Article
Hyperoxia-induced changes in mouse lung mechanics: forced
oscillations vs. barometric plethysmography
PETAK, Ferenc, et al.
Abstract
Hyperoxia-induced lung damage was investigated via airway and respiratory tissue
mechanics measurements with low-frequency forced oscillations (LFOT) and analysis of
spontaneous breathing indexes by barometric whole body plethysmography (WBP). WBP was
performed in the unrestrained awake mice kept in room air (n = 12) or in 100% oxygen for 24
(n = 9), 48 (n = 8), or 60 (n = 9) h, and the indexes, including enhanced pause (Penh) and
peak inspiratory and expiratory flows, were determined. The mice were then anesthetized,
paralyzed, and mechanically ventilated. Airway resistance, respiratory system resistance at
breathing frequency, and tissue damping and elastance were identified from the LFOT
impedance data by model fitting. The monotonous decrease in airway resistance during
hyperoxia correlated best with the increasing peak expiratory flow. Respiratory system
resistance and tissue damping and elastance were unchanged up to 48 h of exposure but
were markedly elevated at 60 h, with associated decreases in peak inspiratory flow. Penh was
increased at 24 h and sharply elevated at 60 h. These results indicate no adverse effect [...]
Reference
PETAK, Ferenc, et al. Hyperoxia-induced changes in mouse lung mechanics: forced oscillations
vs. barometric plethysmography. Journal of Applied Physiology, 2001, vol. 90, no. 6, p.
2221-30
PMID : 11356786
Available at:
http://archive-ouverte.unige.ch/unige:72631
Disclaimer: layout of this document may differ from the published version.
Hyperoxia-induced changes in mouse lung mechanics:
forced oscillations vs. barometric plethysmography
Ferenc Peták, Walid Habre, Yves R. Donati, Zoltán Hantos and Constance
Barazzone-Argiroffo
J Appl Physiol 90:2221-2230, 2001.
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J Appl Physiol
90: 2221–2230, 2001.
Hyperoxia-induced changes in mouse lung mechanics:
forced oscillations vs. barometric plethysmography
FERENC PETÁK,1,2 WALID HABRE,3 YVES R. DONATI,4,5 ZOLTÁN HANTOS,4
AND CONSTANCE BARAZZONE-ARGIROFFO4,5
1
Division of Anesthesiologic Investigations, Departments of 4Pathology and 5Pediatrics,
University of Geneva, 1211 Geneva; 3Division of Anesthesia, Geneva Children’s Hospital,
1205 Geneva, Switzerland; and 2Department of Medical Informatics and Engineering,
University of Szeged, 6720 Szeged, Hungary
Received 23 October 2000; accepted in final form 1 February 2001
OXYGEN IS USED TO TREAT a variety of lung injuries, such
as respiratory distress syndrome, although long exposure to high oxygen concentrations has been shown to
be deleterious. The lung damage that develops during
hyperoxia consists of lung tissue and alveolar edema
(2, 3, 8, 14, 15, 34), surfactant dysfunction (10, 11, 13,
14, 21), lung inflammation (2–5, 10, 11), and subsequent deterioration of the lung function (2, 8, 10, 11,
13, 14, 25, 26, 31, 34, 35). Studies on oxygen toxicity
have primarily focused on parenchymal damage (2–5,
10, 11, 25), whereas the potential adverse effects of
prolonged oxygen exposure on the airways have not
been characterized. Numerous studies have demonstrated decreases in the compliance of the lungs (2, 8,
10, 11, 13, 14, 25) or of the total respiratory system (26,
35) and in the vital capacity (13) after oxygen exposure.
We argue, however, that the previously reported
marked increases in the resistance of the total respiratory system (Rrs) or of the lungs (RL) (10, 11, 13, 14,
26, 31, 34, 35) do not necessarily reflect alterations in
the airway function because these global resistive parameters incorporate a significant component related
to the respiratory tissues (Rti) (1, 16, 18, 19, 23, 29, 30).
Because of the technical difficulties in the measurement of air flow in small animals, lung functions have
mostly been evaluated in large mammalian species.
Although the study of mouse lung pathophysiology has
gained ground in asthma models and other pathological lung conditions, including hyperoxia (2–5, 25), very
few techniques are available to estimate the lung function in mice. The low-frequency forced oscillation technique (LFOT) and model-based evaluation of respiratory system impedance (Zrs) data have been used in
various mammals to partition RL into airway and parenchymal components under control conditions and
during lung constriction (1, 16, 19, 23, 29, 30). Small
laboratory animals also have been investigated extensively by the wave-tube version of this technique, and the
results were successfully validated in rats (23, 29). Therefore, adaptation of the LFOT for mice appears to be
appropriate to characterize the hyperoxia-induced
changes in the lungs because it provides direct and separate assessment of the airway and the tissue mechanics.
Barometric whole body plethysmography (WBP) has
been used in unrestrained mice to detect changes in
lung function indirectly from the characteristic times
Address for reprint requests and other correspondence: F. Peták,
Division of Anesthesiologic Investigations, Univ. of Geneva 1, rue
Michel-Servet, CH-1211 Geneva, Switzerland (E-mail: ferenc.petak
@medecine.unige.ch).
