British Journal of Anaesthesia 1996; 76: 111–115
A perfusion system for in vitro measurement of human cilia beat
frequency†
D. A. SELWYN, A. GYI, J. H. RAPHAEL, A. KEY AND J. A. LANGTON
Summary
We have designed and built a perfusion system and
perfusion chamber to allow investigation of the
effects of anaesthetic agents on human cilia in vitro.
Using this system, samples of human respiratory
cilia can be maintained in a stable and controlled
environment for several hours. We measured cilia
beat frequency of nasal respiratory epithelium from
10 healthy volunteers; cilia beat frequency was
constant over a 4-h period, and measurements were
found to be in good agreement with previously
published work [1]. In a separate study we
investigated the effect of a sleep dose of propofol
on cilia beat frequency in samples from six patients
undergoing minor surgery ; samples were obtained
before and immediately after induction of anaesthesia with propofol 2–3 mg kg91. There was no
statistically significant difference in cilia beat frequency between data obtained before and after
induction with propofol. (Br. J. Anaesth. 1996 ; 76 :
111–115)
Key words
Anaesthetics i.v., propofol. Lung, trachea. Equipment, perfusion
system.
After routine surgery, impairment of respiratory
epithelial cilia function may predispose patients to
develop areas of atelectasis and postoperative chest
infection; the overall incidence of chest infection
after upper abdominal surgery may be as high as
20 % [2]. It has been demonstrated that volatile
anaesthetic agents cause a decrease in mucus transport rate [3] which is an important host defence
mechanism. Respiratory epithelial cilia have a beat
cycle which is specialized to facilitate transport of
mucus and inhaled particles to the oropharynx,
where they may be swallowed or expectorated.
Mucus transport rate is dependent on the interrelationship between cilia beat frequency (CBF) and
the viscoelastic properties of mucus [4, 5]. A previous pilot study from our laboratory has shown that
halothane has a significant depressant effect on CBF
[6], but the mechanism of this effect is not known.
In order to study the effects of anaesthetic agents
on human respiratory epithelial cilia in a controlled
environment, it was necessary to build a new
perfusion chamber, as chambers described previously did not satisfy our requirements for the
investigation of anaesthetic agents. This new per-
fusion chamber enabled us to investigate the survival
of respiratory cilia in vitro and the effect of repeated
measurements of CBF.
Materials and methods
PERFUSION CHAMBER
The perfusion chamber was constructed from aluminimum in two halves, with an integral perfusion
channel that allowed fluid exchange (fig. 1). The cilia
sample was placed between a lower fixed cover slip
and an upper removable cover slip. The upper cover
slip was manufactured specially with a diameter of
45 mm and was sealed using a silicone impregnated
rubber washer. It is this large cover slip which allows
a wide plane of movement by the microscope
objective lens. The chamber depth was 0.85 mm,
producing a small internal volume of 1.1 ml.
Cilia beat frequency has been shown to be
dependent on temperature [9] and therefore the
perfusion system incorporated several features to
maintain a constant temperature. The perfusate was
kept in a heated water bath maintained at 40 ⬚C. The
perfusion channels upstream of the chamber passed
through a countercurrent water jacket maintained at
40 ⬚C. The chamber incorporated an integral thermostatically controlled heating element set to 37 ⬚C (fig.
2). The system was calibrated with a thermocouple
located in the middle of the external surface of the
cover slip and the measured temperature varied by
<0.1 ⬚C. The whole chamber was closed with an
annular threaded screw and a composite locking
device.
The cilia sample was perfused with Hanks balanced salt solution (HBSS, Gibco, Life Technologies) from an aerated reservoir. This was connected
to the chamber using metallic tubing. The medium
reservoir was maintained at a height of 25 cm above
the microscope stage to allow gravity feed of the
medium at a rate of 0.5 ml min91. A calibrated
photometric drip counter enabled a constant flow
rate to be maintained.
D. A. SELWYN, MB, BS, FRCA, A. GYI*, MB, BS, FRCA, J. H.
RAPHAEL, MB, CHB, FRCA, A. KEY, BSC (HON), J. A. LANGTON, MB,
BS, MD, FRCA, University Department of Anaesthesia, Leicester
Royal Infirmary, Leicester LE1 5WW. Accepted for publication:
August 25, 1995.
* Present address: Albert Einstein Medical Center,
Philadelphia, USA.
† A preliminary account of this work was presented at the
Anaesthetic Research Society, Liverpool Meeting, March 25–26,
1994 (British Journal of Anaesthesia 1994; 73: 269P).
112
British Journal of Anaesthesia
Figure 1
Figure 2
Section through the perfusion chamber.
Schematic diagram of the cilia perfusion system.
MEASUREMENT OF CILIA BEAT FREQUENCY
CBF was measured using a television–video modification of the transmitted light technique described
originally by Teichtahl, Wright and Kirsner [7].
This technique uses a pinhole photodiode placed
against a television image of ciliated epithelium.
