Invasive versus noninvasive measurement of allergic and cholinergic

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Invasive versus noninvasive measurement of allergic and cholinergic

airway responsiveness in mice

Glaab1,2, a Ziegert1, Ralf Baelder1, Regina Korolewitz1,

Armin Braun1, Jens M Hohlfeld1,2, Wayne Mitzner3, Norbert Krug1 and

Heinz G Hoymann*1

Address: 1Fraunhofer Institute of Toxicology and Experimental Medicine (ITEM),

Nikolai-Fuchs Str.1, 30625 Hannover, Germany, 2Hannover

Medical School, Department of Respiratory Medicine, Carl-Neuberg Str.1, 30625

Hannover, Germany and 3Division of Physiology, Bloomberg

School of Public Health, s Hopkins University, Baltimore, land 21205,

USA

Email: Glaab -thomasglaab@...; a Ziegert

-michaela@...; Ralf Baelder -ralf.baelder@...;

Regina Korolewitz -korolewitz@...; Armin Braun

-braun@...;

Jens M Hohlfeld -hohlfeld@...; Wayne Mitzner -wmitzner@...;

Norbert Krug -krug@...;

Heinz G Hoymann* -hoymann@...

* Corresponding author

Published: 25 November 2005 Received: 18 January 2005

Respiratory Research 2005, 6:139 doi:10.1186/1465-9921-6-139

Accepted: 25 November 2005

This article is available from: http://respiratory-research.com/content/6/1/139

© 2005 Glaab et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative

Commons Attribution License (http://creativecommons.org/licenses/by/2.0),

which permits unrestricted use, distribution, and reproduction in any medium,

provided the original work is properly cited.

Abstract

Background: This study seeks to compare the ability of repeatable invasive and

noninvasive lung

function methods to assess allergen-specific and cholinergic airway

responsiveness (AR) in intact,

spontaneously breathing BALB/c mice.

Methods: Using noninvasive head-out body plethysmography and the decrease in

tidal

midexpiratory flow (EF50), we determined early AR (EAR) to inhaled Aspergillus

fumigatus antigens

in conscious mice. These measurements were paralleled by invasive determination

of pulmonary

conductance (GL), dynamic compliance (Cdyn) and EF50 in another group of

anesthetized,

orotracheally intubated mice.

Results: With both methods, allergic mice, sensitized and boosted with A.

fumigatus, elicited

allergen-specific EAR to A. fumigatus (p < 0.05 versus controls). Dose-response

studies to

aerosolized methacholine (MCh) were performed in the same animals 48 h later,

showing that

allergic mice relative to controls were distinctly more responsive (p < 0.05)

and revealed acute

airway inflammation as evidenced from increased eosinophils and lymphocytes in

bronchoalveolar

lavage.

Conclusion: We conclude that invasive and noninvasive pulmonary function tests

are capable of

detecting both allergen-specific and cholinergic AR in intact, allergic mice.

The invasive

determination of GL and Cdyn is superior in sensitivity, whereas the noninvasive

EF50 method is

particularly appropriate for quick and repeatable screening of respiratory

function in large numbers

of conscious mice.

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Respiratory Research 2005, 6:139

Background

Asthma is a complex disease associated with reversible airway

obstruction of variable degree, airway inflammation,

airway hyperresponsiveness (AHR) and airway remodeling.

These hallmarks of asthma are being examined in

murine models, with the goal of understanding the basic

cellular and genetic mechanisms of allergic inflammation

that underlie the immunologic basis of the disease [1]. To

investigate the functional consequences of in vitro findings

in the lung in vivo, determination of pulmonary

function is an essential tool. Existing methods for measuring

respiratory function in mice in vivo include invasive

and noninvasive approaches [2,3]. The invasive recording

of pulmonary resistance (RL) or pulmonary conductance

(1/RL), and dynamic compliance (Cdyn) is the gold

standard for precise and specific determinations of pulmonary

mechanics [2,3]. Limitations of traditional invasive

methodologies commonly involve surgical

tracheostomy, anesthesia, and mechanical ventilation, all

of which are procedures that may generate significant artifacts

[2]. In addition, when tracheostomy is done, this

method is limited to single-point measurements only,

usually precluding the possibility of performing follow-

up studies. A novel modification to this invasive technology

has enabled repetitive invasive recordings of pulmonary

mechanics in conjunction with local aerosol delivery

in anesthetized, orotracheally intubated, spontaneously

breathing mice [4].

Noninvasive determination of respiratory parameters in

conscious mice is a convenient, repeatable approach for

screening respiratory function in large numbers of animals.

Here, the application of the empiric variable

enhanced pause (Penh) has gained widespread popularity.

A recent correspondence written by leading experts [5]

has emphasized the danger of the increasing uncritical use

of Penh, with potentially misleading assessment of pulmonary

function in animal models of lung disease.

