Abstract
OBJECTIVE To determine the minimum alveolar concentration that blunts adrenergic responses (MACBAR) for isoflurane and evaluate effects of fentanyl on isoflurane MACBAR in sheep.
ANIMALS 13 healthy adult Dorset-cross adult ewes.
PROCEDURES In a crossover design, each ewe was anesthetized 2 times for determination of isoflurane MACBAR. Anesthesia was induced with propofol administered IV. Sheep initially received fentanyl (5 μg/kg, IV, followed by a constant rate infusion of 5 μg/kg/h) or an equivalent volume of saline (0.9% NaCl) solution (control treatment). After a washout period of at least 8 days, the other treatment was administered. For MACBAR determination, a mechanical nociceptive stimulus (ie, sponge forceps) was applied at the coronary band for 1 minute. The MACBAR values of the 2 treatments were compared by means of a paired t test. During MACBAR determination, blood samples were collected for measurement of plasma fentanyl concentration.
RESULTS Mean ± SD isoflurane MACBAR of the fentanyl and control treatments was 1.70 ± 0.28% and 1.79 ± 0.35%, respectively; no significant difference was found between the 2 treatments. Plasma concentration of fentanyl reached a median steady-state concentration of 1.69 ng/mL (interquartile range [25th to 75th percentile], 1.47 to 1.79 ng/mL), which was maintained throughout the study.
CONCLUSIONS AND CLINICAL RELEVANCE Administration of fentanyl at 5 μg/kg, IV, followed by a constant rate infusion of the drug at 5 μg/kg/h did not decrease isoflurane MACBAR. Further studies to determine the effect of higher doses of fentanyl on inhalation anesthetic agents and their potential adverse effects are warranted. (Am J Vet Res 2016;77:119–126)
Sheep are commonly anesthetized in clinical and research settings for surgical procedures. Whenever surgery is performed on an animal, analgesia, unconsciousness, and immobility are paramount. Lack of movement during stimulation is the basis for the MAC, which is defined as the concentration that prevents movement in response to a noxious stimulus in 50% of the population.1 Previous studies2–4 have revealed that the MAC for isoflurane in adult sheep is between 1.42% and 1.58%. An optimal plane of anesthesia should prevent movement and provide cardiovascular stability. Therefore, it is important to minimize autonomic stimulation during general anesthesia, considering that increases in heart rate and blood pressure may reflect activation of the neuroendocrine stress response.5 The MACBAR is the concentration of inhalation anesthetic required to prevent an autonomic response to a standard noxious stimulus in 50% of the population.6 In humans,7–10 goats,11 dogs,12,13 and cats,14 MACBAR is higher than MAC.
Investigators of previous studies2–4,15–17 of sheep and lambs have used various methods to investigate the mechanical pain threshold. In these studies, the pain stimulus consisted of a pair of hemostat forceps or a clamp applied to the pinna3,4,15,16 and vigorously twisted2 or a metal pin strapped to the distal aspect of the radius to generate pressure against the limb.17 Calibrated forceps have been developed to apply a standardized force during mechanical nociceptive threshold testing in rats18 and dogs19; however, to the authors' knowledge, this type of forceps has not been used in sheep.
Fentanyl can have analgesic-, MAC-, and MACBAR- sparing effects. Fentanyl decreases the MAC of isoflurane in a variety of species, including dogs,20,21 cats,22 rats,23 and goats.24 However, in some species, such as horses, fentanyl has had no effects on isoflurane MAC.25 This was in contrast with findings of a study26 in which fentanyl administered at a high dose decreased isoflurane MAC. Additionally, it has been found that fentanyl decreases MACBAR in humans,8,10 including children.9 To the authors' knowledge, no studies have been conducted to evaluate the effects of fentanyl on isoflurane MACBAR in sheep.
The purpose of the study reported here was to determine MACBAR of isoflurane in sheep and to evaluate the effect of fentanyl on MACBAR. The hypothesis was that isoflurane MACBAR in sheep would be higher than MAC determined in other studies and that fentanyl would decrease isoflurane MACBAR.
Materials and Methods
Animals
Thirteen healthy Dorset-cross adult ewes between 1 and 3 years of age with a body weight between 30 and 84 kg were enrolled in the study. Sheep were assessed as healthy on the basis of results of physical examination and determination of PCV and total protein concentration. Sheep were housed in groups of 3 and were allowed to acclimate for a minimum of 7 days in an animal housing facility at the University of Georgia College of Veterinary Medicine. The study was approved by the University of Georgia Institutional Animal Care and Use Committee.
Experimental design
A prospective, randomized, within-subjects crossover experimental design was used for the study. Each sheep was anesthetized 2 times for MACBAR determination. Sheep were initially assigned by a random number generator to receive fentanyl or an equivalent volume of saline (0.9% NaCl) solution. After a washout period of at least 8 days, sheep were again anesthetized and the other treatment administered.
Anesthesia
Food was withheld from the sheep for 20 to 24 hours before anesthesia. A 16-gauge, 3.25-inch catheter was inserted in a jugular vein. This catheter was used for collection of a preanesthetic blood sample and administration of fluids and anesthetic and analgesic agents used in the study. Anesthesia was induced with propofola (5 to 10 mg/kg, to effect) administered IV as a bolus. Sheep were then orotracheally intubated with a cuffed Murphy-type endotracheal tube. The cuff of the endotracheal tube was inflated as necessary until no leakage was detected at an inspiratory pressure of 20 cm H2O. Anesthesia was maintained with isofluraneb delivered in oxygen through a semiclosed circle system. The rebreathing system was connected to a mechanical ventilator,c and positive-pressure ventilation was instituted to maintain PETCO2 between 35 and 45 mm Hg. An 18-gauge, 1.25-inch catheter was inserted in a saphenous vein for collection of blood samples used for determination of plasma concentrations of propofol and fentanyl.
