INTRODUCTION

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TO CONSTRICT, intraparenchymal airways have to overcome several mechanical loads, including that offered by the parenchymal attachments tethering the airway wall (4, 12, 13). When airways constrict, the parenchyma surrounding the airway undergoes stretch and distortion. In addition, the parenchyma itself may respond to contractile agonists via the stimulation of myofibroblasts and/or smooth muscle within the alveolar duct (7).

The forces opposing airway narrowing include the impedance offered by the resistance of the parenchyma to uniform expansion [bulk modulus (k)] as well as the resistance to isovolume shape distortion [shear modulus (µ)] (20). Because the resistance to shape change will have a greater impact in determining the local factors opposing narrowing of a given intraparenchymal airway (8), µ likely represents the greater load countering airway smooth muscle shortening. Although data are available in the literature for k and µ in the unconstricted state (6, 8, 9, 21), relatively little has been published on the effects of smooth muscle activation on these parameters (17). If, indeed, the parenchyma can respond to contractile activation resulting in alteration of the elastic moduli, this has important implications for the factors modulating airway narrowing, in terms of the load impeding airway smooth muscle shortening.

With regard to the response at the parenchymal level, many authors have shown that both quasi-static and dynamic elastance increase after the administration of exogenous constriction (2, 16, 18). By implication, k should change. However, whether µ is also affected is less clear. We have reported morphological data showing substantial distortion at the parenchymal level after both endogenous and exogenous constriction (14-16). More recently, Adler and colleagues (1) have shown in isolated rat lung explants that parenchymal distortion around constricting airways is quite heterogeneous in nature. This implies that shear properties after induced constriction may vary markedly at the local level.

To address the question of changes in material properties after induced constriction, we excised rat lungs and measured these moduli before and after methacholine (MCh) stimulation. Measurement was made of k during small-volume oscillations on the deflation limb of a pressure-volume curve. By using the punch-indentation test, µ was measured.

	MATERIALS AND METHODS

Animal preparation. Male Sprague-Dawley rats, weighing ~400 g, were studied. After anesthesia was induced by pentobarbital sodium (50 mg/kg ip), animals were tracheotomized by inserting a metal cannula (internal diameter of 2 mm) into the trachea. The abdomen was opened, and animals were exanguinated by severing the inferior vena cava. The thorax was widely opened by means of a midline sternotomy, the lungs were excised, and a piezoresistive microtransducer (Endevco 8510B-2) was placed in the lateral port of the tracheal cannula.

Measurement of k. Three slow inflation-deflations from 0 to 25 cmH₂O transpulmonary pressure (Ptp) were performed by using a graduated syringe. During the third deflation, a standard pressure-volume curve was obtained. Ptp was measured during 0.5- to 1-ml step volume decrements after 60 s of stabilization. Lungs were than slowly reinflated to 25 cmH₂O, a small-animal ventilator (model 683, Harvard Apparatus, South Natick, MA) was connected to the tracheal cannula, and the lungs were slowly deflated to 10 cmH₂O Ptp. Volume oscillations of 0.3 ml were superimposed at a frequency of 60 breaths/min. Tracheal flow was measured by means of a Fleisch pneumotachograph (no. 00, Instrumentation Associates, New York, NY). A 30-s recording was obtained of tracheal pressure and volume (calculated by integration of the flow signal). Ptp was then reduced to 4 cmH₂O, and tidal volume oscillations were repeated. The lungs were then deflated to 0 cmH₂O Ptp, and the absolute lung volume at 0 Ptp was measured by water displacement (tissue volume was subtracted after the lungs were weighed, assuming that tissue specific weight = 1.06 g/cm²).

Measurement of µ. Lungs were connected to a continuous airflow source, slowly inflated to 25 cmH₂O, and deflated to either 4 or 10 cmH₂O Ptp in random order. Lungs were positioned on a balance (Mettler-Toledo, BB120 CH-8606, Greifensee, Switzerland, with a readability of 0.01 g, reproducibility of 0.003 g, and linearity of 0.01 g), and the indentation test was performed by positioning a flat punch (diameter 0.45 cm) on the lung surface. Approximately 1 g of indentation force was applied, and, after 3 min of stabilization, incremental 0.25-mm displacements were imposed with the punch. Force was recorded after 30 s of stabilization. Lungs were reinflated to 25 cmH₂O Ptp and deflated to the other Ptp (4 or 10 cm H₂O), and recordings were repeated.

