INTRODUCTION

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Keywords: rabbit; explants; smooth muscle; lungs; airway

Previous studies have demonstrated that methacholine (MCh)- induced bronchoconstriction in vivo produces greater maximal increases in pulmonary and airway resistances in immature than in mature rabbits (1). Because maximal tension generation is greater in tracheal smooth muscle (TSM) strips from mature than immature rabbits (3), and because the velocity of TSM shortening is reported to be similar in both age groups (6), the greater airway narrowing in immature rabbit lungs may be secondary to maturational differences in the elastic load that resists airway narrowing. We have recently demonstrated that the shear modulus of the lung parenchyma is lower in immature than mature rabbit lungs (7) and that intraparenchymal airways are more distensible in immature than in mature rabbit lung (8). In this current study, we use explant lung slices from immature and mature rabbits to assess maturational differences in the dynamics of airway narrowing. In contrast to in vivo studies in which maximal agonist delivery may be limited by aerosol or intravenous delivery, the explants allow direct delivery to the airway smooth muscle (ASM). In addition, use of explants enables direct visualization of both the rate and magnitude of narrowing of numerous intraparenchymal airways in living tissue, in contrast to the assessment of only maximal airway narrowing in lungs fixed after maximal constriction. We hypothesized that if the elastic load that resists airway narrowing was lower in immature than mature rabbit lungs, then maximal stimulation of lung explants should result in a greater peak velocity of shortening as well as a greater magnitude of airway narrowing in the immature lung.

	METHODS

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Animals

All procedures were reviewed and approved by the McGill University Animal Ethics Committee. Mature (3 mo; 2.8 to 3.8 kg, n = 5) and immature (3 wk; 400 to 600 g, n = 3) male New Zealand White rabbits were obtained from Charles River (St. Constant, QC) and housed in a conventional animal facility at our laboratory.

Preparation of Explants

The preparation of rabbit lung explants was similar to that previously described for rats, mice, and human lungs (9). After deep anesthesia of the rabbit with sodium pentobarbital (40 to 60 mg/kg; MTC Pharmaceuticals, Cambridge, ON), a midline incision from the neck to the lower abdomen was made and the trachea exposed and cannulated with an appropriate size endotracheal tube. The abdominal wall was incised and the animal exsanguinated by cutting the inferior vena cava. The anterior chest wall was opened and the heart-lung block removed. The lungs were inflated to TLC with warm (37° C) agarose-BCM (bicarbonate-buffered culture medium; 1% wt/vol). The agarose-inflated lungs were refrigerated at 4° C for 30 min to gel the agar. Right and left lung were then separated, and each was embedded in a modified syringe tube (60 ml for mature and 20 ml for immature) filled with 4% (wt/vol) agarose and allowed to gel at 4° C. The blocks of agarose-embedded lungs were progressively extruded from the syringe and cut into 0.5-mm-thick explant slices. Explant slices were examined under a dissecting microscope, and intraluminal agar was removed from the airway by gently folding the tissue slice to extrude the plug from the airway lumen. Each slice was cultured in 1 ml BCM at 37° C with 5% CO₂ overnight in a 6-well plate. The next day, cultured lung explants were transferred in 1 ml N-2-hydroxyethylpiperazine- N'-2-ethanesulfonic acid (HEPES)-buffered culture medium (HCM) in each well, washed slowly, and placed on the temperature-controlled stage (37° C) of the inverted microscope equipped with a video camera (model CCD-200-R; Videoscope, Washington, DC). Each slice was inspected and a single airway was selected for study; we excluded airways with a major to minor diameter ratio > 2 by visual inspection and airways that still contained agarose.

Culture Medium

BCM was prepared with 9.52 g/L Earle's minimum essential medium (MEM) powder containing Earle's salts and L-glutamine (GIBCO, Burlington, ON, Canada), supplemented with 2.2 g/L sodium bicarbonate (Fisher, Nepean, ON, Canada), 20 ml/L MEM amino acid solution (50×; GIBCO), 10 ml/L sodium pyruvate (100×; GIBCO), 10 ml/L multi-vitamin solution (100×; GIBCO), 1.0 µg/ml bovine insulin (Sigma Chemical, St. Louis, MO), 0.1 µg/ml vitamin A (GIBCO), 0.1 µg/ml hydrocortisone (Sigma), and 50 µg/ml gentamicin (GIBCO). A BCM with double amount of the supplements was also prepared for diluting of 2% agarose as 1% agarose-BCM to inflate the lungs. The supplemented culture medium was adjusted to pH 7.25, filter sterilized (0.22-µm filter), and stored at 4° C. HCM was prepared in a manner identical to the BCM preparation except that 5.96 g/L HEPES (Sigma) was substituted for sodium bicarbonate. Agarose solutions (2% and 4%, type VII, Sigma) were prepared with BCM without supplements, then autoclaved and stored at 4° C.

