Comparison of elastic properties and contractile responses of isolated airway segments from mature and immature rabbits

R. Ramchandani, X. Shen, S. J. Gunst, R. S. Tepper

Abstract

Immature rabbits have greater maximal airway narrowing with bronchoconstriction in vivo compared with mature animals. As isolated immature lungs have a lower shear modulus, it is unclear whether the greater airway narrowing in the immature lung is secondary to less tethering between the airways and the lung parenchyma or to differences in the mechanical properties of the mature and immature airways. In the present study, we compared the mechanical properties of fluid-filled, isolated, intraparenchymal airway segments of the same generation from mature and immature rabbits. Stimulation with ACh resulted in greater airway narrowing in immature than mature bronchi. The immature bronchi were more compliant, had a lower resting airway volume, and were more collapsible compared with the mature bronchi. When the airways were contracted with ACh under isovolume conditions, the immature bronchi generated greater active pressure, and they were more sensitive to ACh than were mature bronchi. Our results suggest that maturational differences in the structure and function of the airways in the absence of the lung parenchyma can account for the greater maximal narrowing of immature than mature airways in vivo.

  • airways
  • maturation
  • mechanics

our laboratory has previously demonstrated that immature rabbits exhibit greater maximal airway narrowing in response to bronchoconstrictors in vivo, as well as in lung tissue explants (4, 21, 22, 25, 26). We have also demonstrated greater airway distensibility in isolated, nonconstricted immature rabbit lungs compared with isolated mature rabbit lungs (22). The greater airway narrowing in the immature animal, both in vivo and in vitro, could result from a lower elastic load from the airway wall, greater airway smooth muscle force generation, or lower forces of interdependence between the airway and the lung parenchyma. The immature rabbit lung has a lower shear modulus compared with the mature rabbit lung; this lower elastic load from the immature lung parenchyma could contribute to greater airway narrowing (28). In addition, there are structural differences between immature and mature airways; the airway wall of immature rabbits has proportionately less cartilage and more smooth muscle (17). These structural differences could result in a more compliant airway wall with a lower elastic load to resist smooth muscle shortening, as well as greater pressure generation by the smooth muscle to overcome the elastic loads that limit airway narrowing. A theoretical model of airway narrowing for mature and immature rabbits suggests that the maturational difference in the force of interdependence between lung parenchyma and the airway wall is not adequate to account for the greater airway narrowing of the immature airway (11). However, previous physiological studies assessing maturational differences in airway narrowing have not isolated intraparenchymal airways from the surrounding lung parenchyma; thus it has not been demonstrated that, in the absence of the lung parenchyma, immature airways narrow more than mature airways. In addition, although there are differences in the anatomic composition of the wall of intraparenchymal airways of immature and mature rabbits, it has not been demonstrated that the mechanical properties or contractile responses of these intraparenchymal airways differ.

The objective of the present study was to evaluate whether isolated, intraparenchymal airway segments from immature rabbits demonstrate greater airway narrowing than isolated, intraparenchymal airway segments from mature rabbits. In addition, we evaluated whether differences in the airway wall structure could account for the increased airway narrowing observed in immature rabbit lungs. We hypothesized that the lower cartilage content and the increased smooth muscle that we previously reported in immature rabbit airways would result in greater airway compliance and greater maximal pressure generation relative to airways from mature rabbits. In addition, we proposed that the lower elastic load from the immature airway wall and the greater pressure generation would result in greater narrowing of immature than mature airways that were isolated from the surrounding lung parenchyma. To address these questions, we compared the passive mechanical properties and contractile responses of isolated, intrapulmonary airway segments from immature and mature rabbit lungs. We evaluated proximal and distal intraparenchymal airway segments because the structural composition of the airway wall varies with generation: the proportion of the airway wall that is cartilage decreases, and the proportion that is muscle increases, going from proximal to distal airways (17).

