The effect of nitrous oxide on the minimum alveolar concentration (MAC) and MAC derivatives of isoflurane in dogs

Abstract

This study investigated the effects of 70% nitrous oxide (N₂O) on the minimum alveolar concentration (MAC) of isoflurane (ISO) that prevents purposeful movement, the MAC of ISO at which there is no motor movement (MAC_NM), and the MAC of ISO at which autonomic responses are blocked (MAC_BAR) in dogs.

Six adult, healthy, mixed-breed, intact male dogs were anesthetized with ISO delivered via mask. Baseline MAC, MAC_NM, and MAC_BAR of ISO were determined for each dog using a supra-maximal electrical stimulus (50 V, 50 Hz, 10 ms). Nitrous oxide (70%) was then administered and MAC and its derivatives (N₂O-MAC, N₂O-MAC_NM, and N₂O-MAC_BAR) were determined using the same methodology. The values for baseline MAC, MAC_NM, and MAC_BAR were 1.39 ± 0.14, 1.59 ± 0.10, and 1.72 ± 0.16, respectively. The addition of 70% N₂O decreased MAC, MAC_NM, and MAC_BAR by 32%, 15%, and 25%, respectively.

Introduction

The minimum alveolar concentration (MAC) of an inhalational anesthetic is defined as the alveolar concentration at sea level at which there is no purposeful movement in 50% of patients in response to a supra-maximal stimulus (1–3). Recent studies in dogs have investigated MAC derivatives, such as the MAC at which there is no motor movement (MAC_NM) (4) and the MAC at which the autonomic response to noxious stimuli is blocked (MAC_BAR) (5,6).

Nitrous oxide (N₂O) is a colorless, non-flammable gas that is used in humans for its analgesic, immobilizing, and anxiolytic effects (7–9). Compared with other inhalational anesthetics, however, N₂O is low in potency and is less potent in dogs than in humans. Reported MAC values for N₂O in dogs vary from 188% (2) to 222% (10) and N₂O is used primarily as an adjunct to volatile anesthetics for its MAC-decreasing properties. In a recent study, the authors found that 70% N₂O decreased the MAC, MAC_NM, and MAC_BAR of sevoflurane in dogs by 24%, 25%, and 35%, respectively (5).

The purpose of this study was to evaluate the effects of 70% N₂O on the MAC, MAC_NM, and MAC_BAR of isoflurane (ISO) in dogs. It was hypothesized that 70% N₂O would significantly decrease the MAC, MAC_NM, and MAC_BAR of ISO.

Materials and methods

Animals

Six adult (2 to 3 y of age), purpose-bred, mixed-breed, intact male dogs (14 ± 1 kg) were determined to be healthy based on physical examination. Food was withheld for 12 h before anesthesia, but access to water was allowed. Each dog was anesthetized once. The MAC, MAC_NM, and MAC_BAR were determined in that order for ISO alone (baseline) and then for ISO with 70% N₂O (treatment). This sequence of determination of MAC and its derivatives was used to expedite the process and is standard procedure in our laboratory.

The study protocol was approved by the Institutional Animal Care and Use Committee at the University of Tennessee and was carried out in accordance with the Guide for the Care and Use of Experimental Animals.

Anesthesia

Anesthesia was induced with ISO (IsoFlo; Abbott Animal Health, Abbott Park, Illinois, USA) in oxygen delivered via mask from a circle breathing system. After tracheal intubation, anesthesia was maintained with ISO in oxygen (2 L/min) using a small animal anesthetic machine (North American Drager, Telford, Pennsylvania, USA). Ventilation was controlled to maintain the end-tidal carbon dioxide partial pressure (Pe′CO₂) at between 35 to 45 mmHg. Arterial blood samples were drawn from each subject at the time of each MAC, MAC_NM, and MAC_BAR determination to ensure that arterial carbon dioxide tension (PaCO₂), arterial partial pressure of oxygen (PaO₂), and acid-base status were within normal limits. Dogs were placed in lateral recumbency, a 20-ga cephalic catheter (MILA International, Erlanger, Kentucky, USA) was placed, and lactated Ringer’s solution (Abbott Animal Health) was infused (3 mL/kg body weight per h).

