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1Pneumologia-Fisiopatologia Respiratoria and 2Riabilitazione Cardiorespiratoria, Azienda Ospedaliera S. Luigi, Orbassano, Torino, Italy; 3Fisiopatologia Respiratoria, Dipartimento di Medicina Interna, Università di Genova, Genova, Italy; 4Department of Physiology and Biophysics and Internal Medicine, Mayo Clinic and Mayo Foundation, Rochester, Minnesota; and 5Centro di Fisiopatologia Respiratoria e di Studio della Dispnea, Azienda Ospedaliera S. Croce e Carle, Cuneo, Italy
Submitted 4 April 2005 ; accepted in final form 21 February 2006
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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bronchial challenge; mechanics of breathing; lung volumes; airway caliber; deep inhalation; airway smooth muscle
In theory, lowering lung volume could increase airway responsiveness in different ways. For instance, the airway smooth muscle (ASM) could shorten more during breathing at low lung volume, because the extent of shortening for a given force development is inversely related to the magnitude of the elastic load imposed, which is less at low volume (21). Another mechanism could be the greater force generated by the ASM when stimulated at the shorter length accompanying decreased lung volume (15, 37). There is also the possibility that, at low lung volume, airway narrowing may be more severe than at normal volume, because the airways become more resistant to stretching as occurs with large breaths (12, 15, 22). The relative importance of these mechanisms in vivo in humans is unknown.
This study was designed to investigate if breathing at low lung volumes caused by chest wall strapping (CWS) increased airway response to inhaled methacholine (MCh) and whether this occurred due to a decrease in lung elastic recoil, or because the ASM was able to generate greater force. Two sets of experiments were performed. In the first set, the results of three different bronchial challenges were compared. One was performed with subjects breathing at their natural operative lung volume during both MCh inhalation and lung function measurements (CTRL), another with CWS during both MCh inhalation and measurements (CWSon/on), and a third one with MCh inhaled during temporary removal of CWS with measurements made after CWS was reapplied (CWSoff/on). We hypothesized that any increased responsiveness in the CWSoff/on challenge compared with the CTRL would be consistent with a prevalent role of lung elastic recoil in determining airway responsiveness. However, a difference between CWSon/on and CWSoff/on challenges would suggest an effect of the length at which ASM was stimulated. Because the bronchoconstrictor response was increased with CWSon/on but not CWSoff/on, a second set of experiments was undertaken to confirm the hypothesis that the enhanced airflow obstruction with CWS was the result of ASM stimulation at shorter length. This was accomplished by rechallenging a subgroup of subjects with CWS during MCh inhalation and removing CWS before and after making the measurements (CWSon/off). An elevated responsiveness in the CWSon/off sequence would support the importance of the length at which the ASM was stimulated.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Eight male volunteers (Table 1) participated in the study after giving informed consent, as approved by the local ethics committee. Seven subjects considered themselves healthy, and one reported a history of asthma, with no symptoms in the last 6 yr.
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On a prestudy day, spirometry was obtained according to the American Thoracic Society recommendations (1), and a standard MCh challenge was conducted by inhaling doubling doses of a solution of MCh chloride dry-powder (Laboratorio Farmaceutico Lofarma, Milan, Italy) in distilled water from 300 to 5,000 µg. Aerosols were generated by an ampoule-dosimeter system (MB3 MEFAR, Brescia, Italy), delivering particles with a median mass diameter ranging between 1.53 and 1.61 µm. Aerosols were inhaled during quiet tidal breathing in a sitting position. The doses of MCh causing a 10% and 20% decrease of forced expiratory volume in 1 s (FEV1) (PD10 and PD20, respectively) were calculated by linear interpolation between two adjacent points of the (log)dose-response curve.
Study 1.
