Introduction

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Human and porcine fetal airways have been shown to contract spontaneously from the first trimester, the latter also contracting in response to neural stimulation (1). Real-time video imaging of contracting airways revealed that the epithelial tubules are contractile from the proximal airways to the base of the terminal sacs. This phenomenon has been well documented in fetal pig lungs in the first and second trimesters, where spontaneous narrowing and relaxation of the airways move the lung liquid back and forth (2). The narrowing of the airways of fetal pigs in response to neural stimulation was blocked by atropine and by tetrodotoxin, indicating that functional cholinergic nerves were present (2, 3). The presence of nerves and the capacity of the airways to contract indicates that innervated, functional smooth muscle is present in the newly forming airways early in gestation. The objective of the present study was to map the development of the nerves and their relationship to the airway smooth muscle (ASM) in the human fetal lung.

The human lung develops in the embryonic period as an outgrowth of the foregut, an epithelial tube, which is destined to become the trachea. As it elongates, it invades the surrounding mesenchyme and at 4.5 wk in the human embryo comprises five tiny saccules, two on the left and three on the rightthe future lobar bronchi and corresponding lung lobes (4). These epithelial tubules, which terminate in blind sacs, have the appearance of glands when examined in section, and this stage of lung development is termed the pseudoglandular phase. It continues for about another 12 wk with subsequent dichotomous branching giving rise to about 20 generations, with two more added in the subsequent canalicular phase (5). This nearly completes the formation of the bronchial tree, which is 24 generations in the adult, inclusive of the trachea.

Recently Weichselbaum and colleagues (6) showed not only that the growing airways in first-trimester fetal pig lungs were covered in a well-formed layer of smooth-muscle cells, but also that an extensive nerve plexus comprising nerve trunks and ganglia invested the airways, with fine bundles innervating the smooth muscle. To stain the developing nerves, an antibody to protein gene product 9.5 (PGP 9.5) was used that stains all nerves and neurons (7). In first-trimester fetal pigs, PGP 9.5 was also found to stain the epithelial cells diffusely (6). This appears to be a property of the undifferentiated epithelial cells, as has been shown in human fetal lung younger than 16 wk gestation (8). In the present study, this diffuse staining has been used to image the growing tips of the epithelial tubules, thereby facilitating the mapping of the innervation of the developing airways. PGP 9.5 has also been used to stain pulmonary neuroendocrine cells (9). The colocalization of PGP 9.5 and other neuroendocrine markers has been investigated in cross sections of human fetal lung (8, 10) and explants of cultured rat lungs (11).

The aim of the present study was to map immunohistochemically the nerves and the smooth muscle of the bronchial tree in the human fetal lung from early gestation with antibodies to the nerve marker PGP 9.5 and the smooth-muscle protein -actin (12). The latter gives identical staining compared with a polyclonal antibody specific for smooth-muscle myosin in fetal human and porcine airways (3, 6). In addition, Schwann cells have been revealed using S-100, which has been found useful as a means of demonstrating the supporting glial cells of pulmonary ganglia and the Schwann cells of peripheral nerves in the adult respiratory tract (13). Whole mounts of the bronchial tree at 7.5 to 8 wk (early pseudoglandular stage), and 16 to 18 wk (early canalicular stage) were imaged using confocal laser scanning microscopy to reveal the neural, ASM, and vascular structures of the airway wall. Projections of optical sections cut through the thickness of the airway wall have been used to produce overviews of the innervation along the length of the bronchial tree, as well as to provide structural detail. The study has been extended into postnatal life using lungs from infants who died of Sudden Infant Death Syndrome (SIDS).

