INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

There is abundant evidence in the literature that airway resistance in both the unconstricted and constricted state depends on lung volume (1). Studies in humans (1) and animals (4, 5) have shown that induced bronchoconstriction is modulated or reversed at higher lung volumes and/or transpulmonary pressures (Ptp). A modification of the load against which the airway smooth muscle (ASM) contracts has been hypothesized as the mechanism responsible for this finding (6, 7). In order for ASM to shorten, it must overcome different loads, specifically the load imposed by parenchymal attachments, and the load imposed by the airway wall and the extracellular matrix (ECM) surrounding the ASM (8).

There are interspecies differences in the extent to which constriction-induced increases in airway resistance are affected by changes in Ptp. We have recently shown that constricted guinea pig (GP) airways are less sensitive to changes in Ptp than are those of the rat (5). Parenchymal tethering forces on the ASM could potentially be different in the two species because of differences in the viscoelastic properties of the parenchyma or differences in the mechanical properties of the airway wall itself. If parenchymal and airway-wall mechanical characteristics are similar in these two species, then differences in the contractile properties of the ASM may be the mechanism responsible for the difference in sensitivity to changes in transpulmonary pressure. Differences in the morphology or structure of the lung between species may also contribute to this difference.

Colebatch and colleagues (9) demonstrated several years ago that in asthmatic individuals, specific conductance is less dependent on Ptp than in normal subjects and patients with emphysema. A lack of mechanical interdependence could explain the excessive bronchoconstriction typically seen in asthmatic subjects exposed to contractile agonists. GPs similarly display excessive bronchoconstriction and lack of a plateau response (10). Investigating the mechanisms for the modest volume dependence of GP airways might provide some insights into the mechanisms responsible for excessive bronchoconstriction in asthmatic individuals.

We investigated whether the in vivo observation of differential effects of Ptp on airway resistance (Raw) in the GP and rat could be partly explained by differences in the lung-parenchymal properties of the two species. Specifically, we characterized the oscillatory mechanics of fluid-filled GP- and rat-lung parenchymal strips. Several investigators have successfully used lung-parenchymal strips to examine the tensile and viscoelastic properties of the pulmonary parenchyma (11- 14). In addition, we generated quasistatic stress-strain curves to determine whether there were interspecies differences in static mechanical properties of the lung parenchyma. We also examined the GP parenchymal strip morphometrically to determine whether its anatomic constitution was similar to that previously reported for the rat (14).

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Male Hartley guinea pigs (n = 11) and Sprague-Dawley rats (n = 10) weighing 460 ± 46 g (mean ± SD) and 435 ± 69 g, respectively, were obtained from Charles River Inc. (St. Constant, Quebec), and were housed in a regular animal facility at McGill University. Each animal was anaesthetized with a peritoneal injection of sodium pentobarbital (30 mg/kg). After tracheostomy, a metal cannula (internal diameter = 2 mm) was inserted into the trachea and tightly bound. Through an abdominal incision, the diaphragm was cut and a bilateral pneumothorax was induced. To degas the lung, the animal was mechanically ventilated at a frequency of 1.5 Hz with 100% O₂, using a small-animal respirator (Model 683; Harvard Apparatus, South Natick, MA). The thorax was opened and a positive end-expiratory pressure (PEEP) of 5 cm H₂O was applied. After 10 min the tracheal cannula was clamped and the O₂ remaining in the lungs was absorbed into the bloodstream. The animal was then exsanguinated and the heart, lungs, and trachea were carefully resected en bloc, ensuring that the lungs were not punctured. The lungs were then filled and rinsed via the trachea with a modified Krebs solution (in mM: NaCl 118, KCl 4.5, NaHCO₃ 25.5, CaCL₂ 2.5, MgSO₄ 1.2, KH₂PO₄ 1.2, glucose 10; Sigma Chemical, Inc., St. Louis, MO), with a pH 7.40 at 6° C. Lung parenchymal strips (1.5 × 1.5 × 10 mm) were cut in an orientation parallel to the pleural surface, and after the pleura was dissected, the unloaded or resting length (lr) and wet weight (Wo) of each strip were recorded. The strips were kept in a recirculating bath of iced solution, which was continuously bubbled with 95% O₂ 5%  CO₂.

