INTRODUCTION

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A deep inspiratory maneuver is one of the most severe dynamic stresses that lungs normally experience. It is typically a very transient phenomenon. When one takes a sigh, the timing is such that the whole maneuver is over in about 1 to 3 s. This means that the inflation to TLC lasts only on the order of about 1 to 2 s. The airway response to such a deep inspiration has been shown to be different in asthmatic and normal subjects (1). When airway smooth muscle (ASM) is contracted in normal subjects, a deep inspiration results in a subsequent dilation of the airways. However, in asthmatic subjects, a deep inspiration often results in little change in airway function, and sometimes in an even further contraction of ASM (8, 10). The mechanism underlying this different response is still not well understood.

In considering the effects of a deep inspiration, an implicit assumption is that all of the airways are distending in concert with the lung. However, with such rapid lung inflation, it seems quite reasonable to ask if the airways have enough time to distend. For example, if the contracted airways were much more viscous than the lung parenchyma, their dilation could lag substantially behind the inflation of the lung. Additionally, the dynamics associated with stretching or disruption of the actin- myosin bonds of contracted ASM might differ considerably from those of stretching the passive structural elements of the lung parenchyma. Such dynamic behavior may play a role in the pathophysiology of asthma, but there have been no studies to date that provide insights into this differential mechanical behavior. In the present study we designed an experiment to determine how well matched the dynamic response of airways is to that of the lung parenchyma. The results clearly show that contracted airways not only dilate to a lesser extent than does lung parenchyma, but also do so at a substantially slower rate.

	METHODS

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Our study protocol was approved by The Johns Hopkins Animal Care and Use Committee. Six dogs weighing approximately 20 kg each were anesthetized with thiopental (15 mg/kg induction dose followed by by 10 mg/kg/h intravenous maintenance dose). After induction of anesthesia, the dogs were paralyzed with 0.5 mg/kg of succinylcholine, with occasional supplemental doses given as required to ensure the absence of respiratory motion during imaging. After being endotracheally intubated with an 8.0-mm I.D. endotracheal tube, the dogs were placed in the supine position and their lungs were ventilated with room air with a volume-cycled ventilator (Harvard Apparatus, Millus, MA) at a tidal volume (VT) of 15 ml/kg and a rate of 18 breaths/min. A stable depth of anesthesia was maintained by monitoring heart rate changes and eyelash reflex.

To induce a stable state of airway tone, we gave the dogs a continuous intravenous infusion of 67 µg/min methacholine (MCh) (Sigma Chemical, St. Louis, MO). For measurement of the response to lung inflation in relaxed airways, the dogs received 0.2 mg/kg of atropine. Previous work has shown that this dose of atropine abolishes baseline smooth and cholinergic muscle tone in dogs (17).

Imaging and Analysis of Airways

High resolution computed tomography (HRCT) scans were obtained with a Somatom Plus Scanner (Siemens, Iselin, NJ), using a timed mode to acquire tomographic images every 2 s at a fixed location at 137 kVp and 165 mA. The images were reconstructed as 2-mm-thick slices with a 256 × 256-pixel matrix, using a maximum zoom of 4.0 (12-cm field of view) and a high spatial frequency (resolution) algorithm that enhanced edge detection, at a window level of 450 HU and a window width of 1,350 HU. These settings have been shown to provide accurate measurement of airway lumen size in airways as small as 2 mm (18, 19). For repeated airway measurements in a given dog within each experimental protocol, we defined adjacent anatomic landmarks, such as airway or vascular branching points, and matched and measured the airways on the basis of these adjacent landmarks.

The HRCT images were analyzed with the airway analysis module of the Volumetric Image and Display Analysis (VIDA) software package (Department of Radiology, Division of Physiologic Imaging, University of Iowa, Iowa City, IA) as previously described and validated (17, 20). The HRCT images were transferred to a UNIX-based Sun (Palo Alto, CA) workstation. An initial isocontour was drawn within each airway lumen, and the software program then automatically located the perimeter of the airway lumen by sending out rays in a spokewheel fashion to a predesignated pixel intensity level that defines the luminal edge of the airway wall. We (19) and others (20) have previously shown the intra- and interobserver accuracy and variability of the software program with use of this HRCT technique on phantoms consisting of rigid tubes, for the purpose of measuring known areas, to be highly resistant to operator bias.

