special communication

Am J Physiol Lung Cell Mol Physiol 280: L565 L573, 2001. special communication Tidal midexpiratory flow as a measure of airway hyperresponsiveness in allergic mice THOMAS GLAAB, 1 ANGELIKA DASER, 2 ARMIN BRAUN, 3 ULRICH NEUHAUS-STEINMETZ, 2 HELMUT FABEL, 1 YVES ALARIE, 4 AND HARALD RENZ 3 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 Received 15 June 2000; accepted in final form 10 October 2000 Glaab, Thomas, Angelika Daser, Armin Braun, Ulrich Neuhaus-Steinmetz, Helmut Fabel, Yves Alarie, and Harald Renz. Tidal midexpiratory flow as a measure of airway hyperresponsiveness in allergic mice. Am J Physiol Lung Cell Mol Physiol 280: L565 L573, 2001. 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 (EF 50 ) 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 EF 50 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 EF 50 baseline values 24 h after challenge in allergic mice. The decline in EF 50 to MCh challenge correlated closely with simultaneous decreases in pulmonary conductance and dynamic compliance. The decrease in EF 50 was partly inhibited by pretreatment with the inhaled 2 -agonist salbutamol. We conclude that measurement of EF 50 to inhaled bronchoconstrictors by head-out body plethysmography is a valid measure of airway hyperresponsiveness in mice. body plethysmography; allergy; lung function method 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 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@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. http://www.ajplung.org 1040-0605/01 $5.00 Copyright 2001 the American Physiological Society L565

L566 TIDAL FLOW AS A MEASURE OF AIRWAY REACTIVITY IN MICE 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 (EF 50 ). In this investigation, we extend previous studies (18, 23) in further defining the EF 50 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 EF 50 measurement as a valid indicator of airway hyperreactivity in a well-established murine model of allergeninduced airway inflammation (19). Accordingly, we determined the changes in breathing patterns to several stimulants and the responses to a bronchodilator on EF 50 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. MATERIALS AND METHODS 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 allergenchallenged (Ova/Ova) group, six animals were sensitized on days 1, 14, and 21 with three intraperitoneal injections of 10 g 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 l. 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- m 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. For airflow measurement, a calibrated pneumotachograph (Fleisch 00000, Hugo Sachs Electronics, March-Hugstetten, Germany) and a differential pressure transducer (8T-2, Gaeltec, Dunvegan, UK) coupled to an amplifier (Gould Universal Amplifier, Dietzenbach, Germany) 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 (DAS-16, Keithley, Germering, Germany) at a sampling rate of 2,000/s and integrated with time to obtain tidal volume (VT). From these signals, all the variables listed in Table 1 [EF 50, inspiratory time (TI), expiratory time (TE), VT, and respiratory rate (f)] were calculated for each breath except for f, which is the number of breaths during a collection period of 15 s converted to breaths per minute. EF 50 (in ml/s) represents the flow at midexpiratory VT and was used as a measure of airway obstruction to Ova and MCh. Analysis of tidal flow values at 25, 50, and 75% VT during expiration had revealed that EF 50 was the most appropriate value to detect airway obstruction (23). Both nonspecific AR to MCh and allergen-specific AR to Ova were Fig. 1. Schematic drawing of the head-out body plethysmograph illustrating the exposure system and the equipment used for the measurement of tidal midexpiratory flow (EF 50 ), tidal volume (VT), expiratory time (TE), inspiratory time (TI), and breathing frequency (f) and for monitoring of dynamic lung compliance (Cdyn) and pulmonary conductance (GL). The system was operated with the addition of a catheter-tip differential pressure transducer to measure transpulmonary pressure (Ptp). The conscious animal was placed in a glass plethysmograph that was attached to a head exposure chamber. The output of a jet nebulizer was directed to the inlet of the head exposure chamber that was continuously ventilated with a bias flow of 0.2 l/min. Before data collection, mice were allowed to acclimatize for 15 min in the body plethysmograph.

