Differences in the pattern of bronchoconstriction induced by intravenous and inhaled methacholine in rabbit

Respiratory Physiology & Neurobiology 189 (2013) 465–472

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Differences in the pattern of bronchoconstriction induced by intravenous and inhaled methacholine in rabbit Satu Strengell a,∗ , Liisa Porra a , Anssi Sovijärvi b , Heikki Suhonen c , Pekka Suortti a , Sam Bayat d a

Department of Physics, University of Helsinki, Finland Department of Clinical Physiology and Nuclear Medicine, Helsinki University Central Hospital, Finland c European Synchrotron Radiation Facility, Grenoble, France d Université de Picardie Jules Verne, Inserm U1105 – GRAMFC Laboratory and Amiens University Hospital, France b

a r t i c l e

i n f o

Article history: Accepted 28 August 2013 Keywords: Asthma Bronchial responsiveness Computed tomography Regional lung ventilation Xenon Synchrotron

a b s t r a c t We measured bronchoconstriction in central bronchi, and in small peripheral airways causing the emergence of ventilation defects (VD), through two delivery routes: intravenous (IV) and inhaled MCh, in 2 groups of rabbits (A: n = 5; B: n = 4), using synchrotron imaging of regional lung structure and ventilation. We assessed the effect an initial IV challenge on a subsequent inhaled challenge in group B. Inhaled MCh decreased central airway cross-sections (CA) by 13–22%, but increased VD area by 25–49%. IV MCh decreased CA by 44% but increased the area of ventilation defects (VD) by 13% only. An initial IV MCh challenge reduced regional ventilation heterogeneity following a subsequent inhaled MCh challenge, suggesting the role of agonist–receptor interaction in the response pattern. Heterogeneous agonist distribution due to uneven aerosol deposition could explain the different patterns of response between IV and inhaled routes. This mechanism could participate in the emergence of ventilation heterogeneities during bronchial challenge, or exposure to allergen in asthmatic patients. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Inhaled methacholine (MCh) is commonly used to assess bronchial hyperresponsiveness (BHR) for the diagnosis of asthma (Juniper et al., 1981; Crapo et al., 2000). This is usually accomplished by measuring the overall airway response by spirometry, following MCh aerosol inhalation. The most common variable used to assess airway responsiveness in the clinical setting is forced expiratory volume in one second, which mainly reflects changes in central bronchi, but is rather insensitive to small airway constriction and the spatial heterogeneity of bronchoconstriction. However, bronchial smooth muscle constriction shows substantial regional heterogeneity following airway challenge in asthmatic patients, resulting in a heterogeneous regional ventilation distribution. The spatial heterogeneity of regional ventilation in response to airway challenge, has been demonstrated in human asthmatics by positron emission tomography (PET) (Harris et al., 2006) and magnetic resonance imaging (MRI) using hyperpolarized 3 He (Costella et al., 2012; Samee et al., 2003). Few preclinical studies have

∗ Corresponding author at: Department of Physics, Gustaf Hällströmin katu 2a, PL 64, 00014 Helsingin yliopisto, Finland. Tel.: +358 9 191 506833; fax: +358 9 191 50639; mobile: +358 50 3639128. E-mail address: satu.strengell@helsinki.fi (S. Strengell). 1569-9048/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.resp.2013.08.024

assessed the changes in regional ventilation distribution caused by MCh inhalation, using functional imaging techniques. Bronchoconstriction induced by MCh inhalation has been shown to produce clustered ventilation defects and a significant ventilation heterogeneity in sheep by PET imaging (Vidal Melo et al., 2005), in rabbit, using synchrotron imaging (Bayat et al., 2009a), and in mouse, using MRI with hyperpolarized 3 He (Thomas et al., 2012). Data both from these experimental studies and previous human studies suggest that clustered areas of poor regional ventilation, or “ventilation defects” (VD) arise from collective constriction or closure of peripheral airways. The mechanisms of the emergence of such ventilation defects remain poorly understood, despite their crucial importance not only for lung mechanics and gas exchange, but also for their potential impact on the distribution of inhaled medications. Theoretical studies based on integrative computational models of the entire human bronchial tree, suggest that ventilation defects occur beyond a critical level of airway smooth muscle constriction, even with a homogenous activation of the airway smooth muscle (Venegas et al., 2005; Winkler and Venegas, 2012). These studies have suggested that the uneven distribution of the constricting agonist may not be an essential mechanism in the emergence of peripheral ventilation defects. In a previous study in rabbit, we observed that the route of constricting agonist delivery changes the pattern of airway response distribution, despite similar overall respiratory mechanical changes (Bayat et al., 2009a). In that study,

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Fig. 1. Principles and experimental setup of K-edge subtraction imaging. At the Xe K-shell absorption edge (EK ), the attenuation coefficient of Xe increases by a factor of 5.4, while the changes in the attenuation coefficients of other elements remain negligible. The absorption CT images below and above the K-absorption edge show the anatomical structures. The logarithmic difference of these images yields the Xe density on an absolute scale, while features due to other structures are removed. The quantitative density due to either tissue or to Xe can therefore be calculated separately in each voxel, which provides both structural and functional data in the same set of images.