Protocol

The tubing from the ventilator to the endotracheal tube of the animals had an added large-bore Y-connector. One branch of the Y went to the ventilator and the other branch was connected to a constant-pressure source at 37 cm H₂O. This source consisted of an underwater overflow fed by a line from a high-flow oxygen supply. At the start of scanning, the ventilator was shut off while a solenoid valve to the ventilator was simultaneously closed and another solenoid to the pressure source was opened to the dog. The rapid airflow to the lungs was measured with a pneumotachograph in the limb of the respiratory circuit in which pressure was constant. To measure the airway responses to dynamic lung deflation, the solenoid was switched to suddenly expose the trachea to atmospheric pressure.

Dynamic HRCT Measurements

To make measurements of airway size over a period time during lung inflation, one must be able to clearly find the same airway location over the course of inflation. One problem in meeting this requirement is that with change in airway pressure, the airway and lung not only inflate, but the lung also expands caudally as it inflates. Measurement of a specific airway during inflation therefore requires repeated scanning at different positions along the spinal axis of the dog during the inflation maneuver. To accomplish this scanning over time and space we repeated the sudden lung inflation maneuver multiple times, while moving the fixed scanning position after each rapid inflation and deflation (i.e., at each fixed scanner position we acquired HRCT scans every 2 s during the rapid lung volume changes). This was then repeated as often as needed, with the scanner position moved sequentially in a caudal direction until the airways were scanned at each time point of inflation and deflation. This procedure thus enabled us to acquire a complete three-dimensional block of slices at discrete 2-s time points.

Static HRCT Measurements

To quantify the absolute lung volumes at FRC and TLC, we obtained conventional 8-mm-thick computed tomographic slices with the airway pressure at 0 cm H₂O and 37 cm H₂O. The procedure we used was described in previous communications (17, 21). Briefly, the area of the lung on each HRCT scan was defined as the area within the pleural border, excluding the heart and diaphragm. The edge of the lung was defined by the VIDA edge-detection algorithm. All lung slice areas were then multiplied by the slice thickness and summed to calculate the total lung volume for each pressure.

Data Analysis

The completely relaxed airway, after administration of atropine at 37 cm H₂O was defined as 100% (relaxed state, maximum size), and airway luminal areas were expressed as percents of this maximally relaxed area. A plateau was defined as a nonstatistical change in airway area at the subsequent time point. Each airway in each dog served as its own control. A paired t test was used to compare the airway size during inflation and deflation during MCh-induced constriction and after relaxation with atropine, with the Bonferroni-Dunn correction applied for multiple pairwise comparisons. A value of p  0.05 was considered significant.

	RESULTS

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Figure 1 shows the mean time response of lung volume after the sudden increase in airway pressure and its subsequent release. Since our system for rapidly inflating the lungs utilized a stepwise change in airway pressure, the speed at which the lung inflated depended entirely on the mechanical time constant of the respiratory system. The atropine-relaxed lung increased in volume with about a 1-s time constant on both inflation and deflation (0.91 ± 0.08 [mean ± SEM] s and 1.19 ± 0.22 s, respectively). With MCh, the inflation and deflation time constants were 1.2 ± 0.18 s and 0.96 ± 0.06 s, respectively. None of these time constants was significantly different from the others. With a 1 s time constant, it takes 3 to 4 s for the lung volume changes to be completed. Thus, our "rapid" inflations and deflations were substantially slower than might normally occur in humans or dogs, and the impact of this will be subsequently discussed further.

View larger version (11K): [in this window] [in a new window]   Figure 1.   Percent changes in lung volume with a stepwise increase and decrease in airway pressure averaged over all six study dogs. In each animal the 100% value is taken as the value after 20 s at an airway pressure of 37 cm H2O. The absolute FRC and TLC in atropine-relaxed lungs (mean ± SEM) were 1,190 ± 130 ml and 2,759 ± 380 ml, respectively. During MCh infusion the absolute FRC and TLC were 1,149 ± 122 ml and 2,552 ± 265 ml, and these were not significantly different from the values with atropine. The mean values for inspiratory capacity with atropine and MCh were 1,388 ± 230 ml and 1,347 ± 217 ml, respectively.

