Anesth Analg 2003;96:1674-1678
© 2003 International Anesthesia Research Society
ANESTHETIC PHARMACOLOGY
The Effects of Enflurane, Isoflurane, and Intravenous Anesthetics on Rat Diaphragmatic Function and Fatigability
Kahoru Nishina, MD,
Katsuya Mikawa, MD,
Shun-ichi Kodama, MD,
Tetsuro Kagawa, MD,
Takanobu Uesugi, MD, and
Hidefumi Obara, MD
Department of Anaesthesia & Perioperative Medicine, Kobe University Graduate School of Medicine, Kobe, Japan
Address correspondence and reprint requests to Dr. Katsuya Mikawa, Department of Anaesthesia & Perioperative Medicine, Faculty of Medical Sciences, Kobe University Graduate School of Medicine, Kusunoki-cho 7, Chuo-ku, Kobe 650-0017, Japan. Address e-mail to katz{at}post.med.kobe-u.ac.jp
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Abstract
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We examined the effect of isoflurane, enflurane, midazolam, ketamine, propofol, and thiopental on diaphragmatic functions under unfatigued and fatigued conditions in 228 rat isolated muscle strips. Diaphragmatic twitch characteristics and tetanic contractions were measured before and after muscle fatigue, which was induced by repetitive tetanic contraction with or without exposure to one of the anesthetics at clinically relevant plasma concentrations, and at 10 and 100 times this concentration, or at 1, 2, and 3 minimum alveolar anesthetic concentration (MAC). Isoflurane, midazolam, ketamine, propofol, and thiopental did not induce a direct inotropic or lusitropic effect under unfatigued and fatigued conditions. Enflurane did not change contraction or relaxation in fresh isolated diaphragm, but enflurane at 2–3 MAC enhanced diaphragmatic fatigability itself and fatigue-induced impairment of twitch characteristics and tetanic tensions. These effects were greater at 3 MAC than at 2 MAC. Our findings suggest that the reduction of diaphragm function previously reported in in vivo experiments using propofol, midazolam, and isoflurane is not related to a direct effect on intrinsic diaphragmatic contractility. Our results also indicate that large concentrations of enflurane may impair the diaphragmatic function at sites other than excitation-contraction coupling.
IMPLICATIONS: Enflurane did not change contraction or relaxation in fresh isolated rat diaphragm, but enhanced diaphragmatic fatigability itself and fatigue-induced impairment of twitch characteristics and tetanic tensions. Isoflurane, midazolam, ketamine, propofol, and thiopental had no direct effects on diaphragmatic functions under unfatigued and fatigued conditions. Isoflurane and these IV anesthetics may be advantageous over enflurane to anesthetize and/or sedate patients who are predisposed to diaphragmatic fatigue.
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Introduction
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In several animal in vivo studies, volatile (halothane, enflurane, isoflurane, and sevoflurane) and IV (propofol and midazolam) anesthetics cause diaphragmatic contractile dysfunction which probably contributes acute respiratory failure (1–6). The plausible mechanisms underlying anesthetic-induced diaphragmatic dysfunction include reduction of blood flow, failure of neuromuscular transmission, and impairment of membrane excitation and excitation-contraction (E-C) coupling. However, a whole body experiment impedes specification of these responsibilities for diaphragmatic dysfunction. Direct electrical stimulation of diaphragm strips enables us to assess the effect of anesthetics on the diaphragm itself (E-C coupling). Using this in vitro preparation, we previously documented that sevoflurane and halothane augment fatigue-induced impairment of contractile and relaxant properties (7). However, the effects of enflurane and isoflurane were not examined. Furthermore, no information is available about whether the IV anesthetics directly alter diaphragmatic functions. In the current in vitro study, therefore, we assessed and compared the direct effects of isoflurane, enflurane, midazolam, ketamine, propofol, and thiopental on twitch characteristics and tetanic tensions in unfatigued and fatigued diaphragm and on fatigability itself.
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Methods
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The current study was approved by the animal care review board of Kobe University Graduate School of Medicine. Diaphragm strips (5-mm wide) were isolated from 228 male Sprague-Dawley rats (200–250 g) under general anesthesia with sevoflurane, and mounted in an organ bath containing 20 mL of oxygenated Krebs-Henseleit solution (pH 7.40, 22°C) with pancuronium. Strips in the bath were stimulated with supramaximal currents (0.2-ms duration) delivered by an electrical stimulator (DPS-1100D; Dia Medical System, Tokyo, Japan). The muscle isometric tension was measured by using a force transducer (T7-15; NEC San-ei, Tokyo, Japan) and an AC strain amplifier (AS1202; NEC San-ei).
