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Effect of halothane on cilia beat frequency of ciliated human respiratory epithelium in vitro†
British Journal of Anaesthesia 1994; 73: 507-510
LABORATORY INVESTIGATIONS
Effect of halothane on cilia beat frequency of ciliated human respiratory epithelium in vitro\ A. GYI, C. O'CALLAGHAN AND J. A. LANGTON
Summary
Key words Anaesthetics volatile, halothane. Lung, cilia.
Mucus transport rate (MTR) is dependent on the inter-relationship between the integrity of ciliated columnar epithelium, the viscoelastic properties of the mucus, cilia beat frequency (CBF) and co-ordination of the cilia beat. Impairment of mucociliary clearance after general anaesthesia may predispose patients to atelectasis and postoperative basal zone collapse leading to impairment of gas exchange and predisposing to chest infection [1,2]. Decreased MTR has been demonstrated in areas of postoperative atelectasis [1]. Previous work has shown that general anaesthetic agents depress MTR in humans [3], dogs [4] and sheep [5]. The mechanism by which general anaesthetic agents reduce MTR may be depression of ciliary function or an effect on the production and physical properties of respiratory mucus. However, Rubin and colleagues [6] demonstrated in humans that general anaesthetic agents do not alter the viscoelastic properties of respiratory mucus. Nunn, Wills and McPherson [7] showed that general anaesthetic agents depressed swim velocity in ciliated protozoa Tetrahymena pyriformis. The effect of individual modern inhalation anaesthetic agents on human CBF has not been studied. The aim of this study was to examine the effects of halothane on human CBF in vitro. The technique of measurement of CBF using transmitted light was first described by Dalhamm
Subjects and methods After Ethics Committee approval and with informed consent, we studied 24 volunteers aged 22-35 yr with no history of asthma, atopy or smoking. None had suffered an upper respiratory tract infection within the previous 2 weeks and none had been exposed to halothane for at least 24 h. A small bronchoscopy brush was used to obtain a nasal brushing from each volunteer (without local anaesthesia). The nasal brushings were placed into MDE 199 cell culture media and taken to the laboratory for measurement within 20 min of collection. Cilia have been shown previously to remain beating at a normal frequency for periods in excess of 24 h after collection from volunteers [9]. The 24 nasal brushings were divided into four groups of six nasal brushings. Three groups were exposed to either 0.9 %, 1.8 % or 5.7 % halothane. One group of six was used as a control and was exposed to air only. Nasal brushings for CBF measurements after exposure to halothane were divided into two test tubes, each containing 5 ml of MDE 199 medium and placed in a water bath maintained at 37 °C. One test tube was exposed to halothane in air and the other was used as a matched control sample and exposed to air alone (Fig. 1).
HALOTHANE EXPOSURE AND CBF MEASUREMENT
The Fluotec 3 plenum halothane vaporizer (Cyprane) was calibrated using a Capnomac gas analyser. At dial settings 1, 2 and 5, the vaporizer consistently delivered halothane concentrations of 0.9%, 1.8% and 5.7%. The concentration of halothane delivered was stable over 3 h. The carrier gas was air from a cylinder and the flow was adjusted A. GYI, MB, BS, FRCA, J. A. LANGTON, MB, BS, FRCA (University Department of Anaesthesia); C. O'CALLAGHAN, BMEDSCI, MRCP,
DM (University Department of Child Health); Leicester Royal Infirmary, Leicester LEI 5WW. Accepted for publication: March 18, 1994. t Presented in pan at the Anaesthetic Research Society, Oxford Meeting, July 9-10, 1993 (British Journal of Anaesthesia 1993; 71: 757P-758P).
Downloaded from http://bja.oxfordjournals.org/ at New York University on September 1, 2015
The effect of halothane on human ciliated nasal epithelium was studied in vitro. Samples from 24 healthy adult volunteers were exposed to halothane in varying concentrations and cilia beat frequency was measured using the transmitted light technique. Mean cilia beat frequency was measured at 30-min intervals. There was a significant decrease in cilia beat frequency at 2 h in samples that were exposed to halothane (mean 8.4 (SD 2.5) Hz, 9.18 (2.6) Hz and 6.99 (4.9) Hz) compared with air (10.8(2.7) Hz, 11.6(2.1) Hzand12.1 (2.3) Hz) (P <0.01). The coefficient of variation of cilia beat frequency measurements increased after exposure to halothane. There was no change in the cilia beat frequency of controls exposed to air over a 3-h period. (Br. J. Anaesth. 1994; 73: 507-510)
and Rylander 1962 [8]; this is the most widely used technique for measuring CBF. Transmitted light techniques have been shown to produce reproducible results, are convenient to use and require minimal subjective assessment.