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. Section 1734
solely to indicate this fact.
respiratory mechanics; airway resistance; lung elastance;
enhanced pause
http://www.jap.org
8750-7587/01 $5.00 Copyright © 2001 the American Physiological Society
2221
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Peták, Ferenc, Walid Habre, Yves R. Donati, Zoltán
Hantos, and Constance Barazzone-Argiroffo. Hyperoxia-induced changes in mouse lung mechanics: forced oscillations vs. barometric plethysmography. J Appl Physiol 90:
2221–2230, 2001.—Hyperoxia-induced lung damage was investigated via airway and respiratory tissue mechanics measurements with low-frequency forced oscillations (LFOT) and
analysis of spontaneous breathing indexes by barometric
whole body plethysmography (WBP). WBP was performed in
the unrestrained awake mice kept in room air (n ⫽ 12) or in
100% oxygen for 24 (n ⫽ 9), 48 (n ⫽ 8), or 60 (n ⫽ 9) h, and
the indexes, including enhanced pause (Penh) and peak inspiratory and expiratory flows, were determined. The mice
were then anesthetized, paralyzed, and mechanically ventilated. Airway resistance, respiratory system resistance at
breathing frequency, and tissue damping and elastance were
identified from the LFOT impedance data by model fitting.
The monotonous decrease in airway resistance during hyperoxia correlated best with the increasing peak expiratory flow.
Respiratory system resistance and tissue damping and elastance were unchanged up to 48 h of exposure but were
markedly elevated at 60 h, with associated decreases in peak
inspiratory flow. Penh was increased at 24 h and sharply
elevated at 60 h. These results indicate no adverse effect of
hyperoxia on the airway mechanics in mice, whereas marked
parenchymal damage develops by 60 h. The inconsistent
relationships between LFOT parameters and WBP indexes
suggest that the changes in the latter reflect alterations in
the breathing pattern rather than in the mechanical properties. It is concluded that, in the presence of diffuse lung
disease, Penh is inadequate for characterization of the mechanical status of the respiratory system.
2222
LUNG MECHANICS DURING HYPEROXIA
METHODS
Mice
C57BL/6 mice were purchased from IFFA Credo (Labresle,
France) and were kept in the animal facility before the
experiments. The measurements were performed on 6- to
8-wk-old female mice weighing 20–23 g.
Oxygen Exposure
The protocol was approved by the institutional ethics committee for animal experiments (Office Vétérinaire Cantonal
de Genève). Mice were randomly assigned to one or other of
the following four protocol groups: the mice in the control
group were kept in room air (n ⫽ 12), and the mice in the
other three groups were exposed to 100% oxygen for 24 (n ⫽
9), 48 (n ⫽ 8), or 60 (n ⫽ 9) h. Because preliminary experiments revealed that anesthesia and mechanical ventilation
eventually led to mortality in mice exposed to hyperoxia for
72 h, the length of oxygen exposure was limited to 60 h. The
animals were exposed to oxygen in a sealed (8-liter) Plexiglas
chamber under minimal oxygen inflow and outflow (0.5
l/min). The CO2 level in the box was maintained at 1% by
using a CO2 absorber (Drägersorb 800, Dräger Medizintechnik, Lübeck, Germany). Food and water were available ad
libitum.
Barometric WBP
The characteristic parameters of spontaneous breathing
were recorded with a whole body plethysmograph (Buxco,
Troy, NY), as described in detail previously (17). Briefly, the
unrestrained, spontaneously breathing mice were placed in
the main chamber of the plethysmograph. The changes
caused by the pressure difference between the main chamber
and the reference chamber (PB) by spontaneous breathing
were recorded for 5 min. The whole body plethysmograph
with its accessories was placed in a large Plexiglas cylinder.
Inspiratory (TI), expiratory (TE), and relaxation (TR, defined
as the time until 36% of the total box pressure decay during
expiration) times, peak inspiratory and expiratory pressures,
tidal volume (VT), minute ventilation (V̇E), and respiratory
rate (RR) were extracted from the PB recordings. To estimate
the flow and volume units from the PB signal, the plethysmograph was calibrated by injecting 0.5 ml of air before the
measurements. Accordingly, peak expiratory (PEF) and inspiratory (PIF) flows were calculated from peak inspiratory
and expiratory pressures, respectively (17). Pause (⫽ [TE ⫺
TR]/TR) and Penh (⫽ pause PEF/PIF) parameters were also
calculated (17).
Forced Oscillatory Measurements
Animal preparations. After the WBP measurements, the
mice were anesthetized with an intraperitoneal injection of
pentobarbital sodium (50 mg/kg). Tracheostomy was performed, and a polyethylene cannula (30 mm long, 1.17 mm
ID) was inserted into the trachea. The mice were then mechanically ventilated (model 683, Harvard Apparatus, South
Natick, MA) with a VT of 10 ␮l/g at a frequency of 180/min.
