1 Department of Respiratory Medicine, Hannover Medical School, 30625 Hannover; 2 Institute of Laboratory Medicine and Pathobiochemistry, Charité Campus Virchow Clinic, Humboldt University, 13353 Berlin; 3 Institute of Clinical Chemistry and Molecular Diagnostics, Philipps-University, 35043 Marburg, Germany; and 4 Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15238
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ABSTRACT |
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A method for the noninvasive measurement of
airway responsiveness was validated in allergic BALB/c mice. With
head-out body plethysmography and the decrease in tidal
midexpiratory flow (EF50) as an indicator of airway
obstruction, responses to inhaled methacholine (MCh) and the allergen
ovalbumin were measured in conscious mice. Allergen-sensitized and
-challenged mice developed airway hyperresponsiveness as measured by
EF50 to aerosolized MCh compared with that in control animals. This response was associated with increased allergen-specific IgE and IgG1 production, increased levels of interleukin-4 and interleukin-5 in bronchoalveolar lavage fluid and eosinophilic lung
inflammation. Ovalbumin aerosol challenge elicited no acute bronchoconstriction but resulted in a significant decline in
EF50 baseline values 24 h after challenge in allergic
mice. The decline in EF50 to MCh challenge correlated
closely with simultaneous decreases in pulmonary conductance and
dynamic compliance. The decrease in EF50 was partly
inhibited by pretreatment with the inhaled 2-agonist
salbutamol. We conclude that measurement of EF50 to inhaled
bronchoconstrictors by head-out body plethysmography is a valid measure
of airway hyperresponsiveness in mice.
body plethysmography; allergy; lung function method
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INTRODUCTION |
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ALLERGIC ASTHMA IS CHARACTERIZED by an allergen-specific immune response with IgE antibodies, the presence of airway inflammation, and airway hyperresponsiveness (AHR). Because the contribution of each of these components to the pathogenesis of bronchial asthma remains incompletely understood and because extensive in vivo investigations in human subjects are limited due to ethical reasons, there has been considerable interest in the development of animal models that mimic the immunopathogenesis of the disease as closely as possible (3, 15). Mice develop AHR to several bronchoconstrictors after systemic and local allergen sensitization followed by allergen challenge via the airways. During the last several years, we (9, 20) and others (2, 6, 11) have developed and characterized murine models that resemble several of the immunologic key factors present in human bronchial asthma. However, additional studies are necessary to further investigate the kinetics and mechanisms underlying AHR. The most commonly employed tests of lung function in animal models have included invasive and in vitro measurements (13, 14). Such tests are not applicable in conscious animals and, as single-point measurements, do not allow follow-up studies. With the recent emphasis on the benefits of noninvasive technologies, there has been growing interest in the analysis of tidal flow patterns as a tool in the assessment of airway obstruction. The analysis of tidal flow patterns has been shown to be a useful indicator of bronchial obstruction in adults and children and has also been employed in toxicological studies with mice and guinea pigs (17, 22, 23). Head-out body plethysmography, which allows the continuous on-line recording of tidal flow patterns in conscious mice, is advantageous because it is technically easy to perform, allows measurement of airway responsiveness (AR) to aerosolized stimulants, and provides a technique for repeated and long-term measurements of airway reactivity. With this method, airway obstruction induces characteristic alterations of the airflow pattern in mice, which are revealed by a decrease in tidal midexpiratory flow (EF50). In this investigation, we extend previous studies (18, 23) in further defining the EF50 method as a noninvasive approach to measure lung function in mice. The primary objective of the present study was to comprehensively evaluate the applicability of the EF50 measurement as a valid indicator of airway hyperreactivity in a well-established murine model of allergen-induced airway inflammation (19). Accordingly, we determined the changes in breathing patterns to several stimulants and the responses to a bronchodilator on EF50 measurements. Moreover, the changes in noninvasive indexes were directly correlated with simultaneous invasive recordings of pulmonary conductance (GL) and dynamic compliance (Cdyn) in conscious mice, thus avoiding the drawbacks associated with anesthesia or with invasive and noninvasive measurements of pulmonary function at different time points.
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MATERIALS AND METHODS |
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Animals. Pathogen-free female BALB/c mice (Bomholtgaard, Ry, Denmark) 6-8 wk of age and weighing 18-22 g were used in all experiments. The animals were kept under standard housing conditions, fed an ovalbumin (Ova)-free diet, and given water ad libitum. The mice were housed for at least 1 wk before investigation. All experimental animal studies were done in accordance with the National Research Council Guide and under a protocol approved by the appropriate governmental authority (Landesamt für Arbeitsschutz, Gesundheitsschutz und Technische Sicherheit, Berlin, Germany). The mice were killed at the end of the experiments by an overdose of intravenous pentobarbital sodium.
