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Canadian Journal of Anesthesia 53:242-249 (2006)
© Canadian Anesthesiologists' Society, 2006
General Anesthesia

Differential effects of intravenous anesthetics on ciliary motility in cultured rat tracheal epithelial cells

[Les effets différentiels des anesthésiques intraveineux sur la motilité ciliaire de cellules cultivées d’épithélium trachéal de rat]

Hiroko Iida, MD, Shogo Matsuura, MD, Gotaro Shirakami, MD, Keiji Tanimoto, MD and Kazuhiko Fukuda, MD

From the Department of Anesthesia, Kyoto University Hospital, Kyoto, Japan.

Address correspondence to: Dr. G. Shirakami, Department of Anesthesia, Kyoto University Hospital, Kyoto 606-8507, Japan. Phone: 81-75-751-3516; Fax: 81-75-752-3259; E-mail: gshi{at}kuhp.kyoto-u.ac.jp


   Abstract
TOP
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Purpose: It has been shown that airway ciliary function is impaired by several anesthetic or sedative drugs, which may predispose anesthetized or intensive care patients to respiratory complications, such as hypoxemia, atelectasis and pulmonary infection. We studied the effects of midazolam, propofol, dexmedetomidine, ketamine, fentanyl, thiopental and pentobarbital on ciliary beat frequency (CBF) in isolated and cultured rat tracheal epithelial (RTE) cells, to investigate their direct CBF action removing influences of non-epithelial cells.

Methods: Rat tracheal epithelial cells were purely isolated from tracheas of adult male Sprague-Dawley rats. After 14 to 21 days of culture, the images of motile cilia were videotaped using a phase-contrast microscope. Baseline CBF and CBF 30 or 50 min after administration of vehicle or one of the above agents were computer-analyzed.

Results: Midazolam (0.3–10 µM), propofol (1–100 µM), dexmedetomidine (1–100 nM), fentanyl (0.1–10 nM) and thiopental (30–300 µM) had no effect on CBF. Ketamine at a supraclinical dose (1000 µM) increased CBF (22 ± 13, mean ± standard deviation, % increase from baseline; baseline = 100%) significantly (P < 0.01). Fentanyl at a high clinical dose (100 nM) increased CBF significantly (10 ± 9%). Pentobarbital decreased CBF dose-dependently (100 µM, –2 ± 6%; 300 µM, –14 ± 18%; 1000 µM, –75 ± 5%) and reversibly (P < 0.01).

Conclusion: These results show that midazolam, propofol, dexmedetomidine and thiopental have no direct action on CBF in isolated RTE cells, whereas high doses of ketamine and fentanyl have direct ciliostimulatory actions and pentobarbital has a direct cilioinhibitory action.


   Introduction
TOP
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
AIRWAY ciliary function plays an important role in the primary pulmonary defense mechanism to remove foreign particulates, such as dust, microorganisms and dead cells from the tracheobronchial tree.1–3 Impaired ciliary motility in anesthetized or intensive care patients may predispose to respiratory complications such as hypoxemia, atelectasis and pulmonary infections. It is clinically important to determine the factors that inhibit airway ciliary beat frequency (CBF), an important determinant of respiratory clearance rate, to decrease perioperative and intensive care morbidity. Numerous reports demonstrate that various anesthetic and sedative agents decrease CBF or overall airway mucus transport rate.2–17 Inhaled anesthetics (halothane, enflurane and isoflurane),2–8 benzodiazepines (diazepam and tenazepam),9,10 propofol,11 ketamine,12 opioids (morphine and codeine),4,13–15 and barbiturates (thiopental, thiamylal and pentobarbital)5,6,16,17 have been shown to decrease mucociliary function. However, the results of earlier studies were affected more or less by interactions between epithelial cells and other cells or systems. This is because previous studies were accomplished using whole animal or human models in vivo, or organ explant preparations in vitro, which include various types of cells other than airway epithelial cells, such as smooth muscle cells, fibroblasts and vascular cells, and nerve endings. Therefore, direct effects of anesthetics and sedatives on CBF in airway epithelial cells remain unclear. To better understand the mechanism of cilioinhibitory action (an undesirable side effect) of various anesthetic-sedative drugs, it is important to investigate their CBF effects independently from the influences of non-epithelial cells.

