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Department of Physiology, National Taiwan University College of Medicine, Taipei, Taiwan
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ABSTRACT |
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Mice have been widely used in immunologic and other research to study the influence of different diseases on the lungs. However, the respiratory mechanical properties of the mouse are not clear. This study extended the methodology of measuring respiratory mechanics of anesthetized rats and guinea pigs and applied it to the mouse. First, we performed static pressure-volume and maximal expiratory flow-volume curves in 10 anesthetized paralyzed C57BL/6 mice. Second, in 10 mice, we measured dynamic respiratory compliance, forced expiratory volume in 0.1 s, and maximal expiratory flow before and after methacholine challenge. Averaged total lung capacity and functional residual capacity were 1.05 ± 0.04 and 0.25 ± 0.01 ml, respectively, in 20 mice weighing 22.2 ± 0.4 g. The chest wall was very compliant. In terms of vital capacity (VC) per second, maximal expiratory flow values were 13.5, 8.0, and 2.8 VC/s at 75, 50, and 25% VC, respectively. Maximal flow-static pressure curves were relatively linear up to pressure equal to 9 cmH2O. In addition, methacholine challenge caused significant decreases in respiratory compliance, forced expiratory volume in 0.1 s, and maximal expiratory flow, indicating marked airway constriction. We conclude that respiratory mechanical parameters of mice (after normalization with body weight) are similar to those of guinea pigs and rats and that forced expiratory maneuver is a useful technique to detect airway constriction in this species.
static compliance; dynamic compliance; forced expiratory maneuver; airway reactivity
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INTRODUCTION |
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MICE HAVE BEEN WIDELY USED in immunologic research to investigate the influence of different diseases on the lungs. However, functional analysis of the airway in mice is often limited by their small dimensions. The pressure-volume (P-V) (7, 13) and maximal expiratory flow-volume (MEFV) curves (13) were rarely evaluated in the mouse. Therefore, the first aim of this study was to establish a method to perform the P-V and MEFV curves.
In addition to resistance and dynamic compliance (2, 5, 8, 14, 18),
values of forced expiratory volume (FEV) and maximal expiratory flow
(
max) are useful in estimation of airway
constriction (10, 22). The second aim of this study was to compare
values of FEV and
max with dynamic
compliance before and after a bronchoconstrictor-induced airway constriction.
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MATERIALS AND METHODS |
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Determination of respiratory mechanics.
Ten 8- to 12-wk-old C57BL/6 mice weighing 20.6 ± 0.4 g were used in
this study. After anesthesia with pentobarbital sodium (70 mg/kg ip),
each animal's trachea, carotid artery, and jugular vein were
cannulated with an 18-gauge needle, PE-10 tube, and PE-10 tube,
respectively. After being paralyzed with gallamine triethiodide (1 mg/kg), the animal was artificially ventilated with a tidal volume
(VT) of 8-10 ml/kg and frequency of 120 breaths/min by
using a small animal ventilator. It has been shown that arterial acid-base balance was maintained in the normal condition under this
type of artificial ventilation (8, 14). To ensure that the animal was
anesthetized during paralysis, we administered gallamine according to
the following plan. 1) Gallamine was only given when its active
period (40 min) (17) was within the effective duration of pentobarbital
(1-2 h) (17). If it was necessary to administer gallamine beyond
this effective period of the anesthetic, supplemental doses of
pentobarbital were given before any more gallamine treatment.
