EFFECTS OF CHEST WALL STRAPPING ON MECHANICAL RESPONSE TO METHACHOLINE IN HUMANS

Transcription

1 Articles in PresS. J Appl Physiol (February 23, 2006). doi: /japplphysiol EFFECTS OF CHEST WALL STRAPPING ON MECHANICAL RESPONSE TO METHACHOLINE IN HUMANS Roberto Torchio, M.D. 1, Carlo Gulotta, M.D. 1, Claudio Ciacco, M.D. 1, Alberto Perboni, M.D. 1, Marco Guglielmo, M.D. 1, Flavio Crosa, M.D. 1, Mario Zerbini, M.D. 2, Vito Brusasco, M.D. 3, Robert E. Hyatt, M.D. 4, Riccardo Pellegrino, M.D. 5 From: 1 Pneumologia-Fisiopatologia Respiratoria, Azienda Ospedaliera S. Luigi, Orbassano (Torino), Italy; 2 Riabilitazione Cardiorespiratoria, Azienda Ospedaliera S. Luigi, Orbassano (Torino), Italy; 3 Fisiopatologia Respiratoria, Dipartimento di Medicina Interna, Università di Genova, Genova, Italy; 4 Department of Physiology and Biophysics, and Internal Medicine, Mayo Clinic and Mayo Foundation, Rochester, MN 55901, USA; and 5 Centro di Fisiopatologia Respiratoria e di Studio della Dispnea, Azienda Ospedaliera S. Croce e Carle, Cuneo, Italy. Address for correspondence: Riccardo Pellegrino, M.D. Centro di Fisiopatologia Respiratoria e dello Studio della Dispnea Azienda Ospedaliera S. Croce e Carle, Ospedale A. Carle Via Carle CONFRERIA-CUNEO (Italy) Phone: +39 (0171) ; Fax: +39 (0171) ; pellegrino.r@ospedale.cuneo.it Running title: Effects of chest wall strapping on airway responsiveness Copyright 2006 by the American Physiological Society.

2 2 ABSTRACT We examined the effects of chest wall strapping (CWS) on the response to inhaled methacholine (MCh) and the effects of deep inspiration (DI). Eight subjects were studied, one day with MCh inhaled without CWS (CTRL), one day with MCh inhaled during CWS (CWS on/on ), and one day with MCh inhaled during temporary removal of CWS (CWS off/on ). On the CWS on/on day, MCh caused greater increases in pulmonary resistance, upstream resistance, dynamic elastance, residual volume, and greater decreases in maximal expiratory flow than on the CTRL day. On the CWS off/on day, the changes in these parameters with MCh were not different from the CTRL day. Six of the subjects were again studied using the same protocol on CTRL and CWS on/on days, except that on a third day MCh was given after applying the CWS but the measurements before and after the inhalation were made without CWS (CWS on/off ). The latter sequence was associated with more severe airflow obstruction than during CTRL, but less than with CWS on/on. The bronchodilator effects of a DI were blunted when CWS was applied during measurements (CWS on/on and CWS off/on ) but not after it was removed (CWS on/off ). We conclude that CWS is capable of increasing airway responsiveness only when it is applied during the inhalation of the constrictor agent. We speculate that breathing at low lung volumes induced by CWS enhances airway narrowing because the airway smooth muscle is adapted at a length at which the contractile apparatus is able to generate a force greater than normal. Key words: Bronchial challenge, mechanics of breathing, lung volumes, airway caliber, deep inhalation, airway smooth muscle.

3 3 INTRODUCTION Airway hyperresponsiveness is regarded as a characteristic feature of bronchial asthma and, to a lesser extent, of chronic obstructive pulmonary disease. However, it has been also reported in neuromuscular disorders (38), idiopathic scoliosis (4), obesity (41), sleep (22), recumbent position (35), and late pregnancy (18). All these non-asthmatic conditions have in common a decrease in lung volume. That breathing at low lung volume may be sufficient to increase airway responsiveness was first demonstrated by Ding et al (9). 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 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 (CWS on/on ), and a third one with MCh inhaled during temporary removal of CWS with measurements made after CWS was reapplied (CWS off/on ). We hypothesized that