British Journal of Anaesthesia
508 One nasal brushing from each x 24 volunteers
0.9% Halothane Six nasal brushings
Each nasal brushing
Paired control 1 1.8% Halothane Six nasal brushings
Six nasal brushings
Each nasal brushing
Each nasal brushing
1 Paired control 1 5.7% Halothane ~*fPaired control |
Six nasal brushings
Air controls J
Figure 1 Study design showing division of 24 nasal brushing into four groups. A portion from each nasal brushing exposed to halothane was used as matched control.
Video camera t
Television monitor
Phase contrast microscope oil immersion lensx 100 >
Sample slide
Computerized analysis Anadat data analysis program 10-s digital signal t
Data storage
\
r
Power spectrum (mean CBF)
Figure 2 Flow chart of measurement technique for cilia beat frequency (CBF). A television-video modification of the transmitted light technique was used.
through a dry gas bobbin flowmeter. This air was humidified and divided equally into two channels. One channel passed directly to the surface of the control sample test tube, the other flowed through the vaporizer to the surface of the other test tube. The surface of the media in the test tubes containing ciliated samples was therefore exposed to either halothane in air at 100 ml min~l or air only at 100mlmin~'. Using gas chromatography we determined that the time required for halothane concentrations in the media to equilibrate with the carrier gas under these conditions was 15min. Because of physical limitations of slide preparation and the amount of nasal cilia in the sample, CBF of the matched samples was measured after the test tube had been placed in the water bath for 5 min and 2 h later. CBF of the samples exposed to halothane were measured at 30, 60, 90 and 120 min after which the vaporizer was turned off and the sample was exposed to air alone, for a washout period of 50 min. CBF was measured at the end of this washout period. Six nasal brushings were exposed to air alone and CBF measurements were made hourly for 3 h. Throughout the experiment small amounts of distilled water were added to the sample to allow for evaporation. The osmolality of the stock (MDE 199 culture medium) and the osmolalities of the solutions from the test tubes at the end of the experiment were measured. After the above procedure, osmolalities of the solutions in the two test tubes at the end of the
experiment did not differ by more than 3 % from the stock solution. For CBF measurements, samples were retrieved from the test tube and mounted on to a glass slide cover slip chamber. A temperature probe was attached on to the chamber and the heating stage was adjusted to maintain the slide temperature at 37 + 0.5 °C. Five minutes was allowed for temperature equilibration before measurement of CBF. METHOD OF CBF MEASUREMENT
CBF was measured using a television-video modification of the transmitted light technique, described by Teichtahl, Wright and Kirsner [10]. This is an accurate and reproducible method of measuring CBF in vitro, producing data in good agreement with other published data [8, 11]. Samples of human ciliated nasal epithelium were obtained by nasal brushing and examined, at body temperature (37 °C), using a phase contrast microscope with an oil immersion x 100 lens. Ciliated cells are columnar (20 um x 7 um) [12]. A ciliated columnar cell contains 100-250 cilia. Each cilium is 6 um x 0.3 um [12]. The image of the ciliated edge of the epithelium was projected onto a high resolution monitor by an sVHS video camera (fig. 2). The criteria for measurement of CBF were as follows: (1) CBF measurements were made from the first four undisrupted edges of ciliated epithelium sheets; (2)
Downloaded from http://bja.oxfordjournals.org/ at New York University on September 1, 2015
Pinhead photodiodef — * • Oscilloscope
Effect of halothane on cilia beat frequency
Table 1 Mean (SD) cilia beat frequency (Hz) at different times for each group. *Immediately after a 50-min air washout period Time (min)
0.9% Halothane
Air
11.0(2.3)
10.4(1.5) 8.0(2.0) 11.1 (2.2) 8.8(2.1) 8.5 (2.5) 11.1 (2.4) 8.4 (2.5) 11 1(1 9) 50minAW* 8.6 (2.8) Matched air 10.8 (2.7) controls at 120 min 0 30 60 90 120 180
1.8% Halothane
5.7% Halothane
11.5(2.0) 9.9(2.1) 8.8 (2.7) 9.4 (2.4) 9.2 (2.6)
12.1 (2.2) 9.7 (2.9) 10.1(3.8) 8.1 (4.0) 7.0 (4.9)
9.2 (2.5) 11.6(2.1)
7.9 (4.5) 12.1(2.3)
701 60 •3 50
STATISTICAL METHODS
