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1 Asthma, Allergy, and Immunological Disease Cooperative Research Center, Section of Pulmonary and Critical Care Medicine, Department of Medicine, and Committees on Clinical Pharmacology, Cell Physiology, and Comparative Medicine and Pathology, Division of Biological Sciences, The University of Chicago, Chicago, Illinois 60637; and 2 Krankenhaus Grosshansdorf, Zentrum für Pneumologie und Thoraxchirurgie, LVA Hamburg, D-22927 Grosshansdorf, Germany
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Mitchell, R. W., E. Rühlmann, H. Magnussen, N. M. Muñoz, A. R. Leff, and K. F. Rabe. Conservation of
bronchiolar wall area during constriction and dilation of human
airways. J. Appl. Physiol. 82(3):
954-958, 1997.
We assessed the effect of smooth muscle
contraction and relaxation on airway lumen subtended by the internal
perimeter
(Ai)
and total cross-sectional area (Ao)
of human bronchial explants in the absence of the potential lung
tethering forces of alveolar tissue to test the hypothesis that
bronchoconstriction results in a comparable change of
Ai and
Ao.
Luminal area (i.e.,
Ai) and
Ao
were measured by using computerized videomicrometry, and bronchial wall
area was calculated accordingly. Images on videotape were captured;
areas were outlined, and data were expressed as internal pixel number
by using imaging software. Bronchial rings were dissected in 1.0- to
1.5-mm sections from macroscopically unaffected areas of lungs from
patients undergoing resection for carcinoma, placed in microplate wells
containing buffered saline, and allowed to equilibrate for 1 h.
Baseline, Ao
[5.21 ± 0.354 (SE)
mm2], and
Ai
(0.604 ± 0.057 mm2) were
measured before contraction of the airway smooth muscle (ASM) with
carbachol. Mean
Ai
narrowed by 0.257 ± 0.052 mm2
in response to 10 µM carbachol (P = 0.001 vs. baseline). Similarly, Ao
narrowed by 0.272 ± 0.110 mm2
in response to carbachol (P = 0.038 vs. baseline; P = 0.849 vs. change in
Ai).
Similar parallel changes in cross-sectional area for
Ai
and
Ao
were observed for relaxation of ASM from inherent tone of other
bronchial rings in response to 10 µM isoproterenol. We demonstrate a
unique characteristic of human ASM; i.e., both luminal and total
cross-sectional area of human airways change similarly on contraction
and relaxation in vitro, resulting in a conservation of bronchiolar
wall area with bronchoconstriction and dilation.
airway smooth muscle; cross-sectional area; auxotonic contraction; spontaneous tone; carbamylcholine; isoproterenol
INTRODUCTION THE LOADS imposed by pulmonary tethering structures on
airway smooth muscle (ASM) as it shortens in vivo remain
undefined. It is agreed that, as ASM narrows the bronchi,
the deformation of the cartilage and connective tissues imposes a
progressively increased load on the muscle (8, 9); this contraction is defined as auxotonic (6, 7). Contractions of bronchi and bronchioles
deform the lung parenchyma, which further imposes an additional load on
the ASM of constricting airways. The ASM must be attached either
directly to the cartilage (9) or by highly elastic connective tissue
for deformation of the cartilaginous plaques, lung parenchyma, and
bronchoconstriction to occur. However, histological studies of canine
(5) and human (13) airways have failed to demonstrate such structures.
Previous studies using a high-resolution video camera lens and computer
imaging have shown in isolated intact canine airways that, as the lumen
constricts to near-occlusion in response to activation of the ASM, the
external perimeter of the airway does not decrease significantly (12).
Stephens and Jiang (12) concluded, therefore, that this behavior is
possible only if the connection between the ASM and the cartilaginous
plaques is highly compliant. Under these circumstances,
they also concluded that the parenchyma did not impose a load on ASM
and that the negative pressure caused in the airway wall by
constriction of the ASM would result in engorgement of the blood and
lymph vessels.
It has been suggested that in disease states such as asthma or chronic
obstructive pulmonary disease, decreases in preload on the smooth
muscle from either cartilage or alveolar tissue may lead to
augmentation of bronchial narrowing by mediators of contraction (8).