End-tidal ISO (E′_ISO), end-tidal N₂O (E′n₂o), and Pe′CO₂ were monitored continuously with an infrared gas analyzer (Criticare Systems, Waukesha, Wisconsin, USA). Samples were drawn from the proximal end of the endotracheal tube at a rate of 150 mL/min. At the beginning of the study, the monitor was calibrated with the calibration gases supplied by the manufacturer (1% ISO in 5% CO₂ and 60% N₂O; Criticare Systems). Body temperature was monitored using an esophageal probe (Criticare Systems). A circulating warm water blanket and a warm air blanket (Bair Hugger; Arizant Healthcare, Eden Prairie, Minnesota, USA) were used to maintain body temperature within the normal range (37.5°C to 38.5°C). Arterial blood pressure was monitored continuously from a 20-ga catheter placed in a dorsal pedal artery, using a monitor (Criticare Systems) and a disposable transducer (Baxter Healthcare Corporation, Deerfield, Illinois, USA). The middle of the sternum was taken as the zero point for blood pressure measurement. Heart rate and electrocardiogram (ECG) were monitored continuously using a 3-lead system and hemoglobin saturation (SpO₂) was monitored continuously using a tongue probe (Criticare Systems).

Determination of baseline MAC

The determination of baseline MAC began approximately 45 min after induction of anesthesia and with the E′_ISO held constant at 1.5% for at least 15 min. A supra-maximal stimulus (50 V, 50 Hz, 10 ms) was delivered (Grass Instrument Company, West Warwick, Rhode Island, USA) via two 25-ga electrode needles inserted subcutaneously 5 cm apart over the mid-ulnar area. Two single stimuli with a 5-s interval were delivered initially, followed 5 s later by a continuous stimulus of 5 s duration, which was repeated after 5 s (11). Purposeful movement was defined as gross movement of the head or extremities. Twitching of the stimulated limb, coughing, swallowing, rigidity, tail movement, or chewing were not considered purposeful movements. If purposeful movement occurred, the E′_ISO was increased by 0.1% or 0.2% depending on the magnitude of the response; otherwise, it was decreased by 0.1% and the stimulus was reapplied after a 15-min equilibration period. The MAC was defined as the mean of the lowest E′_ISO at which purposeful movement did and did not occur. All MAC values were determined in duplicate and the mean value was taken as the baseline MAC for that animal. If the difference between these values was greater than 10%, a third value was determined and the mean of these 3 values was taken as the baseline MAC for that animal.

Determination of baseline MAC_NM

After MAC was determined, the E′_ISO was maintained at 1.5% for at least 15 min before the baseline MAC_NM was determined using the same methodology as for MAC. The MAC_NM was defined as the lowest E′_ISO at which there was no motor movement, purposeful or non-purposeful, in response to the noxious stimulus. Twitching of the stimulated limb was not considered a positive response.

Determination of baseline MAC_BAR

After baseline MAC_NM was determined, the E′_ISO was maintained at 1.5% for at least 15 min before initiating baseline MAC_BAR determination. During each pre-stimulus period, heart rate (HR) and mean arterial pressure (MAP) values were recorded from the arterial line and were stable for at least 5 min, varying by less than 1%. The greatest HR and MAP values during this time period were taken as the baseline. The baseline MAC_BAR was determined using the same methodology as for MAC and MAC_NM. MAC_BAR was defined as the lowest E′_ISO that prevented a ≥ 15% increase in baseline MAP and HR in response to the noxious stimulus during the 60-s period beginning at the time of the first stimulus.

Administration of N₂O

After baseline MAC, MAC_NM, and MAC_BAR were determined, administration of 70% N₂O began. After a 15-min equilibration period with the E′n₂o maintained at 70% and the E′_ISO at 1.5%, the treatment MAC endpoints (N₂O-MAC, N₂O-MAC_NM, and N₂OMAC _BAR) were determined using the same methods previously described for the baseline MAC and its derivatives.

Time recording began immediately after the initial equilibration period and time to determination of MAC, MAC_NM, and MAC_BAR was cumulative. The dogs were evaluated for tissue damage, lameness, and pain for 24 h after recovery.