Muscle paralysis was accomplished through the intraperitoneal administration of pancuronium bromide (1 mg/kg). To
avoid acute hypoxia in the hyperoxic mice, the animals were
kept in 100% oxygen during the surgical preparation and
mechanical ventilation.
Forced oscillatory setup. The wave-tube technique (36) was
adapted to measure forced oscillatory Zrs in mice. This technique was specially designed to measure the input impedance of rats (29, 30) and guinea pigs (1) without the need for
estimation of the oscillatory flow. We miniaturized the setup
to reduce the ventilatory dead space (40 ␮l) and to adjust its
impedance to the expected high load impedance of the mouse.
The forced oscillatory setup was described in detail previously (29). Briefly, a three-way tap (Becton-Dickinson, model
394600, Helsinborg, Sweden) was used to switch the tracheal
cannula from the respirator to a loudspeaker-in-box system
at end expiration. A 28-cm-diameter woofer (250 W) enclosed
in a 16-liter plastic box served as the pressure generator. The
loudspeaker generated a small-amplitude pseudorandom signal (25 integer-multiple frequency components between 1
and 25 Hz) through a 100-cm long, 1.17-mm ID polyethylene
tube (Becton-Dickinson, model 6253, Rutherford, NJ). Two
identical pressure transducers (model 33NA002D, ICSensors, Milpitas, CA) were used to measure the lateral pressures at the loudspeaker (P1) and at the tracheal end (P2) of
the wave tube. The P1 and P2 signals were low-pass filtered
(5th-order Butterworth, 25-Hz corner frequency) and sampled with an analog-digital board of a computer at a rate of
256 Hz. Fast Fourier transformation with 1-s time windows
and 90% overlapping was used to compute the pressure
transfer functions (P1/P2) from the 3-s recordings. The P1/P2
spectra were used to calculate Zrs as the load impedance of
the wave tube (36). Four to six Zrs values were collected in
each mouse for averaging. To avoid possible bias in the
impedance calculation due to the accumulated oxygen in the
wave tube, the oxygen administration was suspended and
the lungs were washed with room air for 30 s before each
recording. Intervals of at least 2 min were interposed between each two Zrs measurements.
Separation of airway and parenchymal mechanics. On the
basis of the different frequency dependencies of the two
compartments, the airway and parenchymal properties were
quantified by fitting a model incorporating an airway resis-
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and flows of spontaneous breathing (17). The empirically derived parameter enhanced pause (Penh) determined by means of WBP has been demonstrated to
increase during a constrictor challenge (9, 12, 17, 22,
32), and the increases paralleled those in RL in mice
during a methacholine challenge (17). Consequently, it
has been concluded that the use of Penh is suitable for
detection of a transient response of the respiratory
system after a constrictor challenge. However, the relevance of Penh for characterization of the mechanical
status of the respiratory system afflicted by a generalized lung disease has not been validated. Accordingly,
it remains unclear whether Penh is an appropriate
index for characterization of the mechanical properties
of the lungs, i.e., whether it provides an objective
estimation of the altered RL or merely reflects changes
in the ventilatory pattern.
In the present study, we adapted the LFOT for mice
to investigate how hyperoxia-induced lung damage is
manifested in the airways and the respiratory tissues.
We additionally aimed to determine separately the
time courses of the lung damage for the airways and
the respiratory tissues. Finally, we related the parameters obtained by means of WBP to direct measures of
airway, tissue, and total respiratory mechanics within
individual mice, to validate the usefulness of WBP in
the assessment of the respiratory function in a diseased lung.
2223
LUNG MECHANICS DURING HYPEROXIA
tance (Raw) and inertance in series with a constant-phase
tissue model (19), including damping (G) and elastance (H),
to the Zrs spectra by minimizing the differences between the
measured and modeled impedance values. Impedance data at
frequencies coinciding with the heart rate and its harmonics
were omitted from the fitting if the cardiac activity caused a
low signal-to-noise ratio at these frequencies.
Rti was calculated from the model parameters at the rate
of spontaneous breathing (RR), recorded by WBP, as Rti ⫽
G/(2␲RR/60)␣, where ␣ ⫽ (2/␲)arctan(H/G). Rrs at RR was
calculated as Rrs(RR) ⫽ Raw ⫹ Rti(RR). The contribution of
the measurement apparatus including the tracheal cannula
was 0.118 cmH2O 䡠 s 䡠 ml⫺1 to the reported Raw and Rrs
values.
Lung Weight and Microscopy
Statistical Analysis
Scatters in the parameters were expressed in SE values.
Kruskal-Wallis one-way ANOVA on ranks with Dunn’s multiple comparisons was applied to compare the mechanical
parameters between the protocol groups. The Spearman
rank-order correlation test was used to assess the strengths
of associations between parameters derived from LFOT and
WBP. Statistical tests were performed with a significance
level of P ⬍ 0.05.
