Subjects demographic, spirometric, and PC₂₀ data are shown in Table 1. On the basis of these data it is difficult to unambiguously classify the asthmatics subjects. Some (e.g., Subjects 4, 10, and 11) had FEV₁/FVC > 80% but were highly reactive (PC₂₀ < 1.0), whereas others had FEV₁/FVC near or less than 80% but were less reactive (e.g., Subjects 1 and 9). Subjects 2, 5, 6, and 10 presented at baseline with the most acute hyperreactivity (PC₂₀ < 0.01, except for Subject 2 who was so severely constricted at baseline that a challenge could not be performed) as well as distinctly elevated Rl and El data compared with other asthmatics (Figure 2). On the basis of the impedance and PC₂₀ data, we chose to refer to these four subjects as presenting with “severe” baseline asthmatic constriction and all other asthmatics as mild to moderate. (Note that although our criteria for labeling the patients with severe asthma were quantitative, they are not meant to infer a clinical classification, i.e., they were not fully consistent with the NAEPP and GINA guidelines.)

Fig. 2. Rl and El data at baseline from all seven healthy subjects (left panel ) and all asthmatics subjects (right panel ). Note scale differences on two plots. The four asthmatics we classified as severe are shown with dashed lines.

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The data from all subjects are shown in Figure 2. Most data from subjects with mild-to-moderate asthma at baseline were qualitatively similar to the healthy data, although, in general, they showed evidence of slightly elevated Rl at all frequencies and an El that was elevated primarily at higher frequencies (> 2 Hz). The data from the four subjects labeled as severe asthmatics were quite distinct. The Rl was highly elevated, particularly at low frequencies with a large frequency-dependent drop from 0.1 to 2 Hz. The El was nearly double that of other subjects even at 0.1 Hz, and, unlike subjects with mild-to-moderate asthma, the frequency dependent increase in El began immediately and continued through our 8-Hz range.

The key spectral features for all three groups at baseline are compared in Figure 3, whereas Figure 4 tracks the averages within each group throughout the protocol. Also shown in Figure 3 are the E_st values. Specific examples for the three types of subject data (healthy, mild-to-moderate asthmatic, and severe asthmatic) before and after a DI at baseline are provided in Figure 5A.

Fig. 3. Summary comparison of key spectral and spirometric features at baseline for healthy subjects, subjects with mild-to-moderate asthma, and those with severe asthma. Spectral features are Rl at the lowest frequency (R_low) and the highest frequency (R_high); and El at the lowest and highest frequency (E_low and E_high). We also show static elastance (E_st).

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Fig. 4. Four spectral features from Figure 4 tracked throughout the protocols averaged for each of the subject groups separately. Protocol includes baseline (BL), baseline-post-DI (BL-DI), postchallenge (PC), postchallenge-post-DI (PC-DI), and after albuterol from the challenged condition (ALB). Data plotted for standard challenge day (solid line) and modified challenge day separately (dashed line, no DIs permitted). Note, only one subject with severe asthma could perform a modified challenge. Numbers not in parentheses above or below data points indicate p values (paired t test) for a given challenge day as follows: BL-DI versus BI, PC versus BL, and PC-DI versus PC. Also shown boxed are p values comparing modified challenge with standard challenges.

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Fig. 5. (A) Lung resistance and elastance (Rl and El) versus frequency at baseline (solid line) and after (dashed line) a DI for representative healthy subjects, those with mild-to-moderate asthma, and those with severe asthma at baseline. Post-DI response is shown with same open symbol and dashed line. Subject Nos correspond to those in Table 1. (B) Same three subjects after a modified methacholine challenge and before (solid line) and after (dashed line) a DI. Doses for the challenge were 25 mg/ml, 10 mg/ml, and 0.01 mg/ml for the healthy subjects, the subjects with mild-to-moderate asthma, and those with severe baseline asthma, respectively.

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From Figure 3, it is evident that although E_low (i.e., El at 0.15 Hz) progressively rises with more severe asthma, E_st is essentially identical for all three types of subjects. Thus, asthma does not cause a stiffening of lung tissue, and the rise in dynamic El even by 0.1 Hz is due to an airway phenomenon.