A 20-gauge, 1.25-inch catheter was placed in a caudal auricular artery of anesthetized sheep for invasive monitoring of blood pressure. The arterial catheter was connected by a 121.9-cm-long piece of fluid-filled noncompliant tubing to a pressure transducer placed at the level of the heart with the manubrium as a landmark. The pressure transducer was calibrated and then set to 0 prior to the first measurement, as directed by the monitor manufacturer. A multivariable monitord was used to invasively measure blood pressure, heart rate, body temperature, hemoglobin oxygen saturation, and respiratory rate and to display an ECG (lead II). Body temperature was monitored with an esophageal probe, and normothermia was maintained with a forced-air warming devicee with an over-the-body blanket.
A sidestream infrared gas analyzerf was used to measure the fraction of inspiratory oxygen and isoflurane, ETISO, and PETCO2. Samples were collected from the proximal end of the endotracheal tube at a rate of 150 mL/min. The gas analyzer was calibrated prior to the study with calibration gases (60% nitrous oxide and a mixture of 1% isoflurane in 5% carbon dioxide) supplied by the manufacturer and also was calibrated against a refractometer gas analyzerg as described elsewhere.27 Briefly, gas samples were collected at the common gas outlet via closed tubing systems connected to both gas analyzers. The oxygen flow was set at 2 L/min, and 2 minutes were allowed for stabilization of the measurements. After this period, the infrared gas analyzer was calibrated against the refractometer at isoflurane concentrations of 0, 1, 2, and 3 vol%. Reference limits for the monitored variables were defined as MAP of 60 to 100 mm Hg, heart rate of 70 to 110 beats/min, body temperature of 37.0° to 38.5°C, hemoglobin oxygen saturation of 98% to 100%, respiratory rate of 10 to 15 breaths/min, and PETCO2 of 35 to 45 mm Hg.
All sheep received a balanced electrolyte solution (lactated Ringer solution) IV at a rate of 5 mL/kg/h throughout each anesthetic period. Every 2 hours, the urinary bladder of each sheep was manually palpated and emptied as necessary to prevent stimulation from overdistention. Bladder palpation and emptying were performed immediately after mechanical stimulation and data recording and at least 20 minutes before the subsequent nociceptive stimulus was applied.
Treatment administration
Approximately 30 minutes after induction of anesthesia, a fentanylh bolus (5 μg/kg) or equivalent volume of saline solution (control treatment) was administered IV over a 30-second period. The bolus was immediately followed by CRI of fentanyl (5 μg/kg/h, IV, diluted with saline solution) or saline solution, both of which were infused at a rate of 10 mL/h. Syringes were prepared immediately before each anesthetic episode and were not labeled to ensure the investigator determining MACBAR was not aware of the treatment administered to each sheep. The CRI was discontinued after all data were collected and before the sheep were allowed to recover from anesthesia.
MACBAR determination
Hair around the coronary band of the third digit of the right and left thoracic limbs of each anesthetized sheep was clipped. Approximately 1 hour after induction of anesthesia (30 minutes after administration of fentanyl or saline solution), ETISO was held constant at 2% for at least 20 minutes before the first mechanical (noxious) stimulation. Heart rate and MAP were stable for 5 minutes with < 1% variation prior to the stimulation. During this time, the highest values for these variables were recorded as the baseline data. At the conclusion of this period, a mechanical nociceptive stimulus was applied by an investigator (SAK), who was unaware of the treatment administered to each sheep. A pair of sponge forceps was clamped (to the first notch of the ratchet) to the coronary band of a thoracic limb for 1 minute; the clamp was alternately applied to the right and left limbs. Response of a sheep was considered positive and the recording stopped when a 15% increase from the baseline value for heart rate or MAP was detected during or up to 30 seconds after the mechanical stimulus. When there was a positive response, the vaporizer setting was adjusted so that ETISO increased by 0.1%. Conversely, for a negative response, isoflurane administration was adjusted so that ETISO decreased by 0.1%. Once the new ETISO was achieved and maintained continuously for 20 minutes, mechanical stimulation was repeated. For all sheep, MACBAR was determined in duplicate and the mean calculated; however, when the variation between the 2 values was > 10%, a third MACBAR value was determined, and the mean of the 3 determinations was calculated. Blood samples for drug plasma concentrations were collected immediately after each mechanical stimulation.
Force of mechanical stimulus
The diameter of the third digit of the right and left thoracic limb of each sheep was measured at the level of the coronary band. These measurements were used to determine the force of the mechanical stimulus applied to the coronary band with the sponge forceps. A protractor was glued to one of the arms of the forceps. The jaws of the forceps were opened until the diameter measured at the level of the coronary band of the sheep was reached, and a mark was made on the protractor. Jaws of the forceps were then connected to a testing machine,i and they were opened until they reached the mark on the protractor. The testing machine was calibrated to 0; the forceps then were closed to the first notch of the ratchet, and the amount of force generated was recorded. Three measurements for each measured diameter were obtained, and the mean was calculated.