Sensitization protocol. The following experimental groups were investigated. In the allergen-sensitized and allergen-challenged (Ova/Ova) group, six animals were sensitized on days 1, 14, and 21 with three intraperitoneal injections of 10 µg of Ova (grade VI; Sigma, Deisenhofen, Germany) emulsified with 1.5 mg of aluminum hydroxide (Pierce Chemical, Rockford, IL) and diluted with sterile PBS (Seromed, Berlin, Germany) to a total volume of 150 µl. They were then exposed to aerosolized Ova (grade V, 1% wt/vol) diluted with sterile PBS twice a day on 2 consecutive days (days 26 and 27). In the sham-sensitized and allergen-challenged (PBS/Ova) group, six animals received three intraperitoneal injections of PBS on days 1, 14, and 21. They were challenged with aerosolized Ova twice a day on days 26 and 27 as described above. In the allergen-sensitized and sham-exposed (Ova/PBS) group, six animals were sensitized with three intraperitoneal injections of Ova plus aluminum hydroxide on days 1, 14, and 21. They were challenged twice a day with nebulized PBS on days 26 and 27.
Mice were challenged via the airways with Ova (1% in PBS) or PBS for 15 min by jet aerosolization (Pari-Master, LC Star, 2.8-µm mass median diameter, Pari-Werke, Starnberg, Germany) into the head chamber. All agents used in this study, including n-propranolol (Sigma), were nebulized by jet aerosolization except for 2-chlorobenzylchloride (CBC; Sigma), which was generated as a vapor by a Pitt no. 1 generator (Crown Glass, Somerville, NJ) (26). Assessment of AR to methacholine (MCh; Sigma) followed by determination of immunoglobulins, analysis of bronchoalveolar lavage (BAL) fluid, and histological examination was performed on day 28, 24 h after the last aerosol exposure.Noninvasive measurement of AR to MCh in conscious mice.
AR was measured with head-out body plethysmography (23).
Briefly, the system consisted of a glass-made body plethysmograph to
which a head exposure chamber was attached (Forschungswerkstaetten, Hannover Medical School, Hannover, Germany) (Fig.
1). The mouse was positioned in the body
plethysmograph while the head of the animal protruded through a neck
collar (9-mm ID, dental latex dam; Roeko, Langenau, Germany) into the
head exposure chamber, which was ventilated by a continuous airflow of
200 ml/min. Small leaks, if present, were not phasic with respiration
and over a period of several breaths were considered to be constant.
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Invasive measurement of AR to MCh. To compare results from noninvasive measurements directly with invasive indexes without performing a tracheostomy in anesthetized ventilated mice, AR was further assessed with a method previously described (4, 23). Briefly, mice were anesthetized with pentobarbital sodium (intraperitoneally, 50 mg/kg), and a needle-mounted catheter-tip differential pressure transducer (8 CT/SS, Medical Measurements, Hackensack, NJ) was inserted in the pleural cavity to measure transpulmonary pressure (Ptp). The surgical incision sites were closed with sutures and infiltrated with 0.25% bupivacaine, a long-lasting local anesthetic. Before baseline values were measured and the MCh challenge was initiated, the mice were allowed to recover from the systemic anesthesia. The spontaneously breathing animal was then placed in the body plethysmograph coupled to a pressure transducer and constructed with ports for the pneumotachograph and catheter cable access tubing. The system was operated to continuously measure tidal flow (V) and VT from the body plethysmograph; Ptp was determined by a second pressure transducer. The primary signals (Ptp, flow, and VT) were calibrated against known pressures, flow rates, and volume displacements over the physiological range studied. The flow, volume, and Ptp signals were then continuously displayed and digitized as noted in Noninvasive measurement of AR to MCh in conscious mice. Pulmonary resistance (RL) and Cdyn were calculated according to the method of Amdur and Mead (1). Cdyn was calculated by relating the volume changes to the concomitant elastic recoil pressure changes between end inspiration and end expiration (instance of zero flow). For better comparability of data, RL was calculated as GL (1/RL) from the difference in Ptp and the difference in flow between points of equal volume (50% VT) in the respiratory cycle. After a stable baseline airway pressure (<5% variation over 2.5 min) was reached, PBS and MCh were administered via a jet nebulizer into the head chamber as described above. Minimum values for GL and Cdyn were determined and are expressed as percent change from the baseline value. The PD causing a decrease in Cdyn to 55% of the baseline value or a decrease in GL to 200% of the baseline value was determined and is expressed as PD55Cdyn and PD200GL, respectively.