2) If an additional dose of gallamine was needed after a single
dose, we first examined the level of anesthesia and made sure that the
expected anesthesia could be maintained longer than the effective
duration of gallamine. The second injection of gallamine was then given
to the animal. The anesthetized paralyzed and ventilated animal was
placed supine inside a whole body plethysmograph (6 cm ID and 15.25 cm
length) for the mouse (Buxco Electronics, Troy, NY). According to Buxco
Electronics, the frequency response characteristics of the
plethysmograph were flat to >60 Hz. The flow rate was monitored with
a Validyne DP45 differential pressure transducer as the pressure
dropped across three layers of 325-mesh wire screen in the wall of the
plethysmograph. Between a flow rate of 0 and 20 ml/s, we found that
there was a linear relationship between the flow rate and pressure
signal. Lung volume change was obtained via integration of flow. Airway
opening pressure (Pao) was measured with a pressure transducer
(DTX/plus, Viggo-Spectramed). MEFV and static P-V maneuvers were
performed according to our previous method (11). Briefly, for the MEFV
maneuver, the lungs were inflated three times to total lung capacity
(TLC; lung volume at Pao = 30 cmH2O). At peak
volume during the third inflation, the inflation valve was shut off,
and immediately another solenoid valve for deflation was automatically
turned on. The deflation valve was connected to a 20-liter container
with a negative pressure of 40 cmH2O. This negative
pressure produced the
max. The changes in flow, volume, and Pao were traced on a polygraph (model TA11, Gould), and the MEFV plot was also stored on a cathode-ray storage oscilloscope (VC-6025, Hitachi). The flow-volume curve reproduced fairly well for each animal but varied a great deal among animals.
10 cmH2O
or lower. During artificial ventilation (between the interval of the
MEFV or P-V maneuvers), VT and its accompanying Pao
difference (
Pao) were used to calculate dynamic respiratory compliance (Crs = VT/
Pao).
Pao was measured between
end inspiration and end expiration (i.e., instances of no flow).
Before and after each MEFV or P-V maneuver, functional residual
capacity (FRC) (the lung volume at Pao = 0) was determined by using a
modified neon dilution method (11). Starting from FRC, the lungs were
inflated with a standard neon (0.5%) gas mixture to 50% vital
capacity (VC). Gas in the lungs, in the dead space of the instrument,
and in the syringe was mixed thoroughly by repeating the injection and
withdrawing the gas mixture 10-20 times. The equilibrated gas
mixture was withdrawn and analyzed with a Varian gas chromatograph
(model 3300). The total volume (including FRC and instrumental dead
space) was calculated. The FRC was obtained by subtracting the
instrumental dead space from the total volume.
In addition, systemic arterial blood pressure was monitored from the
carotid artery with a pressure transducer. Body temperature of the
animal was estimated from the temperature detected from the rectum by
using a thermister.
Test of bronchial function before and after methacholine challenge.
To induce airway constriction, 10 anesthetized paralyzed animals
weighing 23.8 ± 0.4 g were challenged with intravenous injection of
methacholine (2 mg/kg). This methacholine dose was relatively high
compared with the doses used by Levitt and Mitzner (15) and by Martin
et al. (18) in C57L/6 mice. Immediately after the injection,
VT decreased and Pao increased gradually. These changes in
VT and Pao reached a maximum 40.5 ± 1.8 s after the injection (see RESULTS). At the baseline (before
methacholine challenge) as well as at the time of the maximal response
to methacholine, the MEFV maneuver was performed and the Crs value was
calculated in each animal. Values of FEV in 0.1 s (FEV0.1),
max at 50% VC
(
max 50), and
max at 30% VC
(
max 30) were obtained from tracings
of the MEFV curve.
Statistical analysis. All values are reported as means ± SE. ANOVA was used to establish the statistical significance of differences among groups. If significant differences among groups were obtained by using the ANOVA, then Duncan's multiple-range test was used to differentiate the differences between groups. Differences were considered significant if P < 0.05.
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RESULTS |
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In 20 anesthetized paralyzed mice weighing 22.2 ± 0.4 g, averaged blood pressure and respiratory parameters were as follows: mean systemic blood pressure, 74.4 ± 2.0 mmHg; body temperature, 29.4 ± 0.4°C; FRC, 0.25 ± 0.01 ml; TLC, 1.05 ± 0.04 ml; VC, 0.95 ± 0.03 ml; static lung compliance, 0.075 ± 0.004 ml/cmH2O; and Crs, 0.021 ± 0.001 ml/cmH2O. Therefore, FRC occurred around 24% TLC.
Respiratory mechanics.