Kruskal-Wallis one-way analysis of variance was used to determine statistical differences in CBF measurements of control samples over 3 h. The Mann-Whitney U test for distribution-free observations was used for statistical analysis of CBF between nasal cilia exposed to halotfiane and matched air controls at 2 h. Results Data are shown in table 1. The initial CBF of samples before exposure to halothane in the study groups were mean 10.4 (SD 1.5) Hz, 11.5 (2) Hz and 12.1 (2.2) Hz. The initial control group CBF and at hourly intervals were 11.0 (2.3) Hz, 11.1(2.2), 11.1 (2.4) Hz and 11.1 (1.9) Hz, respectively. There were no significant differences in the control group CBF measurements. These CBF measurements were in good agreement with other published data [8, 10]. There was a decrease in mean CBF after exposure to halothane at all concentrations used. When compared with CBF of matched controls at 2 h (10.8 (2.7) Hz, 11.6 (2.1) Hz and 12.1 (2.3) Hz), there was a significant decrease in CBF (8.4 (2.5) Hz, 9.18 (2.6) Hz and 6.99 (4.9) Hz) of samples exposed to all concentrations of halothane (0.9%, 1.8% and 5.7 %) (P < 0.001, Mann-Whitney). Because of the physical limitations of the technique we could only perform matched air control measurements at 2 h. The samples exposed to 5.7 % halothane showed the greatest increase in coefficient of variation and lower CBF measurements (6.99 (4.9) Hz) at 2 h compared with the other samples exposed to smaller concentrations of halothane (0.9% and 1.8%). This reflects the fact that large areas of ciliated epithelium became immotile after exposure to the highest concentration of halothane (5.7%). Mean CBF did not return to pre-exposure values after the 50-min washout period (8.6 (2.8) Hz, 9.2 (2.5) Hz and 7.85 (4.5) Hz (table 1). There was an increase in the coefficient of variation of mean CBF in samples exposed to halothane. This
03
> 40 |3
60
90 120 Time (min)
150
180
Figure 3 Coefficient of variation of cilia beat frequency (CBF) after exposure to halothane 0.9% (O), 1.8% (A), 5.7% ( • ) or air (•)• Each point represents CBF measurements from six samples. Washout = air washout period. Isolated points = matched controls of samples exposed to halothane.
was most marked in those samples exposed to 5.7 % halothane. The controls and matched controls at 2 h showed little change in the coefficient of variation (fig. 3). Discussion We have found that after in vitro exposure of human ciliated respiratory epithelium to 0.9%, 1.8% and 5.7% halothane in air, mean CBF was depressed at 2 h and did not recover to pre-exposure levels after a washout period of air alone for 50 min. Depression of CBF was greatest at the highest concentration of halothane (5.7%). We are unable to comment on either the onset of CBF depression or the dose relationship of the effect of halothane. Because of the physical limitations of the method we had matched control measurements only at 2 h and therefore we also cannot comment on what happened to CBF beyond the 50-min recovery period. It is unlikely that samples exposed to 5.7% halothane would recover to normal CBF, as many of the ciliated epithelium edges had no visible movement after 2 h of exposure. The increase in coefficient of variation of the CBF measurements with time after exposure to halothane is an indicator of dyskinesia of the cilia. The effect on individual cilia cannot be qualified from these results
Downloaded from http://bja.oxfordjournals.org/ at New York University on September 1, 2015
die ciliated epithelium edges were free of mucus covering; (3) the ciliated epithelium edges were in excess of 60 urn in length; (4) four separate CBF measurements were made from each of the four edges and a mean calculated; (5) single cells and cell groups partially denuded of cilia were not measured. To measure CBF, a pinhead photodiode was placed on the image of the ciliated epithelium transmitted from the microscope on to a high resolution video monitor screen. The photodiode system was calibrated using flashing spots of known frequency displayed on the screen of a BBC computer. The beat frequency signal was transmitted to an oscilloscope. When a good quality signal was seen clearly on the oscilloscope the signal was recorded on to a computer and a power spectrum was calculated using the Anadat data analysis system. This allowed generation of a power spectrum from which CBF was measured. Each epithelial edge chosen was recorded on to a sVHS video.