	Materials and Methods

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Lung Tissues

Human fetal lungs from the first (7.5 to 8 wk) and second (16, 18, and 24 wk) trimesters from therapeutic terminations of pregnancy were obtained from the Department of Pediatrics, University of Iowa Hospitals and Clinics (Iowa City, IA). The tissues were received fixed in 4% buffered formaldehyde. Gestational age was estimated by foot-length measurement (14). In the first trimester, lobes from four lungs at 53, 54, 56, and 58 d gestation were studied. The only complete lung obtained was at 53 d and contained the distal half of the trachea. In the second trimester, lobes from lungs of 16-, 18-, and 24-wk gestation were obtained. The lobar bronchi of these lobes had been removed before they were made available for this study. The results of airways in the 24-wk lung lobe were qualitatively similar to those of the 18-wk lobe and have not been included in the present work because of inferior image quality. The study was approved by the Institutional Review Board of the University of Iowa. The left upper lobes of two infants aged 5 and 10 mo who had died of SIDS were obtained from the Victorian Institute of Forensic Medicine (VIFM), Melbourne, Australia. The time that elapsed between the estimated time of death and the fixing of the lungs in 4% buffered formaldehyde at autopsy was 22 and 51 h, respectively. A period of about 16 wk had elapsed prior to dissection. This study was approved by the VIFM and by the Human Ethics Committee of the University of Western Australia (Nedlands, Australia).

Immunohistochemistry

The parenchyma and vasculature in the lobes from the first-trimester fetal lungs were carefully teased away from the airways using a dissecting microscope, isolating most of the bronchial tree. In the lobes from the second-trimester fetal lungs the segmental bronchus and its branches were separated from the pulmonary arterial tree, leaving both largely intact. In the left upper lobes from the SIDS lungs, distal airways of ~ 1 mm diameter were dissected free of pulmonary arteries and parenchyma. It was difficult to separate the airways from the surrounding tissue without damaging their adventitia, particularly the proximal bronchi of the lobe. The lobe obtained 51 h postmortem could not feasibly be imaged with sufficient quality. In the lobe obtained 22 h after death, the proximal bronchi did not image clearly; however, the distal airways could be imaged satisfactorily. Following dissection, the specimens from fetal and postnatal lung were cleared in dimethyl sulfoxide three times for 10 min each (15). After washing in phosphate-buffered saline (PBS), pH 7.2, twice for 10 min each, nonspecific binding was blocked by an additional washing step in PBS that contained 1% bovine serum albumin before application of the primary antibodies. To stain for all nerves, a rabbit polyclonal antibody to PGP 9.5 (Protein Gene Product 9.5; Ultra Clone, Isle of Wight, UK) was used. Schwann cells were stained with a rabbit polyclonal S-100 antibody (Dako, Sydney, Australia). Smooth muscle was identified with mouse monoclonal anti--actin (Sigma Chemical Co., St. Louis, MO). The final dilutions of the respective antibodies from the stock solutions obtained from the above suppliers were: PGP 9.5, 1:100; S-100, 1:200; -actin, 1:250.

The preparation was then incubated overnight in a humidified chamber at 4°C. After being washed several times with PBS over a 4-h period, the samples were incubated for 12 h at room temperature with the fluorochrome-labeled secondary antibodies. The secondary antibodies (antimouse and antirabbit) were conjugated to either fluorescein isothiocyanate (FITC) (Silenus, Melbourne, Australia), rhodamine red (RITC) (Cappel; Organon Teknika Corp., West Chester, PA), or Cy5 (Amersham, Sydney, Australia). Cy5 was used in Figures 2e and 2f in conjunction with FITC and ethidium bromide, allowing for registration of nonoverlapping fluorescence emission. Control experiments to test for autofluorescence and nonspecific staining due to incomplete washout of the secondary antibody were carried out using nonimmune rabbit and mouse sera. Ethidium bromide staining was performed before mounting by exposing the tissues for 2 min to a 0.1 mg/ml ethidium bromide solution. After further washing with PBS, the preparations were mounted in 90% glycerol containing p-phenylethylenediamine (1 mg/ml) to reduce bleaching of the fluorochromes. Each specimen was mounted on a separate glass slide with individual branches spread out to prevent overlap. The coverslips were raised with custom-made Teflon rings in order to minimize compression of the airways.

