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Keywords:

  • lung development;
  • glucocorticoids;
  • alveolarization;
  • capillary fusion

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Postnatal glucocorticoid treatment of preterm infants was mimicked by treating newborn rats with dexamethasone (0.1–0.01 μg/g, days 1–4). This regimen has been shown to cause delayed alveolarization. Knowing that microvascular maturation (transformation of double- to single-layered capillary networks in alveolar septa) and septal thinning prevent further alveolarization, we measured septal maturation on electron photomicrographs in treated and control animals. In treated rats and before day 10, we observed a premature nonreversing microvascular maturation and a transient septal thinning, which both appeared focally. In vascular casts of both groups, we observed contacts between the two capillary layers of immature alveolar septa, which were predictive for capillary fusions. Studying serial electron microscopic sections of human lungs, we were able to confirm the postulated fusion process for the first time. We conclude that alveolar microvascular maturation indeed occurs by capillary fusion and that the dexamethasone-induced impairment of alveolarization is associated with focal premature capillary fusion. Developmental Dynamics 233:1261–1271, 2005. © 2005 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Glucocorticoids are widely used in perinatology either to accelerate surfactant production in imminent premature birth (Meadow et al., 2003) or postnatally in the context of severe respiratory distress syndrome and chronic lung disease in the newborn (St. John and Carlo, 2003; Truffert et al., 2003). Their widespread use has been questioned by increasing concerns about short-term negative side effects such as gastrointestinal perforation, hyperglycemia, and hypertension (Halliday et al., 2001; The Vermont Oxford Network Steroid Study Group, 2001) as well as negative effects on neurological abnormalities and impaired growth (Gibson et al., 1993; Papile et al., 1998; O'Shea et al., 1999; Murphy et al., 2001; for review see Halliday et al., 2003a–c). Furthermore, postnatal lung development also appeared to be altered by this treatment. Dexamethasone, a potent and often-used glucocorticoid, has been shown to impair alveolarization permanently when given to rats 10–14 days during the first 2 weeks of life (Massaro et al., 1985; Tschanz et al., 1995) or temporarily when given during postnatal days 1–4 (Tschanz et al., 2003). Furthermore, dexamethasone alters cell proliferation and apoptosis (Luyet et al., 2002), as well as the expression of various proteins such as vascular endothelial growth factor receptor-2 (angiogenesis; Clerch et al., 2004) or cyclin-dependent kinase inhibitor (cell cycle; Corroyer et al., 2002).

Alveolarization is characterized by the outgrowth of new (secondary) septa from the saccular wall (primary septa) to subdivide the existing distal airspaces into smaller units, the alveoli. During the lifting off of new septa, the underlaying capillary leaflet grows by intussusceptive angiogenesis and, by this process, moves into the new septum, resulting in a septum that again contains two capillary layers. According to this model of lung alveolarization (Burri, 1974), new septa can only be formed as long as a double capillary network is still present inside the inter-airspace septa. Alveolarization starts in humans at around 36 weeks of gestation. In rats, the bulk of alveolarization lasts from postnatal days 4 to 13 (Burri et al., 1974). For a review, see Schittny and Burri (2004).

Together with the enlargement of the alveolar surface and with the outgrowth of new septa, the vascular bed has to increase in size, too. This process is called angiogenesis. During postnatal lung development and growth, the mechanism of intussusceptive angiogenesis is dominant over sprouting angiogenesis (Caduff et al., 1986; Risau, 1997). Intussusceptive angiogenesis is characterized by a splitting of existing capillaries to form new capillary meshes. Typically, splitting starts by an invagination of parts of the capillary wall into the lumen, followed by a fusion of the invagination with the opposing capillary wall resulting in the formation of an interstitial pillar. The newly formed pillar finally divides the capillary and gives rise to a new capillary mesh (for review, see Djonov et al., 2003). The importance of angiogenesis during alveolarization was emphasized by the finding that inhibition of angiogenesis not only leads to a reduction of pulmonary arterial density but also to a major reduction of the formation of alveoli (Jakkula et al., 2000).