509
British Journal of Anaesthesia
510
In conclusion, we have found that in vitro exposure to halothane caused depression of CBF in human
nasal epithelium and that co-ordination of ciliary beat may also be adversely affected. References 1. Gamsu G, Singer MM, Vincent HH, Berry S, Nadel JA. Postoperative impairment of mucous transport in the lung. American Review of Respiratory Disease 1976; 114: 673-679. 2. Wanner A. Clinical aspects of nasociliary transport. American Review of Respiratory Disease 1977; 116: 73-125. 3. Lichtiger M, Landa JF, Hirsch JA. Velocity of tracheal mucus in anesthetized women undergoing gynecological surgery. Anestheswlogy 1975; 42: 753-756. 4. Forbes AR, Gamsu G. Mucociliary clearance in canine lung during and after general anesthesia. Anesthesiology 1979; SO: 26-29. 5. Landa JF, Hirsch JA, Lebeaux MI. Effects of topical and general anesthetics on tracheal mucous velocity of sheep. Journal of Applied Physiology 1975; 38: 946-948. 6. Rubin BR, Finegan B, Ramirez O, King M. General anesthesia does not alter the viscoelastic properties of human respiratory mucus. Chest 1990; 98: 1101-1104. 7. Nunn JF, Wills EJ, McPherson CK. The effects of inhalational agents on the swimming velocity of Tetrahymena pyriformis. Journal of Cellular Sciences 1974; IS: 537-554. 8. Dalhamm T, Rylander R. Frequency of cilia beat measured with a photosensitive cell. Nature {London) 1962; 196: 592-593. 9. Rutland CJ, Griffin W, Cole PJ. An in vitro model for studying the effects of pharmacological agents on human cilia beat frequency: effects of lignocainc. British Journal of Pharmacology 1982; 13: 679-683. 10. Teichtahl H, Wright PL, Kirsner RLG. Measurement of in vitro cilia beat frequency: a television—video modification of the transmitted light technique. Medical and Biological Engineering and Computing 1986; 24: 193-196. 11. Puchelle E, Zahm JM. Influence of rheologica] properties of human bronchial secretions on the ciliary beat frequency. Biorheology 1984; 21: 265-272. 12. Breeze RG, Wheeldon EB. The cells of pulmonary airways. American Review of Respiratory Disease 1977; 116: 705-777. 13. Robson DAM, Smallman LA, Drake-Lee AB. Factors affecting ciliary function in vitro: a preliminary study. Clinical Otolaryngology 1992; 17: 125-129. 14. Forbes EAR, Horrigan RW. Mucociliary flow in the trachea during anesthesia with enflurane, ether, nitrous oxide, and morphine. Anesthesiology 1977; 46: 319-321. 15. Pizov R, Takahashi M, Hirshman CA, Croxton T. Halothane inhibition of ion transport of the tracheal epithelium Anesthesiology 1992; 76: 985-989. 16. Blanck TJJ, Thompson M. Calcium transport by cardiac sarcoplasmic reticulum: Modulation of halothane action by substrate concentration and pH. Anesthesia and Analgesia 1982; 61: 142. 17. Wheeler DM, Rice RT, Hansford RG, Lakarta EG. The effect of halothane on the free intracellular calcium concentration of isolated rat heart cells. Anesthesiology 1988; 69: 578. 18. Malinconico ST, McCarl RL. Effect of halothane on cardiac sarcoplasmic reticulum Ca^-ATPase at low Ca++ concentrations. Molecular Pharmacology 1982; 22: 8.
Downloaded from http://bja.oxfordjournals.org/ at New York University on September 1, 2015
as our method measures CBF of a group of nasal cilia. If in vivo exposure to high concentrations of halothane produces similar effects on respiratory epithelium then impairment of ciliary function throughout the airways would cause disruption of mucus transport mechanisms. In our study the influence of mucus can be excluded because CBF was measured in vitro, from mucus free edges of ciliated epithelium. Robson, Smallman and Drake-Lee [13] have shown that responses of human nasal cilia to adrenergic drugs suggest the presence of a and perhaps P receptors on ciliated epithelium. Also, ionic fluxes across the ciliated membrane may be important in ciliary activity which may resemble nervous tissue in having excitatory and resting membrane potentials [13]. Forbes and Horrigan [14] showed that halothane and enflurane depressed mucociliary flow in dogs whereas ether did not. It is of interest that ether has sympathomimetic properties. Neither halothane which is a hydrocarbon nor enflurane which is a halogenated ether have sympathomimetic properties. A study on dog respiratory epithelium by Pizov and colleagues [15] showed that halothane significantly decreases ion and water transport and that impaired fluid secretion may contribute to decreased mucus clearance in the perioperative period [11]. In isolated sarcoplasmic reticulum (SR) vesicles [16] and in isolated myocytes [17], decreased Ca2+ accumulation in the SR occurs in response to inhaled anaesthetics. At Ca2+ concentrations similar to those used in studies of skinned fibres, isolated SR ATPase activity is also inhibited by halothane [18]. This decreased accumulation of Ca2+ or reduced ATPase activity may influence the microtubules of the cilia axoneme which are capable of cyclically changing shape as they bind and hydrolyse adenosine triphosphate (ATP). Studies on canine lungs [4] and humans undergoing general anaesthesia for gynaecological operations [3] have demonstrated depression of MTR by halothane in combination with other anaesthetic agents. These observations suggest that halothane may have a directly toxic effect on ion channels in the ciliated epithelium. The dose relationship and reversibility of this effect during clinical use of halothane requires further investigation before determining its contribution to postoperative pulmonary atelectasis and chest infection.