Movement of the beating cilia across the photodiode
produces a characteristic signal which can be viewed
on an oscilloscope before a 15-s sample is downloaded to a 386 sx computer (Dell Computer
Corporation, Bracknell, UK). Having undergone
low-pass filtering (25 Hz) to reduce interference, the
signal was sampled at 200 Hz by an analogue to
digital converter, stored and displayed. The signal
was then processed using mathematica 2.2 software
(Wolfram Software, USA) to produce a power
spectrum using fast Fourier transforms. CBF was
taken as the peak of the power spectrum.
After specimens of cilia were loaded into the
perfusion chamber, two readings were obtained from
as many edges as possible during a 20-min sampling
period, edge selection criteria being those described
previously [6]. In summary these comprised:
measurements made only from free edges of ciliated
epithelium; the edge of the epithelium must not be
disrupted and it must be greater than 60 ␮m in
length ; the edge must not be contaminated with
mucus deposits ; and single cells and cell groups
partially denuded of cilia were not included.
The samples were viewed under a high resolution
(600) microscope (Nikon Diaphot), using the
Normaski polarized light technique. The image
obtained from the microscope was transmitted to a
high resolution video monitor and recorded onto
videotape. CBF was measured at hourly intervals for
4 h.
Table 1 Exclusion criteria for volunteers or patients recruited
for measurement of cilia beat frequency
History of asthma, hay fever or atopy.
History of smoking or cessation of smoking within the past
2 yr.
History of upper respiratory tract infection within the
preceding month.
History of coagulation or bleeding diathesis.
History of cystic fibrosis, bronchiectasis or documented cilia
motility problems.
VALIDATION OF CHAMBER PERFORMANCE
To study the time taken for drug concentrations to
change within the chamber and to ensure that flow
streaming did not occur, we perfused the chamber
with HBSS at a steady rate through a chamber
mounted on a light box. Photographs were taken at
30-s intervals from a camera (Pentax UK Limited) at
a fixed height (41.5 mm) above the light box using a
shutter speed of 1/15 s with an aperture of f11 on to
ISO 100 film (Kodak Limited, USA). After a 2-min
stabilization period, the perfusate was changed to
HBSS containing a known concentration of Dylon
ivory black dye. After 5 min the perfusate was
changed back to HBSS only. The change in dye
colour or wash-in effect was analysed using a
densitometer (LKB, Bromma), performing a line
density plot at three cuts across the chamber
photographs ; an inlet cut, a central cut and an outlet
cut.
CILIA SURVIVAL EXPERIMENTS
This study was conducted in two parts. In the first
part of the study, after obtaining Ethics Committee
approval and written informed consent, we recruited
10 healthy volunteers (mean age 28.8 yr). All volunteers satisfied our admission criteria (table 1). Nasal
A perfusion system for in vitro measurement of human cilia beat frequency
sampling is a relatively painless procedure which
does not require local anaesthesia. In all cases the
method of sampling was identical and we used a
small wire bronchoscopy brush to scrape ciliated
epithelium from the inferior nasal turbinate or
anterior nasal wall, having previously wetted the
sample brush in HBSS. The sample obtained was
placed immediately into Hanks nutrient medium at
room temperature before being transferred to the
perfusion chamber within 1 h.
In the second part of the investigation and after
obtaining Ethics Committee approval and written
informed consent, we recruited six unpremedicated
ASA I patients, undergoing removal of wisdom teeth
under general anaesthetic. Nasal sampling, as previously described, was performed immediately before induction of anaesthesia and again after a sleep
dose of propofol 2–3 mg kg91. A separate wire
bronchoscopy brush was used for each specimen.
The samples obtained were placed into nutrient
medium, at room temperature, before being transferred to the perfusion chamber within 1 h.
Baseline CBF measurements were obtained when
the specimen had been placed within the chamber,
and this represented time point zero. CBF readings
were repeated 1 h later. The chambers were perfused
with nutrient medium throughout the experiment
and maintained at 37 ⬚C (<0.1 ⬚C).
Statistical analysis was performed using SPSS for
windows v 5.01 to give analysis of variance for
repeated measures using MANOVA (multiple analysis of variance). Statistical significance was taken at
P 0.05.
113
Figure 4 Cilia beat frequency (CBF) vs time for volunteer
nasal brushings (mean, SD).
Table 2 Beat frequency (mean (SD)) in respiratory epithelial
cilia before and 1 h after induction of anaesthesia with propofol
CBF (Hz)
at 1 h
Before propofol
induction
After propofol
induction
CBF (Hz)
at 2 h
11.38 (2.33) 11.40 (2.25)
11.54 (2.17) 11.97 (1.89)
Results
VALIDATION OF THE PERFUSION SYSTEM
The results of the line density studies are shown in
figure 3. The densitometer performed a vertical line
density plot across the chamber at three points. The
change in density of the perfusion dye (mean density,
arbitrary units) was plotted against time in seconds.