METHODS

Tissue preparation and experimental apparatus. New Zealand White mature rabbits (6 mo, 2.5–2.7 kg) and immature rabbits (4–6 wk, 0.5–0.6 kg) were studied. This study was approved by the Institutional Animal Care and Use Committee. Animals were anesthetized with thiopental sodium (50 mg/kg) and exsanguinated by severing the abdominal artery. The left lobe of the lung was excised and placed in physiological saline solution [PSS; which contains (in mM) 110 NaCl, 3.4 KCl, 2.4 CaCl2, 0.82 MgCl2, 25.8 NaHCO3, 1.2 KH2PO4, and 5.6 glucose] and aerated with 95% O2 and 5% CO2. With the use of a stereomicroscope, the parenchymal tissue was dissected away from the airways along the main axial pathway. Airway generations were defined as the portion of the main pathway between two major branches (Fig. 1). Intraparenchymal segments corresponding to generations 3–4 (proximal segment) and generations 6–7 (distal segment) along the main axial pathway were isolated. All of the branches on each airway segment were tied off with silk sutures to produce leak-free bronchial segments.

Fig. 1.

Rabbit airway tree with parenchyma removed. Airway generations (nos. 1–8) were defined as the portion of the main axial pathway between 2 major branches, with the trachea being generation 1. Intraparenchymal segments corresponding to generations 3–4 (proximal segment) and generations 6–7 (distal segment) were isolated from mature and immature animals.

Each end of the bronchial segment was mounted and securely tied to a stainless steel cannula with a diameter comparable to that of the end of the bronchial segment. The bronchial segment with the inserted cannula was mounted in a 20-ml tissue bath, which is a Plexiglas chamber filled with PSS solution at 37°C. The bronchial segment was flushed and filled with PSS with the use of a stopcock and syringe connected to the cannula on the distal, smaller end of the segment. The cannula on the proximal end of the bronchial segment was connected to a differential pressure transducer (model 156PC05GW, Honeywell Sensing), which measured transmural pressure, and a microsyringe, which measured changes in bronchial volume. The syringe was attached to a servocontrolled motor that could adjust bronchial volume and pressure. The equipment and methodology were similar to that previously described for studies of canine bronchi (7, 8). The pressure and volume signals were amplified, digitized (DT 2801-A, Data Translation, Marlborough, MA) and recorded on a personal computer for real-time visualization of signals, as well as for subsequent data analysis with the use of commercial software (RHT Infodat, Montreal, PQ).

Passive deflation pressure-volume curves. The passive deflation pressure-volume curves of proximal and distal bronchi from immature and mature rabbits were measured. Each bronchial segment was slowly cycled over a 2-min period between pressures of -25 and +20 cmH2O, until a constant pressure-volume curve was obtained; this usually required three to five cycles. Airway volumes at pressures of -25 and +20 cmH2O were defined as zero volume and maximal volume, respectively. The next inflation and deflation cycle was then used for pressure-volume measurements. Bronchial volumes were measured at pressures of -20, -15, -10, -8, -5, -3, -2, -1, 0, 1, 2, 3, 5, 8, 10, 15, and 20 cmH2O, and the deflation pressure-volume curve for each airway segment was normalized by expressing bronchial volume as a fraction of maximum volume (22).

Maximal isobaric contractions. Maximal airway narrowing was evaluated at transmural pressures of 2, 5, 8, and 12 cmH2O in proximal and distal airways from mature and immature rabbits (22). Each bronchial segment was slowly cycled over a 2-min period between pressures of -25 and +20 cmH2O, until a constant pressure-volume curve was obtained. This usually required three to five cycles. Then, starting from a pressure of 20 cmH2O (maximal volume), the bronchus was deflated to -25 cmH2O (zero volume) and then inflated to one of the four transmural pressures (2, 5, 8, or 12 cmH2O) that were selected in a random order. At the selected transmural pressure, a single maximal dose of ACh (10-4 M) was added to the tissue bath, and the decrease in bronchial volume at constant transmural pressure was recorded. After each constriction, the ACh was washed from the airway segment and the water bath, and the segment was cycled between -25 and 20 cmH2O until the pressure-volume curve returned to baseline. Airway constriction was expressed as the volume of the constricted bronchus after narrowing, normalized to its nonconstricted maximal volume.

Isovolume contraction to ACh. Sensitivity to ACh and maximal pressure generation were evaluated by using isovolumetric contractions to increasing concentrations of ACh. As the contractile properties of ASM are length and load dependent, and the compliance of the airway depends on volume, we assessed the contractile response of the bronchi at both 40 and 80% of maximal volume. The lower volume is near the resting volume of the airway segment, whereas 80% of maximal volume is in the volume range in which the airway becomes stiffer. Airway segments were first inflated to 40% of maximum volume. The ACh concentration in the tissue bath was increased sequentially from 10-9 M to 10-4 M to obtain a cumulative dose-response curve. At each concentration of ACh, pressure generation at constant volume was recorded until a plateau in pressure was achieved. ACh was washed out, and the cumulative dose-response curve was repeated with the volume of the airway segment inflated to 80% of maximum volume.

