Materials and Methods

Animals—The study was approved by the Animal Care Committee of the Universidade Estadual Paulista. Six healthy adult mixed-breed dogs (5 males and 1 female) were used in the study; mean ± SD weight of the dogs was 27.8 ± 4.1 kg. Health status was assessed by means of physical examination, a CBC, measurement of venous blood gases, and serum biochemical analyses.

Study design—Food, but not water, was withheld for 12 hours prior to each experiment. The dogs were anesthetized on 2 occasions in a prospective design. During the first anesthetic procedure (phase 1), the effects of progressively increasing CRIs of remifentanil (0.15, 0.30, 0.60, and 0.90 μg/kg/min) on ISO_MAC were evaluated. After at least a 1-week washout period, the stability of ISO_MAC during a 6-hour CRI of remifentanil (0.15 μg/kg/min) was assessed by determining ISO_MAC at 2, 4, and 6 hours after the start of the remifentanil infusion (phase 2).

Instrumentation—Anesthesia was induced with isoflurane,^a delivered by means of a face mask and an adult-size circle breathing circuit attached to an anesthesia machine.^b During the induction phase, the precision vaporizer was adjusted to deliver 5% isoflurane with an oxygen flow rate of 5 L/min; this concentration was administered until orotracheal intubation could be accomplished. Following placement of a cuffed orotracheal tube, oxygen flow rates were reduced to 2 to 3 L/min. Dogs were positioned in lateral recumbency and mechanically ventilated.^b The inspiration-to-expiration ratio was held constant (1:2), whereas peak airway pressure and respiratory rate were adjusted to maintain eucapnia (PaCO₂, 35 to 45 mm Hg). Samples of airway gases were collected from the distal end of the orotracheal tube at a constant rate (200 mL/min) into an infrared gas analyzer^c to monitor ET_ISO concentrations. The analyzer was calibrated with a standard calibration gas mixture^d before and during each experiment. During instrumentation, isoflurane concentrations delivered by the vaporizer were adjusted to maintain a moderate depth of anesthesia on the basis of clinical evaluation.

Twenty-gauge catheters were placed in the cephalic vein and in the dorsal pedal artery. The venous access was used for administering lactated Ringer's solution (3 mL/kg/h) by use of a peristaltic pump^e and for infusion of remifentanil. The arterial catheter was connected to a pressure transducer system^f filled with heparinized physiologic saline (0.9% NaCl) solution for displaying SAP, DAP, and MAP on the monitor.^g The zero reference level of the pressure transducer was set at the manubrium. Blood samples were collected from the arterial catheter into heparinized syringes at the time of each ISO_MAC determination and immediately analyzed with an automated blood gas system^h for displaying the pH, PaCO₂, PaO₂, and HCO₃⁻ values. Blood gases were corrected on the basis of esophageal temperature, which was obtained by means of a probe with tip positioned at the thoracic portion of the esophagus. During MAC determinations, body (esophageal) temperature was maintained within a narrow range (37.5° to 38.5°C) by means of a forced warm air blanketⁱ and an electric heating pad. Adhesive surface electrodes were attached to the skin according to a lead II ECG to monitor HR.^g

ISO_MAC determination and remifentanil administration—The ET_ISO was maintained constant for 15 minutes before a supramaximal noxious stimulus (50 V and 50 Hz for 10 milliseconds) was delivered. The stimulus was administered over the lateral aspect of the proximal portion of the radius by use of an electrical stimulator^j connected to 2 subcutaneous 25-gauge stainless steel needles placed 5 cm apart. The stimulation protocol consisted of 2 single stimuli and 2 continuous stimuli of 3 seconds' duration with a 5-second interval between all stimuli.³ Stimulation was discontinued if purposeful movement occurred. Purposeful movement was defined as gross movement of the head, trunk, or limbs. Hyperextension of the limbs or neck, shivering, tail movement, swallowing, and spontaneous breathing efforts were not considered to be purposeful movement. One observer classified the motor response as positive or negative (presence or absence of purposeful movement, respectively) on all occasions. In phase 1, the right forelimb was stimulated, and the left forelimb was used in phase 2. In the absence of purposeful movement, the ET_ISO was decreased by 0.2% and the procedure was repeated after a 15-minute equilibration period until purposeful movement occurred. The ET_ISO was then increased by 0.1% until purposeful movement was abolished. In the event of an initial purposeful movement, the ET_ISO adjustments were performed in a reverse order. The ISO_MAC was calculated as the arithmetic mean of the ET_ISO values that allowed and abolished purposeful movement. Typical barometric pressure at our laboratory, located at 785 m above sea level, is 680 mm Hg; ISO_MAC values were corrected to sea level (barometric pressure, 760 mm Hg), according to the formula:

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All experiments started at 7:00 AM. During phase 1, after determination of baseline ISO_MAC, remifentanil^k was administered at progressively increasing CRIs of 0.15, 0.30, 0.60, and 0.90 μg/kg/min by means of a syringe pump,^l and ISO_MAC was determined for each infusion rate. No washout period was allowed between infusions. A 15-minute equilibration period was allowed before beginning MAC determination for each infusion rate. After determination of ISO_MAC for the highest infusion rate (0.90 μg/kg/min) was completed, remifentanil infusion was stopped and ISO_MAC was redetermined approximately 80 minutes later. The times to completion of ISO_MAC measurements at baseline, during each infusion rate of remifentanil, and after the infusion was stopped were recorded.

During phase 2 experiments, ISO_MAC was determined at specific time points during a 6-hour CRI of remifentanil. Following the instrumentation period, remifentanil was infused IV at 0.15 μg/kg/min and ISO_MAC determinations were performed at 3 target time points: 120, 240, and 360 minutes after the beginning the remifentanil CRI. Actual times for completion of ISO_MAC determinations at each targeted time point were recorded.

Assessments—During phase 1 and phase 2 studies, cardiovascular data (HR, SAP, DAP, and MAP), arterial blood gases (pH, PaCO₂, PaO₂, and HCO₃⁻), and esophageal temperature were recorded immediately before noxious stimulations. The parametric variables corresponding to each ISO_MAC were calculated as the arithmetic mean of the values observed at the ET_ISO concentrations used for determining ISO_MAC.

After the end of each experiment, a single dose of meloxicam (0.3 mg/kg, IV) was administered before isoflurane was discontinued and the dogs were allowed to recover from anesthesia. The times until orotracheal tube removal (moment of return of the swallowing reflex), sternal recumbency, and standing position were recorded. These times were measured as the time elapsed from discontinuation of isoflurane administration until the observation of those events.

Statistical analysis—Normal distribution of data was checked with a Kolgomorov-Smirnov test. All values are presented as mean ± SD, unless otherwise stated. During phase 1, ISO_MAC values and cardiovascular data obtained during each MAC determination were compared by use of a 1-way ANOVA followed by a Tukey test. During phase 2, ISO_MAC values obtained at each time point (2, 4, and 6 hours) were compared as described for phase 1 data. Duration of anesthesia, times to recovery, and the total dose of remifentanil recorded in phases 1 and 2 were compared by use of a Mann-Whitney U test. Significance was set at a value of P < 0.05.

Each remifentanil infusion rate was plotted against its percentage reduction in ISO_MAC from baseline (obtained during phase 1 study) by use of a nonlinear regression model²¹:

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where E is the percentage reduction of ISO_MAC, E_MAX is the maximum achievable reduction of ISO_MAC during remifentanil administration, D is the remifentanil infusion rate, ED₅₀ is the remifentanil infusion rate that results in a 50% decrease in ISO_MAC, and γ is the dimensionless coefficient that defines the slope of the dose × response curve.

Least square linear regression analysis was performed to study the correlation between the duration of each infusion rate of remifentanil and its percentage reduction in ISO_MAC.

Results

Mean arterial blood gas variables were maintained within reference ranges for mechanically ventilated dogs breathing inspired O₂ percentage > 90%. Blood gas–derived values recorded during ISO_MAC determinations (pooled data from phases 1 and 2) were as follows: pH = 7.41 ± 0.03, PaCO₂ = 39 ± 3 mm Hg, PaO₂ = 479 ± 39 mm Hg, and HCO₃⁻ = 24.3 ± 1.5 mmol/L. Esophageal temperature was maintained between 37.9° and 38.4°C during MAC determinations.

During phase 1 experiments, the mean (range) of time elapsed from induction of anesthesia until determination of ISO_MAC at baseline was 99 (62 to 131) minutes. Minimum alveolar concentration determinations were concluded in 99 (62 to 128), 117 (98 to 140), 120 (78 to 149), and 70 (66 to 78) minutes after commencing the 0.15, 0.30, 0.60 and 0.90 μg/kg/min infusion rates of remifentanil, respectively. Isoflurane MAC was redetermined at 79 (58 to 113) minutes after the end of the highest infusion rate.