LABORATORY INVESTIGATIONS
Effect of halothane on cilia beat frequency of ciliated human respiratory epithelium in vitro\ A. GYI, C. O'CALLAGHAN AND J. A. LANGTON
Summary
Key words Anaesthetics volatile, halothane. Lung, cilia.
Mucus transport rate (MTR) is dependent on the inter-relationship between the integrity of ciliated columnar epithelium, the viscoelastic properties of the mucus, cilia beat frequency (CBF) and co-ordination of the cilia beat. Impairment of mucociliary clearance after general anaesthesia may predispose patients to atelectasis and postoperative basal zone collapse leading to impairment of gas exchange and predisposing to chest infection [1,2]. Decreased MTR has been demonstrated in areas of postoperative atelectasis [1]. Previous work has shown that general anaesthetic agents depress MTR in humans [3], dogs [4] and sheep [5]. The mechanism by which general anaesthetic agents reduce MTR may be depression of ciliary function or an effect on the production and physical properties of respiratory mucus. However, Rubin and colleagues [6] demonstrated in humans that general anaesthetic agents do not alter the viscoelastic properties of respiratory mucus. Nunn, Wills and McPherson [7] showed that general anaesthetic agents depressed swim velocity in ciliated protozoa Tetrahymena pyriformis. The effect of individual modern inhalation anaesthetic agents on human CBF has not been studied. The aim of this study was to examine the effects of halothane on human CBF in vitro. The technique of measurement of CBF using transmitted light was first described by Dalhamm
Subjects and methods After Ethics Committee approval and with informed consent, we studied 24 volunteers aged 22-35 yr with no history of asthma, atopy or smoking. None had suffered an upper respiratory tract infection within the previous 2 weeks and none had been exposed to halothane for at least 24 h. A small bronchoscopy brush was used to obtain a nasal brushing from each volunteer (without local anaesthesia). The nasal brushings were placed into MDE 199 cell culture media and taken to the laboratory for measurement within 20 min of collection. Cilia have been shown previously to remain beating at a normal frequency for periods in excess of 24 h after collection from volunteers [9]. The 24 nasal brushings were divided into four groups of six nasal brushings. Three groups were exposed to either 0.9 %, 1.8 % or 5.7 % halothane. One group of six was used as a control and was exposed to air only. Nasal brushings for CBF measurements after exposure to halothane were divided into two test tubes, each containing 5 ml of MDE 199 medium and placed in a water bath maintained at 37 °C. One test tube was exposed to halothane in air and the other was used as a matched control sample and exposed to air alone (Fig. 1).
HALOTHANE EXPOSURE AND CBF MEASUREMENT
The Fluotec 3 plenum halothane vaporizer (Cyprane) was calibrated using a Capnomac gas analyser. At dial settings 1, 2 and 5, the vaporizer consistently delivered halothane concentrations of 0.9%, 1.8% and 5.7%. The concentration of halothane delivered was stable over 3 h. The carrier gas was air from a cylinder and the flow was adjusted A. GYI, MB, BS, FRCA, J. A. LANGTON, MB, BS, FRCA (University Department of Anaesthesia); C. O'CALLAGHAN, BMEDSCI, MRCP,
DM (University Department of Child Health); Leicester Royal Infirmary, Leicester LEI 5WW. Accepted for publication: March 18, 1994. t Presented in pan at the Anaesthetic Research Society, Oxford Meeting, July 9-10, 1993 (British Journal of Anaesthesia 1993; 71: 757P-758P).
Downloaded from http://bja.oxfordjournals.org/ at New York University on September 1, 2015
The effect of halothane on human ciliated nasal epithelium was studied in vitro. Samples from 24 healthy adult volunteers were exposed to halothane in varying concentrations and cilia beat frequency was measured using the transmitted light technique. Mean cilia beat frequency was measured at 30-min intervals. There was a significant decrease in cilia beat frequency at 2 h in samples that were exposed to halothane (mean 8.4 (SD 2.5) Hz, 9.18 (2.6) Hz and 6.99 (4.9) Hz) compared with air (10.8(2.7) Hz, 11.6(2.1) Hzand12.1 (2.3) Hz) (P <0.01). The coefficient of variation of cilia beat frequency measurements increased after exposure to halothane. There was no change in the cilia beat frequency of controls exposed to air over a 3-h period. (Br. J. Anaesth. 1994; 73: 507-510)
and Rylander 1962 [8]; this is the most widely used technique for measuring CBF. Transmitted light techniques have been shown to produce reproducible results, are convenient to use and require minimal subjective assessment.
