INTRODUCTION

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Emphysema is induced in hamsters and other rodents by the intratracheal instillation of porcine pancreatic elastase (PPE), human neutrophil elastase, and other elastolytic enzymes (1). Biochemical studies of the lung after PPE treatment show a sharp decrease in total lung elastin within the first 24 h (2, 3). Lung elastin content starts to increase soon after injury and reaches normal or above normal levels by 30 to 40 d (1). Morphometric ultrastructural studies show a decrease in elastic fibers in alveolar septal tips at 1 wk after PPE treatment, the earliest time studied; there are fewer breaks in the elastic fiber continuum of alveolar entrance rings at 4 and 12 wk than at 1 wk, suggesting that repair has occurred (4). Scanning electron microscopy shows abnormal air spaces formed by progressive dilation of alveolar ducts with shortening and occasionally effacement of interalveolar septa; interalveolar fenestrae are occasionally enlarged (5). However, little is known of the precise nature of the repair process.

Light microscopy reveals that within 24 h after intratracheal PPE treatment of the hamster, alveolar wall destruction and air space enlargement are extensive (6); alveolar hemorrhage and inflammation are largely cleared by Day 21 and air space enlargement has progressed (7). The total number of alveoli is decreased to about 45% of normal, the mean linear intercept doubles, and the internal surface area is decreased to about 70% of normal (8). Ultrastructurally, at 4 to 48 h, many alveolar walls are without elastic fibers. Collagen fibrils are present in swollen connective tissue spaces. Complete digestion of elastic fibers is more certain in many areas of pleura. At 4 d, small, fibrillar, newly formed elastic fibers are seen, mainly in pleura and blood vessel walls, adjacent to fibroblasts containing large amounts of rough endoplasmic reticulum and a prominent Golgi apparatus (9).

Most previous work on the elastase model of emphysema has focused on the factors that cause elastin degradationan increase in the elastase burden or a decrease in the antielastase shield of the lungs. We hypothesize that whether or not slow-paced elastin degradation gives rise to emphysema depends on whether or not elastin damage is balanced by elastin repair. This study explores the process of repair of elastin and Type I collagen in vivo after elastase-induced lung injury. We used the PPE animal model of emphysema because the model has been extensively described biochemically, physiologically, and by light and electron microscopy (1, 4).

	METHODS

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Preparation of ³⁵S-labeled Riboprobes

Single-stranded sense and antisense RNA probes are transcribed from a transcription vector containing a complementary DNA (cDNA) fragment coding for type I collagen or elastin using the Riboprobe System (Promega, Madison, WI). The elastin probe used was a 1,600 bp fragment from the 3' terminal portion of rat tropoelastin messenger RNA (mRNA) (12). The collagen probe used in these experiments was a 600 bp fragment from the 3' untranslated region of the rat ₁(I) collagen cDNA (13). This probe specifically identifies murine ₁(I) collagen transcripts (14). The probes are radiolabeled with [³⁵S]uridine triphosphate (Dupont NEN, Boston, MA). After incubation at 37° C for 2 h, the DNA templates are digested with 1 unit of RQ1 ribonuclease (RNase)-free deoxyribonuclease (DNase) (Promega) at 37° C for 15 min. The sense and antisense probes are subjected to alkaline hydrolysis to decrease their average fragment length to 150 bases. The probes are then extracted with phenol-chloroform and precipitated in ethanol.

Animal Treatment

Male VAF/Plus hamster (Mesocricetus auratus, LVG) from Charles River Breeding Laboratories (Wilmington, MA) were anesthetized by CO₂ inhalation and given a transoral intratracheal instillation of 0.5 ml of physiological saline or 200 µg of porcine pancreatic elastase (Elastin Products, Owenville, MO) in 0.5 ml of saline solution.

Preparation of Tissue

At 1, 2, 3, 5, 7, and 30 d after treatment animals were anesthetized with pentobarbital sodium and exsanguinated by cutting the abdominal aorta. The pulmonary vessels were perfused with freshly prepared paraformaldehyde and the lungs were inflated with fixative and excised. At the end of the day the lungs were placed in fresh fixative and stored at 4° C overnight. Slabs of tissue were cut from the lung and dehydrated and embedded in Paraplast Plus (Oxford Laboratories, St. Louis, MO) embedding medium. Four-micron serial paraffin sections were cut, individually handled and numbered, and transferred to Superfrost Plus slides (Fisher Scientific, Springfield, NJ) for in situ hybridization or staining.

