INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Our group has recently demonstrated that healthy subjects sustain potent bronchoconstrictive responses to inhaled methacholine (MCh) if the provocation is performed with prohibition of deep inspirations (DIs) (1). Under these conditions, the sensitivity to this spasmogen is, in several cases, equal to that observed in patients with asthma (i.e., a PC₂₀ of < 8 mg/ ml), whereas when deep breaths are involved, lung function is barely reduced in these subjects, even when they inhale 75 mg/ ml of MCh. Thus, it appears that lung inflation has a protective effect against airway narrowing. In contrast to the healthy state, patients with asthma appear to be deficient with respect to the effect of lung inflation. These observations led us to hypothesize, in agreement with other investigators (2, 3), that the lack of an airway relaxant effect of deep inspiration may underlie the pathophysiology of airway hyperresponsiveness in asthma. Because this observation is important enough to substantially alter the convention on hyperresponsiveness, we designed this study to confirm the airway narrowing in normal subjects with a noninvasive imaging technique, using high resolution computed tomography (HRCT).

HRCT has gained wide acceptance as a diagnostic and investigational tool for the evaluation of airway pathophysiology in humans (4); however, most of the physiologic work has been performed in animals (10). Unfortunately, previous physiologic imaging studies in humans were limited by either a single dose challenge (5), single airway measurements in an individual subject (4), nonautomated airway measurements (5), multiple breathhold maneuvers for each set of CT scans (5), uncontrolled lung volume (4), or the need to transport the subject back and forth between laboratory and clinical patient area for multimodality testing.

In the current study, in order to achieve our objective, we designed an algorithm that allows us to measure changes in pulmonary function using spirometry and concurrent changes in individual airway lumenal area using HRCT. A technical element of this protocol is that it accounts for the changes in lung volume with repeated bronchoprovocations. Thus, we are able to overcome several of the above-mentioned technical problems, make repeated measurements of the same airway in humans, and localize the site of airway narrowing.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Subjects

The study was approved by our Institutional Review Board. Informed written consent was obtained from each subject prior to enrollment. We studied five healthy volunteers. They reported no symptoms consistent with asthma and had never received a diagnosis of asthma from a physician. All subjects were screened by completing an asthma symptom questionnaire and undergoing allergy skin testing with 12 common aeroallergens and routine MCh inhalation challenge (20). All had received the highest concentrations of MCh (75 mg/ml) with no more than 15% reduction in FEV₁. All subjects were nonsmokers and had been free of upper respiratory infection for at least 4 wk before evaluation. Their demographic characteristics, as well as their baseline pulmonary function and allergy skin test results, are presented in Table 1.

Study Design: Methacholine Inhalation Challenge

The study required three visits. On the first visit, plethysmographic determinations of thoracic gas volume (equivalent to FRC) and of expiratory reserve volume (ERV) were first performed using a MedGraphics Body Plethysmography system that utilizes a constant-volume plethysmograph (MedGraphics Corp., St. Paul, MN).

Next, all subjects underwent a modified MCh challenge, a procedure that had two characteristics. First, after each dose of MCh, both spirometry and body plethysmography were performed. Second, the maneuvers used to monitor lung function in the course of the provocation did not involve DIs. Thus, when spirometry was performed, subjects began their forced expiratory effort from the end of a tidal inspiration instead of TLC. Also, during plethysmography, after the determination of FRC with the conventional panting maneuver, a slow expiratory maneuver from end-tidal inspiration to residual volume (RV) was performed. The outcomes of these partial maneuvers were designated with the suffix (p). The outcomes we have determined include FEV₁p, FVCp, FEV₁/FVCp, MMEFp, MMEF/FVCp, and RVp. In addition, we assessed the average time constant () of the middle portion of the forced partial expiration (1). This index is directly related to the product of the average values of lung compliance and resistance to flow between the alveoli and the site of flow limitation in the airways (1).

During the modified challenge, subjects inhaled four increasing concentrations of MCh (0.075, 0.25, 0.75, and 2.5 mg/ml). Each dose was administered as five tidal breaths from a Rosenthal-French dosimeter (0.6-s actuation) attached to a DeVilbiss 646 nebulizer (DeVilbiss Co., Somerset, PA).

