Airway closure in microgravity

Respiratory Physiology & Neurobiology 148 (2005) 97–111

Airway closure in microgravity Brigitte Dutrieue a,∗ , Sylvia Verbanck b , Chantal Darquenne c , G. Kim Prisk c b

a Laboratoire de Physique Biom´ edicale, Route de Lennik, 808, CP 613/3, B-1070 Brussels, Belgium Department of Pneumology, Akademisch Ziekenhuis, Vrije Universiteit Brussels, 1090 Brussels, Belgium c Department of Medicine, University of California, San Diego, La Jolla, CA 92093-0931, USA

Accepted 20 May 2005

Abstract Recent single breath washout (SBW) studies in microgravity and on the ground have suggested an important effect of airway closure on gas mixing in the human lung, reflected particularly in the phase III slope of vital capacity SBW and bolus tests. In order to explore this effect, we designed a SBW in which subjects inspired 2-l from residual volume (RV) starting with a 150 ml bolus of He and SF6 . In an attempt to vary the pattern of airways closure configuration before the test, the experiments were conducted in 1G and in microgravity during parabolic flight allowing the pre-test expiration to RV to be either in microgravity or at 1.8G, with the actual test gas inhalation performed entirely in microgravity. Contrary to our expectations, the measured phase III slope and phase IV height and volume obtained from seven subjects in microgravity were essentially identical irrespective of the gravity level during the pre-test expiration to RV. The results suggest that airway closure configuration at RV before the test inspiration has no apparent impact on phases III and IV generation. © 2005 Elsevier B.V. All rights reserved. Keywords: Single breath washout; Phase III slope; Phase IV; He; SF6 ; Gas mixing; Airway closure; Closing volume

1. Introduction Single breath washin maneuvers (SBW) provide indices of inhomogeneities of ventilation in the lung (Fowler, 1949), particularly through phase III slope and phase IV height. The inhomogeneities are generated by convective processes which can be gravity dependent as predicted by the onion skin model described by Milic-Emili et al. (1966), or non-gravity dependent ∗ Corresponding author. Tel.: +32 2 555 61 35; fax: +32 2 555 61 62. E-mail address: [email protected] (B. Dutrieue).

1569-9048/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2005.05.015

as indicated by ventilation distribution experiments in microgravity (Guy et al., 1994; Prisk et al., 1998), and also by an interaction between diffusive and convective processes in the acinar lung zone (Paiva and Engel, 1987). The purely convective processes (which are also influenced by the closure or near closure of airways) are related to ventilation differences between lung units sufficiently widely separated from each other for diffusion not to be an effective mechanism of abolishing concentration differences generated between them. By contrast, the interaction between convection and diffusion is supposed to occur where both transport mechanism are of the same order of magnitude, i.e.,

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in the human lung at/or slightly peripheral to the entrance of the acinus. Adding tracer gases of different diffusivity to the test gas mixture (usually He and SF6 , with diffusion coefficients that differ by a factor of six) allows the investigation of the diffusion-convection dependent ventilation processes occurring at different levels within the lung periphery (Georg et al., 1965). In adult humans, the He phase III slope is generally considered as an index of inhomogeneity of gas mixing occurring around the acinar entrance while the SF6 phase III slope characterizes gas mixing more peripherally inside the acinus (Paiva and Engel, 1981; Dutrieue et al., 2000). Any changes in the SF6 –He slope difference are assumed to be caused in the lung periphery since purely convective processes would be expected to produce the same contributions to He and SF6 phase III slopes. Gas mixing is also known to be critically dependent on the lung volume excursions that occur during the ventilation distribution test under study. Two SBW maneuvers are relevant to the discussion here: the tidal volume SBW (TV-SBW) in which the subject inhales approximately one liter of test gas from functional residual capacity (FRC), a maneuver close to normal breathing, and the vital capacity maneuver (VC-SBW) involving inhalation of test gas over the full volume range between residual volume (RV) and total lung capacity (TLC). For both maneuvers, previous experimental observations have indicated a substantially steeper phase III slope for SF6 than for He in normal subjects (Prisk et al., 1998; Lauzon et al., 1997) leading to a positive SF6 –He slope difference. These experimental results are in agreement with model simulations of intra-acinar gas mixing in a realistic acinar structure (Dutrieue et al., 2000). A tool to assess the influence of gas mixing at different lung volumes is the single breath bolus test which uses the same lung volume maneuver as a SBW, but where air is breathed for the entire inspiration except for ∼150 ml containing test gas (typically Ar or He and SF6 ), which is inserted at different lung volumes during the test inspiration. We previously hypothesized that the SBW test could be decomposed into several bolus tests in which the bolus is inhaled at different lung volumes in the inspiration. This hypothesis was validated by showing that the sum of He and SF6 bolus phase III slopes on the ground and in microgravity during parabolic flight (Dutrieue et al., 1999) matched those

Fig. 1. Averaged bolus slopes over the indicated lung volume (LV) ranges. He and SF6 are represented in both 1G and transient ␮G conditions. TLC1G and CC1G : total lung capacity and closing capacity, respectively, as measured in 1G condition. Data are mean ± S.E. * Significant differences at P < 0.005 between 1G and ␮G (figure from Dutrieue et al., 1999).

of VC-SBW phase III slopes obtained on the same subjects in the same conditions (Lauzon et al., 1997). The bolus phase III slopes as a function of lung volume at which the boluses were inhaled (Fig. 1), showed that in the extreme lung volume ranges, i.e., below closing capacity (CC) or above 80% of total lung capacity, the bolus phase III slopes were characterized by the largest absolute value (with opposite sign between the two lung volume extremities) (Anthonisen et al., 1978; Dutrieue et al., 1999). On the other hand, boluses inhaled in the middle lung volume range produce relatively small bolus phase III slopes. In summary, bolus tests showed that almost all of the phase III slope seen in conventional VC-SBW tests results from events occurring below CC or near to TLC, with little slope generated by gas boluses inspired in the middle lung volume range. 1.1. Gravity dependent phase III slope generation mechanism If the ventilation distribution was homogeneous in the lung and the acinus structure symmetrical, no phase III slope would be observed for any test (Bolus test or SBW). Fig. 2A and C present schematically washout curves in such a simplified model in the case of a SBW (Panel C) or a respiratory maneuver in which a bolus in inspired at the beginning of the test inspiration (Panel A). For simplicity, let us consider only a gravity