TIDAL FLOW AS A MEASURE OF AIRWAY REACTIVITY IN MICE L567 Table 1. Respiratory parameters derived from tidal flow Baseline Values Respiratory Parameter Definition Unanesthetized mice After recovery from anesthesia VT, ml Amount of air inhaled or exhaled per breath 0.15 0.01 0.14 0.01 TE, s Time from maximum to minimum VT 0.14 0.01 0.13 0.01 TI, s Time from minimum to maximum VT 0.08 0.01 0.07 0.01 f, breaths/min No. of breaths per minute 268 29 275 25 EF 50, ml/s Airflow during expiration at 0.5VT 1.92 0.19 1.89 0.16 Values are means SD obtained during a 10-min control period ( 2,500 3,000 breaths/mouse); n 48 unanesthetized chest-intact mice and 23 mice with an inserted intrapleural catheter after recovery from anesthesia. VT, tidal volume; TE, expiratory time; TI, inspiratory time; f, respiratory rate; EF 50, tidal midexpiratory flow. No significant differences were observed between groups. investigated while the animal was being held in the body plethysmograph. Each investigation consisted of a 10-min (40 periods of 15 s each) baseline period to obtain control data followed by exposure to aerosolized PBS for 2.5 min followed by exposure to increasing concentrations of aerosolized MCh (ranging from 5 to 25 mg/ml). All responses for the respiratory parameters listed in Table 1 were compared with the corresponding baseline values, which were taken as 100%. Minimum values for EF 50 were determined and are expressed as percent changes from baseline values. Dose-response curves to MCh were determined, and the provocative dose (PD) causing a decrease in EF 50 to 55% of the baseline value was calculated from linear interpolation and is expressed as PD 55 EF 50.PD 55 was selected to provide PD values for all groups examined. 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 longlasting 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 PD 55 Cdyn and PD 200 GL, respectively. Effects of inhalative pretreatment with salbutamol on AHR. To investigate the effects of an inhaled 2 -agonist on EF 50 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 allergensensitized 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. Twentyfour 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-. 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- m 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

L568 TIDAL FLOW AS A MEASURE OF AIRWAY REACTIVITY IN MICE 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). RESULTS 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 EF 50 during airway obstruction. Correlation of EF 50 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 Fig. 2. Characteristic modifications to the normal breathing pattern in unanesthetized BALB/c mice. A: normal breathing pattern of BALB/c mice while breathing room air. B: characteristic pattern of airway obstruction during aerosol challenge with MCh, illustrating the decline in EF 50. A and B, top tracings: pneumotachograph airflow signals recorded at 40 mm/s. A and B, bottom tracings: corresponding integrated VT signal as calculated by the computer program from the collected voltage digitalizations. A horizontal line at 0 flow separates inspiratory (Insp; upward; ) from expiratory (Exp; downward; ) airflow. V, tidal flow. 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 EF 50 values correlated with decreased GL and decreased Cdyn values, EF 50 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 EF 50 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 EF 50,GL, and Cdyn (Fig. 3). The PD 55 EF 50 was significantly lower in the Ova/Ovatreated 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 EF 50 values, although Cdyn was less sensitive in detecting enhanced AR (PD 55 Cdyn: 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; PD 200 GL: 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 EF 50 and GL values (r 0.916; P 0.01) and between decreased EF 50 and Cdyn values (r 0.969; P 0.01) in allergen-sensitized and -challenged mice. The peak EF 50 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. EF 50 values decreased proportionally with increasing values of TE and decreasing values of VT and f (Fig. 4). EF 50 is independent of alterations in f, VT, and TE. MCh challenge of mice induced parallel decreases in Cdyn, GL, and EF 50 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 EF 50 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, EF 50 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/m 3 ), 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, orvt were not necessarily accompanied by changes in EF 50, indicating that EF 50 is an independent measure of airway obstruction. Inhibition of increased AR by a 2 -agonist. To investigate the effect of a bronchodilatative 2 -agonist on EF 50, 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 EF 50 values but signifi-