After a DI, the healthy subject in Figure 5A showed virtually no change in El at any frequency, no change in Rl at higher frequencies, and a small decrease in Rl below 1 Hz. Of the 14 measurements at baseline (seven healthy subjects, each on 2 d), eight showed this pattern and six showed no change in Rl even at lower frequencies after a DI. The decrease in R_low after a DI over the whole sample of healthy subjects was not significant (Figure 4). Compared with healthy subjects, subjects with mild-to-moderate asthma present at baseline with elevated Rl at all frequencies (e.g., the mean R_low and R_high were 4.0 ± 1.6 and 2.8 ± 0.1 compared with 3.4 ± 1.3 and 1.8 ± 0.4, respectively, for the healthy (Figure 3)), slightly but significantly elevated El < 1 Hz (e.g., E_low 7.4 ± 1.9 versus 6.1 ± 1.4 for the healthy), and then a more noticeable frequency-dependent rise in El primarily above 2 Hz (e.g., E_high of −16.4 ± 18.2 for subjects with mild-to-moderate asthma versus −31 ± 13.5 for the healthy). In most of the subjects with mild-to-moderate asthma, there was little evidence of a change in the data post-DI. Indeed, over the entire sample, none of the four key spectral features changed in a statistically significant fashion after a DI at baseline (Figure 4).

The baseline pattern of the patients with mild-to-moderate asthma (e.g., Figure 5A) is consistent with a pattern of widespread milder baseline constriction sufficient in some cases to invoke some airway wall shunting (i.e., the dynamic El increase at higher frequencies). Because El at 0.1 Hz is only slightly elevated compared with that in healthy subjects and there is little if any abnormal frequency-dependent increase in El from 0.1–2 Hz, one could infer that there is some heterogeneity causing E_low > E_st by a greater degree than in healthy subjects, but there was not much if any heterogeneous closure in the lung periphery. Moreover, since few if any airways were highly narrowed prior to the DI, after a DI the El at 0.1 Hz is unaffected (i.e., there is communication to the same amount of lung tissue).

Data from the patient with severe asthma is quite distinct in several ways (Figure 5A). At baseline, the R_high represents primarily Raw alone (2) and for the subject shown, the R_high is more than 300% larger than that of the healthy subject's. Second, between 0.1 and 2 Hz, there was now a very large frequency-dependent drop in Rl and a rapid rise in El. Third, the E_low was nearly double that of healthy lungs. Fourth, between 2 and 5 Hz there was another distinct rapid rise in El. Referring to the simulated data of Figure 1, one could presume that at baseline the patient with severe asthma presents with substantial constriction that is sufficiently widespread throughout the lung to induce airway wall shunting but yet still highly heterogeneous in that there are random airway closures or near closures creating elevated El at 0.1 Hz and a large frequency-dependent increase in dynamic El between 0.1 and 2 Hz. Despite presenting with severe airway constriction, there is very little if any bronchodilatory impact after a DI. The Rl data remain unchanged, whereas the El data are slightly lower, but remain extremely elevated compared with mild-to-moderate asthma. Thus, at baseline, a DI was unable to alleviate the constriction level or homogenize the pattern of constriction.

Examples of the modified responses pre- and post-DI for the same three subject types from Figure 5A are shown in Figure 5B. In healthy subjects the Rl and El response to a modified challenge was amplified over that of a standard challenge (Figure 4). However, while preventing a DI amplified the response, it did not do so to the level of even subjects with mild-to-moderate asthma during their standard challenge (Figure 4), i.e., typically modified responses for healthy subjects at 25 mg/ml remained much lower than the asthmatic response at their lower PC₂₀ dose during a standard challenge. The discrepancy was even larger when comparing the healthy modified responses to the asthmatic modified responses.

Healthy subjects. For healthy subjects and a standard challenge, there is a noticeable response at the maximum dose of 25 mg/ml. The Rl tended to rise evenly at all frequencies. Among the groups (Figure 4), there was a small increase in E_low (from 6.4 ± 2 to 7.9 ± 2) and a larger increase in E_high (from −34 ± 9 to 6.6 ± 47). The modified challenge produced a more amplified response at all frequencies, but primarily at lower frequencies. The Rl and El data postmodified challenge now suggest an enhanced degree of heterogeneous constriction involving airway closures or severe narrowing randomly throughout the peripheral airway tree (e.g., as in Figure 1). The increase in R_low (p < 0.01), E_low (p < 0.1), and E_high (p < 0.001) were significantly greater during the modified challenge (Figure 4). In fact, over the whole group, the increase in E_low and R_low were significant for the modified challenge (p < 0.004 and p < 0.01, respectively), but not statistically significant for the standard challenge (Figure 4). Also, there is now a greater frequency dependence of El and Rl, from 0.1 to 2 Hz.

Despite the amplified response during a modified challenge, a DI was dilatory to the same degree as it was in a standard challenge (i.e., the spectra all decreased nearly to the same levels regardless of which challenge preceded the DI; e.g. Figure 5B). Also, the Rl below 2 Hz decreased far more than the Rl at 8 Hz. Note that the DI did not reduce the Rl or the E_high entirely back to the baseline prechallenged levels (Figure 4). The E_low at 0.1 Hz decreased more than half way back to the prechallenged level (e.g., see insert of Figure 5B). The reduction in E_low indicates a combination of substantially reduced heterogeneity and that many formally closed airways became open, producing communication to more tissue.