Both existing (primary) and newly formed (secondary) septa mature during the last step of lung development, during the stage of microvascular maturation (Burri, 1999). As a result, the alveolar septa of the adult lung are significantly thinner compared with the immature ones and contain only a single-layered capillary network in the center of the alveolar septum. Now, gas exchange takes place on both sides of the capillaries. On one side of the septum, the air–blood barrier consists only of the epithelium, a fused basement membrane and the endothelium. This side shows a higher gas exchange rate than the opposite one, which in addition to the aforementioned structures contains the layer of the remaining connective tissue.

Dexamethasone may temporarily or even permanently impair alveolarization in rats. Tschanz et al. postulated a premature microvascular maturation on day 4 and a reversal of this process by day 10 in rats treated with dexamethasone during days 1–4. This hypothesis fits very well with the current view of the mechanisms involved in alveolarization. However, it is based on morphological observations only and not on morphometric data. Therefore, we used the same animal model as our colleagues and measured alveolar wall thickness and microvascular maturity. After 72 hr of dexamethasone treatment, we observed a premature and transient reduction of the thickness of the interalveolar walls and a premature but nonreversing focal acceleration of microvascular maturation.

Based on vascular casts, it was postulated that the reduction from a double- to a single-layered capillary network involves a combination of capillary fusion and differential growth (Caduff et al., 1986). However, this hypothesis was never verified in developing lungs using electron microscopic serial sections (Burri, 1992). We now supply this evidence. Furthermore, we used vascular casts to show that the dexamethasone-induced focal premature microvascular maturation follows the same mechanisms as normal microvascular maturation.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Morphology

On low-power electron microscopic images, we compared the lung parenchyma of treated and untreated rats. On day 4, inter-airspace walls of dexamethasone-treated animals appeared significantly thinner (Fig. 1a,b). In addition, we observed a reduced number of septa with an immature double-layered capillary network in treated animals compared with controls. This premature maturation appeared focally in small areas of the inter-airspace walls that contained only a single capillary layer (Fig. 1a,b). At postnatal day 10, wall thickness and microvascular maturation seemed to be identical for both groups (Fig. 1c,d). This result was confirmed by studying vascular casts using scanning electron microscopy. On day 4, focal areas with a single-layered capillary network were observed in both groups; however, such areas were more frequently detected after dexamethasone treatment (Fig. 2a,b). Over time, the amount of single-layered capillary networks increased. On day 21, only a smaller number of areas containing an immature capillary network were still seen (Fig. 2c).

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Figure 1. Morphological alterations of the lung parenchyma after dexamethasone treatment. a–d: At postnatal days 4 (a,b) and 10 (c,d) lung sections of dexamethasone-treated rats (Dex, a,c) and of controls (Ctrl, b,d) were studied by electron microscopy. a,b: On day 4, the inter-airway walls of the treated animals appeared to be significantly thinner than the ones of the controls. In addition, in these thinner septa, an elevated number of areas containing a mature, single-layered capillary network (arrow) was observed. c,d: On day 10, and afterward, we observed a similar thickness and maturation of the septa in both groups. Arrow, mature single-layered capillary network inside the septum; double arrow, immature, double-layered capillary network. Scale bar = 50 μm.

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Figure 2. Scanning electron microscopic images of single and double capillary networks. a,b: Mercox casts of the vascular bed inside the inter-airspace septa are shown for treated (a) and untreated (b) animals on day 4. c: For comparison, a control lung is shown on day 21. Single arrow, mature single-layered capillary network; double arrow, immature, double-layered capillary network. Scale bar = 50 μm.