Because of the development of a method for measurement of ASM
contraction and high-resolution measurement of airway lumen subtended
by the internal perimeter
(Ai)
and total bronchiolar cross-sectional areas
(Ao)
(1, 4), we were able to test the hypothesis that bronchoconstriction
results in a comparable change of
Ai
and external perimeter. We reasoned that if
Ao
varied directly with
Ai,
then as the ASM contracted in vivo, the change in
Ao
would result in deformation of lung parenchyma, which would add to the
load imposed on ASM during bronchoconstriction. We found that both
Ai
and
Ao
are reduced and augmented similarly on contraction and relaxation in
vitro. Our data suggest that there is conservation of airway wall area
with bronchoconstriction, which may have implications for alveolar
tissue tethering forces on ASM contraction in vivo.
METHODS
9 to
104 M. After a 30-min
equilibration to establish stable baselines, video images were
captured. Carbachol was added to the microwell, and, after 10 min,
another image was captured. Changes in area were measured and expressed
as mean percent change for each explant for each concentration of
carbachol.
Airway cross-sectional area changes with contraction.
After equilibration (~30 min) to ensure stable baselines of
Ai
and
Ao,
video images were captured (before agonist). Carbachol (10 µM final bath concentration) was added to the microwell. This concentration of muscarinic-receptor agonist was determined to be
maximal for these tissues under conditions of auxotonic contraction. After 10 min, another video image was captured (after agonist); Ai
and
Ao
of each image were delineated, and areas before and after agonist were
compared by using a two-tailed paired
t-test. A
P value of < 0.05 in mean areas was
considered to be statistically significant.
Airway cross-sectional area changes with relaxation.
After equilibration (~30 min) to ensure stable baselines of
Ai
and
Ao,
video images were captured (before agonist). Isoproterenol (10 µM
final bath concentration) was added to the microwell. This concentration of 
-adrenergic-receptor agonist was determined to be
maximal for these tissues under conditions of auxotonic relaxation.
After 10 min, another video image was captured (after agonist);
Ai
and
Ao
of each image were delineated, and areas before and after agonist were
compared by using a two-tailed paired
t-test (see Airway
cross-sectional area changes with
contraction).
RESULTS
10 µM; therefore, 10 µM were used for subsequent studies.
Airway cross-sectional area changes in response to carbachol. Computer-enhanced images of human bronchial rings demonstrated luminal narrowing in response to 10 µM carbachol (Fig. 2). Before the addition of carbachol, mean Ai for 16 rings from 9 additional patients was 0.604 ± 0.057 mm2 and Ao was 5.21 ± 0.354 mm2. Carbachol caused Ai to decrease from 0.604 ± 0.057 to 0.347 ± 0.066 mm2 (P = 0.0011; Fig. 3). Ao decreased from 5.21 ± 0.354 to 4.94 ± 0.353 mm2 (P = 0.0382). Although both differences were statistically significant, the absolute decrease in Ai caused by carbachol (0.257 ± 0.052 mm2) was comparable to the decrease in Ao (0.272 ± 0.110 mm2; P = 0.849); hence, both Ai and Ao changed comparably. These data demonstrated a significant correlation coefficient with r = 0.763 (Fig. 4).
Airway cross-sectional area changes in response to isoproterenol. After ~30 min of equilibration to achieve a stable, sustained Ai (see METHODS), 10 µM isoproterenol caused dilation of human bronchial rings (Fig. 5). This relaxation of spontaneous airway smooth muscle tone was observed in these isolated bronchial ring preparations in the absence of exogenously induced active tension. Addition of isoproterenol caused an increase in mean Ai from 0.632 ± 0.083 to 0.777 ± 0.080 mm2 (P = 0.0019) in six rings from six patients (from the same cohort of 9 patients from whom tissues were used for contraction data). The Ao increased from 4.72 ± 0.293 to 4.97 ± 0.283 mm2 after isoproterenol (P = 0.0020). The difference for Ai (0.160 ± 0.024 mm2) and Ao (0.252 ± 0.047 mm2) was not statistically significant (P = 0.0647; Fig. 5).