Eight subjects attended the laboratory on 3 random study days to undergo different bronchial challenges. The sequence of interventions and measurements is shown in Fig. 1. On all days, two MCh doses were used: one equal to PD10 and the other to PD20. The inhalation time for both doses was of 
60 s. On 1 day, subjects inhaled MCh and had lung function measured while breathing at their control operational lung volume (CTRL). On another day, the procedure was repeated while subjects were always breathing at reduced lung volume, i.e., with CWS obtained with two elastic and two inelastic corsets extending from axillae to lower abdomen (CWSon/on). On another day, the procedure was similar to the CWSon/on occasion, with the only difference that the corset was temporarily removed for 3 min to allow MCh inhalation (CWSoff/on). All subjects were instructed to avoid larger than regular breaths during the challenge. On all days, lung function measurements after MCh were made at 3, 6, and 9 min after each dose, as shown in Fig. 1.
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Mouth flow was measured by a mass flowmeter (SensorMedics, Yorba Linda, CA), and volume was obtained by numerical integration of the flow signal. Spirometry and flow-volume curves were obtained in a body plethysmograph (Autobox, SensorMedics, Yorba Linda, CA) as follows. After six to eight regular breaths, thoracic gas volume was measured with the subjects panting against a closed shutter at a frequency slightly <1 Hz while supporting their cheeks with their hands. Soon after tidal breathing was resumed, the subjects were asked to perform a forced partial expiratory maneuver from 70% of their forced vital capacity (FVC), as measured in the prestudy day, to residual volume (RVpart). This was followed by a sustained full inspiration and, without breath hold, by a forced maximal expiratory maneuver to residual volume (RV). Care was taken that the duration of both forced expirations was 
6 s. Mouth flow was plotted against expired volume and measured at constant lung volume of control TLC on both maximal (
max) and partial (part) flow-volume loops. In addition, compression-free forced expiratory flow (max CF) was also obtained by plotting mouth flow against plethysmographic volume (32). This allowed us to compute upstream lung resistance (Rus) (25).
Functional residual capacity (FRC) was obtained from thoracic gas volume corrected for any difference between the volume at which the shutter was closed and the average end-expiratory volume of the four preceding regular tidal breaths. TLC was obtained by adding the inspiratory vital capacity to RVpart. Predicted values for spirometry and lung volumes were taken from Quanjer et al. (31).
Quasi-static transpulmonary pressure-volume (Ptp-V) curves were obtained during intermittent, brief interruptions of flow during a relaxed expiration from TLC. Esophageal pressure (Pes) was measured by a 10-cm-long balloon placed in the lower third of the esophagus after topical anesthesia of nose and throat. The balloon contained 1 ml of air and was connected to a piezoelectric pressure transducer (Microswich, ±200 cmH2O). Mouth pressure (Pm) was measured by a catheter connecting the mouthpiece to a piezoelectric pressure transducer (Microswich, ±200 cmH2O). Ptp was the difference between Pm and Pes. Placement of the balloon was considered correct if the changes in Pes and Pm with gentle inspiratory and expiratory efforts against a partially occluded airway were similar, thus leaving Ptp stable at a given lung volume (3). Volume and Ptp values were measured at the points of zero flow.
Lung resistance (RL) and dynamic elastance (Edyn) were measured by a DIREC System 200/201 (Raytech Instruments, Vancouver, Canada). Flow was measured by a Hans Rudolph pneumotachograph connected to a full-scale differential pressure transducer (±5 cmH2O, flow range: 0–400 l/min, Validyne). Pes and Pm were sensed by two DP15 Validyne differential pressure transducers (±150 cmH2O). Flow, volume, and pressure signals were fed into dedicated software (DR9, Raytech Instruments) and then processed to calculate RL and Edyn with the aid of a program written in SCILAB 3.0 (INRIA and ENPC). Irregular breaths, sighs, and breaths with negative Ptp were manually discarded. For each breath, the pressure difference in phase with volume was subtracted, so that the slope of Ptp vs. flow was RL (26). Edyn was the difference in Ptp at zero flow between end inspiration and end expiration divided by tidal volume (VT). Measurements were taken at least 60 s before and 90 s after a deep inspiration (DI).