MCh-induced constriction. Lungs were inflated to 25 cmH₂O Ptp, deflated to 4 cmH₂O, and reconnected to the ventilator, and MCh (100 mg/ml) was delivered by aerosol (Ultra-Neb 100, DeVilbiss, Somerset, PA) for 90 s at a flow rate of 0.5 l/min. A standard pressure-volume curve and measurement of k were obtained as described above. Absolute lung volume at 0 cmH₂O Ptp was also measured. Lungs were then reinflated to 25 cmH₂O Ptp, and deflated to 4 cmH₂O, and measurements of µ were repeated. (During these measurements, supplemental MCh was not delivered, so there may have been some decline in the level of induced constriction. Nonetheless, the lungs were stiffer than before constriction.)

Calculations. At 4 and 10 cmH₂O Ptp, k was calculated from small-volume oscillations as where V is the absolute lung volume and dP and dV are the changes in transpulmonary pressure and lung volume during small-volume perturbations, respectively. Calculation of µ was made from where µ is shear modulus, G is force, w is displacement of the punch, and D is diameter of punch. To define the G/w ratio in a given lung, we fit a linear regression to the G vs. w data.

Data analysis. Paired t-tests were used to analyze the difference in µ and log k measured at 4 and 10 cmH₂O Ptp under baseline conditions and at 4 cmH₂O Ptp before and after MCh aerosol. Values are reported as means ± SE.

	RESULTS

Figure 1 shows the absolute lung volumes at 0, 4, and 10 cmH₂O Ptp under baseline conditions and after induced constriction. These data are derived from the pressure-volume curves. Increasing Ptp caused an expected increase in lung volume. Although the amount of air remaining in the lungs at 0 cmH₂O Ptp was increased after MCh, compared with control conditions, the volume of air in the lungs at 4 cmH₂O Ptp was equivalent to that under control and constricted conditions.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Lung volume at 0, 4, and 10 cmH2O transpulmonary pressure (Ptp) under baseline conditions and after induced constriction. MCh, methacholine.

Changes in k with increasing Ptp and induced constriction are shown in Figure 2. Increasing Ptp caused a significant increase in k. The increase in k at 4 cmH₂O Ptp after induced constriction was also statistically significant.

View larger version (9K): [in this window] [in a new window]   Fig. 2.   Bulk modulus (k) at different Ptp values and after MCh-induced constriction. * P < 0.001 vs. baseline, 4 cmH2O. # P < 0.05 vs. baseline, 4 cmH2O.

Figure 3 shows typical force-displacement curves obtained during measurement of µ in a single lung. As predicted, G vs. w is described by a linear relationship. The effects of Ptp and induced constriction on µ are presented in Fig. 4. Both increasing Ptp and administration of MCh caused significant increases in µ.

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Force (G) vs. displacement (w) curves under different experimental conditions in a single lung sample.

View larger version (9K): [in this window] [in a new window]   Fig. 4.   Shear modulus (µ) at different Ptp values and after induced constriction. * P < 0.02 vs. baseline, 4 cmH2O; # P < 0.05 vs. baseline, 4 cmH2O.

	DISCUSSION

In this study we have shown that the k and µ of the lung parenchyma increase after smooth muscle activation. This has important implications for the mechanisms limiting airway narrowing.

Before going on to discuss the findings of our study, some technical issues deserve consideration. Most of the studies published to date in which µ has been measured with the punch-indentation technique have used larger animals, i.e., dogs or rabbits (6, 8, 9, 21). The measurements reported in this study are the first in a small animal, such as the rat. As such, the size of the punch relative to the overall lung deserves some comment. One must balance the requirement for a punch that is large enough to minimize the contribution of the pleural membrane to the measurement of µ but small enough so that the apposition between the punch and the pleural surface of the lung is flat and smooth (6). Moreover, the punch-contact area should be two to three punch-diameter distances from the lobe edge to minimize the edge effects on the measurement of µ (3). We used a punch with a diameter of 0.45 cm. After preliminary trials with punches of varying diameters, this represented a diameter that was sufficiently small compared with the surface area of the lobe and with which there was flat contact with the lung surface but sufficiently large so as to minimize the effects of the pleural membrane. We believe the size of the punch was appropriate because our measurements at baseline were similar to those reported in other species (6, 21).

To make meaningful measurements of k and µ before and after induced constriction, it is necessary that comparisons be made at similar lung volumes because both k and µ increase with lung volume (8). At residual volume, there was substantially more trapped air in the constricted lungs, and at 10 cmH₂O Ptp lung volume was different, in a comparison of baseline with constricted conditions. Therefore, we made measurements at 4 cmH₂O only because, at this Ptp, the overall lung volumes were equivalent. We cannot be sure, however, that the volume was homogeneously distributed in the constricted lung. There was a minimum of gross heterogeneity in the lung, and we performed indentations in areas that appeared to be homogeneously inflated. Nonetheless, we cannot exclude the possibility that, in the regions we were sampling, there may have been some heterogeneity in the volume distribution or that distortion was of a heterogeneous nature. This could potentially cause higher estimates of k and µ. Alternatively, one may regard this heterogeneity as an important part of the signal, as suggested by the data of Adler et al. (1).