Although noninvasive measurement of murine respiratory

function has virtually become synonymous with the

recently questioned Penh method [5-9], a variety of other

noninvasive methods have been established [10-12]. We

and others have described the utility of midexpiratory

flow, as measured by head-out body plethysmography, as

a physiologically meaningful, noninvasive parameter of

bronchoconstriction for mice and rats [13-17]. No report

has as yet directly investigated the ability and utility of

repetitive invasive and noninvasive lung function methods

to assess allergen-specific EAR and cholinergic airway

hyperresponsiveness (AHR) in intact mice. The primary

objective of this study in a mouse model of fungal asthma

was to compare the capability of noninvasive EF50 measurements

to reflect the allergen-specific and cholinergic

AR as observed with invasive determination of pulmonary

mechanics. Moreover, to support the argument that non

http://respiratory-research.com/content/6/1/139

invasive EF50 measurement is more valid than Penh we

sought to examine whether EF50, unlike Penh [18], parallels

the actual changes in pulmonary mechanics in

response to hyperoxia in C57BL/6 mice. Our results

showed that, while the noninvasive measurement of EF50

presented greater variability than the classical invasive

measurements of RL and Cdyn, the correlation was sufficiently

strong to support the use of such noninvasive testing

in repetitive measurements in invividual mice.

Methods

Animals and sensitization protocol

Pathogen-free, female BALB/c mice, 12–14 weeks of age,

and female C57BL/6 mice (used only for hyperoxia exposures),

7–8 weeks of age ( River, Sulzfeld, Germany),

were kept in a pathogen-free rodent facility and

were provided food and water ad libitum. All animal

experiments conformed to NIH guidelines and were

approved by the appropriate governmental authority

(Bezirksregierung Niedersachsen, Germany). Allergic

BALB/C mice (n = 8) received an intraperitoneal and subcutaneous

injection of soluble A. fumigatus antigens (5 µg

each, Greer Laboratories Inc, Lenoir, NC, USA), dissolved

in incomplete Freund's adjuvant in a volume of 0.1 ml

given on day 0 and were boosted noninvasively by inhalation

over 10 min in a closed chamber with 1 % of A.

fumigatus aerosol dissolved in saline on day 14 (jet nebulizer,

LC Star, 2.8 µm mass median aerodynamic diameter

(MMAD), Pari GmbH, Starnberg, Germany).

On day 21, allergic mice were challenged once with aerosolized

A. fumigatus followed by methacholine (MCh,

Sigma, Deisenhofen, Germany) dose-response exposure

48 h later (d 23). The control group (n = 8) received the

same treatment schedule but was boosted and challenged

with saline before MCh exposure. This protocol was chosen

to maximize the difference between allergic and control

groups. For the noninvasive measurement of

pulmonary function separate groups of A. fumigatus-sensitized

and control mice were used (n = 8 each group).

Noninvasive measurement of pulmonary function in

conscious mice

Noninvasive respiratory function was assessed with a

glass-made head-out body plethysmograph system for

four mice as previously described [14,17,19]. Briefly, mice

were placed in the body plethysmographs while the head

of each animal protruded through a neck collar (9 mm ID,

dental latex dam, Roeko, Langenau, Germany) into a ventilated

head exposure chamber. Monitoring of respiratory

function was started when animals and individual measurements

settled down to a stable level. For airflow measurement,

a calibrated pneumotachograph (capillary tube

PTM 378/1.2, HSE-Harvard, March-Hugstetten, Germany)

and a differential pressure transducer (Validyne DP

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45-14, range ± 2 cm H2O, HSE-Harvard) coupled to an

amplifier were attached to the top port of each plethysmograph.

For each animal the amplified analog signal from

the pressure transducer was digitized via an analog-to-digital

converter (DT 302, Data Translation, Marlboro, MA).

The pneumotachograph tidal flow signal was integrated

with time to obtain tidal volume (VT). From these signals

the parameters tidal midexpiratory flow (EF50), time of

expiration (TE), tidal volume (VT) and respiratory rate (f)

were calculated for each breath and were averaged in 5 s

segments with a commercial software (HEM 3.4, Notocord,

Paris, France).

During airway constriction the main changes in the tidal

flow signal occur during the midexpiratory phase. We

defined EF50 (ml/s) as the tidal flow at the midpoint (50

%) of expiratory tidal volume, and we used this as a measure

of bronchoconstriction [12,14,17]. A reduction in

EF50 of more than 1.5 Standard deviation (SD) of mean

baseline value (which translates to a reduction of more

than 20% versus baseline) is considered to indicate airway

constriction. The degree of bronchoconstriction to inhalation

challenge was determined from minimum values of

EF50 and was expressed as percent changes from corresponding

baseline values.