Effects of inhalative pretreatment with salbutamol on AHR.
To investigate the effects of an inhaled 2-agonist on
EF50 in a separate group of allergen-sensitized and
-challenged mice, salbutamol (5 mg/ml; Glaxo Wellcome, Hamburg,
Germany) was aerosolized for 5 min followed by inhalation challenges to
increasing concentrations of MCh (5-25 mg/ml) 15 min later. The
control group consisted of allergen-sensitized and -challenged mice
aerosolized for 5 min with PBS instead of salbutamol.
Measurement of serum immunoglobulins. Total IgE and allergen-specific IgE and IgG1 antibody concentrations were measured by ELISA as previously described (20). The antibody titers of the samples were compared with pooled standard serum and are expressed as units per milliliter. Total IgE levels were compared with a known IgE standard. Rat anti-mouse IgE and IgG1 monoclonal antibodies and standards were obtained from PharMingen (Hamburg, Germany).
Assessment of leukocyte distribution in BAL fluid.
Twenty-four hours after the last aerosol challenge with allergen or
PBS, the tracheae were cannulated and the lungs were lavaged two times
with 0.8 ml of PBS. The mean recovery volume was 1.3 ± 0.2 ml.
The BAL fluid from each animal was pooled and centrifuged. Total cell
numbers were determined with a standard hemacytometer. BAL fluid cells
were resuspended, cytocentrifuged at 900 rpm, and differentially
stained with Diff-Quik (Baxter Dade, Duedingen, Switzerland) after
fixation. Cell types were identified by light microscopy with standard
morphological criteria. Differential cell counts of 200 leukocytes were
performed in duplicate. Cell-free lavage fluids were stored at 70°C
until further analysis.
Measurement of cytokines in BAL fluid.
Cytokine levels were measured by ELISA as previously described
(9). The cytokines interleukin (IL)-4, IL-5, IL-2, and
interferon (IFN)- as well as rat anti-mouse IL-4, IL-5, IL-2, and
IFN-
monoclonal antibodies were obtained from PharMingen.
Sensitivities were 10 pg/ml for IL-4, 30 pg/ml for IL-5, 10 pg/ml for
IL-2, and 100 pg/ml for IFN-
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Histology. To assess airway inflammation, the lungs were fixed in situ with 4% (wt/vol) formaldehyde via the trachea, removed, and stored in 4% formaldehyde. Lung tissues were paraffin embedded, and 3-µm sections were stained with hematoxylin and eosin.
Statistical analysis. ANOVA was used to determine significant differences between groups. Single pairs of groups were compared by the Student's t-test. Comparisons for all pairs were performed by Scheffé's test. Correlations were calculated with Pearson's correlation coefficient. Values for all measurements are expressed as means ± SD. P values < 0.05 were considered significant. All statistical analyses were performed with GB-STAT 6.5 (Dynamic Microsystems, Silver Spring, MD).
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RESULTS |
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Breathing patterns and baseline values for respiratory parameters in BALB/c mice. Table 1 presents the baseline values for respiratory parameters obtained from untreated BALB/c mice placed in head-out body plethysmographs with and without transpulmonary measurement. No significant difference in the variables measured was observed between chest-intact conscious spontaneously breathing mice and animals with an inserted intrapleural catheter after recovery from anesthesia.
Figure 2 illustrates the normal breathing pattern of spontaneously breathing BALB/c mice and the characteristic changes to the airflow pattern due to MCh exposure, showing the decrease in EF50 during airway obstruction.
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Correlation of EF50 with direct measurement of
GL and Cdyn in AR to MCh.
Nonspecific AR to MCh in conscious, spontaneously breathing mice was
investigated with head-out body plethysmography. Dose-response relationships to inhaled MCh were compared in Ova/Ova-, PBS/Ova-, and
Ova/PBS-treated mice. To study AR, mice were exposed to increasing doses of aerosolized MCh (ranging from 5 to 25 mg/ml) administered via
the head chamber 24 h after the last aerosol challenge with Ova or
PBS. Baseline values did not differ significantly between groups of
mice after aerosol challenge with PBS. To determine whether decreases
in EF50 values correlated with decreased GL and
decreased Cdyn values, EF50 was measured and compared with simultaneously recorded Cdyn and GL in the same animals.