British Journal of Anaesthesia
508 One nasal brushing from each x 24 volunteers
0.9% Halothane Six nasal brushings
Each nasal brushing
Paired control 1 1.8% Halothane Six nasal brushings
Six nasal brushings
Each nasal brushing
Each nasal brushing
1 Paired control 1 5.7% Halothane ~*fPaired control |
Six nasal brushings
Air controls J
Figure 1 Study design showing division of 24 nasal brushing into four groups. A portion from each nasal brushing exposed to halothane was used as matched control.
Video camera t
Television monitor
Phase contrast microscope oil immersion lensx 100 >
Sample slide
Computerized analysis Anadat data analysis program 10-s digital signal t
Data storage
\
r
Power spectrum (mean CBF)
Figure 2 Flow chart of measurement technique for cilia beat frequency (CBF). A television-video modification of the transmitted light technique was used.
through a dry gas bobbin flowmeter. This air was humidified and divided equally into two channels. One channel passed directly to the surface of the control sample test tube, the other flowed through the vaporizer to the surface of the other test tube. The surface of the media in the test tubes containing ciliated samples was therefore exposed to either halothane in air at 100 ml min~l or air only at 100mlmin~'. Using gas chromatography we determined that the time required for halothane concentrations in the media to equilibrate with the carrier gas under these conditions was 15min. Because of physical limitations of slide preparation and the amount of nasal cilia in the sample, CBF of the matched samples was measured after the test tube had been placed in the water bath for 5 min and 2 h later. CBF of the samples exposed to halothane were measured at 30, 60, 90 and 120 min after which the vaporizer was turned off and the sample was exposed to air alone, for a washout period of 50 min. CBF was measured at the end of this washout period. Six nasal brushings were exposed to air alone and CBF measurements were made hourly for 3 h. Throughout the experiment small amounts of distilled water were added to the sample to allow for evaporation. The osmolality of the stock (MDE 199 culture medium) and the osmolalities of the solutions from the test tubes at the end of the experiment were measured. After the above procedure, osmolalities of the solutions in the two test tubes at the end of the
experiment did not differ by more than 3 % from the stock solution. For CBF measurements, samples were retrieved from the test tube and mounted on to a glass slide cover slip chamber. A temperature probe was attached on to the chamber and the heating stage was adjusted to maintain the slide temperature at 37 + 0.5 °C. Five minutes was allowed for temperature equilibration before measurement of CBF. METHOD OF CBF MEASUREMENT
CBF was measured using a television-video modification of the transmitted light technique, described by Teichtahl, Wright and Kirsner [10]. This is an accurate and reproducible method of measuring CBF in vitro, producing data in good agreement with other published data [8, 11]. Samples of human ciliated nasal epithelium were obtained by nasal brushing and examined, at body temperature (37 °C), using a phase contrast microscope with an oil immersion x 100 lens. Ciliated cells are columnar (20 um x 7 um) [12]. A ciliated columnar cell contains 100-250 cilia. Each cilium is 6 um x 0.3 um [12]. The image of the ciliated edge of the epithelium was projected onto a high resolution monitor by an sVHS video camera (fig. 2). The criteria for measurement of CBF were as follows: (1) CBF measurements were made from the first four undisrupted edges of ciliated epithelium sheets; (2)
Downloaded from http://bja.oxfordjournals.org/ at New York University on September 1, 2015
Pinhead photodiodef — * • Oscilloscope
Effect of halothane on cilia beat frequency
Table 1 Mean (SD) cilia beat frequency (Hz) at different times for each group. *Immediately after a 50-min air washout period Time (min)
0.9% Halothane
Air
11.0(2.3)
10.4(1.5) 8.0(2.0) 11.1 (2.2) 8.8(2.1) 8.5 (2.5) 11.1 (2.4) 8.4 (2.5) 11 1(1 9) 50minAW* 8.6 (2.8) Matched air 10.8 (2.7) controls at 120 min 0 30 60 90 120 180
1.8% Halothane
5.7% Halothane
11.5(2.0) 9.9(2.1) 8.8 (2.7) 9.4 (2.4) 9.2 (2.6)
12.1 (2.2) 9.7 (2.9) 10.1(3.8) 8.1 (4.0) 7.0 (4.9)
9.2 (2.5) 11.6(2.1)
7.9 (4.5) 12.1(2.3)
701 60 •3 50
STATISTICAL METHODS