In summary, inhibiting the DI in healthy subjects amplifies the response (but not to the levels of asthmatics) primarily by permitting a greater degree of a heterogeneous constriction in which some airways close or nearly close. Nevertheless, the healthy subject can stretch these airways open via a DI, and they remain sufficiently open immediately after the expiration so as to homogenize some of the pattern of constriction. In one subject we repeated the OVW 1 min post-DI and found (not shown) that the Rl and El spectra returned to the pre-DI level. This indicates that the post-DI dilatation is transient and that the random airway closure or near closure will reoccur without bronchodilator intervention.

Subjects with mild-to-moderate asthma. These subjects are hyperreactive during a standard challenge. The response during a modified challenge tended to be amplified compared with the standard challenge, but to a less significant extent than in healthy subjects (Figure 4). In both kinds of challenges, the frequency-dependent increase in El begins immediately. For the subject in Figure 5B, note that the modified response in Rl and El after a dose of only 10 mg/ml substantially exceeds that of the healthy response to 25 mg/ml (Figure 5B). There is a substantial dilatory impact of a DI reducing the Rl and El across all frequencies for both types of challenges (Figures 4 and 5). The post-DI reduction in E_Low at 0.1 Hz suggests that some formally closed airways or highly narrowed airways are now open. Unlike that in healthy subjects, in neither challenge did the DI reduce the Rl and El back to baseline. Moreover, compared with the DI poststandard challenge, the DI postmodified challenge is less effective at ablating the impact of the constriction; i.e., the Rl and El post-DI are higher than those found after a DI during a standard challenge (Figure 4), particularly the R_low, E_low, and E_high.

These data suggest that compared with healthy subjects: (1) during a standard or a modified challenge, the response is more heterogeneous and inclusive of more closed airways or nearly closed at a lower dose; (2) comparing the magnitude of the response, especially during a modified challenge, to previous simulations with morphometric models that had challenged only the (< 2 mm) peripheral airways (e.g., Figure 1), it is likely that the constriction and closures are occurring in airways even larger than 2 mm in diameter; and (3) prohibiting a DI during the challenge makes it more difficult to ablate the extreme constriction induced by invoking a DI postchallenge. Nevertheless, administering a bronchodilator does completely ablate it (Figure 4).

Subjects with severe asthma. Because of their baseline conditions, we could perform standard challenges in only three of the four patients with severe asthma and a modified challenge in only one. The patients with severe asthma responded greatly at the lowest dose of 0.01 mg/ml resulting in an amplification of all features of the already abnormal baseline condition (Figures 4 and 5B). When performing a DI postchallenge, there is either no significant dilatory impact or, in the case of this subject, on the standard challenge day evidence of further bronchoconstriction. Finally, while albuterol significantly reduced the Rl and El, perhaps even below baseline conditions, the values postalbuterol were still well above the levels of typical healthy values (Figure 4). Thus, the albuterol could remove the impact of the 0.01 mg/ml dose, but not all the residual constrictive conditions that existed at baseline. This is consistent with a challenge permitting the occurrence of increasing amounts of closed and/or extremely narrowed airways higher in the airway tree that are not dilating during a DI. Thus, the inhaled albuterol is likely not to be delivered to a substantial fraction of constricted smooth muscle and its impact is less.

By measuring Rl and El spectra, we provide data that (1) directly represent the mechanical load against which pressures must be generated for breathing and (2) provide a window on the mean level and pattern of airway constriction. Comparing static with dynamic El (Figure 3) showed that subjects with mild-to-moderate asthma have a small but significant elevation in baseline El even by 0.15 Hz and that the elevation is much larger for those with severe asthma. Nevertheless, these elevations are not due to stiffening of lung tissue, but rather are a consequence of heterogeneous airway constriction because the E_st was the same for all groups.

Does preventing a DI in healthy subjects mimic the hyperreactive constriction response of asthmatics? Qualitatively, the answer is yes, in that preventing a DI during a challenge amplified the low frequency response of Rl and El in a manner consistent with excessive heterogeneity inclusive of randomly distributed severe airway narrowing. Asthmatics show similar low frequency changes even without preventing a DI. Nonetheless, there are two substantial differences between healthy and asthmatic subjects. First, the Rl and El response of asthmatics at their PC₂₀ dose during a standard challenge far exceeds that seen in healthy subjects (Figures 4 and 5). Consistent with the findings of Burns and Gibson (8) and Brusasco and colleagues (9), one cannot make a healthy subject achieve the degree of airway hyperresponsiveness seen in asthmatics simply by prohibiting DIs during a challenge. Second, in healthy subjects, the impact of a DI poststandard or postmodified challenge was bronchodilatory and returned the Rl and El spectra nearly completely back to their prechallenged level. In contrast, subjects with mild-to-moderate asthma showed a substantially diminished DI response, especially for the modified challenge, and those with severe asthma showed no response to a DI with either challenge, or even had a paradoxical bronchoconstriction after a DI.

