment. Anesthetic monitoring was performed: pulse rate, arterial oxygen hemoglobin saturation (through pulse oximetry), and respiratory rate were recorded (Datex-Ohmeda SA5 monitor, General Electric, Helsinki, Finland). Rectal temperature was also monitored and maintained between 37.0°C and 38.0°C using a water-circulating warming blanket (Heat Therapy Pump, Model TP-220; Gaymar, Orchard Park, NY) and a heating light.
Determination of the MAC
The MAC is a standard measure of volatile anesthetic potency and has been defined as the concentration required to prevent gross purposeful movement in 50% of subjects in response to a noxious stimulus. However, in this study, the individual MAC value from every rat was determined by means of intratracheal gas sampling to measure the anesthetic gas concentration. A 20-gauge needle was inserted through the endotracheal catheter with the needle tip located at its entrance, and gas samples were assayed using a side-stream infrared analyzer (Datex-Ohmeda SA5 monitor, General Electric).
The MAC determinations were evaluated by the same investigator (DRP). A supramaximal noxious stimulus was applied with a long hemostat (8-inch Rochester Dean Hemostatic Forceps; Martin, Tuttlingen, Germany) clamped to the first ratchet lock on the tail for 60 seconds or until positive response was observed. To standardize the application of the noxious stimulus, the tail was always stimulated proximally to a previous test site when the previous response was negative, or it was stimulated more distally if the response was positive, starting 6 cm from the tail base. A positive response was considered to be a gross purposeful movement of the head, extremities, or body. A negative response was considered to be a lack of movement or grimacing, swallowing, chewing, or tail flicking. When a negative response was seen, the sevoflurane concentration was reduced in decrements of 0.2 %vol until the negative response became positive. Similarly, when a positive response was seen, the sevoflurane concentration was increased by 0.2 %vol until the positive response became negative. The MAC was considered as a mean of the 2 highest concentrations that did not prevent movement when the stimulus was performed and the 2 lowest concentrations that prevented such movement. Two independent crossover positive–negative or negative–positive responses were used to set the MAC value as the mean of those 4 calculations. Determination of the MAC was performed in a laboratory 650 m above sea level, which lowers the barometric pressure and results in MAC values that are higher than those obtained at sea level. Therefore, MAC values were corrected to the barometric pressure at sea level using the following formula: MAC (%vol) at sea level barometric pressure (760 mm Hg; altitude adjusted MAC) = measured MAC (%vol) × measured ambient barometric pressure (700 mm Hg in the laboratory)/sea level barometric pressure (760 mm Hg).
Experimental Design and Drug Groups
Rats were randomly assigned to 1 of 5 treatment groups (n = 7 per group × 5 groups = 35 rats; Fig. 1). The treatments administered were 10 mg/kg ibudilast intraperitoneally, 240 μg/kg/h remifentanil IV, remifentanil plus ibudilast (240 μg/kg/h IV and 10 mg/kg IP, respectively), remifentanil plus naloxone (240 μg/kg/h IV and 10 ng/kg/h with a loading dose of 10 ng/kg, 2 ng/mL IV, respectively), or saline (equal volumes; control group). The ibudilast dose was selected based on its antinociceptive effects in rat models of peripheral and central neuropathic pain.20,21 The remifentanil constant rate of infusion dose was selected based on its ability to produce opioid-induced hyperalgesia and tolerance in rats.5 The naloxone dose was selected based on its ability to block opioid-induced hyperalgesia in rats.6,22 Ibudilast (100 mg, TCI Europe N.V., Amsterdam, the Netherlands) was dissolved in 2% Tween and 20% phosphate-buffered saline at 5 mg/mL. Remifentanil (Ultiva) was obtained from Glaxo-Smith-Kline (Madrid, Spain), naloxone was obtained from Kern Pharma (Terrasa, Spain), and sevoflurane (Sevorane) was obtained from Abbott Laboratories (Madrid, Spain).