Statistical analysis. The parameters calculated from the deflation pressure-volume curves, as well as the maximal pressure generation and sensitivity to ACh for airway segments obtained from immature and mature rabbits, were compared by using the Mann-Whitney rank sum test. The effect of transmural pressure and age on maximal airway narrowing to ACh was evaluated by using an ANOVA. A P value ≤0.05 was considered statistically significant.

RESULTS

Comparison of airway narrowing. The airways for immature rabbits constricted to smaller volumes than airway segments from mature animals (P < 0.01) (Fig. 2A). A significant difference between the two groups was present at a transmural pressure of 5 cmH2O (P < 0.05), and the greater narrowing of the immature segments approached statistical significance at a transmural pressure of 12 cmH2O(P < 0.06). There was also a significant effect of transmural pressures on airway narrowing (P < 0.01); airway narrowing was significantly less at transmural pressures of 2, 5, and 8 cmH2O compared with 12 cmH2O. The comparisons of airway narrowing for mature and immature proximal and distal airway segments are illustrated in Fig. 2, B and C, respectively. The distal segments of immature airways narrowed more than the comparable mature airway segments; there was a significant difference at a transmural pressure of 12 cmH2O (P < 0.05). In addition, there was a significant effect of transmural pressure on airway narrowing for the proximal segments; airway narrowing was significantly greater at a transmural pressure of 2 than at 12 cmH2O (P < 0.05). Because of the large variability in the responses of animals grouped by age and size, some trends in differences between mature and immature airways did not achieve statistical significance. The proximal segments from the immature animals tended to narrow more, but the difference did not reach statistical significance. The effect of transmural pressure on the distal airway segments also did not achieve statistical significance.

Fig. 2.

Bronchial volume (as %maximal volume at 20 cmH2O) after maximal constriction with ACh (10-4 M) at transmural pressures of 2, 5, 8, and 12 cmH2O for mature (open bars) and immature (solid bars) rabbits. Values are means ± SE. A: combined proximal and distal airway segments for immature rabbits constricted to smaller volumes than segments from mature animals (P < 0.01); there was also a significant effect of transmural pressures on airway narrowing (P < 0.05). B: for the proximal segments, there was a significant effect of transmural pressure on airway narrowing (P < 0.05); however, the difference in narrowing between mature and immature segments did not reach statistical significance. C: for the distal segments, immature airways narrowed more than those of mature animals (P < 0.05); however, the effect of transmural pressure did not achieve statistical significance. Nos. above bars, n rabbits.

Comparison of airway pressure-volume curves. The relationships between bronchial volume vs. transmural pressure for proximal and distal bronchi from immature and mature rabbits are illustrated in Fig. 3. The pressure-volume curves for the immature and mature airway segments were significantly different for proximal and distal segments. The differences in the elastic properties of the mature and the immature bronchi, as assessed by the deflation pressure-volume curves, can be assessed by comparing the compliance of the bronchi, the resting volume of the bronchi, and the collapsibility of the bronchi under negative pressure. The compliance of the proximal bronchi, which was measured over the linear portion of the pressure-volume curve (-1–2 cmH2O), was greater for immature than mature animals, although the compliance of the distal bronchi was not significantly different (Fig. 4A). The resting bronchial volume at transmural pressure = 0 cmH2O was a significantly lower fraction of the maximal volume for immature than mature airway segments (Fig. 4B). Under conditions of negative pressure, the proximal and distal bronchi from immature animals collapsed at a lower volume than bronchi from mature animals (Fig. 4C). These differences in the deflation pressure-volume curves of immature and mature rabbit airways indicate that lower transmural pressures are required to cause narrowing and collapse of immature than mature isolated, intraparenchymal airway segments.

Fig. 3.

Deflation pressure-volume curves from proximal (A) and distal (B) airway segments from mature (○) and immature (•) rabbits. Values are means ± SE; n, no. of rabbits. The bronchial volume is normalized to maximal volume (20 cmH2O). *Mature and immature pressure-volume curves differed when compared by ANOVA with repeat measures, P < 0.05. A: for the proximal segments, the fractional volumes were significantly greater for mature than immature airways at all transmural pressures, except 20, 15, -15, and -20 cmH2O. B: for the distal segments, the fractional volumes were significantly greater for mature than immature airways at transmural pressures of 0, -1, -2, and -3 cmH2O.