Collection of blood samples
A blood sample was collected from the catheter in the jugular vein immediately before the induction of anesthesia (time 0). This sample was used to measure PCV and total protein concentration, and plasma was harvested and used as a negative control sample for assay of the propofol and fentanyl plasma concentrations. All subsequent samples were collected from the catheter in the saphenous vein; those samples were collected after the predetermined ETISO was reached and maintained for 20 minutes (the time points were approximately 25 minutes apart). Only the samples collected at time 0 and time 1 (approx 1 hour after induction of anesthesia) were submitted for analysis of propofol plasma concentration. For each sample, 3 mL of blood was discarded and 6 mL of blood then was collected and transferred to blood tubesj containing lithium heparin. Samples were immediately placed on ice and then centrifuged at 1,960 × g for 10 minutes within 15 minutes after collection. The plasma portion was separated, homogenized via pipetting, dispensed into 1.5-mL cryogenic vials, and frozen at −80°C.
Anesthetic recovery and postoperative monitoring
After data collection, sheep were positioned in sternal recumbency and allowed to recover from anesthesia. The endotracheal tube was removed once a sheep was able to swallow and resisted the endotracheal tube. All indwelling catheters were removed, and the sheep were returned to their stalls in the housing facility. Sheep were monitored for 1 week after the study for lameness of the stimulated limbs and for skin lesions in the coronary band area where the stimulus was applied.
Fentanyl and propofol analysis
Fentanyl and propofol concentrations were quantitated in sheep plasma with liquid chromatography-mass spectrometry analysis of protein-precipitated samples by use of modifications of methods published elsewhere.28,29 Fentanyl-D5 and propofol-D17 were used as the internal standard for fentanyl and propofol analyses, respectively. For fentanyl analysis, plasma calibrators were prepared by dilution of the working standard solutions with drug-free plasma obtained from sheep; diluted concentrations were 0.025, 0.05, 0.1, 0.4, 0.8, 1, 2, 5, 8, and 12 ng/mL. Plasma calibrators for propofol analysis were prepared similarly, with concentrations of 10, 50, 75, 100, 150, and 200 ng/mL. Calibration curves were prepared fresh for each quantitative assay. In addition, quality control samples at concentrations within the standard curve were included with each sample set as an additional assessment of accuracy. The curves for both fentanyl and propofol were linear, with nearly perfect correlation (R2 ≥ 0.99). For fentanyl analysis, accuracy (percentage of nominal concentration) and precision (percentage of relative SD) were 112% and 113% and 5% and 3% for concentrations of 0.15 and 4 ng/mL, respectively. For propofol analysis, accuracy was 97%, 99%, and 110% and precision was 7%, 4%, and 2% for concentrations of 30, 75, and 150 ng/mL, respectively. Accuracy and precision for both assays were considered acceptable on the basis of FDA guidelines for bioanalytic method validation. The technique was optimized to provide a minimum limit of quantification of 0.05 ng/mL and limit of detection of 0.01 ng/mL for fentanyl and a limit of quantification of 10 ng/mL and limit of detection of 1 ng/mL for propofol.
Statistical analysis
Normal distributions of data for age, body weight, dose of propofol required to enable endotracheal intubation, propofol plasma concentrations at time 1, duration of anesthesia, force applied at the coronary band, and MACBAR of fentanyl and control treatments were determined by means of the D'Agostino-Pearson test. A Wilcoxon test was used to compare propofol plasma concentrations of fentanyl and control treatments at time 1, and a paired t test was used to compare the dose of propofol administered, duration of anesthesia, and MACBAR of the 2 treatments. Differences in the force applied to the coronary band by forceps closed to the first notch of the ratchet were calculated by use of 1-way repeated-measures ANOVA. The Kruskal-Wallis test was used to identify differences in fentanyl plasma concentrations among the various time points, and AUC of the fentanyl plasma concentrations were calculated. Data for time points 9, 10, and 11 were excluded for determination of plasma concentrations of fentanyl and calculation of AUC because of an insufficient number of samples.
All analyses were performed with commercially available statistical software.k Parametric values were expressed as mean ± SD and nonparametric values were expressed as median and IQR. Values were considered significant at P < 0.05.
Results
Mean ± SD age of the sheep was 2.4 ± 1.0 years, and mean body weight was 60.3 ± 21.9 kg. Induction of anesthesia with propofol and orotracheal intubation was successfully performed in all animals, except for 2 sheep when receiving the fentanyl treatment. In both of those sheep, the endotracheal tube was placed in the esophagus, but it was immediately repositioned correctly without further complications. Regurgitation during intubation occurred in 2 sheep during the control treatment. In both of those sheep, gastric material was suctioned from the oropharynx, and the endotracheal tube was placed. No further complications during and after the experiment were noticed for either sheep. Regurgitation of copious amounts of gastric material was observed for 2 sheep, which were different sheep from the ones that were initially intubated in the esophagus. Both of these sheep regurgitated approximately 90 minutes after anesthetic induction during fentanyl treatment. No other complications were observed during the study, and all monitored variables (eg, blood pressure, body temperature, hemoglobin oxygen saturation, heart rate, respiratory rate, and PETCO2) remained within reference limits. Only heart rate and MAP increased above the upper limit of the reference range in several sheep during mechanical stimulation; however, both variables returned to baseline values within 60 seconds after the forceps were removed. None of the sheep had overdistention of the urinary bladder, as determined via abdominal palpation. All sheep recovered from anesthesia without complications, and no lameness or visible tissue trauma at the site of application of the forceps were noticed during the monitoring period.