Apparatus

Metal clips were glued to either end of the tissue strip with cyanoacrylate cement. Steel music wires (0.5 mm diameter) were attached to the clips, and the strip was suspended vertically in an organ bath. A mercury bead was placed in the bottom of the organ bath, allowing the wire to pass through the bath but preventing the Krebs solution from leaking out. The bath was filled with 15 ml of Krebs solution, maintained at 37° C and continuously bubbled with the 95%/O₂/5%CO₂. One end of the strip was attached to a force transducer (Model 400A; Cambridge Technologies, Watertown, MA) which had an operating range of ± 10 g, resolution of ± 200 µg, and compliance of 1 µm/g, and the other end was connected to a servo-controlled lever arm (Model 300B; Cambridge Technologies). The lever arm was capable of peak-to-peak length excursions of 8 mm and a length resolution of 1 µm, and was in turn connected to a function generator (Model 3030; B & K Precision, Dynascan Corporation, Chicago, IL) that controlled the frequency, amplitude (), and waveform of the oscillation. The resting tension (T) was set by movement of a thumbwheel screw system that effected slow vertical displacements of the force transducer. Length and force signals were converted with an analogue-to-digital converter (DT2801-A; Data Translation Inc., Marlborough, MA) and recorded on an A/T-compatible computer.

The linearity and hysteresis of the system were tested by measuring the moduli of a steel spring of stiffness comparable with that of the tissue strip. The spring was suspended in the bath by music wire in the same manner as the strip. The frequency- and amplitude-dependence of the system were assessed over a range of frequencies (0.1 to 10 Hz). The spring stiffness did not show any dependence on oscillatory frequency below 5 Hz. The hysteresivity of the system was independent of frequency and had a value < 0.003.

Protocol

Baseline measurements. Each lung-parenchymal strip was preconditioned by slowly cycling the tension applied to it from 0 to 2 g two times; one group of strips (n = 20) was unloaded on the third cycle to a tension of 1.25 g and allowed to stabilize for 60 min. The final resting tension was approximately 1.0 g. After stress adaptation, strips were oscillated at an  of 0.5% of lr and a frequency of either 0.3, 0.6, 1.0, or 3.0 Hz. Twenty-second recordings were made at every frequency. Following this  was changed to 1%, 2.5% or 5% lr, and recordings at each frequency were repeated. The second group of strips (n = 20) was unloaded to 0.75 g and again allowed to stress-adapt for 60 min; the final resting tension was approximately 0.5 g. Recordings were again made at 0.3, 0.6, 1.0, and 3.0 Hz at  of 0.5%, 1.0%, 2.5%, and 5.0% lr.

Postchallenge measurements. Following baseline recordings, strips from the two groups of resting tensions were further divided into four groups (n = 5 per group). Strips were challenged with acetylcholine (ACh) (1 mM) (Sigma) and measurements were repeated on a given strip at a single  (0.5%, 1%, 2.5%, or 5% lr) at all four frequencies (0.3, 0.6, 1.0, and 3.0 Hz). Measurements were obtained roughly 5 min after addition of ACh. (In preliminary experiments, tension increased to a plateau value within 3 to 5 min; the plateau persisted for at least an additional 5 min.)

Quasistatic length-tension measurements. Strips from an additional four guinea pigs and seven rats were studied. Each lung-parenchymal strip was preconditioned by slowly cycling the tension applied to it from 0 to 2 g two times; on the third cycle strips were loaded to a T of 1.25 g and allowed to stabilize for 20 min. Strips were then stretched to a tension of 5 g and measurements of length and T were obtained after 3 min. Length was then decreased in 0.4-mm decrements until approximately zero load. T was recorded at each step after 3 min of stress relaxation. Measurements on four of the rat and four of the GP strips were repeated after basal tone was ablated by preincubation for 20 min with isoproterenol (100 µM).

Measurement of Strip Mechanics

Elastance (E) and resistance (R) were estimated by applying the recursive least-square algorithm to the equation of motion (15):

(1)

where 1 = length, l/t is the length change per unit time, and K is a constant term reflecting the baseline tension. Baseline results were standardized for strip size by multiplying the values of E and R by lr/ Ao. Ao is the unstressed cross-sectional area of the lung-parenchymal strip, obtained from the formula:

(2)

where  is the mass density of the tissue, and is taken as 1.06 g/cm³.

Quasistatic length-tension data are presented in terms of stress versus strain, where stress is defined as T/Ao and strain as (l  lr)/lr. Quasistatic E was calculated from small stepped changes in length, as stress/strain. All data manipulations were performed with the ANADAT software package (RHT-InfoDat, Montreal, Quebec, Canada).