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Morphometry

To verify our morphological observations, we measured the septal thickness and capillary maturation by morphometric methods. During the first 3 postnatal weeks, regular lung development led to a 50% reduction of the observed mean wall thickness from 13.01 μm on day 4 to 6.94 μm on day 21 (Fig. 3a). Between postnatal day 21 and adulthood (postnatal day 60), no significant changes in septal thickness were detected. In the treated group, a small significant increase of the septal thickness existed between day 21 and day 60 (P = 0.027).

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Figure 3. Thickness of septa. On days 4–60, the thickness of the septa was measured on electron microscopic lung images of treated and untreated rats, as shown in Figure 1. On day 4, the observed mean thickness of the septa of the treated lungs was 22% thinner than the one of the control rats. a: Later, no difference was observed. b: A histogram of the thickness measured on day 4, using a class width of 2.4 μm. In the small classes (4.8–9.6 μm), a larger number of measurements was counted in the treated group, whereas in the larger classes (19.2–33.6 μm), the control group showed higher counts. Significance was defined as P < 0.05 using the Student's t-test (asterisk).

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On day 4, after dexamethasone treatment, the mean septal wall thickness was significantly decreased by 22% compared with the placebo group (10.1 μm vs. 13.0 μm; P < 0.05; Fig. 3a). A histogram of the measured thickness was calculated for day 4. It revealed a shift from the classes of thicker measurements (19.2–33.6 μm) to thinner measurements (4.8–9.6 μm) in the dexamethasone group compared with the control (Fig. 3b). At the later time points (days 10, 21, 60), no significant differences were observed anymore between the two groups (Fig. 3a).

The fraction of mature, single-layered capillary networks inside the inter-airspace walls was also determined by morphometry. In control lungs, a distinct increase of the relative amount of septa containing a single capillary layer was detected between days 4 and 10 (16.4% vs. 29.4%, P < 0.05). The bulk of maturation took place between day 10 and day 21 with an increase of mature septa to 78%. At postnatal day 60, almost all the septa were matured (94.6%; Fig. 4).

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Figure 4. Fraction of mature capillary network. The intersection counting was used to determine the percentage of single (mature) and double (immature) capillary layers of the inter-airway septa. In treated animals, 27.2% (±3.3%) of the septa showed mature, single-layered capillaries by day 4, compared with 16.4% (±2.9%) in the placebo group. The difference disappeared on day 10, when the septa of untreated lungs reached the maturity of the dexamethasone group. Afterward, both groups developed in parallel and reached microvascular maturity past day 21. Significance was defined as P < 0.05 using the Student's t-test (asterisk).

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After 3 days of dexamethasone treatment, a significant increase in maturation was observed in comparison to controls: 27.2% of the septa contained a single-layered capillary network compared with 16.4% in the untreated group (P = 0.013; Fig. 4). The differences in microvascular maturation disappeared by day 10, first, because the degree of maturity which is normally observed on day 10 (29.4%) had already been reached in the treatment group on day 4, and second, because in the treated group capillary maturation did not progress further between days 4 and 10 (27.2% vs. 26.0%; P = 0.585). Afterward, the septa of both groups matured in parallel. In other words, during dexamethasone treatment, microvascular maturation progressed rapidly and, by day 4, reached a stage comparable to that seen on day 10 in untreated animals. After the end of the treatment, microvascular maturation ceased temporally until it matched the normal maturation. No reversal of the maturation was observed (Fig. 4).