RESULTS
WBP Data During Hyperoxia
Table 1 lists the hyperoxia-induced changes in the
parameters obtained by means of WBP. Significant
changes already occurred in RR, Penh, V̇E, and the parameters relating to the expiratory phase of the spontaneous breathing (TE, TR, and PEF) by 24 h of hyperoxia.
The significant increases observed in RR and V̇E at
Respiratory System Impedances
The real (Rrs) and imaginary (Xrs) parts of Zrs
obtained in three representative mice, one each from
the control group, the 24-h hyperoxia group, and 60-h
hyperoxia group, together with the corresponding
model fits, are shown in Fig. 1. The Rrs curves fall with
increasing frequency as a result of the decreasing contribution of Rti, and at higher rates they reach plateaus corresponding to Raw. The quasi-hyperbolic rise
in Xrs reflects the elastic behavior of the respiratory
tissues. The 24-h hyperoxia caused a minor change in
Zrs. The marked increases in the frequency dependences of Rrs and Xrs in the mouse exposed to hyperoxia for 60 h suggest significantly elevated values of G
and H. Although the changes in the Rrs spectra at high
frequencies are far smaller, the Rrs values for the
hyperoxia-exposed mice tend to a lower level. Apart
from data points corrupted by cardiac artifacts, the
model fitted well to all Zrs spectra without detectable
systematic fitting errors.
LFOT Data During Hyperoxia
The hyperoxia-induced changes in the forced oscillatory airway and tissue parameters are demonstrated in
Fig. 2. A mild but statistically significant monotonous
decrease was observed in Raw at 24 h (⫺39%, P ⬍
0.001). Because the inertive contribution of the intrathoracic gas to Xrs was negligible (the resonant
frequency in Zrs was above the frequency range measured), the airway inertance values are fairly scattered
and exhibit no obvious change during hyperoxia (P ⫽
Table 1. Effects of hyperoxia on parameters determined by barometric whole body plethysmography
TI, ms
TE, ms
TR, ms
Pause
PIF, ml/s
PEF, ml/s
Penh
VT, ml
V̇E, ml/min
RR, 1/min
Control
24-h Hyperoxia
48-h Hyperoxia
60-h Hyperoxia
57.0 ⫾ 1.7
196 ⫾ 27
138 ⫾ 24
0.61 ⫾ 0.04
8.79 ⫾ 0.40
5.42 ⫾ 0.38
0.387 ⫾ 0.026
0.294 ⫾ 0.020
93.5 ⫾ 5.5
325 ⫾ 22
58.1 ⫾ 1.9
135 ⫾ 12*
77.4 ⫾ 5.1*
0.90 ⫾ 0.07
9.90 ⫾ 0.44
7.71 ⫾ 0.31*
0.805 ⫾ 0.10*
0.326 ⫾ 0.008
127 ⫾ 8.5*
381 ⫾ 19*
57.5 ⫾ 3.2
124 ⫾ 11*
58.5 ⫾ 2.4*
1.46 ⫾ 0.41*
10.0 ⫾ 0.53
8.31 ⫾ 0.37*
1.74 ⫾ 0.85*
0.306 ⫾ 0.012
126 ⫾ 7.9*
406 ⫾ 11*
145.4 ⫾ 16.2*
239 ⫾ 15
40.0 ⫾ 2.1*
5.26 ⫾ 0.56*
4.76 ⫾ 0.32*
8.36 ⫾ 0.36*
13.0 ⫾ 2.0*
0.253 ⫾ 0.0059
48.1 ⫾ 3.6*
183 ⫾ 11*
Values are means ⫾ SE. TI, inspiratory time; TE, expiratory time; TR, relaxation time, defined as the time of the box pressure decay to 36%
of the total box pressure during expiration; pause ⫽ (TE ⫺ TR)/TR; PIF, peak inspiratory flow; PEF, peak expiratory flow; Penh, enhanced
pause defined as pause ⫻ PEF/PIF, VT, tidal volume; V̇E, minute ventilation; RR, respiratory rate. * Significant change from control
(P ⬍ 0.05).
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To estimate pulmonary edema and morphological changes
after hyperoxia, C57BL/6 mice with similar body weights
were investigated in control conditions (n ⫽ 5) and when
exposed to 48 (n ⫽ 3) and 60 h (n ⫽ 3) of hyperoxia. Mice were
bled by opening the abdominal aorta under anesthesia, and
the thorax was opened. The lungs were dissected and
weighed. The lungs were then inflated and fixed by instilling
5% formalin (in 80% ethanol) into the trachea with hydrostatic pressure of 20 cm. Transhilar horizontal sections were
embedded in paraffin and processed for light microscopy (3).
The sections were stained with hematoxylin and eosin.
oxygen exposures of 24 and 48 h were followed by sharp
and statistically significant decreases when hyperoxia
was maintained for 60 h. The increases in Penh were
relatively moderate but statistically significant in the
mice exposed to hyperoxia for 24 and 48 h, whereas the
elevation was more than 30-fold at 60 h. Significant
changes in the parameters characterizing inspiration
(PIF and TI) were observed only at 60 h. Although
there were tendencies to increase at 24 and 48 h and a
decrease at 60 h, no statistically significant changes
occurred in VT.
