Anesth Analg 2005;100:404-408
© 2005 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000142423.87593.CE
ANESTHETIC PHARMACOLOGY
Large Concentrations of Nitrous Oxide Decrease the Isoflurane Minimum Alveolar Concentration Sparing Effect of Morphine in the Rat
Martín Santos, DVM, PhD*
,
Viviana Kuncar, DVM*,
Fernando Martínez-Taboada, DVM*, and
Francisco J. Tendillo, DVM, PhD, DipECVA*
*Department of Experimental Surgery, University Hospital Puerta de Hierro; and Department of Anesthesiology, Complutense University Veterinary School, Madrid, Spain
Address correspondence and reprint requests to Francisco J. Tendillo, DVM, PhD, DipECVA, Servicio de Cirugía Experimental, Hospital Universitario Puerta de Hierro, San Martín de Porres 4, 28035 Madrid, Spain. Address e-mail to ftendillo.hpth{at}salud.madrid.org.
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Abstract
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Many adjuvant drugs have demonstrated anesthetic-sparing properties when combined with volatile anesthetics. Nitrous oxide is combined with volatile anesthetics to reduce the concentrations of volatile anesthetics required to produce anesthesia. Analgesic doses of opioids clearly reduce the requirement for inhaled anesthetics in both human patients and experimental animals. We performed this study to determine whether the combination of nitrous oxide and morphine decreased isoflurane minimum alveolar anesthetic concentration (MAC) even further in the rat. Fifty-eight female rats were used. The rats were divided into 8 groups: isoflurane in 4 possible nitrous oxide concentrations (0%, 30%, 50%, or 70%) with saline or morphine (1 mg/kg). Then the MAC of isoflurane (MACISO)was determined from alveolar gas samples at the time of tail clamp. The MAC of isoflurane was significantly different at each nitrous oxide concentration, and increasing nitrous oxide concentrations reduced anesthetic requirements for isoflurane. The administration of morphine reduced the MACISO when used with 0% or 30% nitrous oxide. This MACISO by morphine reduction was less with 50% nitrous oxide and nonexistent at 70% nitrous oxide. However, with morphine present the MACISO was independent of the nitrous oxide concentration in the 30%–70% range.
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Introduction
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Many adjuvant drugs have demonstrated anesthetic-sparing properties when combined with volatile anesthetics. Nitrous oxide (N2O) has formed the basis for more general anesthetic techniques than any other inhaled anesthetic. Its widespread use resulted from many desirable properties, including low solubility, limited cardiovascular and respiratory system depression, and minimal toxicity (1). It is often combined with volatile anesthetics to reduce the concentrations of volatile anesthetics required to produce anesthesia (2–4). Analgesic doses of opioids clearly reduce the requirement for inhaled anesthetics in both human patients and experimental animals (5).
Based on the evidence that N2O analgesia is mediated, at least in part, by the endogenous opioid system (6–8), the purpose of the current study was to determine whether the combination of N2O and morphine decreased isoflurane MAC (MACISO) even further in the rat.
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Methods
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Fifty-eight female Wistar rats with a mean body weight of 200 ± 10 g (range, 190–210 g) were allowed to acclimatize for at least 1 wk before the experimental procedure. Rats were housed in groups of 8–10 in Plexiglas cages, with free access to food and water, and maintained on a 12 h light:12 h dark cycle (light on at 7 am) under controlled environmental conditions (relative humidity, 50%–70%; Temperature, 20°C ± 2°C). To control for known diurnal fluctuations in responsiveness to nociceptive stimuli, experiments were performed during the morning (9 am to 12 pm). All rats were handled according to the guidelines set in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. The institutional animal care and use committee approved the study.
The unmedicated rats were placed in an induction chamber to which 5% isoflurane in a continuous oxygen flow of 3 L/min was directed. Once the rats were anesthetized, tracheal intubation was performed with a 16-gauge polyethylene catheter using the otoscope method (9). Once the correct catheter position was ascertained, it was connected to a small T piece of minimal dead space (<0.2 mL). Fresh gas flow to the T piece was adjusted to 1 L/min, and isoflurane concentration was adjusted as required after anesthetic reflex assessment. During the study, the rats were breathing spontaneously.
The carotid artery was catheterized with a 24-gauge polyethylene catheter via surgical cut down. This access allowed for arterial blood sampling and arterial blood pressure measurement via a calibrated pressure transducer. Arterial blood pressure was recorded continuously. Arterial blood gases were measured, occasionally during MAC assessment, and at the end of the study period to ensure values were within normal limits of pH (7.30–7.40), pressure of oxygen (Pao2) (>90 mm Hg), and pressure of carbon dioxide (Paco2) (35–45 mm Hg). Rectal temperature also was monitored and maintained at normothermia (between 37°C and 38°C) by means of a total temperature management system. A caudal tail vein was cannulated using a 24-gauge polyethylene catheter for the administration of drugs. Inspired isoflurane concentrations were further decreased to 1.5%, a value close to the average MACISO for rats before the first MACISO determination.