View larger version (114K): [in this window] [in a new window]   Figure 2.   Innervation and ASM in the developing airways of a fetal human lung at 58 d of gestation. The field in a through c shows branching epithelial tubules in the periphery of a lobe; d through f show a single optical section taken at high power that is cut lengthwise through a nerve trunk and ganglion to reveal neural tissue, Schwann cells, and nuclei in the same whole mount as a through c. Left panels: (a) Nerves and ganglia stained for PGP 9.5 (green) form a network ensheathing the future airways. (b) Same field, showing the ASM stained for -actin (red), which is arranged circumferentially around the epithelial tubule and lies essentially perpendicular to the long axis of the tubule. (c) Nerve and smooth-muscle images are merged to show that the main nerves form a sheath that lies above the ASM; in some places as far as ~ 40 µm (arrows). Smaller nerve bundles can be discerned (arrowheads) that, at higher power, are seen to descend to the surface of the smooth muscle. Right panels: A single optical section (thickness < 1 µm) cut through a slightly larger, more proximal nerve than shown in a. Images are progressively merged from d to f. The nerve trunk and the adjoining ganglion are stained for: (d) Schwann cells with S-100 (yellow). The S-100 stains the nuclei of the developing Schwann cells as well as the processes extending from their ends. (e) Neural tissue with PGP 9.5 (blue) and Schwann cells with S-100 (yellow). The nuclei of the neurons occupy most of the cell and exhibit nucleoli. ( f ) Nucleic acid with ethidium bromide (red) show the nuclei present in the tissues in the composite picture. Two round nuclei of unknown identity stain up brightly in the nerve trunk.

Confocal Microscopy

Fluorescent images of the nerves and smooth muscle in the double-stained (FITC/RITC) whole mounts were obtained using a confocal laser scanning microscope (MRC 1000; Bio-Rad, Hemel Hempstead, UK) with COMOS software (version 7.0; Bio-Rad). The excitation wavelengths of the krypton/argon laser for FITC and ethidium bromide were 488 nm. RITC and Cy5 were excited at 568 and 645 nm, respectively. Green emission by FITC was recorded with a 522-nm bandpass filter. Red emission by RITC and ethidium bromide was registered with a 585-nm long-pass filter. Far-red emission by Cy5 was recorded with a 685-nm long-pass filter. The whole mounts were optically sectioned by scanning at increasing depths of focus (typically, in steps of 1 µm) to follow the path of the nerves in relation to the smooth muscle. The maximum intensity of the corresponding pixels in each optical section was used to generate a single image (two-dimensional projection) from a stack of images obtained at varying depths. The FITC image of the nerves was merged with that of the smooth muscle (RITC) to form a composite nerve/muscle image. Image processing (merging and montaging fields) was done with Adobe Photoshop 4.0 software (Adobe Systems, Inc., San Jose, CA). In multistaining experiments, single fields were scanned for each marker, then colorized and superimposed. Measurements of cell sizes in ganglia were made from single optical sections through ganglia. Typically, a ×60 objective was used which, in conjunction with the corresponding settings for laser power, iris width, and photomultiplier gain, allowed sections of less than 1 µm thickness to be obtained. All airway diameters refer to the external diameter of the smooth-muscle layer unless stated otherwise. Cell diameters refer to their largest diameter. The distance separating the nerve plexus from the ASM in the airways of the 58-d gestation fetal pig (Figure 2c) was determined from the number of confocal sections separating the nerve trunks and the surface of the ASM. Data are shown as means ± SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Fetal Lung, 7.5 to 8 wk Gestation

Four lungs from 53 to 58 d of gestation were studied. Figure 1a (color plate) shows a lung at 53 d of gestation with well-developed lobes composed of epithelial tubules that appear as delicate, translucent, tubular branching structures, with each tubule terminating in a budlike blind end (Figure 1b) and supported in a loose extracellular matrix of mesenchyme. The growing tips, which comprise a single layer of epithelial cells, are referred to here as terminal sacs. The pan-neuronal marker PGP 9.5 stained nerves in all tissues examined. Control experiments conducted with nonimmune rabbit sera demonstrated no staining. PGP 9.5 revealed a network of nerve trunks overlying the adventitial surface of the tubules, with finer nerves reaching toward the terminal sacs. Many forming ganglia were present at the divisions of nerve bundles.