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Fig. 2. Stylized washout curves for a bolus inspired at RV (Panels A and B) and vital capacity single breath washins (Panels C and D) as affected by two mechanisms: airway closure with homogeneous ventilation distribution (Panels A and C) or airway closure with the closing airways better ventilated than the un-closing airways (Panels B and D). In Panels B, C and D, the thin lines represent the phase IV obtained with the first mechanism on a Bolus curve (Panel A), the arrows shows how it is affected in each represented conditions. See text for details.

dependent vertical gradient of ventilation distribution between two lung compartments and , based on the onion-skin model of Milic-Emili et al. (1966). In this case, imagine unit in the lower lung region and unit in the upper lung region with therefore unit better ventilated than unit and the best ventilated unit filling last and emptying first. In this case, during a SBW test inspiration, unit receive more test gas than unit resulting at the end of inspiration in a higher test gas concentration in the better ventilated unit ( ) than in the upper lung unit ( ). During the following expiration, the concentration at the mouth is a mixture of the gas coming from both units. Due to the sequence of emptying between both units, the concentration at the mouth is initially dominated by the concentration of unit , richer in test gas and emptying first, and progressively decreases as unit empties preferentially generating a negative slope as shown in Fig. 2D. During expiration, phase III slope will depend on both

the concentration difference between units, and on the sequence of emptying between them. In the case of a Bolus inspired at the beginning of the test inspiration, the bolus test gas will preferentially enter the upper lung unit ( ) that is better ventilated at the beginning of inspiration. This will lead to a concentration difference at the end of inspiration that is reversed in comparison with the SBW case. Therefore, a positive slope will be generated during expiration as shown in Fig. 2B. Other mechanisms generating a phase III slope such as the interaction between diffusion and convection in the asymmetrical acinus, not being directly gravity dependent, are not discussed here. 1.2. Phase III slope and microgravity Microgravity (␮G) was expected to result in a significant reduction in ventilatory inhomogeneity, by abolishing its gravity dependent component, leaving

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other mechanisms unaffected. It was therefore hypothesized that phase III slopes from gases with differing diffusivities would be affected to a similar extent by the removal of gravity. In order to investigate this hypothesis, studies were conducted on the ground (for reference) and in ␮G during parabolic flight (“transient ␮G”) or in spaceflight (“sustained ␮G”). During parabolic flight, the ␮G phase lasts only 22–25 s and is preceded by a hyper-gravity phase (1.8-Gz ) while sustained ␮G lasts for several days. For the VC-SBW maneuver, the short period of transient ␮G requires that the expiration to RV preceding the test occurs during the hyper-gravity phase, as the maneuver itself takes ∼20 s to perform. In contrast to VC-SBW, TV-SBW requires less time and it is possible to perform the entire maneuver, including the initial expiration to RV, within the ␮G phase available during parabolic flight. During spaceflight, both maneuvers are obviously performed entirely in ␮G. Observations of both TV-SBW and VC-SBW performed during parabolic flight (Lauzon et al., 1997; Dutrieue et al., 2003) and in spaceflight (Guy et al., 1994; Prisk et al., 1996, 1998), summarized in Fig. 3, showed significant decreases from 1G to ␮G for all gases in both respiratory maneuvers, irrespective of whether ␮G was transient or sustained. These slope reductions can be partially attributed to the elimination of the gravity dependent convective component of the gas mixing process that affects all gases to the same extent. In particular, the decrease in SF6 slope from 1G to ␮G was similar in all situations (irrespective of ␮G condition or SBW maneuver) suggesting a consistent (or perhaps no) effect of ␮G on the acinar periphery. While the effect on SF6 slope was consistent for all maneuvers, the phase III slope difference between gases of low and high diffusivity () decreased from 1G to ␮G, except for the VC-SBW performed in transient ␮G (compare Fig. 3A, C and D with Fig. 3B). This result was in contradiction with our hypothesis of purely convective effects resulting from the removal of gravity. Gas mixing at lung volumes near FRC (TV-SBW maneuver) produced a comparable  decrease in both transient and sustained ␮G (Fig. 3C and D), especially considering that in transient ␮G, the more diffusive gas was the CH4 , which is a gas of lower diffusivity than the He usually used (in a ratio 4:6) (Dutrieue et al., 2003). While there was also a decrease in  for the VC-SBW maneuver in sustained

Fig. 3. Comparison between 1G and ␮G data obtained in different ␮G conditions and from different test maneuvers. Panel A: vital capacity single breath washout (VC-SBW) phase III slopes obtained in 1G standing position and in sustained ␮G during space flight mission (data from Prisk et al., 1996). Panel B: VC-SBW phase III slopes obtained in 1G sitting position and in transient ␮G during parabolic flight (data from Lauzon et al., 1997). Both vital capacity phase III slope were normalized to 100% background and 0% inspired gas giving slope in %/liter. Panel C: tidal volume phase III slope (TVSBW) obtained in 1G standing position and in sustained ␮G during space flight (data from Prisk et al., 1998). Panel D: TV-SBW phase III slope obtained in 1G sitting position and in ␮G during parabolic flight (data from Dutrieue et al., 2003). Both tidal volume phase III slope were normalized by the end-expiratory concentration and then expressed in l−1 (see text for details). Note the difference in units between VC-SBW and TV-SBW. Data are mean ± S.E. * Significant difference between slope of different gas diffusivity (P < 0.05). (
) SF6 ; () CH4 ; (䊉) He (figure from Dutrieue et al., 2003).

