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Evidence and structural mechanism for late lung alveolarization

¹Institute of Anatomy, University of Bern, Bern, and ²Swiss Light Source, Paul Scherrer Institut, Villigen, Switzerland

Submitted 27 July 2007 ; accepted in final form 19 November 2007

	ABSTRACT

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According to the current view, the formation of new alveolar septa from preexisting ones ceases due to the reduction of a double- to a single-layered capillaries network inside the alveolar septa (microvasculature maturation postnatal days 14–21 in rats). We challenged this view by measuring stereologically the appearance of new alveolar septa and by studying the alveolar capillary network in three-dimensional (3-D) visualizations obtained by high-resolution synchrotron radiation X-ray tomographic microscopy. We observed that new septa are formed at least until young adulthood (rats, days 4–60) and that roughly half of the new septa are lifted off of mature septa containing single-layered capillary networks. At the basis of newly forming septa, we detected a local duplication of the capillary network. We conclude that new alveoli may be formed in principle at any time and at any location inside the lung parenchyma and that lung development continues into young adulthood. We define two phases during developmental alveolarization. Phase one (days 4–21), lifting off of new septa from immature preexisting septa, and phase two (day 14 through young adulthood), formation of septa from mature preexisting septa. Clinically, our results ask for precautions using drugs influencing structural lung development during both phases of alveolarization.

septation; capillary remodeling; lung development

View larger version (46K): [in this window] [in a new window]   Fig. 1. Formation of new septa/classical alveolarization and microvascular maturation. Smooth muscle cell precursors, elastic fibers, and collagen fibrils (green spot) accumulate in immature alveolar septa at sites where new septa will be formed (arrows; A). While the new septa grow, 1 of the 2 layers of the capillary network (red) of the preexisting septa folds up (B). As a result, the newly formed septa subdivide preexisting air spaces, and new alveoli are born (C). A–C, modified from Burri (8, 9). The preexisting and the newly formed septa still contain a double-layered capillary network. During the following phase of microvascular maturation, the capillary layers fuse and form a central capillary layer (D).

The large clinical relevance of late alveolarization inspired us to follow two directions. First, we applied a design-based stereological method by estimating the length density of the alveolar entrance rings (53) and developed a novel approach to follow the anlage (formation) of new alveolar septa throughout lung development and growth. Second, we were wondering how the requirement of a double-layered capillary network inside the existing alveolar septa may be overcome. We studied vascular casts of lungs in three-dimensional (3-D) visualizations obtained by synchrotron radiation X-ray tomographic microscopy (SRXTM; Ref. 46). SRXTM represents the only high-resolution imaging method that allows us to visualize non-destructively the capillary network during the formation of new alveolar septa unrestrictedly from different angles.

	METHODS

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Animals and tissues. Sprague Dawley rats were used for all experiments. For the stereological measurements and for the 3-D visualizations of the tissue, the lungs were prepared as previously described (42, 43) at postnatal days 4, 6, 10, 21, 36, and 60. Briefly, the air space was filled with 2.5% glutaraldehyde in 0.03 M potassium phosphate buffer (pH 7.4, 370 mosmol/kgH₂O) at a constant pressure of 20 cmH₂O. At this pressure, the lung reaches roughly its total lung capacity. To prevent a recoiling of the lung, the pressure was maintained during fixation (minimum of 2 h). The individual lung lobes were separated, and their volumes were determined by fluid displacement (40). For the stereological measurements, the lung lobes were dehydrated in a graded series of ethanol and paraffin-embedded using Histoclear (Life Science International, Frankfurt, Germany) as intermedium. X-ray tomographic microscopy samples were postfixed with 1% OsO₄ in 0.1 M sodium cacodylate (pH 7.4, 340 mosmol/kgH₂O), stained en bloc with 0.5% uranyl acetate in 0.05 M maleate buffer, dehydrated in a graded series of ethanol, and embedded in Epon 812.