Both control groups (PBS/Ova and Ova/PBS) showed similar dose-dependent decreases in EF50 in response to aerosolized MCh, whereas
PBS aerosol had no effect on breathing patterns. Compared with control mice, Ova/Ova mice showed significantly decreased values for
EF50, GL, and Cdyn (Fig.
3). The
PD55EF50 was significantly lower in the
Ova/Ova-treated mice compared with that in the control animals (Ova/Ova, 12.59 ± 2.37 mg/ml; PBS/Ova, 21.55 ± 2.68 mg/ml;
Ova/PBS, 23.59 ± 2.43 mg/ml). Decreases in Cdyn and
GL paralleled the decreases in EF50 values,
although Cdyn was less sensitive in detecting enhanced AR
(PD55Cdyn: 14.65 ± 2.44, 23.51 ± 3.94, and
25.72 ± 3.96 mg/ml in Ova/Ova-, PBS/Ova-, and Ova/PBS-treated
groups, respectively; PD200GL: 7.47 ± 2.15, 18.66 ± 3.52, and 20.47 ± 3.39 mg/ml in Ova/Ova-,
PBS/Ova-, and Ova/PBS-treated groups, respectively). Correlation
analysis showed a close correlation between decreased EF50
and GL values (r = 0.916; P < 0.01) and between decreased EF50 and Cdyn values
(r = 0.969; P < 0.01) in allergen-sensitized and -challenged mice. The peak EF50
responses to MCh challenge varied between 1.5 and 3 min after challenge with aerosolized MCh, whereas recovery to baseline values occurred within 3-8 min after exposure. EF50 values decreased
proportionally with increasing values of TE and decreasing
values of VT and f (Fig. 4).
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EF50 is independent of alterations in f,
VT, and TE.
MCh challenge of mice induced parallel decreases in Cdyn,
GL, and EF50 values when measured on-line.
Simultaneously, an increase in both TI and TE
was observed as well as decreased values for f and VT. To
investigate the influence of f, TE, and VT on
EF50 inhalational challenge, studies with various model
agents (CBC, PBS, and n-propranolol) were performed. In
contrast to Cdyn, which showed a slight but significant decline during
n-propranolol exposure, EF50 and GL
were not affected by airborne substances, leading to a decrease in f
and elongation of TE (CBC generated as a vapor at a
concentration of 380 mg/m3), increase in f
(n-propranolol concentration of 15 mg/ml), or decrease in
VT (n-propranolol; Table
2). These data show that decreases or
increases in f, TE, or VT were not
necessarily accompanied by changes in EF50, indicating that
EF50 is an independent measure of airway obstruction.
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Inhibition of increased AR by a 2-agonist.
To investigate the effect of a bronchodilatative
2-agonist on EF50, salbutamol was
aerosolized for 2.5 min, 15 min before MCh challenge in chest-intact
allergen-sensitized and -challenged mice. Pretreatment with salbutamol
did not affect baseline EF50 values but significantly
reduced the decreases in EF50 values during challenge to
increasing concentrations (5-25mg/ml) of MCh (Fig.
5). Pretreatment with PBS aerosol instead
of salbutamol had no inhibitive effect on EF50 readings
during baseline and exposure periods. These data indicate that maximal
EF50 responses during MCh aerosol exposure were distinctly
reduced after pretreatment with a
2-agonist.
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Effect of allergen challenge on serum immunoglobulins and cytokine
levels in BAL fluid and EF50.
Allergen-sensitized and -challenged mice had enhanced serum levels of
total IgE and allergen-specific IgE and IgG1 compared with control mice
receiving no allergen sensitization or no subsequent Ova airway
challenge (Table 3). In addition, only
allergen-sensitized and -challenged mice revealed significant increases
in IL-4 and IL-5 levels in BAL fluid, with no significant changes in
IFN- and IL-2 (Fig. 6). No responses
were observed in control mice that were either allergen
sensitized without subsequent airway challenge to Ova or were
nonsensitized with subsequent airway challenge to Ova. For the
Ova/Ova-, PBS/Ova-, and Ova/PBS-treated groups, all respiratory
parameters listed in Table 1 were continuously monitored during the
20-min aerosol challenge with Ova or PBS twice a day on days
26 and 27. No significant modifications to the normal
breathing patterns of any of these animals were observed during such
challenges. The EF50 baseline readings were similar for the
control groups (PBS/Ova and Ova/PBS) 24 h after the last challenge
but were significantly reduced in Ova/Ova-treated mice [EF50:
1.815 ± 0.08 (P < 0.05),
1.965 ± 0.12, and
2.113 ± 0.17 ml/s in Ova/Ova-,
PBS/Ova-, and Ova/PBS-treated groups, respectively]. These data
indicate that EF50 values are decreased in Ova/Ova-treated animals.