Total dose of propofol used to enable endotracheal intubation and plasma propofol concentration at time 1 were 5.7 ± 1.1 mg/kg (95% CI, 5.0 to 6.4 mg/kg) and 70.46 ng/mL (IQR, 49.15 to 98.31 ng/mL; 95% CI, 51.68 to 102.10 ng/mL) for the fentanyl treatment and 5.7 ± 1.5 mg/kg (95% CI, 4.9 to 6.6 mg/kg) and 59.85 ng/mL (IQR, 50.94 to 87.88 ng/mL; 95% CI, 52.04 to 90.03 ng/mL) for the control treatment (Figure 1). No significant difference was found between the 2 treatments for the dose of propofol administered (P = 0.93) or the plasma concentration of propofol (P = 0.95).
Mean ± SD dose of propofol used for anesthetic induction (A) and median and IQR for plasma propofol concentration 1 hour after induction (B) in 13 healthy Dorset-cross adult ewes. In a randomized crossover design, each ewe was anesthetized 2 times and received fentanyl (5 μg/kg, IV, followed by CRI of 5 μg/kg/h) or an equivalent volume of saline (0.9% NaCl) solution (control treatment). No difference was found in the amount of propofol administered (P = 0.93) or in the plasma propofol concentration (P = 0.95) between the 2 treatments.
Citation: American Journal of Veterinary Research 77, 2; 10.2460/ajvr.77.2.119
Mean ± SD duration of anesthesia was 233 ± 72 minutes (95% CI, 189 to 277 minutes) and 242 ± 94 minutes (95% CI, 185 to 298 minutes) for the fentanyl and control treatments, respectively. No significant (P = 0.78) difference was found between the 2 treatments.
Mean ± SD diameter of the third digit of the right and left thoracic limbs at the level of the coronary band was 18.9 ± 1.1 mm (95% CI, 18.3 to 19.6 mm) and 18.8 ± 1.0 mm (95% CI, 18.2 to 19.4 mm), respectively. Amount of force generated to the coronary band area of the right and left limbs when the forceps were closed to the first notch was 25.1 ± 1.5 N (95% CI, 24.2 to 26.0 N) and 24.9 ± 1.6 N (95% CI, 24.0 to 25.9 N), respectively. No significant difference was found between the mean force applied when forceps were closed to the first notch in the coronary band area of the right (P = 0.20) and left (P = 0.35) limbs.
Mean ± SD MACBAR of the fentanyl and control treatments was 1.70 ± 0.28% (95% CI, 1.5% to 1.9%) and 1.79 ± 0.35% (95% CI, 1.6% to 2.0%), respectively; MACBAR did not differ significantly (P = 0.37) between the 2 treatments. Plasma concentration of fentanyl reached an apparent steady-state concentration at time 1, with a median of 1.39 ng/mL (IQR, 1.26 to 1.72 ng/mL; 95% CI, 1.26 to 1.68 ng/mL). A median concentration of 1.69 ng/mL (IQR, 1.47 to 1.79 ng/mL; 95% CI, 1.53 to 1.80 ng/mL) was maintained throughout the study (Figure 2). No significant (P = 0.29) differences in plasma fentanyl concentrations were detected among the various time points. Mean ± SD AUC at the various time points was 18.43 ± 1.36 ng/mL (95% CI, 17.29 to 19.57 ng/mL).
Median and IQR plasma concentration of fentanyl in 13 healthy Dorset-cross adult ewes anesthetized with isoflurane in oxygen. Time points are as follows: 0 = before induction of anesthesia; 1 = 1 hour after induction of anesthesia; and 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 = after predetermined ETISO was reached and maintained for 20 minutes and a noxious mechanical stimulus was applied (time points are approx 25 minutes apart). Data for times 9, 10, and 11 were excluded because of an insufficient number of samples obtained at each of those times.
Citation: American Journal of Veterinary Research 77, 2; 10.2460/ajvr.77.2.119
Discussion
Isoflurane MAC in sheep is reportedly between 1.42% and 1.58%,2–4 but to the authors' knowledge, isoflurane MACBAR has not been studied in sheep. Several authors have investigated MACBAR of isoflurane in various species, and reported values are greater than the corresponding MAC. The MACBAR is 2.6× MAC in goats,11 1.1× MAC in cats,14 and 1.3× MAC in humans.10 In the present study, isoflurane MACBAR in sheep for the control treatment was 1.79%, which is approximately 1.2× isoflurane MAC.
