Spatial distribution in the lungs ROBERT

R. MERCER

of collagen and elastin fibers

AND

JAMES

D. CRAP0

Departments of Medicine and Pathology, Duke University Durham, North Carolina 27710

MEEKER, ROBERT R., AND JAMES II. CRAPO. Spatial distribution of collagen and elastin fibers in the lungs. J. Appl. Physiol. 69(Z): 756-765, 1990.~Surface tension forces acting on the thin-wall alveolar septa and the collagen-elastin fiber network are major factors in lung parenchymal micromechanics. Quantitative serial section analysis and morphometric evaluations of planar sections were used to determine the spatial location of collagen and elastin fibers in Sprague-Dawley rat and normal human lung samples. A large concentration of connective tissue fibers was located in the alveolar duct wall in both species. For rats, the tissue densities of collagen and elastin fibers located within 10 pm of an alveolar duct were 13 and 9%, respectively. In human lung samples, the tissue densities of collagen and elastin fibers within 20 pm of an alveolar duct were 18 and 16%, respectively. In both species, bands of elastin fibers formed a continuous ring around each alveolar mouth. In human lungs, elastin fibers were found to penetrate significantly deeper into alveolar septal walls than they did in rat lungs, The concentration of connective tissue elements in the alveolar duct walls of both species is consistent with their proposed roles as the principal load-bearing elements of the lung parenchyma. alveoli; alveolar ducts; collagen fibrils; connective tissue; interdependence; lung anatomy; lung elasticity; morphometry; serial section

ELASTIN AND COLLAGEN FIBERS are the major elements of the connective tissue network within the lungs. Because of the elastic properties of the elastin-containing fibers and the nondistensible character of straight collagen fibers, it has been suggested that elastin-containing fibers account for lung compliance changes occurring in the normal breathing range, whereas collagen, a nondistensible connective tissue element, accounts for the limiting lung volume (17). As a consequence of such behavior, one would expect these two elements to operate independently of each other. This has been substantiated, in part, by studies that used elastase and collagenase digestion to alter the mechanical properties of the lungs (14, 26). Although the lack of absolute selectivity of these enzyme digestions may preclude simple interpretations, the results are in line with this proposed model. To further evaluate these functional models, morphological investigations are needed to define the arrangement of collagen and elastin fibers in the lung. Lightmicroscopic observations of thick sections reveal an apparent spiral arrangement of dense connective tissue 756

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fibers in alveolar duct walls (33). Pierce and Ebert (24) made similar observations and also noted that there was a close association of collagen and elastin based on lightmicroscopic examinations of alkaline-extracted lung specimens. These authors concluded that lung inflation was not associated with significant extension of individual collagen fibers. Such studies have suggested that uncoiling and coiling of connective tissue fibers is responsible for the elasticity of the lungs. In general, anatomic studies of connective tissue in the lungs have not addressed the relative roles of collagen and elastin fibers. The conclusions based on mechanical properties of collagen- and elastin-rich tissue samples such as tendons and nuchal ligaments (1) as well as observations on whole lung mechanical properties after protease digestion suggest that the anatomic arrangement of the collagen and elastin fibers results in independent roles for each. The dissimilar mechanical properties of the two elements suggest that the normal structural arrangement of collagen and elastin in the lungs must allow relatively independent functioning of the two connective tissue elements. However, electron-microscopic examinations of the lung have revealed that the elastin fibers are commonly present in close proximity to collagen fibers (11). Given this close anatomic proximity, apparently minor alterations in their arrangement induced by a disease process might prevent independent functioning and significantly alter the lung’s mechanical properties. Such disorder or rearrangement after remodeling in connective tissues has been suggested as a major factor in lung diseases such as emphysema (15) and fibrosis (11). These studies highlight the fact that knowledge of the arrangement of connective tissue fibers in the lung is critical to understanding their normal function and identifying critical sites of injury. To adequately define these relationships, we have used a combination of morphometry and three-dimensional reconstructions of lung tissue to determine the distribution and relative arrangement of collagen and elastin fibers. METHODS Experimental groups. To determine the relationship between collagen and elastin as well as their placement within alveoli and alveolar ducts, normal human lung specimens obtained from resected lobes and the lungs of normal rats were studied. The three human lung resec-