At baseline of each study day, measurements of lung mechanics were obtained in the following order: at least three sets of partial and maximal maneuvers, two sets of RL and Edyn before and after DI, and at least three quasi-static Ptp-V curves (Fig. 1). After each MCh dose, one set of maximal and partial maneuvers, one set of RL and Edyn before and after a DI, and one Ptp-V curve were taken in the same order. With this order, the effects of DI on RL, Edyn, and partial flow at 60% of TLC (part 60) were minimized. The interval between maneuvers was always 2 min.
Data Reduction and Statistical Analysis
RL and Edyn before DI were computed by averaging the values of at least 10 regular tidal breaths and referred as to pre-DI. As in a previous study after exposure to the constrictor agent (30), RL and Edyn increased almost linearly following the resumption of tidal breathing after DI. Thus all values measured from the end of DI to the point at which a clear plateau was observed were submitted to a linear regression analysis, the intercept and slope of which were taken as estimates of the bronchodilator effect of DI (see below) and ASM reshortening, respectively. Rus was estimated by the ratio of Ptp to max CF at 60% control TLC (25).
The bronchomotor effects of DI before and after MCh were assessed by linearly regressing the values of maximal flow at 60% of TLC (max 60) vs. part 60, and those of RL and Edyn pre- vs. post-DI (30). In this analysis, an increase in slope or decrease of intercept of max 60 vs. part 60, or an increase in slope or intercept of RL or Edyn pre- vs. post-DI would suggest an impaired bronchodilator effect of DI.
A mixed between-within-groups ANOVA with Duncan post hoc comparisons was used. Values of P < 0.05 were considered statistically significant. Data are presented as means (SD).
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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max 60/part 60 (Table 1), but not by RL and Edyn. These data are from study 1. Effects of CWS on Lung Function Before MCh
See Table 3. Compared with CTRL, CWS caused consistent reductions of FVC (P < 0.001), FEV1 (P < 0.001), TLC (P < 0.001), and FRC (P < 0.001), but not in RV (P = 0.18) (Table 3). Similar changes were seen in study 2. At 70% of TLC, both Ptp and max CF were significantly increased (P = 0.009 and P = 0.03, respectively) (Table 4). Ptp at FRC tended to be less during CWS than during CTRL conditions [2.9 (SD 1.9) vs. 4.3 cmH2O (SD 2.3), P = 0.09], an effect that was consistent with the decrease in lung volume, but Ptp was increased at high lung volume. A typical pressure-volume curve is shown in Fig. 2. max 60 did not change with CWS, while part 60 increased significantly (P = 0.03). No significant changes were observed in RL, Edyn, Rus, or breathing pattern. We interpret the lack of changes in RL with decreasing FRC as a result of a decrease in tissue resistance, offsetting the increase in airway resistance.
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See Tables 4 and 5. Results obtained with PD10 and PD20 did not differ qualitatively. Therefore, only the latter are reported. The functional responses to MCh of the subject with prior history of asthma were similar to those of all other subjects, and his data were included in the analysis.
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max 60 (P = 0.017), while part 60 achieved absolute values similar to CTRL (Table 4). Similar results were obtained when the test was repeated in study 2 (Table 5). TLC remained stable during the challenge as did Ptp at any lung volume. FRC remained consistently lower than during CTRL (P = 0.008). VT tended to decrease, and breathing frequency (BF) to increase (P = 0.054).
In study 1, when CWS was applied before and after but not during inhalation of MCh (CWSoff/on), airway narrowing was less than with CWSon/on and not different from CTRL, as indicated by no change in RV (P = 0.97), RVpart (P = 0.97), Edyn (P = 0.38), RL (P = 0.27), and max 60 (P = 0.99) (Table 4).