Air-liquid interface (ALI) cultures have been used to purify the airway epithelial cells and to differentiate ciliated epithelial cells.18,19 In this study, to investigate the direct effects of iv anesthetic-sedative agents used frequently in clinical and/or experimental settings on epithelial cells, we studied the effects of midazolam, propofol, dexmedetomidine, ketamine, fentanyl, thiopental and pentobarbital on CBF using the ALI cultures or purely isolated rat tracheal epithelial (RTE) cells.


   Methods
TOP
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Preparation of rat tracheal epithelial cell culture
Cultured RTE cells were prepared according to the methods described by Kaartinen et al. with some modifications.18,19 The protocol was approved by the Animal Research Committee, Graduate School of Medicine, Kyoto University. Tracheas were removed under sterile conditions from male Sprague-Dawley rats (body weight, 300–350 g; Japan SLC, Hamamatsu, Japan) euthanized by CO2 asphyxiation. They were incubated overnight at 4°C in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Rockville, MD, USA) and Ham’s nutrient F–12 medium (F–12; Gibco-BRL) (1:1) with 0.05% type XIV protease (Sigma, St. Louis, MO, USA). Fetal bovine serum (FBS; Gibco-BRL; last concentration 10%) was then added to incubation medium (DMEM/F–12) and the RTE cells were flushed out. The cells were collected by centrifugation (150 g, 4°C, one minute) and washed twice with DMEM/F–12 containing 10% FBS.

Growth medium for RTE cells consisted of DMEM/F–12 supplemented with 10 mg·mL–1 insulin (Gibco–BRL), 0.1 mg·mL–1 hydrocortisone (Sigma), 0.1 mg·mL–1 cholera toxin (Sigma), 5 mg·mL–1 transferrin (Gibco–BRL), 50 mM phosphoethanolamine (Sigma), 80 mM ethanolamine (Gibco–BRL), 25 ng·mL–1 epidermal growth factor (EGF; Peprotech, Rocky Hill, NJ, USA), 25 mg·mL–1 bovine pituitary extract (Gibco–BRL), 3 mg·mL–1 bovine serum albumin (BSA; Gibco–BRL), 5 x 10–8 M retinoic acid (Sigma), 100 U·mL–1 penicillin (Sigma) and 100 U·mL–1 streptomycin (Gibco–BRL). Polyester permeable membranes (12–mm–diameter, 0.4 mm–pore–size, No. 3460; Corning–Coster, Cambridge, MA, USA) were coated with 40 µL of 3 mg·mL–1 bovine type I collagen (Cellmatrix Type I–P; Nitta gelatin, Osaka, Japan) and gelled as described by the supplier. Membranes with culture inserts were conditioned overnight with 1.5 mL of growth medium with 10% FBS in the lower (basal) compartment of 12 well cultured plates before plating.

Rat tracheal epithelial cells were plated onto the apical (gelled) surface of the membranes 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 hr, 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 seven (at which point the membranes were confluent or almost confluent), medium was removed from the apical compartment to establish an ALI culture (Figure 1Go). Very little or no medium leaked onto the apical surface of the cultures. From day seven 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 movements were visible (seven to 14 days).


Figure 1
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  		FIGURE 1 Schematic drawing of the experimental set-up for measurement of ciliary beat frequency (CBF) in air-liquid interface culture of rat tracheal epithelial (RTE) cells.

 
Experimental protocol
After seven to 14 days of incubation, the inserts with membrane on which RTE cells were grown were placed on 12 well culture plates and washed several times with Hank’s balanced salt solution (HBSS; Gibco–BRL) with 10 mM N–(2–hydroxyl–ethyl)–piperazineethanesulfonic acid (HEPES; Gibco–BRL), pH 7.4 (HBSS/HEPES). Then, 900 to 950 µL observation medium was filled only in the basal compartment. The observation medium was HBSS/HEPES except thiopental sodium for which it was HBSS/HEPES with 5% BSA (pH 7; Nacalai Tesque, Kyoto, Japan).