␮G, the VC-SBW in transient ␮G led to a surprising increase in  from 1G to ␮G, owing to a relatively greater He slope decrease in this particular ␮G condition. This larger He than SF6 slope reduction in the VC-SBW maneuver during transient ␮G suggested that additional conformational changes were occurring around the acinar entrance and that they were crucially affected by lung volumes below FRC or close to TLC (outside the TV-SBW breathing range). When considering the role of lung volume, reduction (in absolute value) of bolus phase III slopes (Fig. 1), as well as of the bolus slope difference, between 1G and ␮G was indeed most marked at lung volumes below CC and near TLC (Dutrieue et al., 1999). This study also showed that if only the low lung volumes were to contribute to the ␮G effect on phase III slope, the overall VC-SBW phase III slope would

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actually be increased in ␮G because of a reduction in the negative slope contribution of bolus inhaled near RV in ␮G. However, the counteracting effect of ␮G induced slope reductions with positive sign at lung volumes near TLC results in an overall VC-SBW phase III slope reduction (Lauzon et al., 1997). Note that the decreases found for boluses inspired near RV in (Dutrieue et al., 1999) (29–36% of their 1G value) were consistent with the results from Michels and West (1978) and Guy et al. (1994) both using argon boluses inhaled at RV in VC-SBW maneuvers and showing absolute argon bolus phase III value reduced by 39% (transient ␮G) and 29% (sustained ␮G) of their 1G value, respectively. Thus, the decrease in He and SF6 SBW phase III slopes from 1G to ␮G can only be partially attributed to the elimination of the gravity dependent convective component of the gas mixing process, and that airways around the acinar entrance are probably affected by ␮G, irrespective of the SBW breathing maneuver. In addition, the bolus studies suggest that extreme lung volumes, below CC and near TLC, play an important role in the gas mixing inhomogeneities taking place in VC maneuvers and are also the ones preferentially affected by the removal of gravity (Dutrieue et al., 1999). The clearly different result in the above observations (Fig. 3) is the peculiar behavior of the VC-SBW in transient ␮G which showed a greater He than SF6 slope decrease, which we hypothesized could be due to the potential effects of the preceding period of hyper-gravity on measurements performed in transient ␮G. In VC-SBW tests performed in transient ␮G, the expiration to RV preceding the test occurred during the transition between hyper-gravity and ␮G phase, potentially generating more airway closure since airway closure is known to be gravity dependent (Milic-Emili et al., 1966). Consistent with the relatively different He slope behavior under these conditions, the airways most susceptible to closure or near closure are expected to be situated around the acinar entrance as opposed to deeper within the acinar air space (which would have affected SF6 slopes more). Based on these observations it appeared useful to perform a dedicated microgravity study focused on the effect of airway closure on ventilation distribution at low lung volumes. To do so we used a bolus inserted at RV, followed by an air inspiration that extended to

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only 2 l above RV (in order to avoid any contribution to phase III slope from lung volumes near TLC). We also examined phase IV as a complementary tool to quantify airway closure. We will first reiterate how phase IV is generated and summarize experimental reports of its influence from ␮G. 1.3. Airway closure and phase IV generation The generation of a phase IV in the presence of airway closure is crucially linked to ventilation distribution. Let us first consider two compartments and , where is the only one to be ventilated below CC, and both units and are equally ventilated (homogeneous ventilation distribution) throughout the rest of inspiration between CC and TLC. Therefore, a bolus of test gas inhaled at RV will only enter the opened unit . Test gas will then only be diluted with air in this unit as inspiration proceeds. Consequently, at the end of inspiration, bolus gas concentration is higher in unit than in unit . During expiration, the horizontal alveolar phase III resulting from mixing of gas from both compartments is followed by a phase IV rise due to the closure of unit below CC and emptying of unit richer in bolus test gas, Fig. 2A shows such a washout curve. During inspiration when the bolus gas dilutes, once the lung volume reaches CC, test gas may also diffuse from unit to unit if the two units are close enough, this will result in a non-zero concentration in unit , but remaining smaller than in unit , and will lead to a smaller phase IV height. Based on the onion-skin model of Milic-Emili et al. (1966), let us now superimpose a gradient of ventilation distribution between the same units and , considering that unit is closed below CC, but is better ventilated than unit above CC with this best ventilated unit emptying first. As in the case when ventilation distribution between units and was homogeneous above CC, unit remains closed during bolus inspiration and all bolus test gas enters the opened unit , however in the present case, the following inspired air dilutes the bolus gas relatively less due to the less ventilation of unit . Hence, at the end of inspiration the difference in concentration between units and will be greater than in the case of homogeneous ventilation distribution and during expiration, phase III slope will be followed by a consequently larger phase IV increase (Fig. 2B).

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Note however that in this case we considered that the better ventilated unit above CC is also the one closing below CC, mimicking the assumptions for a purely gravitation-dependent topographical model of airway closure and ventilation distribution. Indeed, Engel et al. has demonstrated that there were more closing units in the lower part of the lung, i.e., in the better ventilated units of the lung (Engel et al., 1975). However, this does not exclude a patchy distribution of airway closure, in which case inhomogeneous ventilation distribution will not reinforce the phase IV. In fact if an inhomogeneous ventilation distribution, generating a sloping phase III, only occurs between units that are either never closed below CC or both always closed below CC, no additional phase IV rise will ensue with respect to the homogeneous distribution case (Fig. 2A). If only top-to-bottom distributed airway closure and inhomogeneous ventilation distribution were to occur, this should produce the same phase IV height for He and SF6 . However, when measurements were made in parabolic flight (Dutrieue et al., 1999), bolus He phase IV height was significantly less than SF6 phase IV height both in 1G and in ␮G suggesting that at least some of the closing airways are subtending lung units that are close to lung units that remain open below CC, so that He diffusion is an effective mechanism at reducing concentration gradients between them, consistent with Engel’s concept of “patchy” airways closure, (Engel et al., 1975). During a single breath washout with test gas administered during the entire inspiration in a lung with a homogeneous distribution of ventilation, airway closure alone would also generate a phase IV rise as in the case of a bolus. Fig. 2C shows the washout curve in the SBW case. In this case, the phase IV rise is smaller than in the Bolus case because the test gas enters both units throughout the inspiration. This results in a smaller concentration difference between units at the end of inspiration and therefore a smaller phase IV rise. When associated with the inhomogeneous ventilation distribution pattern as in the case of Fig. 2B, a full test gas inspiration could result in a phase IV decrease instead of a phase IV increase, depending on how long the test gas has been able to enter the better ventilated unit to compensate for the originally lower concentration in below CC. If this effect overrules the distribution generated by closure itself, this produces a phase IV decrease in the