During the course of the challenge, we assessed pulmonary function by using the ratio of the volume expired in the first second of the forced partial expiration (FEV₁p) to the total expired volume (FVCp). For each dose level, the maneuver with the highest FEV₁p and that with the highest FVCp were used to determine the FEV₁p/ FVCp. We used this ratio rather than either absolute volume alone because we were concerned that the absolute volumes are dependent on the end-tidal volume that was under voluntary control of the subject. If the FEV₁p/FVCp fell below 0.5 or if uncomfortable chest symptoms occurred before this level was reached, our protocol called for no further inhalations of MCh. All five subjects, however, received the above four doses of MCh without reaching these cutoff points.

On another visit (within 1 wk of the first), in conjunction with HRCT, the subjects underwent a second MCh challenge, in the supine position, while lying on the CT gantry. Four sets of scans were acquired: at baseline and after 0.25, 0.75, and 2.5 mg/ml of MCh provocation (no scanning took place after the 0.075 mg/ml dose). Partial, supine spirometry, but no plethysmography, was performed after each set of HRCT scans was acquired.

To compare spirometric measurements in the supine and the sitting positions, the subjects were again challenged with the same doses of MCh using the modified protocol (absence of DIs) during a third visit. After each dose of MCh, body plethysmography was performed first, followed by three sets of partial spirometric maneuvers, each set comprising a supine and a sitting maneuver, for a total of six expiratory maneuvers after each dose. The best of the three spirometric measurements for each MCh dose in the supine and in the sitting position was accepted.

Image Acquisitions

All scans were performed using spiral CT (Somatom Plus 4; Siemens, Erlangen, Germany) with settings of 140 kVp, 200 mAs, 2 mm slice thickness, rotation feed of 2 mm/s, and a reconstruction interval of 2 mm (total, 19 scans per set). Scanning began approximately 5 cm above the diaphragm, at FRC, and moved caudally. A reference scan was acquired prior to each spiral CT to ensure reproducible scanning at the same location in the lung. The images were reconstructed as 16-bit 512 × 512 matrix using a zoom of 2.5. Images were reconstructed with the use of a high-spatial frequency (resolution) algorithm that enhanced edge detection, at a window level of 450 Hounsfield units (HU), and a window width of 1,350 HU. All airways visualized approximately perpendicular to the scan plane (long to short axis ratio less than 1.5:1) were measured. For repeated airway measurements in a given subject within each experimental protocol, adjacent anatomic landmarks such as airway or vascular branching points were defined on the baseline HRCT image. After each challenge step, the same airways were located from these adjacent landmarks and measured.

Maintenance of Constant Lung Volume

By controlling and adjusting the amount of inspired air, lung volume at each scan series was maintained approximately constant. Prior to each series, subjects exhaled to RV and then inspired all air delivered from a respiratory bag connected to the mouthpiece through a pneumatic valve. Once all air was inspired, the pneumatic valve was closed, so that the subject would not inadvertently exhale or inhale any air during scanning. At baseline, the fixed amount of air delivered from the bag was equal to the ERV determined during the first visit, also at baseline. Therefore, baseline scanning was performed at FRC (RV + ERV = FRC). Because airway constriction is associated with increases in RV ("air trapping"), if the original ERV was to be delivered through the respiratory bag at every step of the protocol prior to scanning, the lung volume at which each scanning series would be performed would increase with the progression of the challenge. This could create methodologic problems in terms of lumenal area measurements with the assumption that changes would reflect the effect of the MCh challenge. To maintain the subject's lung volume constant during all scan series, the amount of air delivered from the respiratory bag was reduced by the individually estimated amount of increase in RV after each step of the challenge. The estimation of the increase in RV after each dose of MCh was based on regression lines that were constructed for each subject during the first visit. These lines correlated the changes in various partial spirometric outcomes and the change in RV during MCh challenge. The regression with the best ability to predict the change in RV from a partial spirometric outcome (i.e., the highest r²) was used during the HRCT visit (Figure 1). During that visit, after each dose of MCh, the change in RV was estimated from the change in the spirometric measurements. To maintain lung volume constant, the amount of inspired air in the respiratory bag was decreased prior to each scanning by the calculated increase in RV in an individually tailored fashion. The relationships between the changes in RV and the changes in the partial spirometric outcomes were obtained in the sitting position, whereas spirometry during the HRCT protocol was, by necessity, performed in the supine position. To assess the reliability of our approach, we added a third visit in which the changes in partial spirometric outcomes in the sitting and in the supine positions, in relation to the changes in RV, during another MCh challenge, were calculated.