4 4 any increased responsiveness in the CWS off/on challenge compared to the CTRL would be consistent with a prevalent role of lung elastic recoil in determining airway responsiveness. However, a difference between CWS on/on and CWS off/on challenges would suggest an effect of the length at which ASM was stimulated. Because the bronchoconstrictor response was increased with CWS on/on but not CWS off/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 re-challenging a subgroup of subjects with CWS during MCh inhalation and removing CWS before and after making the measurements (CWS on/off ). An elevated responsiveness in the CWS on/off sequence would support the importance of the length at which the ASM was stimulated. METHODS Subjects 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, one reported a history of asthma, with no symptoms in the last 6 years. Study protocol On a pre-study day, spirometry was obtained according to the ATS 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 1.61µm. Aerosols were inhaled during quiet tidal breathing in a sitting position. The doses of MCh causing a 10% and 20% decrease of FEV 1 (PD 10 and PD 20, respectively) were

5 5 calculated by linear interpolation between two adjacent points of the (log)dose-response curve. Study 1. Eight subjects attended the laboratory on three random study days to undergo different bronchial challenges. The sequence of interventions and measurements is shown in figure 1. On all days, two MCh doses were used, one equal to PD 10 and the other to PD 20. The inhalation time for both doses was of about 60 s. On one 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 (CWS on/on ). On a another day the procedure was similar to the CWS on/on occasion with the only difference that the corset was temporarily removed for about 3 min to allow MCh inhalation (CWS off/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 Figure 1. Study 2. Six subjects were re-challenged on three days with the same MCh doses of study 1. On two occasions the tests were conducted with and without CWS as in study 1 (CWS on/on and CTRL, respectively). On a third occasion, the corset was applied 1 min before inhaling MCh and removed 1 min after (CWS on/off ). The measurements of lung function before and after MCh were collected without CWS. The study days were conducted in random order with number and order of maneuvers exactly as in study 1 (Figure 1). Definitions of abbreviations of the protocols are reported in table 2. Lung function measurements Mouth flow was measured by a mass flow-meter (SensorMedics Inc., Yorba Linda, CA) and volume was obtained by numerical integration of the flow signal. Spirometry and

6 6 flow-volume curves were obtained in a body plethysmograph (Autobox, SensorMedics Inc., Yorba Linda, CA) as follows. After 6-8 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 about 70% of their forced vital capacity (FVC), as measured in the pre-study 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 (V & max) and partial (V & part) flowvolume loops. In addition, compression-free forced expiratory flow (V & max CF ) was also obtained by plotting mouth flow against plethysmographic volume (32). This allowed 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 endexpiratory 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 cm H 2 0). Mouth pressure (Pmo) was measured by a catheter connecting the mouthpiece to a piezoelectric pressure transducer (Microswich, ±200 cm H 2 0). Ptp was the difference between Pmo and

7 7 Pes. Placement of the balloon was considered correct if the changes in Pes and Pmo 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 (R L ) and dynamic elastance (Edyn) were measured by a DIREC TM System 200/201 (Raytech Instruments Inc., Vancouver, Canada). Flow was measured by a Hans Rudolph pneumotachograph connected to a full-scale differential pressure transducer (±5 cm H 2 O, flow range: L/min, Validyne). Pes and Pmo were sensed by two DP15 Validyne differential pressure transducers (±150 cm H 2 O). Flow, volume, and pressure signals were fed into dedicated software (DR9, Raytech Instruments Inc., Vancouver, Canada) and then processed to calculate R L and Edyn with the aid of a program written in SCILAB 3.0 (INRIA and ENPC, France). 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 R L (26). Edyn was the difference in Ptp at zero flow between end-inspiration and end-expiration divided by tidal volume (V T ). 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 R L and Edyn before and after DI, and at least three quasi static Ptp-V curves (figure 1). After each MCh dose, one set of maximal and partial maneuvers, one set of R L 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 R L, Edyn, and V part 60 were minimized. The interval between maneuvers was always 2 min.