test gas curve (Fig. 2D) (the equivalent of a phase IV increase in a N2 washout). Since experimentally, N2 VC-SBW always showed positive phase IV heights (Michels and West, 1978), the associated preferential ventilation of the units subtended by closed airways below CC appears to be a dominant effect. The fact that absolute phase IV height was smaller for N2 after a full O2 inspiration, than for the simultaneous Ar bolus inspired at RV (Michels and West, 1978), supports the fact that inhomogeneous ventilation distribution has a subtractive effect in the SBW test, and an additive effect on the bolus test when the bolus is inhaled at RV. 1.4. Airway closure and gravity Removing gravity would be expected to abolish the vertical gradient of ventilation distribution in the lung and thus attenuate the extent of airway closure. In fact, if phase IV only had a gravity-dependent interregional basis (both in terms of closure and vertical gradient of ventilation distribution), phase IV height resulting from a bolus inhaled at RV would become zero in ␮G. If only airway closure (without associated preferential ventilation of the closed airways below CC) were to persist, phase IV would dramatically decrease without complete disappearance in the case of bolus and might even invert (negative phase IV height) in the case of SBW. Microgravity studies showed that for boluses inhaled near RV, a significant He and SF6 phase IV height persists in transient ␮G, reducing it by 51% compared to 1G (Dutrieue et al., 1999), consistent with the 78% reduction in Ar bolus phase IV height found by Michels and West (1978). Guy et al. (1994) reported a 60% reduction in Ar bolus phase IV height and a 82% reduction in N2 VC-SBW phase IV height, under sustained ␮G. The persistent phase IV height in ␮G for boluses inhaled at RV is consistent with the concept that the previously hypothesized patchy airways closure (Engel et al., 1975) still occurs in ␮G. Such patchy closure without associated preferential ventilation of the closed airways would indeed still show a phase IV rise in the bolus but less so in the SBW maneuver. Since He and SF6 phase IV heights persisted in being different from each other in ␮G (as was the case in 1G) a similar diffusion mechanism reducing concentration gradients between closing and open lung units is expected to occur in ␮G (Dutrieue et al., 1999).

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It might be argued that in transient ␮G (Michels and West, 1978; Dutrieue et al., 1999) airway closure could have been facilitated by the fact that the expiration to RV prior to the bolus test inspiration partially occurred in hypergravity phase. However, that is clearly not the case for maneuvers performed in sustained ␮G where a phase IV height was also seen to persist for the Ar bolus (Guy et al., 1994). 1.5. Near residual volume He and SF6 boluses in transient microgravity The objective of the present study was to assess the airway closure contribution to gas mixing at low lung volumes in transient ␮G, including the effect of the different gravity phases during the expiration preceding the test inhalation. Because previous bolus data covering the entire lung volume range between RV and TLC had indicated a ␮G influence on He and SF6 mixing occurring close to TLC (Dutrieue et al., 1999), we deliberately chose a maneuver inhaling a bolus from RV followed by only 2 l of air. We hypothesized that with a pre-test expiration performed in hypergravity and the actual bolus test in ␮G, phase IV height will be greater compared to the corresponding test with pre-test expiration in ␮G, due to an increase in airway closure in the hypergravity phase. In addition, if inhomogeneous ventilation distribution between top and bottom of the lung is a major contributor to phase IV at 1G, phase IV height for both ␮G maneuvers are still expected to be smaller than phase IV height obtained in 1G. Since airway closure also affects phase III slope for boluses inhaled near RV (Dutrieue et al., 1999), we expected a similar phase III slope behavior to that predicted for phase IV height, but smaller in magnitude because other mechanisms unrelated to airway closure also contribute to phase III slope. 2. Methods 2.1. Experimental system The system used was a prototype of that developed for spaceflight (Pulmonary Function System, PFS, Damec, Odense, Denmark). Briefly, it comprised a differential pressure flowmeter connected to the mouthpiece which in turn connected to a computer-controlled rotary valve to direct the breathing path. The subject

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was responsible for actuation of the valve at predetermined points in the test maneuver. Gas was sampled from the mouthpiece by a mass spectrometer gas analyzer (GASMAP, NASA). Data were acquired at 100 Hz (200 Hz for flow) by the data management system of the PFS and displayed on an IBM Thinkpad (Model 760-ED). A flow display was mounted in front of the subject for flow control. Ambient pressure and temperature (used for BTPS correction) were given by the PFS module. Two-point gas calibrations were performed for each session. Gas analyzer transit time was determined by measuring the time required for a sharp puff containing CO2 to be detected by the gas analyzer and the data were then aligned accordingly. This maneuver was repeated daily and the average delay time was 1.73 ± 0.09 s with only inconsequential differences in the delays for different gases. Flow was calibrated by integration of the flow strokes of a 3-liters calibration syringe (Model 5530, Hans Rudolf), and flow drifts were corrected in post-processing by selecting a 2 s interval of imposed zero flow before or after the maneuver. All the reported data (ground and flight data) were collected with the above mentioned system installed aboard the A300 ZERO-G aircraft (Novespace Centre National d’Etudes Spatiales, CNES) during the 34th ESA parabolic flight campaign (March 2003). A typical flight profile consisted of a climb to an altitude of ∼6000 m with the cabin pressurized to ∼600 mmHg. The aircraft was pitched up at 1.8-Gz to a 50◦ nosehigh attitude resulting in a hyper-gravity (HG) phase lasting ∼20 s. Then, the nose was lowered to abolish wing lift, and thrust was reduced to balance drag (thus maintaining ␮G). A ballistic flight profile resulted and was maintained until the nose of the aircraft was 40◦ below the horizon. In this manner, the HG phase was first smoothly decreased from ∼1.8-Gz to ∼1.5Gz (this transition lasted 4.8 ± 1.4 s), followed by, an abrupt transition from HG to ␮G lasting 5.9 ± 0.7 s. The ␮G phase was maintained for a period of 18.1 ± 0.7 s. A 1.8-Gz pullout of ∼20 s followed the ␮G phase until the aircraft returned to the horizontal, which was followed by 1G flight conditions for at least 90 s. The duration values were computed from 15 parabola profiles. The G-profiles of two parabola samples are presented on Fig. 4 (G-level curves). The cycle was repeated for 31 successive parabolas.