Image Processing

Images were recorded with a videodisc recorder (Panasonic TQ 2026F, Osaka, Japan). The recorded images were digitized by using a 80386 Intel-based microcomputer equipped with a frame grabber board (Matrox PIP1024B, Montreal, QC) and custom-made image-acquisition software. The digitized images were analyzed with the NIH Image 1.62a Software (National Institutes of Health, Bethesda, MD) on a Macintosh G3 computer (Apple Computer, Inc., Cupertino, CA). For each airway, we measured the area (Ai) enclosed by the epithelial luminal border as well as the major and minor axes. Calibration was performed using a 1-mm graticule image.

Dynamics of Airway Narrowing

After recording a baseline image, 15 µl of 10² M MCh solution was dropped directly on the airway of interest. Only one airway was imaged per slice. Preliminary experiments demonstrated that this MCh dose induced a maximum contraction. Images were sequentially recorded at frequencies of 2.0 Hz for 0 to 16 s and then single images at 20, 30, 40, 50, and 60 s. The internal perimeter (Pi) was calculated from the measure of the area by assuming a circular lumen with Pi = 2r and r = (Ai/)^1/2. We calculated the velocity of shortening of the lumen perimeter by numerically differentiating the values of Pi versus time, and then normalizing the velocity to the baseline Pi. Results are therefore reported in units of % baseline Pi/s.

Statistical Analysis

The nonparametric Kolmogorov-Smirnov (KS) test was used to compare cumulative frequency distributions for the variables of mature and immature rabbits. T tests were used to compare velocity and airway narrowing at several time points. A probability level of p < 0.05 was considered statistically significant. Statistics were carried out using Statview (ver. 5.0; SAS Institute Inc., Cary, NC).

	RESULTS

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A total of 196 airways were studied. Of these airways, six airways had a baseline major/minor diameter ratio > 2 when measured from the digital images and we excluded these airways from further analysis (four mature, two immature airways). There were 110 airways in the mature group (10 to 29 airways per rabbit) and 80 airways in the immature group (25 to 28 airways per rabbit). Based on the cumulative frequency distribution, the baseline airway area was significantly smaller for immature than mature rabbits (p = 0.003, KS, Figure 1). There were no significant differences in the distributions of airway sizes among individual rabbits within each group (size range: 0.048 to 2.05 mm² for the immature and 0.120 to 3.48 mm² for the mature). No differences were found by KS tests between animals of each group except for one animal in the immature group, which was smaller (Im 3).

View larger version (18K): [in this window] [in a new window]   Figure 1.   Cumulative frequency distribution of baseline airway lumen area for all mature and immature airways studied. The immature airways (open symbols) were significantly smaller than the mature airways (closed symbols) (p < 0.005, KS).

Airway Narrowing

Representative images of an airway at baseline and during narrowing after MCh application are illustrated in Figure 2A. In addition, the measured absolute airway area and the calculated velocity of shortening versus time using all images obtained for the same airway are presented in Figures 2B and 2C. These figures demonstrate that there is a rapid early decline in the area of the airway lumen with an associated rapid velocity that peaks within a few seconds. In addition, maximal airway narrowing is achieved within 60 s.

View larger version (49K): [in this window] [in a new window]   Figure 2.   (A) Four images of an airway from an immature lung explant, obtained at baseline (t = 0 s), and 10, 20, and 60 s after direct application of 102 M MCh. (B) The measured lumen area is expressed as a function of time for all data images collected. (C ) The corresponding velocity of narrowing of the airway internal perimeter.

The time course of airway narrowing for pooled airways from mature versus immature rabbits (mean ± SE) is illustrated in Figure 3, which shows the airway lumen area expressed as a percentage of the baseline area. The smaller the area, the greater was the airway narrowing. Similar to the individual curve illustrated in Figure 2, the average data demonstrate a rapid decrease in airway narrowing during the first 5 s and more than 85% of the final response occurred during the first 20 s. At 60 s after MCh application, immature compared with mature airways narrowed to a smaller percentage of baseline area (21.9 ± 1.6 and 42.2 ± 1.8%, respectively). The curves for mature and immature airways are significantly different as soon as 0.5 s after MCh stimulation (t test; p < 0. 01).