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DISTRIBUTION

OF

COLLAGEN

tions were from cases of nonsmoking individuals with normal pulmonary function tests and normal chest radiographs other than the small lesions that led to lobar resection. Two of the resected human lobes were from a 37-yr-old Caucasian man and a 62-yr-old Caucasian woman who had small adenocarcinomas with no evidence of metastasis. The third lobe was from a 47-yr-old Negro woman who had a small alveolar cell carcinoma with no evidence of metastasis. Samples from these lobes were taken from unaffected regions that were normal under gross examination. The sampled regions were also qualitatively normal under subsequent light- and electronmicroscopic examination. All lobes were fixed within 30 min of resection by intrabronchial instillation of 2% glutaraldehyde in a 0.84 M sodium cacodylate buffer at pH 7.4 and at a pressure of 30 cm of fixative as previously described (5). Samples (2 X 2 mm) from these lungs were taken and en bloc stained for 2 h in a 2% osmium tetroxide-saline solution adjusted to 350 mosM with sucrose. The unreacted osmium was removed by a saline solution adjusted to 350 mosM, and the blocks were stained for 8 h in a 4% tannic acid- (Gallotannin, Pfaltz and Bauer, Waterbury, CT) saline solution, pH 7.0, adjusted to 350 mosM. The blocks were then rinsed and stained in a 2% uranyl acetate-sucrose solution (350 mosM) for 8 h before they were embedded in epoxy resin. Preembedding staining with tannic acid at a pH of 7.0 results in an electron-dense staining of both elastin and collagen fibrils (28) and eliminates the need for tannic acid staining of sections (l3), which tends to form precipitates and gives less intense staining. The lungs of four male specific pathogen-free SpragueDawley rats [ CD( SD)BR, Charles River, Wilmington, MA] weighing 292 t 23 (SE) g were fixed while being inflated with air at a transpulmonary pressure of 5 cmH20. Vascular perfusion of fixative was followed by perfusion dehydration (23, 29). To carry out vascular perfusion fixation of the lungs, procedures similar to those previously described were used (20). The rats were anesthetized with pentobarbital sodium (60 mg/kg) by intraperitoneal injection. A tracheal cannula was inserted and attached to a pressure reservoir that maintained the lungs at a transpulmonary pressure of 5 cmH*O. In quick succession, a midline incision of the thorax was made, the pulmonary artery was cannulated by puncture of the right ventricle, and the left atrium was connected to a large-bore tube, the open end of which was 5 cm above the heart. Vascular perfusion of the lungs was begun by clearing blood from the lungs at a perfusion pressure of 15 cmH20 by use of a physiological salt solution containing heparin (100 U/ml) and adjusted to 350 mosM with sucrose. The lungs were then inflated to 30 cmHg0 and deflated to 5 cmHzO three times. The lungs were then inflated to 30 cmH20 and deflated to the holding pressure of 10 cmH20. At this point the perfusate was switched to a fixative solution containing 2% glutaraldehyde, 1% formaldehyde, and 1% tannic acid in cacodylate buffer adjusted to pH 7.4 (475 mosM). After 5 min of perfusion with this fixative, the lungs were cleared of fixative with the physiological salt solution. The lungs were then perfused for 5 min with a

AND

ELASTIN

757

2% osmium tetroxide-saline solution adjusted to 350 mosM with sucrose and then perfused again with the physiological salt solution. After removal of the unreacted osmium, the lungs were perfused with a 4% tannic acid-saline solution. This solution was held in the lungs for 30 min to allow adequate time for diffusion of the tannic acid into the interstitium before perfusion with a 2% uranyl acetate-sucrose solution (350 mosM). After 15 min of uranyl acetate perfusion, the lungs were perfusion dehydrated by a series of graded alcohols from 20 to 90% ethanol. Five milliliters of each were perfused through the lungs, and 50 ml of 100% ethanol were used in the final perfusion. Samples from these lungs were then embedded in epoxy resin without rehydration. Connective tissue fiber reconstructions. Three-dimensional reconstructions of the connective tissue elements were carried out by use of serial step sections (0.1 pm thick). Every third section of a total of 200-400 sections (20-40 pm deep into the block) was collected on a Formvar-coated hole grid and stained with uranyl acetate and lead citrate. To avoid potential bias in selection of connective tissue fibers because of size or length, the collagen and elastin fibers were reconstructed from alveolar septa of alveoli selected by use of the unbiased sampling criteria previously described (20). Photographs were taken of three and six adjacent step sections in the middle region of the series of rat and human lungs, respectively. These sections were then printed at ~300 final magnification on 11 x 14-in. photographic paper, and a selector box was drawn 1 in. from the outer margins of each print. Unbiased sampling was carried out by identification of those alveoli that opened up into the adjacent alveolar duct in the first print but not in the last print. Of the identified alveoli, alveoli were selected for connective tissue fiber reconstruction if the point at which they initially opened into the adjacent alveolar duct was contained inside the selector box. Two to three alveoli in the rat lung and one to two alveoli in the human lung were typically selected per series. The alveolar septal walls of the selected alveoli were then examined with a Phillips CM10 electron microscope. The alveolar septal wall of each selected alveolus was photographed and printed at a final magnification of x2,000 on 11 X 14-in. photographic paper. In some cases, the photography was extended to cover extensions of the connective tissue fibers into adjacent alveoli, as will be discussed. The prints of the series were examined to identify the collagen and elastin fibers, interstitial cells, epithelial surface, and endothelial cell profiles. These structures were entered into the computer for three-dimensional reconstruction by implementation of methods previously described (19). A total of 20 serial section reconstructions were carried out (4 per lung in 3 rat lungs and 2-3 per lung in 3 human lungs). Collagen fibril reconstructions. Collagen fibril reconstructions were carried out to determine the structural arrangement of the fibrils forming collagen fibers. Ribbons of 60-100 serial sections (35 nm thick) were cut and broken up into three to five groups and placed on 1.5 x 2.0-mm slotted Formvar-coat grids in a manner