In study 2 (Table 5) when CWS was applied during inhalation of MCh but not during measurements (CWSon/off), airflow obstruction was more severe than during CTRL for maximal flow at 70% of TLC (max 70) (P = 0.016), RV (P = 0.035), and Edyn (P = 0.039), but not for RL. However, the changes from CTRL were less than with CWSon/on for RV (P = 0.019), Edyn (P = 0.0002), and RL (P = 0.005). In summary, the bronchoconstriction in study 2 with CWSon/off was significantly greater than at CTRL, as reflected by changes in RV, max 70, and Edyn, but less than CWSon/on.
Effects of CWS on the Effects of DI
See Tables 6 and 7 and Fig. 3. In study 1 (Table 6), the bronchodilator effect of DI was reduced during induced bronchoconstriction with either CWSon/on or CWSoff/on, as suggested by similar increments in the slopes of RL post- vs. pre-DI (P < 0.001) and decrements in the intercept of max 60 vs. part 60 (P = 0.022 with CWSon/on, and P = 0.11 with CWSoff/on). The only difference observed between CWSon/on and CWSoff/on in response to DI was a faster recovery of Edyn with the former (P = 0.013) (Table 6 and Fig. 3).
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max 60 vs. part 60 (P = 0.0044), and a strong tendency for the slope of RL post- vs. pre-DI to increase (P = 0.058). However, the effect of DI was not significantly different between CWSon/off and CTRL conditions. The response to CWS of the asthmatic subject was similar to that of all other subjects.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Exaggerated Airway Narrowing
Examples of exaggerated airway responsiveness to constrictor agents in subjects breathing at low lung volume have been reported in the literature (8, 9, 35). Consistent with these observations, the present study demonstrated that MCh inhalation at reduced lung volume (CWSon/on) consistently worsened all indexes of intraparenchymal airway patency, except part 60. These changes were much greater than those observed when the MCh challenge was conducted at a normal lung volume (CTRL).
One possible cause for an increased bronchoconstrictor response during low lung volume breathing could be a different deposition of MCh within the airways. With CWS, VT tended to decrease and BF to increase, which might have caused preferential deposition of MCh in central airways. This would be consistent with the greater increase of RL observed with CWSon/on than at CTRL. However, it would not explain the remarkable constriction of the intraparenchymal airways distal to the flow-limiting segments, as reflected by the changes in maximum flows, Rus, Edyn, and RV. It is possible that, because of the reduced airway size at low lung volume, a greater amount of MCh was deposited per unit bronchial surface, thus reaching ASM cells in a greater concentration. This possibility cannot be excluded.
However, for the reasons discussed below, we favor the hypothesis that the increase in airway response with CWS was due to dynamic length adaptation of ASM at low lung volume. First, the tendency to reduce the amplitude of tidal breathing with CWS would tend to increase ASM tone (12) and thus exaggerated airway narrowing, were it not for the concomitant increase in BF, which is capable of offsetting in vitro the increase in bronchial tone that occurs when cyclic lengthening is reduced (36). More likely it could have been the plastic adaptation of the ASM to shorter length at low lung volume that worsened airway narrowing. An experimental in vitro study (37) provides evidence that, in tracheal smooth muscle stimulated at short length, the contractile filaments rearrange in a way that they best accommodate to that length and generate high pressure and tone. In contrast, when stimulated at long length and then suddenly shortened, the filaments are unable to adapt to the new length and thus generate less force. The results of the present study in humans are strikingly similar to those of Shen et al. (37) in animals. When challenged at 1 liter below natural FRC during CWSon/on, our subjects developed much greater airway narrowing than when challenged at their natural FRC (CTRL). In contrast, when challenged at their natural FRC and strapped soon after (CWSoff/on), they only developed airflow obstruction similar to that under CTRL conditions. This similarity to the results of Shen et al. suggests that, in humans, the magnitude of airway response to a constrictor agent strongly depends on the ASM length at which they are stimulated. This hypothesis is further supported by comparing the results of CWSon/off of study 2 with CWSoff/on of study 1. The fact that the CWSon/off was associated with greater response than at CTRL, but less than with CWSon/on, suggests that, to enhance airway narrowing, lung volume must be reduced when the airways are exposed to the constrictor agent, and that the longer the lung volume remains low before the exposure to the constrictor agent, the greater the response. Although it is not known how quickly ASM adapts to changes in lung volume in vivo, an in vitro study provides evidence that, after passive shortening, ASM recovers tension in an exponential fashion, with an increase in force of 30% over the first 3 min and is almost complete within 40 min (40). Thus, if ASM adaptation was the mechanism underlying the greater airflow obstruction observed when CWS was applied during inhalation of MCh, then its gradual tension recovery when FRC was reduced could explain the greater airway narrowing observed with CWSon/on compared with CWSon/off.