2224
LUNG MECHANICS DURING HYPEROXIA
these parameters. Figure 3 illustrates the closest associations between the parameters determined via the
LFOT and WBP.
The relationships between the forced oscillatory parameters and Penh are demonstrated in Fig. 4. The
changes in Raw and Penh were fairly parallel but
opposite, and the relatively small decreases in Raw
(maximum 39%) were associated with massive (⬃25fold) increases in Penh. The associations between the
changes in the tissue parameters (Rti, G, and H) and
those in Penh were rather poor because Penh exhibited
marked and statistically significant increases in the
mice exposed to hyperoxia for 24 or 48 h, whereas no
changes in tissue mechanics were apparent. Exposure
to oxygen for 60 h caused marked increases in both
tissue parameters and Penh, although the changes in
the latter were far greater. Because Rti accounted for
the majority of Rrs (71 ⫾ 1.8%), the relationship between Rrs and Penh resembles that between the tissue
parameters and Penh.
Histology and Lung Weights
DISCUSSION
0.2). Hyperoxia for 24 or 48 h caused no change in G, H,
Rti, or Rrs, whereas marked and statistically significant increases were noted after 60 h (64, 63, 168, and
95% for G, H, Rti, and Rrs, respectively; P ⬍ 0.0001 for
all). It should be noted that, although Rti is derived
from the model parameter G, it is also determined by
RR (see METHODS above). Because RR was decreased at
60 h, the changes in Rti, and thus in Rrs, are greater
than those in G.
Relationships Between LFOT and WBP Parameters
We also examined how the altered WBP indexes
reflect the hyperoxia-induced changes in the airway
and tissue mechanical parameters. The correlation between the WBP indexes and LFOT parameters are
listed in Table 2. Raw exhibited a rather poor association with parameters determined by WBP, the closest
relationship being that with PEF. PIF was found to
correlate best with the LFOT parameters G and H.
Concerning the independent parameters only, Rti and
Rrs correlated most satisfactorily with the TI and flow
indexes. Trivially, Rti and Rrs are not independent of
RR (and thus V̇E) because RR was used to calculate
In the present study, the effects of hyperoxia on the
mouse respiratory system were investigated by measuring passive respiratory mechanics and analyzing
indexes of spontaneous breathing patterns. The partitioning of Rrs into airway and tissue components revealed a decrease in Raw by 24 h of hyperoxia, whereas
the tissue properties remained unchanged up to 48 h
and were then increased significantly at 60 h of oxygen
exposure. Variable changes were detected in the indexes derived from the spontaneous breathing: Penh
increased significantly as early as by 24 h of hyperoxia,
and changes were observed in other parameters relating to the expiratory phase of spontaneous breathing.
The parameters relating to the inspiratory phase, however, were observed to be changed significantly only at
60 h of hyperoxia. Changes in lung weights or histology
indicated no lung injury until 48 h of hyperoxia,
whereas marked edema and parenchymal damage
were evident after longer oxygen exposures.
Mechanical Parameters During Hyperoxia
Very little is known about the mechanical properties
of the mouse respiratory system, primarily because of
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Fig. 1. Real (Rrs) and imaginary (Xrs) parts of respiratory system
input impedances in representative mice from the control group
(circles) and from groups exposed to hyperoxia for 24 (squares) or
60 h (triangles). Symbols with bars represent mean ⫾ SD values
from 4–6 consecutive measurements. Lines indicate the corresponding model fits (solid for control, dotted and dashed for 24 and 60 h of
hyperoxia, respectively). Open symbols, data points that were excluded from model fits because of corruption by cardiac artifacts.
Figure 5 illustrates lung histology in representative
mice exposed to room air and to hyperoxia for 48 and
60 h. No pathological alterations were observed in the
airways at any time of oxygen exposure. In agreement
with previous findings (3, 4), no histological changes in
the tissues were present in the mice exposed to 48 h of
hyperoxia, whereas exposure to oxygen for 60 h induced interstitial edema and thickening of alveolar
septa. Lung weights were not significantly higher in
the mice exposed to hyperoxia for 48 h (0.185 ⫾ 0.017
g) than those in the control group (0.175 ⫾ 0.016 g, P ⫽
0.69), whereas significant increases were observed after longer oxygen exposures (0.25 ⫾ 0.015 g, P ⬍ 0.02).
LUNG MECHANICS DURING HYPEROXIA
2225
the small size of the animal. We therefore adapted the
wave-tube technique of forced oscillations, the lowfrequency version of which has been validated in rats
(23, 29) and has been shown to provide a separate
estimation of airway and tissue parameters (1, 16, 19,
23, 29, 30). These previous investigations demonstrated that lung mechanical parameters identified
from low-frequency input impedance data characterize
the mechanical conditions of the airways and the respiratory tissues accurately in a healthy and moderately or homogeneously constricted lung (23, 29). In the
severely constricted lung, enhanced interregional ventilation resulting from heterogeneously constricted peripheral airways may lead to a virtual increase in
tissue damping, whereas the elastance remained rela-
tively unaffected by this phenomenon (23, 29). Because
the increases in G and H were proportional during
hyperoxia in this study, the development of severe
inhomogeneities was unlikely to occur.