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the study. They had undergone medical examinations equivalent to FAA Class II and reported no pulmonary problems on questioning. They were well trained in these respiratory maneuvers and only tests meeting predefined quality control criteria (see below) were retained, therefore results come from 7 of the subjects (4 females and 3 males) aged 39 ± 5 (mean ± S.D.) years, weight 66 ± 15 kg and a height of 173 ± 15 cm. All subjects took anti-motion sickness drugs (0.4 mg scopolamine, 5 mg dexedrine) approximately 1 h before flight. The data were collected with the subjects sitting in a standard aircraft seat restrained with a lap belt during all phases (1G, ␮G, HG) of the parabolic flights. All ground data were collected on the ground under sea level conditions without medication. The flight data were collected at a pressure of ∼600 mmHg. All experiments were reviewed and approved by the ESA Medical Board and all subjects were fully informed and provided written consent. 2.3. Test maneuvers

Fig. 4. Respiratory maneuver during a parabola. SF6 concentration, volume and G-level. HG: hyper-gravity, ␮G: microgravity. The subject expired to residual volume (RV) during ␮G phase (Panel A) or during HG phase (Panel B), performed a short breath hold (BH1 ), then inspired ∼1.2 l, performed a short breath hold (BH2 ) and finally expired back to RV before the end of ␮G phase. The dotted vertical lines indicate the beginning and the end of the expiration to RV prior to the test. The dashed line indicates the onset of the test inspiration (coinciding with the beginning of the ␮G phase in Panel B).

2.2. Subject and data collection Nine healthy subjects, non-smokers for at least 5 years before the start of data collection, participated in

Bolus test maneuver as shown in Fig. 4 began with quiet breathing through the mouthpiece. On command, the subject expired to residual volume and performed a breath hold (BH1 , see Table 1) waiting for a signal given by the operator before starting the test. During this time, the rotary valve turned, connecting the subject to a 150 ml tube containing the Bolus test gas composed of 40% SF6 , 40% He, balance O2 with a one-way inspiratory valve at its distal end. On command from the operator, the subject inspired at a controlled flow rate of ∼0.4 l/s (Table 1) the Bolus gas followed by air with a total preset inspiratory volume of typically 1.94 l (Table 1). At the end of inspiration, the subject performed a short breath hold (BH2 ∼2 s, see Table 1) allowing the valve to be actuated prior to expiration. The expiration was performed at the same controlled flow rate until the subject reached RV. On the aircraft during flight, the Bolus tests were performed entirely in 1G condition (1G–1G test) or in one of the two following variant gravity conditions (see also Fig. 4): (1) in the ␮G–␮G test (Fig. 4A), the start of the preliminary expiration to RV was performed at the beginning of the ␮G phase so that the entire maneuver (expiration – BH1 – inspiration – BH2 – expiration) was in ␮G. (2) In the HG–␮G test (Fig. 4B), the pretest expiration to RV was performed during the HG

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Table 1 Respiratory bolus test maneuver parameters

Vinsp (l) Flow (l/s) BH1 (s) BH2 (s)

Ground data

Flight data

1Gground

1G–1G

1.85 0.46 3.24 1.85

± ± ± ±

0.24 0.04 0.84 0.35

1.95 0.46 2.18 1.58

± ± ± ±

␮G–␮G 0.21 0.04 0.69 0.64

1.99 0.45 1.78 1.55

± ± ± ±

HG–␮G 0.23 0.05 1.21 0.35

1.94 0.49 7.23 1.70

± ± ± ±

0.27 0.06 1.33* 0.58

Data are mean ± S.D. Vinsp : inspired volume, BH: breath-hold, BH1 : BH at residual volume before test inspiration, BH2 : BH between test inspiration and expiration. * Significantly different with other data sets (P < 0.001).

phase preceding the ␮G phase, and BH1 was extended as necessary until a stable ␮G level was reached. In both cases, the remainder of the test itself (inspiration, BH2 , expiration) was performed in ␮G and so tests differed only by the gravity condition during the expiration to RV preceding the test. On board the aircraft (during flight), all subjects repeated the Bolus test at least once in each of the three conditions (1G–1G, ␮G–␮G, HG–␮G). On the ground, Bolus tests were also performed at least 3 times by each subject (1Gground ). In addition, ground experimental conditions allowed the time to perform a traditional SBW test maneuvers in order to compare data from the present subjects to previously published data. Those SBW tests followed basically the same procedure as the Bolus tests with the major difference that the entire test inspiration was performed from a bag containing the SBW test gas composed of 5% SF6 , 5% He, balance O2 . The two types of SBW maneuvers (TV-SBW and VC-SB) were performed at least 3 times for each subject. For the TV-SBW, the subject began a ∼1.2 l test gas inspiration from FRC followed by expiration to RV. For the VC-SBW, the subject inspired test gas from RV to total lung capacity followed by expiration to RV. 2.4. Data analysis Data were first corrected for gas analyzer transit time and volume was obtained from flow integration corrected for zero offset, flowmeter calibration and corrected to BTPS conditions. Bolus Tests were then selected according to the following inclusion criteria: (1) the mean inspiratory and expiratory flow rate had to be in a consistent window of ±0.15 l/s around the mean for all tests of that subject (collected in the differing

gravity conditions). (2) Inspired volume (Vinsp ) had to be in a consistent window of ±0.3 l around the mean for that subject. (3) Breath hold between inspiration and expiration had to be in a consistent window of ±0.5 s around the mean for that subject. (4) Tests performed in the aircraft had to be well synchronized with respect to the targeted gravity profile of each one of the three protocols (1G–1G, ␮G–␮G, HG–␮G). For the HG–␮G protocol, successful timing had to insure that: (I) the pre-test expiration to RV was performed in HG condition (and not during the transition from HG to ␮G); (II) the test inspiration occurred in ␮G; (III) the test expiration was completed within the available ␮G timeframe. As a result, this breath hold time at RV (BH1 ) was much longer for tests performed in HG–␮G condition (∼7 s) than in ␮G–␮G condition (∼2 s) (Table 1). Only subjects with at least one test in every gravity conditions after test selection were included in the study. The resulting Vinsp , Vexp , BH at RV prior to test (BH1 ), BH between inspiration and expiration (BH2 ), and flow are presented in Table 1 (mean ± S.D.) for the four variants of Bolus test conditions. SF6 and He were analyzed by considering the pretest gas concentration in the lung as 0% and the inspired (bolus or SBW) test gas concentration as 100%. For bolus test, this means also that the following air inspired was considered as 0% concentration. Both for bolus and SBW, the resulting phase III slopes and phase IV height were positive. For each test, the gas concentration was plotted against expired volume and the phase III slope was computed from linear regression on the alveolar plateau. For TV-SBW, the linear regression was computed between 0.7 and 1.2 l of expired volume. The TVSBW slope was divided by the concentration at 1.2 l of expired volume (Dutrieue et al., 2003).