Mechanism of Microvascular Maturation

While studying the maturation of the capillary network using vascular casts, we observed sites that were predictive for capillary fusion. Such a site is shown in Figure 5 (arrow 1). The lumen of the capillaries of the two layers get in very close contact to each other. Typically, the capillaries are separated by less than 1 μm. Before filling the capillaries with resin and subsequent digestion of the tissue, this gap was filled by tissue. Furthermore, we observed sites, where this gap was interrupted by a bridge of Mercox (Fig. 5, arrow 2). Because the Mercox resin represents the lumen of the capillaries, it is believed that this interruption represents the first opening connecting the two capillaries during fusion. Such sites of proposed capillary fusions were detected more than once in all lungs investigated and in both groups between days 2 and 21. The observed numbers were small, because scanning electron microscopy of vascular casts allows the investigation of only a very small volume of a whole lung (only the first 20–30 μm of the cut block could be studied). Therefore, a correct morphometric determination of the number of sites of capillary fusion was not possible. However, in samples of treated lungs obtained on days 2–5, these sites were encountered more often than in any other stage of normal development.

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Figure 5. Scanning electron microscopic images of capillary fusion. Representative Mercox casts of lung capillaries of dexamethasone-treated newborn rats on day 4 (a) and day 6 (b). a: Arrow 1 points to a contact of a width of less than 1 μm and arrow 2 to a location where the capillaries are already fused in the central part of the contact. b: Capillary fusion was very often observed at multiple sites located very close to each other. Scale bar = 10 μm.

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This fusion process is apparently not limited to rat, because the same sites of capillary fusions were also observed in human and mouse lungs in our laboratory (data not shown; Caduff et al., 1986; Zeltner and Burri, 1987). However, all of these studies were based on vascular Mercox casts and potential filling artifacts could not be excluded. To verify these results, we searched through electron microscopic serial sections of human lungs and detected sites of capillary fusion (Fig. 6). We chose human lungs due to its clinical importance. The lungs had been fixed in the phase of microvascular maturation. Below and above the site of fusion, the capillaries were only separated by a layer of less than 1 μm of tissue, consisting of the endothelium of both capillaries (Fig. 6c,e,j,l). In the center of the fusion, a small connection between the two capillaries was detected (Fig. 6d,k). Therefore, these serial sections confirmed the scanning electron microscopic observations (Fig. 5), which we and others interpreted as sites of capillary fusion. The intercellular space between the two endothelial cells is closed by tight junctions to prevent a leakage of the capillaries (Fig. 6i–l). Sites of fusion were observed more than once in all human lungs investigated.

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Figure 6. Capillary fusion as seen by transmission electron microscopy in human developing lungs. Capillary contacts and fusion were also studied on electron microscopic serial sections. a–g: An overview of a site of fusion. h–l: An enlargement of the zone of fusion. k: A still small opening between the two capillaries is shown. j,l: Below (j) and above (l) the opening, the capillaries are only separated by two endothelial cells, which are interconnected by tight junctions. An interesting observation is the fused basement membrane between the upper endothelial cell and an extension of a type 1 alveolar epithelial cell inside the septum as shown in h. b,i; In three dimensions, it represents an extension of the air–blood barrier as see in the next section. A, airspace of an alveolus; BM, basement membrane (asterisk, fused basement membrane of epithelium and endothelium); C, lumen of a capillary; EN, endothelium; EP, type 1 alveolar epithelial cell; FC, fibrocyte. The distance to the first section (a,h) is given in micrometers at the right upper corner. Scale bar = 2 μm.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Dexamethasone is known for its negative impact on developing lungs. Given postnatally over a period of 10 to 14 days in a low dose, it has been shown to impair secondary septation in rats with the irreversible result of fewer and larger airspaces and a diminished alveolar surface area compared with controls (Massaro et al., 1985). In contrast to this low-dose but long-term treatment protocol, a regimen with a higher dose of dexamethasone but given only for a period of 4 days in newborn rats would better reflect the situation of steroid treatment in premature babies. Such a protocol was shown to impair secondary septation only temporarily with no long-term injury at the adult age. It led transitionally to a lung parenchyma of decreased complexity, with fewer and larger alveoli, especially on day 10 (Schwyter et al., 2003). These differences did not exist any longer on day 36 and 60. Furthermore, in rats treated for 72 hr with dexamethasone, Tschanz et al. found a reduced septal volume as a result of a reduction of interstitial volume already on day 4 (Tschanz et al., 2003).