The vascular casts of the alveolar microvasculature were prepared according to Haas et al. (18). Briefly, lungs were perfused with PBS (containing 1% procaine, 20 U/ml heparin, and 10 mM EDTA) immediately followed by freshly prepared Mercox (0.1-ml accelerator per 5-ml resin/Mercox = methylmethacrylate of the Japan Vilene; Ref. 47). Constant pressure was maintained until the resin jellified. Lungs were removed and transferred into 15% potassium hydroxide solution for 3–10 wk. After digestion of tissue, the casts were washed with distilled water, stained with iodine, dehydrated in a graded series of ethanol, dried in a vacuum desiccator, and mounted on top of a rodlike sample holder of a diameter of 3.0 mm.

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.

Stereology/anlage of septa. A series of 10–13 serial step sections of 4-µm thickness was obtained along the longitudinal axis of the left lungs. For the lung parenchyma, we may assume that the structures are isotropic (15). By using the same axis for every sample, we were able to assure random sampling in a well-standardized setup. The gap between the sections (length of the step) was constant for all lobes obtained at the same postnatal day, but it increased with increasing size of the left lungs due to lung development and growth. Sections were transferred onto silanized microslides, air-dried over night at 37°C, and stained with fuchsine. Approximately 40 images per animal were taken according to a systematic random sampling scheme (15). The images were recorded using a Leica DM RB light microscope (Glattbrugg, Switzerland) equipped with a motorized Maerzhaeuser XY stage (Wetzlar, Germany). Images were obtained using a JVC 930 three-chip color video camera (Oberwil, Switzerland) and the software analySIS (Münster, Germany). They were printed at a magnification of x250. All stereological measurements were done in randomly sampled reference areas (15).

The volume density of the lung parenchyma (air spaces and septal tissue, excluding bronchi, bronchioli, and blood vessels, >20 µm in diameter) was estimated by point counting. The absolute parenchymal volume was calculated as the product of the parenchymal volume density and the lung volume for each animal and time point (23, 53).

Stereologically, any length appearing in a 3-D volume may be estimated by counting the number of points cutting the plane of 2-D sections (23, 53). In 3-D, the entrance rings of the alveoli are equal to the network of all free edges of the alveolar septa (Fig. 2; also, see RESULTS). In 2-D sections, these free edges appear as end points (tips) of the cut septa (Fig. 2). We employed theses two principles and estimated the length of the free edge of the septa by counting the end points of the septa in a reference space on 2-D paraffin sections. We estimated the length density based on the lung parenchyma (length per parenchymal volume). The total length was calculated by multiplying the length density by the parenchymal volume.

View larger version (71K): [in this window] [in a new window]   Fig. 2. Estimation of the anlage of new septa. A shows a scanning electron micrograph of lung parenchyma. The cut surface of this sample is colored in purple to show how a 2-dimensional (2-D) section taken at this surface would look like. A similar lung section is shown in B (paraffin section stained with hematoxylin-eosin). The free edge of a septum (yellow dashed line; A) is represented by the tip of a cut septum in a 2-D section (yellow asterisk; A and B). To estimate the length density (length per volume) and the total length of the free edge (Fig. 3, A and B), the number of the tips of the cut septa was counted in a reference area (red-green rectangle) on (2-D) lung sections (B). Based on the measured length density, the amount of newly formed septa (anlage of septa) was estimated after a mathematical correction for the growth of the lung itself (Fig. 3C).

In a model of lung development of the rhesus monkey (24), we compared our estimation of the anlage of new septa to the number of alveoli. The data sets correlate very well throughout lung development (number of alveoli compared with the total length of the free septal edge, separately calculated for males and females, n = 13 for both groups; data not shown). Therefore, both methods are well-suited to study alveolarization.

Statistical analysis. For all morphometric measurements, 3–5 animals per time point were used. The Gaussian distribution of the data was shown using the Kolmogorov-Smirnov test (1, 37). Differences between groups were assessed by one-way ANOVA followed by Bonferroni-Holm-corrected post hoc t-tests (1, 37). Statistical significance was defined as P < 0.05.