Our Zrs spectra are qualitatively similar to those
obtained in previous studies in rats (29, 30), in guinea
pigs (1), and in mice (16). In agreement with previous
findings (1, 16, 19, 23, 29, 30), the model involving an
airway and a constant-phase tissue compartment was
consistent with the frequency dependence of Zrs. Hessel et al. (20) measured Zrs data in mice at frequencies
above the rate of spontaneous breathing. Their Rrs
values at 16 Hz (590 ⫾ 51 cmH2O 䡠 s 䡠 l⫺1) are slightly
lower than those obtained in the present study at the
same frequency (720 ⫾ 29 cmH2O 䡠 s 䡠 l⫺1 in the control
Downloaded from on May 18, 2015
Fig. 2. Forced oscillatory airway and tissue
parameters obtained in the control group
(Ctrl) and in mice exposed to 24, 48, or 60 h of
hyperoxia. Each symbol represents the parameter value of an individual mouse. Lines
with bars denote the group mean ⫾ SE values. Raw and Iaw, airway resistance and inertance, respectively. G and H, tissue damping and elastance, respectively. Rti and Rrs,
tissue and total respiratory system resistances, respectively, at the frequency of spontaneous breathing. *P ⬍ 0.05 compared with
Ctrl.
2226
LUNG MECHANICS DURING HYPEROXIA
Table 2. Spearman correlation coefficients between
tidal breathing indexes obtained by means of
whole body plethysmography and passive
respiratory mechanical parameters
determined by forced oscillations
Raw
TI
TE
TR
Pause
PIF
PEF
Penh
VT
V̇E
RR
⫺0.37*
⫺0.11
0.68‡
⫺0.64‡
0.10
⫺0.69‡
⫺0.67‡
0.02
0.08
0.15
G
0.45†
0.27
⫺0.54‡
0.44*
⫺0.54‡
0.22
0.49†
⫺0.46†
⫺0.50†
⫺0.45†
H
0.46†
0.34*
⫺0.48†
0.39*
⫺0.49†
0.21
0.41*
⫺0.36*
⫺0.51†
⫺0.51†
Rti
0.77‡
0.75‡
⫺0.26
0.40*
⫺0.76‡
⫺0.00
0.42*
⫺0.55‡
⫺0.85‡§
⫺0.86‡§
Rrs
0.74‡
0.75‡
⫺0.24
0.35*
⫺0.79‡
⫺0.08
0.36*
⫺0.62‡
⫺0.89‡§
⫺0.86‡§
Raw, airway resistance; Rti and Rrs, tissue and total respiratory
system resistance, respectively; G and H, tissue damping and elastance, respectively. * P ⬍ 0.05, † P ⬍ 0.005, ‡ P ⬍ 0.001; § pairs of
not independent variables.
Fig. 3. Closest associations between parameters determined by low-frequency forced oscillatory technique and whole body plethysmographic measurements, according to the
Spearman rank-order correlation test. Symbols with bars represent mean ⫾ SE values in
Ctrl mice and in mice exposed to hyperoxia
for 24, 48, or 60 h. PEF and PIF, peak expiratory and inspiratory flows, respectively.
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mice). Besides the obvious difference in the animal
preparation (face mask vs. tracheostomy), Hessel et al.
determined Zrs during spontaneous breathing at
higher mean airway pressures (20), at which Raw, and
thus Rrs, is expected to be lower (16). More recently,
Gomes et al. (16) identified airway and constant-phase
tissue parameters in anesthetized mechanically ventilated mice. Although our Raw values in the control
mice are similar to their estimates, their measurements of G and H are ⬃20% lower than our findings.
This discrepancy is likely to be attributed to the higher
oscillatory amplitude applied by Gomes et al. (35% of
the tidal volume), which results in lower tissue impedance because of the highly nonlinear characteristics of
the chest wall (18).
We observed a striking decrease in Raw as early as
by 24 h of oxygen exposure. To our knowledge, only one
earlier study has reported hyperoxia-induced changes
in the airway properties in vivo (13). In that study,
Raw measured with a body plethysmograph in adult
volunteers showed no statistically significant change
at 6–11 h of hyperoxia. Nevertheless, it is difficult to
compare their results with our findings because, besides the difference in species, the oxygen exposure of
the humans was terminated quickly after the development of symptoms related to oxygen toxicity. Other
authors found that the smooth muscle area measured
in isolated bronchi was not affected by exposure to
hyperoxia for 1 or 3 days (33). The lack of structural
changes in the airways is in accordance with our finding that Raw did not increase but rather decreased
further, even after 60 h of hyperoxia. Many factors may
LUNG MECHANICS DURING HYPEROXIA
2227
have contributed to the Raw decrease observed in the
present study. Hyperoxia may have induced bronchodilation via neural pathways that were missing in the
isolated bronchial preparations. The increase in functional residual capacity during hyperoxia was also suggested to lead to a decrease in Raw, although the
changes in functional residual capacity were modest
(13, 14). Moreover, hyperoxia might increase the levels
of endogenous catecholamines in consequence of pain
and discomfort (13) and thereby relax the bronchial
smooth muscles (24).