To quantify our visual impression of premature septal thinning after dexamethasone treatment (Fig. 1) and the morphological observation of Tschanz et al. (2003), we here directly measured the thickness of the interalveolar wall. We were able to show that dexamethasone given to newborn rats indeed reduced the interalveolar wall thickness after 3 days of treatment (Fig. 3a). In agreement with our data, Massaro et al. found in another rodent model a similar reduction of the wall thickness (Massaro and Massaro, 1986). At later time points, no statistically significant difference could be observed any longer. Despite that the difference on day 4 reached statistical significance, the measured mean thickness differed only by 2.9 μm or 22% between controls and treated animals (13.01 μm vs. 10.14 μm), which hardly corresponded to the visual impression of more areas with clearly thinner septa in the dexamethasone group. Therefore, we hypothesized that the mean wall thickness might not be reduced due to an overall reduction of thickness, but because of a focal wall thinning. To better demonstrate the distribution of the septal thickness, we assigned each measured septum to a specific thickness group (Fig. 3b). As expected, this histogram did not reveal any differences in the overall range, but a shift toward a higher number of areas with thinner septa in the treatment group, underlining the hypothesis that the wall thinning might be due to multiple focal events.

Using the same dexamethasone treatment of rats, Tschanz et al. (2003) postulated “a precocious microvascular maturation resulting in single capillary network septa in the first 4 postnatal days. By postnatal day 10, the lung morphologic phenotype showed a step back in the maturational state, with an increased number of septa with double capillary layer.” This “step back” represents a very interesting result; however, their findings were purely based on morphological observations. Furthermore, to the best of our knowledge, the maturation of the inter-airspace septa was never followed by direct stereological measurements that included the phases of alveolarization and of microvascular maturation.

Therefore, we used intersection counting on transmission electron photomicrographs as shown in Figure 1 to determine the percentage of double (immature) and single (mature) -layered capillary networks inside the inter-airspace septa between days 4–60. In the control group, we found that, at the very beginning of alveolarization (postnatal day 4), 16% of the inter-airspace septa possessed a mature capillary bed with only one layer (Figs. 2, 4). At postnatal day 10, this value roughly doubled to 29%. This finding confirms that there is an overlap between the periods of microvascular maturation and of alveolarization—an overlap that is larger than expected. Most likely microvascular maturation already starts during the first half of the phase of alveolarization. The bulk of vascular maturation happened during the next days as proposed by Burri (1974). By day 21, a total of 78% of the area showed a mature vascular network. However, a significant fraction of septa only matured within the next 40 days, leading to an almost complete microvascular maturation by the age of 60 days (95%).

In addition to the findings of accelerated wall thinning, we measured an accelerated microvascular maturation with a significant increase of areas containing a monolayered capillary network in the dexamethasone group on day 4 (Fig. 4). Vascular casts illustrating this result are shown in Figure 2. During the following 6 days, the microvascular maturity stayed constant, resulting in the same maturity for both groups by day 10. Our result was unexpected in reference to Tschanz et al. (2003). The postulated splitting of single-layered capillary networks into double-layered ones was not observed. Instead, there was a temporary arrest of microvascular maturation in treated rats until day 10. Afterward, no difference in microvascular maturation was detected anymore between the two groups—suggesting that ∼70% of double-layered capillary networks were not only sufficient for normal alveolarization but also for the rescue of the dexamethasone-induced delay of alveolarization. This finding result may be explained by the focal nature of the premature microvascular maturation. We would like to postulate for both groups that, after day 10, the formation of new septa takes place outside of the focal areas of the (premature) microvascular maturation and that the remaining immature double-layered areas are large enough to allow the formation of an adequate number of new septa.