The strips were randomly allocated into seven groups: nonanesthetic control, enflurane, isoflurane, midazolam, ketamine, propofol, and thiopental. Each anesthetic group was subdivided into three groups according to their concentrations as listed in the Tables. The 3 concentrations of each anesthetic corresponded to 1 minimum alveolar anesthetic concentration (MAC), 2 MAC, and 3 MAC for the volatile anesthetics and clinical plasma concentrations, and 10, and 100 times these concentrations for the IV anesthetics. For the volatile anesthetics groups, enflurane or isoflurane was delivered with 95% O2/5% CO2 mixed gas through agent-specific vaporizers (Datex-Ohmeda, Helsinki, Finland). The concentration of the volatile anesthetics in the gas phase was adjusted with a calibrated gas analyzer (Capnox CX-2Sp; Colin, Aichi, Japan). To apply the desired concentrations of enflurane or isoflurane in the bath, the Krebs solution in the bath was gently replaced with Krebs solution that had been equilibrated with the concentration of the anesthetic to be studied. After replacement of the solution, the volatile anesthetic was supplied to maintain the concentration at the same level until the end of the experiment. Preliminary studies revealed that dissolved volatile anesthetic concentrations in the organ bath after solution replacement showed a linear correlation to the bubbled concentration of enflurane (1%–5%) and isoflurane (0.5%–4%) in our preparation, which were assayed by using gas chromatography (8) (GC-8A; Shimadzu, Kyoto, Japan). For nonanesthetic control and IV anesthetics groups, Krebs solution was not replaced and bubbled with 95% O2/5% CO2 mixture alone.
Resting tension of the strips was 5 g and the strips were measured with a micrometer. We initially determined the twitch contractile equivalence among the groups before the application of anesthetics at the end of the 15-min equilibration period. After a 15-min incubation in Krebs solution containing each concentration of the volatile or IV anesthetics, muscle twitch characteristics (maximal rate of muscle tension development [dp/dtmax], half-relaxation time [HRT; the time required for the peak tension to decrease by 50%], and peak tension during a single twitch contraction) were assessed in the unfatigued state. The strips were sequentially stimulated at frequencies of 1, 10, 20, 50, and 100 Hz (800 ms at 5-s intervals) to obtain the force-frequency relationships. Thirty seconds after the force-frequency relationship was determined, muscle fatigue was induced by rhythmic repetitive contractions produced by trains of 20 Hz stimuli (500-ms train duration, 0.50 duty cycle, 60 trains/min) over a period of 5 min. Muscle fatigability was assessed by reduction of tension generated over this period and the time until tension decreased to 50% of the initial value (T50%). Five seconds after the end of the fatigue trial, twitch characteristics and the force-frequency relationship were again measured in the fatigued state. The muscle strip was then weighed.
Force generation was normalized as force per unit cross-sectional area (kg/cm2) as previously described (7). Data (mean ± SD) of dp/dtmax, HRT, and T50% were statistically analyzed using one-way analysis of variance (ANOVA) followed by Tukey-Kramer post hoc test. The whole data of fatigability force-time curves were statistically analyzed using repeated-measures ANOVA to compare the dose/MAC effects of the different treatments, and the changes in effects with time, and to assess an interaction of the effects. In the enflurane treatment groups, there were significant differences within and between groups. However, because an interaction between the two factors (time and MAC) was observed, multiple comparison tests could not be performed. Because our interest was particularly in the MAC effect of enflurane, data of 5 measuring points (1, 2, 3, 4, and 5 min) in 4 doses (0, 1, 2, and 3 MAC) of enflurane were analyzed using one-way ANOVA followed by the Tukey-Kramer post hoc test. Likewise, the whole data of the force-frequency relationship were analyzed using repeated-measures ANOVA and one-way ANOVA followed by the Tukey-Kramer post hoc test. P < 0.05 was deemed statistically significant.
The sample size of the current study (n = 12 each) is sufficient to detect large differences (effect size = 1.2) with a 0.05 two-sided significance level and a power level of 0.8 (9). However, a possibility of a type II error remains because the sample size (n = 12) is not large enough to detect a moderate effect size ( 1.0) with high power.
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Results
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Mean (±SD) muscle strip length (7.6 ± 0.8 mm) and weights (35 ± 8 mg), and basal contractile properties were comparable among 19 subgroups. Compared with the nonanesthetic control, the two volatile anesthetics and the four IV anesthetics did not change dp/dtmax, HRT, twitch tensions, or force-frequency relationships before the fatigue trial (Table 1). Rhythmic repetitive contraction produced rapid diaphragmatic fatigue in the control group. Enflurane at 2 and 3 MAC further shortened T50% and decreased tensions during the fatigue trial although enflurane at 1 MAC did not change these variables of fatigability (Table 2). These effects were the most at 3 MAC. Isoflurane or the four IV anesthetics had no effect on T50% and tension generated during the fatigue test. In the control group, the fatigue trial reduced dp/dtmax, twitch contraction, and tetanic tensions, and prolonged HRT (Table 3). Enflurane at 2 and 3 MAC augmented fatigue-induced impairment of the twitch characteristics and tetanic tensions (Table 3). Enhancement was the greatest at 3 MAC. However, enflurane at 1 MAC, isoflurane at any MAC, or the 4 IV anesthetics had no effects on these variables after the fatigue trial (Table 3).