Once this concentration was achieved, rats were randomly allocated to 1 of 8 anesthetic groups as follows: (1 [n = 7] and 2 [n = 8]) isoflurane in 100% oxygen and 0% N2O with or without morphine; (3 [n = 7] and 4 [n = 7]) isoflurane in 70% oxygen and 30% N2O with or without morphine; (5 [n = 8] and 6 [n = 8]) isoflurane in 50% oxygen and 50% N2O with or without morphine; and (7 [n = 6] and 8 [n = 7]) isoflurane in 30% oxygen and 70% N2O with or without morphine. The rats in groups without morphine received an IV bolus of 0.5 mL of saline solution, and the rats in groups with morphine received an IV bolus of 1 mg/kg of morphine diluted in 0.5 mL of saline solution. Both morphine or saline IV bolus were administered IV in 3–5 min to reduce cardiovascular and respiratory effects if administered more quickly. MACISO was determined 20 min after drug administration.
Intratracheal gas sampling was used to measure anesthetic gas concentration for determination of the MAC. Inspired and end-tidal isoflurane concentrations were obtained continuously from gas drawn from a fine tubing inserted through the endotracheal catheter over a hole in the T piece and with the tip located at the level of the carina. The proximal end of the catheter was connected to a calibrated infrared-absorption analyzer with a 60-mL/min aspiration flow of the gas sample. After every step change in isoflurane concentration delivered by the anesthetic circuit, at least 15 min were allowed for equilibration maintaining a constant alveolar concentration and an alveolar-to-inspired ratio (FA/FI) more than 0.95.
The MACISO value was established according to the method described by Eger et al. (10). A painful noxious stimulus was applied with an 8-in. hemostat clamped to the first ratchet lock on the tail for 60 s. The tail was always stimulated proximal to a previous test site. A positive response was considered when a gross purposeful movement of the head, extremities or body, or both was observed, whereas a negative response was the lack of movement, swallowing, chewing, or tail flick. The isoflurane concentration was then reduced in decrements of 0.1%–0.2% until the negative response became positive. The MACISO was defined as the average of the smallest concentration preventing a positive response and the largest concentration allowing a positive response to the supramaximal painful stimulus. For each rat, MAC was determined in duplicate. The person assessing the response was blinded to the drugs administered at each group.
Statistical analysis of data was performed using the SPSS 10.0 software program (SPSS Inc, Chicago, IL). All data were grouped and summarized as mean ± sd. To compare the effect on MACISO of different N2O concentrations with or without morphine, an analysis of variance was performed, and post hoc comparison of the groups was performed using the Tukey test. A P value <0.05 was considered statistically significant. A regression analysis was applied to determine the correlation between isoflurane MAC at the 0%, 30%, 50%, and 70% N2O concentrations.
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Results
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The average MAC value for isoflurane in 0% N2O was 1.59% ± 0.10%. The addition of 30%, 50%, and 70% N2O significantly reduced the isoflurane MAC to 1.26% ± 0.04% (P < 0.001), 1.16% ± 0.10% (P < 0.001), and 0.95% ± 0.09% (P < 0.001), respectively (Fig. 1). The MAC of isoflurane was significantly different at each N2O concentration, and increasing N2O concentrations reduced in a dose-dependent manner the anesthetic requirements for isoflurane. Regression analysis was applied to determine the correlation coefficient and slope of the line extrapolated through the data sets. The regression equation derived was Y = 1.572 – 0.00886X. The R2 value for this line was 0.98, and the predicted MAC of N2O by extension of the linear regression line was 177% (Fig. 2).
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Figure 1. Isoflurane minimum alveolar anesthetic concentration (MACISO) after the administration of either saline or morphine (1 mg/kg) in combination with 0%, 30%, 50%, and 70 % nitrous oxide (N2O) in rats. Values are mean ± sd. *Statistically significant (P < 0.001) with respect to 0% N2O. Statistically significant (P < 0.05) with respect to the without morphine groups.
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Figure 2. The mean of isoflurane minimum alveolar anesthetic concentration (MACISO) determined at 0%, 30%, 50%, and 70% N2O are plotted, and the solid line represents the linear regression equation for these data points. The fine extension of the line to the x-axis represents the predicted MAC of N2O (177%).
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The administration of morphine reduced the MACISO when used with 0% or 30% N2O (from 1.59% ± 0.10% to 1.35% ± 0.08% [P < 0.001] and from 1.26% ± 0.04% to 0.99% ± 0.08% [P < 0.001], respectively). This MACISO by morphine reduction was less with 50% N2O (from 1.16% ± 0.10% to 1.02% ± 0.09% [P = 0.011]) and nonexistent at 70% N2O (from 0.95% ± 0.09% to 0.98% ± 0.11%) (Figs. 1 and 3). However, with morphine present, the MACISO was independent of the N2O concentration in the 30%–70% range (0.99% ± 0.08%, 1.02% ± 0.09%, and 0.98% ± 0.11%, respectively) and showed a significant reduction (P < 0.001) with respect to 0% N2O in the morphine group (1.35% ± 0.08%) (Fig. 1).
