INTRODUCTION

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The effects of a deep inspiration (DI) in individuals with asthma differ from those observed in healthy subjects (1). In healthy subjects, a DI acts both as a bronchoprotector and as a bronchodilator (9, 10), but bronchoprotection by DI appears to be lost in asthma (9, 11). The bronchodilatory ability of a DI is somewhat reduced in asthma, but the differences between healthy subjects and subjects with asthma are not striking (10).

It has been postulated that the beneficial effect of lung inflation is mediated by airway stretch (7). One hypothesis that has been proposed to explain the defects in the function of lung inflation in asthma is that, in this condition, DI may be unable to stretch the airways. This may result from attenuation of the tethering forces between the airways and the surrounding parenchyma either because of increased airway wall stiffness or because of "loosened attachments" between the alveolar septa and the airway walls. Any of these abnormalities could be attributed to inflammatory changes observed in the airways of patients with asthma, such as remodeling or edema.

In vivo airway imaging provides a direct approach to test the hypothesis that airway stretch may not take place during lung inflation. Using high-resolution computed tomography (HRCT), we have previously demonstrated the ability to measure airway constrictor responses in animals and the effects of lung inflation (12) in conducting airways at specific points during inspiration and expiration.

In the current study, we used HRCT to examine the ability of a deep inspiration to distend the airways of subjects with asthma compared with healthy subjects at baseline and after increasing airway tone with methacholine (MCh). In both conditions, we found that a DI can increase the airway area to a similar extent in healthy subjects and in subjects with asthma. However, after increased airway tone was induced and after the DI maneuver was performed, scanning at FRC showed attenuation of airway constriction in the healthy subjects but accentuation in subjects with asthma.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Subjects

The study was approved by our Institutional Review Board, and informed written consent was obtained from each subject prior to enrollment. We studied nine healthy volunteers and 10 volunteers with asthma. All subjects were screen with a questionnaire, allergy skin testing, and a routine MCh inhalation challange (15) (Table 1).

Study Design

During the first visit, all subjects underwent a modified MCh challenge (prohibition of deep inspiration) while partial spirometry and thoracic gas volume (equivalent to functional residual capacity, FRC) and expiratory reserve volume (ERV) were determined. The outcomes of the partial maneuvers were designated with the suffix p. Subjects were challenged with four increasing concentrations of MCh (0.075, 0.25, 0.75, and 2.5 mg/ml). If the FEV₁p/FVCp fell below 0.5 or if uncomfortable chest symptoms occurred before the highest dose (2.5 mg/ml) of MCh was reached, the challenge was terminated. On another visit, the subjects underwent a second modified MCh challange, while supine on the computed tomography (CT) gantry.

HRCT Image Acquisitions

All scans were performed by spiral CT (Somatom Plus 4; Siemens, Iseline, NJ) as previously described (16). All airways visualized approximately perpendicular to the scan plane were matched and measured (Figure 1).

View larger version (170K): [in this window] [in a new window]   Figure 1.   Matched HRCT scans from one individual at baseline (left panels) and after challenge with aerosolized methacholine (MCh, right panels). Images were acquired at low lung volume (FRC, top right) and at high lung volume (TLC, bottom right). The arrows show the same airways matched under all conditions.

Maintenance of Constant Lung Volume

By controlling the amount of inspired air, lung volume at each scan series was maintained approximately constant at FRC (RV + ERV = FRC) (16). Before each scan series, subjects exhaled to RV and then inspired a specified amount of air based on regression lines that were constructed for each subject during the first visit (16). To measure airway area at TLC, the subject was instructed to take a maximal deep breath.

Airway Measurements with HRCT

Airway lumenal area. The airway lumenal area measurement methodology has previously been described (17) and validated (18) and is based on the use of the airway analysis module of the Volumetric Image and Display Analysis (VIDA) software package (Division of Physiologic Imaging, Department of Radiology, University of Iowa, Iowa City, IA).