View larger version (22K): [in this window] [in a new window]   Figure 1.   Linear regression line (solid line) and 95% Confidence Intervals (dashed lines) for each subject depicting the relationship between the best fit spirometric index and the change in residual volume on the first visit. For Subject #2, no significant correlation was obtained between any of the spirometric indices and the change in RV. For this subject, no adjustment was made in the volume of inspired air during MCh challenges during HRCT visit (see text).

Airway Measurements with HRCT

The airway lumenal area measurement methodology has previously been described and validated (16, 21). Briefly, the HRCT images were transferred to a UNIX-based work station and analyzed using the airway analysis module of the Volumetric Image and Display Analysis (VIDA) software package (Dept. of Radiology, Division of Physiologic Imaging, Univ. of Iowa, Iowa City, IA). To measure airway areas, the operator drew a rough isocontour estimate of the lumen of the airway. The software program automatically located a precise isocontour perimeter of the airway lumen by sending out rays in a spoke-wheel fashion to a predesignated pixel intensity level that defined the lumenal edge of the airway wall. The length of the rays was set at six pixels. The software program used an algorithm for edge detection based on the "full-width: half-maximum" principle. The edge of the wall was defined by the program by the points along the lines where the pixel intensity changed to half its maximum through the wall. All full and partial pixels (full pixel size equals 0.1537 mm² with our settings) within the adjusted isocontour were counted and represented the airway area.

Data Analysis

All airways that were visualized under all conditions were matched and measured. HRCT data were expressed as a percent of baseline and were averaged at baseline and after each MCh dose. The data from spirometry and body plethysmography acquired during the first visit and the spirometry and HRCT data from the second visit were analyzed using one-way ANOVA with Bonferroni correction for multiple comparisons. In addition, linear regression models were constructed to relate the partial spirometry and the HRCT airway lumenal area data on the second visit as well as the partial spirometry and the plethysmography data on the first visit at each dose of the MCh provocation. Finally, linear regression models were constructed to compare the sitting and supine spirometric data acquired during the third visit. Statistical significance was accepted at p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

During the first visit, we observed reductions in all partial spirometric parameters induced by the MCh inhalation challenge, but these changes did not reach statistical significance. However, RVp at the 2.5 mg/ml dose of MCh was 127 ± 11% (mean ± SEM) of baseline, a change that was significant (p = 0.01). The best relationships between the changes in RVp and a spirometric parameter for each subject are shown in Figure 1. Subjects 1 and 4 had changes in RVp that correlated best with the changes in MMEFp (r² = 0.34, and r² = 0.81, respectively). Subject 3 had changes in RVp that correlated best with the changes in FEV₁p (r² = 0.72). Subject 5 had changes in RVp which correlated best with the changes in FVCp (r² = 0.84). These spirometric parameters were chosen as those to be used in adjusting the volume of inspired air during the subsequent MCh challenge during the HRCT scanning. For Subject 2, RV did not change with the inhalation of MCh, and therefore no volume adjustment was implemented during HRCT scanning. During the third visit, for the group, we observed similar changes in the spirometric indices in the sitting and the supine position. We also obtained highly significant correlation for the group, between the sitting and the supine partial spirometric indices in the course of MCh challenges (FEV₁p: r² = 0.33, p = 0.004; FVCp: r² = 0.48, p = 0.0002; FEV₁p/ FVCp: r² = 0.82, p = 0.0001; MMEFp: r² = 0.37, p = 0.002; p: r² = 0.82, p = 0.0001). Furthermore, we compared the calculated slopes of the regression lines between the upright and the supine positions, for the various spirometric values obtained during the third visit. Because baseline lung function measurements were significantly reduced when moving from the upright to the supine position, we calculated the slopes of the regressions between the percent changes in the respective outcomes rather than the absolute values. These slopes approached 1 in all cases (for FEV₁: 1.1, for FVC: 1.2, for FEV₁/ FVC: 1.1, for MMEF: 1.2). Therefore, for the group, the changes in spirometric parameters induced by MCh, when measured in the upright position, are highly predictive of those measured in the supine position. However, individual subjects showed variability in the sitting versus supine relationships.