8 8 Data reduction and statistical analysis R L 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), R L 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 V max CF at 60% control TLC (25). The bronchomotor effects of DI before and after MCh were assessed by linearly regressing the values of V max 60 vs V part 60, and those of R L and Edyn pre- vs post-di (30). In this analysis, an increase in slope or decrease of intercept of V max 60 vs V part 60, or an increase in slope or intercept of R L or Edyn pre- vs post-di would suggest an impaired bronchodilator effect of DI. A mixed between-within groups analysis of variance (ANOVA) with Duncan post-hoc comparisons was used. Values of p<0.05 were considered statistically significant. Data are presented as mean (SD). RESULTS Baseline lung function was within the normal limits in all subjects except in the one with past asthma in whom FEV 1 /FVC was Baseline function did not change between study days. Taking a DI had a small yet significant (p=0.049) bronchodilator effect when assessed as V max 60 /V part 60 (Table 1), but not by R L and Edyn. These data are from study 1.

9 9 Effects of CWS on lung function before MCh (Table 3) Compared to CTRL, CWS caused consistent reductions of FVC (p<0.001), FEV 1 (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 V & 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±1.9 cm H 2 O vs. 4.3±2.3 cm H 2 O, 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 figure 2. V max 60 did not change with CWS, while V part 60 increased significantly (p=0.03). No significant changes were observed in R L, Edyn, Rus or breathing pattern. We interpret the lack of changes in R L with decreasing FRC as a result of a decrease in tissue resistance offsetting the increase in airway resistance. Effects of CWS on response to MCh (Tables 4 and 5) Results obtained with PD 10 and PD 20 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. In study 1 when CWS was applied during both inhalation of MCh and lung function measurements (CWS on/on ), the airflow obstruction was worse than at CTRL, as documented by greater increments in RV (p=0.003), RVpart (p=0.01), Edyn (p<0.001), R L (p<0.001), and Rus (p =0.017), and by greater decrement in V max 60 (p=0.017), while V 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). Tidal volume (V T ) tended to decrease and breathing frequency (BF) to increase (p=0.054).

10 10 In study 1 when CWS was applied before and after but not during inhalation of MCh (CWS off/on ), airway narrowing was less than with CWS on/on and not different from CTRL, as indicated by no change in RV (p= 0.97), RVpart (p= 0.97), Edyn (p=0.38), R L (p=0.27), and V max 60 (p=0.99) (Table 4). In study 2 (Table 5) when CWS was applied during inhalation of MCh but not during measurements (CWS on/off ), airflow obstruction was more severe than during CTRL for V max 70 (p=0.016), RV (p=0.035), and Edyn (p=0.039), but not for R L. However, the changes from CTRL were less than with CWS on/on for RV (p=0.019), Edyn (p=0.0002), and R L (p=0.005). In summary, the bronchoconstriction in study 2 with CWS on/off was significantly greater than at CTRL as reflected by changes in RV, V max 70, and Edyn, but less than CWS on/on. Effects of CWS on the effects of DI (Tables 6 and 7, Figure 3) In study 1 (Table 6), the bronchodilator effect of DI was reduced during induced bronchoconstriction with either CWS on/on or CWS off/on as suggested by similar increments in the slopes of R L post- vs pre-di (p<0.001) and decrements in the intercept of V max 60 vs V part 60 (p=0.022 with CWS on/on, and p=0.11 with CWS off/on ). The only difference observed between CWS on/on and CWS off/on in response to DI was a faster recovery of Edyn with the former (p=0.013) (Table 6 and Figure 3). In study 2 (Table 7), the bronchodilator effect of DI was also reduced during induced bronchoconstriction with CWS on/on, as suggested by an increment of the slope of Edyn postvs pre-di (p=0.011), a decrement of the intercept of V & max 60 vs V & part 60 (p=0.0044), and a strong tendency for the slope of R L post- vs pre-di to increase (p=0.058). However, the effect of DI was not significantly different between CWS on/off and CTRL conditions. The response to CWS of the asthmatic subject was similar to all other subjects.