Clinical effects of fentanyl have been studied in several species, but there are only a few reports17,30–32 for sheep. Various doses ranging from 5 to 20 μg/kg, administered as an IV bolus to awake subjects, have been used in some of these studies. Investigators of 1 study17 reported an increase in mechanical threshold for > 30 minutes when a low dose (5 μg/kg) was administered. These results were in contrast with those of another study32 for which investigators found that a dose of 10 μg/kg was required to increase the mechanical threshold. At this dose, some adverse effects were evident, such as excitement and ataxia during the first 25 to 30 minutes after the administration of fentanyl and a significant increase in Paco2 for the first 5 minutes and decrease in Pao2 for the first 10 minutes after administration. Only 1 sheep received the highest dose (20 μg/kg).32 A study33 conducted to evaluate the pharmacokinetic effects of fentanyl in sheep revealed that a dose of 2.5 μg/kg, IV, resulted in plasma concentrations of 1.65 ng/mL at 2 minutes and 0.48 ng/mL at 10 hours after injection. In the present study, a bolus of 5 μg/kg, IV, followed by CRI of 5 μg/kg/h resulted in plasma fentanyl concentrations of 1.39 ng/mL at time 1 (30 minutes after IV administration of the bolus of fentanyl), and plasma concentrations of 1.69 ng/mL were maintained during the IV infusion. When these plasma concentrations were compared, no difference was found at any time points. This was also confirmed by the AUCs for plasma fentanyl concentration, which yielded similar values at the various time points, with a small SD (< 10%) and narrow 95% CI.
The plasma fentanyl concentration that correlates with analgesic- or MAC-sparing effects in sheep is unknown. In humans, plasma concentrations of 0.9 to 2.0 ng/mL are effective for providing analgesia.34,35 Fentanyl concentrations of 0.6 ng/mL35 and 1.18 ng/mL36 have been found to have analgesic efficacy in dogs. Plasma concentrations that result in analgesia in cats are not as clearly defined. In 1 study,37 plasma concentrations in cats did not consistently correlate with clinical effects. In another study,38 plasma fentanyl concentrations of 1.14 to 2.22 ng/mL resulted in analgesia. Investigators of that latter study38 reported that the same dose of fentanyl used in the present study (5 μg/kg, IV, followed by CRI of 5 μg/kg/h, IV) induced a 27% reduction in isoflurane MAC in goats; however, plasma concentrations for this dose were not analyzed. In the present study, plasma concentrations of 1.69 ng/mL did not have significant isoflurane MACBAR– sparing effects in sheep. This was evident because the MACBAR of sheep receiving fentanyl was compared with the MACBAR when the sheep received the control treatment, and no difference was found between the 2 treatments.
Stimulation with ear clamps or hemostatic forceps has been the technique most commonly used to assess mechanical threshold in sheep.2–4,15,16 A more standardized approach was used by investigators in another study.17 In that study,17 sheep receiving systemically administered analgesic agents were stimulated with a mechanical device. For that technique, a metal pin was strapped to the distal aspect of the radius to generate pressure against the limb. The metal pin was attached to a scale, which was correlated to a set force. Those authors found that 3.9 N was sufficient to stimulate signs of pain in nonsedated sheep, and approximately 9.8 N was necessary to stimulate the same reaction when a bolus of fentanyl (5 μg/kg, IV) was administered to the same animals.17 A similar device was also used in other studies conducted to assess mechanical threshold in sheep after application of fentanyl patches and intraperitoneal injection of medetomidine31 and in dogs and cats after administration of various opioids.39 Force used in these studies ranged from 5.3 ± 2.6 N to 8.3 ± 5.8 N in sheep and from 5.5 ± 1.4 N to 15.0 ± 5.1 N in dogs and cats. For mechanical nociceptive testing in rats, large blunt forceps have been clamped on a paw. The forceps were equipped with a strain gauge connected to a modified electronic dynamometer. Use of these forceps was found to be safe and effective to stimulate signs of pain in rodents.18 In dogs, a pair of modified Kelly forceps was used for mechanical threshold testing.19 The ratchet was removed from the forceps, and a digital load cell was secured to the jaw. The load cell was calibrated at 9.8, 19.6, 29.4, and 39.3 N against a tensiometer. These forceps were found to be effective in nociceptive testing in dogs.19 Vulsellum forceps, clamped at the coronary band, have been used for isoflurane MAC determination in goats, but the force applied was not measured or standardized.24,40 In the present study, sponge forceps were used at the level of the coronary band of the third digit for mechanical threshold testing. A testing machine was used to quantify the force applied during the experiment, and 3 measurements for the right side and 3 measurements for the left side were obtained for each sheep. Comparison of the mean of these 3 measurements for either side revealed no significant differences, which confirmed that a constant and repeatable stimulus was applied. The force used in other studies to stimulate pain response in awake sheep17 and dogs19 was 9.8 and 14.7 N, respectively. In the present study, a mean force of 25.0 N was applied, which was greater than the force used in other studies, but it was < 49 N, which was considered the limit above which tissue damage can potentially occur.19 The small SD and 95% CI for the study reported here confirmed that the force applied to the coronary band during stimulation was similar and differed little among the sheep.
Inhalation anesthesia involves the concept of MAC to define the level of anesthesia necessary to prevent movement. For anesthesia achieved by IV administration of drugs, a similar concept is the effective plasma concentration of the IV-administered anesthetic at which 50% of humans will not respond to a noxious stimulus.41 In humans, the effective plasma concentration of propofol for loss of response to painful stimulus was 4.0 μg/mL for squeezing of the trapezius muscle42 and 15.0 μg/mL for a skin incision.41 In goats, plasma concentrations of propofol between 1.5 and 3.1 μg/mL reduced isoflurane MAC by 16% when Vulsellum forceps were clamped at the coronary band.40 In the present study, propofol was used to induce anesthesia, and the plasma concentrations at time 1 were not significantly different between the fentanyl and control treatments. These concentrations were 70.46 and 87.88 ng/mL, respectively, and the plasma concentrations required to decrease isoflurane MAC in goats are approximately 30 to 40 times as high. It is reasonable to assume that the dose of propofol in the present study was too small to influence isoflurane MACBAR of sheep.