Morphometric Analysis

Parenchymal strips from six guinea pigs were fixed in 10% formalin under 1 g of tension for morphometric analysis. Formalin-fixed strips were processed through alcohol and embedded in paraffin. Serial longitudinal sections, 5 µm thick, were cut on a microtome, and every 20th section was stained with hematoxylin and eosin (H&E). Sections were examined with light microscopy at ×100 magnification, and an ocular equilateral triangular grid (Weibel type 2) was applied to measure the fractional area of tissue constituents using point counting. Fractional areas were measured for alveolar wall (AW), blood-vessel wall (BVW), and bronchial wall (BW). All points falling on these components were counted in consecutive nonoverlapping fields of view until all sections from each strip were counted. BVW and BW were counted when a point fell on the epithelial layer, the endothelial layer, the smooth muscle, or its associated connective tissue. Approximately 1,000 points were counted per strip (after the points falling on alveolar air space, blood-vessel lumen, and bronchial lumen were excluded). Mean relative SE values were 0.008 for AW, 0.175 for BVW, and 0.196 for BW.

Statistical Analysis

Analysis of variance (ANOVA) was used to determine the effect of frequency, amplitude, and T on E and R within a species, and to assess whether the effect in the two species was different. ANOVA was also used to determine the effect of ACh challenge on E and R, and whether this effect was different in GP and rat. Comparisons between morphologic data were made with an unpaired t test. Results were considered statistically significant at p  5%. Values are reported as mean ± SE.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Baseline Mechanics

Values of E and R as a function of resting T, f, and  in the GP and rat are shown in Figures 1 and 2. In both species, E increased at higher resting T (p < 0.001), and in the GP decreased at higher  (p < 0.001); R increased at higher  and resting T, and fell with f (p < 0.001). GP parenchymal strips, as compared with those of rats, had significantly higher baseline values of E and R (p < 0.001). The dependence of E and R on resting T was less in the GP than in the rat (p < 0.001). The effect of f and  on R was larger in the GP (p < 0.001 and p < 0.05, respectively).

View larger version (20K): [in this window] [in a new window]   Figure 1.   Effect of resting tension, frequency, and amplitude of oscillation on elastance under baseline conditions. The effect of tension on elastance was significantly different between rat and guinea pig (p < 0.001).

View larger version (18K): [in this window] [in a new window]   Figure 2.   Effect of resting tension, frequency, and amplitude of oscillation on resistance under baseline conditions. The effects of tension, amplitude, and frequency on resistance were significantly different between rat and guinea pig (p < 0.001, p < 0.05, and p < 0.001, respectively).

Agonist Response

T, E, and R significantly increased in response to ACh challenge in both the GP and rat (comparison at 0.3 Hz). The increase in T was larger in the GP parenchymal strips (p < 0.05), whereas the increase in R was larger in rat parenchymal strips (p < 0.01).

Mechanics in the Constricted State

GP parenchymal strips, as compared with those of rats, had significantly higher values of E and R (p < 0.001). Values of E and R as a function of resting T, f, and  in the two species are shown in Figures 3 and 4. Again, in both species, E increased with resting T (p < 0.001); R increased with resting T and decreased with f (p < 0.001). In the GP, R also increased with  (p < 0.05). The effect of resting T on E was larger in the rat (p < 0.01), whereas the effect of f on R was larger in the GP (p < 0.01).

View larger version (21K): [in this window] [in a new window]   Figure 3.   Effect of resting tension, frequency, and amplitude of oscillation on elastance after ACh (1 mM)-induced contraction. The effect of tension on elastance was significantly different between rat and guinea pig (p < 0.01).

View larger version (18K): [in this window] [in a new window]   Figure 4.   Effect of resting tension, frequency, and amplitude of oscillation on resistance after ACh (1 mM)-induced contraction. The effect of frequency on resistance was significantly different between rat and guinea pig (p < 0.01).

Quasistatic Stress-Strain Curves

Figure 5 shows stress-strain curves in individual rat and GP parenchymal strips. The relationship between quasistatic elastance and stress in rat and GP parenchymal strips was comparable (Figure 6). Tension in both GP and rat parenchymal strips was modified by preincubation with isoproterenol (data not shown). However, the relationship between E and stress was unaffected in parenchymal strips from both species (data not shown).

View larger version (18K): [in this window] [in a new window]   Figure 5.   Individual quasistatic deflation stress-strain curves in guinea pig and rat parenchymal strips.

View larger version (19K): [in this window] [in a new window]   Figure 6.   Quasistatic elastance versus stress in guinea pig and rat parenchymal strips. Second-order equations were fit to the data. Dashed lines represent 95% CIs.