11 11 DISCUSSION This study was designed to determine whether decreasing lung volume with CWS altered the response of airways to a bronchoconstrictor agent in healthy humans. We found that, despite a significant increase in lung elastic recoil above control FRC, CWS was associated with consistent and exaggerated bronchoconstriction. However, this occurred only when there was a significant decrease in the operative lung volume at the time the MCh was inhaled. CWS also impaired the ability of DI to decrease the bronchomotor tone, thus contributing to increase airway narrowing. 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 (CWS on/on ) consistently worsened all indexes of intraparenchymal airway patency, except V & 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 tidal volume tended to decrease and breathing frequency to increase, which might have caused preferential deposition of MCh in central airways. This would be consistent with the greater increase of R L observed with CWS on/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

12 12 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 breathing frequency, 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. in animals (37). When challenged at about 1 L below natural FRC during CWS on/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 (CWS off/on ), they only developed airflow obstruction similar to that of under CTRL conditions. This similarity to the results of Shen et al. (37) suggests that in man 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 CWS on/off of study 2 with CWS off/on of study 1. The fact that the CWS on/off was associated with greater response than at CTRL, but less than with CWS on/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

13 13 constrictor agent, the greater the response. Though 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 about 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 CWS on/on compared to CWS on/off. Reducing FRC with CWS before inhalation of MCh as in CWS off/on did not increase airway response compared to CTRL conditions. These data are in keeping with the findings of Moore et al (27), who showed that repeated deep expirations to residual volume 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 non-uniform airway constriction and closure, as suggested by studies based on functional imaging (5, 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 R L. 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 breathing

14 14 frequency and decrease in V T with CWS could have also contributed to increase Edyn on the CWS on/on and CWS on/off days, though this seems unlikely if we consider that similar changes in breathing pattern on the CWS off/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 transpulmonary pressures was above 10 cm H 2 O (16). In the present study, Ptp with CWS was significantly increased at 70% control TLC. Below that value, the Ptp-V curves gradually converged towards the same intercepts as at CTRL, as shown in the typical example of figure 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 CWS off/on compared to CTRL, this eliminates elastic recoil as a cause of the heightened constriction in the CWS on/on conditions. Analysis of forced expiratory flows deserves consideration. First, imposing CWS prior to MCh inhalation caused an increase in V max CF and V part 60 but not V 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 R L and/or Edyn, which further increased with MCh after CWS, V & part 60 remained surprisingly unchanged. The fact, however, that RVpart increased more on CWS on/on than on CTRL day suggests that at lower lung volumes even partial flow

15 15 followed the same trend as R L and Edyn. Thus, the unchanged V & max CF70 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 re-constriction We examined the effects of DI by linearly regressing R L 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 R L, 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, R L, 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

16 16 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, R L, 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 CWS on/on than under other conditions. A similar pattern, though not significant, was observed for R L. In vivo, increased recovery of airway function parameters after DI has only been reported in patients with bronchial asthma (6, 30, 34) and in sheep after reducing lung volumes with a corset for 4 weeks (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 (4), cervical spine cord injury (38), and late

17 17 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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23 23 Table 1. Subjects main anthropometric and functional data of study 1. Subjects, n 8 Age, yr 42 (6) Height, cm 176 (7) BMI, Kg m (4) FEV 1, % of predicted 109 (10) FVC, % of predicted 115 (11) TLC, % of predicted 105 (13) FRC, % of predicted 110 (28) RV, % of predicted 101 (25) V & max 60 /V & part 60, % 114 (16) * R L post-di/pre-di, % 105 (29) Edyn post-di/pre-di, % 88 (19) BMI, body mass index; FEV 1, forced expiratory volume in 1 s; FVC, forced vital capacity; TLC, total lung capacity; FRC, functional residual capacity; RV, residual volume; V & max 60 and V & part 60, maximal to partial flow ratio at 60% of TLC, respectively; R L, pulmonary resistance; Edyn, lung dynamic elastance. Predicted values are from Quanjer et al. (31). Data are mean (SD). *, significantly different from 1.00 (p =0.049).

24 24 Table 2. Definitions of abbreviations of the protocols. Protocol MCh Measurements CTRL No CWS No CWS CWS on/on CWS CWS CWS off/on No CWS CWS CWS on/off CWS No CWS CWS, chest wall strapping; MCh, methacholine; CTRL, control day without CWS; CWS on/on, day with CWS applied during MCh inhalation and lung function measurements; CWS off/on, day with CWS applied before and after but not during MCh inhalation; CWS on/off, day with CWS applied only during MCh inhalation but not during measurements.