Airway wall area. We measured airway wall area as described previously (19) by first measuring mean wall thickness in each airway. At least three lines were randomly drawn through the airway wall. From the measured lumenal area and wall thickness, we then calculated the total airway area and wall area.

Protocols

Protocol 1. During the second visit, HRCT scans were acquired at baseline and after the administration of the highest doses of MCh, at both FRC and at TLC, in 10 subjects (5 healthy and 5 asthmatic).

Protocol 2. In nine subjects (healthy and asthmatic), HRCT scans were acquired at baseline (FRC) and after the administration of the highest doses of MCh at FRC and at TLC, and then again immediately after a DI, when lung volume returned to FRC.

Data Analysis

The airway area at baseline FRC was defined as 100%, and all subsequent measurements are presented as a percent of baseline. The data were analyzed by one-way analysis of variance (ANOVA) with the Bonferroni/Dunn correction. In addition, linear regression models were constructed to relate the airway wall area and airway lumenal area. Significance was accepted at a two-tailed p  0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The healthy subjects and subjects with asthma were similar in age and baseline pulmonary function (Table 1). During their screening, the healthy volunteers received the highest concentration of MCh (75 mg/ml) with no more than 15% reduction in FEV₁. The subjects with asthma received MCh up to a dose that caused a 20% reduction in FEV₁. The average (± SEM) provocative concentration of MCh that caused a decrease in the FEV₁ of 20% (PC₂₀) for the asthmatic group was 2.7 ± 1.2 mg/ml. The mean age was 30 ± 2 (mean ± SEM) and 31 ± 3 yr for the 9 healthy subjects and 10 subjects with asthma, respectively (p = 0.77). Mean (± SEM) baseline FEV₁ was 95 ± 3% and 94 ± 3% of predicted for the healthy and asthmatic groups, respectively (p = 0.84). Mean baseline FVC was 101 ± 3 and 110 ± 3% of predicted for the healthy and asthmatic groups, respectively (p = 0.04). The mean baseline FEV₁/FVC was 82 ± 2 and 76 ± 2% of predicted for the healthy and asthmatic groups, respectively (p = 0.06).

In the sitting position, during the modified MCh challenges (DIs withheld), all healthy subjects completed the challenge protocol up to the maximum dose of 2.5 mg/ml, which caused a decrease in the mean FEV₁p to 75 ± 5% of baseline (p < 0.0001), a decrease in the mean FVCp to 87 ± 3% (p < 0.0001), and an increase in RVp to 148 ± 17% (p < 0.0001) of baseline. Eight subjects with asthma stopped the MCh challenge at the second highest dose of 0.75 mg/ml, and two subjects with asthma stopped at a dose of 0.25 mg/ml because either the FEV₁p/FVCp ratio decreased to approximately 0.50 or the subjects complained of chest tightness. These doses caused a mean decrease in FEV₁p to 67 ± 5% of baseline (p < 0.0001), a mean decrease in FVCp to 83 ± 4% of baseline (p < 0.0001), and an increase in RVp to 168 ± 14% of baseline (p < 0.0001). The changes in the pulmonary function measurements in the sitting position were not different between the healthy subjects and the subjects with asthma for either the FEV₁p (p = 0.27), the FVCp (p = 0.39), or the RVp (p = 0.36) measurements.

In the supine position in the HRCT scanner, during the MCh challenges in which DIs were withheld, all the healthy subjects again completed the challenge protocol up to the maximum dose of 2.5 mg/ml. This caused a decrease in the mean FEV₁p to 65 ± 6% of baseline (p < 0.0001), and a decrease in the mean FVCp to 76 ± 6% of baseline (p < 0.0001). Among the nine subjects with asthma, two received the maximum dose of 2.5 mg/ml, four received 0.75 mg/ml, and three received 0.25 mg/ml. In one subject with asthma, the aerosol challenge was stopped after the first dose of MCh (0.075 mg/ ml). In the subjects with asthma, these doses caused a decrease in FEV₁p to 64 ± 6% of baseline (p < 0.0001), and a decrease in FVCp to 84 ± 4% of baseline (p < 0.0001). While the doses delivered to the subjects with asthma were lower than those delivered to the healthy individuals (p < 0.0001), the changes in partial spirometry and in airway size (see below) were not different between these groups. Namely, while in the HRCT scanner, the MCh-induced changes in FEV₁p and FVCp from baseline did not differ significantly, i.e., the p values for the comparisons between the healthy subjects and the subjects with asthma were p = 0.90 and p = 0.31, respectively.