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Association of EF50 with airway inflammation.
To study the association between AR and airway inflammation, we
investigated leukocyte populations in BAL fluid in Ova/Ova-treated animals compared with those in control animals. Although total cell
number and leukocyte populations from control animals were not
different from those in untreated mice (data not shown), allergen challenge in allergen-sensitized mice uniformly caused significant increases in eosinophils, lymphocytes, and neutrophils (Table 4). In contrast, alveolar macrophages
comprised a significantly smaller proportion of total BAL fluid cells.
Histological analysis of fixed lung specimens revealed a perivascular
mononuclear infiltrate with numerous eosinophils in the Ova/Ova-treated
group that was not present in control animals.
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DISCUSSION |
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We found that EF50 as measured by head-out body plethysmography can be used as an index of airway obstruction in conscious spontaneously breathing mice. A variety of methods for studying AR in mice have previously been used (5), the major disadvantages being invasive procedures, the need for anesthesia with potential drug-induced changes in AR or in sensitivity to cholinergic challenge, mechanical ventilation, systemic rather than local administration of drugs, inability of repeated on-line measurements, or extended challenges with aerosols. Recently, noninvasive methods have been developed that permit measurement of respiratory function and, particularly, the development of AR in conscious intact mice. Hamelmann et al. (8) described a noninvasive whole body plethysmography (WBP) technology that uses the dimensionless variable enhanced pause to empirically monitor bronchoconstriction in conscious animals. In comparison, head-out body plethysmography obviates efforts to compensate for the adiabatic conditions that occur through temperature and humidity changes by inspired and expired air of mice placed in a WBP chamber. A continuous bias flow through the head chamber further allows continuous acute and long-term measurements of pulmonary function with no need to replace the air inside the plethysmograph. The intraindividual variability in respiratory function measured by head-out body plethysmography was acceptable. We showed that the mean values of f, TE, TI, VT, and EF50 were highly reproducible and comparable with values previously reported for BALB/c mice (21). To evaluate the sensitivity of head-out body plethysmography to detect changes in pulmonary function, we measured the bronchoconstrictive response to aerosolized MCh and differentiated between normal levels of AR in control animals and AHR in allergen-sensitized and -challenged mice. Sensitization without allergen challenge or airway challenge with allergen in nonsensitized mice had no effect on the EF50 values compared with those in untreated animals. Consistent with data from this and other studies (6, 8, 18, 20), specific allergen challenge with Ova via the airways had no acute bronchoconstrictive effect on allergen-sensitized mice. The fact that this group of animals had decreased EF50 baseline values 24 h after challenge indicates that the specific allergen challenge had a delayed effect on pulmonary function. Our results differ from those of Cieslewicz et al. (2), who showed that BALB/c mice immunized by Ova display early- and late-phase AHR in response to inhaled Ova. This may result from different protocols (e.g., additional 5% Ova aerosol provocation after systemic sensitization and three aerosol challenges with 1% Ova), particularly because they also found no allergen-specific AHR in allergen-sensitized and -challenged mice that had not received subsequent 5% Ova aerosol provocation.
The EF50 response to MCh in allergen-sensitized and
-challenged animals was both shifted to the left and amplified compared with that in control animals. This is similar to AHR in patients suffering from bronchial asthma as well as to measurements of AHR with
invasive and noninvasive methods in other animal models (24). A decrease in EF50 was
characteristically accompanied by an elongation in TE and
by a decrease in both f and VT. The changes in TE
and f during bronchoconstriction were similar to the results
obtained with the WBP method (8). In contrast, we observed
a decline in VT during airway obstruction, which has been
confirmed by a recent study of respiratory mechanics in mice (12). That EF50 measurements are independent
of f, TE, and VT was determined in studies with
various airborne stimuli (CBC and n-propranolol) in
conscious animals in which EF50 values were unaffected by
drug-induced changes in f, VT, and TE,
suggesting that EF50 does not correlate simply with changes
in breathing patterns. The MCh-related decrease in EF50 is
presumably mediated by specific muscarinic M3 receptor
activation on airway smooth muscles (7). Although our data
do not provide proof of this mechanism, the rapid onset and resolution
of the response to aerosolized MCh makes alternative mechanisms of
obstruction such as edema or mucus hypersecretion rather unlikely.
