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Anesth Analg 2006;102:1703-1708 © 2006 International Anesthesia Research Society doi: 10.1213/01.ane.0000216001.36932.a3
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Abstract |
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Halothane and isoflurane potently depress airway ciliary motility. We compared the effect of sevoflurane on ciliary beat frequency (CBF) with that of halothane and isoflurane using purified and cultured rat tracheal epithelial cells. Rat tracheal epithelial cells were isolated from adult male Sprague-Dawley rats to establish an air-liquid interface culture. Apical surfaces of the cells were exposed to a fresh gas containing humidified and warmed (25°C) air (vehicle) with or without sevoflurane (0%–4%), halothane (0%–2%), or isoflurane (0%–2%). The images of motile cilia were videotaped and CBF was analyzed using a computer. Baseline CBF (= 100%) and CBF 30 min after the exposure were measured. CBF 30 min after vehicle exposure was 101% ± 4% (mean ± sd). Exposures to 0.25%–2% sevoflurane did not change CBF significantly, although exposures to 0.25%–2% halothane or isoflurane decreased CBF dose-dependently. CBFs 30 min after exposures to 2% of sevoflurane, halothane, and isoflurane were 97% ± 9%, 56% ± 14%, and 47% ± 6%, respectively (n = 5 each). Sevoflurane 4% reduced CBF significantly but slightly (84% ± 2%, n = 5). These results show that sevoflurane has a direct cilioinhibitory action but its action is much weaker than that of halothane and isoflurane in isolated rat tracheal epithelial cells.
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Introduction |
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Airway ciliary motility plays an important role in the host defense mechanism that clears foreign particles, such as dust, debris, and bacteria, from the respiratory tract (1–3). Patients with impaired ciliary function, such as those with Kartagener syndrome, chronic obstructive pulmonary diseases, and airway injury, including smoking, are predisposed to perioperative pulmonary complications such as arterial hypoxemia, atelectasis, and pulmonary infection. The volatile anesthetics, halothane, enflurane, and isoflurane, inhibit mucociliary clearance in experimental animals and humans in vivo and depress ciliary beat frequency (CBF) in explanted airway tissues in vitro (2–13). It has been assumed that volatile anesthetics may contribute to perioperative pulmonary complications, especially in prolonged exposure and/or pulmonary compromised patients, although this has not been confirmed (2,3). An anesthetic without a CBF-reducing effect may be of benefit to surgical patients.
The lower solubility of sevoflurane compared with halothane, enflurane, and isoflurane in blood and other tissues provides for rapid induction of anesthesia, prompter changes in anesthetic depth after changes in delivered concentration, and faster emergence after discontinuation of drug administration (14,15). Because sevoflurane is not irritating to the airway, it is also a useful anesthetic for inhaled induction. The effect of sevoflurane on CBF has not been reported.
Because previous studies of the effect of volatile anesthetics on ciliary function used whole animals or airway tissue explant preparations that included various types of cells and nerve endings other than airway epithelial cells (4–13), it is possible that the results were affected by interactions between epithelial and nonepithelial cells or nerve terminals, and the direct effects of volatile anesthetics on CBF were not fully understood. To purify airway epithelial cells and differentiate them from a ciliated phenotype, air-liquid interface (ALI) cultures have been used (16,17). The purpose of our study was to investigate the direct action of sevoflurane on CBF without the influence of nonepithelial cells and nerve endings and to compare it with that of halothane and isoflurane using isolated and cultured rat tracheal epithelial (RTE) cells.
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Methods |
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Cultured RTE cells were prepared according to the methods previously described by Kaartinen et al. (16) and Davenport and Nettesheim (17) with some modifications. The study was approved by the animal research committee of Kyoto University. Tracheas were removed under sterile conditions from male Sprague-Dawley rats (body weight, 300–350 g; Japan SLC; Hamamatsu, Japan) killed by CO2 asphyxiation. The tracheas were incubated overnight at 4°C in Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL, Rockville, MD) and Ham's nutrient F-12 medium (F-12; Gibco-BRL) (1:1) with 0.05% type XIV protease (Sigma, St. Louis, MO). Fetal bovine serum (FBS; Gibco-BRL; final concentration 10%) was then added to the incubation medium (DMEM/F-12) and the RTE cells were flushed out. The cells were collected by centrifugation (150g, 4°C, 1 min) and washed twice with DMEM/F-12 containing 10% FBS.