The timings for concluding ISO_MAC determinations for each infusion rate in phase 1 were variable among individual dogs; therefore, there were differences in the cumulative doses of remifentanil administered until completion of ISO_MAC determinations for each CRI. To determine that this did not affect the results, least square linear regression analysis was performed to study the correlation between the duration of each infusion rate of remifentanil and its percentage reduction in ISO_MAC. Although there was a positive correlation between the duration of remifentanil infusion and the percentage of ISO_MAC reduction at the 0.15 μg/kg/min infusion rate (P = 0.04), no correlation could be detected at higher infusion rates (P = 0.8, 0.7, and 0.7 at remifentanil infusion rates of 0.3, 0.6, and 0.9 μg/kg/min, respectively).

The baseline ISO_MAC was 1.24 ± 0.18% (Figure 1). Increasing remifentanil infusion rates significantly decreased ISO_MAC, compared with baseline values. The infusion rates of 0.15, 0.30, 0.60, and 0.90 μg/kg/min decreased ISO_MAC values by 43 ± 10%, 59 ± 10%, 66 ± 9%, and 71 ± 9%, respectively. The ISO_MAC determined during the 0.30, 0.60, and 0.90 μg/kg/min infusion rates did not differ from each other, but these values were significantly lower than ISO_MAC recorded during the lowest infusion rate of remifentanil (0.15 μg/kg/min). After the infusion of remifentanil was stopped, the ISO_MAC (1.09 ± 0.18%) did not differ from the baseline ISO_MAC (1.24 ± 0.18%) but was higher than ISO_MAC values obtained during all infusion rates of remifentanil. The nonlinear regression model, which resulted in coefficients of determination (R²) values close to identity, predicted a maximum ISO_MAC reduction (E_MAX) of 70.5%. According to the same model, the infusion rate of remifentanil that reduced ISO_MAC by 50% (ED₅₀) was 0.20 μg/kg/min.

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Effects of remifentanil on ISO_MAC in dogs. A—Mean ± SD values of ISO_MAC in 6 dogs before remifentanil administration (baseline [BL]), during administration of progressively increasing infusion rates of remifentanil (0.15, 0.30, 0.60, and 0.90 μg/kg/min), and after completion of remifentanil infusion (Final). B—Relationship between remifentanil infusion rates and percentage reductions in ISO_MAC (mean ± SD). Notice the fitted regression line, R² (coefficient of determination), ED₅₀ (infusion rate that resulted in a 50% reduction in ISO_MAC), and E_MAX (maximum achievable reduction in ISO_MAC). C—Heart rates (mean ± SD) in the same dogs as in panel A. D—Systolic, mean, and diastolic pressures (SAP, MAP, and DAP [mean ± SD]) recorded in the same dogs as in panel A. *Significant (P < 0.05) difference from BL. †Significant (P < 0.05) difference from Final. §Significant (P < 0.05) difference from 0.15 μg/kg/min.

Citation: American Journal of Veterinary Research 71, 2; 10.2460/ajvr.71.2.150

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Heart rates were significantly lower during remifentanil infusions than at baseline; however, HR returned to baseline values after the remifentanil infusion was stopped (Figure 1). Five to 10 minutes after remifentanil infusion was discontinued, HR returned to values that were equal to or greater than baseline in all dogs. Furthermore, HR was lower at 0.90 μg/kg/min, compared with HR at 0.15 μg/kg/min. Bradycardia (defined as HR < 60 beats/min) was not observed during baseline measurements. Heart rates < 60 beats/min were recorded in 2 dogs in the 0.15 and 0.30 μg/kg/min remifentanil groups and in 4 dogs in the 0.60 and 0.90 μg/kg/min groups; lowest HR values recorded during opioid infusion were 51, 37, 39, and 34 beats/min after dogs received 0.15, 0.30, 0.60, and 0.90 μg/kg/min, respectively.

Systolic arterial pressure was higher during all infusion rates of remifentanil than at baseline and higher during the 0.60 and 0.90 μg/kg/min infusion rates, compared with SAP during the lowest infusion rate. After completion of the remifentanil infusion, SAP returned to values not significantly different from baseline. Mean arterial pressure was higher during the 3 higher infusion rates (0.30, 0.60, and 0.90 μg/kg/min) than MAP at baseline (Figure 1). Hypotension (defined as MAP < 60 mm Hg) did not occur at any time.