Three MAC values (MAC-A or baseline, MAC-B, and MAC-C) were determined, each day, on days 0, 2, and 4, and in every animal within each treatment group (total of 9 MAC determinations in every rat). Once anesthetized and instrumented, 30 minutes of equilibration were allowed, and the baseline sevoflurane MAC (MAC-A, baseline) was determined. Each animal acted as its own control. Then, on day 0, 1 of 5 treatments was randomly administered to every animal, and a second MAC value (MAC-B) was calculated starting 30 minutes after drug administration. Finally, a third MAC value (MAC-C) was calculated starting 30 minutes after MAC-B determination. Each MAC determination lasted between 40 and 60 minutes, and the entire experiment lasted around 4.0 to 4.5 hours. The same treatments were administered to rats on days 2 and 4. On day 4, animals were killed at the end of the experiment with 200 mg/kg pentobarbitone IV while deeply anesthetized.
Acute tolerance effect was considered to be a decreased ability of remifentanil to reduce the MAC soon after their administration (MAC B-A versus MAC C-A), and a delayed opioid tolerance resulted from a lower reduction in the MAC B-A by this same opioid on days 2 and 4 compared with day 0.
Two types of statistical analysis were performed. First, multiple comparisons (analysis of variance) were performed for the 5 means of MAC-A values on day 0 (baseline values) to ensure that there were no significant differences between the means of the 5 treatment groups at baseline. Second, for each 1 of the 5 treatment groups, the following paired Student t test comparisons were performed: (1) between MAC-A and MAC-B on each of the days 0, 2, and 4, to assess the effects of drugs on the MAC within each group; (2) between MAC-B and MAC-C on each of the days 0, 2, and 4, to assess the tolerance to remifentanil; and (3) between MAC-A on days 0 and 2 and on days 0 and 4, to determine changes in the baseline MAC over the time (hyperalgesia). In all of these tests, P values are reported to evaluate the significance of the comparisons.
The requirements for estimating the sample size for each group were established by assuming a risk of false positives (significance level) α = 5%, a risk of false negatives β = 20% and considering significant a minimum difference of 2 times the SD in the mean MAC values between groups. With these settings, the sample size needed to compare simultaneously the (normal) means of 5 groups is n = 7 for each group. Rats in each experiment were randomly allocated using a random number generator (www.randomization.com). The results are presented as the mean value of MAC ± SD.
To check the normality of the data, the standardized skewness and kurtosis and the Shapiro-Wilk test (P > 0.05) were used. The homogeneity of the variance through the 5 groups has been tested with the Levene test (P > 0.05). All analyses were performed using Statgraphics Centurion XVI (Statpoint Technologies, Inc., Warrenton, VA).
There were no differences in body weight between group treatments or studied days (372 ± 65 g, day 0, n = 35, P = 0.852). No adverse (side) effects were observed during the experimental treatment days.
Multiple Comparisons for MAC-A on Day 0
There were no differences in the baseline mean MAC value between the 5 groups (first MAC measurement on the first day in the untreated rat; MAC-A, day 0; analysis of variance for multiple comparisons, P = 0.5192) with an overall mean baseline MAC-A of 2.4 ± 0.2 %vol (mean of 5 groups × 7 rats).
Ibudilast Reduced the Sevoflurane MAC (MAC-B)
The MAC-A was not modified significantly when compared with the first MAC determination after treatments (MAC-B) in the control group. However, the MAC was reduced significantly (MAC-B) with ibudilast, remifentanil, remifentanil plus ibudilast, and remifentanil plus naloxone (Table 1 and Fig. 1).