Values for k and µ have been reported by Lai-Fook and colleagues (6, 8, 9) and Stamenovic and Yager (21) in dog, pig, horse, and rabbit lungs. Values for k in dog lungs were slightly lower than those reported here in rats; values in rabbit lungs were quite similar. Values for µ were almost equivalent (within 2-fold) in all species studied. In addition, the relationship among k, µ, and Ptp was similar in the rat lung to that previously reported in other species (8, 21). After induced constriction, values of k and µ increased significantly. Under control conditions, the µ-to-Ptp ratio was equal to 0.96; after induced constriction, the µ/Ptp ratio corresponded to 1.52. One potential mechanism to explain the increase in µ and k involves constriction of contractile elements at the parenchymal level. Kapanci et al. (7) were the first to describe myoepithelial cells at the alveolar level that had the capacity to contract. Other investigators have subsequently made similar observations (5). These cells contain contractile elements and respond to hypoxia as well as contractile agonists. They have been postulated to account for observed increases in tissue resistance after contractile stimulation (16, 18) and could well account for increases in the elastic moduli of the parenchymal tissues. A second mechanism relates to constriction-induced heterogeneous distortion at the parenchymal level. We have shown in morphological studies that stimulation with contractile agonists, administration of hyperpnea challenge to guinea pigs, and delivery of aerosolized allergen to sensitized rats can all result in substantial parenchymal distortion (14-16). More recently, Adler et al. (1) have shown, by using isolated lung explants, that airway constriction results in heterogeneous distortion of the surrounding alveolar architecture. They did not address the issue of whether this reflects direct contraction of the parenchyma or is simply a heterogeneous response of the connective tissue matrix to constriction of subtended airways due to local differences in the structural makeup of the parenchyma. Under either circumstance, such heterogeneity would be predicted to cause local variations in the bulk and shear properties of the alveolar tissues. Finally, constriction-induced alterations in parenchymal geometry could cause increases in µ and k secondary to effects on surface tension (21).

That k and µ can change in response to contractile stimulation has important implications for constriction-induced airway narrowing. The airway wall is set within the connective tissue matrix, in a "parenchymal tunnel," tethered by parenchymal attachments. The tethering force of the lung parenchyma will be determined by the lung volume, which will, in part, determine on what portion of the length- tension curve the parenchyma is positioned, the number of airway parenchymal attachments, and the bulk and shear properties of the lung parenchyma. In determining peribronchial pressure (8), µ is the key factor. Whenever an airway does not expand uniformly with the remainder of the lung, local shape distortions will occur in the parenchyma (19). With increasing lung volume, the stress accompanying shape distortion should be relatively small. However, if µ increases with induced constriction, the stress secondary to local deformation may be enhanced. Sasaki and colleagues (19) reported several years ago that parenchymal recoil in regions surrounding constricting airways could be substantially greater than that of the overall lung. Moreover, if the airway diameter is equal to or smaller than the parenchymal tunnel, increases in µ would reduce the bronchoconstriction that would occur with smooth muscle contraction and increase the bronchodilating effects of an increase in lung volume.

Recently, Lambert and Paré (10) have modeled the relationship between the airway wall and parenchymal µ and concluded that, at low recoil pressures, µ plays a key role in determining airway narrowing. Any stimulus that augments the degree of local nonuniformities will have a further impact on the modulating role of µ on airway narrowing. The data of Adler et al. (1) showing heterogeneous parenchymal distortion, our previous morphological data during induced constriction (14-16), and the data of the present experiment demonstrating an increase in µ with MCh stimulation all further establish the importance of µ in modulating airway narrowing.

These data also have implications for published studies in which the effect of external load on airway smooth muscle shortening is modeled (10, 12). In such studies, µ has been modeled as either a constant (12) or as a variable (10). Our data suggest that the latter approach is one that more closely approximates experimental conditions. Indeed, in the model of Lambert and Paré (10), when the lungs were at a Ptp of 4 cmH₂O, changing µ by a factor of only one-third altered the maximal increase in airway resistance by a factor of more than twofold. This suggests that modest changes in µ can have a major impact on changes in airway caliber induced by constriction.

In conclusion, the material properties of the lung parenchyma change with induced constriction. If load is a key factor limiting bronchoconstriction in normal airways compared with those in asthmatic subjects (11), differences in the bulk and especially the shear properties of normal vs. asthmatic lungs, and/or differences in the responsiveness of the parenchymal tissues to contractile stimulation, may contribute to the inability of asthmatic airways to stop narrowing.