Growth medium for RTE cells consisted of DMEM/F-12 supplemented with 10 µg/mL insulin, 0.1 µg/mL hydrocortisone, 0.1 µg/mL cholera toxin, 5 µg/mL transferrin, 50 µg/mL phosphoethanolamine, 80 µg/mL ethanolamine, 25 ng/mL epidermal growth factor, 25 µg/mL bovine pituitary extract, 3 mg/mL bovine serum albumin, 50 nM retinoic acid, 100 U/mL penicillin, and 100 U/mL streptomycin. All of the supplemental reagents were obtained from Gibco-BRL except hydrocortisone, cholera toxin, phosphoethanolamine, and retinoic acid (Sigma), and epidermal growth factor (Peprotech, Rocky Hill, NJ). Polyester permeable membranes on culture inserts (12-mm-diameter, 0.4-µm-pore-size; No. 3460; Corning-Coster, Cambridge, MA) were coated with 40 µL of 3 mg/mL porcine type I collagen (Cellmatrix Type I-P; Nitta gelatin, Osaka, Japan) and gelled as described by the supplier. The culture insets were conditioned overnight with 1.5 mL of growth medium with 10% FBS in the lower (basal) compartment of a 12-well culture plate before plating.
RTE cells were plated onto the apical (gelled) surface of the inserts with 0.5 mL of growth medium without serum in the upper (apical) compartments of the culture plates (2.5 x 104 cells/membrane). Cultures were grown in 95% air and 5% CO2 at 37°C. After 24 h, media in both compartments were removed and replaced with growth medium without serum. The medium was changed every other day using 1.5 and 0.5 mL growth medium without serum in the basal and apical compartments, respectively. On day 7 (at which point the membrane were confluent or almost confluent), medium was removed from the apical compartment to establish an ALI culture (Fig. 1). Very little or no medium leaked onto the apical surface of the cultures. From day 7 the medium (2.0 mL growth medium without serum) was changed every day only in the basal compartment and cultures were grown until RTE cells were well differentiated and ciliary movement was visible (7–14 days).
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The culture inserts with membrane on which RTE cells were grown were placed in another 12-well culture plate and washed several times with Hank's balanced salt solution (HBSS; Gibco-BRL) with 10 mM N-(2-hydroxy-ethyl)-piperazineethanesulfonic acid (HEPES; Gibco-BRL), pH 7.4 (HBSS/HEPES), and then 1 mL HBSS/HEPES was filled only in the basal compartment. The plate was placed in a special exposure chamber (80 x 120 x 20 mm, polystyrene box) mounted on a phase-contrast microscope (IMT-2, Olympus, Tokyo, Japan). Warmed and humidified air (vehicle) with or without sevoflurane, halothane or isoflurane was passed through the chamber at a total gas flow rate of 2 L/min using an anesthesia gas machine, a vaporizer (Tec3; Datex-Ohmeda, Helsinki, Finland) and a water bottle immersed in a water bath with a thermostatically controlled heat exchanger (Fig. 1). The temperature in the chamber was maintained at 25°C ± 0.5°C throughout the experiment. The anesthetic concentrations in the chamber were maintained constant using an anesthesia gas monitor (Capnomac; Datex-Ohmeda).
The cells were viewed at 400x magnification. All observations were monitored and recorded for analysis using a 3CCD color videocamera (DXC-C33; Sony, Tokyo, Japan), a DVCAM video cassette recorder (DSR-30; Sony), and a Trinitron color monitor (CVM-1271; Sony). After >90-min equilibration and 5-min baseline periods, air (vehicle) with or without a volatile anesthetic (sevoflurane 0%–4%, halothane 0%–2%, and isoflurane 0%–2%) was introduced into the chamber (Fig. 2). After a 30-min exposure, the anesthetic was washed out by air (vehicle), and the image was recorded for the subsequent 10 min (washout period). The culture plates (membrane) were used once for each experiment and not reused.
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To determine CBF, video images were later captured at 30 frames per second and digitized using a Macintosh computer and iMovie software (Apple Computer, Cupertino, CA). Light intensities derived from a single pixel region of interest were picked up as a time-amplitude waveform by ImageJ software (Rasband WS, National Institutes of Health, Bethesda, MD). Power spectrum analysis of the signal waveform was performed and the dominant frequency was determined using the maximum entropy method (MEM software; Ishikawa Y, Saitama, Japan). A simple average of the dominant frequency of 3 to 5 regions of interest from a single cell was regarded as the CBF of the cell. The CBF value of one plate (membrane) was the average of CBF values for 4 cells that were not adjacent and separated each other.
To confirm the concentration of anesthetic dissolved in HBSS/HEPES in the basal compartment, the solution was sampled using a headspace sampler (No.7694; Agilent Technologies, Palo Alto, CA) at the end of the exposure period, and analyzed using a gas chromatograph (No.6890N GC; Agilent Technologies).