the inhaled route of bronchial challenge produced a significantly more heterogeneous distribution of regional ventilation with dramatically larger ventilation defects. We have recently found that aerosol particles of identical aerodynamic diameter than in our previous study, show significant deposition heterogeneity in rabbit (Bayat et al., 2011). These observations suggested that uneven deposition of the inhaled particles carrying a constricting agonist could substantially modify the spatial distribution of bronchoconstriction. This hypothesis is significant in the clinical setting, since uneven agonist distribution could contribute to the emergence of ventilation defects during bronchial challenge, or exposure to irritants or allergen, in patients. In the present study, we tested the hypothesis that the distribution of the constricting agonist plays a role in the emergence of ventilation defects. To this end, we compared the magnitude of bronchoconstriction in central bronchi of different sizes, and in small peripheral airways causing the emergence of ventilation defects, through two routes of MCh challenge: intravenous (IV) infusion and inhaled. To assess whether the observed patterns of response to MCh are specifically determined by agonist–receptor interactions, we compared the patterns of central and peripheral airway response within the same subject, through subsequent challenges through the infused and inhaled routes. A reduced patchiness of regional ventilation, following an initial IV infusion challenge, or tolerance to MCh, would be in favor of this hypothesis.

2. Methods Animal care and procedures of the experiment were in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Research Council and approved by the local institutional authorities. The experiments were performed on nine male New Zealand rabbits (average weight: 2.5 ± 0.1 kg; Elevage Scientifique des Dombes, Chatillon sur Chalaronne, France).

2.1. Animal preparation A catheter was inserted in the marginal ear vein (22-gauge, Cathlon IV, Ethicon, Rome, Italy) after local anesthesia using 5% topical lidocaine (Emla, Astra-Zeneca, Rueil, France). Anesthesia was induced by IV injection of thiopental sodium (25 mg/kg IV, Nesdonal, Rhone-Poulenc-Rohrer, Paris, France). The animal was tracheostomized, and an endotracheal tube (no. 3.5, Portex, Berck sur Mer, France) was inserted and secured with a gas-tight seal. A catheter (22-gauge) was inserted into the left carotid artery for blood pressure monitoring and arterial blood sampling for blood gas analysis (Radiometer model 77, Acid Base Laboratory, Copenhagen, Denmark). The lower extremities were wrapped with bandage, and the animal was immobilized in a custom-made plastic holder in the vertical position. The chest wall and diaphragmatic motion were free of constraint. The forelimbs were fixed to the holder to keep them out of the image field and the lower limbs were securely maintained in the holder using foam. Anesthesia was maintained by inhaled isoflurane (0.5–1.5%, Forene, Abbott, Paris, France). Paralysis was induced by IV vecuronium bromide (1.0 mg/h, Norcuron, Organon, Puteaux, France). A custom-made mechanical ventilator was used (Monfraix et al., 2005). The animals were ventilated in pressure-control mode, with a tidal volume (VT) of 7 ml/kg, and a respiratory rate adjusted in order to maintain a PaCO2 close to 40 mmHg, as monitored by arterial blood gas analysis. Mean end-expiratory pressure was 1.5 cmH2 O. Gas flows were measured continuously and recorded using mass flow meters (Aalborg, Orangeburg, NY). During imaging, the inhaled gas mixture was automatically switched to xenon (Xe, 70%) and oxygen (O2 , 30%) using electromagnetic valves controlled by the image acquisition software. Airway pressure (Prs ) was monitored continuously. Ventilatory gas flows were measured using a heated pneumotachometer (Hans Rudolph, Kansas City, MO). All monitored signals were amplified, digitized at 1000 Hz (PowerLab, ADInstruments, Oxfordshire, UK), and recorded on a computer. At the end of the experiment, the

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animals were euthanized by IV anesthetic overdose (Dolethal, 5 ml, Vetoquinol, Lure, France). 2.2. K-edge subtraction imaging A detailed description of the K-edge subtraction (KES) imaging has been published previously (Bayat et al., 2001; Porra et al., 2004; Suhonen et al., 2008). In this technique, two images are simultaneously obtained using two beams of synchrotron radiation, focused on the animal and tuned to two different energies, above and below the Xe K-absorption edge (34.56 keV). Above the K-edge, the attenuation coefficient of Xe increases by a factor of 5.4, while the change in the attenuation coefficients of other elements is negligible. Using the dual energy KES imaging method, the quantitative density due to either tissue or to Xe can be calculated separately in each voxel in the image using a specifically developed computer algorithm (Elleaume et al., 2002). An example of a “Xe-density” image is shown in Fig. 1. X-rays from a synchrotron radiation source are required since, as opposed to standard X-ray sources, they allow the selection of monochromatic beams from the full X-ray spectrum while conserving enough intensity for imaging with sufficient temporal resolution. The experiments were performed at the Bio-Medical beamline ID17 of the European Synchrotron Radiation Facility (ESRF, Grenoble, France). The experimental setup is described in detail elsewhere (Elleaume et al., 1999; Bayat et al., 2006). Briefly, two mono-energetic beams with an energy difference of 250 eV were produced from the continuous synchrotron radiation spectrum by a bent silicon crystal. A liquid nitrogen-cooled high-purity germanium dual-line detector with pixel width of 0.35 mm (Eurisys Measure, Lingolsheim, France) was used. The horizontal beam width was 150 mm, and the vertical beam height 0.7 mm. Quantitative Xe and tissue density images were reconstructed with the filtered-back-projection algorithm, using the Interactive Data Language (IDL, RSI, Boulogne-Billancourt, France). 2.3. Experiment protocol The study protocol is shown in Fig. 2. Each animal served as its own control. In group A (n = 5) baseline imaging was followed by