Morphometry

Table 1 shows results of the morphometric analysis of GP parenchymal strips obtained in this experiment, and of those of rats drawn from our previous work (14). Although the amount of bronchial wall was similar in the two species, rat parenchymal strips had significantly less alveolar wall (p = 0.01) and significantly more blood-vessel wall (p < 0.01).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The results of this study show that the oscillatory mechanics of rat and GP parenchymal strips differ. Elastance, which represents the load against which ASM contracts, was less dependent on changes in tension in the GP parenchymal strip than in that of the rat. This difference could potentially contribute to interspecies differences in maximal bronchoconstriction and lung-volume dependence.

Before going on to a discussion of the specific results of this experiment, it is worth considering the comparability of GP and rat parenchymal strips as proxies for the peripheral lung. Strips were obtained from lungs of similarly sized animals. There are data to support the idea that similar-size GPs and rats have similar-sized lungs. Crossfill and Widdicombe (16) reported values for FRC of 6.2 ml/kg in rats and 6.9 ml/kg in guinea pigs. We have reported values for interalveolar wall distance (Lm) in GP and rat lung fixed at transpulmonary pressures of 4 and 3 cm H₂O, respectively, which were equivalent (64.7 ± 0.2 µm in GPs and 65.5 ± 0.7 µm in rats) (17, 18). Because Lm is related to lung volume, these data also argue that at similar Ptp values, lung volumes in the rat and GP are similar.

Although the size of these lungs was similar, the question remained of whether the composition of the peripheral lung was also comparable. To address this question we examined peripheral lung tissues morphometrically and quantitated the volume proportion of small airways, vessels, and alveolar wall in GP parenchymal strips. We compared these results with those that we previously reported in the literature (Table ). Although there was no difference in the amount of bronchial wall in the two preparations, there were significant differences in the amount of alveolar and blood-vessel wall.

Rat and GP parenchymal strips in the unconstricted state showed dependence of E and R on , f, and T, in agreement with data reported in previous studies. Hildebrandt (19, 20), as well as other investigators (21), have shown in excised lungs, as well as in fluid-filled parenchymal strips, that R depends on f and lung volume or T, and that E depends on lung volume or T. In our study, we also found a positive effect of  of oscillation on R. After ACh-induced constriction, the dependence of E on T and the dependence of R on f and T persisted.

Although GP and rat parenchyma demonstrated qualitatively similar oscillatory behavior, some quantitative differences were present. E and R in GP parenchymal strips were higher than those measured in rat parenchymal strips in both the unconstricted and constricted state. This finding is potentially important, since the external load on the ASM may be critical in determining the resting length of the smooth muscle and, thereby, where the muscle will be disposed on its force- length curve. The effect of f on R was larger in the GP during both baseline conditions and after induced constriction, whereas the effect of  on R was larger in GP parenchymal strips only during baseline measurements. However, the effect we believe to be most pertinent to understanding the interspecies difference in volume dependence of induced bronchoconstriction is that of tension, since changes in T in vitro are analogous to changes in transpulmonary pressure, or, more indirectly, in lung volume, in vivo. The effect on E of changing T (during both baseline conditions and after induced constriction) and R (at baseline) was significantly greater in the rat.

The T values at which oscillatory measurements were made in GPs and rats corresponded to stresses of roughly 25 and 45 g/cm² (stress equals tension/cross-sectional area). We used relatively small strips, since in larger strips or strips that are not directly subpleural, the volume proportion of small airways increases, and their contribution to contractile responses becomes significant (14). It is difficult to translate in vitro stresses into their in vivo equivalents. Mead and colleagues (6) have argued that the stress sustained by the parenchyma in an isotropic lung is equivalent to Ptp. However, after induced constriction, peribronchial pressure may be as much as 50 cm H₂O higher than Ptp (24). Furthermore, there is no contribution from surface tension in the in vitro fluid-filled preparation. Surface tension is important in stabilizing alveolar geometry and thereby modulating local tension (25). Nonetheless, we chose to examine mechanics at the lowest stresses technically feasible, which we believe were within the higher range of loads to which the airway wall and parenchyma would be exposed in vivo.

The relationship between E and T in these strips was qualitatively similar to corresponding previous data obtained in vivo (5). The percentage increase in dynamic elastance (Edyn) as Ptp was increased from 3 to 11 cm H₂O under baseline conditions and after induced constriction in intact rats and GPs is shown in Figure 7. Note the similar differential in the relative dependence of Edyn in the two species, as demonstrated in the current in vitro study. In the current study, rats showed a greater relative increase in E at the higher resting tension (ANOVA for all amplitudes and frequencies, p < 0.001; see RESULTS). In the in vivo study, the relative increase in E at the higher Ptp in rats was greater than in GPs under baseline conditions (p < 0.05). After induced constriction, the difference was not significant, but a similar trend was observed. It is likely that after induced constriction, airway heterogeneities contribute to the increase in Edyn (26). Hence, the in vivo signal would not measure purely "tissue" properties as in the current study. Nevertheless, these data demonstrate a similar differential in the relationship between E and Ptp or T in vivo and in vitro.