The above leads to two additional questions. (1) Why are asthmatics who present with mild-to-moderate asthmatic conditions at baseline hyperreactive? (2) Why are asthmatics with severe baseline constriction showing no bronchodilatory impact of a DI before or after challenge? Consider first the potential role of airway wall-thickening and smooth muscle biological state. We speculate that preventing a DI might promote the airway smooth muscle to transition toward a state in which more force would be created during bronchoprovocation. Such a state could be consistent with either the latch hypothesis (28, 29) or a plastic remodeling of the muscle filament lattice (31). Regardless of how it gets there, once provoked the muscle now can constrict more and become stiffer once constricted. Our healthy subjects did constrict more when a DI was prevented, but never to the level of an asthmatic. Combined with the fact that healthy subjects have no standing inflammatory or thickened airway walls, a DI should be more effective at transmitting the forces necessary to reopen and dilate constricted airways, and this is what we found. Alternatively, the asthmatics at baseline are more likely to have some elevations in inflammatory cells (32) and/or thicker walls not only within a given airway but throughout a greater portion of the airway tree, i.e., extending higher up in the tree (33). Such conditions might facilitate less fluctuations in the smooth muscle length during tidal breathing or a DI, which would facilitate the smooth muscle being closer to a latch or remodeled state that allows more reactivity when provoked. If so, the response to Mch would be greater throughout the airway tree. Certainly, the asthmatic responses suggested a substantially more constricted airway smooth muscle than that in healthy subjects. It is likely that in asthmatics the muscle is so stiff that a DI is simply less effective at generating the forces to open and dilate these airways. Extending this scenario, asthmatics presenting with severe spontaneous constriction likely have substantial wall thickening surrounded by highly stiff muscles. Hence, a DI becomes essentially completely ineffective. Indeed, Johns and colleagues (34) have recently provided evidence that asthmatic airways are less distensible than are healthy airways.

If indeed airway closures of near closures are occurring during asthmatic constriction, we should consider the phenomenon of airway reopening during a DI. Generally, the threshold pressure necessary to reopen a closed airway is P_th = Kγ/r, where r is airway radius to which the airway would “pop open” (or the r from which these airways closed during constriction), γ is surface tension, and K is a constant that depends on the local geometry and the airway wall stiffness (35). At baseline, in asthma, the radius would be smaller, and K would be larger because of both increased muscle tension and wall-thickening. Thus, it is possible that during a DI, patients with severe asthma simply cannot generate sufficient transmural pressures to reopen the closed airways. Nevertheless, it is uncertain from our data whether asthmatics have a reduced capacity to dilate their airways or whether dilation occurs, but the reconstriction during the exhalation from TLC is so rapid as to not provide any residual reduction in the measured Rl and El spectra.

There is certainly evidence of airway closure in asthmatics with provocation, especially in the small airways, or with provocation in healthy subjects administered from a lower lung volume (36-39). Indeed, Gunst and colleagues (30, 40) had already reported that closure is possible. This phenomenon has been the subject of increased attention, but not in the context we propose; namely, to produce a dangerous pattern of heterogeneous peripheral constriction. We showed evidence that when DIs are prohibited during induced constriction, airway closures and severe airway narrowing are more likely to occur. Such closure also reduces the volume of lung tissue communicating with the airway opening, so that the effective tissue elastance and resistance, as measured from the mouth, increase. We do not suspect, or have any evidence of, substantial real increases in tissue elastance during asthma. Evidence of acute heterogeneity with closure or near closure occurred in our asthmatics, or when one attempts to suppress dynamic modulation of smooth muscle by preventing a DI (i.e., a modified Mch challenge).

Extreme heterogeneity of constriction represents an important phenotype of the clinical defect during a severe asthmatic condition. Certain forms of heterogeneous constriction can establish excessive elevations in dynamic Rl and El at typical breathing rates. Our data suggest that preventing a DI during bronchoprovocation amplifies the heterogeneity of the constriction response. By implication, inflammation may serve to exacerbate the effects of increased tone by permitting random airway closures or near closures and preventing residual reopening via DI. In fact, if inflammation also inhibits stretching of these airways, such patients are in double jeopardy. First, smooth muscle stimulation now creates random closures or near closures with consequent ineffective ventilation distribution at typical breathing rates (10). Second, DIs by the patient cannot result in a reopening of these airways and consequent improvement of ventilation. In short, the patient becomes locked in this heterogeneous and dysfunctional state. Only bronchodilators acting directly on airway smooth muscle will “unlock” them. Effective treatment protocols should then focus on distributing the bronchodilator to all airway smooth muscle, including those subtended by closed airways.