View larger version (15K): [in this window] [in a new window]   Figure 3.   Percentage of baseline lumen area as a function of time (s) during MCh-induced constriction. Each time point is the mean and standard error of 80 airways obtained from three immature rabbits (open diamonds) and 110 airways from five mature rabbits (filled circles). The degree of airway narrowing is significantly greater for immature than mature airways starting at 0.5 s after MCh challenge (t test).

As the response for maximal airway narrowing was heterogeneous both within an animal as well as among animals within a group, we compared the cumulative frequency distributions of the maximal airway narrowing for individual animals (Figure 4). Three airways did not constrict more than 10% (90% of baseline). Airway closure (0% of baseline) was relatively infrequent; however, there were more immature than mature airways that closed (6 of 80 versus 1 of 110 respectively, p < 0.05, Fisher exact test). The intragroup variability was greater in mature than in immature rabbits. No differences were found between the immature animals; between the mature animals only the extremes were different by KS test, and after adjusting for multiple testing and ordering, this difference was not significant. Although there is heterogeneity, there is no overlap in the individual distribution for the mature and immature groups. In addition, the distribution of airway narrowing for the most responsive mature animal (Ma 4) demonstrated significantly less narrowing than the distribution from the least responsive immature animal (Im 2) (p < 0.001, KS test).

View larger version (28K): [in this window] [in a new window]   Figure 4.   Cumulative frequency distributions of airway narrowing expressed as a percentage of baseline area for the individual mature (Ma; filled symbols) and for the immature rabbits (Im; open symbols). Each distribution of airway narrowing from immature animals was significantly different from that of mature animals (KS).

Velocity of Shortening

The mean (± SE) values for the velocity of shortening versus time for mature and immature airways are illustrated in Figure 5. The group mean peak velocity is significantly greater for immature than mature airways (6.98 ± 0.32 versus 4.22 ± 0.18, p < 0.0001, t test); these mean values are slightly greater than those depicted in Figure 5, secondary to small variations in the time to peak values among animals. The frequency distributions for the peak velocity of shortening for individual animals are illustrated in Figure 6. Again, there is a wide variability among the airways within each animal, as well as among animals in each group. In general, the distributions for the immature airways are shifted to the right of the distributions for mature airways, that is, to higher peak velocities. There is overlap of the distribution of the fastest mature rabbit (Ma 4) and the distribution for the airways from the slowest immature animal (Im 2); these two distributions are not significantly different (p > 0.15). However, all other comparisons between mature and immature animals are significantly different.

View larger version (20K): [in this window] [in a new window]   Figure 5.   Velocity of shortening expressed as a percentage of baseline perimeter versus time (s) during MCh-induced constriction. Each time point is the mean and standard error of 80 airways obtained from the three immature rabbits (open diamonds) and 110 airways from the five mature rabbits (filled circles). The peak velocity of shortening is significantly greater for immature than mature airways (p < 0.0001, t test).

View larger version (28K): [in this window] [in a new window]   Figure 6.   Cumulative frequency distributions of the peak velocity of shortening expressed as a percentage of baseline perimeter/s for the individual mature (Ma: filled symbols) and immature rabbits (Im: open symbols). All distributions of mature airway peak velocities are significantly different from distributions of the immature peak velocities, except for the comparison of the fastest mature rabbit (Ma 4) and the slowest immature rabbit (Im 2), which did not reach statistical significance (p > 0.15, KS).

Influence of Airway Size on Airway Narrowing

To evaluate whether maturational differences in airway narrowing and velocity of shortening were related to airway size, we compared the responses of airways from immature and mature animals that we matched for size. For this purpose, we reanalyzed the data after eliminating airways from both groups that were less than 0.07 or more than 2 mm² (33 airways removed). With this exclusion criterion, there was no statistical difference between the cumulative distributions of mature and immature airway area (p > 0.6). After matching for airway size, immature compared with mature airways still had greater maximal airway narrowing (18.6 ± 1.6 versus 42.3 ± 2.1% of initial area; p < 0.0001) and greater peak velocity of shortening (6.63 ± 0.28 versus 4.23 ± 1.19%/s; p < 0.0001). With each age group analyzed separately, we found no correlation between baseline airway area and maximal airway narrowing or peak velocity of shortening for airways from mature animals (p > 0.30). In contrast, among immature airways both maximal airway narrowing and velocity of shortening increased with decreasing airways size (p < 0.005 and p < 0.001); however, airway size accounted for only 10% of the interairway variability of either maximal narrowing or peak velocity.