The MCh-induced percent changes in pulmonary function were not different in the supine position compared with the sitting position for either the healthy subjects (p = 0.18, p = 0.15 and p = 0.84 for FEV₁p, FVCp, and FEV₁p/FVCp, respectively) or the subjects with asthma (p = 0.66, p = 0.73, and p = 0.41 for FEV₁p, FVCp, and FEV₁p/FVCp, respectively).

Protocol 1

On HRCT, 191 airways were matched and measured (106 in the five healthy subjects and 85 in the five subjects with asthma, range 14-28 airways per subject). The airways ranged in size from 1.2 to 12.2 mm in diameter. The frequency distribution of airway sizes measured on HRCT images in all healthy subjects and subjects with asthma (subjects pooled from both protocols), at baseline FRC, are shown in Figure 2.

View larger version (32K): [in this window] [in a new window]   Figure 2.   The frequency distribution of airway sizes measured from HRCT images of 9 healthy subjects and 10 subjects with mild asthma at baseline FRC. The majority of airways in both groups were in the range of 2-6 mm in diameter. The mean (± SEM) airway size was 4.7 ± 0.2 and 4.0 ± 0.1 mm in diameter for the healthy subjects and the subjects with asthma, respectively (p = 0.002). The variance (F value) of the distributions of the two groups was not different (p = 0.06).

At baseline, lung inflation from FRC to TLC increased airway area in the five healthy subjects and five subjects with asthma. At TLC, the mean airway area in the healthy subjects was 146 ± 4% of baseline FRC size. In the subjects with asthma, at TLC, the mean airway area was 157 ± 5% of baseline FRC size. These percent increases in mean airway areas induced by the DI to TLC were not different between the healthy subjects and the subjects with asthma (p = 0.07). To analyze the effect of lung inflation by airway size, we arbitrarily divided the airways into four size categories: < 3, 3-5, 5-7, and > 7 mm in diameter. In the baseline state, there was greater distensibility in the smaller compared with the larger airways in the subjects with asthma (Figure 3, p < 0.05), whereas there was no difference in distensibility among the various size airways in the healthy subjects (Figure 3, p > 0.05). When the airways of the two groups were compared by size category, the < 3-mm (p = 0.02) and 5- to 7-mm airways (p = 0.02) of the subjects with asthma were more distensible than the airways of the healthy subjects.

View larger version (16K): [in this window] [in a new window]   Figure 3.   The increase in airway area with a deep inspiration (distensibility) in five healthy (open columns) and in five subjects with asthma (solid columns) (protocol 1). Scanning was performed at TLC, under baseline conditions (before administration of methacholine). The data have been stratified on the basis of airway diameters and are presented as means ± SEM. The airways < 3 mm in diameter and 5-7 mm in diameter were more distensible in the subjects with asthma than in the healthy subjects (*p < 0.05).

We also measured the airway wall areas in the five healthy subjects and the five subjects with asthma at baseline FRC. Figure 4 shows that airway wall area, expressed as a fraction of total airway area, is greater in the subjects with asthma compared with the healthy subjects throughout the entire airway size range (p = 0.004). However, we found no predilection by size for thickened airways in the subjects with asthma compared with the healthy subjects; that is, the slopes of the regression lines between wall area and airway diameter were not different in the range of airways that we measured (p = 0.79).