Active regurgitation occurred in 2 sheep during induction of anesthesia for the control treatment, most likely as a result of a premature attempt for orotracheal intubation and an inadequate plane of anesthesia. Passive regurgitation occurred in 2 other sheep during fentanyl treatment approximately 90 minutes after induction and 1 hour after the IV bolus of fentanyl and CRI were started. Anesthesia can inhibit hindbrain reflex centers responsible for evoking primary and secondary cycle contractions of the reticulorumen and can cause relaxation of the cardia, which can initiate passive regurgitation.43,44 Fentanyl can evoke a centrally mediated blockade of the cyclic contractions of the reticulum of sheep, which results in inhibition of stomach motility.45 It is reasonable to assume that fentanyl exacerbated the gastrointestinal adverse effects associated with anesthesia and increased the chance of passive regurgitation in the present study.
Studies46,47 have revealed that withholding food from cattle for 48 hours can cause bradycardia; however, this response has not been confirmed in sheep. In the present study, we adhered to general guidelines for unfed small ruminants43,48 to avoid bradycardia induced by prolonged food deprivation, which could have interfered with MACBAR determination. A baseline heart rate was obtained prior to placement of a catheter in the jugular vein, which confirmed the absence of bradyarrhythmias in all sheep.
In the study reported here, distention of the urinary bladder was monitored via abdominal palpation. Overdistention of the bladder can affect heart rate in anesthetized humans and domestic animals, causing tachycardia by increasing the efferent cardiac sympathetic pathway49,50 or bradycardia via the vagovagal reflex.51,52 None of the sheep had an overdistended urinary bladder, and fluctuations in heart rate were not observed during the absence of mechanical stimulation.
The present study had some limitations. Use of propofol as the anesthetic induction agent could have interfered with MACBAR determination. Alternatively, anesthesia could have been induced with isoflurane via face mask. This technique was considered by the authors to be too stressful for the animals and would have prolonged the anesthetic induction time, which would have increased the risk of regurgitation and aspiration pneumonia. One hour after anesthetic induction, when the first MACBAR determination was recorded, the plasma concentrations of propofol were minimal and most likely insufficient to have affected MACBAR.
Isoflurane MAC was not determined in the present study; therefore, a direct comparison between MAC and MACBAR was not possible. Because of the lack of these data, MACBAR from the present study was compared with published values for isoflurane MAC in sheep.
The nature of the present study resulted in some missing data points for fentanyl plasma concentration. Blood samples were collected when the mechanical stimulus was applied, and the study ended when a duplicate MACBAR was obtained. This time point, which differed among sheep and between treatments, influenced the duration of anesthesia and the number of blood samples collected. A small number of blood samples were collected at times 9, 10, and 11; thus, these time points were excluded from statistical analysis and the data treated as nonparametric.
Analysis of the results of the present study indicated that isoflurane MACBAR in sheep was 1.79%, which was approximately 1.2× MAC in this species, as determined on the basis of published values. Use of fentanyl at 5 μg/kg, IV, followed by CRI of 5 μg/kg/h, did not decrease the MACBAR of isoflurane, which confirmed the lack of an isoflurane-sparing effect. It is possible that fentanyl might increase the risk of passive regurgitation in anesthetized sheep. Further studies to determine the effect of higher doses of fentanyl on inhalation anesthetic agents and their potential adverse effects are warranted.
ABBREVIATIONS
| AUC | Area under the curve |
| CI | Confidence interval |
| CRI | Constant rate infusion |
| ETISO | End-tidal concentration of isoflurane |
| IQR | Interquartile range |
| MAC | Minimum alveolar concentration |
| MACBAR | Minimum alveolar concentration that blunts adrenergic responses |
| MAP | Mean arterial blood pressure |
| PETCO2 | End-tidal partial pressure of carbon dioxide |
Footnotes
Propofol, Abbott Laboratories, Abbott Park, Ill.
Isoflow, Abbott Laboratories, Abbott Park, Ill.
Model 2000, Hallowell Engineering and Manufacturing Corp, Pittsfield, Mass.
Surgident Advisor vital signs monitor, Smiths Medical, Saint Paul, Minn.
Bair Hugger warming unit, Augustine Medical Inc, Eden Prairie, Minn.
POET IQ 602, Criticare Systems Inc, Waukesha, Wis.
Riken optical gas indicator, model FI-21, Riken Keiki Co Ltd, Tokyo, Japan.
Fentanyl citrate, Hospira Inc, Lake Forest, Ill.
Instron 3367, Instron, Norwood, Mass.
BD Vacutainer, lithium heparin, 95 USP units, 6.0 mL, Becton Dickinson Co, Franklin Lakes, NJ.
Prism, version 6.0, Graph Pad Software Inc, La Jolla, Calif.
References
1. Eger EI II. Age, minimum alveolar anesthetic concentration, and minimum alveolar anesthetic concentration—awake. Anesth Analg 2001; 93: 947–953.
2. Bernards CM, Kern C, Cullen BF. Chronic cocaine administration reversibly increases isoflurane minimum alveolar concentration in sheep. Anesthesiology 1996; 85: 91–95.
3. Okutomi T, Whittington RA, Stein DJ, et al. Comparison of the effects of sevoflurane and isoflurane anesthesia on the maternal-fetal unit in sheep. J Anesth 2009; 23: 392–398.