Relationship between Maximal Airway Narrowing and Peak Velocity of Shortening

Airway narrowing, expressed as a percentage of baseline perimeter, was related to the peak velocity of shortening for both mature and immature airways (Figure 7). Those airways with greater peak velocity narrowed to a smaller percentage of their baseline size.

View larger version (25K): [in this window] [in a new window]   Figure 7.   Maximal airway narrowing (percentage of baseline perimeter) versus peak velocity of shortening (percentage of baseline perimeter/s) for airways from mature (closed circles) and immature (open diamonds) rabbit airways. Airways that shortened at a greater velocity had greater airway narrowing.

	DISCUSSION

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In this study, we found greater maximal airway narrowing and greater peak velocity of shortening in immature compared with mature airways in rabbit lung explants. The greater maximal airway narrowing is consistent with our previous in vivo findings of greater increases in airway and pulmonary resistances after MCh challenge, as well as the greater airway narrowing observed in histologic sections from lungs fixed after constriction (1). Our new observation of a greater velocity of airway narrowing is consistent with our hypothesis of a lower elastic load limiting airway narrowing in immature compared with mature airways.

Wang and colleagues (11) and Duguet and coworkers (9) have previously used lung explants to demonstrate differences in both velocity and magnitude of airway narrowing between different strains of rats (11) and mice (9). Explants have several advantages. This in vitro technique using living tissue enables the assessment of multiple intraparenchymal airways, particularly for small animals in which studies are often otherwise limited to isolated extrathoracic airways. In addition, the agonist can be applied directly to the airway to ensure maximal stimulation, in contrast to heterogeneous distribution of the agonist delivered by the intravenous or aerosol routes in vivo. Furthermore, the airway response can be visualized directly and measured continuously as a function of time.

In this study, we sampled a wide distribution of intraparenchymal airways from mature and immature animals, which would correspond approximately to generations 4 to 9 (8, 12). As expected, the distributions of airway sizes were different for the two age groups, with the immature having smaller airways (Figure 1). However, this difference in size did not account for the greater response of the immature airways, as airways matched for size still demonstrated similar maturational differences.

The degree of airway narrowing is a balance between ASM force generation and the loads that resist ASM shortening (13, 14). The greater airway narrowing observed in immature airways could result either from greater ASM force generation or a lower elastic load, or both. Among different species there have been conflicting results with respect to findings of maturational differences of ASM contractility. In pigs, maximal stress of TSM strips decreased and shortening velocity increased with maturation (15). In contrast to pigs, TSM strips from guinea pigs demonstrated no difference in maximal stress generation but a decrease in shortening velocity with maturation (16). Several studies have suggested that in rabbits, maturational differences in ASM are not likely to account for the greater airway narrowing that we observed in the immature animals. A previous study demonstrated lower and not higher maximal stress generation by TSM strips from immature than mature rabbits (3), whereas other investigators found no maturational difference in TSM stress generation (6, 17). In addition, Roepke and coworkers found no maturational difference in the velocity of shortening of rabbit TSM strips or in the myosin isoforms (6). A previous study has demonstrated that in intrathoracic airways, the relationship between airway size and ASM area was similar for immature and mature rabbits; smaller airways having proportionately more ASM area relative to the size of the airway (2). As immature animals had smaller airways than mature animals, the immature animals had airways with proportionately more ASM relative to the airway size. However, in that study, there was not a significant relationship between airway responsiveness in vivo and the quantity of ASM relative to airway size for individual animals, which suggested that the quantity of ASM relative to airway size was not the primary determinant of the observed maturational differences in airway narrowing. That study is also consistent with our current findings that maturational differences in the velocity and the degree of airway narrowing persisted when the distributions of airway sizes were matched for mature and immature rabbits. Therefore, there are currently no data suggesting that immature ASM has greater force generation or velocity of shortening nor that differences in the amount of ASM account for the observed maturational difference in airway narrowing in rabbits.