Before each experiment, the cultures were allowed to stabilize and warm at 26.5°C for > 30 min (equilibration period). The plate was placed on an inverted phase-contrast microscope (IMT–2; Olympus, Tokyo, Japan), (Figure 1Go). Following equilibration and ten-minute baseline periods, 50 to 100 µL of vehicle or test drug solution were added and observed 30 or 50 min (administration period). The vehicle solution of test drugs dissolved, except propofol and thiopental, was HBSS/HEPES. Propofol was dissolved in dimethyl sulfoxide (DMSO; Wako Pure Chemical, Osaka, Japan) and pluronic F–127 (Sigma) in HBSS/HEPES: final DMSO and pluronic concentrations in the observation medium were 0.1 and 0.05%, respectively. Vehicle solutions with or without anesthetic-sedative drug except thiopental were adjusted to pH 7.4 using 0.1 mM sodium hydroxide or 0.1 mM hydrogen chloride and maintained at 26.5°C. Thiopental was dissolved in distilled water (26.5°C). The observation medium solution containing vehicle/anesthetic was also adjusted to, and maintained at pH 7.4 ± 0.1 and 26.5 ± 1°C throughout the experimental periods.

In the pentobarbital 1000 µM experiment, some of the plates were washed twice with 2 mL observation medium after a 30–min administration period, and were observed for a subsequent 20 min (washout period).

The anesthetic-sedative agents used were midazolam maleate (Sigma), propofol (Aldrich, Milwaukee, WI, USA), dexmedetomidine (Abbot Japan, Osaka, Japan), ketamine hydrochloride (Sigma), fentanyl citrate (Sankyo, Osaka, Japan), thiopental sodium potassium salt (Sigma), and pentobarbital sodium salt (Nacalai Tesque, Kyoto, Japan).

Measurement of ciliary beat frequency
The cells were viewed at 400 x magnification. All observations were monitored and videotaped for analysis using a 3CCD colour videocamera (DXC–C33; Sony, Tokyo, Japan), a DVCAM video cassette recorder (DSR–30; Sony), and a Trinitron colour monitor (CVM–1271; Sony). To determine CBF, video images were later captured at 30 frames per second and digitized using a Macintosh computer and iMovie software (Apple Computer, Santa Clara, CA, USA). Light intensities derived from a single pixel region of interest were picked up as a time-amplitude waveform by ImageJ software (Wayne R., Bethesda, MD, USA). Frequency analysis of the signal waveform was performed by maximum entropy method20 (MEM software; Ishikawa Y., Saitama, Japan). Three regions of interest from a single cell were analyzed and the dominant frequency was regarded as the CBF of the cell. The CBF value of one plate was the average of values for five independent cells. The CBF were measured in the same cells during the experimental periods.

Statistical analysis
Values are expressed as mean ± standard deviation. Values of n represent the number of plates. Comparisons of group-time effects were performed using repeated-measures analysis of variance. Time-matched values in the groups were compared using the Bonferroni test after one-way analysis of variance. P < 0.05 was considered statistically significant.


   Results
TOP
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Midazolam at a dose of 10 µM administration did not change CBF during the experimental periods, compared with vehicle administration (Figure 2AGo). Midazolam (0.3–10 µM) revealed no concentration effect on CBF (Figure 2DGo). Propofol (1–100 µM) and dexmedetomidine (1–100 nM) also did not change CBF (Figures 2B, C, E and FGo).


Figure 2
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  		FIGURE 2 Time course of effect of (A) midazolam, (B) propofol and (C) dexmedetomidine on ciliary beat frequency (CBF) in cultured rat tracheal epithelial (RTE) cells (n = 8 each). After the baseline period (-ten minutes to minute zero), one of the drugs or its vehicle was added to the medium at time zero (arrow), and CBF was observed during the subsequent 50 min. Values are mean ± standard deviation. P values compare responses over time with treatments (repeated measures analysis of variance). Concentration effects of (D) midazolam, (E) propofol and (F) dexmedetomidine on CBF (n = 8 each). Ciliary beat frequency was measured at baseline (minute zero) and 50 min after administration of one of the drugs or its vehicle. Values are expressed as a percentage of baseline CBF (baseline = 100%) and represent mean ± standard deviation. P values compare responses between doses (one-way analysis of variance).