Invasive measurement of pulmonary function

AR was assessed as an increase in RL or decreases in Cdyn

and EF50 in response to aerosolized A. fumigatus or MCh

in anesthetized, spontaneously breathing mice as previously

described in detail [4]. Briefly, mice were anesthetized

with intraperitoneal injections of metomidate (total

dose: 38–60 mg/kg) and fentanyl (total dose: 0.02 – 0.06

mg/kg) with minimal supplementations as required.

When an appropriate depth of anesthesia was achieved,

mice were suspended by their upper incisors from a rubber

band on a Plexiglas support. The trachea was transilluminated

below the vocal cords by a halogen light source

and a standard 20G × 32 mm Abbocath®-T cannula

(Abbott, Sligo, Ireland) was gently inserted into the tracheal

opening. The intubated, spontaneously breathing

animal was then placed in supine position in a thermostat-

controlled whole-body plethysmograph (type 871,

HSE-Harvard, designed in cooperation with Fraunhofer

ITEM). The orotracheal tube was directly attached to a

pneumotachograph (capillary tube PTM T16375, HSE-

Harvard) installed in the front part of the chamber. Tidal

flow was determined by the pneumotachograph connected

to a differential pressure transducer (Validyne DP

45-14, HSE-Harvard). To measure transpulmonary pressure

(PTP) a water-filled PE-90 tubing was inserted into

the esophagus to the level of the midthorax and coupled

to a pressure transducer (model P75, HSE-Harvard). The

amplified analog signals from the pressure transducers

http://respiratory-research.com/content/6/1/139

were digitized as described above for noninvasive measurements.

Pulmonary resistance (RL) and dynamic compliance

(Cdyn) were calculated over a complete respiratory cycle

using an integration method over flows, volumes and

pressures as previously described [4,20]. The resistance of

the orotracheal tube (0.63 cm H2O·s·ml-1) was subtracted

from all RL measurements. RL, Cdyn, EF50 together

with other basic respiratory parameters were continuously

recorded with a commercial software (HEM 3.4, Notocord).

For easier comparison of trends among all variables,

RL was expressed as pulmonary conductance GL (GL

= 1/RL).

Respiratory parameters were averaged in 5 s segments and

minimum GL, Cdyn and EF50 values were taken and

expressed as percent changes from corresponding baseline

values. After the measurements on day 21, mice were

removed from the chamber and extubated as soon as they

began recovering from anesthesia.

Administration of aerosols

After recording of baseline values, airway responsiveness

(AR) to A. fumigatus 2 % or saline (control group) was

determined in separate groups of conscious and intubated

mice on day 21. On day 23, dose-response studies to aerosolized

MCh were performed in the same mice.

For intubated mice, dried aerosols of A. fumigatus 2 %

(inhaled dose: 8 µg) and MCh 5 % (inhaled doses: 0.05–

2.5 µg) were generated by a computer-controlled, jet-

driven aerosol generator system (Bronchy III, particle size

2.5 µm MMAD, Fraunhofer ITEM, licensed by Buxco,

Troy, NY) as previously described (15, 21).

Conscious mice placed in the head-out body plethysmographs

were exposed noninvasively to A. fumigatus (2 %,

inhaled dose 32 µg) and MCh aerosols (0.5–3 %, cumulative

inhaled doses: 3–14 µg) delivered by a Pari jet nebulizer

as previously described [13,14,22]. In both systems,

aerosol concentrations were determined by a gravimetrically

calibrated photometer. The total inhalation doses of

A. fumigatus and MCh were calculated based on the continuously

measured aerosol concentrations and respiratory

volume per min [4,21]. The results of the

bronchoconstrictor response to MCh were expressed as

PD50 which is the dose of MCh required to reduce either

GL, Cdyn or EF50 to 50 % of their respective baseline values

and was calculated from the dose-response curves.

Exposure to oxygen

C57BL/6 mice were randomly assigned to two groups: The

mice in the control group (n = 8 each) were kept in room

air whereas the other group of 8 mice was exposed to 100

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Table 1: Baseline values for respiratory parameters from allergic and control

BALB/c mice

Respiratory

parameters

Definition Control mice

conscious

Allergic mice

conscious

Control mice

anesthetized

Allergic mice

anesthetized

VT, ml

f, breaths/min

TE, s

EF50, ml/s

GL, ml·s-1·cmH2O-1

Cdyn, ml·cmH2O-1

tidal volume

respiratory frequency

time of expiration

tidal midexpiratory flow

pulmonary conductance

dynamic compliance

0.21 ± 0.05

198 ± 41

0.17 ± 0.06

2.05 ± 0.89

-

-

0.19 ± 0.04

220 ± 23

0.14 ± 0.02

2.26 ± 0.46

-

-

0.14 ± 0.02

129 ± 20*

0.3 ± 0.04*

0.93 ± 0.14*

1.05 ± 0.36

0.037 ± .007

0.13 ± 0.02

124 ± 29*

0.3 ± 0.05*

1.12 ± 0.43*

1.29 ± 0.69

0.030 ± .008

Baseline values are means ± SD obtained from 8 animals per group during a 5 min

control period from conscious and anesthetized, orotracheally

intubated BALB/c mice. In comparison with conscious mice, EF50, TE and f values

were significantly altered in anesthetized mice. No difference was

found between allergic animals and control groups when separated into conscious

and anesthetized mice. *P < 0.05 versus conscious mice.

