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.
- capillary remodeling
- lung development
during lung development, the airways and an extensive gas-exchange area have to be formed. Development starts with the appearance of two lung buds. At the terminal ends of the buds, a repetitive process starts where elongation of the future airways alternates with branching. After ∼20 rounds of outgrowth and branching, the conducting and parts of the respiratory airways are formed (rats, embryonic days 11–20; humans, 4–26 wk preterm; Ref. 27). During alveolarization, the gas-exchange area is further enlarged by a subdivision of the terminal air spaces by the formation of new septa. One leaflet of the double-layered capillary network inside the existing septa folds up and gives rise to a new double-layered capillary network within the newly forming septa (Fig. 1, A–C; rats, postnatal days 4–14; humans, 36 wk preterm to 1–2 yr). This process requires smooth muscle cells, elastic fibers, and collagen fibrils, which are concentrating at the site where the new septum will be formed. During the upfolding, these elements stay close to the free edge of the newly forming septum just underneath the folding of the capillary network (10, 28, 41).
Later, during microvascular maturation, the double-layered capillary network of the alveolar septa is reduced to a single-layered one (Fig. 1D; rats, postnatal days 14–21; humans, 0–3 yr). Currently, it is believed that the lifting off of new septa from preexisting septa is excluded due to the missing of the second capillary layer (7, 13). Consequently, after microvascular maturation is completed, the enlargement of the gas-exchange area will be achieved by lung growth and not by a considerable addition of new alveolar septa. By the same token, a mature alveolar septum, which, once lost, will most likely not be reformed. Therefore, a noteworthy amount of lung regeneration is excluded according to the current view (7, 10, 13, 41). However, it has been postulated that, in rats, the number of alveoli is still increasing after the lung parenchyma has reached maturity (4, 31, 33, 36, 54). In humans, the time point when alveolarization stops has also not been well-defined and discussed in decades. Currently, many agree on an age of 2–3 yr (7, 13), whereas older data suggested that the formation of new alveoli ceases at ∼8 yr or even at 16–18 yr of age (16, 21, 22, 48, 52). Furthermore, new alveoli may be formed after starvation and refeeding (mice and rats; Refs. 30, 35) and during compensatory lung growth after pneumonectomy or lobectomy (rats and dogs; Ref. 6). Nevertheless, one question remained open: how may new alveoli be formed at a later time point? It has been proposed that 1) late alveolarization may take place in subpleural areas where a double-layered capillary network is not required (2, 32), or 2) late alveolarization may follow a different unknown mechanism (31, 54). So far, alveolarization after the phase of microvascular maturation is on debate, and the question how any form of “late” alveolarization may take place remains open.
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.
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 ×250. 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.
During lung development, we were interested in the amount of septa that were newly formed in addition to the gain of length due to the isometric growth of the existing septa. If a given volume contains a given length, the length density decreases during an isometric growth of volume and length because the volume increases with r3 and the length “only” with r1. The decrease of the length density may be corrected by multiplying the length density with , where V0 represents the volume at the start of the growth and Vx the one at the time point X. The resulting “growth-corrected length density” will stay constant throughout the isometric growth as long as no additional length is added. If new septa are formed, e.g., during lung alveolarization, new lengths will be added and the growth-corrected length density will increase. The increase represents a measure for the addition of new septal length or septa, respectively. We call this increase “anlage of new septa” or “formation of new septa.” Vice versa, a decrease of the growth-corrected length density stands for a loss of original septa during lung growth, which may happen due to aging and/or to structural lung diseases. We followed the increase of the growth-corrected length density throughout lung development and estimated the formation or anlage of new septa, respectively.
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.
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.
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 (ΔE/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 ×10, 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.
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).
Furthermore, we calculated the amount of newly formed septa (anlage of septa) and normalized it to day 4 (Fig. 3C). We observed that roughly half of the septa of a young adult lung (day 60) are lifted off of existing septa after the maturation of the alveolar microvasculature is completed (day 21 in rats). Therefore, half of the septa are formed during a time period when most of the existing septa contain only a single-layered capillary network (Fig. 3C).
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.
Local capillary duplications.
Our result that alveoli are formed throughout lung development is contradictory to the current view that a double-layered capillary network is required inside the existing septa for the lifting off of new septa. During microvascular maturation (days 14–21 in rats), the double-layered capillary networks of the alveolar septa fuse to a single-layered network. Therefore, further alveolarization seemed to be excluded by the end of the third postnatal week (7, 13). To resolve this contradiction, we decided to study vascular Mercox casts of the alveolar capillary network after microvascular maturation is completed (day 21). Using SRXTM, we were able to study blocks of 0.3–0.5 mm3 at a pixel size of 700 nm and to search for alveoli that contained an upfolding capillary network as shown in Fig. 5, A, C, and E and in the supplementary video (available in the data supplement online at the AJP-Lung Cellular and Molecular Physiology web site). These upfoldings are indicative for the formation of new septa. In addition, SRXTM enabled us, for the first time, to visualize the backside of the septa in question. As indicated by the dashed blue line, we observed local duplications of the capillary network at the basis of the newly forming septa (Fig. 5, B, D, and F and supplementary video). Whereas Fig. 5, D and F, shows examples where the duplications are already completed, Fig. 5B shows an example where it appears that the duplication is still forming.
The question of if and how alveoli may be formed in young adults has been debated for many years (4, 7, 13, 31, 33, 36, 41, 54). The question is very important due to its clinical relevance. Acute lung injury and acute respiratory distress syndromes are common causes of morbidity and mortality in intensive care units. Irrespective of the initial cause of the lung injury, both diseases are characterized by a diffuse damage of the lung parenchyma, which includes a reduction of the diffusion capacity and may lead to a loss of alveolar septa (45).
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).
We conclude that the postulated requirement of a double-layered capillary network at the site of septation (7) is still valid (see Introduction). However, the two layers do not have to be preexisting as currently postulated, but they may be formed rapidly and locally by angiogenesis when needed. Because microvascular maturation takes place during alveolarization, we call the entire time when new septa/alveoli are formed during lung development and growth “developmental alveolarization.” This term distinguishes the developmental processes from any kind of lung regeneration (6, 30, 35). We call the latter “regenerative alveolarization.” To account for the role of the microvasculature, we define two phases of developmental alveolarization. Phase one represents the formation of new septa/alveoli starting from septa containing a double capillary network. This phase blends over into phase two where the new septa/alveoli are formed from mature preexisting septa. To account for the large overlap between the two phases (Fig. 3A), we define phase one as days 4–21 and phase two as day 14 through young adulthood. Comparing our new suggestion with the current classification of alveolarization, phase one represents classical alveolarization (days 4–14) and phase two late alveolarization (late alveolarization defined as any kind of alveolarization after the phase of microvascular maturation, after day 21; Ref. 11).
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.
This work was supported by Swiss National Science Foundation Grants 3100.068256.02 and 3100A0-109874.
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.
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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