SRXTM. The Epon-embedded samples were shaped down to rods of a diameter of 1.3 mm on a watchmaker's lathe. They were glued on a rodlike sample holder of a diameter of 3.0 mm using a two-component epoxy resin-based glue (Araldite Rapid; Novartis, Basel, Switzerland). Special care was taken so that the samples were mounted perpendicularly to the surface of the sample holder to fit exactly into the window of the camera. Vascular casts were cut into cubes of 2–4 mm edge length and glued onto specimen holders for scanning electron microscopy of a diameter of 13 mm (Provac, Balzers, Lichtenstein) using Araldite Rapid.

The samples were scanned at an X-ray wavelength of 1 Å (corresponding to an energy of 12.398 keV) at the microtomography station of the Materials Science Beamline at the Swiss Light Source of the Paul Scherrer Institut (Villigen, Switzerland; Ref. 46). The monochromatic X-ray beam (E/E = 0.014%) was tailored by a slits system to a profile of 1.4² mm². After penetration of the sample, X-rays were converted into visible light by a thin Ce-doped YAG scintillator screen (Crismatec Saint-Gobain, Nemours, France). Projection images were further magnified by diffraction limited microscope optics and finally digitalized by a high-resolution CCD camera (Photonic Science, East Sussex, United Kingdom; Ref. 46). For the tissue samples, the optical magnification was set to x10, and on-chip binning was selected to improve the signal-to-noise ratio, resulting in isotropic voxels of 1.4³ µm³ for the reconstructed images. The vascular casts were scanned without binning, resulting in a voxel size 0.7³ µm³. For each measurement, 1,001 projections were acquired along with dark and periodic flat field images at an integration time of 4 s each without binning and 2 s each after binning. Scanning time for 1 volume of interest summed up to 88 min (binned) to 148 min (without binning) and resulted in 2–8 gigabytes (GB) of raw projection data. Data were postprocessed and rearranged into flat field-corrected sinograms online. Reconstruction of the volume of interest was performed on a 16-node Linux PC Farm (Pentium 4, 2.8 GHz, 512 megabytes RAM) using highly optimized filtered back-projection routines and taking <20 min for a full 2 GB data set. We used a global thresholding approach for surface rendering. For 3-D visualization and surface rendering, we applied the software Imaris (Bitplane, Zürich, Switzerland) installed on an Athlon 64 3500-based personal computer.

	RESULTS

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Anlage of new septa. Most studies characterizing lung development are focusing on the appearance of new alveoli and/or the enlargement of the alveolar surface (7, 24, 33). Alveoli are formed by subdividing existing air spaces due to the lifting off of new septa. Therefore, the latter represents the primary event during alveolarization. To the best of our knowledge, the present study is the first one characterizing alveolarization based on the anlage of new alveolar septa, meaning that, for the first time, the length or length density of the free septal edges was measured throughout lung development, and the amount of newly formed septa was calculated.

Every alveolar septum that is in contact to the alveolar entrance ring is characterized by one free edge, by a number of edges that are in contact to the neighboring septa ("bound" edges), and by its surface. All free septal edges taken together are equal to the network of alveolar entrance rings (Fig. 2A). During the lifting off of a new alveolar septum, its free edge will be the first structure detectable. In classical 2-D sections of lungs, the free edges are recognized as the tip of a cut septum (Fig. 2B). Stereologically, the length density is reliably determined by counting the number of these tips in a reference area (Fig. 2B). To follow the formation of new alveolar septa (anlage of septa), we estimated the length density (length per volume) of the free septal edges and calculated the total length of the free septal edges (23, 53). As shown in Fig. 3, A and B, the total length of the free septal edge increases throughout the entire period of observation. During growth of the lung and even without the addition of new septa, the total length of the alveolar septa will increase. Based on the estimated value obtained at day 4 (start of alveolarization), we calculated the total length of the free septal edge assuming that the lungs are growing isometrically without the addition of new septa and compared it to the estimated values (Fig. 3, A and B). We observed the formation of new septa between days 4–60 throughout the entire period of observation. This result shows that septation does not stop at the end of classical alveolarization (in rats days 4–14) but continues during adolescence into young adulthood and that alveolarization really takes place after the maturation of the alveolar microvasculature (Fig. 3, A and B).