In Situ Hybridization

The sections were deparaffinized in xylene, hydrated, and fixed with 4% paraformaldehyde-phosphate-buffered saline. A 5-min incubation in 20 µg/ml proteinase K at 37° C or room temperature followed and the sections were then fixed briefly again in 4% paraformaldehyde. Sections were treated with acetic anhydride (0.026 M) in a 0.1 M triethanolamine bath, dehydrated and air dried for a minimum of 2 h before hybridization. The riboprobes were heated to 80° C for 2 min prior to addition to ice cold hybridization buffer (containing 50% formamide, 0.3 M NaCl, 20 mM Tris-HCl, 5 mM disodium ethylenediaminetetraacetate, 10 mM NaH₂PO₄, 10% dextran sulfate, 1× Denhardt's, 0.5 mg/ml transfer RNA (tRNA), and 10 mM dithiothreitol). Aliquots of 30 µl of ³⁵S-labeled riboprobe at a concentration of 50,000 cpm/µl in hybridization buffer were spread over each section and then covered with coverslips. Slides were placed in plastic slide boxes containing a sponge soaked with 50% formamide and 4× standard sodium citrate (SSC) solution and sealed with electrical tape to provide humid conditions, and allowed to hybridize at 52° C. After 16 h of hybridization, the sections were sequentially washed in 5× SSC containing 10 mM dithiothreitol at 50° C and then in 50% formamide, 2× SSC, and 10 mM dithiothreitol at 65° C, to reduce nonspecific binding by the probe. Next, sections were incubated in 20 µg/ml RNase A at 37° C for 1 h to digest unbound probe. A second wash in 50% formamide, 2× SSC, and 10 mM dithiothreitol was done to further reduce background activity followed by washes in 2× SSC and 0.1× SSC. Slides were then dehydrated in graded ethanol in the presence of 0.3 M NH₄Ac and air dried. Slides were dipped in Kodak NTB-2 (Eastman Kodak, Rochester, NY) emulsion and exposed for 3 d at 4° C in light-tight boxes containing a desiccant. Emulsion-coated slides were developed in Kodak D19 and fixed with Kodak fixer.

Staining

Hybridized sections were either stained with Gill's hematoxylin and eosin B, or left unstained. Paraffin sections, neighboring those used for in situ hybridization, were deparaffinized and stained with Harris hematoxylin and eosin Y or with Verhoeff's elastic stain with and without the van Gieson counterstain (Sigma Chemical, St. Louis, MO).

Tissue Section Analysis

Lung tissue from 44 animals was analyzed. The animals were distributed among the treatment groups as follows: 10 animals treated with saline, and 4, 4, 8, 3, 8, and 7 animals killed at 1, 2, 3, 5, 7, and 30 d, respectively, after PPE treatment. Each microscopic slide contained 1 slab of tissue from the left lung and 1 or 2 slabs of tissue from right lung lobes. The mean linear intercept was measured by projection microscopy on 20 randomly selected fields from each animal's lung sections.

An average of 4 slides per animal were hybridized for elastin and another 4 slides were hybridized for 1(I) collagen. Stained sections were observed and photographed with bright-field optics on a Leitz Orthoplan microscope (Ernst Leitz GMBH, Wetzlar, Germany). Unstained hybridized tissue was observed and photographed with darkfield and phase optics with Leitz NIP Fluotar phase objectives. Identification of the cells and tissues was aided by comparison with neighboring serial sections that were stained for elastin and collagen. For comparison of elastin and ₁(I) collagen mRNA expression, neighboring serial sections, each hybridized with one of the probes, were used. Color photomicrographs of serial sections were taken to aid in the analysis. Montages of photographs of alveolar ducts between terminal bronchiole and pleura were also studied.