25 25 Table 3. Spirometry and lung volume data on each day of study 1 before methacholine. CTRL CWS on/on CWS off/on P (ANOVA) FVC, L 5.42 (0.63) 4.68 (0.65) 4.59 (0.65) <0.001 FEV 1, L 4.19 (0.51) 3.55 (0.45) 3.49 (0.45) <0.001 TLC, L 7.33 (0.99) 6.50 (0.92) 6.42 (0.86) <0.001 FRC, L 3.77 (1.08) 2.68 (0.64) 2.66 (0.61) <0.001 RV, L 1.89 (0.67) 1.82 (0.49) 1.83 (0.43) 0.18 Abbreviations as in tables 1 and 2. Data are mean (SD), n= 8.

26 26 Table 4. Lung mechanics on each study day of study 1 before and after methacholine (MCh). RV, L RVpart, L Ptp, cm H 2 O V & max CF, L s -1 V & max 60, L s -1 V & part 60, L s -1 R L, cm H 2 O L -1 s Rus, cm H 2 O L -1 s Edyn, cm H 2 O L -1 BF, min -1 V T, L Condition Baseline MCh PD 20 CTRL CWS on/on CWS off/on CTRL CWS on/on CWS off/on CTRL CWS on/on CWS off/on CTRL CWS on/on CWS off/on CTRL CWS on/on CWS off/on CTRL CWS on/on CWS off/on CTRL CWS on/on CWS off/on CTRL CWS on/on CWS off/on CTRL CWS on/on CWS off/on CTRL CWS on/on CWS off/on CTRL CWS on/on CWS off/on 1.89 (0.67) 1.82 (0.49) 1.83 (0.43) 2.05 (0.61) 1.93 (0.46) 1.95 (0.40) 7.6 (2.1)* 10.8 (3.1)*,# 9.8 (2.8)# 7.92 (2.61)* 8.31 (1.73)* 8.20 (1.66) 3.86 (1.45) 3.81 (1.23) 3.89 (1.00) 3.54 (1.04)* 4.06 (0.86)* 4.28 (0.81) 2.20 (0.66) 2.90 (1.31) 2.33 (0.50) 1.78 (0.70) 1.46 (0.93) 1.19 (0.38) 4.44 (1.09) 5.59 (2.17) 5.17 (1.47) 12.5 (2.9) 15.1 (1.8) 14.6 (3.7) 0.72 (0.06) 0.61 (0.09) 0.68 (0.08) 2.46 (0.80)* 3.09 (0.72)* 2.65 (0.80) 3.07 (0.93)* 3.50 (0.91)* 2.89 (0.91) 7.5 (2.2)#* 10.5 (2.3)# 11.4 (2.8)* 4.55 (1.92) 3.69 (2.19)* 5.25 (2.55)* 1.80 (0.94)* 1.01 (0.50)* 1.58 (0.88) 0.87 (0.57)* 0.85 (0.78) 1.51 (0.96)* 8.46±4.35* 12.99±3.80* 8.43± (2.53)* 6.06 (4.46)* 4.25 (3.20) 8.89 (2.64)* (4.06)* (4.69) 12.0 (3.0)* 16.7 (2.5) 18.2 (5.1)* 0.74 (0.13) 0.65 (0.11) 0.67 (0.10) P (ANOVA) Conditions MCh 0.18 < < < < <0.001 <0.001 < <0.001 < RV and RVpart, residual volumes achieved after a maximal and partial forced expiratory maneuver, respectively; Ptp and V & max CF, transpulmonary pressure and maximum expiratory flow corrected for gas compression at 70% of control TLC, respectively; Rus, upstream lung resistance; BF, breathing frequency; V T, tidal volume. Other abbreviations as in tables 1 and 2. Data are mean (SD), n=8. Pairs of symbols indicate statistically significant differences by post-hoc analysis (see text for p values).