Hyperoxia had no detectable effect on the resistive or
elastic properties of the respiratory tissues at 48 h of
hyperoxia, whereas oxygen exposure for 60 h was followed by marked increases in both G and H. According
to our histological findings and in agreement with
previous observations (2–5, 8, 14, 15, 34), these increases can be attributed to the development of interstitial or alveolar edema. In addition, lung inflammation (2–5, 10, 11) and/or the impaired surface activity
of the surfactant (10, 11, 13, 14, 21) might have contributed to the impairment in parenchymal mechanics.
The physiopathological changes in the surfactant function may affect the viscoelastic properties of the parenchyma or may lead to disperse atelectasis in the alveoli
(10, 11, 13, 14, 21). Although our data do not allow to
estimate the degree of a possible surfactant damage,
our findings may provide an insight into the characteristics of the tissue processes. Heterogeneous lung injury
leads to an additional rise in G, whereas it affects H to a
far lesser degree (23, 29). Accordingly, the same magnitude of relative increases in G (64%) and H (63%) in the
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Fig. 4. Relationship between parameters estimated from the forced oscillatory impedances of the respiratory system and the enhanced pause (Penh) determined by means of
whole body plethysmography. Symbols with
bars represent mean ⫾ SE values in the Ctrl
mice and in mice exposed to 24, 48, or 60 h of
hyperoxia.
2228
LUNG MECHANICS DURING HYPEROXIA
Fig. 5. Lung histology of representative
mice exposed to room air (Ctrl) or to 100%
oxygen for 48 and 60 h. Sections were
stained with hematoxylin and eosin. B,
bronchus; V, vessel; E, edema fluid. Note
the absence of pathological changes in the
bronchi at any time of hyperoxia exposures
and normal alveolar structure at 48 h hyperoxia. The presence of edema and thickening of alveolar septa (arrowheads) are
obvious only in the mouse exposed to hyperoxia for 60 h. Magnification: ⫻20.
many factors including age, gender, and strain of the
particular species. We studied 6- to 8-wk-old female
C57BL/6 mice inbred in a pathogen-free environment
because the effects of oxygen toxicity are well characterized in these animals (3–5).
WBP Indexes During Hyperoxia
We used WBP to assess hyperoxia-induced changes in
the respiratory system. This approach has been demonstrated to detect changes in the spontaneous breathing
pattern, which was proved (17) or assumed (9, 12, 22, 32)
to reflect an altered lung resistance after a pulmonary
constrictor challenge. In the present study, two different
phases can be distinguished in the parameter changes
during hyperoxia. In the first phase, up to 48 h of oxygen
exposure, RR and PEF increased, whereas TE and TR
decreased. We also observed a marked (fivefold) increase
in Penh during this first phase. In agreement with the
current findings, it has been demonstrated that a short
exposure to hyperoxia stimulates breathing by increasing
RR and V̇E (6, 7). These increases are reflected in the
decreases in the expiratory time intervals (TE and TR).
The increases in PEF either may be attributed to active
expiration or may reflect changes in the dynamic mechanical properties of the respiratory system. However,
the shape of the flow-time curves did not indicate qualitative changes between the control condition and that at
48 h of hyperoxia, which suggests that the expiration
remained a passive process (Fig. 6). Therefore, it is more
likely that the increases in PEF can be attributed to a
bronchodilation effect, as also indicated by the decreases
in Raw. Because Penh is proportional to PEF and TE ⫺
TR and inversely proportional to TR and PIF, we conclude that it was the superposition of the changes in the
ventilatory pattern that resulted in the markedly in-
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present study indicates that the parenchymal damage
was rather uniform throughout the lungs. Such pathological changes may be more characteristic of an edematous process, because atelectasis is expected to lead to a
highly heterogeneous lung structure. The fairly uniform
presence of edema evidenced from histology also confirms
this conclusion. The restrictive changes in the lungs,
causing increases in the parenchymal parameters but
with no consequent increase in Raw, suggest that the
pathological changes were limited to the alveolar compartment, without the development of obstructive
changes in the conducting airways.