All values were expressed as mean ± sd. Values of n represent the number of plates (membranes). Comparisons of trends over time of two groups were performed with use of two-factor repeated-measures analysis of variance. Time-matched values in the groups were compared using Dunnett multiple comparison tests after one-way analysis of variance. All statistical analyses were performed using Prism 4 software (GraphPad Software, San Diego, CA). P values <0.05 were considered to be statistically significant.
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Results |
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Exposure of 4% sevoflurane (n = 5) decreased CBF slightly but significantly (16%) during the exposure period, compared with vehicle (air) administration (n = 5) (Fig. 3A). In contrast, 2% halothane and isoflurane (n = 5 each) significantly decreased CBF (44% and 53%, respectively) (Figs. 3B and 3C). CBF returned to baseline levels within 5 min after washout of the anesthetics.
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In the groups exposed to 0% (vehicle), 0.25%, 0.5%, 1%, 2%, and 4% sevoflurane, baseline CBFs (CBF at 0 min) were 7.7 ± 0.6, 8.0 ± 1.2, 8.1 ± 1.1, 7.8 ± 0.7, 7.6 ± 0.8, and 7.4 ± 0.7 beats/s, respectively, and did not differ statistically among the 6 concentration groups (n = 5 each). CBFs 30 min after sevoflurane exposure were 102% ± 5%, 100% ± 5%, 100% ± 3%, 100% ± 6%, 97% ± 9%, and 84%± 2% of baseline (= 100%), respectively, and differed significantly among the 6 groups (P = 0.003; Fig. 4A). However, there was no statistical difference among 0%–2% groups (P = 0.7107).
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P < 0.05 versus sevoflurane at the same concentration. B, Plotting % baseline CBF versus minimal alveolar concentration (MAC).Baseline CBFs in the groups exposed to 0%, 0.12%, 0.25%, 0.5%, 1%, and 2% halothane were 7.9 ± 0.6, 8.3 ± 0.6, 8.6 ± 0.7, 7.7 ± 0.6, 8.0 ± 0.7, and 7.4 ± 1.0 beats/s, respectively, and did not differ statistically (n = 5 each). Halothane decreased CBF in a concentration-dependent manner (101% ± 1%, 99% ± 5%, 97% ± 3%, 89% ± 6%, 82% ± 6%, and 56% ± 14% of baseline at 30 min, respectively; P < 0.0001; Fig. 4A). Baseline CBFs in the groups exposed to 0%, 0.12%, 0.25%, 0.5%, 1%, and 2% isoflurane were 8.0 ± 1.1, 6.9 ± 0.6, 8.7 ± 0.5, 7.7 ± 0.6, 6.9 ± 0.8, and 7.9 ± 0.6 beats/s, respectively, and were not different (n = 5 each). Isoflurane also decreased CBF in a dose-dependent manner (101% ± 4%, 98% ± 4%, 95% ± 4%, 90% ± 4%, 79% ± 11%, and 47% ± 6% of baseline at 30 min, respectively; P < 0.0001; Fig. 4A).
Concentrations of anesthetics in the basal compartment solutions 30 min after administration of 1% of sevoflurane, halothane and isoflurane were 132 ± 12, 423 ± 3, and 276 ± 1 µmol/L, respectively (n = 3 samples, each). Those after administration of 2% of sevoflurane, halothane and isoflurane were 265 ± 54, 899 ± 14, and 589 ± 27 µmol/L, respectively (n = 3 samples, each).
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Discussion |
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In the current study using isolated and cultured RTE cells, we confirmed previous reports demonstrating that volatile anesthetics have a cilioinhibitory action (4–13). In addition, our study demonstrates that the CBF-depressant effect of sevoflurane is much weaker than that of halothane and isoflurane. Although the earlier studies used whole animals or explant airway tissues, we chose ALI cultures that contain few nonepithelial cells and no nerve terminals. Vagal or sympathetic nerve stimulation increase CBF (1–3,18). Volatile anesthetics may affect the autonomic nervous systems and work on CBF indirectly. Our study clearly demonstrates that volatile anesthetics act directly on cultured RTE cells and inhibit CBF.
In our study, 2% halothane and isoflurane inhibited CBF by 40% and 50%, respectively. Previous reports demonstrated that halothane decreased CBF by 34% or 44% (2.25% halothane exposure) (11,12), 14% (1.8% exposure) (9), and 20% (2% exposure) (10) and that isoflurane depressed CBF by 33% or 19% (3.6% isoflurane exposure) (11,12) using explanted human nasal tissues. The extent of inhibition in our study is larger, especially for isoflurane, than that in earlier reports. This may be attributable, at least in part, to different species, cell types, and experimental conditions. In explant preparations, indirect effects or cell-cell interactions could not be negligible. In isolated and cultured conditions cell characters may be different more or less from intact tissues (2).