The primary elastic loads that limit ASM shortening are the lung parenchyma, the airway wall, and the internal resistance to shortening of the smooth muscle cell (13, 14, 18). The lung parenchyma functions as an elastic load to airway narrowing by the alveolar attachments to the airway wall. The lung volume at which the explants are prepared will affect the elastic load, which includes both the static recoil pressure as well as the local tissue distortion produced by airway narrowing. The pressure-volume curves of immature and mature rabbit lungs do not differ when normalized for lung volume; this suggests that the static elastic load of the parenchyma does not differ for mature and immature animals (8). Inflating mature and immature rabbit lungs with a volume of agarose that equals TLC should produce comparable elastic loads in both groups. Adler and coworkers (19) have previously demonstrated that 2% agarose-filled lungs have a shear modulus similar to that of the air-filled lungs, thus suggesting that airway- parenchymal interdependence may be similar in the explants and in vivo. Immature rabbit airways have approximately 15% fewer alveolar attachments compared with airways from mature animals (12). In addition, the shear modulus for the nonconstricted lung is approximately 15% lower in immature than mature rabbit lungs (7). Therefore, the parenchyma may provide a lower elastic load to airway narrowing and contribute to the greater airway narrowing in the immature lung.

Application of MCh onto the lung explant produces not only constriction of airway smooth muscle, but also constriction of smooth muscle in the parenchyma that could alter the forces of interdependence between the airways and the lung parenchyma. There are conflicting results as to whether the shear modulus of the lung is altered with bronchoconstriction. Okazawa and colleagues found no increase in the shear modulus when mature rabbit lungs were constricted (20); however, Salerno and Ludwig reported an increase in the shear modulus of rat lungs with bronchoconstriction (21). Although the punch indentation test has been used to measure the shear modulus of the lung parenchyma, this technique does not directly measure the forces of interdependence between the airways and the lung parenchyma. In addition, the methodology assumes that all airways remain patent during bronchoconstriction, an assumption that may not be correct. Maturational differences in the effect of bronchoconstriction upon the local forces of parenchymal deformation and the shear modulus of the lung parenchyma have not been evaluated, and maturational differences in this elastic load may contribute to greater narrowing of the immature airway.

The elastance of the airway wall is the other important elastic load that resists ASM shortening and airway narrowing. The simple model of airway narrowing proposed by Macklem (13) illustrates that the passive length-tension curve of the airway influences airway narrowing; a more compliant airway will narrow more for the same ASM force generation. Immature rabbit intraparenchymal airways are more distensible and have proportionally less cartilage than anatomically matched airways from mature animals (8, 12). Therefore, a lower elastic load within the airway wall of the immature animal may contribute to greater airway narrowing. The cytoskeleton of the smooth muscle cells within the airway wall also provides an elastic load as the cell shortens. This internal resistance to cell shortening is most prominent during the later 50% of maximal shortening. Because the velocity of airway narrowing we observed in the explants was greater in immature than mature airways even during the early course of the contraction (Figure 5), and because there are not maturational differences in velocity of shortening of TSM (6), the internal resistance to shortening of the ASM most likely does not account for our observed maturational differences in the lung explants.

Velocity of shortening of ASM is very sensitive to external load (22, 23). The relationship between velocity of shortening and load is hyperbolic; the highest velocity is observed in unloaded smooth muscle and decreases dramatically with relatively small increases in load. We found a relationship between velocity and airway narrowing; higher velocities were associated with greater narrowing. Both mature and immature lungs were similarly inflated with agar to TLC so that we anticipate that at this lung volume there was a significant load resisting ASM shortening. The presence of a significant load is consistent with the low frequency of airway closure observed in our rabbit explant preparation. However, the value of peak velocity of airway narrowing that we calculated in our lung explants is similar in magnitude to that reported in rabbit TSM strips (6). The similarities in the velocity of shortening between the explant and the isolated TSM, and the significant degree of airway narrowing that we observed, suggest that our preparation is not on the flat portion of the load-velocity relationship but is likely to be in the load-sensitive region. Therefore, our observed maturational difference in velocity and magnitude of airway narrowing is likely to be secondary to differences in the loads resisting ASM shortening.

In summary, we found greater velocity of shortening and greater maximal airway narrowing in the lung explants from immature than mature rabbits. These findings are consistent with the hypothesis that the elastic load limiting ASM shortening is less in the immature animals. In view of available data that do not support maturational differences in ASM contractility in rabbits, our present findings strongly suggest that elastic loads limiting ASM shortening are lower in immature than mature rabbit lungs.