Ibudilast and Naloxone Did Not Prevent the Reduction of Remifentanil Efficacy to Reduce the MAC in the Short Term (Acute Tolerance; MAC-C)
Remifentanil effects on the MAC were blunted in the short term (1.5 hours) with an increase in MAC-C compared with MAC-B, while maintaining the opioid infusion constant (P = 0.0007, 0.0016, and 0.0001 on days 0, 2, and 4, respectively). This effect was not blocked when this opioid was administered combined with ibudilast (P = 0.0170,a 0.0020, and 0.0014 on days 0, 2, and 4, respectively) or naloxone (P = 0.0025, 0.0054, and 0.0004 on days 0, 2, and 4, respectively; Fig. 1)
Ibudilast and Naloxone Blocked the Delayed Sevoflurane MAC Increase Observed with Remifentanil
Remifentanil administered alone (group remifentanil) increased the MAC-A on days 2 and 4, compared with day 0, by 0.20 ± 0.16 %vol and 0.42 ± 0.19 %vol, respectively (P = 0.0190b and 0.0012, respectively). However, when ibudilast (P = 0.6947 and 0.9854 on days 2 and 4, respectively) or naloxone (P = 0.2999 and 0.44581 on days 2 and 4, respectively) was coadministered with remifentanil, this delayed increase in the MAC was prevented (Fig. 2). A detailed summary of the results can be found in the Supplemental Digital Content (http://links.lww.com/AA/B358).
The opioid, remifentanil, not only induced an immediate reduction of the sevoflurane requirements (50%) but also a delayed increase, within 4 days, close to 20%. This delayed increase in the baseline MAC was effectively prevented by the TLR4 antagonist, ibudilast, and also, as previously described, by naloxone.6 Interestingly, ibudilast, but not naloxone, also promoted a reduction in the MAC of sevoflurane, although not potentiating the effects of remifentanil. However, in this study, ibudilast and naloxone did not prevent the loss of remifentanil efficacy in the short term, suggesting a lack of effect on the tolerance to this opioid.
Analgesic treatment with opioids has a major effect on anesthetic requirements by reducing them.23,24 This reduction can be measured by determining the associated change in the MAC and has clinical relevance. However, the observed changes in the MAC cannot be considered a measure of analgesia but just the interaction of these drugs with inhaled anesthetics.25
When combined with sevoflurane in the nonoperated rat, an acute tolerance to remifentanil is observed,5 which reduces anesthetic potency by one-half only 90 minutes after the start of the opioid infusion. Findings in this study suggest that ibudilast and naloxone would not prevent the loss of remifentanil efficacy (increased MAC requirements) or tolerance in the short term. There are conflicting results concerning the potential preventive actions of ibudilast on opioid-induced tolerance. Coadministration of ibudilast with morphine did not attenuate the development of tolerance to this opioid, whereas, in morphine-tolerant rats, ibudilast partly restored morphine-induced antinociception.7 With the exception of gabapentin26 and paracetamol,27 drugs such as ketamine,28 amitriptyline,29 naloxone,6 or ibudilast failed to block opioid tolerance, although the preventive effect might be a consequence, at least in part, of the potentiation of sevoflurane by these drugs. This tolerance effect was determined only acutely but not 2 (or 4) days later, when the remifentanil-sparing actions were those observed on the first day of opioid administration.