TIDAL FLOW AS A MEASURE OF AIRWAY REACTIVITY IN MICE L569 cantly reduced the decreases in EF 50 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 EF 50 readings during baseline and exposure periods. These data indicate that maximal EF 50 responses during MCh aerosol exposure were distinctly reduced after pretreatment with a 2 -agonist. Fig. 4. Representative time-response analysis of an allergen-sensitized and -challenged mouse showing the decrease in EF 50 during aerosol exposure to MCh (25 mg/ml), including baseline, PBS exposure, and postexposure periods as well as the elongation in TE and the decreases in both f and VT during airway obstruction. Each parameter is plotted as percent change from the control value, the control value being the mean for all the breaths collected for this animal during the baseline period and made equal to 100%. Mean baseline values were EF 50, 1.85 0.09 ml/s; TE, 0.14 0.01 s; f, 284 25 breaths/min; and VT, 0.15 0.01 ml. Effect of allergen challenge on serum immunoglobulins and cytokine levels in BAL fluid and EF 50. 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 Fig. 3. Dose-response relationship after challenge with increasing doses of aerosolized methacholine (MCh; 5 25mg/ml) in 3 different groups of mice: allergen sensitized and allergen challenged [ovalbumin/ovalbumin (Ova/Ova)], sham sensitized and allergen challenged (PBS/Ova), and allergen sensitized and sham exposed (Ova/PBS). Aerosolized PBS was used as negative control (0) before MCh challenge. Measurements of GL (C) and Cdyn (B) were recorded simultaneously with EF 50 (A). Values are means SD from 6 animals. Baseline values were 1.81 0.10, 2.02 0.18 ml/s, and 1.93 0.14 ml/s for EF 50 in Ova/Ova-, PBS/Ova-, and Ova/PBS-treated groups, respectively; 0.72 0.09, 0.79 0.08, and 0.87 0.11 ml s 1 cmh 2 O 1 for GL in Ova/Ova-, PBS/Ova-, and Ova/PBStreated groups, respectively; and 0.020 0.001, 0.022 0.002, and 0.023 0.001 ml/cmh 2 O for Cdyn in Ova/Ova-, PBS/Ova-, and Ova/PBS-treated groups, respectively. EF 50 was significantly decreased in a dose-related manner in Ova/Ova-treated mice compared with that in control animals (PBS/Ova and Ova/PBS). *P 0.01 between Ova/Ova- and Ova/PBS-treated groups (5 mg/ml of MCh). There were parallel decreases in Cdyn and GL, respectively, after MCh aerosol challenge in the Ova/Ova-treated and control groups. Significant difference between Ova/Ova and control (PBS/Ova and Ova/PBS) groups: *P 0.01; **P 0.05.

L570 TIDAL FLOW AS A MEASURE OF AIRWAY REACTIVITY IN MICE Table 2. Effects of various stimuli on respiratory parameters Substance f VT TE EF 50 GL Cdyn CBC 72 15* 29 9* 139 18* 7 4 2 4 5 4 n-propranolol 35 10* 41 12* 30 7* 8 4 5 3 19 7 PBS 9 4 2 7 2 4 5 5 6 4 4 3 Values are means SD expressed as peak percent change from baseline value; n 4 untreated mice/group. CBC, 2-chlorobenzylchloride; GL, pulmonary conductance; Cdyn, lung dynamic compliance. Baseline values with CBC were f, 294 8 breaths/min; VT, 0.16 0.01 ml; TE, 0.13 0.01 s; EF 50, 2.11 0.22 ml/s; Cdyn, 0.022 0.001 ml/cmh 2 O; and GL, 0.84 0.11 ml s 1 cmh 2 O 1. Baseline values with n-propranolol were f, 268 15 breaths/min; VT, 0.14 0.01 ml; TE, 0.14 0.01 s; EF 50, 1.89 0.17 ml/s, Cdyn, 0.023 0.002 ml/cmh 2 O; and GL, 0.91 0.10 ml s 1 cmh 2 O 1. CBC nebulization resulted in a major increase in TE and decrease in f, with a modest decrease in VT, whereas EF 50, Cdyn, and GL were unaffected. n-propranolol aerosol challenge caused a slight but significant decrease in Cdyn that correlated with increasing values for f but with decreasing values for VT and TE. A preceding PBS aerosol challenge had no significant effect on respiratory parameters except for a small increase in f. *P 0.01 compared with respective PBS value. P 0.05 compared with CBC and PBS values. 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 EF 50 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 [EF 50 : 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 EF 50 values are decreased in Ova/Ova-treated animals. Association of EF 50 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. DISCUSSION We found that EF 50 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, Fig. 5. Salbutamol pretreatment reduced EF 50 decreases in Ova/ Ova-treated mice after MCh aerosol challenge. After baseline EF 50 values were obtained, mice were treated with either aerosolized salbutamol or PBS for 2.5 min. After 15 min, mice were challenged with PBS aerosol (0) and were then exposed to increasing concentrations of aerosolized MCh (5 25 mg/ml) for 2.5 min each, and EF 50 was recorded. Ova/Ova-treated mice with salbutamol (n 4) and PBS (n 4) pretreatment are compared. Values are means SD. Baseline values were 1.79 0.09 ml/s after salbutamol pretreatment and 1.83 0.08 ml/s after PBS pretreatment for EF 50 in the Ova/Ova-treated group. *P 0.01. Table 3. Total and Ova-specific Ig levels in serum Ova/PBS PBS/Ova Ova/Ova Total IgE, ng/ml 1,202 508 1,077 613 2,411 1,538 Ova-specific IgE, U/ml 31 12* 16 8* 44 17* Ova-specific IgG1, U/ml 893 369 774 438 3,733 2,052 Values are means SD; n 6 mice/group. Ova/PBS, allergen (ovalbumin) sensitized and sham exposed (control); PBS/Ova, sham sensitized and allergen challenged (control); Ova/Ova, allergen sensitized and allergen challenged. On day 28, 24 h after the last inhalational challenge, serum was obtained from the tail vein and assayed for total IgE as well as for Ova-specific IgE and IgG1. *P 0.01 between the Ova-sensitized (Ova/PBS and Ova/Ova) and shamsensitized groups.