View larger version (21K): [in this window] [in a new window]   Figure 4.   Individual airway wall areas expressed as a fraction of total airway area and plotted against airway size (diameter) in the five healthy subjects (open squares) and five subjects with asthma (solid diamonds) who participated in Protocol 1. The relationship between the two plotted outcomes is reflected in the regression lines: solid line for the healthy subjects and dashed line for the subjects with asthma. The airway wall areas are greater in the subjects with asthma throughout the range of airway sizes (the slopes of the two lines are not different, p = 0.79).

The MCh aerosol challenges decreased airway area at FRC in both the five healthy and the five subjects with asthma (Figure 5). At FRC, methacholine decreased the mean airway area in the healthy subjects to 85 ± 2% of baseline size (p < 0.0001 compared to baseline). In the subjects with asthma, MCh decreased the mean airway area to 88 ± 2% of baseline size (p < 0.0001 compared with baseline). The decreases in airway area with the MCh challenges did not differ between the two groups (p = 0.38). In addition, in the healthy subjects, there was a predilection for the smallest airways to constrict more intensely in response to Mch compared with the larger airways (Figure 5, p < 0.002). However, we did not observe a predilection by airway size for constriction with Mch in the asthmatic group (Figure 5, p > 0.13).

View larger version (16K): [in this window] [in a new window]   Figure 5.   The decrease in airway lumenal size in five healthy subjects (open columns) and five subjects with asthma (solid columns) after methacholine (MCh) aerosol challenge (Protocol 1). Scanning was performed at FRC. The data have been stratified on the basis of airway diameters and are presented as means ± SEM. In the healthy subjects, there was a predilection for the smallest scanned airways to constrict with MCh, compared with the larger airways (*p < 0.002).

The MCh aerosol challenge also caused a decrease in airway area at TLC in the five healthy subjects and the five subjects with asthma who participated in Protocol 1. In the healthy subjects, after MCh challenge, the mean airway area at TLC was 129 ± 3% of baseline (p < 0.0001, compared with 146 ± 4%, which was the value at TLC before the MCh challenge). In the subjects with asthma, airway area at TLC was 126 ± 3% of baseline (p < 0.0001, compared with 157 ± 5%, the value before the MCh challenge). However, even after the airways were constricted with Mch, there was no difference in mean airway areas with a deep inspiration to TLC between the healthy subjects and the subjects with asthma (Figure 6, p = 0.44). After the airways were narrowed with MCh, there was no difference between the large and small airways in either the subjects with asthma or the healthy subjects (p > 0.05).

View larger version (15K): [in this window] [in a new window]   Figure 6.   The increase in airway area over baseline (premethacholine FRC) with a deep inspiration (distensibility) after methacholine (MCh) aerosol challenge in the five healthy subjects (open columns) and five subjects with asthma (solid columns) who participated in Protocol 1. Scanning was performed at TLC. The data have been stratified on the basis of airway diameters and are presented as means ± SEM. In contrast to the data presented in Figure 3, which show distensibility at baseline, the data here show that, after increased airway tone was induced with MCh, no difference in distensibility between the large and small airways in either the subjects with asthma or the healthy subjects could be detected.

Protocol 2

On HRCT, 195 airways were matched and measured (92 in four healthy subjects and 105 in five subjects with asthma, range 14-24 airways per subject). The airways ranged in size from 1.6 to 11.4 mm in diameter.

As in Protocol 1, the MCh aerosol challenge caused a decrease in the airway area at FRC in both the five healthy subjects and the four subjects with asthma. At FRC, the spasmogen decreased the mean airway area in the healthy subjects to 80 ± 2% of baseline size (p < 0.0001 compared with baseline). In the subjects with asthma, MCh decreased the mean airway area to 84 ± 2% of baseline size (p < 0.0001 compared with baseline). The decreases in airway area induced by the MCh challenges were not different between the healthy subjects and the subjects with asthma (p = 0.24). In addition, in the healthy subjects, as described in Protocol 1, there was a predilection for the smallest airways to constrict in response to MCh more so than the larger airways (p = 0.0004). No such predilection was found in the subjects with asthma (p = 0.8).