Fig. 2. Experimental protocol. Protocol A (n = 5): the baseline imaging at 3 axial levels, was followed by airway challenge with inhaled MCh (Ainhaled ). Protocol B (n = 4): baseline imaging at 3 axial levels, was followed by MCh infusion at 10 ␮g/kg/min (Biv and a recovery period of 45–105 min. After full recovery, airway provocation was repeated with inhaled MCh (Binhaled ). Gray shading indicates the MCh challenge periods.

inhaled MCh and immediate imaging thereafter (Ainhaled ). In group B (n = 4) baseline imaging was followed by IV infusion of MCh (Biv ), followed by a recovery period. In order to assess the effect of an initial MCh challenge on a subsequent challenge in the same subject in protocol B, an inhaled MCh challenge (Binhaled ) was performed after full recovery from the previous IV challenge. Criteria for appropriate recovery were the return of endotracheal pressure and blood pressure to the baseline level. Methacholine was infused via the peripheral ear vein catheter at 40 ␮g/kg/min, using an infusion pump (Fresenius Vial, Pilot A2, Paris, France). Three minutes were allowed for the effect of infused MCh to stabilize. Image acquisition during MCh infusion lasted 14 ± 7 min (Biv ). Inhaled MCh (125 mg/ml) was administered during 60 s using an ultrasonic nebulizer (SAM LS2000, Villeneuve-sur-Lot, France), via the endotracheal tube. Images were taken approximately one minute after the inhalation of MCh in both Ainhaled and Binhaled . The mass median aerodynamic diameter (MMAD) of the aerosol particles was 3.5 ␮m with a geometrical standard deviation of 2.0 ␮m, as determined by laser optical diffraction (Malvern, Mastersizer X, Worcestershire, UK). The doses of MCh through both routes were chosen to produce similar effects on the respiratory system resistance (Rrs ). In order to compare the response to MCh in central airways of different sizes, imaging was performed at three axial slice levels selected at the fourth (apical), sixth (middle), and eighth (caudal) thoracic vertebral levels, based on an initial scout projection image.

Fig. 3. Definitions of ventilated alveolar area (VAA) and ventilation defect (VD), including poorly or non-ventilated regions. Left: baseline, right: after inhaled MCh. Upper panels: Xe density in the apical level in one representative animal; lower panels: the corresponding distributions of Xe density, within the same slice. A log-normal function was fitted to the histogram of high Xe density values, and the portion above the median () minus 2SD threshold was defined as VAA. Voxels with values below this Xe concentration threshold represent ventilation defects (VD). After inhaled MCh the VD was markedly increased (right panel).

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Fig. 4. CT images from two animals at 3 axial levels (apical, middle, caudal). Left: protocol A; right: protocol B. Tissue-density images are shown in the upper panels. The corresponding Xe-density images are shown in the lower panels; square indicates a main bronchus; upper left corner: 3× magnification of the bronchus.

Due to the monopodial structure of the bronchial tree in rabbit, the internal diameters of the 2 main bronchi (left and right) decrease as the image plane evolves from the apical to the caudal end. The CT images were acquired at end-expiration during an apnea of 4 s. The duration of an imaging sequence from the switch to the Xe–O2 gas mixture to the switch back to air was approximately 20 s. The same sequence of image acquisition was repeated at baseline, during IV MCh challenge, upon recovery and following inhaled MCh challenge.

of Xe density values was calculated for each Xe-image, and fitted with a log-normal function. The ventilated alveolar area (VAA) was defined as the area where the Xe density was higher than the median of the fitted distribution minus two standard deviations: median-2SD (Porra et al., 2011). The remaining lung regions were defined as ventilation defects (VD). These regions include both poorly ventilated and non-ventilated areas. Within-subject heterogeneity of ventilation was calculated from the coefficient of variation of Xe density (CV). Fig. 3 demonstrates typical histograms of Xe density.

2.4. Image analysis 2.5. Respiratory mechanical parameters The reconstructed CT-images were processed using the MatLab programming package (Mathworks Inc., MI, USA). The crosssectional areas (CA) of the two largest bronchi in each image slice were calculated by fitting an ellipse to the bronchus observed in the tissue-density image. The ellipse fit was performed semiautomatically using specific software developed for this purpose. The minor axis of the ellipse was used to compute the radius of the bronchus. The lung was selected within the tissue-density CT images, using a Region Growing segmentation algorithm. Xedensity images were smoothed with a 5 × 5 pixel moving average. In order to quantify the well-ventilated lung zones, a histogram

Respiratory mechanical parameters were determined breath by breath, using a multiple linear regression method based on a model fit of selected parts of the flow signal (Eberhard et al., 2003): Prs = P0 + Ers V + Rrs V 

(1)

where Prs (cmH2 O) is the airway opening pressure, P0 (cmH2 O) is the dynamic end-expiratory pressure, Ers (cmH2 O/ml) is respiratory system elastance, V (ml) is volume, Rrs , is respiratory system resistance and V (ml/s) is respiratory flow.

