Institute of Anatomy, University of Bern, CH-3000 Bern 9, Switzerland
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ABSTRACT |
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Prematurely born babies are often treated with glucocorticoids. We studied the consequences of an early postnatal and short dexamethasone treatment (0.1-0.01 µg/g, days 1-4) on lung development in rats, focusing on its influence on peaks of cell proliferation around day 4 and of programmed cell death at days 19-21. By morphological criteria, we observed a dexamethasone-induced premature maturation of the septa (day 4), followed by a transient septal immatureness and delayed alveolarization leading to complete rescue of the structural changes. The numbers of proliferating (anti-Ki67) and dying cells (TdT-mediated dUTP nick end labeling) were determined and compared with controls. In dexamethasone-treated animals, both the peak of cell proliferation and the peak of programmed cell death were reduced to baseline, whereas the expression of tissue transglutaminase (transglutaminase-C), another marker for postnatal lung maturation, was not significantly altered. We hypothesize that a short neonatal course of dexamethasone leads to severe but transient structural changes of the lung parenchyma and influences the balance between cell proliferation and cell death even in later stages of lung maturation.
apoptosis; glucocorticoids; tissue transglutaminase
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INTRODUCTION |
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MAMMALIAN LUNG DEVELOPMENT is characterized by two principally different mechanisms: by branching and by septation. In humans and various laboratory animals, branching is completed shortly before birth. Further enlargement of the gas-exchange surface is achieved by the formation of new septa inside the existing air spaces. Roughly, most of the septation (alveolarization) takes place in the terminal third of the airway tree between postnatal days 4 and 13 in rats (phase of alveolarization) (6).
Before septation of the air spaces starts, the lung expands for a short period of time (from birth to day 4 in rats), and the cells of the interairway walls show a peak of proliferation at day 4 (14). At this point, the interairway walls (primary septa) contain two capillary layers that are located underneath the gas-exchange surfaces of both sides and that are separated by a sheet of connective tissue (1, 9). The new septa are formed by lifting off tissue ridges from the existing interairway walls. These ridges include one of the capillary layers, which folds up and forms again two sheets of capillaries inside the new septum (secondary septum). These capillary networks are also separated by connective tissue (5, 10, 19).
Both the primary and the secondary septa mature during the phase of microvascular maturation (3rd postnatal week in rats). The maturation includes a transformation of the bilayered capillary network into a single centrally located network. The former central layer of connective tissue is reduced to a fibrous meshwork interwoven with the capillaries (4, 8). The process results not only in a reduction of the absolute mass of the interstitial tissue, but also in a reduction of the absolute number of fibroblasts by 10-20% and the absolute number of epithelial cells by >10% (14). It was shown, first by Schittny and coworkers (24) and shortly afterward, but independently, by Bruce and coworkers (3), that the surplus of fibroblasts is eliminated by classical apoptosis, which is defined by a typical pattern of morphological changes including ultimate fragmentation of the cell into membrane-enclosed vesicles (apoptotic bodies) (15, 27). The surplus of epithelial cells, mainly type II epithelial cells, is eliminated by programmed cell death without the appearance of apoptotic bodies. Most likely apoptotic type II epithelial cells are phagocytosed by alveolar macrophages in an early stage of programmed cell death before apoptotic bodies are formed. Both cell types are eliminated without an inflammatory reaction (24). The elimination of the cells distinguishes programmed cell death and apoptosis from necrosis, where cells disintegrate and inflammation is induced by cellular lysis (17).
Lung development is highly susceptible to various influences, like partial pressure of oxygen, glucocorticoids, thyroid hormones, and retinoids (7, 20). In rats, postnatal administration of minute amounts of glucocorticoids within the first 2 wk of life significantly impairs the formation of alveoli (18, 19, 23). Tschanz and coworkers (26) postulated that under the influence of glucocorticoids microvascular maturation occurs too early. Because a double capillary network is probably required for the formation of secondary septa (9), fewer alveoli will be formed under these circumstances. Interestingly, these authors also showed that after drug withdrawal some recovery in septal formation occurred. The lung parenchyma appeared partially reverted to a more immature state (i.e., higher number of septa with a double capillary layer), and, as a consequence, additional septal buds could thus be formed (26).