Elastin mRNA expression was scored on 22 individual subpleural air spaces for 3 hamsters each, of saline control, 3-d PPE-treated and 30-d PPE-treated groups. The perimeter of each air space section was calculated by measuring the major and minor axes and assuming that the air spaces were elliptical in shape. Air spaces of different sizes were picked that were representative of the sizes seen in their immediate area. The pleura and septal wall segments of the air spaces were scored separately on a ranking scale as follows:

1. 0  no silver grains above background
2. 1  density of silver grains at least double background over some portion of the segment of interest
3. 2  density of silver grains focally at least quadruple background (focal is defined as < 20% of length of segment of interest)
4. 3  density of silver grains at least quadruple background over > 20% of length of the air space wall
5. 4  focal, dense cluster of grains (many silver grains overlapping)

5  dense clustering of grains over > 20% of the length of segment of interest

Statistics

The Spearman rank order correlation test was used on the air space wall elastin mRNA scoring versus air space section perimeter data. The Kruskal-Wallis and Mann-Whitney rank sum tests were used to compare grouped data.

	RESULTS

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The mean linear intercept values (mean ± SE) for the saline-treated control hamsters and for the hamsters studied 1, 2, 3, 5, 7, and 30 d after PPE treatment were 46 ± 5, 89 ± 4, 111 ± 45, 103 ± 15, 126 ± 16, 130 ± 22, and 135 ± 35 µm, respectively, and are shown in Figure 1.

View larger version (15K): [in this window] [in a new window]   Figure 1.   The evolution over time of mean linear intercept (MLI) values for hamsters given saline or 50 µg PPE in saline. Groups of hamsters were studied 1, 2, 3, 5, 7, and 30 d after treatment. Data from saline-treated hamsters are plotted at 0 d. The values shown are mean ± SE. The data suggest that the increase in MLI is nearing an asymptote by Day 5, and that much of the increase in MLI occurs in the first 24 h.

In saline-treated control hamsters, weak signals for elastin mRNA were seen in the pleura and in medium and large intrapulmonary arteries and veins. Weak signals for ₁(I) collagen mRNA were occasionally detected in the adventitia of large airways, arteries, and veins. Neither message was readily detected in respiratory air space walls.

One day after PPE treatment, destruction of lung tissue with enlargement of respiratory air spaces was extensive. Verhoeff's elastic stain showed loss of elastic fibers in respiratory air space walls and pleura, and in the basement membrane of airway epithelium; the elastic lamellae of large and small blood vessels were disrupted. Elastin mRNA expression was minimally and focally increased in the pleura and adventitia of airways with a more general increase in the large arteries; ₁(I) collagen mRNA was focally increased in the adventitia of airways, and large and medium sized arteries.

Elastin mRNA expression was increased on Day 2 over Day 1 and reached an asymptote by Day 3 that was maintained until Days 5 to 7; the signal for ₁(I) collagen mRNA also reached an asymptote at 3 d but appeared to decrease after 5 d. Elastin and ₁(I) collagen mRNA expression in the pleura were especially intense in areas of pleural thickening (Figure 2). Elastin mRNA expression occurred throughout the walls of large arteries, especially in the media (Figure 3), but tended to be focal in large veins. In blood vessels ₁(I) collagen message was confined to the adventitia. In large airways, elastin mRNA expression occurred on both sides of the muscularis. Elastin, and especially ₁(I) collagen expression were particularly intense in the adventitia between abutting large arteries and airways (Figure 3). Elastin and ₁(I) collagen message were often seen in the walls of medium sized blood vessels, but only elastin message was frequent in the walls of small vessels.

View larger version (113K): [in this window] [in a new window]   View larger version (135K): [in this window] [in a new window]   View larger version (139K): [in this window] [in a new window]   Figure 2.   Three 4-µm serial sections from pleura and underlying alveoli of hamster, 3 d after PPE treatment. Panel a is from a Verhoeff-van Gieson-stained section in which elastin stains black and collagen red. Panel b is from the second section, hybridized with elastin mRNA probe, and panel c is from the third section, hybridized with the 1(I) collagen mRNA probe. Panels b and c were photographed using phase contrast optics. Panel a shows thickened pleura (P) with missing and abnormal, granular elastic fibers (arrowheads). An alveolar septum (S) from its origin in the pleura to the septal edge is shown. Panels b and c show intense elastin and high 1(I) collagen mRNA expression in the pleura but no expression in the septum. The horizontal bar represents 20 µm.