The previously reported time course of hyperoxiainduced lung tissue damage exhibits great variability
and reveals a substantial species dependence. Significant decreases in lung compliance were reported by
merely 11 h of oxygen exposure in humans (13), within
24 h in preterm rabbits (8), and at 48 h in rabbits (26)
and piglets (10, 11). Adult rabbits, however, exhibited
slight increases in Rrs and decreases in respiratory
system compliance only after 69 h (35), and the increase in RL became significant only after 96 h in sheep
(14), whereas guinea pig pups displayed no change in
Rrs after even 96 h of hyperoxia (31). Nevertheless, the
results of the few studies in which mice were exposed
to hyperoxia are in accordance with the current findings on lung morphology and oscillatory mechanics: no
lung damage had occurred in this species by 48 h,
whereas symptoms of oxygen toxicity were present at
72 h after exposure (2–5, 25). In addition, the results of
the present study demonstrate that changes occur as
soon as by 60 h of hyperoxia in mice, and the pathological changes in lung morphology are reflected in the
compromised parenchymal mechanics. It should be
noted that, besides the species dependence, the temporal changes during hyperoxia may also depend on
LUNG MECHANICS DURING HYPEROXIA
creased Penh during the first 48 h of hyperoxia and that
no pathological changes in lung function or structure
were reflected by the altered Penh.
In the second phase, at 60 h of hyperoxia, the
changing trends in RR, TE, V̇E, VT, and PIF were
abruptly reversed, whereas TR continued its decreasing tendency and TI, pause, and Penh were
dramatically increased. All of these changes, i.e., the
sharp decreases in PIF, RR, VT, and V̇E and the
increase in TE, suggest the occurrence of a profound
change in respiration, induced by the prolonged hyperoxia, that was also obvious in morphology and in
the marked increases in the tissue mechanical parameters measured by the LFOT.
Relationships Between LFOT and WBP Parameters
As a result of its demonstration of significant increases in Penh after administration of methacholine
(17, 22, 32), histamine (9), or an allergen (12), WBP
was proposed as a means of detecting altered lung
resistance via the recording of the changes in the
spontaneous breathing pattern in unrestrained, awake
mice. The changes in spontaneous breathing, however,
can also be associated with a number of other phenom-
ena, such as modified neural control, pain, or discomfort
(28), besides altered respiratory mechanics. Although
it has been established clearly that a constrictor challenge increases Penh (9, 12, 17, 22, 32), the inverse
question of whether an increased Penh is a consequence of changes in the mechanical properties of the
respiratory system has not been investigated thoroughly. In particular, the validity of Penh in the estimation of the respiratory mechanical consequences
during a generalized pulmonary disease affecting the
airway and parenchymal compartments has not been
tested. In the present study, we obtained an impressive
(fourfold) and statistically significant increase in Penh
at 48 h of hyperoxia, which was not accompanied by
histological alterations or changes in tissue parameters, whereas Raw even slightly decreased. Although
these opposite changes resulted in some correlation
between Raw and Penh, it is very difficult to interpret
this relationship because increases in Penh have been
suggested to reflect a constrictor response (9, 12, 17,
22, 32). It should be stressed again that, in agreement
with our lung weight, histology, and oscillatory data,
there is a consensus in the literature that no lung
injury develops in mice within the first 48 h of oxygen
exposure (2–5, 24). Hence, the current study not only
justifies the concerns regarding the interpretation of
the quantitative changes in Penh (27) but also demonstrates that the changes in Penh do not even qualitatively follow the changes in the respiratory mechanical
properties during oxygen exposure.
We additionally examined which indexes measured
by means of WBP reflect the changes observed in the
oscillatory mechanical parameters under the conditions of generalized lung disease. The correlation analysis revealed the strongest relationship between Raw
and PEF; moreover, the tissue parameters extracted
from the LFOT measurements exhibited the best correlations with the length (TI) and the intensity (PIF) of
the inspiration. Whereas an increased PEF associated
with a decreased Raw may be explained on the basis of
passive mechanical properties, the correlations between the inspiratory indexes and both G and H are
not plausible, and the relationships between Penh and
the Zrs parameters are rather puzzling.
In conclusion, partitioning of the airway and tissue
mechanical properties of the respiratory system during
normobaric hyperoxia in mice revealed distinct responses of the two compartments. Mild bronchodilation was detected as early as by 24 h of oxygen exposure. In contrast, no adverse effect of hyperoxia was
evident in the respiratory tissues during the first 48 h,
whereas marked alveolar edema associated with increases in tissue damping and elastance had developed
by 60 h. Comparison of the forced oscillatory and plethysmographic parameters revealed that the changes
in the direct measures of the airway and tissue mechanical properties during hyperoxia are not reflected
by those in Penh, and the statistical relationships with
other plethysmographic indexes are not easily interpretable either. We conclude that, as a shape factor
characterizing the breathing pattern via the plethys-
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Fig. 6. Traces of box pressure signals in the whole body plethysmograph in representative mice from the Ctrl group and from the
groups exposed to 48 or 60 h of hyperoxia. Inspirations are in
negative directions.
2229
2230
LUNG MECHANICS DURING HYPEROXIA
mographic pressure fluctuations, Penh may become
unrelated to changes in lung function and, in particular, airway resistance in the case of chronic lung disease.
We thank P. Rimensberger, J. Savoy, and P. F. Piguet for pertinent advice.
This study was supported by the Swiss National Science Foundation Grants 056717.99/1 and 056949.99/1 and Hungarian Scientific
Research Grant OTKA T30670.
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