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758 TABLE

DISTRIBUTION

OF

COLLAGEN

1. Tissue density of collagen and elastin fibers Rat Collagen fibers Elastin fibers Collagen/elastin n

0.031f0.003 0.01640.001 1.94kO.2 4

Human 0.102+-0.011 0.067kO.020 1.46kO.16 3

Values are means k SE. Data are expressed as volume or elastin fibers per unit volume of parenchymal tissue contents of capillary lumina).

of collagen (excluding

FIG. 1. Electron micrograph of an alveolar septal edge from a rat lung demonstrating close proximity and high concentration of collagen (cl) and elastin (el) fibers in alveolar septal edges forming walls of alveolar ducts.

similar to that described by Bundgaard (4). Six series were cut from sites selected at random from the four perfusion-fixed rat lungs. The large collagen fibers near the alveolar septal edges were photographed in these series at ~32,000 and printed on 11 X 14-in. photographic paper at a final magnification of X150,000. Each of the collagen fibrils present in the middle print of the series was assigned a number, and a random number table was used to select eight fibrils for serial section reconstruction. The centers of mass for the profiles of each fibril were ‘computed, and the length of the path taken along the centers of mass was determined in three dimensions. The ratio of this distance to the straight-line distance in three dimensions from the first to last profile of each fibril in the series was then computed as a measure of the percentage of length extension that the fibrils could undergo without stretching the fibrils. Collagen and elastin fiber distribution. Our goal in the determination of the collagen and elastin fiber distribution was to detect how the content of elastin and collagen fibers varied as one moved radially outward from the alveolar duct wall into the alveolar septal region. This is most accurately done by quantitative serial section reconstruction techniques. However, because of the potential variability between animals and regions of the lung, the number of required reconstructions was prohibitive. We therefore chose to use a combination of serial section methods and single section analysis, which allowed a sufficiently accurate and rapid analysis that multiple sites could be studied in a number of lungs. For this

AND

ELASTIN

purpose we made use of the proximal alveolar duct isolation technique previously described (3) and a modified selector (7) to select, in an unbiased manner, alveolar ducts with central axis perpendicular to the plane of sectioning. Ten serial step sections (60 nm thick) were taken at 5-pm intervals from four to six randomly sampled blocks of the left lungs from the four rats and from the four human lung specimens. From low-magnification pictures (x60) of the series, the alveolar duct boundaries were marked on the prints as described previously (20). The center of the alveolar ducts that were within 10” of perpendicular to the plane of sectioning were then determined in the middle print of the series. The alveolar ducts with their centers contained in the selection area of the middle print (a rectangular box placed on the print) were then selected for analysis of the connective tissue fiber distribution vs. distance from the alveolar duct wall. The photography for study of the connective tissue fiber distribution was done by placing a randomly oriented line over each of the selected alveolar duct centers and photographing any alveolar tissue present along the line for 110 and 210 pm in rat and human lungs, respectively, starting at the point where the previously drawn alveolar duct boundary was intersected by the randomly oriented line. These distances were chosen to cover half the average distance between alveolar ducts based on measurements to be described subsequently. The final printing magnification of this set of prints was ~5,500, with an average of three to four alveolar ducts selected per block. For each of the selected alveolar duct profiles, the area and perimeter were measured and the radius computed. The area and perimeter of collagen fibers, elastin fibers, and tissue (excluding capillary lumen contents) were then determined for each lo-pm-deep compartment beginning at the alveolar duct wall and continuing outward along the alveolar septa in these prints. The tissue volume fractions for collagen and elastin fibers were then determined for each compartment. The volume fractions for each fiber type and compartment were corrected for the effect of finite section thickness as described by Weibel (31). For both collagen and elastin fibers, this correction was small and resulted in a lo-12% reduction between the original and corrected means for volumes of collagen and elastin fibers. The distribution of collagen and elastin fibers was then expressed as the fraction of collagen or elastin fibers per unit of tissue volume for the tissue present in each lo-pm-deep compartment. Tissue density vs. distance from the alveolar duct wall.