View larger version (16K): [in this window] [in a new window]   Fig. 3. Anlage of alveolar septa. The total length of the free edges of the alveolar septa (length of alveolar entrance rings) was estimated as explained in Fig. 2 and plotted over the age (A; closed diamonds/estimated). We expected that the formation of new alveolar septa will cease at the latest after day 21 in rats (A; gray dotted line/expected), but we detected the formation of new septa throughout the entire time period of observation (days 4–60). It has to be noted that more than half of the septal length is formed later than day 21. In addition to septation, the total length of the septa increases due to isometric growth of the lung. Based on day 4, the septal length increases by only a factor of 2 due to isometric lung growth (A; open squares/isometric lung growth, calculated). This value is small compared with a 10x increase due the formation of new septa. B shows a double logarithmic plot of the estimated (closed diamonds) and the expected (gray dotted line) total length of the free edges over the parenchymal volume. The observed slope of the estimated increase of the length of the free septal edges is at every time larger than the slope of the calculated increase of the septal length due to the isometric growth of the lung (B; open squares). It shows the continuous formation of new septa during the growth of the lung. Furthermore, we calculated the amount of newly formed septa (C; closed diamonds) and compared it to the growth of the lung parenchyma (C; closed squares). The observed anlage of septa was normalized to the amount of septa present at day 4 because these septa are formed by branching morphogenesis and not by septation or alveolarization respectively. Until day 21, the formation of new septa occurs at a higher speed than the growth of the lung parenchyma. Afterwards, the parenchymal growth exceeds the anlage of new septa.

Visualization of newly forming alveolar septa. At days 6 and 10, low ridges are observed that are lifting off of existing septa (Fig. 4, B and C). These ridges are representing newly forming septa. They are subdividing existing terminal air spaces resulting in the formation of new alveoli. As predicted by the estimation of the length of the free septal edges (Fig. 3), the same low ridges were also frequently detected between days 21–60 (Fig. 4, D and E). These observations show that alveolarization takes place not only before, but also after the maturation of the alveolar microvascular network. The observed anlage of new septa was found everywhere in the growing lung parenchyma and were not limited to subpleural areas or areas facing larger airways and vessels.

View larger version (118K): [in this window] [in a new window]   Fig. 4. 3-D visualization of terminal air spaces. After electron microscopical embedding, rat lungs were scanned in an X-ray tomographic microscope. 3-D visualizations of terminal air spaces were obtained throughout lung development and lung growth. At postnatal day 4 (A), we observed large sacculi that were formed by branching morphogenesis. Between days 6–36 (B–E), the formation of new septa was abundant (arrows). Between days 4–21 (A–D), we 1st observed a decreasing, afterwards an increasing, size of the terminal air spaces (days 36 and 60; E and F). Bar, 50 µm at the surface of the sample only (the image represents a perspective view).

View larger version (124K): [in this window] [in a new window]   Fig. 5. 3-D visualizations of the capillary network of single alveoli. Vascular casts of 21-day-old rat lungs were obtained using Mercox. The images show the lumen of the capillaries, which is identical to the inner surface of the capillaries. Inside of the cavity of an alveolus, an upfolding of the single-layered capillary network was observed (blue dashed lines in A, C, and E). The folding is indicative for the formation of a new septum. The 3-D visualization enabled us, for the 1st time, to look at the backside of the same septum (B, D, and F). At the basis of the folding, we detected a local duplication of the existing capillary network (covering of the blue dashed line in B, D, and F). Whereas most of the duplications are already formed in these examples (arrowhead), 1 is most likely just forming by sprouting angiogenesis (arrow in B). In addition, (forming) tissue posts inside of the capillary network (holes in the vascular cast, green asterisk) are indicative for intussusceptive angiogenesis (the growth of the capillary network to allow the upfolding; Refs. 13, 17). The entrances of the alveoli are labeled with a yellow dotted line. Bar, 10 µm (the magnification varies inside the image due to the foreshortened view). The data of A and B are also shown in the supplementary video.