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Table 1. Effects of Anesthetics on Diaphragmatic Twitch Characteristics and Tetanic Tensions Before the Fatigue Trial
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Table 3. Effects of Anesthetics on Diaphragmatic Twitch Characteristics and Tetanic Tensions After the Fatigue Trial
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Discussion
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Midazolam and propofol reduce contractility of unfatigued and fatigued diaphragm as assessed by transdiaphragmatic pressures (Pdi) using a canine whole body preparation (1–4). Contrary to these reports, in the current study, propofol and midazolam had no effects on twitch characteristics or tetanic tensions of rat unfatigued and fatigued diaphragm strips. We assessed the responses to direct electrical stimulation which reflects E-C coupling of the muscle. Thus, our findings indicate that midazolam or propofol may reduce Pdi through mechanisms other than E-C coupling failure. There are few reports concerning whether thiopental and ketamine alter diaphragmatic function. We have also shown that thiopental and ketamine had no effect on diaphragmatic contractility under unfatigued and fatigued conditions. Furthermore, the four IV anesthetics in the current study did not alter diaphragmatic fatigability itself. Enflurane depresses contractility of unfatigued diaphragm in a MAC-dependent manner (1–2 MAC) in a canine in vivo preparation (5). It is speculated that this depression may occur mainly through failure of neuromuscular transmission and partly through impairment of the E-C coupling (5). Inconsistent with this previous study, we have shown that enflurane (1–3 MAC) did not change fresh diaphragmatic functions in an in vitro preparation. Thus, enflurane also seems to impair diaphragmatic contractility at sites other than the E-C coupling. In a canine whole body experiment, isoflurane (1–2 MAC) depressed contractile response to tetanic stimulation, probably at the prejunctional site, although it did not change the twitch characteristics of unfatigued diaphragm (6). However, isoflurane (1–1.5 MAC) has diverse effects on diaphragmatic contractility in the rat in vivo system: Pdi increases and decreases at 20- and 100-Hz stimulation, respectively (10). These conflicting results may be explained by differences in species and the concomitant use of pentobarbital. Enflurane and isoflurane at 1–3 MAC have no effect on twitch contractile response to direct stimulation in isolated lumbrical muscle (11). The effects of volatile anesthetics have been reported to be more pronounced in the diaphragm (12). Limited data are available regarding the effects of enflurane and isoflurane on fatigue-induced diaphragmatic dysfunction. In the current study, although isoflurane did not influence the fatigue-induced impairment of twitch characteristics and tetanic tensions, enflurane at 2 or 3 MAC enhanced the diaphragmatic fatigability itself and fatigue-induced diaphragmatic dysfunction. In the current study, using an in vitro system, we assessed only intrinsic diaphragmatic contractility. Many factors in the in vivo experimental system may explain the discrepancies in observations between the previous reports and ours, including arterial blood flow of the diaphragm, central nervous system respiratory drive, diaphragmatic shape, and neuromuscular transmission (13). Furthermore, in the in vivo system, change in plasma catecholamine levels probably influences diaphragmatic contractility under fatigued condition through intracellular cyclic adenosine monophosphate.
The various mechanisms for diaphragmatic fatigue include sodium-potassium derangements which cause a decrease in velocity of propagation of muscle action, impairment of Ca2+ uptake and release, and increased oxygen free-radical formation related to cellular energetics (14). We are unable to give a satisfactory explanation for the additive effect of enflurane on fatigue-induced diaphragmatic dysfunction. Metabolic changes in fatigued muscle fibers are associated with decreased Ca2+ uptake in the sarcoplasmic reticulum, contributing to slowed relaxation in the fatigued muscle (15). Because enflurane simultaneously increases both Ca2+ uptake and release, the anesthetic does not seem to change [Ca2+]i in the myoplasm (16,17). Direct measurement of the [Ca2+]i remains to be done.
Diaphragmatic fatigue is likely to occur in various clinical conditions, including sepsis, increased airway resistance (e.g., airway obstruction), decreased lung compliance (e.g., pulmonary emphysema), and nonstandard surgical positions. Use of the four IV anesthetics and isoflurane would not be a disadvantage in patients who are predisposed to diaphragmatic fatigue. Aggravation of fatigue-induced diaphragmatic dysfunction by enflurane may be unfavorable in these patients during spontaneous breathing, or during recovery from enflurane anesthesia. However, the use of enflurane at 2–3 MAC during spontaneous ventilation is not clinically relevant.
In conclusion, enflurane at 2–3 MAC enhanced diaphragmatic fatigability itself and fatigue-induced impairment of twitch characteristics and tetanic tensions, indicating that large concentrations of enflurane may exert action at sites other than E-C coupling. Isoflurane, midazolam, and propofol had no direct effects on diaphragmatic function under unfatigued and fatigued conditions, suggesting that diaphragmatic dysfunction previously demonstrated in in vivo experiments using these anesthetics is not related to the direct mechanism. Although the respiratory effects of enflurane may be of clinical importance, we are unable to simply extrapolate our in vitro findings to the clinical settings.
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Acknowledgments
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This work was supported solely by department funds.
The authors thank Professor N. Maekawa (Department of Anesthesiology and Emergency Medicine, Kagawa Medical University) for his instruction of statistical analysis.
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Accepted for publication January 24, 2003.
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Abstract
Introduction