Kruskal-Wallis one-way analysis of variance was used to determine statistical differences in CBF measurements of control samples over 3 h. The Mann-Whitney U test for distribution-free observations was used for statistical analysis of CBF between nasal cilia exposed to halotfiane and matched air controls at 2 h. Results Data are shown in table 1. The initial CBF of samples before exposure to halothane in the study groups were mean 10.4 (SD 1.5) Hz, 11.5 (2) Hz and 12.1 (2.2) Hz. The initial control group CBF and at hourly intervals were 11.0 (2.3) Hz, 11.1(2.2), 11.1 (2.4) Hz and 11.1 (1.9) Hz, respectively. There were no significant differences in the control group CBF measurements. These CBF measurements were in good agreement with other published data [8, 10]. There was a decrease in mean CBF after exposure to halothane at all concentrations used. When compared with CBF of matched controls at 2 h (10.8 (2.7) Hz, 11.6 (2.1) Hz and 12.1 (2.3) Hz), there was a significant decrease in CBF (8.4 (2.5) Hz, 9.18 (2.6) Hz and 6.99 (4.9) Hz) of samples exposed to all concentrations of halothane (0.9%, 1.8% and 5.7 %) (P < 0.001, Mann-Whitney). Because of the physical limitations of the technique we could only perform matched air control measurements at 2 h. The samples exposed to 5.7 % halothane showed the greatest increase in coefficient of variation and lower CBF measurements (6.99 (4.9) Hz) at 2 h compared with the other samples exposed to smaller concentrations of halothane (0.9% and 1.8%). This reflects the fact that large areas of ciliated epithelium became immotile after exposure to the highest concentration of halothane (5.7%). Mean CBF did not return to pre-exposure values after the 50-min washout period (8.6 (2.8) Hz, 9.2 (2.5) Hz and 7.85 (4.5) Hz (table 1). There was an increase in the coefficient of variation of mean CBF in samples exposed to halothane. This
03
> 40 |3
60
90 120 Time (min)
150
180
Figure 3 Coefficient of variation of cilia beat frequency (CBF) after exposure to halothane 0.9% (O), 1.8% (A), 5.7% ( • ) or air (•)• Each point represents CBF measurements from six samples. Washout = air washout period. Isolated points = matched controls of samples exposed to halothane.
was most marked in those samples exposed to 5.7 % halothane. The controls and matched controls at 2 h showed little change in the coefficient of variation (fig. 3). Discussion We have found that after in vitro exposure of human ciliated respiratory epithelium to 0.9%, 1.8% and 5.7% halothane in air, mean CBF was depressed at 2 h and did not recover to pre-exposure levels after a washout period of air alone for 50 min. Depression of CBF was greatest at the highest concentration of halothane (5.7%). We are unable to comment on either the onset of CBF depression or the dose relationship of the effect of halothane. Because of the physical limitations of the method we had matched control measurements only at 2 h and therefore we also cannot comment on what happened to CBF beyond the 50-min recovery period. It is unlikely that samples exposed to 5.7% halothane would recover to normal CBF, as many of the ciliated epithelium edges had no visible movement after 2 h of exposure. The increase in coefficient of variation of the CBF measurements with time after exposure to halothane is an indicator of dyskinesia of the cilia. The effect on individual cilia cannot be qualified from these results
Downloaded from http://bja.oxfordjournals.org/ at New York University on September 1, 2015
die ciliated epithelium edges were free of mucus covering; (3) the ciliated epithelium edges were in excess of 60 urn in length; (4) four separate CBF measurements were made from each of the four edges and a mean calculated; (5) single cells and cell groups partially denuded of cilia were not measured. To measure CBF, a pinhead photodiode was placed on the image of the ciliated epithelium transmitted from the microscope on to a high resolution video monitor screen. The photodiode system was calibrated using flashing spots of known frequency displayed on the screen of a BBC computer. The beat frequency signal was transmitted to an oscilloscope. When a good quality signal was seen clearly on the oscilloscope the signal was recorded on to a computer and a power spectrum was calculated using the Anadat data analysis system. This allowed generation of a power spectrum from which CBF was measured. Each epithelial edge chosen was recorded on to a sVHS video.