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Fig. 5. (a) The airway cross-sectional area (CA) as percent of the baseline value during inhaled MCh challenge in protocol A (Ainhaled , n = 5), IV MCh challenge in protocol B (BIV , n = 4), recovery (Brec ) and inhaled MCh challenge (Binhaled ). Two airways are measured in each of the 3 axial image slices; (b) area of VD as percent of the studied lung area; dashed line: baseline value; (c) CV of Xe density distribution in all lung zones relative to baseline; (b and c) results for the 3 axial image levels are combined; *p < 0.05 versus baseline; # p < 0.05 versus Biv ; § p < 0.05 versus Binhaled ; & p < 0.05 between axial image levels.

2.6. Statistical analysis Data are expressed as mean ± SD. Differences in the measured parameters between baseline and MCh challenge and in between groups were tested using Two Way ANOVA. A p < 0.05 was considered as significant. 3. Results 3.1. Responses to infused and inhaled MCh in central bronchi Tissue and Xe-density CT images from representative animals at three thoracic levels (apical, middle, caudal) and in the different experimental conditions, are shown in Fig. 4. Tissue-density images are shown in greyscale, whereas the Xe distribution is shown using a color scale. The cross-section of a main bronchus is magnified in the upper left corner of each tissue image. The mean diameter of proximal bronchi at baseline in the 3 axial image slices ranged from 2.3 mm to 3.2 mm. These calibers

correspond approximately to airway generations 4–6 in rabbit (Ramchandani et al., 2000). MCh infusion induced a marked decrease in central airway CA, in the middle and caudal image levels. The relative changes in the CA of central airways of different sizes, are shown in Fig. 5a. There were marked differences in the central airway response to inhaled and infused MCh. Inhaled MCh (Ainhaled ) caused a moderate but significant reduction of 17–29% in CA without significant differences between larger (apical) and smaller-caliber (caudal) bronchi. However, during IV challenge (Biv ) the response to MCh significantly increased in magnitude toward the smaller more caudal central bronchi. The decreases in CA relative to baseline were: 17 ± 22%, 46 ± 24%, and 67 ± 23% in the apical, middle and caudal levels, respectively. Recovery of the proximal airways after MCh infusion was complete, with CA = 105 ± 22%, after 45–105 min. Inhaled MCh (Binhaled ) following an initial IV challenge, induced a significantly smaller bronchoconstriction in the apical and middle image slices compared to inhaled MCh (Ainhaled ) in the unchallenged lung.

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Fig. 7. Changes in respiratory system elastance (Ers ) and resistance (Rrs ) compared to baseline (dashed line); *p < 0.05 versus baseline. Fig. 6. Comparison between cross-sectional areas (CA) of central airways and the area of ventilation defects (VD), taken pair-wise for each animal. Data points represent: in protocol A: 5 animals × 3 image levels (15 observations); protocol B: 4 animals × 3 image levels (12 observations); broken line: baseline values; ellipse: 1.5 SD boundaries; thick line: Biv ; thin line; Ainhaled .

3.2. Effects of infused and inhaled MCh on the regional distribution of ventilation Fig. 4 illustrates the changes in the regional Xe distribution. The Xe distribution became highly heterogeneous after MCh inhalation. The differences in the area of VD and in CV of Xe density were not significant between the three vertical image levels. The values of VD and CV in the different axial image slices were therefore averaged, and are shown in Fig. 5b and c, respectively. The differences between the effects of inhaled and infused MCh challenges on these parameters were large and systematic. Based on the criteria defined in 2.4, about 8% of lung regions were classified as VD at baseline and upon recovery. The VD increased to 57 ± 23% of the total selected lung area in Ainhaled , but only to 33 ± 27% in Binhaled (p = 0.009) and to 21 ± 12% after the initial MCh infusion (Biv ). The heterogeneity of ventilation reflected in the CV of Xe density paralleled the relative increases in VD. 3.3. Effect of the route of MCh challenge on the response in central airways and peripheral lung The relative changes in CA and in VD were compared by pairing the observations for individual animals. Fig. 6 demonstrates the essential differences between the effects of inhaled and infused MCh challenges. The ellipses, which delineate the 1.5 SD boundary of the distributions, are given as guides of the