Statistical analysis

Percent change in MAC, MAC_NM, and MAC_BAR was calculated according to the formula:

$$(\text{treatment\hspace{0.17em}value}-\text{baseline}\hspace{0.17em}\text{value})/(\text{baseline\hspace{0.17em}value})\times 100$$

A mixed-model analysis of variance (ANOVA) (PROC MIXED) was used to determine the effect of treatment on MAC, MAC_NM, and MAC_BAR. Dog was included as a random factor in the model. Dog, treatment, and endpoint were included as class variables. Independent variables included treatment, endpoint, time, and the 2-way interaction between endpoint and treatment. A second mixed-model ANOVA was used to compare the percent change in MAC among endpoints (MAC, MAC_NM, and MAC_BAR). Class variables included in the model were dog and endpoint. Endpoint was the independent variable and dog was included as a random factor in the model. A multiple range test according to the method of Tukey was used to adjust for multiple comparisons. Fit of the models was evaluated using the −2 log likelihood ratio and the fit of residuals from the model to a normal distribution. Residuals were evaluated using the test statistic of Shapiro-Wilk. Effect of treatment on percent change in MAC at each endpoint was evaluated using a paired t-test [PROC UNIVARIATE]. Data are expressed as least squares means (LSM) and standard error of the mean (SEM). A P-value of ≤ 0.05 was considered significant.

Results

The mean baseline values for MAC, MAC_NM, and MAC_BAR were 1.39%, 1.59%, and 1.72%, respectively (Table I). Administering 70% N₂O decreased these values by 32%, 15%, and 25%, respectively. Baseline MAC_NM was not significantly different than N₂O-MAC_NM. While the percent change in MAC_BAR was not significantly different than the percent change in MAC, the percent change in MAC_NM was significantly different than the percent change in MAC and MAC_BAR (Table I). The estimated hemoglobin saturation was > 95% and PaO₂, PaCO₂, and acid-base status were normal at all times before and during administration of N₂O. Recovery from anesthesia was uneventful and the dogs resumed normal activities within 2 to 3 h of recovery. The stimulated limbs appeared normal at all times.

Table I.

Baseline and treatment MAC, MAC_NM, and MAC_BAR values of isoflurane and percent change in each value after adding 70% N₂O in 6 male dogs

MAC endpoint	Baseline	Treatment	% Change
MAC	1.39 ± 0.14a	0.98 ± 0.14b	−31.9 ± 3.31
MACNM	1.59 ± 0.10c	1.37 ± 0.10c	−14.9 ± 3.32
MACBAR	1.72 ± 0.16d	1.31 ± 0.12e	−24.9 ± 3.31

MAC — minimum alveolar concentration; MAC_NM — minimum alveolar concentration at which there is no motor movement; MAC_BAR — minimum alveolar concentration at which autonomic response is blocked.

^a,b,c,d,eValues in the same row with different letters are significantly different.

^1,2Values in the same column with different numbers are significantly different (P ≤ 0.05). All values are presented as least squares mean ± standard error of the mean.

Discussion

In this study, administering 70% N₂O decreased MAC, MAC_NM, and MAC_BAR (Table I). The baseline MAC value of 1.39% was comparable to the values reported for dogs in previous studies: 1.38% (12), 1.28% (13), and 1.34% (11). While interindividual variation in MAC values of 10% to 20% is typical (3), variation was minimized in this study by the use of only 1 observer. The MAC can also be affected by extremes of PaCO₂, PaO₂, body temperature, and arterial blood pressure. These variables were maintained within normal range in each patient throughout the experiment.

The addition of 70% N₂O decreased the MAC by 32%, which is comparable with the MAC-sparing effects of N₂O reported in previous studies. In halothane-anesthetized dogs, 75% N₂O decreased MAC by 34% (10) and in sevoflurane-anesthetized dogs, 70% N₂O decreased MAC by 24% (5). In a clinical study of dogs undergoing ovariohysterectomy, a 37% decrease in requirement for isoflurane was reported when 64% N₂O was included in the anesthetic protocol (14). These results are also consistent with the effects of N₂O in other species. For example, 70% N₂O decreased the MAC of isoflurane in rats by 40% (15) and 75% N₂O decreased the MAC in swine by 38% (16). In desflurane-anesthetized dogs, however, 70% N₂O decreased the MAC by only 16% (17). Differences among studies are likely due to individual variation, sample size, inhalational anesthetic, and experimental design.

The MAC_NM in this study was 1.59% or 1.14 MAC (Table I). This ratio of MAC_NM/MAC is comparable to the reported ratio of 1.16 for sevoflurane MAC_NM/MAC in dogs (5). These data are also in general agreement with a study of human surgical patients, which reported that the E′_ISO that prevented movement in 95% of the population was approximately 25% greater than the MAC (18). In contrast, a comparable endpoint in halothane-anesthetized ponies was equivalent to 1.6 MAC (19), which may reflect differences among species and inhalational anesthetics. The addition of 70% N₂O decreased MAC_NM by 15% (Table I), but there was wide variability among dogs. To the authors’ knowledge, there are no published reports on the effect of N₂O on MAC_NM in dogs. In another study, the authors determined that 70% N₂O decreased sevoflurane MAC_NM in dogs by 25% (5).