4. Palahniuk RJ, Shnider SM, Eger EI II. Pregnancy decreases the requirement for inhaled anesthetic agents. Anesthesiology 1974; 41: 82–83.
5. Love L, Egger C, Rohrbach B, et al. The effect of ketamine on the MACBAR of sevoflurane in dogs. Vet Anaesth Analg 2011; 38: 292–300.
6. Roizen MF, Horrigan RW, Frazer BM. Anesthetic doses blocking adrenergic (stress) and cardiovascular responses to incision—MAC BAR. Anesthesiology 1981; 54: 390–398.
7. Albertin A, Casati A, Bergonzi P, et al. Effects of two targetcontrolled concentrations (1 and 3 ng/mL) of remifentanil on MAC(BAR) of sevoflurane. Anesthesiology 2004; 100: 255–259.
8. Katoh T, Kobayashi S, Suzuki A, et al. The effect of fentanyl on sevoflurane requirements for somatic and sympathetic responses to surgical incision. Anesthesiology 1999; 90: 398–405.
9. Katoh T, Kobayashi S, Suzuki A, et al. Fentanyl augments block of sympathetic responses to skin incision during sevoflurane anaesthesia in children. Br J Anaesth 2000; 84: 63–66.
10. Daniel M, Weiskopf RB, Noorani M, et al. Fentanyl augments the blockade of the sympathetic response to incision (MACBAR) produced by desflurane and isoflurane: desflurane and isoflurane MAC-BAR without and with fentanyl. Anesthesiology 1998; 88: 43–49.
11. Antognini JF, Berg K. Cardiovascular responses to noxious stimuli during isoflurane anesthesia are minimally affected by anesthetic action in the brain. Anesth Analg 1995; 81: 843–848.
12. Seddighi R, Egger CM, Rohrbach BW, et al. Effect of nitrous oxide on the minimum alveolar concentration for sevoflurane and the minimum alveolar concentration derivatives that prevent motor movement and autonomic responses in dogs. Am J Vet Res 2012; 73: 341–345.
13. Yamashita K, Furukawa E, Itami T, et al. Minimum alveolar concentration for blunting adrenergic responses (MAC-BAR) of sevoflurane in dogs. J Vet Med Sci 2012; 74: 507–511.
14. March PA, Muir WW III. Minimum alveolar concentration measures of central nervous system activation in cats anesthetized with isoflurane. Am J Vet Res 2003; 64: 1528–1533.
15. Bokesch PM, Kapural M, Drummond-Webb J, et al. Neuroprotective, anesthetic, and cardiovascular effects of the NMDA antagonist, CNS 5161A, in isoflurane-anesthetized lambs. Anesthesiology 2000; 93: 202–208.
16. Brett CM, Teitel DF, Heymann MA, et al. The cardiovascular effects of isoflurane in lambs. Anesthesiology 1987; 67: 60–65.
17. Nolan A, Livingston A, Morris R, et al. Techniques for comparison of thermal and mechanical nociceptive stimuli in the sheep. J Pharmacol Methods 1987; 17: 39–49.
18. Luis-Delgado OE, Barrot M, Rodeau JL, et al. Calibrated forceps: a sensitive and reliable tool for pain and analgesia studies. J Pain 2006; 7: 32–39.
19. Trumpatori BJ, Carter JE, Hash J, et al. Evaluation of a mid-humeral block of the radial, ulnar, musculocutaneous and median (RUMM block) nerves for analgesia of the distal aspect of the thoracic limb in dogs. Vet Surg 2010; 39: 785–796.
20. Ueyama Y, Lerche P, Eppler CM, et al. Effects of intravenous administration of perzinfotel, fentanyl, and a combination of both drugs on the minimum alveolar concentration of isoflurane in dogs. Am J Vet Res 2009; 70: 1459–1464.
21. Wilson D, Pettifer GR, Hosgood G. Effect of transdermally administered fentanyl on minimum alveolar concentration of isoflurane in normothermic and hypothermic dogs. J Am Vet Med Assoc 2006; 228: 1042–1046.
22. Yackey M, Ilkiw JE, Pascoe PJ, et al. Effect of transdermally administered fentanyl on the minimum alveolar concentration of isoflurane in cats. Vet Anaesth Analg 2004; 31: 183–189.
23. Criado AB, Gomez e Segura IA. Reduction of isoflurane MAC by fentanyl or remifentanil in rats. Vet Anaesth Analg 2003; 30: 250–256.
24. Dzikiti TB, Stegmann GF, Dzikiti LN, et al. Effects of fentanyl on isoflurane minimum alveolar concentration and cardiovascular function in mechanically ventilated goats. Vet Rec 2011; 168: 429.
25. Knych HK, Steffey EP, Mama KR, et al. Effects of high plasma fentanyl concentrations on minimum alveolar concentration of isoflurane in horses. Am J Vet Res 2009; 70: 1193–1200.
26. Thomasy SM, Steffey EP, Mama KR, et al. The effects of I.V. fentanyl administration on the minimum alveolar concentration of isoflurane in horses. Br J Anaesth 2006; 97: 232–237.
27. Rudolff AS, Moens YP, Driessen B, et al. Comparison of an infrared anaesthetic agent analyser (Datex-Ohmeda) with refractometry for measurement of isoflurane, sevoflurane and desflurane concentrations. Vet Anaesth Analg 2014; 41: 386–392.