During phase 2 experiments, the mean (range) of the actual times for determination of ISO_MAC were 123 (113 to 130), 242 (233 to 251), and 354 (344 to 369) minutes after the start of the remifentanil CRI (0.15 μg/kg/min). The ISO_MAC values obtained at these 3 time points were 0.75 ± 0.10%, 0.73 ± 0.12%, and 0.73 ± 0.12% at 120, 240, and 360 minutes, respectively, and these values did not differ significantly from each other. Cardiovascular variables did not change over time during the remifentanil CRI. Recoveries from anesthesia were without complications; however, times recorded for recovery variables were significantly longer in phase 1 than in phase 2 (Table 1).

Discussion

Results of the present investigation indicated that remifentanil caused dose-related reductions of ISO_MAC in dogs. However, a ceiling effect was apparent at infusion rates > 0.30 μg/kg/min because only small decrements in MAC were achieved as the infusion rate was tripled. The present study also revealed that the reduction in ISO_MAC induced by remifentanil was reversed in a short time after termination of a prolonged infusion regimen (phase 1) and remained constant during the course of a 6-hour CRI (phase 2). These results were in agreement with previous studies^11,12 indicating that remifentanil induces stable and predictable reduction of MAC.

Factors that may influence MAC include circadian rhythm, age, methodology for MAC assessment, hypothermia, hypercapnia, hypoxemia, and acidemia or alkalemia.² In the present study, potential confounders were controlled. All experiments were started at 7:00 AM; the method used for MAC assessment was validated³; dogs were mechanically ventilated with oxygen to prevent conditions of hypercapnia, hypoxemia, acidemia or alkalemia; and esophageal temperature was maintained within a narrow range (37.9° to 38.4°C). Although the age of the dogs could not be precisely determined, dogs were determined to be young adults on the basis of physical examination findings. Baseline ISO_MAC (1.24 ± 0.18%) did not differ substantially from ISO_MAC values reported for healthy adult dogs.^3,4

Other potential confounders in studies^8–10 evaluating the effects of drugs that are administered as CRIs on the MAC of inhalational anesthetics include variations in plasma concentrations or drug accumulation during the course of a prolonged CRI. It has been reported that in dogs, steady state plasma concentrations of remifentanil were achieved within 25 minutes of a CRI of 0.36 μg/kg/min without the use of a loading dose.¹⁹ Negligible residual effects are expected from prolonged infusions of remifentanil; in humans, the context-sensitive half-life (the time to a 50% decrease in plasma concentration after a termination of a CRI) of remifentanil is extremely short (3 minutes after a 3-hour CRI) and is independent of the duration of the infusion.^14,15 Because remifentanil is also rapidly eliminated (elimination half-lives of 3 to 6 minutes) in dogs,^18,19 its accumulation in plasma as a result of a prolonged CRI is unlikely in dogs; however, a shortcoming of the study reported here was the fact that remifentanil plasma concentrations were not determined.

Because the procedures for determining ISO_MAC during phase 1 of the study were not completed before 60 minutes after each CRI was commenced, a carry-over effect of previous remifentanil infusions was probably avoided. On the basis of the pharmacokinetic profile of remifentanil in dogs,^18,19 30 minutes at each new infusion rate would be enough to allow almost complete elimination of remifentanil from the previous infusion, while at the same time allowing a new steady state plasma concentration to be achieved without the need for a loading dose.

Administration of 0.9 μg of remifentanil/kg/min caused a 71 ± 9% reduction in ISO_MAC, and this was consistent with the findings of a study^m in which remifentanil (1.0 μg/kg/min) decreased the sevoflurane MAC by 74 ± 6%. The maximum percentage reduction in ISO_MAC reported in the present study was also comparable with the maximum reduction of enflurane MAC (63 ± 10%) induced by remifentanil in dogs.¹¹ Conversely, an unexpected finding in the present study was that the ED₅₀ of remifentanil during isoflurane anesthesia (0.20 μg/kg/min) was substantially lower than the ED₅₀ of remifentanil reported during enflurane anesthesia (0.72 μg/kg/min),¹¹ suggesting that the relative potency of remifentanil, measured by its ability to reduce the MAC of inhalational drugs, is greater during isoflurane anesthesia than during enflurane anesthesia. Other drugs also appear to be more potent in reducing the MAC of isoflurane than in reducing the MAC of enflurane. In 1 enflurane study,²² higher plasma concentrations of ketamine had less effect on MAC than much lower plasma concentrations of ketamine in dogs undergoing isoflurane anesthesia.²³ In dogs undergoing orthopedic surgery, a remifentanil infusion of 0.25 μg/kg/min decreased by 49% the ET_ISO necessary to maintain anesthesia⁶ and this is comparable with the dose of remifentanil (0.20 μg/kg/min) that induced a 50% MAC reduction in the present study.