After MCh challenge, the mean airway area at TLC was 124 ± 4% of baseline (p < 0.0001 compared with FRC post-MCh challenge) and 121 ± 3% of baseline (p < 0.0001 compared with FRC post-MCh challenge) for the healthy subjects and the subjects with asthma, respectively. After the airways were constricted with MCh, there was no difference in mean airway areas with a deep inspiration to TLC between the healthy subjects and the subjects with asthma (p = 0.56). In addition, in the healthy subjects, there was a tendency for the smallest airways to distend more compared with the larger airways (p = 0.05). This was not observed in the subjects with asthma (p = 0.75).

After the DI was released and the lung volume returned to FRC, we found that the airways of the healthy subjects, compared with those of the subjects with asthma, behaved differently. The airways of the healthy subjects were slightly dilated, compared with their pre-DI constricted state. In contrast, the airways of the subjects with asthma constricted further, compared with their pre-DI state (p < 0.0001). In the four healthy subjects, the airway area after MCh challenge, but before the DI, was 80 ± 2% of baseline. The major site of airway dilation occurred in the smallest airways measured (Figure 7). After the DI, the airway area of the healthy subjects increased to 86 ± 3% of baseline (p = 0.03 compared with pre-DI; Figure 7). The opposite occurred in the subjects with asthma. In this group, the airway area after MCh challenge but before the DI was 84 ± 2% of baseline. After the DI, the airway area narrowed further to 74 ± 2% of baseline (p < 0.0001 compared with pre-DI; Figure 7). In the subjects with asthma, there was no predilection by size to constrict after the DI (p > 0.08).

View larger version (15K): [in this window] [in a new window]   Figure 7.   The percent change in FRC airway area caused by the DI on return to FRC. These data are derived from the four healthy subjects (open columns) and the five subjects with asthma (solid columns) who participated in Protocol 2. Scanning was performed at FRC. The data have been stratified on the basis of airway diameters and are presented as means ± SEM. The percent change in FRC airway area by DI was defined as: [(AI post-DI  Ai pre-DI)/(Ai pre-DI)] × 100, where Ai is airway lumenal area expressed as a percent of baseline area at FRC after MCh. In the healthy subjects, the airway area increased after the DI, with the smallest airways showing the greatest increase (*p = 0.03). The opposite occurred in the subjects with asmtha: the airway area further decreased after the DI (p < 0.0001), but no predilection by size was detected.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This study was designed to test the hypothesis that, in asthma, when the lungs become inflated by deep inspiration, the airways do not distend and, therefore, stretch is not exerted on them. If this hypothesis were true, it would have provided us with a first step in explaining the beneficial effects of lung inflation in humans as well as with an explanation of the defective function of lung inflation in asthma. However, the data we have generated do not support this hypothesis; that is, we failed to demonstrate that a defect in the distensibility of the asthmatic airways exists. On the other hand, our findings raise a new hypothesis that an exaggerated excitation-contraction mechanism inhibits the bronchodilatory effect of a DI in subjects with mild asthma.

In the current study, we selected a population of healthy volunteers and clinically stable subjects with asthma with mild to moderate airway hyperresponsiveness who use only sympathomimetic inhalers on an as-needed basis (intermittent asthma). With the exception of small difference in FEV₁/FVC, because of the slightly higher FVC in the subjects with asthma compared with the healthy subjects, the two groups were similar in terms of baseline lung function characteristics. All the healthy subjects received the top dose of MCh, that is, 2.5 mg/ ml, during the provocation during CT imaging. On average, lower doses were delivered to the subjects with asthma, their median dose being 0.75 mg/ml. However, it is striking that, in the absence of DIs, the difference in the doses administered, which achieved a similar degree of obstruction in the healthy subjects and the subjects with asthma, is so small. Our group has previously reported such similarity in airway responsiveness, in the absence of deep inspirations, and these data are confirmatory of our previous report (7). The similarity in MCh responsiveness between the two subject groups in the absence of DIs allowed us to compare them with respect to airway distensibility at baseline, as well as with respect to distensibility during increased airway tone, at the same level of induced obstruction.