	DISCUSSION

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In the present study, we now show that half of the alveolar septa are formed after the phase of the maturation of the alveolar microvasculature is completed (in rats days 14–21) and that alveolarization continues at least until young adulthood in rats (day 60; Fig. 3). We used the well-established animal model of rat lung development (4, 7, 12, 26, 31, 33, 36) because, for ethical reasons, similar investigations are practically impossible in humans. Because this result is contradictory to the current view, we compared the measured data with 3-D lung images obtained by SRXTM. At days 6 and 10, we observed the formation of new alveolar septa, which start to subdivide the existing air space (Fig. 4, B and C). The appearance of these still low septa shows that septation takes place. It was postulated that septation is only responsible for the first rapid phase of septation and that, later, a different mechanism will be predominate (31, 54). However, we observed that the same low septa are very abundant at days 21 and 36 (Fig. 4, D and E). This result is evidence that septation (alveolarization) takes place at least until young adulthood, and most likely no other mechanism is employed. As shown in Fig. 4, we observed a decreasing size of the terminal air spaces between days 4–21, followed by an enlargement of its size at days 36 and 60. This observation reflects our stereological data very well. During the first part of alveolarization, the anlage of new alveolar septa exceeds the growth of the lung parenchyma, whereas afterwards, the growth rates are reversed (Fig. 3).

For the present study, we used the control animals of an investigation describing the effects of an early, high-dose dexamethasone treatment of newborn rats (14, 29, 39, 44, 51). This investigation included a classical stereological characterization of lung development. Using the published numbers for the alveolar surface and the volume of the parenchyma (51), we calculated that between days 21–60, half of the alveolar surface was formed due to isometric growth and half of it due to the formation of new septa by developmental alveolarization.

Our results of rat lung development are also supported by a very recent work of Hyde et al. (24). They counted the number of alveoli in rhesus monkey throughout lung development and observed that new alveoli are formed until young adulthood. This result may not be explained by the classical mechanism of alveolarization (11, 13) because, in the rhesus monkey, microvascular maturation is basically completed around birth (J. C. Schittny, unpublished observations).

How may new alveolar septa be formed of an existing alveolar septum at a time point when most of the alveolar septa are mature and therefore contain only a single-layered capillary network? First, we studied vascular casts (Mercox) by scanning electron microscopy. In a set of roughly 100 samples, we observed upfoldings of the alveolar capillary network that resemble sites where new septa are forming, but we were not able to determine unquestionably the number of layers of the capillary network in this particular area (data not shown). We searched for a different imaging method and decided to use SRXTM because it has been already successfully applied to study other structures owning a complex 3-D architecture like bone (49), capillary networks (20), and fossilized embryos (19). SRXTM gave us a virtually unrestricted 3-D access, meaning that we were able to choose freely any region of interest and any viewing angle. Re-examining the same samples, we observed the same upfoldings of the alveolar capillary network as seen using scanning electron microscopy. In samples obtained at day 21 or later, most of alveolar capillary networks were single-layered (39). Looking at the backside of the upfolding, we detected local duplications of the capillary network (Fig. 5 and supplementary video). The second layer of the capillary network, which is needed for the formation of a new septum, was observed exactly at and only at the site of septation. Before microvascular maturation, a new septum may form while the blood supply of the alveolar surface at the backside of the fold is guaranteed by the second capillary layer (Fig. 6A). However, after the maturation of the alveolar microvasculature, this second layer most likely does not exist and will be formed locally by a duplication of the capillary network (Fig. 6B). For two reasons, it is unlikely that most of the duplications were preexisting or "left over" from the former double-layered capillary network, respectively. First, we observed these kinds of duplications exactly at the concave side of the upfolding. At some locations, we even observed very shallow upfoldings where the duplications were not yet completed (Fig. 5B and supplementary video). Second, roughly half of the alveolar septa form after day 21 (Fig. 3). If new septa would only be formed at sites containing a preexisting double-layered capillary network ("left over immaturity"), a significant number of them should be detectable at day 21 at sites where no new septa are forming. Using light and electron microscopical sections as well as vascular casts (scanning electron microscopy and SRXTM), we searched for them thoroughly, but we were not able to detect a significant number of this kind of capillary duplication. We hypothesize that at least a larger number of these duplications is formed by angiogenesis during the lifting off of the new septa from interalveolar walls containing a single-layered capillary network. The hypothesis of angiogenesis is also supported by the finding that a lot of angiogenesis occurs during lung development and growth at least until day 60 (13). Furthermore, both the alveolar surface and the capillary surface increases about three times between days 21–60 in rats. Because the diameter of the capillaries stays constant, this large increase may only be explained by ongoing angiogenesis (13, 51).