Determinations of the tissue density as a function of distance from the alveolar duct wall were also made. An overlay of 224 points was placed on the low-magnification picture of the middle print of each series. For each of the alveolar ducts selected in the collagen and elastin fiber distribution study, a tally was made of the points over tissue or air at 20-pm intervals from the alveolar duct wall. For sections of the rat lung, points were tallied O-60 pm from the alveolar duct wall. For sections of human lungs, the tally of points was O-220 pm from the alveolar duct wall. By multiplying the tissue density as a

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DISTRIBUTION A

Distance

100

Alveolar

Duct

A

6

“v

0.

FIG. 2. Distribution of collagen and elastin fibers in rat lung parenchyma. Relative tissue fractions (V,) of collagen (A) and elastin (B) as a function of radial distance outward from alveolar duct wall are shown. High concentration of collagen and elastin fibers located in alveolar septal edges forming alveolar duct wall is demonstrated.

ELASTIN

50

from

759

AND ELASTIN

COLLAGEN

6

10

OF COLLAGEN

( pm

)

COLLAGEN

FIG. 3. Distribution of collagen and elastin fibers in human lung parenchyma. Relative tissue fractions (V,) of collagen (A) and elastin (B) as a function of radial distance outward from alveolar duct wall are shown. Same high concentration of connective tissue fibers in alveolar tissue adjacent to alveolar duct is depicted in human lungs as was seen in rat lungs. Unlike the rat, there is also a high concentration of elastin fibers throughout alveolar septal wall.

EI-ASTIN

l-

10

Distance

110

from

Alveolar

210

Duct

(

pm

1

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760

DISTRIBUTION

TABLE

COLLAGEN

2. Rat connective tissue fiber distribution Distance O-20

Tissue Pm3 % Collagen fibers Pm3 % Elastin fibers w-n3 % Alveolar

TABLE

OF

duct

3. Human

Tissue Pm3 % Collagen fibers Pm” % Elastin fibers Pm3 % Alveolar

duct

Duct Wall, Total 60-120

15.2

4,304 37.6

5,404 47.2

154 43.3

7% 22.0

122 34.5

355

13% 76.5

33 18.3

10 17.1

180

46.5 & 2.2 (SE)

11,455

pm. n = 4 specimens.

connective tissue fiber distribution Distance

From Alveolar Pm

O-20

20-120

120-220

19,357 36.2

30,023 56.2

53,424

7.6 874 17.6

1,579 31.7

2,522 50.7

4,146

647 22.0

896 31.5

1,300 45.7

2,843

4,044

radius

Alveolar rum 20-60

1,747

radius

From

136.3

k 16.0 (SE)

pm,

Duct

Wall, Total

n = 3 specimens.

AND

ELASTIN

the space between alveolar ducts was determined by calculating the mean free distance between alveolar ducts (30). The mean free distance was calculated from the volume density of alveolar ducts, and the surface density of the alveolar duct boundary was obtained by point and intercept counts from an overlay placed on the middle print of the low-magnification series used in the connective tissue fiber distribution measurements. To determine the fraction of the alveolar epithelial surface that formed the alveolar duct wall, intercepts with epithelial surface were determined in the middle print of each low-magnification series. Alveolar duct wall surface intercepts were taken as those intercepts with epithelial surface that formed part of an alveolar duct boundary. Collagen and elustin fiber volume density. Collagen and elastin fiber volume density for lung parenchyma was determined from randomly sampled blocks. This was done by taking 20 electron micrographs from 4 randomly selected sites from each of the human and rat lungs. The micrographs were printed at a final magnification of X8,500 on 11 X 14-in. photographic paper. A counting overlay containing 112 &cm lines was used to determine the fraction of points on tissue (excluding capillary lumen contents), collagen fibers, and elastin fibers. RESULTS

The average lung tissue density of collagen and elastin fibers as well as the mean collagen-to-elastin

function of distance from the alveolar duct wall by the collagen or elastin fiber volume fraction of tissue, the absolute amounts of collagen and elastin fibers could be determined as a function of distance from the alveolar duct wall. The average distance through the alveoli occupying

Rat

A

10 I

ratio for

rats and humans based on random sampling of the lungs are given in Table 1. In both species the connective tissue fibers were present in high concentration in the alveolar tissue forming alveolar duct walls (Fig. 1). The results of the determinations of collagen and elastin fiber tissue volume fraction vs. distance from the alveolar duct in rat

FIG. 4. Relative amounts of collagen and elastin fibers vs. distance outward from alveolar duct for rat (A) and human lungs (B). For alveolar septal edges that form alveolar duct walls, collagen-toelastin ratio is - 1. Further out from an alveolar duct, in alveolar septal walls, collagen-to-elastin fiber ratio is -5 in rat and 1.5-2 in human.