In this study, baseline MAC_BAR was 1.72% or 1.24 MAC (Table I). This endpoint is typically greater than the other MAC derivatives, as autonomic responses are activated at lower stimulus levels and are more resistant to blockade than movement responses (20). Suppression of this response may be clinically relevant because autonomic activation can have deleterious effects on the patient (20–22). There is limited information on MAC_BAR in dogs and other veterinary species, and most MAC_BAR studies in humans include N₂O in the baseline anesthetic protocol, which makes it difficult to compare results. Recent studies by the authors reported MAC_BAR values of 1.27 MAC (5) and 1.4 MAC (6) for sevoflurane. Reported MAC_BAR values vary widely in other species. A study of isoflurane-anesthetized goats reported a MAC_BAR of 2.8 MAC (23), but in cats anesthetized with isoflurane, the MAC_BAR was only 1.1 MAC (24). In rats, the MAC_BAR for sevoflurane did not differ significantly from the MAC (25), and MAC_BAR values of 2.58 MAC (26) and 3.9 MAC (27) for sevoflurane have been reported in human female patients. Variations of such magnitude are likely due to the same factors as those discussed previously for MAC.

In the present study, the mean decrease in MAC_BAR with the addition of 70% N₂O was 25%. To the authors’ knowledge, there are no published reports on the effect of 70% N₂O on the MAC_BAR of isoflurane in dogs. In a previous study, however, the authors found that 70% N₂O decreased the MAC_BAR of isoflurane by approximately 35% in dogs (5).

Decreases in MAC and its derivatives with N₂O could be due to its analgesic and/or immobilizing effects. Although the mechanisms of action of N₂O are not completely understood, its analgesic actions are likely separate from its immobilizing effects (9,28). Immobility during general anesthesia is mediated by motor neurons located in the ventral horn of the spinal cord (29,30). Interestingly, it has been shown that neurons in the ventral horn are more sensitive to the depressant effects of N₂O than are neurons in the dorsal horn (31). The immobilizing effects of N₂O may be due to N-methyl-D-aspartate (NMDA) receptor blockade in the ventral horn (32,33), although mechanisms involving monoaminergic pathways have also been suggested (34).

Numerous mechanisms have been proposed to explain the analgesic actions of N₂O. Although still controversial, prevailing evidence supports the involvement of opioid and alpha-2 adrenergic receptors (28,35,36). Specifically, nitrous oxide induces analgesia by activating opioidergic neurons in the periaqueductal gray matter and noradrenergic neurons in the locus coeruleus. This results in modulation of nociceptive transmission at the level of the spinal cord (7,37–39).

In this study, the decrease in MAC and its variants with the addition of 70% N₂O ranged from 15% to 32%. It therefore appears that N₂O provides a clinically important reduction in MAC. The difference in the magnitude of the effect of N₂O on MAC and MAC_NM is surprising because they are both presumably mediated at the level of the spinal cord. This difference may be due to the small sample size and variability among subjects.

In this study, the determination of MAC and its derivatives was not randomized. Determining MAC provides a starting point for the determination of MAC_NM or MAC_BAR, as previously published studies and experience have indicated that MAC_NM and MAC_BAR are usually higher than MAC (5,23,26,27). Determining MAC and its derivatives in this order expedites the process. The current study and a previous study (5) from our laboratory demonstrated that the time to determine MAC and its derivatives has no significant effect on outcome, although this does not completely rule out an effect of order of determination.

The benefits of administering N₂O must be weighed against its potential adverse effects on patients, personnel, and the environment. The patient’s oxygenation must be monitored continuously throughout the perioperative period, as hypoxemia is more likely when using N₂O. Long-term exposure to N₂O can have adverse effects on personnel, including bone marrow suppression, spontaneous abortion, teratogenicity, genotoxicity, and myelinopathies (39–41). In addition, N₂O contributes to ozone depletion in the environment (40,41).

In conclusion, adding 70% N₂O significantly decreased the MAC, MAC_NM, and MAC_BAR of isoflurane (ISO) in dogs by 32%, 15%, and 25%, respectively.