There is an increasing concern about potential complications involving anesthetic interventions in morbidly obese patients.1 An effect of increased body mass index (BMI) on anesthetic pharmacokinetics has been shown.2 Increased adiposity complicates the assessment of anesthetic requirements and creates the possibility of subtherapeutic anesthesia, which is considered to be a primary cause of intraoperative awareness with explicit recall.3,4 Intraoperative awareness remains a feared complication of surgery and is associated with a high incidence of posttraumatic stress disorder.5 A number of risk factors have been suggested for this complication,3 including obesity.6 A retrospective study of intraoperative awareness questioned the role of obesity as a risk factor,3 but there are no data regarding anesthetic requirements as measured by minimum alveolar concentration (MAC) in this population.
Obesity is associated with comorbidities such as diabetes and cardiovascular disease, which together are features of human metabolic syndrome.7,8 A rodent model of metabolic syndrome has been generated that is characterized primarily by low aerobic oxidative capacity,9–11 obesity, and risks for complex diseases.12 In brief, rats from a founder population of genetically heterogeneous rats (N:NIH stock) were artificially selected to diverge into 2 distinct lines: inherent high aerobic capacity runners (HCRs) and inherent low aerobic capacity runners (LCRs).9 As compared to HCR rats, the LCR rats show increased levels of visceral fat, plasma triglycerides, plasma free fatty acids, and fasting insulin levels, as well as higher blood pressure and impaired glucose tolerance (Table 1).12 This is consistent with the finding that aerobic exercise capacity shows a strong negative correlation with the occurrence of morbidities including obesity, diabetes, and cardiovascular disease.13
We used this contrasting animal model system to test the hypothesis that the MAC of isoflurane and sevoflurane varies significantly as a function of maximal oxidative capacity and obesity. To address the potential influence of sex on drug effects,14,15 both males and females were studied.
Model of Metabolic Syndrome
All experimental procedures were approved by the University of Michigan Committee for the Care and Use of Animals and were in accordance with the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals and the Guide for the Care and Use of Laboratory Animals. We studied 5 male and 5 female rats of both HCR and LCR type (total n = 20) from the 26th generation, with ages between 10 to 12 months. The rats were maintained on a 12-hour light: 12-hour dark cycle (lights on at 6:00 AM), and ad libitum food and water were provided.
The development of these rat models using artificial selection and the maintenance of the rat colonies has been described in detail elsewhere.9 Briefly, the rats were tested for endurance running capacity on a treadmill. The treadmill was operated at a constant slope of 15 degrees with a speed of 10 m/min. The rat was run to exhaustion while the speed was being increased at the rate of 1 m/min every 2 minutes. The failure to keep pace with the treadmill resulted in the rats receiving a mild shock (1.2 mA, 3 Hz) from a metal grid placed at the base of the treadmill. The failure to keep pace with the treadmill on 3 consecutive runs was defined as exhaustion. The test was repeated on 5 consecutive days.
The details of the individual body weights, best distance run, and the best time over a set of 5 trials for the animals in this study are provided in Table 2. It should be noted that the capacity of HCRs to run a longer distance in less time is an inherent trait and is not a result of training exercise. Similarly, the LCR rats are inherently characterized by obesity and run significantly shorter distances than do HCRs. The mean body weight of male LCR rats (606.4 g ± 37.38; 95% CI: 560.0 to 652.8) was significantly higher than the mean body weight of the male HCR rats (371.0 g ± 21.30; 95% CI: 344.6 to 397.4) (P = 0.004). Similarly, the mean body weight of female LCR rats (302.6 g ± 8.14; 95% CI: 292.5 to 312.7) was significantly higher than the mean body weight of the female HCR rats (231.0 g ± 7.38; 95% CI: 221.8 to 240.2) (P = 0.004). The mean body weight of the male LCR and HCR rats was also significantly higher than the mean body weight of the female LCR (P = 0.004) and female HCR rats (P = 0.004), respectively.