eye. Inhaled MCh (Ainhaled and Binhaled ) caused marked increase in VD with a wide range, while the decrease of CA in central airways was only moderate and did not correlate with the increase of VD in the same animals. There was no difference between Ainhaled (inhaled only) and Binhaled (infused, then inhaled after recovery) in this two-dimensional analysis. The CA response was more scattered but the variation of VD was smaller after MCh infusion (Biv ) than after inhaled MCh. Infused MCh caused significant reduction in the CA of the central airways, while the increase of VD was moderate and not correlated to changes in CA. 3.4. Respiratory mechanics and blood gases Airway pressure, tidal volume and gas exchange data are summarized in Table 1. Responses to MCh challenge showed substantial variation between animals. Both inhaled and infused MCh administration significantly increased Rrs and Ers (Fig. 7). The increase in respiratory mechanical parameters was reflected in an increase of Prs and reduction of VT. Inhaled MCh (Ainhaled ), produced a larger, albeit not statistically significant increase in Ers than infused Mch (Biv ), reflecting a larger ventilation heterogeneity and airway closures. This outcome is in line with our previous findings in this model (Bayat et al., 2009a). Although Rrs increased significantly, by 361 ± 36% compared to baseline during infusion (Biv ), and by 297 ± 53% following inhalation (Ainhaled ), the rise in this parameter was smaller when the inhaled challenge followed an initial MCh infusion (Binhaled ): 261 ± 36% (NS).

Table 1 Respiratory mechanical and gas exchange parameters under different experimental conditions: tracheal pressure (Ptr ), tidal volume (VT). Data are mean ± SD from 5 animals in protocol A and 4 animals in protocol B. Condition

Ptr (cmH2 O)

VT (ml)

pH

paCO2 (mmHg)

paO2 (mmHg)

A (n = 5) Baseline Inhaled Mch (Ainhaled )

13.6 ± 3.5 21.5 ± 4.3* , §

18.4 ± 2.2 13.5 ± 3.5*

7.45 ± 0.06

29.6 ± 2.1

97.6 ± 12.6

B (n = 4) Baseline i.v Mch (Biv ) Recovery Inhaled Mch (Binhaled )

10.8 15.4 9.1 14.7

± ± ± ±

7.47 ± 0.03

30.8 ± 3.6

98.3 ± 6.6

7.26 ± 0.07

34.5 ± 1.3

81.8 ± 13.1*

* §

p < 0.05 versus baseline. p < 0.05 Binhaled versus Ainhaled .

± ± ± ±

1.3 2.1 1.3 5.1§

17.7 14.8 17.0 15.7

1.6 3.3 2.2 4.1

*

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4. Discussion The main finding of the present study is that the pattern of response to MCh is significantly modified by the route of challenge. Methacholine infusion predominantly reduced the cross-sectional area of central conducting bronchi, but only slightly increased the area of ventilation defects and the heterogeneity of inhaled Xe distribution, in the peripheral lung. Conversely, inhaled MCh produced less constriction in central conducting airways, but more ventilation defects in the lung periphery. In this study, we found that MCh infusion produced significantly more constriction in the smaller caudally located central bronchi. Inhaled MCh however, despite similar overall changes in respiratory system resistance, had a smaller effect on central airways, and unlike infused MCh, the response did not increase with smaller central airway caliber. Regional differences in central airway constriction can arise from differences in local agonist concentration, receptor density and regional differences in the airway wall mechanical properties. The smaller-caliber distal conducting airway wall contains less cartilage and is more compliant (Fujiwara et al., 1988), and airway smooth muscle density is significantly higher in smaller bronchi in rabbits (Smiley-Jewell et al., 2002). These structural differences in the airway wall mechanical properties may explain the increasing effect of infused MCh in smaller-caliber caudally locate central airways. However, none of the above factors can convincingly explain the observed differences in the distribution of central airway constriction between the two routes of administration. The regional density distribution of M3 muscarinic receptors and the airway structures were identical between the two routes of MCh challenge. Regarding local agonist distribution, it can be assumed that during intravenous MCh provocation equal concentrations of MCh reach the bronchial smooth muscle through the bronchial circulation, in the generations of central airways measured here. However, during inhaled MCh challenge, preliminary data from our group using KES to image the deposition of aerosol particles with a similar MMAD, ventilation circuit and settings, suggest significant central airway deposition in rabbit (Bayat et al., 2011). Moreover, bronchoconstriction itself enhances aerosol deposition in the conducting airways (Darquenne, 2012). Considering the structural differences within the central airways discussed above, prominent central airway deposition of MCh should have induced more constriction in the central bronchi, however, this was not the case. An alternative explanation for this finding can be proposed based on the dynamic interdependence of the conducting airways and the small peripheral airways (Winkler and Venegas, 2007). Tidal variations in the transmural pressure can modulate smooth muscle contraction by reducing the mean contractile force or “fluidizing” the airway smooth muscle (Fredberg et al., 1997). Because contiguous ventilation defects can appear without significant constriction in central airways (Bayat et al., 2006), they are likely due to the constriction of clusters of small peripheral airways (Winkler and Venegas, 2012). In the presence of collective small airway closures which give rise to ventilation defects in the lung periphery, increases in the peak transmural pressures during inspiration can limit or diminish constriction in the central conducting airways. This hypothesis could explain the differences in the relation between CA and VD observed in inhaled and IV challenges, which are described in Fig. 6. Previously, Venegas et al. showed that the dynamic interaction between central and small peripheral airways, termed “serial interdependence”, can be simulated in silico using a computational model of the airway tree, where smooth muscle behavior is modulated by tidal variations in transmural pressure (Venegas et al., 2005). Consistent with this hypothesis, in healthy rabbits challenged with inhaled histamine, we previously found that increasing the dose of agonist did not