27 27 Table 5. Lung mechanics on each day of study 2 before and after methacholine (MCh). RV, L V max 70, L s -1 R L, cm H 2 O L -1 s Edyn, cm H 2 O L -1 Condition Baseline MCh PD 20 CTRL CWS on/on CWS on/off CTRL CWS on/on CWS on/off CTRL CWS on/on CWS on/off CTRL CWS on/on CWS on/off 2.23 (0.68) 2.14 (0.61) 2.24 (0.56) 5.18 (1.67) 5.69 (1.93) 5.08 (1.21) 2.57 (0.61) 3.03 (1.25) 2.67 (1.12) 3.87 (0.95) 4.71 (1.77) 3.75 (0.85) 2.71 (0.79)* 3.38 (0.78)* 3.02 (0.74) 3.30 (1.31)* 1.86 (0.43)* 1.89 (1.12) 7.08 (3.36)* 13.7 (5.49)* 8.50 (3.89) 7.02 (1.40)* (5.23)* (1.43) + P (ANOVA) Conditions MCh 0.76 < < < <0.001 Abbreviations as in tables 1 and 2. Data are mea (SD); n=6.

28 28 Table 6. Effects of deep inhalation on lung mechanics on each day of study 1. CTRL CWS on/on CWS off/on ANOVA V max 60 Intercept 1.18 (0.70)* (1.50)* 0.36 (0.38) vs. V part 60 Slope 0.74 (0.22) 0.93 (0.43) 0.82 (0.18) 0.28 R L post-di Intercept 2.06 (0.68) 1.29 (1.06) 1.24 (1.28) 0.23 Vs. R L pre-di Slope 0.04 (0.24)* 0.53 (0.22)* 0.69 (0.24) <0.001 Edyn post-di Vs. Intercept 3.41 (1.27) 2.69 (2.66) 3.05 (3.43) 0.86 Edyn pre-di Slope 0.13 (0.24) 0.35 (0.23) 0.39 (0.48) 0.29 R L recovery post-di, cm H 2 O/L Edyn recovery post-di, cm H 2 O/L/s (0.035) (0.069) (0.034) (0.022)* (0.076)* (0.016) Abbreviations as in tables 1 and 2. Slopes and intercepts were obtained by linear regression analysis. Data are mean (SD), n=8. Pairs of symbols indicate statistically significant differences by post-hoc analysis (see text for p values).

29 29 Table 7. Effects of deep inhalation on lung mechanics on each day of study 2. CTRL CWS on/on CWS on/off ANOVA V max 50 Intercept 0.80 (0.50)* 0.11 (0.15)* 0.40 (0.29) vs. V part 50 Slope 0.62 (0.20) 0.72 (0.22) 0.75 (0.10) 0.40 R L post-di vs. R L pre-di Intercept Slope 1.82 (0.85) 0.23 (0.86) 1.45 (0.75) 0.86 (0.74) 2.28 (0.50) 0.18 (0.15) Edyn post-di vs. Intercept 3.25 (1.65) 1.83 (3.13) 2.87 (1.51) Edyn pre-di Slope 0.06 (0.26)* 0.50 (0.33)* 0.13 (0.16) R L recovery post-di, cm (0.029) (0.050) (0.039) H 2 O/L Edyn recovery post-di, cm H 2 O/L/s (0.012) (0.037) (0.013) Abbreviations as in table 6. Data are mean (SD), n=6.

30 30 Figure 1. Protocol of the study. CTRL, control day with no chest wall strapping; CWS on/on, day with chest wall strapping (CWS) applied during both MCh inhalation and lung function measurements; CWS off/on, day with chest wall strapping applied before and after but not during methacholine (MCh) inhalation (study 1); CWS on/off, day with chest wall strapping applied only during MCh inhalation (study 2); M1, partial and maximal flow-volume loops; M2, lung resistance and dynamic resistance before and after a deep breath; M3, quasi-static lung compliance; PD 10 and PD 20, doses of MCh that caused a decrease in FEV 1 by 10 and 20% of control. Arrows indicate the time at which the measurements were taken.

31 31 Figure 2. Quasi-static transpulmonary pressure(ptp)-volume curves at baseline in a representative subject during the three study days (CTRL, CWS on/on, and CWS off/on ). Note the increase in Ptp at most lung volumes and the decrease in FRC with CWS (arrows).

32 32 Figure 3. Average data for the 8 subjects of study 1 of the time course of pulmonary resistance (R L ) and dynamic elastance (Edyn) before and after a deep inspiration (DI) at baseline (upper panels) and during methacholine (MCh)-induced bronchoconstriction (lower panels). The horizontal lines before DI are the average values of the preceding regular tidal breaths, the oblique lines after DI are the average regression slopes. CTRL, CWS on/on, and CWS offon denote study days as in figure 1.