In the current study we were using a similar rat animal model as Tschanz and coworkers (26), but our rats were only treated with a short pulse of dexamethasone at postnatal days 1-4. Apart from morphological changes we investigated the appearance of cell proliferation and programmed cell death and the expression and action of the enzyme tissue transglutaminase.
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MATERIALS AND METHODS |
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Animals and tissues. Newborn Sprague-Dawley rats were treated with dexamethasone sodium phosphate (Decadron, Merck Sharp & Dohme AG, Glattbrugg, Switzerland) at postnatal day 1 (0.1 µg/g body wt sc), day 2 (0.05 µg/g body wt sc), day 3 (0.025 µg/g body wt sc), and day 4 (0.01 µg/g body wt sc). Control animals received the same volume of 0.9% saline. Lungs were prepared according to Schittny and coworkers (25) at postnatal days 1, 4, 10, 16, 19, 21, 28, and 36. Briefly, the pulmonary blood vessels were perfused with phosphate-buffered saline (PBS = 10 mM sodium phosphate, containing 127 mM sodium chloride, pH 7.4), containing 5 U/ml heparin, 10 mg/ml procaine, and 10 mM EDTA (Fluka Chemie AG, Buchs, Switzerland), and the air space was filled with PBS, containing 4% freshly prepared paraformaldehyde (Merck, Darmstadt, Germany) at a constant pressure of 20 cmH2O. At this pressure, the lung reaches roughly its midrespiratory volume. To prevent a recoiling of the lung, the pressure was maintained during fixation.
Handling of the animals before and during the experiments, as well as the experiments themselves, were approved and supervised by the Swiss Agency for the Environment, Forests, and Landscape and the Veterinary Service of the Canton of Bern.Histochemistry. Lungs were fixed for 1 h with 4% paraformaldehyde freshly dissolved in PBS, briefly washed three times in PBS, and embedded in paraffin (Histosec; Merck) at 60°C after using a graded series of ethanol and Histoclear (Life Science International, Frankfurt, Germany) as intermedium. Under all conditions no recoil of the lungs was observed. Sections (3.5-5 µm) were cut, transferred onto microslides treated with aminopropyltrimethoxysilane, and air-dried overnight at 37°C. All sections were dewaxed in three changes of Histoclear and a graded series of ethanol, followed by two changes of PBS (25).
TdT-mediated dUTP nick end labeling assay and immunofluorescence double staining. The TdT-mediated dUTP nick end labeling (TUNEL) assay and the immunofluorescence staining were performed as described in Schittny and coworkers (24). Briefly, after a digestion with 5 µg/ml proteinase K (21°C, 10 min), the sections were incubated with terminal transferase reaction solution, containing 9 mM digoxigenin-11-dUTP and 0.165 U/ml enzyme (Boehringer Mannheim, Mannheim, Germany) for 50 min at 37°C. The incorporated digoxigenin was detected with anti-digoxigenin (FITC- or rhodamine-anti-digoxigenin from Boehringer Mannheim). For the following immunofluorescence staining, the sections were incubated with the first antibody for 1-15 h and the second one for 30-60 min. The rabbit anti-laminin antiserum (Sigma Chemical, St. Louis, MO) was diluted 1:100, and the monoclonal mouse antibody MNF-116 (Dakopatts, Glostrup, Denmark) was diluted 1:10 in Tris-buffered saline (TBS)/BSA. As secondary antibodies we used rhodamine- or FITC-labeled goat anti-rabbit IgG (Cappel Research Products, Organon Teknika, Durham, NC; 1 mg/ml diluted 1:60 to 1:100 in TBS/BSA) or rhodamine-labeled sheep anti-mouse IgG, F(ab')2 fragments (Boehringer Mannheim; diluted 1:40 to 1:60), respectively .