Determination of MAC
All rats were used for isoflurane and sevoflurane experiments with an interval of at least 3 to 4 weeks between anesthetic exposures. The requirements for both anesthetics were measured as MAC using a bracketing design.16 MAC was chosen as the primary dependent variable of interest because other measures such as time to induction could be subject to pharmacokinetic influence via higher BMI or cardiovascular differences, as opposed to the steady state design of MAC at equilibrium.16 A week before the experiment, each rat was habituated to a clear-walled anesthesia induction chamber (10.0 inches × 4.7 inches × 4.0 inches) on 3 separate days for 15 minutes each day. The habituation and the experimental procedures were conducted between 8:30 AM and 12:00 PM to control for circadian influences. Anesthetic induction was achieved with isoflurane (2.5%) and sevoflurane (4.5%) delivered in 100% oxygen at a flow rate of 0.5 L/min. After the loss of righting reflex as a surrogate for loss of consciousness, the animal was positioned on an inclined polycarbonate platform in dorsal recumbency for endotracheal intubation (Small Animal Intubation Kit, Kent Scientific, Torrington, CT). We used a 16G catheter (Exel Safelet Cath, Exelint International Co., Los Angeles, CA) modified to have a thick sleeve/cuff on the upper one third of the tubing for a sealed connection with the trachea. Thereafter, the rat was connected to a nonrebreathing gas circuit and the lungs artificially ventilated under constant pressure mode (TOPO Dual Mode Ventilator, Kent Scientific, Torrington, CT). Inspiratory pressure was maintained between 10 and 15 cm H2O, and respiration rate was adjusted to maintain end-tidal CO2 between 30 to 45 mm Hg. The end-tidal CO2 and the anesthetic concentration were monitored using an inline Datex Capnomac Ultima analyzer (Datex Medical Instrumentation, Inc., Tewksbury, MA). Rectal temperature was monitored and maintained at 37° ± 1° Celsius using a heat lamp and water-based heating system (TP-500 Heat Therapy Pump, Gaymar Industries Inc., Orchard Park, NY). The heart rate and oxygen saturation were continuously monitored using a MouseOx monitor (Starr Life Sciences, Corp., Oakmont, PA). The animal was equilibrated to either isoflurane (1.7%) or sevoflurane (2.7%) for 30 minutes, after which a noxious mechanical stimulus was applied until a gross purposeful movement (positive response) occurred within 60 seconds. Purposeful movement was defined as what appeared to be voluntary movement of the limbs or movement related to change of body posture (positive response), as opposed to the movement of the whiskers, or slight muscular twitching (negative response). The stimulus consisted of a clamp to the upper third of the tail using a 10-inch hemostat (Roboz RS-7182), with the point of contact at the tip of the hemostat and with the hemostat closed to the first ratchet. During the application of the stimulus, the hemostat was oscillated at 45 degrees at 1 Hz. If there was no response after the application of stimulus for 60 seconds, the anesthesia was decreased by 10%. If a positive response occurred within 60 seconds, then the anesthesia was increased by 10%. The increment or decrement of anesthetic was followed by equilibration for 30 minutes, after which another identical stimulus was applied. Each subsequent stimulus was provided at a site proximal to the first test site. The determination of the positive or negative response was based on the concurrence of 2 experimenters. The MAC was calculated to be an average of (i) the maximum anesthetic concentration that evoked a positive response to the noxious stimulus and (ii) the minimum anesthetic concentration that prevented a positive response to the noxious stimulus. A pilot study to validate the MAC procedure was performed in male Sprague–Dawley rats. The pilot data on the MAC for isoflurane (1.47% ± 0.24% − average of 2) and sevoflurane (2.1%) were consistent with the MAC reported in the literature for isoflurane (1.51% ± 0.12%)17 and sevoflurane (2.4% ± 0.30%)18 (Table 3).
The statistical tests were performed using the software package Graph Pad Prism 5.01 (La Jolla, CA). The MAC data for isoflurane and sevoflurane from the combined pool of LCR and HCR rats were confirmed to be normally distributed (D'Agostino and Pearson test) and were compared using a 2-tailed unpaired t test. A nonparametric test (2-tailed Mann–Whitney) was used to compare the MAC for male and female groups within and between each phenotype. A 1-tailed Mann–Whitney test was used to compare the body weight of the male and female rats within and between each phenotype. The data are reported as mean ± SD (standard deviation) along with the 95% confidence interval (CI). A P value of <0.05 was considered statistically significant.
The MAC of isoflurane and sevoflurane for individual LCR and HCR rats of either sex are shown in Table 2. The mean isoflurane-MAC for the HCR rats (1.90% ± 0.19%; 95% CI: 1.76 to 2.03) was significantly higher than the mean isoflurane-MAC for LCR rats (1.52% ± 0.13%; 95% CI: 1.42 to 1.62) (P = 0.0001, t = 5.019, df = 18) (Table 3; Fig. 1A). Further analysis comparing the MAC between male and female rats of LCR and HCR type showed that the mean isoflurane-MAC for the male HCR rats (1.92% ± 0.1%; 95% CI: 1.78 to 2.05) was significantly higher than the mean isoflurane-MAC for male LCR rats (1.48% ± 0.1%; 95% CI: 1.34 to 1.61) (P = 0.01) and female LCR rats (1.56% ± 0.16%; 95% CI: 1.35 to 1.76) (P = 0.01) (Fig. 1B). Furthermore, the mean isoflurane-MAC for the female HCR rats (1.88% ± 0.26%; 95% CI: 1.54 to 2.21) was also significantly higher than the mean isoflurane-MAC for male LCR rats (1.48% ± 0.1%; 95% CI: 1.34 to 1.61) (P = 0.02) (Fig. 1B). The isoflurane-MAC was not significantly different between the male and female rats within each phenotypic group (Fig. 1B).
In contrast to isoflurane, the MAC for sevoflurane was not significantly different between the HCR (2.76% ± 0.27%; 95% CI: 2.56 to 2.96) and LCR rats (2.53% ± 0.38%; 95% CI: 2.25 to 2.80) (P = 0.13, t = 1.57, df = 18) (Table 3; Fig. 2A). The comparison of sevoflurane-MAC for male and female rats of both LCR and HCR type showed that none of the groups differed significantly from each other (P = 0.13) (Fig. 2B).