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increase constriction in central airways, despite increasing the size of ventilation defects (Bayat et al., 2009a). Similarly, Thomas et al. in ovalbumin-sensitized mice found that MCh challenge caused large ventilation defects but only moderate constriction in central airways (Thomas et al., 2012). Such dynamic serial interactions between central and small peripheral airways could explain the decreased relative constriction in central bronchi with inhaled as opposed to infused MCh challenge observed in this study. Another important finding was that inhaled MCh induced a much larger area of ventilation defects than the IV infusion. This was also manifested by a significantly larger heterogeneity of Xe concentration in the lung periphery. This finding is consistent with our previous observations in the same model (Bayat et al., 2009b). Previous studies have shown that the deposition of aerosol particles with an MMAD similar to the present study is heterogeneous in the peripheral airways in rat and rabbit under mechanical ventilation (Leong et al., 1998; Dahlbäck et al., 1994). Therefore, heterogeneous local agonist concentrations due to uneven deposition are a likely cause of this different pattern of response in small peripheral airways. Small local differences in agonist concentrations can contribute to positive feedback mechanisms between neighboring airways, which can set off the emergence of ventilation defects (Anafi and Wilson, 2001; Venegas et al., 2005), where clusters of small peripheral airways subtending a peripheral lung region can constrict or close, while neighboring pathways remain open. This phenomenon is in agreement with the finding that the distribution of Xe concentration within regions outside of the ventilation defects was very similar to baseline. An initial IV administration of MCh reduced both central and peripheral components of the effect of a subsequent inhaled challenge. The subsequent inhaled challenge was given in order to compare the spatial patterns of response through different routes within the same subject. A potential mechanism of the reduced response may have been tachyphylaxis, or tolerance, to MCh. This phenomenon has been demonstrated in normal humans during multiple challenges with MCh (Beckett et al., 1997; Stevens et al., 1990) and at high MCh concentrations in dogs (Shore and Martin, 1985). Tachyphylaxis to MCh may be due to the release of inhibitory prostaglandins in the airways, a mechanism similar to that observed with histamine tachyphylaxis in humans (Manning et al., 1987) and dogs (Shore and Martin, 1985), since it is inhibited by indomethacin pre-treatment in both. Regardless of its mechanism, this finding suggests that the observed patterns of response to MCh are specifically determined by agonist–receptor interactions. Other mechanisms may have contributed to the blunted response to inhaled Mch following the initial IV challenge. Hypoxia can induce relaxation by limiting the entry of Ca2+ into the airway smooth muscle cells and by opening adenosine triphosphate sensitive-potassium channels (Fernandes et al., 1993; Linderman et al., 1994; Vannier et al., 1995). In addition, extracellular acidosis can relax airway smooth muscle tone (Faisy et al., 2007). However, upon recovery from the initial MCh infusion in group B in our study, Rrs was not significantly reduced, despite a moderate acidosis and hypoxia. Finally, stimulation of the non-adrenergic non-cholinergic inhibitory system may have participated in the inhibition of a subsequent inhaled MCh challenge. In conclusion, our findings indicate that in healthy anaesthetized and mechanically ventilated rabbits, the route of MCh challenge is an important factor in determining the spatial distribution of response among central and small peripheral bronchi. Intravenous MCh infusion induces constriction mainly in central bronchi and less in small peripheral airways, with less and smaller ventilation defects. Inhaled MCh on the other hand, despite a similar overall mechanical response, induces a markedly heterogeneous constriction and closure of small peripheral airways, with large ventilation defects, but less narrowing in central bronchi.