Cell proliferation. For the immunoperoxidase staining the same protocol as for the immunofluorescence staining was used with the following exceptions. 1) The proteinase K treatment was substituted by demasking of the antigen by cooking of the dewaxed sections in a household pressure cooker in 10 mM citrate-buffer, pH 6.0 for 5 min, at 2 bar. 2) Endogen peroxidases were quenched by a treatment with 3% H2O2 (30%) in methanol two times for 20 min. 3) The first antibody was detected using biotinylated, affinity-isolated goat anti-mouse globulin (Dakopatts, diluted 1:200 in TBS), followed by the visualization with avidin-biotin peroxidase complex (ABC, Dakopatts) using diaminobenzidine as substrate. The first antibody, monoclonal antibody MIB-5 (Immunotech, Marseille, France; Refs. 11, 12), was diluted 1:50 in TBS/BSA containing 1% normal goat serum.
Tissue transglutaminase.
To distinguish between cellular and extracellular tissue
transglutaminase, cryosections were immunostained with anti-tissue transglutaminase antibodies either with or without fixation of the
section in acetone at 20°C according to Schittny and coworkers (25). The fixation is important, because without it, the
cellular tissue transglutaminase was washed out and only the
extracellular transglutaminase was stained. After rehydration and
blocking, first antibodies were applied in a dilution of 1:10 for
anti-
-Glu-
-Lys cross-link, 1:25 to 1:30 for anti-tissue
transglutaminase, and 1:100 for anti-dansyl (negative control for the
-Glu-
-Lys cross-link staining). First antibodies were visualized
using peroxidase-conjugated swine anti-rabbit IgG (Dakopatts; diluted
1:100) followed by development with 3-amino-9-ethylcarbazole (Sigma).
Controls. Negative controls were performed with nonspecific mouse or rabbit IgG. None or only little nonspecific background was observed in all negative controls. The shown samples were taken from all parts of the lung. No significant differences between central and peripheral regions of the lung were observed under all conditions.
Counting of positive nuclei. The counting of positive nuclei was done according to Schittny and coworkers (24). Areas of lung parenchyma (1.5 × 1.0 mm) were systematically randomly photographed. The micrographs covered 50-80% of the total area of the parenchyma. The images were enlarged in a slide projector, and positive nuclei (programmed cell death or cell proliferation) were counted. Apoptotic bodies that were very close to each other were counted as one dying cell. In lung tissue the time lag between the first sign of programmed cell death and the disappearance of the dying cells is not known. Furthermore, no information is available on how long the TUNEL assay stays positive. Therefore, it is not possible to determine the total number of dying cells. Using a single image as standardized reference space, we obtained only a relative number of dying cells, which we compared between samples of different postnatal days. Whenever data were compared, the sections were processed the same day in the same batch, to minimize variations. Every batch was repeated two to four times, giving the same outcome. At least one of the repetitions was counted independently by a different person. At every time point the lungs of three to five animals were studied in both the treated and untreated groups. The same restrictions apply for the counting of proliferating cells.
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RESULTS |
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Morphology.
Newborn rats were treated with dexamethasone at postnatal days
1-4 (0.1-0.01 µg/g body wt) just before bulk
alveolarization started. We compared the morphology of the lungs on
paraffin sections between treated and untreated animals and observed
dexamethasone-induced structural alterations at days 4 and
10, but not at day 16 or later. At day
4 (Fig. 1, a and
b) the interairway walls of the treated animals appeared to
be significantly thinner, a finding that implies a premature maturation
of the septa. Furthermore, the individual air spaces seemed to be
enlarged, but this may appear so because of the reduced interstitial
volume. At day 10 (Fig. 1, c and d),
we observed a similar thickness of the primary and secondary septa in
both groups. We studied the morphological appearance of distal air
spaces and found that they were smaller and more frequent in the
controls than in the treated animals. In other words, in the treated
animals the septation of the distal airways appeared to be regressed
compared with the controls. At day 16 (Fig. 1, e
and f) and later (data not shown), no differences were
detectable by eye anymore between the two groups. Our morphological observations are backed by a morphometric study done under the same
experimental conditions by Tschanz SA, Haenni B, and Burri PH
(unpublished observations).