View larger version (97K): [in this window] [in a new window]   View larger version (139K): [in this window] [in a new window]   View larger version (137K): [in this window] [in a new window]   Figure 3.   Three 4-µm serial sections of large intrapulmonary artery and airway of hamster 3 d after PPE treatment. Panel a is from a Verhoeff-van Gieson-stained section in which elastin stains black and collagen red. Panel b is from the second section hybridized with elastin mRNA probe, and panel c is from the third section hybridized with the 1(I) collagen mRNA probe. The panels show a large airway (AW) with an abutting large pulmonary artery (PA). The connective tissue of the artery and airway wall appear normal. Panel b shows elastin mRNA expression in the adventitia of the airway and artery and in the media of the artery. In panel c, 1(I) collagen expression is found in the adventitia of both artery and airway. The horizontal bar represents 20 µm.

Elastin message was sparse in respiratory air space septa and ₁(I) collagen message was almost absent. Focal collections of elastin message were confined to the free margins of respiratory septa (Figures 4-5).

View larger version (109K): [in this window] [in a new window]   View larger version (104K): [in this window] [in a new window]   View larger version (101K): [in this window] [in a new window]   View larger version (110K): [in this window] [in a new window]   Figure 4.   Alveolar walls of hamster 5 d after PPE treatment. The panels are photomicrographs of four consecutive 4-µm serial paraffin sections. Panels a and d show sections stained for elastin and collagen fibers. Panel a lies proximal to an alveolar entrance and shows an alveolar septum (S) and a small vessel (V); panel d cuts across the alveolar entrance and therefore shows two free septal margins (see Figure 7, panels c and d ). Panel b, from a section hybridized with elastin mRNA probe, lies on the free septal margin and shows elastin expression. Panel c, from a section which lies just within the alveolar entrance is hybridized with the 1(I) collagen mRNA probe and suggests some 1(I) collagen message expression at the left free septal margin edge. The horizontal bar represents 20 µm.

View larger version (61K): [in this window] [in a new window]   View larger version (71K): [in this window] [in a new window]   View larger version (63K): [in this window] [in a new window]   View larger version (70K): [in this window] [in a new window]   View larger version (70K): [in this window] [in a new window]   View larger version (61K): [in this window] [in a new window]   View larger version (70K): [in this window] [in a new window]   View larger version (67K): [in this window] [in a new window]   Figure 5.   Alveloar wall of hamster 7 d after PPE treatment. The panels are photomicrographs selected from 10 serial sections, each 4 µm thick. Panels a and b are from the first two consecutive sections and show alveolar wall away from the free margin. Sections c and d, hybridized with 1(I) collagen, revealed no message and are not shown. Panels e through j are from the next six consecutive sections and lie progressively closer to the midline of the alveolar duct, traversing through the septal margin into the alveolar entrance. Panels a, e, and h are from sections stained with Verhoeff-van Gieson; panels b, f, g, i, and j are from sections hybridized with the elastin probe. The sections through the free margin of the alveolar septa show elastin mRNA expression. The intensity of the expression at the free margins varies (see Figure 7) but the intensity always decreases in the septal wall distal to the free margins. The horizontal bar represents 20 µm.