%

Collagen -

5-

Elastic

10

Distance

210

110

from

Alveolar

Duct

(

pm

1

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DISTRIHJTION

OF COLLAGEN

AND ELASTIN

761

FIG. 5. Electron micrographs of collagen and elastin fibers in alveolar region of rat. Connective tissue elements are seen that are present aa one traverses from alveolar entrance ring along one alveolar duct (A) through alveolar septal wall (B) to alveolar entrance ring on an adjacent alveolar duct (C). D region from which A, B, and C were taken.

4

ALVEOLAR DUCT

FIG. 6. Serial section reconstruction of collagen and elastin fibers in alveoIar region view shows entire traversal htween two alveolar ducts. B: reconstruction demonstrates orang] and elastin (el, blue) fibers between adjacent alveolar duck. Example illustrates el and larger cl. which contain 86300 fibrils per fiber, present in duct walls. Also shown 8-20 fib& per fiber, as they weave through capillary lattice (cap, red) deeper in alveolar

and human lungs are given in Figs. 2 and 3, respectively. The absolute tissue volume, connective tissue fiber volume, and percentage of total connective tissue fiber volume for a cylinder containing an alveolar duct and the alveolar septal walls for a distance halfway between adjacent alveolar ducts were determined. These were based on the volume fraction of connective tissue fibers in Figs. 2 and 3, the tissue density vs. distance from the alveolar duct boundary, the aIveolar duct radius, and the average distance between alveolar ducts. The results for rat and human lungs are given in Tables 2 and 3, respec-

of rat. A: low-magnification arrangement of collagen (cl, typical pattern observed for are smaller cl, which contain sepm.

tively. In the rat, the majority of the elastin fiber volume (77%) and -44% of the collagen fiber volume was in the first lo-Frn compartment of alveolar tissue adjacent to the alveolar duct boundary (Table 2). In the human lungs, 22% of the elastin fibers and 18% of the collagen fibers contained in the cylinder were present within 20 pm of the alveolar duct wall. The average tissue density of collagen fibers in the cylinder was 0.031 and 0.093 for rat and human lungs, respectively. The average elastin fiber density was 0.016 and 0.053 for rat and huma lungs, respectively. These results based on a cylindrical

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762

DISTRIBUTION

OF

COLLAGEN

AND

ELASTIN

0.5 pm I lveolar pening

8. Serial section reconstruction of collagen fibrils. A: electron micrograph of one ultrathin section (35 nm) from a series of 95 sections through a main collagen fiber in an alveolar duct wall. Over 3.3~pm interval of series, this fiber was perpendicular to plane of sectioning with no significant curvature. Perpendicular orientation was also confirmed by the fact that collagen fibrils of fiber all had circular profiles, as opposed to elliptical profile one would see in a nonperpendicular cut through a cylinder. B: shaded surface view from a serial section reconstruction of 10 fibrils (l-10), which were arranged in a line on outer edge of fiber at starting section of series. This as well as serial section reconstructions used to determine fibril extensibility (see RESULTS) demonstrated that collagen fibrils of alveolar duct wall collagen fibers have a crimped or wavelike arrangement. FIG.

coliagen 7. Arrangement of connective tissue fibers in alveolar tissue immediately adjacent to alveolar duct. A: scanning electron micrograph of a vascular perfusion-fixed rat lung showing an alveolar duct in cross section as it originates from a bronchiole-alveolar duct junction (BADJ). B: schematic of arrangement of connective tissue fibers in alveolar tissue at boundary of alveolar duct. Elastin fibers are present as a single continuous band that encircles each alveolar entrance ring. Collagen fibers form a meshlike arrangement around entrance. Typically, one or more of collagen fibers (*) is significantly larger than other elements of mesh, which gives spiral pattern of connective tissue fibers observed in thick sections. FIG.

model of connective tissue distribution agree closely with those obtained by random sampling of lung tissue (Table 1). The mean free distance between alveolar ducts was 209 + 24 pm for rat and 416 & 61 pm for human lungs. The percentage of the alveolar epithelial surface that formed the walls of the alveolar ducts was 9.4 + 0.9 and 5.3 + 1.1% in rat and human lungs, respectively. The collagen-to-elastin fiber ratio given in Table 1 is -2 for the rat and 1.5 for the human specimens. The graphs of the distribution of the collagen-to-elastin ratios given in Fig. 4 and the corresponding examples in Fig. 5 demonstrate that this average does not accurately reflect the spatial relationships of these two connective tissue elements. One serial section reconstruction of the connective tissue fibers from a rat lung is shown in Fig. 6. This reconstruction demonstrates the connective tissue-rich region of the alveolar duct wall with the consistent presence of a prominent and continuous band of elastin fibers in the outer edge of the alveolar entrance ring forming the alveolar duct wall. In the reconstruction