% oxygen for 48 h. Exposure to 100 % oxygen was performed

in a sealed (25 L) Plexiglas chamber with a flow of

2 L/min as similarly described earlier [18]. The CO2 level

in the chamber was maintained at 1 % by using a CO2

absorber (Drägersorb 800 plus, Dräger, Lübeck, Germany).

Food and water were provided ad libitum.

Bronchoalveolar lavage (BAL) cell counts

At the end of this protocol, total and differential cell

counts from BAL samples using 2 × 0.8 ml aliquots of

saline were determined as previously described (14),

except that, recovery of BAL fluids was performed from

the distal trachea in intubated animals.

Statistics

Comparisons of baseline values between groups and

intraindividual comparisons were analyzed by the Student's

two-sided t-test, allergic responses of the group of

allergic mice versus control mice were analyzed by one-

sided t-test. P values < 0.05 were considered significant.

Descriptive results were expressed as means ± SE unless

indicated otherwise. Comparison of a new measurement

technique with an established one is needed to see

whether they agree sufficiently. A plot of the difference

against the standard measurements will often appear to

show a relation between difference and magnitude when

there is none. A plot of the difference against the average

of the standard and new measurements is unlikely to mislead

in this way. Accordingly, the agreement between the

invasive and noninvasive lung function methods was analyzed

by the method of Bland and Altman [23]. Graphically,

the difference of each pair of measurement was

plotted against their mean values. Agreement was

expressed as the mean differences over all measurements

and their corresponding 95% confidence intervals (95%

CI). The limits of agreement were expressed as the mean

differences ± 2 SD of the differences, together with their

95% confidence intervals (95% CI). Statistics was performed

with SPSS 11.5.

Results

Baseline values for respiratory parameters in conscious

and anesthetized mice

To illustrate the impact of anesthesia on respiratory function,

baseline respiratory parameters were measured in

anesthetized and conscious mice. Table 1 presents the

baseline values of respiratory parameters obtained from

conscious and anesthetized BALB/c mice. There were significant

differences in f, TE and EF50 values between anesthetized

and conscious animals at baseline. In addition,

no differences in respiratory parameters were observed

between allergic and control mice at baseline when separated

into conscious and anesthetized groups.

Comparison of invasive and noninvasive lung function

measurements of EAR

The allergen-specific early airway response (EAR) to A.

fumigatus was investigated in allergic mice on day 21 (Fig.

1 and 2). To avoid unbalanced challenges with allergen or

saline, each group was separated into two subgroups for

invasive and noninvasive measurement of pulmonary

function.

Invasive recordings of EAR in allergic mice showed significant

decreases in simultaneously measured GL, Cdyn,

and EF50 compared with controls thus indicating an allergen-

specific EAR to A. fumigatus. As shown in Figure 1,

the most prominent alteration was shown for GL with a

reduction by -62.1 ± 5.1 % (P < 0.001 vs. control) compared

with a reduction by -48.8 ± 8.3 % in Cdyn (P <

0.001 vs. control), and a decrease by -34.5 ± 5.1 % in EF50

(P < 0.001 vs. control). The bronchoconstrictive response

started within 7 ± 4 minutes (mean ± SD) after start of

exposure and reached its maximum within 14 ± 3 min

(mean ± SD). Figure 2 illustrates a characteristic time-

response course of the EAR in an anesthetized, orotracheally

intubated allergic mouse.

To determine if decreases in invasively monitored EF50,

relate to changes in GL and Cdyn, we analyzed the agreement

between these measurements by the method of

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Figure 1

Early airway responsiveness. Invasive vs. noninvasive

assessment of early airway responsiveness (EAR) to aerosolized Aspergillus

fumigatus 2 %. Allergic (black columns)

and control mice (white columns) were separated into

groups of invasively and noninvasively monitored animals.

The allergic mice showed significant reductions in simultaneously measured GL,

Cdyn and EF50an (an: anesthetized), compared with control animals. Noninvasive

determination of

EF50con (con: conscious) elicited significant decreases in EF50

to inhaled A. fumigatus compared with control animals. EAR

was expressed as % change from corresponding baseline values, which were taken

as 0 %. Values are means ± SE, n = 8

per group, *p < 0.01 vs. control.

Bland and Altman. Although all three parameters, Cdyn,

GL and EF50, adequately reflected the pronounced EAR in

allergic mice there was enhanced variation between GL vs.