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Heterogeneous local agonist concentrations related to uneven deposition are a likely cause of this different pattern of response, in small peripheral airways. Differences in central airway response however, are more difficult to explain and may be due to dynamic interdependence between central and small peripheral airways. These findings are important for a better understanding of the distribution of airway narrowing between central and small peripheral airways due to inhaled MCh challenges in the clinical setting. Our data also suggest that uneven agonist distribution could participate in the emergence of ventilation heterogeneities during bronchial challenge, or exposure to allergen in asthmatic patients. Regional ventilation heterogeneities could also negatively affect the delivery of inhaled medications. These findings provide a rationale for the development both of techniques to measure ventilation heterogeneity and of therapeutic approaches aiming at reducing ventilation heterogeneity in patients with asthma. Acknowledgements We would like to thank Thierry Brochard, Christian Nemoz, and Herwig Requard for technical assistance and Dominique Dallery for valuable help with the animal care. This work was supported by the Tampere Tuberculosis Foundation (A.S.), by the Academy of Finland (no. 126747), by the Ida Montin foundation (S.S.), by the Conseil Régional de Picardie grant (no. REG08009, S.B.), and by the ESRF. References Anafi, R.C., Wilson, T.A., 2001. Airway stability and heterogeneity in the constricted lung. J. Appl. Physiol. 91, 1185–1192. Bayat, S., Le Duc, G., Porra, L., Berruyer, G., Nemoz, C., Monfraix, S., Fiedler, S., Thomlinson, W., Suortti, P., Standertskjøld-Nordenstam, C.G., Sovijärvi, A.R.A., 2001. Quantitative functional lung imaging with synchrotron radiation using inhaled xenon as contrast agent. Phys. Med. Biol. 46, 3287–3299. Bayat, S., Porra, L., Suhonen, H., Nemoz, C., Suortti, P., Sovijärvi, A.R.A., 2006. Differences in the time course of proximal and distal airway response to inhaled histamine studied by synchrotron radiation CT. J. Appl. Physiol. 100, 1964–1973. Bayat, S., Strengell, S., Porra, L., Jánosi, T.Z., Peták, F., Suhonen, H., Suortti, P., Hantos, Z., Sovijärvi, A.R., Habre, W., 2009a. Methacholine and ovalbumin challenges assessed by forced oscillations and synchrotron lung imaging. Am. J. Respir. Crit. Care Med. 180 (4), 296–303. Bayat, S., Porra, L., Suhonen, H., Suortti, P., Sovijärvi, A.R.A., 2009b. Paradoxical conducting airway response and heterogeneous regional ventilation after histamine inhalation in healthy anaesthetized rabbits studied by synchrotron radiation CT. J. Appl. Physiol. 106, 1949–1958. Bayat, S., Degrugilliers, L., Porra, L., Albu, G., Suhonen, H., Strengell, S., Fodor, G., Peták, F., Suortti, P., Habre, W., Sovijärvi, A.R.A., 2011. K-edge subtraction (KES) synchrotron imaging allows quantitative measurement of regional aerosol deposition, lung ventilation and airway morphology in rabbit. Eur. Respir. J. 38 (55s), 888s, Abstract. Beckett, W.S., Pace, P.A., Sferlazza, S.J., Carey, V.J., Weiss, S.T., 1997. Annual variability in methacholine responsiveness in nonasthmatic working adults. Eur. Respir. J. 10 (11), 2515–2521. Costella, S., Kirby, M., Maksym, G.N., McCormack, D.G., Paterson, N.A.M., Parraga, G., 2012. Regional pulmonary response to a methacholine challenge using hyperpolarized 3 He magnetic resonance imaging. Respirology 17 (8), 1237–1246. Crapo, R.O., Casaburi, R., Coates, A.L., Enright, P.L., Hankinson, J.L., Irvin, C.G., 2000. Guidelines for methacholine and exercise challenge testing 1999. Am. J. Respir. Crit. Care Med. 161 (1), 309–329. Dahlbäck, M., Wollmer, P., Jonson, B., 1994. Selective deposition of inhaled aerosols to mechanically ventilated rabbits. J. Aerosol Med. 7, 315–324. Darquenne, C., 2012. Aerosol deposition in health and disease. J. Aerosol Med. Pulm. Drug Deliv. 25 (3), 140–147. Eberhard, A., Carry, P.Y., Perdrix, J.P., Fargnoli, J.M., Biot, L., Baconnier, P.F., 2003. A program based on a ‘selective’ least-squares method for respiratory mechanics monitoring in ventilated patients. Comput. Methods Programs Biomed. 71, 39–61. Elleaume, H., Charvet, A.M., Berkvens, P., Berruyer, G., Brochard, T., Dabin, Y., Dominguez, M.C., Draperi, A., Fiedler, S., Goujon, G., Le Duc, G., Mattenet, M., Nemoz, C., Perez, M., Renier, M., Schulze, C., Spanne, P., Suortti, P., Thomlinson,