 
Ketamine at a dose of 1000 µM increased CBF significantly, from 8.7 ± 1.1 Hz at time zero (baseline) to 10.7 ± 1.2 Hz (25 ± 21% increase from baseline; baseline = 100%) and 10.4 ± 0.9 Hz (22 ± 13%) at time 40 and 50 min, respectively, compared with vehicle (7 ± 9 and 5 ± 8%, respectively), (Figure 3AGo). Ketamine at doses of 30–300 µM revealed no significant effect on CBF (Figure 3CGo). Fentanyl at the highest dose (100 nM) increased CBF significantly but slightly, from 9.7 ± 1.2 Hz at time zero to 10.3 ± 1.0 Hz (7 ± 7%), 10.9 ± 1.3 Hz (12 ± 15%), 10.5 ± 1.3 Hz (8 ± 6%) and 10.7 ± 1.3 Hz (10 ± 9%) at times 20, 30, 40 and 50 min, respectively, compared with vehicle (1 ± 2, 1 ± 4, 3 ± 6 and 4 ± 3%, respectively), (Figure 3BGo). Fentanyl at doses of 0.1–10 nM had no effect on CBF (Figure 3DGo).


Figure 3
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  		FIGURE 3 Time course of effect of (A) ketamine and (B) fentanyl on ciliary beat frequency (CBF) in cultured rat tracheal epithelial (RTE) cells (n = 8 each). Concentration effects of (C) ketamine and (D) fentanyl on CBF (n = 8 each). Legends are the same as in Figure 2Go. * P < 0.05 vs vehicle (Bonferroni test). *P < 0.05 vs vehicle, {dagger}P < 0.05 vs 30 µM ketamine, {ddagger}P < 0.05 vs 100 µM ketamine (Bonferroni test).

 
Thiopental (30–300 µM) did not change CBF (Figures 4A and CGo). In contrast, pentobarbital at a dose of 1000 µM decreased CBF, from 9.2 ± 1.1 Hz at time zero minute to 2.3 ± 0.6 Hz (–75 ± 5%) at time 30 min, compared with vehicle (2 ± 10%), (Figure 4BGo). The cilioinhibitory action of pentobarbital was dose-dependent (300 µM, –14 ± 18%; 100 µM, –2 ± 6%), (Figure 4DGo). To test the reversibility of the depressant effect of pentobarbital, three of the eight plates were washed, medium was changed, and CBF were measured subsequently. After pentobarbital washout, CBF resumed within ten minutes, from1.9 ± 0.4 Hz (–79 ± 6%) before washout to 9.0 ± 0.9 Hz (–5 ± 7%) and 9.4 ± 1.1 Hz (–2 ± 9%) ten minutes and 20 min after washout, respectively.


Figure 4
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  		FIGURE 4 Time course of effect of (A) thiopental and (B) pentobarbital on ciliary beat frequency (CBF) in cultured rat tracheal epithelial (RTE) cells (n = 8 each). After the baseline period (-ten minutes to minute zero), one of the drugs or its vehicle was added to the medium at time zero (arrow), and observed during the subsequent 30 min. Values are mean ± standard deviation. P values compare changes over time with treatments (repeated measures analysis of variance). *P < 0.05 vs vehicle (Bonferroni test). Concentration effects of (C) thiopental and (D) pentobarbital on CBF. Ciliary beat frequency was measured at baseline and 30 min after administration of one dose of a drug or its vehicle (n = 8 each). Values are expressed as a percentage of baseline CBF (baseline = 100%) and represent mean ± standard deviation. P values represent among the doses (one-way analysis of variance). *P < 0.05 vs vehicle, {dagger}P < 0.05 vs.100 .µM pentobarbital, {ddagger}P < 0.05 vs 300 µM pentobarbital (Bonferroni test).