Maintaining a relatively constant lung volume between the HRCT scans is an important technical aspect of this study. We have developed a methodology that allowed us to achieve this goal with good reliability (16). Before each HRCT scan, subjects exhaled to RV and then inspired a given amount of air that was predetermined as the difference in volume between their RV and FRC. Therefore, baseline scanning was performed at FRC. After the MCh challenges, the amount of air inhaled from RV was individually adjusted to account for the estimated increase in RV with MCh challenges. This increase was determined during the subjects' initial MCh provocation in which body plethysmography was performed. Because the provocation during HRCT was performed in the supine position, in which body plethysmography could not be performed, the change in RV had to be estimated. To achieve this, for each subject, a regression equation was constructed that provided us with the ability to predict the change in RV on the basis of the change in a partial spirometric outcome. These equations were derived from the first provocation series, which involved both partial spirometry and body plethysmography, and have been shown to be reproducible with fairly narrow confidence intervals (16). Also, we have previously demonstrated that regression equations constructed between RV measurements and sitting spirometry or between RV measurements and supine spirometry are similar (16). Multiple airways, 9 to 24, were measured and matched within each subject. The ability to match and measure airways has been demonstrated in previous animal studies in our laboratory (12, 17, 20), and in human subjects by ourselves (16) and others (23, 24). Although the conducting airways of humans, relative to lung size, are smaller compared with those of either dogs or sheep, multiple airways perpendicular to the scan plane were visible in the sections of lung imaged in all our subjects. In addition, the use of spiral acquisition and thin sections (2 mm) facilitated matching of the airways. Finally, the use of spiral methodology allowed complete image acquisition after each maneuver with a single breath hold, assuring more consistent lung volumes.

We found a small difference in the mean baseline size of the airways at FRC between the healthy subjects and the subjects with asthma, the airways of the healthy subjects being slightly larger (4.7 versus 4.0 mm in average diameter). We chose a similar starting location of HRCT scanning in the normal subjects and the subjects with asthma, the airways after the main stem bronchi branch on the right and the left side. We then scanned continuously caudally and were able to identify airways starting at approximately 11 mm in diameter and ranging as small as 1-2 mm in diameter. Given that these airways are third to sixth generation and we scanned at similar locations in both the healthy subjects and the subjects with asthma, it is unlikely that we systematically chose smaller airways to measure in the subjects with asthma. To our knowledge, there are no data indicating that subjects with asthma have smaller airways compared with healthy subjects. One possible explanation for this finding is the increased thickness in airway wall that was observed in the airways of the subjects with asthma compared with the healthy subjects. We found thicker airway walls at baseline FRC throughout the range of airway sizes that we evaluated in this study (Figure 4). Increased wall thickness could result in a smaller airway lumen for a given airway size. Another possibility to account for the difference in measured airway size may be differences in baseline airway smooth muscle tone. In animal models, we have observed dramatic intra- and intersubject variability in the amount of tone (21). It is possible that, in the subjects with asthma, compared with the healthy control subjects, increased baseline tone causes a greater amount of airway constriction at the same generation along the airway tree. Although, during the first visit, the FEV₁/FVC derived from the full maximal expiratory maneuver was not significantly different between the two groups (p = 0.06), on the day of HRCT, the partial forced expiratory maneuver-derived ratio FEV₁p/FVCp was lower in the asthmatic group compared with the healthy group (p = 0.03). This is consistent with the increased airway smooth muscle tone hypothesis.