given in Fig. 6, several of the minor collagen fibers were demonstrated to interconnect between alveolar entrance ring regions of adjacent alveolar ducts. We were unable to determine the fraction of these alveolar septal wall fibers that interconnect adjacent alveolar ducts because of the long and tortuous paths taken by these fibers. However, it is clear that a significant fraction of these fibers do interconnect between adjacent alveolar ducts and that the remainder cross a significant fraction of that distance. In all serial section reconstructions where the alveolar entrance ring was examined, a prominent and continuous band of elastin fibers around the alveolar mouth was found (Fig. 7B). In some regions of the reconstructions, the bandlike collection of elastin fibers subdivided into several bundles that were closely associated with collagen fibers (Fig. 1). In other regions, a single distinct band of elastin fibers was found with a separation of several microns between it and the adjacent collagen fibers. In addition to determining the measurements of parenchymal structure and connective tissue distribution, an effort was made to determine the extent to which the collagen and elastin fibers were mechanically interconnected with each another. As indicated above, the elastin fibers forming the alveolar entrance rings occurred in two particular configurations relative to the adjacent collagen fibers. In one configuration, the elastin fibers were in intimate contact with the adjacent collagen fibers. In serial sections, these elastin-containing fibers were found to be interwoven with numerous collagen fibrils of the contacting collagen fiber. This would indi-

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DISTRIBUTION

OF

COLLAGEN

cate that elastin-containing fibers in close proximity with collagen fibers are to some extent mechanically interconnected. The elastin fibers of the second configuration were in a tight bandlike configuration and were spatially separated from the adjacent collagen fibers. To quantify this, we determined the fraction of the alveolar entrance ring that contained elastin fibers interwoven with the adjacent collagen fibers as in Fig. 1. This was done by taking photographs of all septal edges present in the sections previously used in the determination of the distribution of collagen and elastin. The fraction of the length of the alveolar entrance ring containing elastin fibers that were interwoven with adjacent collagen fibers was then determined from the ratio of septal edges containing elastin fibers in intimate contact with collagen fibers to those septal edges containing elastin fibers spatially separated from the adjacent collagen fibers. The fraction of the elastin band in the alveolar entrance ring that was interwoven with the adjacent collagen fibers was 51% for rat and 41% for human lungs. Collagen fibers present in the alveolar entrance ring demonstrated a wide variation in pattern and width, which was similar to the descriptions of Matsuda et al. (16). This variation corresponded to the partitioning of the main collagen fibers present in the alveolar entrance ring because they subdivided into finer collagen fibers that passed out into the alveolar septal wall. Collagen fibrils of the large collagen fibers near the alveolar septal edge were found to have a significant degree of zigzag or wavelike structure in serial sections (Fig. 8). When the wavelike structure of adjacent fibrils in a given fiber was compared, the fibrils meandered about one another with no apparent order or uniform pattern. The percent extension of the fibrils that could be obtained by straightening of the wavelike structure of the fibrils was 16.1 -c3.2% (SE) (n = 6). DISCUSSION

Oldmixon and Hoppin (22) found volume fractions of 0.08 and 0.03 for collagen and elastin fibers, respectively, in the dog lung, which is higher than the corresponding values for the rat and lower than the values for the human lung. Of particular importance is the fact that the human, unlike the rat, has a significantly higher percentage of elastin fibers. Indeed, we found a two- to threefold greater concentration of elastin fibers in the lung parenchyma of humans. This increase in connective tissue fibers in normal human lungs may be related to the greater interstitial thickness and interstitial cell numbers in human lungs, which has been previously reported (6), or to the greater age of the humans. However, it appears unlikely that the greater age of the humans is responsible for this difference because a number of reports (12, 21, 24) have demonstrated that agerelated increases in human lung elastin are restricted to airways and blood vessels. The ratio of collagen to elastin fiber volume in Table 1 is -30% lower than comparable measurements based on biochemical analysis of slices of lung parenchyma (9, 25). This difference may be due to the fact that our analysis excluded all vascular and airway structures,