Fig. 4.

Comparison of proximal and distal airway segments from mature (open bars) and immature (solid bars) rabbits. Values are means ± SE; n, no. of rabbits. Immature airway segments were more compliant and more collapsible than mature airway segments. A: compliance was significantly greater for proximal segments from immature than mature rabbits (P < 0.5). B: bronchial volumes at transmural pressure (Ptm) of 0 cmH2O were significantly smaller for proximal and distal airway segments from immature than mature rabbits. C: bronchial volumes at Ptm of -2 cmH2O were significantly smaller for proximal and distal airway segments from immature than mature rabbits.

Comparison of airway contractility. Active pressures generated during isovolumetric contractions at 40 and 80% of maximal volume vs. ACh concentration for proximal and distal bronchial segments from immature and mature rabbits are illustrated in Fig. 5. Bronchi from immature animals generated significantly greater pressures compared with bronchi from mature animals. When normalized to maximal active pressure generation, the bronchi from immature animals required a significantly lower ACh concentration than bronchi segments from mature animals to achieve 50% of the maximal response (proximal: 1.63 × 10-7 M for immature airways vs. 1.97 × 10-6 M for mature airways, P < 0.05; distal: 4.97 × 10-8 M for immature airways vs. 8.42 × 10-7 M for mature airways, P < 0.05).

Fig. 5.

Active pressure (cmH2O) vs. ACh dose (M) for proximal and distal airway segments from mature (○) and immature (•) rabbits. A and B: proximal airway segment at 40 and 80% of maximal volume, respectively. C and D: distal airway segment at 40 and 80% of maximal volume, respectively. Values are means ± SE. Immature airway segments generated greater active pressure and were more sensitive to ACh than mature airway segments. *Significant difference (P < 0.05).

DISCUSSION

The objective of the present study was to determine whether differences in the mechanical properties of mature and immature airways, in the absence of the influences of the lung parenchyma, can account for the greater responsiveness and increased narrowing of the airways of immature animals relative to the airway from mature animals that is observed in vivo. In a previous study, we (28) found that the shear stress of parenchymal tissue from the lungs of immature animals is less than that from mature animals; this by itself might account for the greater narrowing of their airways. By studying isolated, intraparenchymal airways, in the absence of the influences of the lung parenchyma, we sought to determine whether the increased airway responsiveness of immature airways could be accounted for by the airways themselves. Our results demonstrate that isolated, intraparenchymal bronchi from immature rabbits generate greater transmural pressures and narrow more than bronchi from mature rabbits. These findings indicate that maturational differences in airway narrowing and airway closure observed in vivo and in isolated lungs could result primarily from functional differences in the airways themselves, rather than from differences in the lung parenchyma.

The immature airways generated much greater transmural pressures during isovolumetric stimulation with ACh than the mature airways. In addition, when the isovolume dose-response curve was normalized to maximal pressure generated, the immature airways were more sensitive to ACh than the mature airways. The degree of airway narrowing in the intact lung is a balance between the airway transmural pressure generated by airway smooth muscle contraction and the distending pressure generated by the lung parenchymal tissue. When the transmural pressure generated by the airway is sufficient to overcome the distending pressure, the airway will close. In the present study, isolated airways from immature animals generated transmural pressures that were as much as two to three times higher than those generated by mature airways. With such marked differences in pressure generation by the two groups of airways, much greater airway narrowing and more airway closure in immature animals would be predicted, even if the properties of the parenchymal tissue were the same. The lower shear modulus for the immature lung would further magnify this difference (28). Thus the higher transmural pressures generated by the immature airways can account for the increased airway resistance and higher frequency of airway closure observed in response to bronchial challenge in vivo and in isolated, intact lungs (21, 22, 25, 26).