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Apoptosis.
Searching for programmed cell death we applied TUNEL. In the control
animals we were able to confirm the first postnatal peak (16), appearing directly after birth, and the second
postnatal peak of programmed cell death (3, 24), appearing
toward the end of the third week (days 19-21). The
dexamethasone treatment suppressed the latter peak of programmed cell
death to a level of a slightly, but not significantly, elevated
baseline (Fig. 2). This result was
surprising, because by morphological criteria the differences between
treated and untreated animals had disappeared before this time point
(Fig. 1, e and f). Interestingly, in the treated group a significant decrease of programmed cell death was
observed from day 3 to day 4 (P = 0.009; Fig. 2), while at all other days no significant differences
between the treated and untreated animals could be observed.
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Cell proliferation.
Kauffman and coworkers (14) described a peak of cell
proliferation at day 4 in rats that steadily declines
afterward. Using the monoclonal antibody MIB-5, which recognizes the
Ki67 antigen (11, 12), we observed the described peak of
cell proliferation in the untreated animals at the same day.
Surprisingly, the number of proliferating cells rose sharply from
day 3 to day 4 and not gradually from day
1 to day 4 as expected from previous studies (14). The sharp rise of cell proliferation takes place
just at the beginning of the septation of the distal airways. In the dexamethasone-treated group cell proliferation was drastically reduced
at days 2-4 (Fig. 4).
Later, at days 10-36, no significant difference between
treated and untreated animals was detected anymore (Fig. 4; data not
shown for days 16-36).
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Tissue transglutaminase.
The extracellular expression and action of the enzyme tissue
transglutaminase is also considered to be a marker for the maturation of the alveolar septa. It takes place toward the end of the third postnatal week in rats at the same days when the peak of programmed cell death was observed (24, 25). At days
4-36 we studied the intracellular and extracellular
expression of tissue transglutaminase as well as the appearance of the
enzyme product, the -Glu-
-Lys cross-link, using the same
antibodies and immunohistochemical techniques as in the original study
(25). Similarly to the lack of structural alteration of
the lung parenchyma in the treated animals toward the end of the third
week, we were not able to detect any significant difference
between the treated and untreated animals in the expression pattern and
action of intra- and extracellular tissue transglutaminase (data not shown).
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DISCUSSION |
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In 1994, the National Institutes of Health Consensus Development Panel on the Effect of Corticosteroids for Fetal Maturation on Prenatal Outcomes recommended the broad use of prenatal glucocorticoids for women at high risk of preterm delivery, because after this treatment a decrease in neonatal morbidity and mortality was observed (22). Later it was shown that multiple courses of antenatal corticosteroids did not improve outcome and were associated with increased mortality compared with a single course (2). In addition, a number of animal studies revealed that a prolonged postnatal treatment of rats with corticosteroids caused structural alterations of the lung that persisted into adulthood (18, 23, 26). These observations suggest that a short course of dexamethasone is more beneficial than a long one. On the basis of these findings, we studied the consequences of a short dexamethasone treatment in rats. The drug was administered at postnatal days 1-4 just before the phase of bulk alveolarization started, which is roughly equivalent to the 34th to 36th weeks of pregnancy in humans.