EF50, GL vs. Cdyn and EF50 vs. Cdyn in response to specific

allergen challenge. As shown in Table 2, EF50 tended to

underestimate the decreases in GL by -27.6 %, and by

14.3 % for Cdyn in allergic animals. In contrast, a very

good agreement between EF50, GL and Cdyn values was

found for control mice, with mean differences ranging

from -2.4 to -6.1 %.

Noninvasive measurements of pulmonary function in

allergic mice also demonstrated a marked allergen-specific

EAR as manifested by a significant decline by -44.6 ± 6.2

% in EF50 compared with that in control animals (P =

0.002, Fig. 1). The magnitude of the response was similar

to the decline observed with invasively recorded EF50.

Reduced EF50values were accompanied by decreased VT

Figure 2

Example of EAR. Example of an early airway response

(EAR) to inhaled A. fumigatus 2 % in an orotracheally intubated allergic mouse.

Decreases in GL, Cdyn, and EF50 values

were associated with small declines in VT, f and TE. The ordinate at the bottom

indicates the photometric signal of the

allergen aerosol challenge.

values and – in contrast to invasive measurements – by

decreased f and increased TE values.

Invasive vs. noninvasive determination of cholinergic AHR

To further characterize the utility of noninvasive vs. noninvasive

pulmonary function tests, AR to increasing doses

of aerosolized MCh, was investigated 48 h after EAR

recordings in the same animals. Baseline GL, Cdyn and

EF50 values were not significantly different from initial

baseline values.

MCh exposure elicited a dose-related reduction in GL,

Cdyn, and EF50 values in the intubated animals that was

significantly enhanced in allergic mice (p < 0.05 vs. control

group). The magnitude of cholinergic AR was significantly

higher for GL and Cdyn compared with

simultaneously measured EF50 (P = 0.027). Accordingly,

the mean PD50 causing a decrease in Cdyn, EF50 and GL

to 50 % baseline was 0.4 ± 0.1 for GL, 0.4 ± 0.1 for Cdyn,

and 1.2 ± 0.4 µg MCh for EF50 in allergic mice (Fig. 3). The

respective mean PD50 values for control animals were significantly

higher: 2 ± 0.4 for GL (P = 0.001), 3.4 ± 0.7 for

Cdyn (P = 0.002), and 4.9 ± 1.2 µg MCh for EF50 (P =

0.008). The dose-related decreases in EF50 were accompanied

by increases in esophageal pressures. At the level of

the 50% decline in EF50 (PD50), the peak esophageal pressure

increased 121 ± 13 % for the allergic mice and 104 ±

16 % for the control group.

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Table 2: Bland-Altman analysis of the differences in GL, EF50 and Cdyn.

Early AR Cholinergic AR

Group Parameters Mean ± SD

(95% CI)

Upper limit (95% CI)

Lower limit (95% CI)

Mean ± SD

(95% CI)

Upper limit (95% CI)

Lower limit (95% CI)

Allergic EF50 vs. GL

GL vs. Cdyn

EF50 vs. Cdyn

-27.6 ± 17.8

(-42.6/-12.7)

13.3 ± 21.9

(-5/31.7)

-14.3 ± 29.5

(-39/10.3)

8.0 (-17.8/33.9)

-63.3 (-89.2/-37.5)

57.1 (25.4/88.8)

-30.5 (-62.2/1.2)

44.7 (2/87.4)

-73.3 (-116/-30.6)

-0.7 ± 0.7

(-1.3/0.1)

0 ± 0.2

(-0.2/0.2)

-0.7 ± 0.9

(-1.4/0)

0.7 (-0.3/1.8)

-2.1 (-3.2/-1.1)

0.4 (0.1/0.7)

-0.4 (-0.7/-0.1)

1 (-0.2/2.3)

-2.5 (-3.7/-1.2)

Control EF50 vs. GL -2.4 ± 9.5

(-10.4/5.5)

16.6 (2.8/30.5)

-21.5 (-35.3/-7.7)

-2.9 ± 3.3

(-5.7/-0.2)

3.7 (-1.1/8.5)

-9.5 (-14.3/-4.7)

GL vs. Cdyn -3.7 ± 10.4

(-12.2/5.1)

17.2 (2.1 to 32.3)

-24.6 (-39.7/-9.4)

1.4 ± 1.8

(-0.2/2.9)

5 (2.4/7.7)

-2.3 (-4.9/0.4)

EF50 vs. Cdyn -6.1 ± 9.1

(-13.8/-1.5)

12.2 (-1.1/25.4)

-24.4 (-37.6/-11.2)

-1.5 ± 3.5

(-4.5/1.4)

5.5 (0.4/10.7)

-8.6 (-13.8/-3.5)

Differences in simultaneous invasive measurements of GL, EF50 and Cdyn for

allergic and control mice during EAR and cholinergic AR. Values are

means ± SD (95 % confidence intervals (CI) in brackets) for 8 animals per

group. The upper and lower limits of agreement (means ± 2 SD) as well

as the corresponding 95 % CI intervals (in brackets) are shown. Values for the

EAR represent the % change from baseline, whereas the values for

cholinergic AR show the absolute PD50 values in µg MCh.