In our study, it took <5 min to recover CBF after washout of the volatile anesthetics. Raphael et al. (12) reported that recovery of CBF after 1-hour exposure of 3 minimum alveolar concentration (MAC) of halothane and isoflurane took 90 and 60 minutes, respectively. The discrepancy in recovery speed is probably a result of the difference in anesthetic washout speed. Our RTE cells were exposed directly to air (2 L/min) with vaporized anesthetic from the apical side, whereas Raphael et al.'s explanted nasal cells were perfused with solutions (HBSS, 0.5 mL/min) with dissolved anesthetics. Anesthetic gas dissolved in liquids (basal compartment) could be quickly swept away to air space (apical compartment) during the initial few minutes of the washout period in our gas exposure preparation. In a liquid exposure preparation the anesthetic concentrations in solutions might decrease very slowly during the washout period because the oil-water partition coefficients of volatile anesthetics (halothane, 310; isoflurane, 170; at 37°C) (19) are far higher than water-gas partition coefficients (halothane, 0.86; isoflurane, 0.63; at 37°C) (20).
Cervin et al. (21,22) demonstrated that halothane and isoflurane increased CBF during the first several minutes after exposure and then decreased through a cholinergic or neurokinin receptor-mediated pathway in the rabbit maxillary sinus in vivo. In our study, no CBF stimulatory effect of sevoflurane, halothane, or isoflurane was observed in purely isolated RTE cells in vitro. This finding corroborates that the short-term CBF stimulatory effects of halothane and isoflurane are not a result of their direct action on ciliated epithelial cells.
To verify anesthetic delivery, we measured anesthetic concentrations not only in the exposure chamber (apical compartment) but also in solutions in the basal compartment. Considering the water/gas partition coefficients (14,20), calculated equilibrium water concentrations of 1% sevoflurane, halothane, and isoflurane are 161, 392, and 277 µM, respectively, at 37°C. Our measured values in solutions were near these calculated values. Although these figures cannot be simply compared because our study was performed in nonequilibrium condition and in HBSS/HEPES at room temperature, our result confirmed that the anesthetics were delivered to the basal compartment without barrier.
It is of interest that sevoflurane is much weaker than halothane or isoflurane in its cilioinhibitory effect, although many pharmacologic actions of these anesthetics are similar (3,15). The mechanism responsible for the regulation of CBF is complicated and not fully understood at present. CBF is regulated by many intrinsic factors (1–3,18,23). Muscarinic or ß-2 adrenergic receptor stimulation, for example, increases CBF. Increase of intracellular cyclic adenosine and/or guanosine monophosphate (cAMP and/or cGMP) concentrations augments CBF. Activations of cAMP-dependent protein kinase and/or cGMP-dependent protein kinase increase adenosine triphosphate (ATP) hydrolase (ATPase) activity in dynein, a motor protein for ciliary movement, leading to CBF increase (2,18). ATPase activity and ATP play a crucial role in spontaneous or basal beating of airway cilia (2,23). To elucidate the mechanism of anesthetic action on CBF and the differences between anesthetics, studies concerning their effects on CBF-related molecules are required.
Cilioinhibitory action is an undesired side effect of volatile anesthetics (1–3). Impaired ciliary function is recognized as one of the risk factors for perioperative respiratory morbidity (2,3). The MACs of sevoflurane, halothane, and isoflurane are 1.97%, 0.88%, and 1.12%, respectively, in rats (24). Calculating from concentration-response curves, 1.5 MAC of sevoflurane, halothane, and isoflurane inhibits CBF by 9%, 29%, and 44%, respectively (Fig. 4B). This result suggests that a clinical dose of sevoflurane has little effect on CBF and sevoflurane may be advantageous compared with the older anesthetics, although we cannot extrapolate our study to clinical conditions. In patients with preexisting airway disease the CBF depressant effect of volatile anesthetics may be of limited relevance, as ciliary function is diminished or virtually absent in such patients. Further investigations are necessary to elucidate whether sevoflurane has less cilioinhibitory action in vivo or has advantages in the clinical setting.
In conclusion, the current study clearly shows that sevoflurane has a direct cilioinhibitory effect, but its effect is much weaker than that of halothane and isoflurane in isolated and cultured RTE cells. Further studies, including clinical trials, are needed to determine whether sevoflurane has a pulmonary advantage in comparison with the older anesthetics.
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Footnotes |
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Accepted for publication January 24, 2006.
Supported, in part, by Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (No. 12671463 and 14571432)
Presented in part at 2004 annual meeting of the American Society of Anesthesiologists in Las Vegas, Nevada.
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