W., Esteve, F., Bertrand, B., Le Bas, J.F., 1999. Instrumentation of the ESRF medical imaging facility. Nucl. Instr. Methods A 428, 513–527. Elleaume, H., Charvet, A.M., Corde, S., Estève, F., Le Bas, J.F., 2002. Performance of computed tomography for contrast agent concentration measurements with monochromatic X-ray beams: comparison of K-edge versus temporal subtraction. Phys. Med. Biol. 47, 3369–3433. Faisy, C., Planquette, B., Naline, E., Risse, P.A., Frossard, N., Fagon, J.Y., Advenier, C., Devillier, P., 2007. Acid-induced modulation of airway basal tone and contractility: role of acid-sensing ion channels (ASICs) and TRPV1 receptor. Life Sci. 81, 1094–1102. Fernandes, L.B., Stuart-Smith, K., Croxton, T.L., Hirschman, C.A., 1993. Role of Ca2+ entry in the modulation of airway tone by hypoxia. Am. J. Physiol. 264 (3), L284–L289. Fredberg, J.J., Inouye, D., Miller, B., Nathan, M., Jafari, S., Raboudi, S.H., Butler, J.P., Shore, S.A., 1997. Airway smooth muscle, tidal stretches, and dynamically determined contractile states. Am. J. Respir. Crit. Care Med. 156, 1752–1759. Fujiwara, T., Itoh, T., Kuriyama, H., 1988. Regional differences in the mechanical properties of rabbit airway smooth muscle. Br. J. Pharmacol. 94, 389–396. Harris, R.S., Winkler, T., Tgavalekos, N., Musch, G., Melo, M.F., Schroeder, T., Chang, Y., Venegas, J.G., 2006. Regional pulmonary perfusion, inflation, and ventilation defects in bronchoconstricted patients with asthma. Am. J. Respir. Crit. Care Med. 174 (3), 245–253. Juniper, E.F., Frith, P.A., Hargreave, F.E., 1981. Airway responsiveness to histamine and methacholine: relationship to minimum treatment to control symptoms of asthma. Thorax 36, 575–579. Leong, B.K., Coombs, J.K., Sabaitis, C.P., Rop, D.A., Aaron, C.S., 1998. Quantitative morphometric analysis of pulmonary deposition of aerosol particles inhaled via intratracheal nebulization, intratracheal instillation or nose-only inhalation in rats. J. Appl. Toxicol. 18, 149–160. Linderman, K.S., Fernandes, L.B., Croxton, T.L., Hirschman, C.A., 1994. Role of potassium channels in hypoxic relaxation of porcine bronchi in vitro. Am. J. Physiol. 226 (3), L232–L237. Manning, P.J., Jones, G.L., O‘Byrne, P.M., 1987. Tachyphylaxis to inhaled histamine in asthmatic subjects. J. Appl. Physiol. 63, 1572–1577. Monfraix, S., Bayat, S., Porra, L., Berruyer, G., Nemoz, C., Thomlinson, W., Suortti, P., Sovijärvi, A.R.A., 2005. Quantitative measurement of regional lung gas volume by synchrotron radiation computed tomography. Phys. Med. Biol. 50, 1–11. Porra, L., Monfraix, S., Berruyer, G., Nemoz, C., Thomlinson, W., Suortti, P., Sovijärvi, A.R.A., Bayat, S., 2004. Effect of tidal volume on distribution of ventilation assessed by synchrotron radiation CT in rabbit. J. Appl. Physiol. 96, 1899–1908. Porra, L., Suhonen, H., Suortti, P., Sovijärvi, A.R.A., Bayat, S., 2011. Effect of PEEP on regional ventilation distribution following airway constriction in rabbit studied by synchrotron radiation imaging. Crit. Care Med. 39 (7), 1731–1738. Ramchandani, R., Shen, X., Elmsley, C.L., Ambrosius, W.T., Gunst, S.J., Tepper, R.S., 2000. Differences in airway structure in immature and mature rabbits. J. Appl. Physiol. 89, 1310–1316. Samee, S., Altes, T., Powers, P., de Lange, E.E., Knight-Scott, J., Rakes, G., Mugler 3rd, J.P., Ciambotti, J.M., Alford, B.A., Brookeman, J.R., Platts-Mills, T.A., 2003. Imaging the lungs in asthmatic patients by using hyperpolarized helium-3 magnetic resonance: assessment of response to methacholine and exercise challenge. J. Allergy Clin. Immunol. 111 (6), 1205–1211. Shore, S., Martin, J.G., 1985. Tachyphylaxis to inhaled aerosolized histamine in anesthetized dogs. J. Appl. Physiol. 59, 1355–1363. Smiley-Jewell, S.M., Tran, M.U., Weir, A.J., Johnson, Z.A., Van Winkle, L.S., Plopper, C.G., 2002. Three-dimensional mapping of smooth muscle in the distal conducting airways of mouse, rabbit, and monkey. J. Appl. Physiol. 93, 1506–1514. Stevens, W.H., Manning, P.J., Watson, R.M., O‘Byrne, P.M., 1990. Tachyphylaxis to inhaled methacholine in normal but not asthmatic subjects. J. Appl. Physiol. 69 (3), 875–879. Suhonen, H., Porra, L., Bayat, S., Sovijärvi, A.R.A., Suortti, P., 2008. Simultaneous in vivo synchrotron radiation computed tomography of regional ventilation and blood volume in rabbit lung using combined K-edge and temporal subtraction. Phys. Med. Biol. 53 (3), 775–791. Thomas, A.C., Kaushik, S.S., Nouls, J., Potts, E.N., Slipetz, D.M., Foster, W.M., Driehuys, B., 2012. Effects of corticosteroid treatment on airway inflammation, mechanics, and hyperpolarized 3He magnetic resonance imaging in an allergic mouse model. J. Appl. Physiol. 112 (9), 1437–1444. Vannier, C., Croxton, T.L., Farley, L.S., Hirshman, C.A., 1995. Inhibition of dihydropyridine-sensitive calcium entry in hypoxic relaxation of airway smooth muscle. Am. J. Physiol. 268 (2 Pt 1), 201–206. Venegas, J.G., Winkler, T., Musch, G., Vidal Melo, M.F., Layfield, D., Tgavalekos, N., Fischman, A.J., Callahan, R.J., Bellani, G., Harris, R.S., 2005. Self-organized patchiness in asthma as a prelude to catastrophic shifts. Nature 434 (7034), 777–782. Vidal Melo, M.F., Harris, R.S., Layfield, J.D.H., Venegas, J.G., 2005. Topographic basis of bimodal ventilation–perfusion distributions during bronchoconstriction in sheep. Am. J. Respir. Crit. Care Med. 171, 714–721. Winkler, T., Venegas, J.G., 2012. Are all airways equal? J. Appl. Physiol. 112 (9), 1431–1432. Winkler, T., Venegas, J.G., 2007. Complex airway behavior and paradoxical responses to bronchoprovocation. J. Appl. Physiol. 103 (2), 655–663.