The results show for the first time that the mean isoflurane-MAC (1.52% ± 0.13%) in LCR rats, which display obesity and symptoms related to human metabolic syndrome, is similar to the published MAC for isoflurane in normal rats (1.51% ± 0.12%).17 Isoflurane has been shown to have a long time constant for equilibration with fat, which exceeds the typical duration of anesthetic delivery.19 The blood perfusion of fat tissue decreases with increasing adiposity.20 Therefore, considered together, the data suggest that the effect of increased adiposity on isoflurane requirements would be minimal. Interestingly, the mean isoflurane-MAC of HCR rats (1.90% ± 0.19%) was significantly higher than the isoflurane-MAC for LCR rats (1.52% ± 0.13%) and was elevated to a similar extent (about 20% increase) in comparison with the isoflurane-MAC published for normal rats (1.51% ± 0.12%)17 (Fig. 1 and Table 3). The mean sevoflurane-MAC was not significantly different between male and female LCR and HCR rats. A further validation of the lack of a significant difference in the mean sevoflurane-MAC between LCR and HCR rats is provided by a recent study in human subjects, which showed that the presence of obesity does not prolong the induction of or emergence from sevoflurane anesthesia.21
It is possible that the difference in the isoflurane-MAC between LCR and HCR rats is a result of differential tolerance to the mechanical noxious stimulus used in this study. However, a previous study found no significant difference in the mechanosensory responses between LCR and HCR rats as quantitatively assessed through von Frey testing.22 The present results encourage future MAC studies using additional sensory modalities.
Naturally occurring genotypic differences affect MAC.23 Furthermore, genetic manipulations affecting ion channels have been shown to alter anesthetic sensitivity.24,25 Recently, Chae et al.26 reported a differential effect of inactivation of the TRESK (2-pore potassium channel) gene on anesthetic sensitivity. The knockout mice lacking TRESK showed decreased sensitivity and hence increased MAC for isoflurane.26 However, TRESK inactivation did not affect the sensitivity to sevoflurane, halothane, and desflurane.26 Collectively, the present results along with the published literature support a genetic basis for differences in anesthetic sensitivity that is agent specific. Future studies involving genetic manipulations of ion channels in LCR and HCR rats may provide further support of an underlying genetic basis for the differential anesthetic sensitivity between LCR and HCR rats. Neither LCR nor HCR rats showed any sex-specific differences in the MAC of either isoflurane or sevoflurane. This is in agreement with a previous report that the genotype rather than sex of the subject is the major determinant of anesthetic sensitivity.23
The current study has a number of limitations. First, caution is warranted when applying interpretations of animal data to clinical care in humans. Second, we did not test all clinically relevant anesthetics; future studies might include drugs such as desflurane. Third, a larger number of animals in the sevoflurane group might have resulted in a significant difference in the MAC of HCR and LCR rats. However, even assuming such significance, the effect size and thus the clinical relevance would likely be minimal. This is also true for conclusions regarding anesthetic sensitivity across males and females.
In conclusion, obesity and associated comorbidities did not affect inhaled anesthetic requirements of isoflurane and sevoflurane, as measured by MAC. These novel results support human observational data suggesting that obesity is not a risk factor for intraoperative awareness.3 Additionally, these data support the view that future anesthesia-related studies of metabolic syndrome in rats will not be confounded by extreme alterations in MAC. HCR rats displayed increased anesthetic requirements for isoflurane; both anesthetic requirements and aerobic fitness are complex, polygenic traits. The differential effects of HCR on isoflurane and sevoflurane support a genetic and drug-specific basis for anesthetic sensitivity. The present finding of strain-specific effects is consistent with studies documenting significant differences between HCR and LCR rats in the response to acute27 and chronic22 nociceptive input. Considered together, these data support the interpretation that this rodent model of metabolic syndrome12 provides a unique resource for phenotyping the mechanisms by which obesity and metabolic syndrome impact anesthesia.
Name: Dinesh Pal, PhD.
Contribution: This author helped design the study, analyze the data, and write the manuscript.
Attestation: Dinesh Pal has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Meredith E. Walton, BA.
Contribution: This author helped conduct the study.
Attestation: Meredith E. Walton has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: William J. Lipinski, MS.
Contribution: This author helped conduct the study.
Attestation: William J. Lipinski has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Lauren G. Koch, PhD.
Contribution: This author helped develop the rodent model.
Attestation: Lauren G. Koch has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Ralph Lydic, PhD.
Contribution: This author helped the intellectual and technical development of the project.
Attestation: Ralph Lydic has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Steve L. Britton, PhD.
Contribution: This author helped develop the rodent model.
Attestation: Steve L. Britton has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: George A. Mashour, MD, PhD.
Contribution: This author helped design the study, analyze the data, and write the manuscript.
Attestation: George A. Mashour has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
This manuscript was handled by: Marcel E. Durieux, MD, PhD.
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