View larger version (59K): [in this window] [in a new window]   Fig. 6. Comparison of phases 1 and 2 of developmental alveolarization. During phase 1 of developmental alveolarization (A–C), new septa are lifted off of immature septa containing a double-layered capillary network (red). During phase 2, new septa are formed from mature septa containing a single-layered capillary network (red; D–F). In both modes of alveolarization, the underlying capillary layer folds up (B and E) and forms a double-layered capillary network inside the newly formed septum (C and F). Because during phase 2 the 2nd capillary layer is most likely missing in the preexisting mature septum, this 2nd layer will be formed by angiogenesis at the basis of the newly formed septum (yellow arrows in E). Green, accumulation of smooth muscle cell precursor, elastic fibers, and collagen fibrils; blue, septum of connective tissue.

Extrapolating the rat and rhesus monkey (24) data to humans would mean that alveolar septa are formed until growth stops and that half of the alveolar septa are formed roughly between an age of 3 to 18 yr. This opinion was already present 30–40 yr ago (16, 21, 22, 48, 52), but, for theoretical reasons, it was changed later to the present view that alveolarization stops after microvascular maturation took place (2–3 yr of age in humans; Refs. 7, 13).

Clinically, this insight has large significance, especially for humans 3–18 yr of age. Steroids are widely used during the treatment of lung diseases like asthma and wheezing illnesses or other diseases like inflammatory bowel diseases (3, 25). Furthermore, retinoids are used for the treatment of psoriasis and severe acne (5). In rats, both drugs are known to alter the lung structure when given neonatally or during the phase of classical alveolarization (34, 50). So far, there was little concern regarding possible side effects of these drugs in children and adolescents due to the view that alveolarization is most likely already completed at this time point. Further studies are necessary to understand the influence of these drugs on the structure of the lungs during the second phase of developmental alveolarization. However, phase two opens also a very positive outlook, because new alveoli may, in principle, be formed at any time lung regeneration is feasible. We define the latter phenomenon as regenerative alveolarization.

In summary, the application of SRXTM was essential for the structural understanding how new alveoli are formed throughout lung development and growth. We have shown that new alveoli are formed not only before, but also after the maturation of the alveolar microvasculature. During the latter, the requirement of a double-layered capillary network at the site where a new septum will be formed is overcome by a local duplication found at the sides of septation. Most likely, many of these duplications are not preexisting. We call the classically described alveolarization phase one of developmental alveolarization and the newly described form phase two of developmental alveolarization. Until now, the understanding of phase two is based on structural evidence only. However, due to its clinical significance, we believe that these structural findings will be the starting point for investigations of the molecular mechanisms involved. The description of phase two will most likely force us to rethink our views 1) of lung regeneration and 2) of side effects on the structure of the lungs during the treatment of children and adolescents, e.g., with glucocorticoids and retinoids.

	GRANTS

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This work was supported by Swiss National Science Foundation Grants 3100.068256.02 and 3100A0-109874.

	ACKNOWLEDGMENTS

 
We thank Bettina de Breuyn, Krystyna Sala, Beat Haenni, and Christoph Lehmann for expert technical assistance, Dr. Amela Groso for help at the beamline, and Dr. Stefan Tschanz for contribution to the animal experiments.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. C. Schittny, Institute of Anatomy, Univ. of Bern, Baltzerstrasse 2, CH3012 Bern, Switzerland (e-mail: johannes.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.

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