The differences in airway narrowing between isolated immature and mature airways might reflect differences in either the anatomy or structure of the airway wall, or the intrinsic responsiveness of the airway smooth muscle, or both. Theoretically, a difference in airway wall thickness relative to airway size could result in differences in both compliance and airway wall narrowing. However, in a previous study, we found no evidence that immature airways have a greater airway wall thickness-to-diameter ratio (17). In the present study, we found that the isolated, nonconstricted, intraparenchymal bronchi from immature rabbits are more compliant, maintain a lower fraction of maximal volume at 0 cmH2O transmural pressure, and are more collapsible than airways from mature rabbits. Similarly, the trachea of the immature rabbit was also found to be more compliant than the trachea of the mature rabbit (1). The differences in the pressure-volume curves of mature and immature airways may reflect differences in airway structure: the walls of airways from immature rabbits have less cartilage than those of mature rabbits (17). The maturational differences in the pressure-volume curves were more prominent in the proximal than in the distal airway segments, which may reflect the greater difference in cartilage content in the proximal airways of mature and immature animals. The cartilage content of the distal airways of the two groups is more similar.

The differences in the structural composition of the airway wall and the greater compliance of the immature airway may lead to greater airway narrowing of the immature than mature airway by decreasing the elastic load against which the airway smooth muscle must shorten. In isolated porcine and canine bronchi, airways with more cartilage are stiffer and narrow less with stimulation than airways with less cartilage; in addition, removing cartilage from the airway wall increases the compliance of the airway and increases the narrowing of the airway in response to contractile stimulation (7, 15).

The airways from immature rabbits exhibited a greater sensitivity and a higher maximal increase in isovolume pressure in response to ACh than did the mature airways. These differences in response might also be related to differences in the anatomic structure and composition of the airway wall. The immature airway has not only proportionately less cartilage within the airway wall but also proportionately more smooth muscle (17). The increased smooth muscle content of the airway wall could result in greater isovolumetric pressure generation. Structural differences in the airway wall that affect the transmission of active stress generated by the smooth muscle might also contribute to the differences in responsiveness. In a previous study, distal canine airways with less cartilage were also shown to generate higher isovolumetric pressures than more proximal airways with more cartilage, despite a lower pharmacological sensitivity of distal airway smooth muscle (7).

The difference in the responsiveness of mature and immature airways might also reflect differences in the pharmacological responsiveness of the airway smooth muscle tissue. There is evidence from a number of studies, including a previous study from our laboratory, that the pharmacological sensitivity of isolated muscle from immature animals from some species is greater than that from mature animals (6, 9, 13, 14, 16, 23, 26). We previously found that isolated strips of rabbit tracheal smooth muscle exhibit increased sensitivity to both ACh and histamine relative to that from mature animals (26). However, most of these studies measured isometric force in trachealis smooth muscle strips in vitro. The magnitude of the differences in pharmacological responsiveness varied significantly, depending on the species, the particular pharmacological agonist used, and its concentration. Furthermore, the pharmacological responsiveness of tracheal and bronchial muscle differs significantly (5, 5a, 20). As no differences in shortening velocity were detected in tracheal muscle from mature and immature rabbits, despite the differences in pharmacological sensitivity, this suggests that differences in the magnitude of the isometric response might not be a sensitive predictor of differences in the magnitude or rate of shortening (18). There are also conflicting reports as to whether there are differences in the maximal velocity of shortening of mature and immature tracheal smooth muscle from swine (2, 10). Thus it is difficult to predict whether increased pharmacological sensitivity of airway smooth muscle would result in significant differences in the magnitude of airway narrowing or pressure generation in intact intraparenchymal bronchi.

There are similarities in the structural and functional differences between the immature and mature rabbits and humans. Not only do both species demonstrate a decline in airway responsiveness with maturation, but in both species the mature animal exhibits a plateau in the airway response to increasing doses of agonist, and the maximal response is relatively small (4, 12, 21, 22, 2427, 29). In contrast, in both species, the immature animals demonstrate progressive airway narrowing and do not appear to limit the maximal airway response. As we observed for rabbit airways, immature humans have greater airway compliance compared with the mature of the species (3). Our findings in rabbits might thus explain the physiological behavior of the airways of immature humans.

In summary, isolated, intraparenchymal airway segments from immature rabbits generate much higher transmural pressures and greater maximal airway narrowing than airway segments of the same generation from mature rabbits. The higher transmural pressures generated by these airways can account for the greater narrowing and increased airway closure exhibited by immature airways in vivo and in intact lungs. Our findings suggest that differences in the properties of the lung parenchyma are unlikely to account for greater airway responsiveness of immature airways in vivo. The greater narrowing of immature airways may reflect the differences in anatomic structure of the airways, the pharmacological sensitivity of the airway smooth muscle, or both. We speculate that alterations in the structure and function of the airway wall with growth and development of the lung may be responsible for the decreased responsiveness with maturation.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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