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For Bolus test and VC-SBW, the limits of the alveolar plateau were determined at the same time as the onset of phase IV by use of an iterative procedure (Guy et al., 1994). Phase IV volume was taken to be the volume from the end of the phase III slope to the end of expiration. Phase IV height was referenced to the extrapolated phase III slope line (Guy et al., 1994). When no clear phase IV was observed, both phase IV volume and height were considered to be zero. Statistical analysis was performed using Statistica (Statsoft Inc., Tulsa, OK), and SPSS (SPSS Inc., Chicago, IL). Comparisons involved ttest, one- or two-way Anova tests with Tukey post-hoc testing. Values are mean ± S.E. and significant differences were accepted at P < 0.05.

sion in combination with the normalization, leads to results exactly comparable to that of the first breath of the MBW data from spaceflight (Prisk et al., 1998) and to previous TV-SBW data from parabolic flight (Dutrieue et al., 2003). Similarly, the method used in VC-SBW analysis leads to VC-SBW tests results exactly comparable to the SBW data from spaceflight (Guy et al., 1994) and from previous parabolic flight campaign (Lauzon et al., 1997). Table 2 also presents phase III slopes obtained by several authors for comparison suggesting that the subjects under study were representative of previously reported sample populations. 3.2. 1G data: ground versus flight Table 3 presents Bolus tests obtained in 1G condition on the ground and during flight: for both gases phase III slope, phase IV height and volume are presented. Data from bolus tests results obtained on the ground were significantly different from those obtained on 1G condition during flight. For both gases, phase III slope decreased significantly from ground to flight condition with He slope decreasing from 0.63 ± 0.18 to 0.49 ± 0.16%/l and SF6 from 0.67 ± 0.19 to 0.48 ± 0.16%/l (Table 3). The SF6 –He phase III slope difference was 0.069 ± 0.018%/l on the ground and fell to zero in flight (−0.014 ± 0.026%/l). For both gases, phase IV height increased significantly in flight compared to ground data with He phase IV height increasing from 1.24 ± 0.23 to 1.76 ± 0.26% and SF6 from 1.41 ± 0.24 to 1.99 ± 0.32%. No sig-

3. Results After quality control criteria test selection (see above), only subjects with at least one Bolus test in every gravity conditions were included in the study, retaining therefore seven of the nine subjects. 3.1. Comparison with previous studies Baseline data obtained on the ground are summarized in Table 2. VC-SBW phase III slopes were 0.77 ± 0.08 and 1.04 ± 0.09%/l for He and SF6 , respectively, and TV-SBW phase III slopes were 0.10 ± 0.01 and 0.12 ± 0.01 l−1 for He and SF6 , respectively. For TV-SBW, the volume limits used for the slope regresTable 2 Comparison with previously published ground SBW phase III slope values

TV-SBW (l−1 )

VC-SBW (%/l)

Present study Lauzon et al. (1997) Prisk et al. (1996) Olfert and Prisk (2004) Dutrieue et al. (2003) Prisk et al. (1998) Crawford et al. (1985)

He

SF6

He

SF6

0.77 ± 0.08 0.88 ± 0.06 0.94 ± 0.07 – – – –

1.04 ± 0.09 1.06 ± 0.08 1.18 ± 0.06 – – – –

0.10 ± 0.01 – – 0.094 – 0.060 ± 0.003 0.087 ± 0.021

0.12 ± 0.01 – – 0.151 0.101 ± 0.007 0.101 ± 0.003 0.114 ± 0.022

Data are mean ± S.E. except for data from (Olfert and Prisk, 2004) in which S.E. were not available. Note that the TV-SBW slopes from Crawford et al. (1985) were normalized by the mean expired concentration instead of the concentration at the end of expiration as for the other TV studies, leading only to a small difference. In the present study, the maneuver parameters were for VC and TV SBW, respectively (mean ± S.D.) 4.87 ± 1.48 and 1.44 ± 0.07 l of inspired volume, 0.44 ± 0.05 and 0.40 ± 0.03 l/s of flow and 2.61 ± 0.68 and 2.24 ± 0.51 s of apnea between inspiration and expiration (BH2 ).

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Table 3 1G-Bolus test results Ground data

Phase III slope (%/l) Phase IV height (%) Phase IV volume (l)

Flight data

He

SF6

He

0.60 ± 0.18 1.24 ± 0.23 0.46 ± 0.05

0.67 ± 0.19 1.41 ± 0.24 0.50 ± 0.05

0.49 ± 1.76 ± 0.26* 0.48 ± 0.05

SF6 0.16*

0.48 ± 0.16* 1.99 ± 0.32* 0.52 ± 0.05

Data are mean ± S.E. * Significant difference between ground and flight data at P < 0.005. Difference between SF and He data were significant only for phase III 6 slope obtained on the ground (P = 0.05).

nificant changes were observed for phase IV volume. Because of the difference in 1G data between ground and flight, comparison of the effects of gravity level on the bolus washouts has been limited to only data collected in flight. 3.3. Inflight Bolus phase III slope The He and SF6 Bolus phase III slopes obtained when pooling all subject together for the three inflight gravity conditions (1G–1G, ␮G–␮G and HG–␮G) are presented on Fig. 5. Both He and SF6 phase III slopes were significantly smaller in ␮G–␮G and HG–␮G conditions compared to 1G–1G. SF6 phase III slope was slightly larger in HG–␮G condition compared to ␮G–␮G whereas He slope showed no significant difference between these two conditions. In each of the three inflight gravity conditions, no significant differences were observed between SF6 and He phase

Fig. 5. Bolus phase III slope obtained in flight in normal gravity condition (1G–1G), or obtained with pre-test expiration to RV performed in hypergravity phase (HG–␮G) or in microgravity (␮G–␮G), with the following of the maneuver being performed in both case in microgravity. Values are the mean over all subjects mean ± S.E. * Significant difference at P < 0.05, for detailed values see Table 4. ( ) He and ( ) SF6 .