The most important finding of this study is that there was no overall difference in the degree of airway distension from FRC to TLC between the healthy subjects, and the subjects with asthma at baseline or after airway tone was increased by MCh. This finding raises serious doubts that, in mild intermittent asthma, a problem of airways-parenchyma interdependence exists. Macklem has suggested that the forces of interdependence of the parenchymal attachments on the airway wall may provide the means of attenuating bronchoconstriction in vivo (25). In studies by Bellofiore and coworkers, the elastic load that lung parenchyma imposes on the airway smooth muscle was identified as a major factor in determining the degree of airway constriction for a given dose of spasmogen. Their work suggested that the interaction between the parenchymal elastic fibers and airway smooth muscle shortening was influenced by the constrictor response to MCh and the dilator response to increased lung volume (26). Bellofiore and coworkers decreased the airway-parenchymal interdependence in the lungs of rats with elastase treatment to induce emphysema, and quantified the decrease in parenchymal tethering by morphometric analysis. They found that disruption of the elastic fiber network increased the airway constrictor response to MCh at the highest doses tested. They also showed that, before the elastase treatment, the airway response to MCh was attenuated when end-expiratory volume was increased above FRC. However, after the elastase treatment, they not only found greater constrictor response to MCh at FRC, but also that increased end-expiratory volume no longer attenuated the constrictor response to MCh. Therefore, with decreased parenchymal interdependence, the effect of increased lung volume to dilate the airways was abolished. These results, however, are not consistent with findings in rats by other investigators. Dolhnikoff and coworkers (27) were unable to demonstrate significant morphological changes in parenchymal distortion after induced airway contriction with increased lung volume in rats. They concluded that parenchymal tethering did not attenuate airway narrowing (27). Results from Okazawa and coworkers in carbachol-challenged rabbit lungs also supported the notion that the elastic loads exerted by the parenchyma are not sufficient to explain the attenuation of smooth muscle shortening by lung inflation (28). Our results, showing no difference in airway size at TLC between the asthma subjects and the healthy subjects at baseline, or between the two groups after MCh challenge, are in agreement with these latter conclusions.

One could argue that differences in distensibility between individuals with asthma and healthy individuals may be present in smaller diameter airways that cannot be visualized by HRCT. If anything, however, in the smallest airways visualized in our study (between 1.2 and 3 mm in diameter), subjects with asthma showed significantly higher distensibility by lung inflation, compared with the healthy control subjects. Another theoretical cause of impairment of airway-parenchyma interdependence, that is, tissue remodeling and/or edema formation, could arguable not be significant in the subjects with mild asthma that we studied. Several authors have proposed that in asthma, edema fluid collects between the airway smooth muscle and the surrounding lung parenchyma and that this fluid should attenuate the forces of radial traction produced by increased lung volume (25, 29, 30). Although HRCT is unable to differentiate water from tissue, we found significantly increased airway wall area in the subjects with asthma compared with the healthy control subjects along the entire range of the bronchial tree that we could image. Despite this finding, however, both at baseline and with increased airway tone, the airways of subjects with asthma and healthy subjects dilated to a similar extent during a DI. It is interesting to note that in a model of bradykinin-induced airway edema in sheep, our laboratory found the bronchodilatory response to a deep inspiration to be unaffected by an increase in airway wall area as great as 50% (13).

It is possible that with greater amounts of airway tone, the airway-stretching effect of DI can substantially diminish. Work from our laboratory has demonstrated that, in healthy individuals, bronchodilation after DI becomes limited with increasing MCh-induced bronchoconstriction (10). In addition, in preliminary experiments, using subjects with moderate to severe asthma, we have observed that chronic moderate airway obstruction prevents airway distensibility and bronchodilation by DI. These findings are consistent with animal and in vitro studies showing that increasing airway smooth muscle tone decreases airway distensibility (13, 14, 31, 32).

It was surprising that the subjects with asthma distended their small airways more than did healthy subjects (Figure 3). Furthermore, although there was no overall difference in airway constriction between the healthy subjects and the subjects with asthma after MCh challenge, the small airways of the healthy subjects constricted more (Figure 5). The fact that the small (< 3 mm in diameter) airways of the subjects with asthma constricted less in response to MCh and distended more during a DI, compared with the healthy subjects, could be viewed as suggestive of localization of airway mechanical alterations in asthma. Interestingly, such localization would point toward the larger airways. An alternative interpretation of the data presented in Figure 5 could be that, in asthma, the constrictor response to MCh is more homogeneous along the various size airways, and this may indicate a more widespread disease process. Whether such a homogeneous response is associated with the phenomenon of airway hyperresponsiveness will require further investigation. Our findings in this respect differ from those of Okazawa and coworkers, who reported that airways 2-4 mm in diameter showed the greatest postmethacholine changes in both healthy subjects and subjects with asthma (24). Differences in both the MCh challenge and the HRCT scanning protocols could account for this discrepancy.