When subjects were supine in the scanner during the second visit, the same doses of MCh similarly reduced the partial spirometric parameters, with some now reaching statistical significance (FEV₁p, p = 0.003, FEV₁p/FVCp, p = 0.0006; MMEFp, p = 0.04).

On the HRCT images, 14 to 23 airways ranging from 2.3 to 10.4 mm in diameter at baseline were matched and measured within each subject under all conditions (Figure 2). Consistent with the changes in partial spirometry, mean airway lumenal area decreased in response to the increasing doses of aerosolized MCh. Airway lumenal area decreased to 91 ± 2%, 88 ± 2%, and 80 ± 2% of baseline at the MCh doses of 0.25, 0.75, and 2.5 mg/ml, respectively (p < 0.001) (Figures 3 and 4). Most importantly, we found significant correlations between the reduction in the mean airway lumenal area as measured by HRCT and the mean partial spirometric outcomes both on the basis of the individual best fit spirometric measurement and change in HRCT for each subject (Figure 5). However, because of the small number of data points, in two subjects the strong correlations were influenced by a single data point. Overall, we found significant correlations between the group spirometric values and airway area as measured by HRCT for all subjects: FEV₁p (r² = 0.46, p = 0.001), FVCp (r² = 0.20, p = 0.05), FEV₁/FVCp (r² = 0.55, p = 0.002), MMEFp (r² = 0.31, p = 0.01), and p (r² = 0.51, p = 0.0004). Within each subject as well as between subjects, considerable heterogeneity in individual airway responsiveness to MCh was observed (Figure 6). To examine whether the heterogeneous responses were a function of the original size of the airways, we arbitrarily divided the airways into four groups by size: < 3.5 mm, 3.5 to 5 mm, 5 to 6.5 mm, and > 6.5 mm in diameter. We found no differences among these four groups of airways in their responsiveness to the spasmogenic stimulus (ANOVA: p = 0.72, p = 0.70, and p = 0.67 for the 0.25, 0.75, and 2.5 mg/ml doses, respectively).

View larger version (28K): [in this window] [in a new window]   Figure 2.   Baseline airway lumenal diameter frequency distribution (in millimeters) of all the airways measured in the five subjects.

View larger version (143K): [in this window] [in a new window]   Figure 3.   Example of matched HRCT scans at baseline (top left) and after each MCh challenge: 0.25 mg/ml (top right), 0.75 mg/ml (bottom left), and 25 mg/ml (bottom right) from one subject. Arrows indicate examples of matched airways that narrowed with increasing doses of methacholine.

View larger version (17K): [in this window] [in a new window]   Figure 4.   Mean (± SEM) airway lumenal area measured by HRCT, expressed as a percent of baseline, after each concentration of the MCh challenge for the five healthy subjects who underwent the provocation in the supine position on the HRCT gantry.

View larger version (17K): [in this window] [in a new window]   Figure 5.   Linear regression line for each subject depicting the relationship between the best fit spirometric index and the change in mean airway lumenal area, as measured by HRCT. All subjects showed good correlation between the change in spirometry and airway area.

View larger version (31K): [in this window] [in a new window]   Figure 6.   Individual airway sizes at baseline and after each successive dose of MCh for each of the five subjects (left axis) and mean (± SEM) airway area (solid diamonds), expressed as a percent of baseline (right axis), at baseline and after the highest dose of MCh. In all subjects, airways showed significant response heterogeneity; however, a decrease in mean airway area after the highest dose of MCh was present in every subject.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Although healthy subjects do not demonstrate significant bronchoconstriction when they inhale spasmogen such as MCh under routine conditions (when TLC maneuvers are involved in the protocol), we have previously shown that, in the absence of DIs, they will respond to MCh in a manner similar to that of asthmatics (1). The current study has confirmed the stepwise airway narrowing induced by MCh in the healthy state and has also demonstrated that the location of airway narrowing includes the conducting airways that can be visualized on HRCT images. Most importantly, the MCh-induced changes in mean airway size closely correlate with the simultaneously measured changes in lung function.

In the current study, induced bronchoconstriction was diffuse and no preferential location was observed, at least within the range of airway sizes visualized and measurable on HRCT images. Whether this is specific to nonasthmatics or to the applied type of spasmogen requires further study. In any case, since we can now measure airway narrowing in healthy subjects, future physiologic imaging studies can focus on the differences between healthy and asthmatic subjects with respect to their ability to dilate or to protect their airways using deep breaths. We and others have proposed that a profound difference in this ability between the two groups may represent a major mechanism of airway hyperresponsiveness (1).