A delayed reduction in the potency of the inhaled anesthetic, sevoflurane,6,30 was prevented by an ultralow dose of naloxone, an opioid antagonist with TLR4 antagonist activity.6 Similar to naloxone, ibudilast prevented the remifentanil-induced delayed increase in the MAC, presumably as a consequence of their TLR4 antagonistic actions and further confirming its involvement. However, ibudilast is somewhat nonspecific, as it is a nonselective phosphodiesterase inhibitor. Therefore, we should be cautious about attributing the observed actions of this drug solely to TLR4 antagonism.14,15
Ibudilast might also have prevented the slight increase in MAC when the MAC method is applied over several days as a consequence of repeated tail clamping.6 Nonoperated naive rats were used; thus, this is a potential limitation of the study; the use of a painful surgical model would better mimic the clinical setting. Furthermore, in this study, a behavioral approach with tests to investigate hyperalgesia, tolerance, or allodynia were not performed. However, in previous studies, the administration of ibudilast at similar doses to those used in this study attenuated mechanical allodynia and injury-induced tactile hypersensitivity.14
The anesthetic-sparing action of ibudilast might be related to its analgesic action where dose-related antinociception has been determined at doses similar to those used in this study.7 However, a sedative action cannot be ruled out as contributing to this action on the MAC.7 Most analgesics may reduce the MAC, but sedatives with no analgesic properties may also interact with inhaled anesthetics, allowing a reduction in the dose of the latter.31,32 Amitriptyline, an antidepressant with strong TLR4 antagonist activity,33 also reduced the sevoflurane MAC to a similar extend in the rat (24%).29
Ibudilast,7 and also amitriptyline,33 may potentiate antinociception in an additive fashion when combined with systemic morphine. The analgesic efficacy produced by morphine,15,34 but also by oxycodone,9,16 can be improved by ibudilast coadministration. A similar additive effect has been found in terms of anesthetic potentiation where amitriptyline and remifentanil each produced a 40% reduction in the CAM, whereas approximately 80% reduction was observed when both drugs were combined.29 However, ibudilast did not potentiate the remifentanil anesthetic-sparing effect, which may suggest that additional mechanisms may be involved in the interaction between these drugs and inhaled anesthetics.
There are potential limitations to the study. MAC determinations were evaluated by the same investigator who administered the drugs, and treatment blinding was not considered for 2 reasons. First, remifentanil produces an initial, and obvious, respiratory depression requiring assistance by gently touching the chest to promote ventilation and prevent hypercapnia. Besides, this opioid produces an initial peak reduction in the sevoflurane MAC, which is rapidly blunted by the acute tolerance effect. Therefore, to more accurately determine the maximum MAC reduction produced by the opioid, an initial reduction close to that expected is required (e.g., 50%). Although the doses used in rodents are relatively higher than those used in patients, it should be noted that clinically effective doses should be tailored to the species-specific metabolic rate by applying allometric escalation.35 According to our results, remifentanil produced a delayed (4 days) increase in baseline MAC of sevoflurane, which has been associated to opioid-induced hyperalgesia.36,37 However, because hyperalgesia was not determined in this study, a direct link between both phenomena cannot be established.
Overall, our results suggest that ibudilast, besides reducing the sevoflurane MAC, prevented the delayed increase in baseline MAC produced by remifentanil but not the increase in the MAC caused by tolerance to remifentanil.
Name: Daniel Ruiz-Pérez, DVM, MS.
Contribution: This author helped design the study, conduct the study, analyze the data, discuss the results, and write the manuscript.
Attestation: Daniel Ruiz-Pérez has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Javier Benito, DVM, MS.
Contribution: This author helped design the study, conduct the study, analyze the data, discuss the results, and write the manuscript.
Attestation: Javier Benito has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Gonzalo Polo, DVM.
Contribution: This author helped with MAC determinations.
Attestation: Gonzalo Polo has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Carlota Largo, DVM, PhD, MS.
Contribution: This author helped with drugs administration and anesthesia.
Attestation: Carlota Largo has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Delia Aguado, DVM, PhD, MS.
Contribution: This author helped analyze the data.
Attestation: Delia Aguado has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Luis Sanz, PhD.
Contribution: This author helped analyze the data and write the manuscript.
Attestation: Luis Sanz has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Ignacio A. Gómez de Segura, DVM, PhD, Dip ECVAA, Dip ECLAM.
Contribution: This author helped design the study, conduct the study, analyze the data, discuss the results, and write the manuscript.
Attestation: Ignacio A. Gómez de Segura has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
This manuscript was handled by: Markus W. Hollmann, MD, PhD, DEAA.
The authors thank the staff from the Experimental Surgery Unit, La Paz University Hospital, for their assistance with the animals and the study.
a Shapiro-Wilk normality test, P = 0.141. Levenne homoscedasticity test, P = 0.933.
b Shapiro-Wilk normality test, P = 0.437. Levenne homoscedasticity test, P = 1.0.
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