View larger version (25K): [in this window] [in a new window]   Figure 7.   Percent increase in dynamic elastance with increasing resting tension (0.5 to 1.0 g) (in vitro) and Ptp (3 to 11 cm H2O) (in vivo) in rats and guinea pigs under baseline conditions (A) and after methacholine-induced constriction (B). (Data from the current study for both rats and guinea pigs at a single amplitude,  = 1%, and frequency of oscillation, f = 1Hz, are presented in a similar format to that of the in vivo study to aid comparison.) *p < 0.05 versus guinea pig. In vivo results are taken from Nagase and colleagues (5). The percent increase in elastance in rat versus guinea pig parenchymal strips was different under both baseline and constricted conditions when data at all frequencies and amplitudes of oscillation were compared (ANOVA, p < 0.001, see RESULTS).

The elastic properties of the lung parenchyma and their alterability with changes in Ptp or tension will be important in determining the lung-volume dependence of Raw. When noncartilaginous airways constrict, the surrounding parenchyma undergoes stretch and distortion. The excess load offered by the parenchymal attachment will depend upon the relationship between parenchymal stiffness and tension or Ptp. The effect will be tempered to some extent by the proportional increase the excess load represents (i.e., whether the extra load is a large or small fraction of the total load). Nonetheless, as shown in this study in vitro and from the results of the previous study in vivo, the relationship between stiffness and load is different in rats and GPs. This could potentially account for observed differences in lung-volume dependence of Raw.

We also made measurements of E during quasistatic conditions, in order to compare dynamic and static characteristics. The effect of stress on quasistatic E was not statistically different in strips from the two species. Thus, the relationship between E and stress or tension varied between the two species under dynamic versus quasistatic conditions. Oscillation, in and of itself, can alter the mechanical characteristics of the smooth muscle present in the preparation (27), and may explain the difference between static and dynamic results. Because tissue normally oscillates during tidal breathing, we believe that the dynamic, rather than quasistatic, characteristics are most pertinent to the in vivo state.

When the parenchymal strips were preincubated with isoproterenol, T decreased, demonstrating the presence of basal tone. However, the relationship between stress and stiffness did not change. The constant relationship between stress and E during dynamic oscillation has been previously demonstrated by Fredberg and associates (13), who showed that the slope of E versus T in GP parenchymal strips was similar whether stress was modified by passive stretch or by smooth-muscle activation or relaxation. Insofar as this relationship was independent of the state of smooth-muscle activation, the different effect of stress on stiffness between the rat and GP should not be due to a difference in resting tone, but should rather reflect an intrinsic difference in the mechanical properties of the parenchymal tissues.

From these experiments we cannot comment on the relationship between E and L, since the variable that we altered was stress or tension. It is possible that interspecies differences in the relationship between elastance and length also contribute to the interspecies difference in volume-or pressure-dependence. Furthermore, the actual starting L of parenchymal elements could be different in the rat and GP. The morphometric data are interesting to consider in this regard. The volume proportion of alveolar wall was significantly greater in GP parenchymal strips than in those of the rat. This difference could play a role in the transmission of stress to the airway wall via the parenchymal attachment.

Another potential source of parenchymally related load operating against smooth-muscle shortening is lung-tissue resistance (Rti) (28). Rti increases after contractile stimulation (2, 28), and is likely to be even higher around constricted airways, since parenchymal attachments will be distorted by airway constriction, with the effect that the parenchyma surrounding the airways will be operating at an effectively higher lung volume, and Rti is increased at higher lung volumes (2). Although the effect of tension on R was greater in rat than in GP parenchymal strips under baseline conditions, this difference did not persist after the strips were constricted with ACh. Moreover, data from the in vivo study showed no difference in the dependence of tissue resistance on Ptp in the two species (5).

In conclusion, we have shown in this study that rat parenchymal strips show a greater increase in dynamic E when tension is increased than do GP parenchymal strips. This could, in part, explain the observation that constricted rat airways are more sensitive to changes in Ptp, since excessive airway narrowing is reversed or modulated through the development of high local impedances. Differences in the elastic properties of the lung parenchyma are therefore one potential source of differences in bronchial hyperresponsiveness and the volume dependence of Raw between species. Further studies, with the goal of characterizing the shear properties of rat and GP parenchyma, are warranted to more completely define how differences in elastic properties between species contribute to differences in interdependence of the airway and parenchyma.