Reducing FRC with CWS before inhalation of MCh as in CWSoff/on did not increase airway response compared with CTRL conditions. These data are in keeping with the findings of Moore et al. (27), who showed that repeated deep expirations to RV taken before inhaling the constrictor agent do not affect the ensuing constriction. Based on the fact that tension recovery from short to high length in vitro is faster than in the opposite direction (40), we speculate that releasing the strapping for 3 min to inhale MCh was sufficient to allow ASM adaptation to a longer length and thus generate normal response to the active agent. More difficult to explain is the lack of effect on airflow obstruction after reapplying CWS 1 min after MCh inhalation was terminated. It is possible that, once initiated, airway narrowing becomes little sensitive to external modulation.
In humans, inhalation of bronchoconstrictor agents is known to result in a nonuniform airway constriction and closure, as suggested by studies based on functional imaging (6, 19, 28, 29), alveolar capsule techniques (11), and mathematical modeling (2, 13, 20). In the present study during CTRL conditions, Edyn, which is generally taken as an index of nonuniform distribution of airflow obstruction, increased with MCh to values similar to RL. In the presence of CWS, Edyn further increased, possibly suggesting that an extra number of airways tended to close with the chest wall constrained. A small increase in BF and decrease in VT with CWS could have also contributed to increase Edyn on the CWSon/on and CWSon/off days, although this seems unlikely, if we consider that similar changes in breathing pattern on the CWSoff/on day resulted in a significantly smaller increase in Edyn.
The observed increase in lung elastic recoil with CWS was not sufficient to counteract the constrictor effect of MCh. In dogs, nebulizing 50% MCh did not cause airway closure if Ptp values were above 10 cmH2O (16). In the present study, Ptp with CWS was significantly increased at 70% control TLC. Below that value, the Ptp-V curves gradually converged toward the same intercepts as at CTRL, as shown in the typical example of Fig. 2, and Ptp at the new, reduced FRC tended to decrease. Thus there are no reasons to believe that Ptp could modulate airway narrowing, because it actually decreased within the tidal breathing range with CWS. Previous studies (7, 39) documented increments of Ptp with CWS greater than in the present study, presumably due to the use of tighter corsets. However, as in the present study, the Ptp-V curves with and without CWS converged near RV, suggesting once again that, owing to the decrease in FRC, the increase in lung elastic recoil with CWS does not represent an important load for the airways during tidal breathing. Indeed, since there was no increase in the degree of constriction with MCh in the CWSoff/on compared with CTRL, this eliminates elastic recoil as a cause of the heightened constriction in the CWSon/on conditions.
Analysis of forced expiratory flows deserves consideration. First, imposing CWS before MCh inhalation caused an increase in max CF and part 60 but not max 60. In agreement with Fairshter et al. (10), this could be explained by a greater alveolar pressure developed by the expiratory muscles during maneuvers initiated from full inflation rather than at volumes below TLC. Second, differently from RL and/or Edyn, which further increased with MCh after CWS, part 60 remained surprisingly unchanged. The fact, however, that RVpart increased more on CWSon/on than on CTRL day suggests that, at lower lung volumes, even partial flow followed the same trend as RL and Edyn. Thus the unchanged max CF with CWS after MCh could have well been due to the higher Ptp than at lower lung volumes. Alternatively, flow interdependence across highly inhomogeneous obstructed regions helped flow to remain constant (24, 42).