Mean static P-V curves of the lung, chest wall, and total respiratory
system are illustrated in Fig. 1. For
volume changes between 100 and 10% TLC, mean pressure changes in the
lung, chest wall, and total respiratory system were 30.1, 4.9, and 35 cmH2O, respectively. The curve of the chest wall was fairly
steep, and its total pressure range was small, indicating a compliant
chest wall. Because of the compliant chest wall, the curve of the total system was nearly similar to that of the lungs.
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max,
max decreased gradually until it reached
residual volume (RV) at ~10% TLC.
max
values were 13.5, 8.0, and 2.8 VC/s at 75, 50, and 25% VC,
respectively. Combining both MEFV and static P-V curves of the lung,
the maximal flow-static pressure curves were plotted in Fig.
3. The fitted correlation curves were
relatively linear, and all slopes were significantly different from
zero.
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Bronchial function before and after methacholine challenge.
Intravenous injection of methacholine induced changes in flow, volume,
and Pao (Fig. 4). Both flow and volume
decreased, whereas Pao increased soon after the challenge. The change
in Pao reached a maximum around 40 s after the challenge. In addition,
methacholine challenge caused a marked change in the flow-volume curve
(Fig. 5). Averaged values of Crs,
FEV0.1,
max 50, and
max 30 before and after methacholine
challenge are listed in Table 1.
Methacholine challenge caused significant decreases in all values,
indicating methacholine-induced marked airway constriction.
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DISCUSSION |
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Averaged TLC and FRC were 1.05 ± 0.04 and 0.25 ± 0.01 ml,
respectively, in anesthetized paralyzed mice. The static P-V curve of
the chest wall was fairly compliant and the FRC-to-TLC ratio (FRC/TLC) was relatively low in the mouse. The peak
max was 16.0 ± 0.7 ml/s and
occurred at 86.0% VC.
max values were
13.5, 8.0, and 2.8 VC/s at 75, 50, and 25% VC, respectively. In
addition, methacholine challenge caused significant decreases in Crs,
FEV0.1,
max 50, and
max 30. Several features of these
results will be discussed below.
Respiratory mechanical properties of the mouse. The peak Pao used for the mean static P-V curve was 25 cmH2O (Fig. 1). The lung volume at this static peak Pao should be fairly close to the peak volume at dynamic peak Pao of 30 cmH2O during the MEFV maneuver because of the following three reasons. 1) There is a resistive pressure loss during the dynamic condition. This resistive pressure loss is required to overcome the airway and lung tissue resistance but cannot help to expand the lung. 2) There is a stress relaxation immediately after the transition from the dynamic to the static condition or flow interruption. 3) The P-V curve is fairly flat at the volume close to TLC. A difference in pressure of 1-2 cmH2O should produce very little change in volume. Accordingly, we believed that TLCs during the static P-V and dynamic MEFV maneuvers should occur at about the same volume.
Compared with the rat (12), the mouse has a similar FRC/TLC and P-V curve characteristics. Compared with normal awake humans, however, the anesthetized paralyzed mouse has a smaller FRC and RV expressed as %TLC. Although anesthesia and/or paralysis might alter respiratory mechanics, the lower FRC and RV may be related mainly to the compliant chest wall. Let us compare compliance in the VT range for the mouse and humans. In the horizontal posture, the mouse's chest wall is much more compliant (23.6 ml · cmH2O
1 · kg
body wt
1) than that of humans (2.9 ml · cmH2O
1 · kg
body wt
1) (21). The P-V curve of the mouse's chest
wall, compared with that of humans, is shifted to the right below
midlung volumes. This shift moves the point of resting balance of
forces between the lung and the chest wall to a lower volume and
results in a lower FRC. Contrary to chest wall compliance, the value of
lung compliance is closer between humans (3.7 ml · cmH2O
1 · kg
body wt
1) (21) and the mouse (3.6 ml · cmH2O
1 · kg
body wt
1 obtained from Fig. 1) within the
VT range. Gillespie (6) reviewed cross-species data showing
the negative correlation trend between chest wall compliance/TLC and
FRC/TLC. In addition, Leith (13) suggests that the mouse's chest wall
is so compliant that mice set their FRC with constant activation of the
inspiratory muscles. Functionally, FRC acts as a buffer for pulmonary
gas exchange (1). To compensate for its lower FRC, the mouse has a much higher breathing frequency than do humans so that fluctuation in
alveolar gas concentration is correspondingly reduced.
max.