AND

ELASTIN

763

whereas biochemical analyses of lung slices include small blood vessels and airway fragments, which have a significantly higher collagen-to-elastin ratio than the lung parenchyma (24). As has been demonstrated by the distribution of collagen-to-elastin ratios given in Fig. 4 and the micrographs of Fig. 5, the collagen-to-elastin ratio is only an average for all locations in the lung parenchyma. The average does not accurately reflect the spatial relationships of these two connective tissue elements. Indeed, the ratio is -1 in the alveolar septal edges forming the alveolar duct walls where connective tissue fibers are most prominent (Fig. 5). If collagen and elastin fibers act as mechanical elements in the lungs, we may ask what roles they take. Mass, springs, and dampers (dashpots) are the three purely translational mechanical elements (27). Viscous or frictional resistance of the connective tissue fibers due to a mass-damper arrangement will determine the work required for dynamic movements of the lung parenchyma and can also be responsible for hysteresis in mechanical properties. The springlike action of connective tissue fibers will alter the static mechanical properties. Because the static mechanical properties of the lung are of significance in interpretations of lung volume changes, we in general consider only the springlike action of connective tissue elements. Thus, when a high concentration of connective tissue fibers is observed in the walls of the alveolar ducts (Figs. 2 and 3), it suggests that this represents a biological response to reduce a high local stress in the walls of the alveolar ducts to some equilibrium level. Such a role for the connective tissue fibers in the walls of the alveolar ducts follows the predictions of the model developed by Wilson and Bachofen (32). Based on experimental and theoretical results, these investigators suggested that the walls of the alveolar ducts correspond to a mechanical load-bearing structure and that the surrounding alveoli correspond to a surface tension-dominated region (composed of the alveoli surrounding alveolar ducts). The results for the rat (Table 2) indicating the high collagen and elastin fiber volume in the first 20 pm of alveolar tissue adjacent to the alveolar duct wall support this model and its more recent modifications (2), which suggest an even greater role for tissue retractive forces. However, in the human lung the connective tissue fibers and in particular elastin are more dispersed throughout the alveolar septal wall (Table 3), which does not correspond to the Wilson and Bachofen model. This greater dispersion of elastin fibers throughout the alveolar septal wall and the larger total quantity of connective tissue fibers in the human lungs were the major differences found between human and rat lungs. The threefold increase in connective tissue fibers and the more disperse distribution of the fibers throughout the larger-radius alveoli of the human lung may reflect the need for greater tissue recoil forces throughout the alveolar septal wall in larger lungs, where the increased radius of curvature will tend to reduce the lung recoil due to surface tension forces. The quantitative results of this study suggest two separate anatomic locations and functions of connective

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764

DISTRIBUTION

OF

COLLAGEN

tissue fibers in the lung parenchyma. The predominant of these is in the alveolar duct wall, as illustrated in Fig. 7B. This consists of the elastin fibers, which band together to form a complete ring around the alveolar mouth openings, and the collagen fibers of the alveolar duct wall, which form a meshlike network between adjacent alveolar openings. This meshlike network of collagen fibers is not uniform and in general contains one or more collagen fibers that are significantly larger than the other elements of the mesh. In thick sections where the finer elements of the mesh are not distinct, this gives the appearance of a single collagen fiber encircling the alveolar duct in a springlike manner as observed by Young et al. (33). The second anatomic location is composed of collagen fibers and elastin fibers that pass in the alveolar septal walls between adjacent alveolar ducts. The arrangement of the elastin fiber bands, which are just below the alveolar septal edges and often in intimate contact with the collagen fibers of the alveolar duct wall, brings into question whether elastin has a stress-bearing role. This same question regarding the role of elastin fibers has been addressed in studies of veins, where it has been suggested by Azuma and Hasegawa (1) that the collagen fiber network is loose or kinked at normal levels of distension and thus allows elastin to undergo stress before the collagen fibers become straight and limit further distension, In our serial section analysis of the collagen and elastin fibers in the walls of the alveolar ducts, both collagen and elastin fibers were close to the curvature of the adjacent epithelial surface. The absence of any loose or kinked arrangement of collagen fibers in the alveolar septal walls indicates that the connective tissue fiber model proposed for veins by Azuma and Hasegawa is not applicable to the lungs. A similar conclusion has been reported by Fung (lo), who found no significant difference between the curvature of collagen and elastin fibers in the alveolar duct wall. These results highlight the role of elastin fibers as mechanical elements in the lung because they indicate that elastin and collagen fibers act as parallel mechanical elements to applied stress or strain. The parallel nature of this arrangement suggests that collagen fibers must have some degree of elasticity for the elastin to function as a stress-bearing element. Results by Diamant et al. (8) demonstrated that collagen fibers in the rat tail tendon may undergo a considerable extension (up to 13% at 2 wk of age) before the traditionally assumed inelastic behavior of collagen fibers is observed. These investigators demonstrated that this highly extensible behavior for initial stretch above the resting length of the fiber was due to the crimped or wavelike structure of the collagen fibrils making up the fiber. In serial section reconstructions, we found that collagen fibrils of alveolar duct wall collagen fibers have a similar wavelike arrangement (Fig. 8). Measurements of percent extensibility created by the wavelike structure indicate that collagen fibrils, at lung volumes comparable to functional residual capacity, may be extended by as much as 16% before they become straight and limit further extension. This is an important functional change because it is comparable in magnitude to the changes expected to occur in