The second major finding from this study is that, following a single deep inspiration, after constriction had already been induced by MCh, bronchodilation occurred in the healthy subjects but further bronchoconstriction occurred in the subjects with asthma. Because we had matched the two groups with respect to the level of constriction, as well as to the degree of distensibility of the airways with lung inflation from FRC to TLC, we surmised that we would observe similar airway dilation by a DI. The bronchodilation seen in the healthy subjects after DI is what would have been expected. However, the fact that the airways of the subjects with asthma constricted further after the DI is suggestive of an altered excitation-contraction mechanism (33). A model of tonically contracted muscle presented by Fredberg and colleagues involves the presence of slowly cycling latch bridges in the airway smooth muscle (33). According to their model, such a state would result in stiff airway muscle with low hysteresis. The total time during which DIs did not take place in both our healthy subjects and subjects with asthma was on the order of 20-25 min, from the time the subject lay on the CT gantry to the time of the DI for the TLC measurement. This time period is consistent with that in the ex vivo work by Fredberg and coworkers (33). Therefore, it is possible that, at least in the subjects with asthma, a latched state with low hysteresis may have been reached. One could then invoke the airway/parenchyma relative hysteresis hypothesis of Ingram and colleagues (34) to offer an interpretation of our finding. If the hysteresis of the smooth muscle is low relative to that of the parenchyma, the distending forces of interdependence exerted on the airways by the parenchyma will be lower at the end of a deep inspiration, compared with their state before the deep inspiration. This will lead to further airway narrowing. The opposite may be true in the healthy airways. The phenomenon becomes obvious only at FRC because the distending forces become overwhelming at TLC. Therefore, airway dilation at TLC does not differ between subjects with asthma and healthy control subjects.

The physiologic significance of our observation needs further exploration. Not having performed spirometry in these subjects after the single deep inspiration in the CT gantry, we cannot comment on the extent to which airflow limitation was worsened in the subjects with asthma by the lung inflation maneuver. It is known, however, that at least some patients with asthma develop bronchoconstriction with deep inspirations. In our hands, in subjects with mild asthma, comparable to those who participated in the current study, five DIs taken at baseline (in the absence of substantial airway tone) do not cause significant bronchoconstriction (9). Also, after the development of significant bronchoconstriction with MCh (average reduction in FEV₁ of about 30%), five DIs clearly induce bronchodilation in patients with mild asthma, albeit of less magnitude than that observed in healthy subjects (11). It is possible, therefore, that two independent but opposing phenomena induced by lung inflation are operating in patients with asthma: through one mechanism, airway stretch induces smooth muscle relaxation, whereas, through another mechanism preexisting smooth muscle tone becomes exaggerated. Our interpretation of the above described findings is that, in patients with mild disease, when a single stretch is imposed, the second phenomenon dominates, but repeated stretches activate the bronchodilatory mechanism.

In summary, both at baseline and after the induction of increased smooth muscle tone with MCh, a DI distended the airways of healthy subjects and subjects with asthma to a similar extent, indicating that abnormal interdependence between the lung parenchyma and the airways is unlikely to play a major role in the loss or in the attenuation of the beneficial effect of lung inflation that characterizes asthma. Furthermore, our findings suggest that an abnormal excitation-contraction mechanism in the airway smooth muscle of subjects with mild asthma counteracts the bronchodilatory effect of a DI. Therefore, the mechanism for reduced bronchodilation after deep inspirations in subjects with mild asthma could be intrinsic to the airway smooth muscle.