28. Boscan P, Rezende ML, Grimsrud K, et al. Pharmacokinetic profile in relation to anaesthesia characteristics after a 5% micellar microemulsion of propofol in the horse. Br J Anaesth 2010; 104: 330–337.
29. Thomasy SM, Mama KR, Whitley K, et al. Influence of general anaesthesia on the pharmacokinetics of intravenous fentanyl and its primary metabolite in horses. Equine Vet J 2007; 39: 54–58.
30. Ahern BJ, Soma LR, Boston RC, et al. Comparison of the analgesic properties of transdermally administered fentanyl and intramuscularly administered buprenorphine during and following experimental orthopedic surgery in sheep. Am J Vet Res 2009; 70: 418–422.
31. Musk GC, Murdoch FR, Tuke J, et al. Thermal and mechanical nociceptive threshold testing in pregnant sheep. Vet Anaesth Analg 2014; 41: 305–311.
32. Waterman AE, Livingston A, Amin A. The antinociceptive activity and respiratory effects of fentanyl in sheep. Vet Anaesth Analg 1990; 17: 20–23.
33. Ahern BJ, Soma LR, Rudy JA, et al. Pharmacokinetics of fentanyl administered transdermally and intravenously in sheep. Am J Vet Res 2010; 71: 1127–1132.
34. Holley FO, van Steennis C. Postoperative analgesia with fentanyl: pharmacokinetics and pharmacodynamics of constant-rate I.V. and transdermal delivery. Br J Anaesth 1988; 60: 608–613.
35. Robinson TM, Kruse-Elliott KT, Markel MD, et al. A comparison of transdermal fentanyl versus epidural morphine for analgesia in dogs undergoing major orthopedic surgery. J Am Anim Hosp Assoc 1999; 35: 95–100.
36. Kyles AE, Hardie EM, Hansen BD, et al. Comparison of transdermal fentanyl and intramuscular oxymorphone on post-operative behaviour after ovariohysterectomy in dogs. Res Vet Sci 1998; 65: 245–251.
37. Franks JN, Boothe HW, Taylor L, et al. Evaluation of transdermal fentanyl patches for analgesia in cats undergoing onychectomy. J Am Vet Med Assoc 2000; 217: 1013–1020.
38. Davidson CD, Pettifer GR, Henry, JD Jr. Plasma fentanyl concentrations and analgesic effects during full or partial exposure to transdermal fentanyl patches in cats. J Am Vet Med Assoc 2004; 224: 700–705.
39. Dixon MJ, Taylor PM, Slingsby L, et al. A small, silent, low friction, linear actuator for mechanical nociceptive testing in veterinary research. Lab Anim 2010; 44: 247–253.
40. Dzikiti BT, Stegmann FG, Cromarty D, et al. Effects of propofol on isoflurane minimum alveolar concentration and cardiovascular function in mechanically ventilated goats. Vet Anaesth Analg 2011; 38: 44–53.
41. Smith C, McEwan AI, Jhaveri R, et al. The interaction of fentanyl on the Cp50 of propofol for loss of consciousness and skin incision. Anesthesiology 1994; 81: 820–828.
42. Puri GD, Mathew PJ, Sethu Madhavan J, et al. Bi-spectral index, entropy and predicted plasma propofol concentrations with target controlled infusions in Indian patients. J Clin Monit Comput 2011; 25: 309–314.
43. Riebold TW. Ruminants. In: Tranquilli WJ, Thurmon JC, Grimm KA, eds. Lumb and Jones' veterinary anesthesia and analgesia. 4th ed. Ames, Iowa: Blackwell Publishing, 2007;731–746.
44. Leek BF. Clinical diseases of the rumen: a physiologist's view. Vet Rec 1983; 113: 10–14.
45. Ruckebusch Y, Bardon T, Pairet M. Opioid control of the ruminant stomach motility: functional importance of mu, kappa and delta receptors. Life Sci 1984; 35: 1731–1738.
46. Clabough DL, Swanson CR. Heart rate spectral analysis of fasting-induced bradycardia of cattle. Am J Physiol 1989; 257: 1303–1306.
47. McGuirk SM, Bednarski RM, Clayton MK. Bradycardia in cattle deprived of food. J Am Vet Med Assoc 1990; 196: 894–896.
48. Hall LW, Clarke KW, Trim CM. Anaesthesia of sheep, goats, and other herbivores. In: Veterinary anesthesia. 11th ed. Philadelphia: Saunders Elsevier, 2014;345–383.
49. de Burgh Daly M, Ward J, Wood LM. Effects of distension of the urinary bladder on the cardiovascular reflexes from the carotid baroreceptors in the dog. J Physiol 1993; 463: 545–564.
50. Hassan AA, Hicks MN, Walters GE, et al. Effect of distension of the urinary bladder on efferent cardiac sympathetic nerve fibres which respond to stimulation of atrial receptors. Q J Exp Physiol 1987; 72: 1–11.
51. Koch HJ, Szecsey A, Raschka C. Severe bradyarrhythmia in a patient with Alzheimer's disease and a patient with cerebral ischaemia, both induced by acute distension of the bladder. Int J Clin Pract 2001; 55: 323–325.
52. Yamaguchi Y, Tsuchiya M, Akiba T, et al. Nervous influences upon the heart due to overdistension of the urinary bladder: the relation of its mechanism to vago-vagal reflex. Keio J Med 1964; 13: 87–99.