The peak responses for GL, Cdyn and EF50 occurred within

1 min after challenge and recovered to within 10–20 % of

the baseline before MCh exposure during 1–3 min. Agreements

between Cdyn, EF50 and GL were excellent, the

mean ranging from 0 to -0.71 µg MCh for the allergic

group and from -2.9 to 1.38 µg MCh for the control group

(Table 2). Figure 4 shows the corresponding Bland-Altman

plots of the differences between EF50 vs. GL and

between EF50 vs. Cdyn against the mean of both values in

allergic animals.

Noninvasive determination of EF50 also showed that allergic

mice were significantly more responsive to MCh, as

indicated by significantly lower PD50 values for EF50

when compared with controls (P = 0.032) (Fig. 3).

Allergic airway inflammation

The A. fumigatus-sensitized and boosted animals showed

significant increases in eosinophils and lymphocytes in

BAL fluid (Table 3) compared with control mice. This

indicates the presence of an inflammatory response in the

lungs of allergic mice. The intubated animals receiving

aerosols directly via the orotracheal tube had slightly

higher numbers of leukocyte populations compared with

conscious mice (statistically not significant).

Impact of hyperoxia on EF50 measurements in C57BL/6

mice

To examine how EF50 correlates with direct lung resistance

measurements, C57BL/6 mice were exposed to 100% oxy

gen for 48 h. Table 4 lists the hyperoxia-induced changes

detected by invasive and noninvasive lung function measurements

compared with control animals. Noninvasive

recordings revealed no significant differences in breathing

rate, TE, VT, and EF50 between control and hyperoxia mice

after 48 h of hyperoxia. Likewise, direct measurements of

pulmonary mechanics in the same animals did not show

any differences in EF50, Cdyn and RL values, thus confirming

the absence of airway constriction in both groups.

Discussion

In the present study we have evaluated the sensitivity and

reliability of repeatable noninvasive versus invasive pulmonary

function tests to sequentially measure AR in

response to specific allergen and cholinergic challenge in

spontaneously breathing mice. Our results demonstrate

that both systems reflect the allergen-specific early AR and

cholinergic AHR of allergic compared with control mice.

The ability to manipulate the mouse genome has opened

up new opportunities to develop mouse models of allergic

asthma that demonstrate spontaneous or chronic disease

[24]. For a proper phenotyping of AR in experimental

models it is crucial to monitor pulmonary function as reliably

as possible. One way to achieve this is a novel in-vivo

method that combines repetitive recordings of classical

pulmonary mechanics with cholinergic aerosol challenges

in orotracheally intubated mice [4]. Despite being an

accurate measurement of classical pulmonary function on

multiple occasions, this invasive method does not readily

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Table 3: Cellular composition of BAL fluid

Control mice conscious Allergic mice conscious Control mice anesthetized

Allergic mice anesthetized

Eosinophils, × 104

Lymphocytes, × 104

Neutrophils, × 104

Macrophages, × 104

< 1

< 1

< 1

12.3 ± 3.3

7.9 ± 5.6*

3.2 ± 2.2*

1.3 ± 1

13.7 ± 3.1

< 1

0.5 ± 0.4

1.9 ± 1.3

22 ± 9.2

13.4 ± 9.3*

1.8 ± 1.6*

2.7 ± 4.1

16.7 ± 6.1

Values are means ± SD from 8 animals per group. Eosinophils and lymphocytes

recovered from bronchoalveolar lavage (BAL) fluid 48 hours after

allergen challenge were increased in both conscious and intubated allergic mice.

*P < 0.05 vs. control mice.

allow for rapid screening of pulmonary function in large

numbers of animals.

In contrast, noninvasive head-out body plethysmography

has been shown to yield stable and reliable on-line measurements

of AR in several conscious mice at a time and

serves as a suitable and valid tool to complement the traditional

measures of pulmonary mechanics

[13,14,16,22,25]. Limitations of previous EF50 validation

studies in mice particularly have included pleural catheterization

with the inability to conduct reproducible

measurements, the contribution of upper airway resistance

and intravenous rather than aerosol challenge

[14,17]. These methodological shortcomings introduced

variability into the results which made them difficult to

compare with other invasive techniques [10].

The current report intended to overcome such problems

in that GL, Cdyn and EF50 were measured simultaneously

in intact mice including local aerosol challenges via an

orotracheal tube. In parallel, noninvasive determinations

of EF50 were performed in allergic and control mice. The

noninvasive experiments relied on methodologies identical

to those used in our previous mice studies to facilitate

comparisons [14,17,22].