III slopes. The complete set of data is presented in Table 4 and shows the between test variability. 3.4. Inflight Bolus phase IV height and volume The He and SF6 phase IV heights and volumes obtained when pooling all subject together for the three inflight gravity conditions (1G–1G, ␮G–␮G and HG–␮G) are presented in Fig. 6A and B, respectively.

Fig. 6. Bolus phase IV height (Panel A) and volume (Panel B) obtained in same condition as in Fig. 5. Format same as Fig. 5.

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Table 4 Reproducibility of bolus test indices Subject # Phase III slope (%/l)

Phase IV height (%)

Phase IV volume (liters)

1G–1G

␮G–␮G

HG–␮G

1G–1G

␮G–␮G

HG–␮G

1G–1G

␮G–␮G

HG–␮G

He 2 3 8 10 11 12 14

0.17 ± 0.08 0.33 ± 0.08 0.65 ± 0.20 0.03 ± 0.11 0.46 ± 0.15 1.36 ± 0.21 0.46 ()

0.10 ± 0.09 0.17 ± 0.02 0.17 ± 0.16 0.09 () 0.24 ± 0.08 0.69 ± 0.17 0.12 ()

0.15 ± 0.10 0.34 ± 0.07 0.18 ± 0.07 0.10 ± 0.03 0.28 ± 0.03 0.59 ± 0.14 0.30 ()

1.06 ± 0.12 0.65 ± 0.09 1.84 ± 0.35 2.57 ± 0.25 2.43 ± 0.36 1.73 ± 0.59 2.04 ()

0.25 ± 0.25 0.07 ± 0.10 0.45 ± 0.02 0.25 () 0.59 ± 0.12 0.87 ± 0.10 0.55 ()

0.08 ± 0.12 0.00 ± 0.00 0.46 ± 0.16 0.68 ± 0.11 0.46 ± 0.07 0.60 ± 0.03 0.55 ()

0.57 ± 0.04 0.40 ± 0.11 0.60 ± 0.11 0.38 ± 0.07 0.36 ± 0.01 0.35 ± 0.06 0.69 ()

0.25 ± 0.23 0.11 ± 0.16 0.45 ± 0.11 0.21 () 0.29 ± 0.07 0.21 ± 0.01 0.33 ()

0.11 ± 0.15 0.00 ± 0.00 0.56 ± 0.13 0.21 ± 0.06 0.26 ± 0.07 0.20 ± 0.07 0.49 ()

All

0.49 ± 0.44

0.23 ± 0.21 0.28 ± 0.16

1.76 ± 0.70 0.43 ± 0.27 0.41 ± 0.26

0.48 ± 0.14 0.27 ± 0.11 0.26 ± 0.20

SF6 2 3 8 10 11 12 14

0.11 ± 0.18 0.43 ± 0.07 0.62 ± 0.13 −0.03 ± 0.12 0.40 ± 0.04 1.30 ± 0.17 0.52 ()

0.10 ± 0.03 0.29 ± 0.05 0.37 ± 0.11 0.12 () 0.16 ± 0.16 0.59 ± 0.06 0.08 ()

1.27 ± 0.19 0.77 ± 0.17 2.05 ± 0.10 3.32 ± 0.37 2.55 ± 0.42 1.69 ± 0.57 2.29 ()

0.63 ± 0.06 0.45 ± 0.10 0.62 ± 0.11 0.43 ± 0.11 0.42 ± 0.00 0.38 ± 0.06 0.70 ()

All

0.48 ± 0.43

0.24 ± 0.19 0.34 ± 0.13

1.99 ± 0.85 0.53 ± 0.23 0.48 ± 0.21

0.52 ± 0.13 0.37 ± 0.09 0.30 ± 0.17

ns

ns

P = 0.015*

ns

=

0.23 ± 0.15 0.39 ± 0.02 0.29 ± 0.07 0.15 ± 0.08 0.36 ± 0.03 0.57 ± 0.22 0.36 ()

0.45 ± 0.19 0.21 ± 0.01 0.34 ± 0.11 0.44 () 0.69 ± 0.15 0.77 ± 0.13 0.78 ()

0.27 ± 0.05 0.15 ± 0.21 0.46 ± 0.02 0.70 ± 0.18 0.48 ± 0.12 0.65 ± 0.11 0.64 ()

0.52 ± 0.07 0.32 ± 0.11 0.42 ± 0.09 0.35 () 0.37 ± 0.13 0.23 ± 0.02 0.40 ()

0.22 ± 0.08 0.09 ± 0.13 0.57 ± 0.09 0.22 ± 0.01 0.27 ± 0.06 0.25 ± 0.10 0.49 ()

= = He

ns

ns

ns

P = 0.002*

ns

Data are mean ± S.D. () Only one data point. In each panel, row marked as (=) significant difference between gravity conditions as obtained from two-way Anova test. In the SF6 panel, (= He) significant difference between SF6 and He data as obtained from two-way Anova test.

Both the He and SF6 phase IV heights were significantly smaller in both ␮G–␮G and HG–␮G conditions with respect to 1G–1G. However, all phase IV heights (for both ␮G conditions and for both gases) were significantly different from zero, and averaged 27% of the 1G flight value (mean of phase IV height values for He and SF6 in ␮G–␮G and HG–␮G). There was no significant phase IV height difference between ␮G–␮G and HG–␮G conditions. A significant difference between SF6 and He phase IV height (larger SF6 ) was observed in 1G condition whereas they showed no significant difference in the two other conditions. For both gases, phase IV volumes were significantly smaller in both ␮G–␮G and HG–␮G gravity conditions with respect to 1G–1G condition. While there were occasional differences in phase IV volume between gases, these

were small and there was no consistent pattern to them (Fig. 6B).