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British Journal of Anaesthesia
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In conclusion, we have found that in vitro exposure to halothane caused depression of CBF in human
nasal epithelium and that co-ordination of ciliary beat may also be adversely affected. References 1. Gamsu G, Singer MM, Vincent HH, Berry S, Nadel JA. Postoperative impairment of mucous transport in the lung. American Review of Respiratory Disease 1976; 114: 673-679. 2. Wanner A. Clinical aspects of nasociliary transport. American Review of Respiratory Disease 1977; 116: 73-125. 3. Lichtiger M, Landa JF, Hirsch JA. Velocity of tracheal mucus in anesthetized women undergoing gynecological surgery. Anestheswlogy 1975; 42: 753-756. 4. Forbes AR, Gamsu G. Mucociliary clearance in canine lung during and after general anesthesia. Anesthesiology 1979; SO: 26-29. 5. Landa JF, Hirsch JA, Lebeaux MI. Effects of topical and general anesthetics on tracheal mucous velocity of sheep. Journal of Applied Physiology 1975; 38: 946-948. 6. Rubin BR, Finegan B, Ramirez O, King M. General anesthesia does not alter the viscoelastic properties of human respiratory mucus. Chest 1990; 98: 1101-1104. 7. Nunn JF, Wills EJ, McPherson CK. The effects of inhalational agents on the swimming velocity of Tetrahymena pyriformis. Journal of Cellular Sciences 1974; IS: 537-554. 8. Dalhamm T, Rylander R. Frequency of cilia beat measured with a photosensitive cell. Nature {London) 1962; 196: 592-593. 9. Rutland CJ, Griffin W, Cole PJ. An in vitro model for studying the effects of pharmacological agents on human cilia beat frequency: effects of lignocainc. British Journal of Pharmacology 1982; 13: 679-683. 10. Teichtahl H, Wright PL, Kirsner RLG. Measurement of in vitro cilia beat frequency: a television—video modification of the transmitted light technique. Medical and Biological Engineering and Computing 1986; 24: 193-196. 11. Puchelle E, Zahm JM. Influence of rheologica] properties of human bronchial secretions on the ciliary beat frequency. Biorheology 1984; 21: 265-272. 12. Breeze RG, Wheeldon EB. The cells of pulmonary airways. American Review of Respiratory Disease 1977; 116: 705-777. 13. Robson DAM, Smallman LA, Drake-Lee AB. Factors affecting ciliary function in vitro: a preliminary study. Clinical Otolaryngology 1992; 17: 125-129. 14. Forbes EAR, Horrigan RW. Mucociliary flow in the trachea during anesthesia with enflurane, ether, nitrous oxide, and morphine. Anesthesiology 1977; 46: 319-321. 15. Pizov R, Takahashi M, Hirshman CA, Croxton T. Halothane inhibition of ion transport of the tracheal epithelium Anesthesiology 1992; 76: 985-989. 16. Blanck TJJ, Thompson M. Calcium transport by cardiac sarcoplasmic reticulum: Modulation of halothane action by substrate concentration and pH. Anesthesia and Analgesia 1982; 61: 142. 17. Wheeler DM, Rice RT, Hansford RG, Lakarta EG. The effect of halothane on the free intracellular calcium concentration of isolated rat heart cells. Anesthesiology 1988; 69: 578. 18. Malinconico ST, McCarl RL. Effect of halothane on cardiac sarcoplasmic reticulum Ca^-ATPase at low Ca++ concentrations. Molecular Pharmacology 1982; 22: 8.
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as our method measures CBF of a group of nasal cilia. If in vivo exposure to high concentrations of halothane produces similar effects on respiratory epithelium then impairment of ciliary function throughout the airways would cause disruption of mucus transport mechanisms. In our study the influence of mucus can be excluded because CBF was measured in vitro, from mucus free edges of ciliated epithelium. Robson, Smallman and Drake-Lee [13] have shown that responses of human nasal cilia to adrenergic drugs suggest the presence of a and perhaps P receptors on ciliated epithelium. Also, ionic fluxes across the ciliated membrane may be important in ciliary activity which may resemble nervous tissue in having excitatory and resting membrane potentials [13]. Forbes and Horrigan [14] showed that halothane and enflurane depressed mucociliary flow in dogs whereas ether did not. It is of interest that ether has sympathomimetic properties. Neither halothane which is a hydrocarbon nor enflurane which is a halogenated ether have sympathomimetic properties. A study on dog respiratory epithelium by Pizov and colleagues [15] showed that halothane significantly decreases ion and water transport and that impaired fluid secretion may contribute to decreased mucus clearance in the perioperative period [11]. In isolated sarcoplasmic reticulum (SR) vesicles [16] and in isolated myocytes [17], decreased Ca2+ accumulation in the SR occurs in response to inhaled anaesthetics. At Ca2+ concentrations similar to those used in studies of skinned fibres, isolated SR ATPase activity is also inhibited by halothane [18]. This decreased accumulation of Ca2+ or reduced ATPase activity may influence the microtubules of the cilia axoneme which are capable of cyclically changing shape as they bind and hydrolyse adenosine triphosphate (ATP). Studies on canine lungs [4] and humans undergoing general anaesthesia for gynaecological operations [3] have demonstrated depression of MTR by halothane in combination with other anaesthetic agents. These observations suggest that halothane may have a directly toxic effect on ion channels in the ciliated epithelium. The dose relationship and reversibility of this effect during clinical use of halothane requires further investigation before determining its contribution to postoperative pulmonary atelectasis and chest infection.