TIDAL FLOW AS A MEASURE OF AIRWAY REACTIVITY IN MICE L571 Fig. 6. Cytokine levels of interleukin (IL)-4, IL-5, interferon (IFN)-, and IL-2 in bronchoalveolar lavage (BAL) fluid 24 h after the last aerosol challenge with Ova or PBS in Ova/Ova-, PBS/Ova-, and Ova/PBS-treated mice. Values are means SD; n 6 mice/group. *P 0.01 between Ova/Ova-treated and control (Ova/PBS and PBS/Ova) groups. 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 EF 50 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 EF 50 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 EF 50 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 EF 50 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 EF 50 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 EF 50 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 EF 50 values were unaffected by druginduced changes in f, VT, and TE, suggesting that EF 50 does not correlate simply with changes in breathing patterns. The MCh-related decrease in EF 50 is presumably mediated by specific muscarinic M 3 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 EF 50 was obtained with a 2 -agonist; pretreatment of allergen-sensitized and -challenged animals with aerosolized salbutamol significantly reduced the decreases in EF 50 due to MCh challenge. Noninvasive measurement of EF 50 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 EF 50 closely reflect the changes observed in Cdyn and GL during bronchoconstriction. Although Cdyn correlated well with measurements of EF 50 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 Table 4. Cellular composition of BAL fluid Ova/PBS PBS/Ova Ova/Ova Eosinophils, 10 3 1 1 523.6 119.9* Lymphocytes, 10 3 5.2 3.3 2.1 1.6 89.3 20.7* Neutrophils, 10 3 1.6 1.3 2.6 1.9 118.8 45.8* Macrophages, 10 3 204.7 98.4 225.4 105.1 102.6 25.2 Values are means SD; n 6 mice/group. Eosinophils, neutrophils, lymphocytes and macrophages were recovered from bronchoalveolar lavage (BAL) fluid 24 h after the last aerosol challenge with Ova or PBS. Significant difference between Ova/Ova-treated and control (Ova/PBS and PBS/Ova) groups. *P 0.01. P 0.05.

L572 TIDAL FLOW AS A MEASURE OF AIRWAY REACTIVITY IN MICE 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 EF 50 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 EF 50 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 EF 50,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 EF 50 can significantly discriminate the degree of AR between allergen-sensitized and -challenged animals and control animals after MCh challenge. The determination of EF 50 thus appears to be a simple and valid indicator of AR in mice. Decreases in EF 50 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 EF 50 method as an indicator of airway obstruction in conscious mice. The measurement of EF 50 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 EF 50 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 EF 50 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. 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. 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