During the whole developmental period, thinning of septal walls went parallel to the reduction of the capillary network from two to one layer. This observation verifies the hypothesis, that dexamethasone might reduce inter-airspace wall thickness by accelerating maturation of the capillary bed as it has been suggested earlier (Tschanz et al., 1995). On the other hand, the observed increase of the volume density of the capillary lumen between days 21 and 60 (Tschanz et al., 2003) is reflected in a small increase of septal thickness between the same days (Fig. 3a).

To further investigate the mechanisms of microvascular maturation, we analyzed scanning electron photomicrographs of Mercox casts of the lung capillary bed (Figs. 2, 5). We searched for very narrow gaps, as well as contacts between the two layers of the capillary network in immature alveolar septa, because these structures were interpreted as sites of fusing capillaries (Caduff et al., 1986; Zeltner and Burri, 1987). In numerous photomicrographs and at all days studied (days 2–21), we found these narrow gaps and contacts (Fig. 5) with a tendency of an elevated number of contacts in the lungs of treated animals on days 2–6. Several of these sites of fusion showed multiple contacts between the two capillary networks (Fig. 5b). We interpreted it as a focal area of microvascular maturation as discussed above.

To the best of our knowledge, the interpretation that these gaps and contacts represent sites of capillary fusion was never verified by serial electron microscopic section in developing lungs (Burri, 1992). We now supply this evidence (Fig. 6) and would like to describe a scenario of how capillary fusion may take place. Due to the thinning of the alveolar septum, two capillaries may get in close contact. To establish the first contact between the basal surfaces of the endothelial cells, their basement membranes have to be removed at the location of the contact (Fig. 6h–l). The first opening between the two capillaries may be formed by the appearance of two corresponding holes in both endothelial cells. The rims of the holes are sealed to each other by tight junctions to prevent a leakage. The holes themselves have a similar appearance as the holes in the endothelium of fenestrated capillaries (Fig. 6d,k). To complete the fusion, the first opening enlarges by remodeling of the shape of the endothelial cells and the structure of the surrounding connective tissue. A similar concept of fusion of capillaries has been observed in chicken chorio-allantoic membranes (Patan et al., 1997). It could be shown by in vivo microscopy that modification of the vascular bed by thinning of the intercapillary walls and finally merging of the two lumina happened within minutes to a few hours (Djonov et al., 2003). This mechanism would allow rapid and focal adaptation of the capillary network to the actual physiologic needs.

In summary, this work confirms that microvascular maturation is already taking place while the bulk of alveoli is still forming. Therefore, alveolarization and microvascular maturation happen simultaneously. The apparent contradiction may be explained by the focal appearance of the fusion of the two capillary layers. Dexamethasone leads to an accelerated and nonreversing fusion of the capillary network in the immature septum. The areas with fused capillaries have a reduced thickness of the inter-airspace wall, which explains the histological appearance of thinner septa in the treated lungs. Because a capillary bilayer is a prerequisite for septation of the airspaces, precocious microvascular maturation may explain the reduced alveolarization.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Animals and Tissues

Newborn Sprague–Dawley rats were treated with dexamethasone sodium phosphate (Decadron, Merck Sharp & Dohme AG, Glattbrugg, Switzerland) at postnatal days 1 (0.1 μg/g body weight, s.c.), 2 (0.05 μg/g), 3 (0.025 μg/g), and 4 (0.01 μg/g). Control animals received the same volume of 0.9% saline. Lungs were prepared according to Schittny and coworkers (1997) at postnatal days 4, 10, 21, and 60 for transmission electron microscopy and at postnatal days 2–21 for vascular casts (scanning electron microscopy). Briefly, the airspace was filled with 2.5% glutaraldehyde in 0.03 M potassium–phosphate buffer (pH 7.4, 370 mOsm) for transmission electron microscopy or with phosphate buffered saline (PBS = 10 mM sodium phosphate, containing 127 mM sodium chloride, pH 7.4) for scanning electron microscopy at a constant pressure of 20-cm water column. 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.