4. Discussion 4.1. 1G data: ground versus flight Both phases III and IV obtained with this particular bolus maneuver were significantly different between 1G data obtained on the ground and in flight, and the SF6 –He phase III slope difference was actually abolished in flight. The differences in experimental conditions between those two 1G data sets were the ambient pressure and the anti-motion sickness medication taken by all subjects. Based on model simulations,

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ambient pressure change is not likely to have produced the observed modifications (Dutrieue et al., 2003, 2000). To the best of our knowledge no effects of the anti-motion-sickness drugs on airway function has been reported but as the present study was designed to evidence effects of airway closure as one particular source of ventilation inhomogeneity in ␮G, it is conceivable that drug induced airway closure effects were already present, but were not picked up by previous ventilation distribution studies. Therefore, the following discussion focuses on the flight data sets for which the experimental conditions were identical, i.e., all tests were acquired during flight at the same barometric pressure and with the same anti-motion sickness drug dosing. 4.2. Bolus tests results versus gravity condition Airway closure is believed to occur during exhalation to residual volume (at lung volumes below CC) and to be gravity dependent (Milic-Emili et al., 1966). We therefore expected that lung configuration would be different when pre-test expiration to RV was performed in HG versus in ␮G (with more airway closure in the HG condition) and that this would show up in differences in phase IV height (as a specific index of airway closure) and/or in phase III slope (as a ventilation distribution inhomogeneity index partly affected by airway closure). Contrary to our expectations however, only minor differences in both phases III and IV indices were observed between ␮G–␮G and HG–␮G conditions (Figs. 5 and 6). There were marginal differences in phase III slope and phase IV volume for SF6 . Considering the difficulty in clearly distinguishing a phase IV on some washout curves, particularly in ␮G, and that a “zero” phase IV height automatically leads to a zero phase IV volume for that test (intrinsic to phase IV height and volume definitions) the observed SF6 phase IV volume difference should not be over-interpreted. Hence, the present results show that for a 2-l SBW starting from RV there is no impact of the gravity condition during pre-test expiration to RV on the airway closure and inhomogeneity of ventilation distribution indices. In an attempt to explain the observed lack of change in airway closure related indices between ␮G–␮G and HG–␮G conditions, we nevertheless consider two possibilities. The first one is related to the experimental

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constraints (transition from HG to ␮G phase) when performing the HG–␮G bolus protocol: in this protocol, the subject expired to RV and then, had to breath-hold at RV until ␮G was achieved (∼7 s) before initiating test inspiration. Hence, it is possible in this maneuver that the lung was initially in a “HG configuration” resulting from the expiration to RV in HG, but that it internally reconfigured itself during the breath-hold and the transition from HG to ␮G. Whether such a reconfiguration to a “␮G like” lung configuration could take place within the confines of a fixed overall lung volume (a breath-hold) and within such a short time span (less than 5 s) remains unclear. Another possibility is related to changes in cardiac output. At RV, intra-thoracic pressure is increased, impeding venous return particularly when the pre-test expiration is performed in HG during which time cardiac output is minimal. A difference in cardiac output and consequently in pulmonary blood flow and pulmonary blood volume between ␮G–␮G and HG–␮G could produce changes in the conformation of the peripheral airways. Whether such a change (if indeed it occurs) alters airway closure is unclear, but such an effect at least seems plausible, although it is not possible to verify this speculation with the data to hand. 4.3. Relation to previously published results In the original context of attempting to explain the difference in the behavior of VC-SBW in transient ␮G (Lauzon et al., 1997) (Fig. 3B) versus in sustained ␮G (Prisk et al., 1996) (Fig. 3A), we speculated on the potential effect of the hyper-gravity phase on VC maneuver measurements performed in transient ␮G. Note that in the previous transient ␮G studies (Lauzon et al., 1997; Dutrieue et al., 1999) there was no breath-hold between the pre-test expiration and the onset of the test inspiration, minimizing the chance for any reconfiguration of the lung at RV. The present results obtained in an exaggerated case where the expiration prior to SBW testing was performed entirely in the hyper-gravity phase (in contrast to previous experiments where pre-test expiration mostly occurred during the transition period between HG and ␮G, without any breath hold at RV), did not show any such effect. Indeed, the airway closure lung configuration resulting from expiration to RV in different gravity condition (HG or ␮G) did not

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induce any change in airway closure indices (phase IV height and volume) nor in the index of inhomogeneity of ventilation distribution (phase III slope). Thus, the present findings tend to invalidate the hypothesis of hyper-gravity configuration at the onset of the VC-SBW in transient ␮G as a cause of the difference between sustained (Fig. 3A) and transient ␮G results (Fig. 3B). 4.4. Airway closure distribution in µG With this ventilation distribution test that was specifically targeted at studying gas mixing at low lung volumes where airway closure may play an important role even in normal subjects, the results confirm that airway closure is greatly reduced but not totally abolished under transient microgravity conditions. Closure of some airways therefore still occurs in ␮G but leads to a phase IV height reduction most probably due to the absence of the associated gravity-dependent ventilation distribution contribution (i.e., case A versus case B in Fig. 2). Those airways closing in ␮G could be the same airways as those closing at 1G. However, since there is clear evidence for preferential basal airways closure in 1G (Milic-Emili et al., 1966) related to the vertical gradient of ventilation distribution that is abolished in ␮G, this explanation seems unlikely to us. Alternatively, non-gravitationally distributed (“patchy”) airway closure could exist. The tendency towards a decreased phase IV height and volume for He versus SF6 (Fig. 6) suggests a proximity of the lung units subtended from closed airways, which favors the “patchy” airway closure model. In summary, the results show that for a 2-l inspiration SBW starting from RV there is no impact of the gravity condition during the pre-test expiration to RV on the airway closure and inhomogeneity of ventilation distribution indices (i.e. unchanged phase III slope and phase IV height and volume). This suggests that the difference in gravity condition during the expiration preceding VC-SBW performed in transient versus sustained microgravity cannot explain the differences observed. On the other hand, the present results confirm that airway closure is greatly reduced, but not totally abolished, under transient microgravity conditions and suggest that in microgravity airway closure occurs in a patchy fashion throughout the lung.

Acknowledgments The authors thank Andr´e Kuipers, John Ives, Marine Le Gouic, Mats Rieschel, Dag Linnarson, St´ephanie Montmerle and Aren Borgdorff for their contribution in performing these experiments. We also acknowledge the team of the A300-ZERO G of the Novespace/CNES for organization of the parabolic flights and Damec for building the PFS system, and support during the flight campaign. This work was supported by European Space Agency (ESA) funding for parabolic flight campaign. B. Dutrieue was supported by contract Prodex with the Belgian Federal Office for Scientific Affairs and S. Verbanck was supported by the Federal Fund for Scientific Research-Flanders (FWO). G.K. Prisk and C. Darquenne were partially supported by NASA contracts NAS9-98124 and NCC9-168.

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