The values for respiratory parameters measured from both

conscious and anesthetized BALB/c mice were reproducible

and comparable with those reported previously for

this strain (Table 1) [4,14,26]. The changes in respiratory

patterns observed in anesthetized mice were associated

with increased expiratory time, decreased f, and decreased

EF50 values, events likely related to anesthetic effects on

neural respiratory control. The independence of EF50

recordings from changes in frequency has been demonstrated

in previous investigations [14,15].

To examine the sensitivity of noninvasive and invasive

indices of bronchoconstriction, we monitored allergen-

specific EAR and, 48 h later, performed MCh dose-

response studies in the same allergic animals compared

with controls. Challenge with aerosolized A. fumigatus

resulted in significant reductions in Cdyn, GL and in EF50

values in allergic mice compared with (sham-exposed)

control animals. Demonstration of allergen-specific EAR

in allergic mice was followed by cholinergic AHR that was

linked with a pronounced influx of neutrophils and eosinophils

in BAL fluid. Consistent with previous results,

invasively recorded EF50 was slightly less sensitive in

detecting the maximum degree of bronchoconstriction to

A. fumigatus and MCh compared with GL and Cdyn

recordings [15].

Agreement between invasively measured EF50, GL and

Cdyn during EAR and cholinergic AHR was good,

although there was increased variability at the time of EAR

in allergic mice (Table 2). This variability may reflect different

sensitivities of GL, EF50 and Cdyn to the airway and

tissue components of total pulmonary resistance [3,16].

Related to this issue, is a previous study indicating that

mice with airway inflammation experience quite heterogeneous

airway narrowing and airway closure during airway

smooth muscle contraction [27].

Nevertheless, despite this variability, it is important to

emphasize that the noninvasive measurement of EF50 still

reflected the enhanced AR to A. fumigatus and MCh in

allergic relative to control mice (Figs. 1, 3). Thus, although

the calculated inhalation doses for A. fumigatus and MCh

in conscious mice may be not as accurate as in intubated

mice, the observed EF50 responses still reflect airway constriction.

These findings indicate that EF50 can distinguish

between different magnitudes of AR and reflects the

changes with GL and Cdyn during bronchoconstriction at

least under the conditions of this study. Moreover, the

relation of the cholinergic EF50 response between allergic

and control animals was similar for invasive and noninvasive

measurements (Figure 3). The higher PD50 values for

EF50 in conscious compared with intubated animals to

MCh challenge can be explained by methodological

issues. Administration of aerosols directly into the lungs

via an orotracheal tube results in aerosol deposition

mainly in the parenchyma. In conscious animals there

will be substantial deposition in the nasal passages and

upper airway, which should lead to the higher PD50 values

observed. The AR, as measured noninvasively by EF50,

may also be partly affected by altered upper airway resistance.

However, because of the rapid onset and resolution

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Respiratory Research 2005, 6:139 http://respiratory-research.com/content/6/1/139

Figure 3

Cholinergic AR. Magnitudes of cholinergic AHR, 48 h after

EAR, expressed as PD50 values, which is the dose of MCh

required to reduce either GL, Cdyn or EF50 to 50 % of their

respective baseline values) of invasively measured GL, Cdyn

and EF50 (A) as well as of noninvasively recorded EF50 (.

Allergic mice (black columns) showed significantly lower

PD50 values compared with controls (white columns). Baseline values were not

significantly different from initial baseline

values 48 h before and were within the means ± SD as listed

in Table 1. Values are means ± SE, n = 8 per group, *p < 0.05

vs. control.

of the response, it seems unlikely that edema or mucus

hypersecretion in these upper airways was responsible for

the increased AR.

In agreement with other investigations, decreases in EF50,

as measured by noninvasive head-out body plethysmography,

were linked with decreased frequency and VT values

and increasing values for TE [12,14,15]. In contrast,

no relevant impact on frequency and TE was found in

anesthetized, intubated mice during bronchoconstriction.

Concerns with noninvasive EF50 recordings include the

uncertainty about the exact degree and localization of

bronchoconstriction as well as the potential contribution

of upper airway resistance. Due to methodological differences,

comparisons between invasive and noninvasive

measures are of indirect, qualitative nature. A quantitative

comparison, however, is directly available from the

intraindividual differences between simultaneously measured

EF50 and GL in unconscious mice. Because EF50 tends

to underestimate the magnitude of bronchoconstriction

(discussed below) it is still unclear whether this limits its

use in detecting less marked changes in airway hyperresponsiveness

than those induced in high-reponder models.

As a result, EF50 measures should be confirmed with

direct assessments of pulmonary resistance under these

circumstances. Despite these methodological restrictions,

the observed EF50 responses still reflected the enhanced

AR to ACh and allergen under the conditions of this study.