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Figure 3. Decrease in isoflurane minimum alveolar concentration (MACISO) produced by 1 mg/kg of morphine in combination with 0%, 30%, 50%, and 70 % N2O in rats. Values are mean ± sd.
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During the MAC determination, there were no differences in heart rate, arterial blood pressure, pH, Paco2, and temperature among the studied groups, but a dose-dependent decrease in Pao2 among different N2O concentration groups was observed (Table 1). A decrease in arterial blood pressure and a transient decrease in respiratory rate were observed during the administration of morphine.
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Table 1. Measured Physiological Data (mean ± sd) at the Moment of the Determination of Isoflurane Minimum Alveolar Concentration After Administration of Either Saline or Morphine (1 mg/kg) in Combination with 0, 30, 50 and 70% Nitrous Oxide in Rats
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Discussion
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N2O reduces, in a dose-dependent manner, the anesthetic requirements for isoflurane. N2O 0% and 30% do not change the effect of 1 mg/kg of morphine on isoflurane MAC, and larger concentrations of N2O decrease the effectiveness of the morphine in a dose-related manner, having no effect at 70% N2O. However, with morphine present, the MACISO was independent of the N2O concentration in the 30%–70% range.
N2O is frequently combined with volatile anesthetics to reduce the concentrations of volatile anesthetics required to produce anesthesia (2–4). A dose-dependent interaction between N2O and volatile anesthetics has been observed in rats (11); however, it did not do so linearly (11,12). We observed that increasing the N2O concentration produced a dose-dependent linear decrease in the MACISO, similar to the findings of Murray et al. in children (3,4). The nonlinear potency of N2O in decreasing the requirement for volatile anesthetics observed by Cole et al. (11) was at sub-MAC concentrations of N2O. The linearity of MACISO reduction by N2O observed in our study might be because of the lack of N2O concentrations in the 0%–30% range, although interpretation of the MAC studies is the subject of debate (13–15).
Regression analysis for the MACISO values associated with the four concentrations of N2O shows a linear decrease in MACISO as a function of the concentration of N2O. Furthermore, the extrapolation of the results to the abscissa indicates a predicted MAC of N2O by extension of the linear regression line of 177%. The extrapolated value may slightly overestimate the MAC because of the effect of altitude on MAC. The average ambient barometric pressure in Madrid is approximately 704 mm Hg. Adjusting the predicted MAC to barometric pressure at sea level by using the following formula—Altitude adjusted MAC (%) = measured MAC (%) x measured ambient barometric pressure (mm Hg)/sea level barometric pressure (760 mm Hg) (16)—the predicted MAC of N2O was 163%. This prediction is supported by the results from the direct determinations of N2O MAC values by Russell and Graybeal (12), who obtained a MAC of 159% for N2O, albeit for other strains of rats.
Analgesic doses of opioids clearly reduce the MAC of inhaled anesthetics (5). We observed that 0% and 30% N2O does not change the effect of 1 mg/kg of morphine on MACISO, and larger concentrations of N2O decrease the effectiveness of the morphine in a dose-related manner, with 70% eliminating the effect (Fig. 3). This might be explained by a common action of N2O and morphine and the saturation of those sites by either drug. Morphine and N2O activate opioid receptors either directly or indirectly (6–8). N2O does not interact directly with opioid receptors (17); it increases the brain tissue concentration of opioid peptides such as ß-endorphin (18) and met-enkephalin (19), which binds to the µ and 
receptors (20). The morphine side effects have been firmly established to result from the stimulation of µ receptors (21).
However, this implies a ceiling effect of both drugs. A ceiling effect on MAC reduction has been described for most narcotics, including morphine (22), which cannot supply MAC by itself, but clearly this is not true of N2O, which can supply MAC by itself. Thus, the interpretation might be that morphine acts on some subset of sites acted on by N2O, so it is possible that morphine has less effect if N2O is already present; however, our study did not allow us to determine the mechanism of action.
The measured MAC values of an anesthetic can be altered by differences in animal physiology. During our MAC determinations, there were no differences in heart rate, arterial blood pressure, pH, Paco2, and temperature among the studied groups. In N2O groups, we observed a dose-dependent significant decrease in Pao2. MAC of halothane in dogs is unaffected by Pao2 of 38–500 mm Hg; at Pao2 38 mm Hg, MAC was still 80% of control, whereas at less than 38 mm Hg, hypoxia induces progressive narcosis (23). The dose-dependent decrease in Pao2 observed in our study never was less than 80 mm Hg.
In summary, our study demonstrates that N2O decreases MACISO in a dose-related manner, and the addition of morphine further decreases MAC in the presence of smaller concentrations of N2O, but at larger (70%) N2O concentrations, the effect of morphine vanishes.
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Footnotes
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Accepted for publication August 2, 2004.
The authors would like to acknowledge Dr. Isabel Millán for statistical assistance.
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Abstract
Introduction