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ELASTIN

alveolar wall surface area during deflation from total lung capacity to functional residual capacity (20). These results indicate that extension of connective tissue fibers in the alveolar duct walls may be in two stages. At low levels of strain, the collagen fibrils are in a wavelike configuration and are easily extended, with most of the stress being borne by the adjacent elastin fibers. At higher levels, the collagen fibrils become straight and act to limit further distension of the alveolar ducts. The transition of collagen fibrils from a wavelike to a straight pattern suggests that collagen fibers may act as dampers in the translation of mechanical forces due to frictional resistance as collagen fibrils deform within the surrounding interstitial matrix. The energy loss due to this frictional resistance would be expected to lead to tissue hysteresis between inflation and deflation of the lungs. Such a nonconservative mechanical element has been proposed by Bachofen et al. (2) based on the fact that measured surface tensions in the lungs are not sufficient to fully account for pressure-volume hysteresis. This study was supported by the US Environmental Protection Agency under cooperative agreement CR813113 and Department of Energy Grant DE-b’G05--88ER60654. Although the research described in this article has been funded wholly or& part by the US Environmental Protection Agency, it has not been subjected to the Agency’s required peer and policy review; therefore, it does not necessarily reflect the views of the Agency, and no official endorsement should be inferred. Address for reprint requests: R. R. Mercer, Box 3177, Duke University, Pulmonary Medicine, Durham, NC 27710. Received

12 April

1989;

accepted

in final

form

5 April

1990.

REFERENCES 1. AZUMA, T., AND M. HASEGAWA. Distensibility of the vein: from the architectural point of view. Biorheology 10: 469-479, 1973. 2. BACHOFEN, H., S. SCHURCH, M. URBINELLI, AND E. R. WEIBEL. Relations among alveolar surface tension, surface area, volume and recoil pressure. J. Appl. Physiol. 62: 1878-1887, 1987. 3. BARRY, B. E., F. J. MILLER, AND J. D. CRAPO. Effects of inhalation of 0.12 and 0.25 ppm ozone on the proximal alveolar region of juvenile and adult rats. Lab. Invest. 53: 692-704, 1985. 4. BUNDGAARD, M. The three-dimensional organization of tight junctions in a capillary endothelium revealed by serial section electron microscopy. J. Ultrastruct. Res. 88: 1-17, 1984. AND E. R. 5. CRAPO, J. D., B. E. BARRY, P. GEHR, M. BACHOFEN, WEIBEL. Cell number and cell characteristics of the normal human lung. Am. Rev. Resoir. Dis. 125: 740-745, 1982. E. K. FRAM; K. E. PINKERTON, B. E. 6. CR~PO, J. D., S. L: YOUNG, BARRY, AND R. 0. CRAPO. Morphometric characteristics of cells in the alveolar region of mammalian lungs. Am. Rev. Respir. Dis. Suppl. 128: S42-S46,1983. 7. CRUS-ORIVE, L. M. Particle number can be estimated using a disector of unknown thickness: the selector. J. Microsc. Oxf. 145: 121-142,1987. 8. DIAMANT, J., A. KELLER, E. BAEP, M. LITT, AND R. G. C. ARRIDGE. Collagen; ultrastructure and its relation to mechanical properties as a function of ageing. Proc. R. Sot. Lord. B Biol. Sci. 180: 293315,1972. 9. FULMER, J. D., AND R. G. CRYSTAL. The biochemical basis of pulmonary function. In: The Biochemical Basis of Pulmonary Function, edited by R. G. Crystal. New York: Dekker, 1976. (Lung Biol. Health Dis. Ser.) 10. FUNG, Y. C. Evaluation of mechanical properties of the lung on the basis of its microstructure (Abstract). FASEB J. 2: A1270, 1988. 11. HANCE, A. J., AND R. G. CRYSTAL. The connective tissue of lung. Am. Rev. Respir. Dis. 112: 657-711, 1975.

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F. TAVASSOLI. The scanning electron microscopy of elastase-induced emphysema: a comparison with emphysema in - _ man. Lab. Invest. 34: 219,- 1976. 16. MATSUDA. M.. Y. C. FUNG. AND S. S. SOBIN. Collagen and elastin fibers in human pulmonary alveolar mouths and ducts. J. Appl. Physiol. 63: 1185-l 194, 1987. 17. MEAD, J. Mechanical properties of lungs. Physiol. Rev. 41: 2&132&,1961. MERCER,

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Spatial distribution of collagen and elastin fibers in the lungs.

Surface tension forces acting on the thin-wall alveolar septa and the collagen-elastin fiber network are major factors in lung parenchymal micromechan...
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