Another finding consistent with results from in vivo studies in dogs (10) and lung explants of rats (22) and humans (23) is the observation of airway response heterogeneity to a bronchoconstrictor. Clearly, some airways both within and between subjects had a more vigorous response to MCh (Figure 6). It is interesting that in every subject at every dose of MCh some airways actually increased in size (Figure 6). This may be a reflex compensatory response. Dandurand and colleagues (22) studied the heterogeneous response of individual airways in vitro. They used a rat lung explant model to measure individual airway responsiveness to methacholine and found that the responsiveness of individual airways in a given animal was far more variable than that between animals. Whether the airways of asthmatics would respond with a similar extent of heterogeneity or with a more homogeneous decrease in airway size requires further study. A hypothesis worth testing is that the lack of a heterogeneous response may contribute to the observed hyperresponsiveness to spasmogens in asthma.

Many requirements need to be fulfilled for multiple imaging measurements of the airways of human subjects to be considered reliable: (1) lung volume during scanning must be maintained approximately constant; (2) subjects must be able to hold their breath for the duration of the scan acquisition; (3) airways must be located, matched, and measured at the same anatomic location; and (4) a relationship between imaging and conventional outcomes of lung function should exist. Each of these requirements was addressed in this protocol.

We designed the HRCT scanning to be performed at what we expected to be a constant lung volume to avoid underestimating the narrowing of the airways with the imaging approach. That is, we were concerned that if we were to keep the volume of air delivered through the respiratory bag constant at each step of the bronchoprovocation at which scanning was to be performed, because of the expected increase in RV, the lung volume at which we were to scan would also increase with every dose of MCh. Although static transpulmonary pressure is an important determinant of airway caliber with changes in lung volume, we previously showed, in animal models, that airway caliber does not change isotropically with lung volume when the airways are relaxed, but does appear to change in a linear fashion with lung volume when the airways have mild tone (12, 14). Because we did not measure transpulmonary pressure, we instead chose to maintain lung volume constant.

We believe that with the employed technique, lung volume was maintained relatively constant throughout the MCh bronchoprovocation. Each subject exhaled to RV and then inspired a given amount of air that was changed at every step of the challenge to account for the estimated increase in RV. Airway constriction is associated with increases in RV ("air trapping"). At the highest dose of MCh administered on the first visit, the average increase in RV was 27%. Given that during the first visit, changes in RV in four of the subjects were correlated with changes in spirometry, we were able to construct regression lines that could predict the increase in RV from the decrease in partial spirometric parameters. We also examined these partial spirometric parameters in the sitting and supine positions. As a group, these parameters were correlated even though individual subjects demonstrated variability. One potential weakness with our methodology is that, although the changes in sitting RV were correlated with the changes in spirometric outcomes induced by MCh in the sitting and the supine position, we cannot state that the same relationship would exist if the RV measurements were made in the supine position as well.

Also, our extrapolations, based on spirometric changes, are hindered by a significant degree of variance in the individual regressions we have constructed (Figure 1). From these individual regressions, two points can be made: First, the number of observations (four to five) from which the regression lines were drawn was small, resulting in relatively wide confidence intervals. One way to increase the precision of the measurement would be to increase the number of points that are used to construct the regression line. This could be done by increasing the number of doses of MCh, e.g., doubling instead of tripling doses. Second, in one of the five subjects (Subject 2), there was essentially no relationship between changes in spirometric parameters and residual volume, i.e., there was no air trapping and increase in RV with the observed decrease in FEV₁p, and no correction of inspired volume of air was made for that subject after MCh challenge. To increase the reproducibility of the measurements, individual subjects with poor reproducibility may not be included in future studies.