Further evidence for airway smooth muscle constriction as the major
mechanism for the decrease in EF50 was obtained with a
2-agonist; pretreatment of allergen-sensitized and
-challenged animals with aerosolized salbutamol significantly reduced
the decreases in EF50 due to MCh challenge.
Noninvasive measurement of EF50 directly correlated with simultaneous invasive measurements of Cdyn and GL in the same animals after recovery from anesthesia. The responses monitored in the two systems under the same physiological conditions were virtually identical, with comparable increases and a similar left shift of the dose-response curve over baseline values. These data indicate that changes in EF50 closely reflect the changes observed in Cdyn and GL during bronchoconstriction. Although Cdyn correlated well with measurements of EF50 and GL, it was less sensitive in detecting enhanced AHR to MCh. This finding is consistent with previous studies (12, 14, 25) that suggested that Cdyn, even more than flow resistance, is mainly determined by the plastoelastic recoil of the lung. With such an influence, Cdyn may be less sensitive than the flow resistance to detect small changes in the bronchial system. In addition, the decrease in Cdyn caused by n-propranolol aerosol may relate to a direct effect on tissue elasticity that is also fully compatible with inducing an increase in f and a decrease in VT (16, 27). One problem of measuring airway hyperreactivity with head-out body plethysmography is the uncertainty of the site of obstruction. We are aware that a part of airway obstruction as measured by EF50 may be related to altered upper airway resistance as shown in other studies using noninvasive parameters for the measurement of AR (4, 8, 10). Neuhaus-Steinmetz et al. (18) addressed this issue recently, demonstrating that the noninvasive determination of EF50 correlates well with the invasive measurement of airway resistance in tracheostomized, ventilated mice. However, because the distribution of airway size versus airway generation is not known in the mouse, it is unclear whether relative changes in EF50, GL, and Cdyn can be used to specifically localize the site of the response, i.e., larger versus smaller conducting airways. Despite these limitations, we found that EF50 can significantly discriminate the degree of AR between allergen-sensitized and -challenged animals and control animals after MCh challenge. The determination of EF50 thus appears to be a simple and valid indicator of AR in mice.
Decreases in EF50 values in allergen-sensitized and -challenged mice were associated with the production of allergen-specific IgE and IgG1 and the development of eosinophil infiltration in the lungs. In addition, increased numbers of eosinophils and lymphocytes as well as elevated titers of the Th2 cytokines IL-4 and IL-5 in BAL fluid were detected after allergen challenge of sensitized mice. These data suggest an association between AHR and allergic inflammation of the airways in this model.
In summary, this study has extended previous investigations
(18, 23) by further defining the EF50 method
as an indicator of airway obstruction in conscious mice. The
measurement of EF50 closely reflected the enhanced airway
response to MCh observed with simultaneously measured GL
and Cdyn in allergen-sensitized and -challenged BALB/c mice. We further
demonstrated that the decline in EF50 to MCh challenge was
partly inhibited by pretreatment with an inhaled
2-agonist and that associated changes in f and VT did not directly influence EF50 values. The
fact that murine respiratory function can be continuously followed
makes this noninvasive technique particularly useful for acute and
long-term in vivo studies that require the on-line measurement of
respiratory signals to determine the onset of airway response and to
follow transient or delayed respiratory reactions to several different
aerosolized bronchial challenge experiments. We conclude that this
technique is potentially attractive in investigating mechanisms of
acute and chronic inflammatory reactions of the airways such as asthma or cystic fibrosis and provides a tool to assess new therapeutic strategies under in vivo conditions.
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ACKNOWLEDGEMENTS |
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The computer programs used in this study are available free of charge to interested researchers from Y. Alarie. The DOS software is year 2000 compatible. We thank M. Ehrhardt and Dr. S. Riedel (Physikalisch-Technische Bundesanstalt Berlin) for excellent technical assistance. Dr. J. Hohlfeld and Prof. N. Krug are gratefully acknowledged for reviewing the manuscript.
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FOOTNOTES |
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This work was supported by the Volkswagen Stiftung and Hochschulinterne Leistungsfoerderung of the Hannover Medical School.
Address for reprint requests and other correspondence: T. Glaab, Dept. of Respiratory Medicine, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany (E-mail: thomasglaab{at}web.de).
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.
Received 15 June 2000; accepted in final form 10 October 2000.
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