 

   Discussion
TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Ciliodepressant actions of a number of iv anesthetic-sedative agents have been demonstrated.2,4–6,9–17 The interpretation of these reports must take into account that a given drug can influence not only epithelial cells but also other types of cells or systems, such as the autonomic nervous systems, that regulate CBF.1,2 In this study we used the isolated RTE cells which have no nerve endings and little influence of non-epithelial cells. A limitation of our study is that the ciliated epithelial cells in vitro and in vivo are not always the same, and we cannot necessarily extrapolate the results to humans. Another limitation is that our study period was relatively finite. Drug exposure time is usually brief in the anesthesia setting, whereas sedatives are often infused over hours or days in the critical care setting. The clinical importance of cilioinhibition may be greater in the latter settings or long exposure, and in severely compromised patients. Despite the limitations of the experimental model, our study provides basic information as to whether a drug can act directly on epithelial cells and induce a change in CBF and mucociliary clearance during early exposure period.

In this study, midazolam 0.3–10 µM revealed no effect on CBF in the RTE cells, consistent with a previous report demonstrating that 20 µM midazolam did not change CBF in human nasal turbinate explants.21 Considering the plasma concentrations required for hypnosis and amnesia (100–200 ng·mL–1 or 0.3–0.6 µM) and peak plasma concentration after anesthesia induction (1–2 µM) in humans,22 midazolam may have no direct effect on CBF clinically. However, these findings are inconsistent with other reports using other benzodiazepine preparations. It is reported that temazepam 10 mg po reduced tracheobronchial clearance of inhaled radioaerosol by 22% in humans in vivo9 and diazepam decreased CBF dose-dependently (0.4–40 mg·mL–1; 17–74% decrease) in human nasal turbinate explants in vitro.10 It is difficult to explain the discrepancy, but indirect effects, such as influences on the autonomic nervous system, may be involved in the benzodiazepine-induced CBF depression.

Plasma levels of propofol required for surgical anesthesia are 2–5 µg·mL–1 (10–30 µM) in human.22 We demonstrated in this study that propofol (1–100 µM) had no CBF effect on purified RTE cells, which is consistent with a previous report demonstrating that propofol at a dose of 70 µM did not change CBF in human nasal turbinate explants.21 However, this is inconsistent with another report demonstrating that propofol (10–300 µM) has a CBF stimulating action via the nitric oxide-cyclic guanosine monophosphate pathway in cultured rat tracheal explants.23 The discrepancy might be explained by fact that the expression of the nitric oxide-cyclic guanosine monophosphate pathway was weak in our RTE cells2 or that propofol stimulates CBF indirectly. It is reported that bronchial mucus transport velocity was decreased in patients anesthetized with propofol, alfentanil and vecuronium.11 Considering the in vitro reports including ours, propofol may not inhibit mucus transport directly.

The therapeutic plasma concentration of dexmedetomidine is 0.3–2 ng·mL–1 (1–8 nM).22 Our study demonstrates that dexmedetomidine, an {alpha}2-selective agonist, at the doses of 1–100 nM has no effect on CBF in the RTE cells. Several studies report that {alpha}-adrenergic agonist affects airway CBF.2,24–27 Xylometazoline or oxymethazoline, an imidazoline derivative that activates both {alpha}1- and {alpha}2-adrenergic receptors, depressed CBF in chicken embryo tracheal explants and cryopreserved human sphenoidal sinus mucosa27 and human nasal turbinate explants.25,26 Phentolamine, a non-specific {alpha}-adrenergic receptor antagonist, blocked xylometazoline-induced CBF depression.25,26 Phenylephrine, an {alpha}1-selective adrenergic agonist, at a low dose (0.01%) increased CBF, but higher doses (0.25 and 0.5%) decreased CBF in harvested human nasal brushings in vitro.24 There are no studies evaluating the effect of {alpha}2-adrenergic agonists on ciliary function in vivo. Since dexmedetomidine affects the autonomic nervous system, it may have indirect CBF effect.2,22