DISCUSSION
The objective of this study was to determine the relationship between
luminal and total cross-sectional area of human bronchi in response to
contraction and relaxation to better understand auxotonic contractile
mechanisms of the airways in vivo. The precise mechanism of airway
narrowing in human airways has not been defined. Previous studies have
noted bronchial diameter changes in animal models (5, 12) or tracheal
rings from nonhuman species (4, 8). However, there are limitations to
the application of findings from animal models to humans based on
anatomy (13), responsiveness to agonists (4-6, 8, 9), and airway
generation (10). Thus airway narrowing may be different in human
conducting bronchi. By utilizing an image-capture system and a
sensitive method of image analysis, we were able to measure
Ai
and
Ao
of human sixth- and seventh-generation bronchi and, by subtraction, to
calculate the bronchial wall area (Fig. 2). We found that as the lumen
narrowed (Ai)
in response to muscarinic-receptor activation or relaxed in response to
-adrenergic-receptor activation, the total area (Ao)
of the bronchus changed similarly, and, by calculation, bronchial wall
area was unchanged (Figs. 3 and 5). We did not note any significant heterogeneity of responsiveness of seventh-generation human bronchi to
either carbachol or isoproterenol; this was in contrast to computed-tomography data reported for canine airways in vivo (2) and
explanted rat airways (3). Our human tissues responded within a narrow
range of dispersion in preliminary studies to determine the optimal
concentration of carbachol (n = 4 tissues/concentration; Fig. 1). Our in vitro observations of minimal
heterogeneity may be species and method specific.
These data also differ from preliminary studies of isolated canine bronchi where it was shown that as the lumen constricts in response to activation of the ASM, the external perimeter did not decrease significantly (12). It was concluded that this behavior is possible only if the connection between the ASM and the cartilage plaques is highly compliant. Under these circumstances, Stephens and Jiang (12) also concluded that the parenchyma would not load ASM and that the negative pressure caused in the airway wall by constriction of the ASM would result in engorgement of the blood and lymph vessels. Our data suggest that, for human sixth- and seventh-generation bronchi, neither of these conclusions likely applies. Although the change in Ao and Ai demonstrated a significant correlation coefficient (r = 0.763), the data were skewed somewhat (Fig. 4). The bronchial ring preparations that demonstrated the greatest changes in both Ai and Ao showed the greatest deviation from a slope of 1.0. The Ao for these tissues demonstrated a greater apparent change than the change in Ai. Potential reasons why the Ao decreased more than the Ai may be that, as the bronchial ring contracts under maximal activation with carbachol, 1) the mucosal tissues are forced into the longitudinal plane of these airway preparations (into the plane of the video camera), resulting in an underestimate in the change in Ai, or 2) because of the folding of the mucosa, the edges may be difficult to resolve with use of videomicrometry. Another important limitation to the interpretation of our data is that these studies were conducted on human bronchi in vitro. In vivo, the vascular and lymphatic vessel beds in the bronchial wall may be significant modulating factors during bronchoconstriction and dilation (12, 13).
Because Ao, as defined by the outer perimeter (1), is reduced similarly to Ai during contraction, our data further suggest that as the airway narrows during bronchospasm, the alveolar tissue in intimate contact with the outer perimeter surface of the bronchioles will be stretched. Thus smooth muscle in human airways during bronchoconstriction must overcome alveolar tissue tethering forces in addition to the resistive forces of the bronchial wall.
Our data, derived from this application of videomicrometry to measure
airway responses by accurately measuring computer-captured video
images, also suggest that spontaneous tone in isolated human bronchi is
inherent and not a manifestation of an imposed preload (5-7, 11,
12); i.e., in the absence of any resting tension that could be imposed
by isometric fixation to force transducers in organ perfusion systems
(3, 8), bronchial rings significantly relaxed in response to the
-adrenergic-receptor agonist isoproterenol (Fig. 5). These tissues
were fully equilibrated (see
METHODS), and luminal perimeters
were stable for at least 15 min before addition of
isoproterenol.
By using videomicrometry of human bronchial rings in vitro, we demonstrate that both Ai and Ao decrease and increase similarly in response to contractile and relaxant agonists, resulting in a conservation of bronchiolar wall area. We also demonstrate relaxation of human bronchial rings from spontaneous tone in the absence of isometric fixation. These data imply that 1) the reduction in Ao with bronchoconstriction will stretch adjacent lung parenchyma in vivo, thus imposing an additional load on the airway smooth muscle from alveolar tissue tethering forces, and 2) spontaneous tone of these bronchi is not a manifestation of in vitro isometric fixation.
ACKNOWLEDGEMENTS
This work was supported by National Heart, Lung, and Blood Institute Grants HL-46368 and HL-35718; National Institute of Allergy and Infectious Diseases Grant AI-34566; Bundesministerium für Forschung und Technologie, Germany (Förderkennzeichen 01 KE 9301); and Specialized Center of Research Grant HL-56399.
FOOTNOTES
Address for reprint requests: R. Mitchell, Sect. of Pulmonary and Critical Care Medicine, MC 6076, The Univ. of Chicago, 5841 S. Maryland Ave., Chicago IL 60637.
Received 22 April 1996; accepted in final form 5 November 1996.
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