Effects of DI on Bronchodilatation and Following Reconstriction
We examined the effects of DI by linearly regressing RL and Edyn against time, with the relevant values post-DI being the slope and the intercept at the time when DI ended. Our data reveal that, on both CWS days of study 1, the effects of DI on the bronchomotor tone were significantly less than on CTRL day. For flow, the intercept of the linear regression was significantly lower than at CTRL, thus suggesting that the bronchodilator effect with CWS was blunted both at baseline and after exposure to MCh. For RL, the slope increased, thus indicating that the dilator effect of DI was gradually lost with the intervening airway narrowing. For Edyn, the slope tended to increase, and this was only significant in study 2. Unable to compare our results with other data due to the absence of similar findings in the literature, we only offer tentative explanations. In humans exposed to a constrictor agent, a series of five breaths of different amplitude ranging from end-tidal inspiration to TLC have been shown to exhibit a variable bronchodilator effect in an amplitude-dependent manner (33). Thus it is legitimate to believe that part of the effects of DI lost with the CWS was due to a decrease in TLC. If this is the case, then the underlying mechanisms should be sought within the frame of the dynamic equilibrium of the contractile proteins of the ASM (12) and/or plastic adaptation internal to the ASM cells (15, 37). By plotting flow, RL, and Edyn pre- and post-DI at baseline and after the two doses of MCh, we were able to abolish the artifacts due to the extent of airway narrowing on the response to DI. Thus the relative inability of the airways to react to DI with CWS was unlikely related to the greater airway narrowing. We feel, however, that the amount of airway closure produced by CWS could have also contributed to blunt the bronchodilator effect of DI. Theoretically, this could be made possible because of the high pressure necessary to open fully closed or near closure rather than simply constricted airways (14). However, we do not have sufficient evidence to support this contention, for no direct relationships were observed between the slopes or intercepts of flow, RL, and Edyn and degree of increase in Edyn or decrease in FRC with CWS. Yet it must be acknowledged that the design of the study was not such as to allow drawing any conclusions on this issue.
Interestingly, Edyn after DI recovered significantly faster with CWSon/on than under other conditions. A similar pattern, although not significant, was observed for RL. In vivo, increased recovery of airway function parameters after DI has only been reported in patients with bronchial asthma (4, 30, 34) and in sheep after reducing lung volumes with a corset for 4 wk (23), which may be likely due to an increased velocity of ASM shortening (17). Our data would bring further evidence that the mechanisms for faster recovery of airway narrowing after DI mostly reside on ASM increased rate of shortening when the airways are stimulated or kept at short length after stimulation, even for brief periods of time. The precise mechanisms of such a potent activity of the internal contractile machinery remain to be elucidated.
Clinical Implications and Conclusions
Our experimental findings show that decreasing FRC and TLC with CWS can clearly enhance the response to a constrictor agent in normal humans when the agent is administered at the reduced FRC. Whether and to what extent such a mechanism explains the reported airway hyperresponsiveness in conditions characterized by chest wall restriction such as sleep (22), obesity (41), restrictive chest wall disorders (5), cervical spine cord injury (38), and late pregnancy (18) is a matter of speculation due to the complexity and diversity of these conditions. Furthermore, the results of this study in subjects with presumably normal ASM contracted with MCh cannot be directly extrapolated to bronchial asthma, where ASM muscle contractility may be different and inflammatory mediators are involved in the bronchospastic response. However, it is tempting to speculate that comorbidities associated with breathing at low lung volume may enhance broncoconstriction during natural asthma attacks.
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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