Transmission Electron Microscopy

Samples of three to five rats per time point (days 4, 10, 21, and 60) were post-fixed in 0.1 M sodium cacodylate (pH 7.4, 340 mOsm), containing 1% OsO4, dehydrated in a graded series of ethanol, and embedded in Epon 812. Sections of ∼80 nm thickness were cut, picked up on Formvar-coated copper grids, double stained with lead citrate (Reynolds, 1963) and uranyl acetate (Frasca and Parks, 1965), and viewed in a Philips 400 electron microscope.

Scanning Electron Microscopy

For vascular casts, three to six lungs per time point (days 2–21 of both groups) were perfused with PBS (containing 1% procaine, 20 U/ml heparin, and 10 mM ethylenediaminetetraacetic acid; Fluka Chemie AG, Buchs, Switzerland) by means of the right ventricle, immediately followed by freshly prepared Mercox (0.1 ml of accelerator/5 ml of resin; Mercox is methylmethacrylate produced by the Japan Vilene Co.; Tano et al., 1981). Constant pressure was maintained until the resin polymerized. Lungs were removed and transferred to 15% potassium hydroxide solution for 3 to 10 weeks. After tissue digestion, the casts were washed with distilled water, dehydrated with a graded ethanol series, dried in a vacuum desiccator, sputter-coated with 10-nm gold (Balzers Union sputtering device; Balzers, Liechtenstein), mounted, and photographed with a Philips XL-30 FEG scanning electron microscope (Patan et al., 1992).

Human Lungs

The human lungs were obtained from two girls and one boy who were between 16 and 18 months of age. The children had no history of previous cardiopulmonary diseases. The cause of death was isolated head injury in two cases and an acute viral encephalitis in the third case. Their origin, sampling, and preparation have been reported in detail in Zeltner et al. (1987). In short, lungs were fixed in the supine position by intratracheal instillation of 2.5% phosphate buffered glutaraldehyde. The fixed lungs were removed and processed for transmission electron microscopy. The experiments were approved and supervised by the authorities of the University of Bern and its Medical Faculty.

Morphometry

Before embedding for electron microscopy, the left lungs were cut completely into slices of 2 mm thickness perpendicular to the longitudinal axis. Four to five slices were taken and diced into cubes of 2 × 2 × 2 mm. Ten blocks were selected and embedded in Epon 812 (see above). Of the 10 blocks, 5 were selected, and on one section of each block, three to four images were taken on 35-mm film at a primary magnification of ×120 using a Philips 300 electron microscope. Every step of the sampling was done randomly (Cruz-Orive and Weibel, 1981). In the treated and the control groups, three to five animals were investigated at each time point (n = 5 for days 4 and 10, n = 3 for days 21 and 60). Films were viewed in a projection unit at final magnification of ×1,200 using a squared lattice containing 100 squares inside the sampling area. At each intersection of the horizontal lines with a lung septum, the septum was classified as containing a single or double capillary network. At the same location, the thickness of the septum was measured perpendicular to the surface of the septum. The mean of the measurements was calculated (sum of measurements divided by number of measurements) and divided by the total magnification of the images. Intersections with lung epithelium adjacent to nonparenchymal structures were not taken into account (Weibel, 1984). Means and standard errors were calculated for each group. Statistical comparisons were made using the Student's t-test. Values for P < 0.05 were considered statistically significant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We thank Mrs. Catherine Allemann, Mrs. Bettina de Breuyn, Mrs. Marianne Hofstetter, and Mr. Beat Haenni for expert technical assistance. P.H.B. and J.C.S. were funded by grants of the Swiss National Science Foundation, and M.R.-K. was funded by Fondation Pittet and Fondation Eugenio Litta, Switzerland, and Fonds de Perfectionnement et de pediatrie du CHUV, Lausanne, Switzerland. T.M.B. received the GLAXO Award 1999.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
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