In the worst case scenario, we would have systematically overestimated the increase in RV induced by the MCh challenge. This would have led us to administer less than an appropriate volume of air through the respiratory bag, resulting in a decreased lung volume and a spurious reduction in airway area with increasing concentrations of methacholine. However, we doubt this was the case. Our main argument against this possibility derives from the relationships depicted in Figure 5 and the overall correlations between the spirometric indicies and the changes in airway area measured by HRCT. In this figure, we show good correlations between partial spirometric parameters and the changes in airway area that were induced by MCh. If this relationship between the spirometry and HRCT-imaged airways was spurious, we would need to accept that the changes in the spirometric measures (which we have no reason to question) came from airways not imaged by HRCT, whereas the airways we imaged were decreasing in size solely as a result of insufficient inflation, i.e., because of our systematic error. We believe that this is very unlikely. We also need to point out that the average volume of air that we delivered through the respiratory bag at baseline to inflate the lungs above RV was 1.7 L, whereas the average adjustment (reduction) in this volume after the highest MCh concentration was 120 ml, a change of less than 7%. The decrease in airway area measured by HRCT, on the other hand, was 20%. It is hard to believe that our adjustment can account for the entire decrease in airway area after MCh challenge, even if there was no change in RV. In fact, because of the lack of a correlation between changes in RV and the spirometric indices on the first visit, we did not adjust the volume of air delivered from the respiratory bag on Subject 2 during the HRCT visit. Still, the MCh challenge reduced airway area by almost 25% in this subject (Figure 6).

When a subject's position changes from sitting to supine, FRC decreases by approximately 25%, whether measured by helium dilution (24) or by body plethysmography (25). The decrease is most likely due to a reduction in the outward recoil of the chest wall because of increased pressure from the abdomen into the chest. The decrease in lung volume in the supine position also increases airway resistance (24). The average baseline FEV₁ and FVC for the five subjects was decreased by 28 and 26%, respectively, when they were supine compared with the sitting position. Ding and colleagues (26) demonstrated that decreased lung volume, under controlled conditions, caused increased airway responsiveness to MCh, whereas increased lung volume diminished airway responsiveness. Although we observed increased airway responsiveness when the subjects were prevented from taking a deep breath in the sitting position similar to our previous results (1), these changes in the current study did not reach statistical significance, likely because of the small sample size. When the subjects were prevented from taking a deep inspiration and were tested in the supine position, some of the changes in lung function with MCh challenge reached statistical significance. It is unlikely that the difference in airway responsiveness observed between the upright and the supine positions was secondary to a reason other than the difference in lung volume. This is supported by the fact that, once baseline lung function differences are taken into account, the mean responsiveness appears to be identical, that is, (1) there is high correlation between the MCh-induced changes from baseline in spirometric outcomes obtained in the upright and the supine position and (2) the slope of the regression lines approach 1 in all cases (for FEV₁: 1.1, for FVC: 1.2, for FEV₁/FVC: 1.1, for MMEF: 1.2). One could also argue that the difference in responsiveness between the two positions could be secondary to a different aerosol distribution. However, when respiratory rate is maintained at a normal rate, positional changes have not been shown to alter aerosol deposition in humans (27).

Subjects were able to hold their breath for the duration of the scans even after the highest dose of MCh was administered (2.5 mg/ml). The duration of the spiral scan acquisition was 20 s, a relatively short amount of time for a healthy person, even in the presence of bronchoprovocation. Furthermore, in this healthy population, no respiratory motion artifact occurred that prevented matching and measuring the airways in any of the subjects. The duration of breathhold time should not need to be shortened for moderate asthmatics during bronchoprovocation. Preliminary experiments using asthmatics under the same protocol support this claim.

Multiple airways, 14 to 23, were measured and matched in each subject. The ability to match and measure airways has been demonstrated in previous animal studies in our laboratory (10, 13), and in human subjects by others (4, 5). Although the conducting airways of humans, relative to lung size, are smaller compared with those in either dogs or sheep, multiple airways perpendicular to the scan plane were visible in the section of lung imaged in all of our subjects. Additionally, the use of spiral acquisition and thin sections (2 mm) facilitated matching of the airways. Furthermore, the use of spiral methodology allowed complete image acquisition after each dose of MCh with a single breathhold, assuring more consistent lung volumes.

In summary, we have demonstrated that, in normal subjects who are prevented from taking a deep breath, the spirometric changes associated with aerosol MCh challenge are reflected in the narrowing of the conducting airways. We have also developed an algorithm by which airway lumenal area can be serially visualized and measured in the course of bronchoprovocation. Individual airway responsiveness as well as regional heterogeneity can thus be assessed in humans and a comparison between the healthy and the asthmatic state can be made in this respect. Also, this imaging technique may enable future studies to delineate the mechanisms of airway hyperresponsiveness as it pertains to the inability of asthmatics to relax their airways with deep inspiration.