The smooth advancement of the wavefront can be
seen in the three areas of study : the inlet of the
chamber, the central portion of the chamber and the
exit region. The outlet region showed a delayed time
Figure 3 Change in dye concentration with time for the three
cuts (inlet (), middle (!) and outlet (") cuts) across the
chamber.
course with respect to the inlet region. The time
taken for change within the chamber to plateau was
approximately 160 s. There was no evidence of flow
streaming or any areas of diminished perfusion.
CILIA SURVIVAL EXPERIMENTS
CBF of nasal respiratory epithelium in healthy
volunteers is shown in figure 4. The initial readings
were taken within 1 h of the specimen being
obtained. The subsequent readings were taken at
hourly intervals for a total of 4 h. The cilia samples
were perfused continuously with Hanks medium.
Each reading represents the mean of data from 10
subjects, of which each subject had at least 10
separate readings of CBF at each time. The 15-s
sampling period and mean beat frequency represents
over 1500 datum points for each subject at each time.
Mean baseline CBF in the volunteer group was
11.3 (SEM 0.774) Hz. The measured CBF was shown
to be constant over a 4-h time period and was in good
agreement with other published work [1]. There was
no statistically significant difference in CBF between
the different times.
CBF for patients before and after induction of
anaesthesia with propofol is shown in table 2. Time
point one represents the baseline readings when the
sample had been placed into the chamber and the
chamber temperature had stabilized. This was within
114
60 min of the sample being obtained from the
patient. The second time represents CBF measured
1 h later. Mean baseline CBF in the propofol group
before induction of anaesthesia was 11.4 (0.93) Hz
and after induction 11.7 (0.83) Hz. There was no
significant difference between the two groups at each
time and CBF was in good agreement with previously published work [1].
There were no anaesthetic complications caused
by sampling of nasal respiratory cilia and neither the
patients nor the volunteers found the sampling
procedure particularly painful.
Discussion
Several factors contribute to mucociliary transport,
including mucus quantity and composition, number
of ciliated cells, CBF and effectiveness and coordination of cilia beating. The exact relationship
between cilia activity or beat frequency and tracheal
clearance rate is unclear. A good correlation between
CBF and mucus transport rate (MTR) has been
reported in some studies [9], whereas in others no
direct relationship has been found [10]. Many of the
studies investigating CBF have been performed on
animal species or have used laboratory apparatus
where specimens can be kept for only short periods.
Consequently, we designed and validated a perfusion
system which allows samples of human respiratory
cilia to be kept in a stable and controlled environment
for many hours while permitting reliable and
repeated measurement of CBF in real time. The
chamber design was based on that of Braga [11], with
a number of significant improvements ; our chamber
had a large viewing area to permit increased
microscope objective travel across the sample, and it
also allowed focusing throughout the whole depth of
the sample which improved sample visualization.
CBF varies markedly with temperature ; increasing
temperature from 25 to 37 ⬚C increases CBF by 50 %
[12]. Consequently, it is important to have accurate
and stable heat control. Our chamber was temperature regulated and maintained a stable sample
environment by preheating the perfusate and using
an integral thermostatic heating element. The
chamber was completely sealed and therefore allowed accurate administration of volatile anaesthetic
agents in a known and reliable concentration [13].
Earlier pilot work revealed that volatile agent was
lost with plastic tubing , and therefore throughout
this system we have used metallic stainless steel
tubing to minimize losses.
We have shown that there was uniform flow and
therefore perfusion throughout the whole chamber
and also that significant flow streaming did not
occur. This allowed us to be confident that cilia
specimens can be maintained in optimal conditions
for long periods of time and that drugs added to the
perfusate system will bathe the whole sample under
investigation.
We chose a 4-h measurement period to enable us
to detect any delayed variation in CBF with time ;
during this period, cilia maintained normal beat
frequencies.
Initial pilot work using a peristaltic perfusion
British Journal of Anaesthesia
pump produced sample movement and disruption,
and was therefore abandoned in favour of a gravity
feed system. We chose a perfusion rate of
0.5 ml min91 as it allowed rapid change in chamber
conditions without producing sample movement or
disruption. Accurate flow of perfusate was ensured
by the use of a calibrated photometric drip counter.
With the aid of a densitometer, we showed that a
change in drug concentration would be expected to
equilibrate within the chamber after 160 s. The
chamber was easy to assemble, robust and reliable,
and easy to clean.
It is known that there are ␣ and ␤ receptors on
ciliated epithelium. Studies have shown that autonomic neurotransmitters and neurohormones have a
significant influence on mucociliary activity. The
action of these neurotransmitters and neurohormones on CBF may be mediated via two different
mechanisms, one involving calcium and one via
cAMP [14–17]. We found that in vivo, after an
induction dose of propofol 2–3 mg kg91, there was no
measurable effect on CBF. Halothane has been
shown in vitro to have a depressant effect on CBF [6]
although the mechanism for this is not known, and
further work is necessary to elucidate the exact
cellular mechanisms involved.
Acknowledgement
This study was supported by an equipment grant from Zeneca
Pharma.
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