During phase 1, the effects of remifentanil on ISO_MAC were effectively reversed by 79 ± 20 minutes. Minimum alveolar concentration values are expected to not differ by > 10% when repeated MAC determinations are performed in the same animal.² The 12% variation observed between mean ISO_MAC values recorded at baseline (1.24 ± 0.18%) and mean ISO_MAC values recorded after completion of remifentanil infusion (1.09 ± 0.18%) was slightly larger than the typical variation reported in the literature,² and this could be attributable to the use of single MAC determinations at each time point; performing MAC determinations in duplicate or triplicate at each time point could have reduced some of this variation.

Results of the present study indicated that the effects of remifentanil on ISO_MAC are not influenced by the duration of the CRI. The lowest infusion rate of remifentanil (0.15 μg/kg/min) used during phase 1 experiments was chosen to be used during phase 2 because the ISO_MAC reduction induced by this dose did not approach the ceiling inhalant-sparing effect. During prolonged opioid infusions, the development of opioid tolerance could cause the MAC of inhalational anesthetics to increase over time,¹⁰ but this was not observed in the study reported here because MAC values remained constant throughout the 6-hour remifentanil CRI.

The longer duration of anesthesia in phase 1 was associated with longer recoveries from anesthesia. These results might be attributable to more prolonged administration of isoflurane and remifentanil. However, a cumulative effect is unlikely because the rapidity of remifentanil elimination is independent of the dose administered; a 100-times increase in remifentanil CRI (from 0.36 to 36.0 μg/kg/min) did not substantially increase the elimination half-lives in dogs.¹⁹ The difference in recovery variables is most likely clinically unimportant because recovery from anesthesia was without complications and relatively fast; dogs regained standing position within 17.8 and 9.2 minutes from discontinuation of isoflurane administration in phases 1 and 2, respectively. These results suggest that remifentanil is suitable for administration during prolonged anesthetic procedures, without adversely affecting recovery. Care must be taken when remifentanil administration is discontinued at the end of anesthesia in surgical patients; the fast termination of its effects will likely result in pain perception if supplemental analgesics are not administered for pain relief upon recovery from anesthesia.

Isoflurane is known to decrease arterial blood pressure in a dose-related fashion.⁴ This effect is attributable mainly to a decrease in systemic vascular resistance.⁴ Thus, the increase in SAP and MAP observed in the present study might be partly attributed to increases in systemic vascular resistance because the isoflurane end-tidal concentrations were progressively decreased during remifentanil infusion.

Although SAP and MAP were increased during the infusion of remifentanil, and none of the dogs developed hypotension (defined as MAP < 60 mm Hg) during opioid infusion, arterial blood pressure may have poor correlation with tissue O₂ delivery. Conversely, bradycardia during anesthesia may reduce tissue O₂ delivery; vagally mediated bradycardia induced by another phenylpiperidine derivative (fentanyl) caused significant decreases in cardiac output and in tissue O₂ delivery, effects that were reversed by atropine administration.²⁴ In the present study, vagally mediated bradycardia was evident during remifentanil infusion; HR values as low as 34 beats/min were recorded in 1 dog during the highest infusion rate of remifentanil. Reversal of remifentanil-induced bradycardia with an anticholinergic agent may be considered to maintain tissue perfusion, even if other indirect signs of poor tissue perfusion (eg, hypotension) are not observed.

Remifentanil induced dose-related decreases in ISO_MAC in dogs, and these effects were reversed within approximately 80 minutes after the administration of a prolonged infusion. Doses up to 0.30 μg/kg/min are recommended for use in combination with isoflurane because higher doses provide only small additional decrements in ISO_MAC. Remifentanil's ISO_MAC-reducing effect was stable during prolonged infusions. These characteristics, combined with fast recovery from anesthesia, suggest that remifentanil is suitable as an adjuvant to isoflurane in dogs undergoing prolonged anesthetic procedures.