In comparison with the widely used Penh method, EF50

differs substantially in several important ways: EF50

decreases with bronchoconstriction and in line with invasively

measured lung resistance or conductance is linked

with a decline in VT during bronchoconstriction [7,28].

Even more importantly, EF50 has a physical meaning (ml/

s), allows direct comparison from one animal to another

and is closely related to airway resistance. Indeed, if it

were possible to know the esophageal pressure in the conscious

animals, one could calculate a precise lung resistance.

If we assume that esophageal pressure does not

change, then changes in the EF50 would be directly proportional

to the lung resistance. However, in the anesthetized

animals, we found that the esophageal pressure

actually increased as the airways constricted, perhaps in

response to the increased resistance and lower air flow.

This suggests that the EF50 in conscious animals may

underestimate the actual changes in lung resistance.

Despite this quantitative limitation, the method seems far

more representative of changes in resistance than other

noninvasive methods, and the approach allows for direct

quantitative comparisons from animal to animal. The

commonly measured Penh has no theoretical linkage to

lung resistance, and its usefulness was further weakened

by recent reports, one of which showed that changes in

Penh were no better than simply measuring TE to assess

AR in common strains of laboratory mice [6]. It is also

known that a decline in noninvasively measured EF50 is

associated with an increase in TE [12,14]. However, it is

important to note that conditions entirely unrelated to

Table 4: Impact of hyperoxia over 48 h on invasively and noninvasively measured

respiratory parameters

Noninvasive measurement

EF50 TE VT f RL Cdyn

Invasive measurement

GL EF50 TE VT* f

Control 2.36 ±

0.12

0.13 ±

0.01

0.20 ±

0.01

251 ± 14 1.44 ±

0.27

0.017 ±

0.004

0.72 ±

0.15

1.01 ±

0.13

0.3 ±

0.03

0.11 ±

0.02

106 ± 9

Hyperoxia 2.30 ± 0.14 ± 0.20 ± 245 ± 41 1.27 ± 0.018 ± 0.85 ± 0.93 ±

0.32 ± 0.14 ± 99 ± 15

0.41 0.02 0.02 0.29 0.007 0.18 0.15 0.04 0.02

Values are means ± SD from 8 C57BL/6 mice per group. *P < 0.05 vs. control

mice. VT: tidal volume, EF50: tidal midexpiratory flow, TE: time of

expiration, f: respiratory rate, RL: pulmonary resistance, Cdyn: dynamic

compliance, GL: pulmonary conductance (GL = 1/RL).

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Respiratory Research 2005, 6:139

Figure 4

Bland-Altman plots. Individual differences in the degree of

MCh-induced bronchoconstriction between invasively measured EF50 and GL and

between EF50 and Cdyn, are plotted

against the average corresponding values (expressed as

PD50, µg MCh). The solid line represents the mean of the

differences, the dashed lines show the upper and lower limits

of agreement.

bronchoconstriction, such as sensory irritation, will also

result in increasing TE values [12,29].

Another report demonstrated that Penh was inadequate

for characterization of pulmonary mechanics in the context

of hyperoxia-induced changes in C57BL/6 mice [18].

These authors pointed out that Penh may significantly

overestimate the actual changes in lung resistance after 24

and 48 h of hyperoxia. Interestingly, increases in Penh

were accompanied by decreased TE and rising VT and f.

This contrasts with the above-mentioned observation of

decreased VT during bronchoconstriction as observed

with EF50 and invasive pulmonary function methods

http://respiratory-research.com/content/6/1/139

[4,28]. Our study in C57BL/6 mice showed a consistent

relationship between EF50 and lung resistance measurements

in reponse to 48 h hyperoxia, thus indicating non-

constricted airways. These data support the concept that

EF50 more reliably reflects airway resistance than Penh,

which is largely a function of respiratory timing.

Conclusion

In conclusion, this study investigated the utility of repetitive

invasive vs. noninvasive techniques to determine AR

to allergen and cholinergic challenge in intact, spontaneously

breathing mice. We demonstrated allergen-specific

EAR to A. fumigatus followed by cholinergic AHR in allergic

mice compared with controls. Our results show that

the noninvasive EF50 method is directly related to lung

resistance, and is thus particularly appropriate for quick

and repeatable phenotyping of airway function in large

numbers of conscious mice.

Competing interests

The author(s) declare that they have no competing interests.

Authors' contributions

TG participated in the design and coordination of the

study and drafted the manuscript. MZ and RB carried out

the lung function experiments. RK participated in the data

analysis of all experiments, AB carried out the cytological

and ELISA tests. WM helped to draft the manuscript. JMH

and NK participated in the coordination and analysis of

the study. HGH conceived of the study, and participated

in its design and analysis. All authors read and approved

the final manuscript

Acknowledgements

We greatly thank Prof. H. Hecker, Biometrics of Hannover Medical School,

for statistical support and Dr. C. Nassenstein, Fraunhofer ITEM, for excellent

technical support.

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