Peak
max was 16.0 ml/s and occurred at
86.0% VC (or 88.5% TLC) in the mouse. The occurrence of the mice's
peak
max, expressed as %TLC, was similar
to that of guinea pigs at 84% TLC (11) and hamsters at 70-95%
TLC (16). After peak
max,
max decreased gradually with reducing
lung volume. No apparent plateau on the MEFV curve was detected. This
may be related to the negative pressure employed to perform the MEFV
curve in this study. In our previous study of
max in guinea pigs, a plateau
on the MEFV curve was usually found when a vacuum pressure of
10
to
20 cmH2O was used but not when a vacuum pressure
of
40 cmH2O or higher was used (11).
max of
the mouse (13.2 VC/s) is similar to that of the rat (18.0 VC/s) (3) and
the guinea pig (17.9 VC/s) (11). However, the mouse's peak
max is much higher than that of humans
with 1.8 VC/s (9). Similarly,
max values
(in VC/s) at other lung volumes are much higher than those of humans
(Table 2). This higher
max would indicate a lower airway
resistance and could help to reduce the work of breathing in this
species with high breathing frequency mentioned in Respiratory mechanical properties of the mouse. In addition, peak
max (VC/s) values have been demonstrated
to be correlated closely with resting metabolic rate among several
mammalian species (13), including the mouse. The linear pressure-flow
relationship (Fig. 3) may indicate that we measured the relationship
over essentially linear parts of both the flow-volume curve and the
static P-V curve within a pressure range of 1-9
cmH2O. Both recoil pressure and
max decreased gradually with the decrease
in lung volume. The slope of this pressure-flow curve indicates simply
the upstream resistance (or conductance) of the pulmonary system (19).
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Bronchial response to methacholine challenge.
Changes in resistance and dynamic compliance are often used as
indicators of airway constriction (4). However, alterations in
max and FEV also provide a reliable
estimation of changes in bronchial dimension (10, 22). In addition,
because of the very compliant chest wall in our animals, that change in
Crs was considered to be mainly the change in dynamic compliance of the lung. Accordingly, changes in Crs, FEV0.1,
max 50, and
max 30 were compared in the mouse in
response to methacholine challenge in this study. This methacholine
challenge caused 61.8, 61.0, 65.1, and 67.1% decreases in Crs,
FEV0.1,
max 50, and
max 30, respectively, indicating
marked airway constriction. It is possible that the change in dynamic
compliance indicates an alteration in the peripheral airway.
max is related mainly to the mechanical property of the portion of the airway upstream from the flow-limiting (equal pressure) point (10, 19). Because the flow-limiting point is
moving to the peripheral portion of the airway when lung volume is
reduced,
max should be related mainly to
the peripheral airway resistance at low lung volume such as FRC, with
the decreased FEV0.1 value reflecting, perhaps, the more
central, and flows at the lower volume the more peripheral, airways.
Accordingly, our findings of similar methacholine-induced decreases in
Crs, FEV0.1, and
max may
indicate the constriction in all airways, although methacholine has
been shown to act mainly in the central airways in humans (23).
max
and FEV0.1 are also useful indicators of airway
constriction in the mouse.
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ACKNOWLEDGEMENTS |
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This work was supported by the National Science Council (NSC88-2314-B-002-222 and NSC88-2314-B002-066M41).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: Y.-L. Lai, Dept. of Physiology, College of Medicine, National Taiwan Univ., No. 1, Sect. 1, Jen-Ai Rd., Taipei, Taiwan (E-mail: tiger{at}ha.mc.ntu.edu.tw).
Received 11 January 1999; accepted in final form 27 October 1999.
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W. MITZNER, R. BROWN, and W. LEE In vivo measurement of lung volumes in mice Physiol Genomics, January 19, 2001; 4(3): 215 - 221. [Abstract] [Full Text] [PDF] |
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