View larger version (82K): [in this window] [in a new window]   Figure 7.   Cartoon of two adjacent alveoli, opening into the same alveolar duct. The depiction of the elastic fibers is based on the descriptions of Wright (28) for human lung. The thick lines represent the elastic fibers in the alveolar entrance ring. The thinner lines represent the elastic fibers in the alveolar septal wall. Panel a shows the normal condition. The arrowheads indicate the free septal margin of the left alveolus. Panel b depicts the lung soon after treatment with elastase. In this schema there is one break in the alveolar entrance ring (large arrow) and many breaks in the elastic fibers of the alveolar wall (small arrows). Panel c shows enlargement of the air spaces with the loss of the structural support of the broken and weakened fibers. The arrowheads indicate the free margin of the septa of the enlarged left alveolus. The tissue at the free septal margin consists partly of the original entrance ring and partly of capillaries, elastic fibers, and collagen fibers (not shown) that were part of the septal wall. This scenario can be thought of as a fenestration coalescing with an alveolar entrance ring. Our data suggest that the major location of elastin production in the parenchyma is in the free septal margin, but it is unclear whether the elastin produced is predominantly in the original entrance ring, possibly at the elastic fiber junctions (largest arrowhead ) at breaks in elastic fibers (medium arrowhead ) or whether it occurs also in the newly created septal margins (small arrowhead ) of fenestrations. The straight line in Panel c indicates a plane of section through the alveolar entrance and surrounding septal wall. The microscopic view of this section would look like panel d, where the arrowheads indicate two parts of the free septal margin; the gap between is the alveolar entrance, and the tissue lateral to the arrowheads is the body of the septal wall. In this scenario we would predict that the left septal edge might show more elastin expression than that of the right septal edge, because it is in the vicinity of a break in the elastic fibers of the original alveolar entrance ring. See Figure 5i for a possible example.

By Day 30, elastin mRNA expression was close to control levels, although occasionally, focal, heavy expression was seen in the pleura and in the walls of large blood vessels and airways. Expression of ₁(I) collagen mRNA had returned to control levels except for occasional foci in large arteries. Hemorrhage and debris were largely absent from the lungs and widespread emphysema was evident. In slides stained for elastin, elastic fiber architecture was not always restored to normal in parenchyma, in the pleura, or in the elastic lamellae of blood vessels.

The ranking of elastin mRNA expression in subpleural air spaces is given in Figure 6. The scores for the 3-d PPE group were significantly higher than that for the 30-d PPE group which were significantly higher than that for the control hamsters. There was a weak but significant correlation (r = 0.28) between score and air space size (length of perimeter) within the controls. The correlation was moderate for the 3-d group (r = 0.67) and weaker for the 30-d group (r = 0.46). The air spaces scored on control lungs had sectional perimeters ranging from 79 to 688 µm. In 3- and 30-d PPE groups, air spaces with perimeters greater than 688 µm had significantly higher scores (p < 0.01 and p < 0.05, respectively) than the air spaces within the normal range. The scores for pleura ranked significantly higher (p < 0.001) than that for septal walls for all groups (data not given).

View larger version (18K): [in this window] [in a new window]   Figure 6.   Elastin mRNA scores for the septa of subpleural air spaces. See METHODS for a description of the six scoring ranks. The number of air spaces receiving scores in each rank is given in parenthesis. No air space septa received the highest score of 5. The Spearman rank correlation (r) of elastin mRNA score for air spaces versus their calculated perimeter is given along with its probability value (p). Panel a shows the scores of 66 subpleural air spaces from hamsters treated with saline. Panel b shows the scoring of 66 subpleural air spaces from hamsters studied 3 d after PPE treatment. Panel c shows the scoring of 66 subpleural air spaces from hamsters studied 30 d after PPE treatment. The scores of air spaces 3 d after PPE are significantly greater than 30 d after PPE, and both are significantly greater than the scores for air spaces of saline control hamsters. There are highly significant positive correlations of elastin mRNA score and air space size 3 and 30 d after PPE treatment.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

With intratracheal PPE treatment of hamsters there was a rapid increase in air space size. The mean linear intercept (MLI) nearly doubled in the first 24 h, as previously seen in treated hamsters (6). The MLI appeared to be reaching an asymptote by Day 5, but we do not know what happens beyond 30 d.

Our finding of minimal expression of elastin mRNA in the normal lung is consistent with other studies indicating a long half-life for elastin (15). Localization of elastin message in large blood vessels may relate to the stress induced by pulsatile blood flow. The elastic fiber damage and enhanced expression of elastin and ₁(I) collagen mRNA in blood vessel walls after PPE treatment of hamsters is not surprising, because vascular wall inflammation and focal thrombosis have previously been described in the first 4 d after PPE treatment (18).