A total of 54 airways in six dogs were matched and measured (range: 1.9 to 12.7 mm I.D.). Figure 2 shows the mean responses of airways to the sudden lung inflation and deflation in MCh-contracted and atropine-relaxed airways. In atropine-relazed airways, the mean airway luminal size at FRC was 63 ± 3% of maximum. When the lungs were inflated to 37 cm H₂O, the luminal size of the relaxed airways increased to 98 ± 1% of their maximum size, which was not significantly different from their maximum size (p = 0.07). Furthermore, all relaxed airways reached a plateau in size within 6 s (p = 0.40 as compared with the next time point) after inflation of the lungs was begun, and remained at a steady plateau for the remainder of the imaging series (18 s). When the pressure was released and the trachea was suddenly opened to atmospheric pressure, the relaxed airways rapidly returned to their preinflation size of 63 ± 3% of maximum size (p = 0.34 compared with preinflation). Moreover, the time course of the deflation of the relaxed airways was at least as short as that during inflation, with all airways returning to preinflation size within 6 s (p = 0.17) after the release of positive pressure. Unfortunately, the dynamic response of the relaxed airways upon inflation and deflation could not be fitted with exponential equations, since most of the change in airway area had already occurred by the time we could make our first measurement at the 4-s time point.

View larger version (14K): [in this window] [in a new window]   Figure 2.   Changes in mean airway area averaged across all dogs and expressed as a percent of maximal airway area. The maximal area for each airway was taken to be the airway area after 18 s at an airway pressure of 37 cm H2O in atropinized animals.

The airway response to rapid inflation during MCh infusion was both diminished in magnitude and of substantially longer duration than when the airways were relaxed. During MCh infusion, the mean airway size at FRC was significantly decreased, to 26 ± 1% of maximum (p < 0.0001 compared with the relaxed airways). When the lungs were inflated to 37 cm H₂O, the area of MCh-constricted airways increased to only 78 ± 4% of maximum, which was significantly less than when the airways were relaxed (p < 0.0001). Furthermore, it took considerably longer for the airways to reach a plateau than in the relaxed state. MCh-constricted airways did not reach a plateau in size until 12 s after the pressure increase. We fitted the airway inflation curves to a single exponential equation and found an average time constant on inflation of 3.7 s. When the airway pressure was suddenly decreased to atmospheric pressure, the MCh-constricted airways returned to their preinflation size of 27 ± 2% of maximum size (p = 0.34 compared with preinflation), and this airway deflation took at least as long as the inflation. The constricted airways returned to preinflation size at 14 s (p = 0.06) after the release of positive pressure. This slower recovery on deflation was manifested in a slightly larger mean time constant of 4.3 s.

	DISCUSSION

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Our findings clearly show that dilation and recovery of contracted airways can substantially lag behind the volume changes associated with inflation and deflation of the lung. Without smooth-muscle tone, airways appear to follow changes in lung volume more closely. Unfortunately, the temporal and dynamic resolution of our HRCT scanning system did not allow us to resolve changes in the first 2 s after the stepwise changes in airway pressure, so we could not quantify any potential lag between relaxed airway and lung responses. However, if there is a difference, it must be very small. This seems logical, since the time constant of changes in lung volume was about 1 s (reaching a steady state in 4 s), and the relaxed airway responses were 90% complete in 4 s.

Technical and mechanical limitations also prevented us from producing inflation of the lungs as rapidly as we would have in order to mimic what might occur in conscious animals. Since we inflated the animals' lungs from a constant-pressure source, the time to full inflation was dependent on the resistive and compliant properties of the lung. With this constraint and a respiratory system time constant of about 1 s, it took 3 to 4 s to completely inflate or deflate the lungs. In human subjects undertaking a voluntary inflation to TLC, the inspiratory muscles do not drive the lung with a constant pressure. Rather they can adjust the pressure to maximize inflation. Although it is experimentally possible to design a system to drive the canine lungs more rapidly (e.g., with knowledge of each animals' inspiratory capacity and a piston rapidly driving in the appropriate volume), such a system was not available to us for animals in the CT scanner. Such rapid inflations near TLC might also result in unacceptable lung damage in the chronic animals studied.