It was demonstrated that ketamine decreased mucociliary clearance, as similar to pentobarbital in the baboon.12 However, our study demonstrates that ketamine has no direct cilioinhibitory action. Considering the therapeutic plasma level of ketamine is 0.7–2.2 µg·mL–1 (3–9 µM) in humans,22 ketamine at clinical doses does not affect CBF directly, though experimental animals often need higher doses of ketamine. Ketamine, a N-methyl-D-aspartate (NMDA) receptor antagonist, is known to have a sympathomimetic action.22 Sympathetic stimulation increases CBF.2 In our experimental system, ß2-adrenergic agonists increase CBF (unpublished observation), which is consistent with previous reports.2,28 Because sympathetic influence can be ignored in our isolated RTE cells, the ciliostimulatory action of the highest dose of ketamine (1000 µM) is not due to sympathetic stimulation. Whether the NMDA receptor is involved in ciliostimulatory action of ketamine or not is a matter for further investigation.

Several studies have reported that opioids depress respiratory mucus transport or ciliary movement.2,4,13–15 The addition of morphine reduced mucociliary flow rate in dogs anesthetized with halothane and nitrous oxide.4 Codeine decreased CBF in rat tracheal or bronchial explants.13,14 Aerosolized ß-endorphin decreased mucociliary clearance in dogs.15 In contrast, Selwyn et al. reported that morphine (10 µM) did not change CBF in human nasal turbinates explants.29 In our study, fentanyl at doses between 0.1–10 nM revealed no effect on CBF, and 100 nM fentanyl tended to increase CBF. Considering the therapeutic plasma concentration of fentanyl (1–30 ng·mL–1 or 3–100 nM),30 clinical doses of fentanyl had no direct inhibitory action on CBF. The depressant effects of opioids on CBF may be indirect actions.

It has been reported that barbiturates decrease CBF and/or mucociliary clearance.5,6,13,16,17 Thiopental depressed mucociliary clearance in dogs5 and rats.6 Pentobarbital decreased mucociliary clearance in sheep,16 dogs17 and rats6 and in rat bronchial explants.13 Our study demonstrates that, thiopental, a thio–barbiturate, has no direct effect on CBF, but pentobarbital, an oxy-barbiturate, has a direct cilioinhibitory effect in the RTE cells. Therapeutic plasma levels of thiopental are 20–80 µg·mL–1 (80–300 µM).31 Therapeutic concentrations of pentobarbital during barbiturate therapy are 30–45 µg·mL–1 (120–180 µM).32 Considering the blood concentration of pentobarbital, clinically relevant doses of pentobarbital may inhibit CBF, whereas CBF depressant effects of thiopental may be an indirect action. In our thiopental experiments, we used an observation medium with 0.5% albumin to dissolve it and adjust pH. We could not investigate a concentration of 1000 µM thiopental due to the limited solubility at this concentration. Thiopental is bound to albumin and other plasma proteins in circulating blood (60–97% protein bound).22,31 Although actual concentrations of unbound (free) thiopental were uncertain in our study, it is hard to consider that the result was affected by limited availability of free thiopental. The mechanism of the difference in the direct CBF action between these drugs is not clear. It is reported that they have opposite effects on platelet aggregation33 and vascular smooth muscle tension.34 Differences in lipid solubility or dynamics of the intracellular signaling molecule, such as the calcium ion,33,34 might be involved.

In conclusion, the present study demonstrates that clinical doses of midazolam, propofol, dexmedetomidine, ketamine and thiopental have no action on CBF, whereas pentobarbital has a direct CBF depressant action in isolated RTE cells. With regards to ketamine and fentanyl, only the highest studied doses stimulated, or tended to stimulate CBF. The cilioinhibitory actions of benzodiazepines, ketamine, opioids and thiopental are probably due to indirect effects, or cell-cell interactions. Further studies are required to clarify direct and indirect effects of iv anesthetic-sedative agents on airway mucociliary clearance.


   Acknowledgments
 
We thank Sankyo Pharmaceutical for providing fentanyl citrate, and Abbot Japan for supplying dexmedetomidine.


   Footnotes
 
The study was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (No. 12671463 and 14571432).

Accepted for publication August 11, 2005 Revision accepted September 16, 2005.


   References
TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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