The expression of elastin mRNA in respiratory air space walls after PPE treatment may relate to the direct passage of PPE through the airway wall or, as we have previously hypothesized, PPE may have spread through the lung via the interstitium during the early hours after its administration (1). We believe the pleura is more heavily labeled than septal walls because of the greater amount of elastic tissue in pleura than in air space walls.

We hypothesized that there would be a correlation between elastin mRNA expression and air space size in PPE-treated lungs because larger air spaces would be seen in regions of greater elastolytic damage; therefore they might show greater evidence of repair. As shown in Figure 6 this was indeed the case. The strength of the association between elastin mRNA score and air space size is influenced by at least two artifacts that work in opposite directions. Peripherally cut air space sections underestimate the size of air spaces and cause a decrease in the association, with high scores for spuriously small air spaces. However, the correlation may be strengthened, in part, due to the ranking method we used to quantify the silver grains that represent elastin mRNA: the longer the segment of air space wall inspected, the more likely there is to be a cluster of grains above background. It is noteworthy that the repair process was still active at 30 d and that there is still a tendency for larger air spaces to express the most elastin mRNA.

Observation of serial lung sections showed that the local and limited repair in the respiratory air space walls after PPE treatment, occurs mainly at free margins of septa. The free septal margins of the enlarged air spaces probably consist of remnants of the original alveolar entrance ring as well as margins newly created by fenestration of alveolar walls (Figure 7). Elastolytic injury may be minimal or absent in well-preserved alveolar walls and conditions may not be favorable for elastin deposition at the newly created septal margins. During development, elastin is synthesized at septal edges as a component of the alveolarization process. The finding that elastin mRNA is localized at selected sites along the alveolar duct is reminiscent of this process. Whether in fact this localization represents a partial reinitiation of the alveolarization program is unclear.

Administration of beta-aminopropionitrile, an inhibitor of lysyl oxidase and therefore of incorporation of elastin and collagen into cross-linked fibers, results in worsening of the emphysema in elastase-treatment animals (19, 20). These results suggest that, despite its highly limited extent, repair of elastic tissue in the respiratory air spaces following elastolytic injury is of functional importance.

Expression of ₁(I) collagen mRNA is greatly increased in the lungs after intratracheal treament with PPE. However, the kinetics and location of type I collagen mRNA expression were different from elastin. Still, we cannot distinguish the relative contribution of either of the matrix substances to overall repair. Increased expression was evident at 1 to 2 d for both, but collagen expression appeared not to persist as long. Collagen mRNA was not expressed over the media of arterial walls and was less evident than expression of elastin mRNA in pleura, alveolar tissue, and medium and small blood vessels. This diversity was probably due to differences in the mechanisms controlling collagen and elastin production, but may also reflect differences in severity and loci of injured collagen as opposed to injured elastin.

PPE does not solubilize cross-linked collagen without prior exposure to mammalian collagenase (21) and in tissue culture, PPE alone solubilize less than 5% of collagen markers (22). In the in vivo PPE model, collagen injury may be mediated by inflammatory cells rather than by the direct action of PPE. Although net collagen content of the lung is little changed by PPE treatment, studies following ¹⁴C-proline administration reveal that synthesis of collagen is increased during the first 2 wk after PPE treatment (2). Transgenic mice that overexpress collagenase in the lung develop emphysema, suggesting that the degradation of collagen itself can result in air space enlargement (23). Smoke-induced emphysema in guinea pigs is associated with morphometric evidence of collagen breakdown and repair (24) and cadmium chloride-induced air space enlargement is associated with an increase in both collagen and elastin in the lungs but no decrease in the neonatally formed elastin (25).

Histochemically collagen concentration is increased in centriacinar, distal acinar, and irregular air space enlargement; biochemically elastin is decreased in all grades of panacinar emphysema and in severe centriacinar emphysema (26). In smokers with emphysema lost respiratory tissue is associated with net increase in collagen mass (27). It is apparent that although elastin degradation is important in the pathogenesis of emphysema, collagen, and probably other components of connective tissue also play important roles.

We believe that the development of emphysema in humans is the result of a balance between two competing processes. Elastin degradation may occur as a result of elastase-antielastase imbalance. If elastin degradation is balanced by elastin repair, emphysema does not result; the failure of elastin repair to keep up with elastin degradation results in emphysema.