We found that the time constants for distention and relaxation of contracted airways were three to four times greater than that of the lung. If this property exists in human lungs, it provides a mechanism that might account for the different responses to deep inspiration in normal and asthmatic subjects (1). With the not unreasonable hypothesis that seasonal low levels of allergen-induced contraction cause asthmatic airways to have increased muscle tone and viscosity, for example, the effect of this increased basal tone might be to minimize the substantial airway distention that would otherwise occur during inflation to TLC with a deep inspiration. Normal deep inspiratory maneuvers can be very rapid, sometimes being completed in just a second or two. Our present results would suggest that in such a short period there may be very little dilation of airways that have smooth-muscle tone, so that with a return to FRC and concomitant decrease in lung recoil pressure, the contracted airways might show further constriction. In our animals, we found an inflation time constant in contracted airways of 3.7 s. If with this single-exponential model of airway distension we could have inflated the lungs to TLC in just 1 s, there would have been an airways dilation of only about 24%.

Lim and colleagues demonstrated that the airways of asthmatic subjects responded differently than normal airways to stretch, whether they were constricted by an exogenous pharmacologic challenge or through intrinsic mechanisms (14). Therefore, an intrinsic cause of bronchoconstriction may also affect both the time and magnitude of airway distention. The other important factor is clearly the duration of the deep inspiratory maneuver itself. A slower inspiratory expansion, or simply more time at TLC during a deep inspiratory maneuver, may lead to substantial dilation even of contracted airways. Indeed, with steady-state changes in lung volume, the airways of subjects with mild asthma were recently shown to distend to a similar extent as those of normal subjects both at baseline and after MCh challenge (22).

One other technical consideration that warrants discussion is the mode of challenge. In the present study we used a continuous intravenous infusion of MCh. We have used this in previous studies and have found that it is well tolerated by animals (17). The advantage of this mode of challenge in these experiments is that it provides a stable level of airway constriction over the time needed for multiple lung inflations. Atropine has also been shown to readily reverse any residual contraction after stopping the MCh infusion. In the cited studies of the effect of deep inspiration in humans, the MCh challenges were always done with an aerosol. The transient nature of this aerosol-induced contraction may also have some impact on the response to deep inspiration. In the study by Lim and colleagues (14), in which the response to deep inspiration was examined after airway contraction induced by an allergic asthmatic attack, the airway contraction might have been more stable than that induced with a single aerosol challenge. Such contraction may be better modeled by the stable kind of challenge that we used in our study.

Interestingly, we did not find significant differences in the inflation time constants of the lung during the MCh infusion as compared with the relaxed state. When the whole lung is challenged with a continuous infusion of MCh, all ASM is contracted, resulting not only in airway narrowing, but also presumably in parenchymal stiffening (23) and increased tissue resistance. Since as a first approximation the time constant is the product of compliance and total resistance in series, this observation suggests that the increases in lung and tissue resistances with MCh infusion were balanced by the decrease in lung compliance (24). Apparently, the lag that we observed in distention of the conducting airways with rapid lung inflation does not alter the lung inflation time constant sufficiently to affect whole-lung inflation dynamics. Whether there is a similar lag in distention of the contracted smaller airways that probably dominate the changes in lung compliance and tissue resistance is not yet known.

We have not been able to locate in the literature any studies comparable to the present study that have attempted to address the relative rates of dynamic distention of airways and lung parenchyma. Indeed, the concept seems not to have even been discussed previously. There are some studies, however, that allow us to speculate on possible mechanisms by which contracted airways respond more slowly than the lung parenchyma. Intuitively, our results seem to make sense if we attribute this dynamic slowness of airways to some intrinsic properties of the smooth-muscle contractile proteins. There has been considerable interest in recent years in the plastic properties of contracted ASM (25). Mechanical plasticity would surely limit the ability of the muscle to be dynamically stretched. Another model of tonically contracted muscle, presented by Fredberg and colleagues, involves the presence of slowly cycling latch bridges in ASM (26). According to their model, such a state would produce stiff airway muscle with low hysteresis. Fredberg and colleagues, did not analyze the situation with regard to such large, rapid stresses as we imposed, but it also makes some intuitive sense to envision that a muscle with a preponderance of latch-state bridges might require extra time to break and disrupt these links. It might also be expected that the slowness of airways to return to their shortened state on deflation results from the additional time needed to reestablish the slowly cycling latch state.

In summary, we have shown that airways contracted with MCh fail to dilate at the same rate as the lung parenchyma during rapid lung inflation. This effect may play a role in the unique response of asthmatic subjects to deep inspiration. The mechanism of this dynamic slowness of contracted airways probably involves intrinsic properties of the smooth-muscle contractile processes.