The alveolar septa of the treated rats appeared significantly thinner and more mature than the ones of the controls at the last day of treatment (day 4; Fig. 1, a and b). Six days later, at day 10 (Fig. 1, c and d), this peculiar difference was completely gone: the septa of the treated animals appeared as thick and immature as the ones of the controls. Now, however, the formation of the secondary septa (new alveoli) was significantly suppressed in the treated animals. Thus the long-term (18, 23, 26) and our short-term treatment produced a similar effect: a premature maturation of the septa during the treatment, followed by a transient septal immatureness and a delayed alveolarization after withdrawal of the drug. Contrary to the findings after long-term treatment, in which the structural alterations persisted into adulthood (18, 23, 26), we observed an apparent repair already at day 16 (Fig. 1, e and f). It is believed that the repair process requires the transient thickening of the interairspace walls, because two separated capillary layers are necessary for the lifting off of new tissue ridges during alveolarization (see introduction) (26). We conclude that an early short-term dexamethasone treatment does not cause permanent structural alterations of the lung due to a remarkable potential of repair at this time point.
During the normal maturation and thinning of the alveolar septa (3rd postnatal week in the control rats), the absolute number of fibroblasts and epithelial cells is reduced by >10% (14) by a peak of programmed cell death (3, 24). During the dexamethasone-induced premature maturation of the interairspace walls, cell death is not significantly altered compared with the control group at the same days (Fig. 2), but cell proliferation is strongly suppressed (days 2-4; Fig. 4). These findings are contrary to the role of cell death and cell proliferation during the normal thinning of the septa during the third week. It may be explained by the observation that cell proliferation is already low in the untreated animals before the normal septal thinning starts (14). Therefore, cell death appears to be the only way to reduce the number of cells in the alveolar septa (3, 24). At the beginning of the dexamethasone-induced premature septal thinning, cell proliferation is relatively high, the lung is expanding, and new cells would be needed for normal development. The dexamethasone-induced structural changes may be explained by a reduction of cell proliferation in combination with the normal rate of programmed cell death. However, cell death is not completely unaltered by the dexamethasone treatment, because it is significantly reduced from day 3 to day 4 (Fig. 2). A similar reduction was also observed 2.5 wk later at the end of the normally observed septal thinning (3, 24), but not at the same days in the control group (Fig. 2).
Our dexamethasone treatment suppressed the normally observed peak of programmed cell death at days 19 and 21. With a half-life of ~1 day, dexamethasone is eliminated at a reasonable speed. Therefore, this effect may not be explained by a direct action of the drug. The following secondary effect may be postulated. During normal postnatal lung development a surplus of cells is produced during the first 2 wk of life. The surplus may serve as a backup to ensure the presence of the appropriate cell at the correct time and location. On maturation of the alveolar septa, the cellular excess is removed by programmed cell death (Fig. 2c) (3, 24). As shown in Fig. 4, the dexamethasone treatment prevented the formation of the cellular surplus during the first week, and therefore a significant removal of cells may not be necessary anymore during the maturation of the alveolar septa (Fig. 2c).
As a side effect we observed that the rate of cell proliferation increased exactly in parallel to the beginning of the phase of alveolarization (day 4; Fig. 4). We would like to postulate that cell proliferation may be a prerequisite for the formation of new septa.
In summary we were able to show that a short dexamethasone treatment before the start of the bulk alveolarization did cause severe, but transient, structural alterations of the lung architecture. The induced premature maturation of the lung parenchyma was rescued, involving a transient septal immatureness and an alteration of the balance between cell proliferation and cell death.
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ACKNOWLEDGEMENTS |
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We thank Dr. D. Aeschlimann for the kind gift of anti-tissue
transglutaminase, anti--Glu-
-Lys cross-link, and anti-dansyl antibodies. We appreciate the expert technical assistance of M. Hofstetter and B. de Breuyn.
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FOOTNOTES |
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This work was financially supported by Swiss National Science Foundation Grant 31.55895.98.
Address for reprint requests and other correspondence: J. C. Schittny, Institute of Anatomy, Univ. of Bern, Buehlstrasse 26, CH-3000 Bern 9, Switzerland (E-mail: schittny{at}ana.unibe.ch).
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.
10.1152/ajplung.00406.2000
Received 13 November 2000; accepted in final form 25 June 2001.
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