View larger version (93K): [in this window] [in a new window]   View larger version (116K): [in this window] [in a new window]   Figure 1.   (a) Videomicrograph of a human fetal lung at 53 d gestation. The left upper lobe has become detached from its bronchus. (b) Low-power view of a whole mount of part of the right upper lobe of this lung taken with a confocal microscope. The neural tissue is stained with an antibody to the pan-neuronal marker PGP 9.5 (green), which reveals a network of nerve trunks and forming ganglia. PGP 9.5 also stains the epithelial cells of the terminal sacs at this early stage of development. The ASM stained for -actin (red) is seen more clearly in the periphery where the mesenchyme is thinner (arrows). Smooth-muscle bundles can be seen encircling the tubules in places. A few remaining pulmonary blood vessels are present that also stain with -actin (arrowheads). (c) ASM in the trachea of the human fetal lung at 53 d of gestation stained for -actin (shown in a). The smooth muscle completely encircles the circumference of the trachea, lying perpendicular to the long axis of the trachea. The muscle fibers are tightly packed into bundles, which can just be distinguished by their slight separation in some places around the circumference (arrows). Cartilage rings are absent at this stage of development.

ASM, stained for -actin, forms an essentially continuous layer over these future airways extending to the base of the terminal sacs. These sacs are stained by the PGP 9.5 antibody (6, 8), thus delineating the collar of smooth muscle terminating at their base. The ASM is most clearly revealed in the periphery (Figure 1b, arrow), where the overlying mesenchyme is less dense. Pulmonary vessels that have not been removed during dissection stained with -actin and are seen to follow the distal tubules closely (Figure 1b, arrowheads). ASM completely surrounds the circumference of the trachea (Figure 1c), lying perpendicular to its long axis. The continuity of the smooth muscle was confirmed by also imaging the other side of the trachea. The arrangement of smooth-muscle fibers into bundles (~ 50 µm wide) is typical for mature smooth muscle seen postnatally (M. P. Sparrow and H. W. Mitchell, unpublished data). The cartilage rings are absent at this stage of development.

The relationship of the neural plexus to the ASM in the distal tubules in a 58-d fetal lung is shown in Figures 2a through 2c. A network of nerve trunks with forming ganglia at their intersections covers the future airways (Figure 2a) with a continuous layer of smooth-muscle cells arranged cylindrically around them (Figure 2b). The pitch of the fibers is essentially at right angles to the long axis of the airways, contrasting with data by Ebina and associates (16), whose electron microscopic study indicated a pitch of approximately 30 degrees in adult human airways. The neural tissue appears significantly separated from the airways because of the unstained mesenchymal tissue surrounding the airways that supports the neural sheath (Figure 2c). An estimate of the average distance (along the z-axis; see MATERIALS AND METHODS) of the nerve trunks to the smooth muscle was 19 ± 1.0 µm (n = 27) in three fields of this 58-d fetal lung. In some places this separation appears considerably greater, as much as 40 µm (Figure 2c, arrows), possibly because of compression of the circular airway during mounting. This field is representative of the innervation and ASM in similar preparations from 53-, 54-, and 56-d fetal lungs. Small nerve bundles were observed descending from the trunks toward the smooth-muscle layer (Figure 2c, arrowheads), and at higher power (not shown) these gave rise to some fine varicosed fibers lying in close proximity (< 1 µm) to the smooth-muscle cells.

A developing Schwann cell population was present in the nerve trunks, and to a lesser extent in the ganglia, revealed using the Schwann cell marker S-100 that stains their nuclei and sheaths. Figure 2d shows a nerve trunk and ganglion cut lengthwise by the confocal microscope. This single optical section shows the Schwann cell nuclei with short processes extending from their ends. Some Schwann cell nuclei and fibrillar material are seen in the adjacent ganglion where dark, rounded nuclei of nerve bodies are present. Concurrent staining with the PGP 9.5 antibody reveals the neural cytoplasm in the nerve trunk and in the nerve bodies of the ganglion (Figure 2e). The dark spots within the nuclei appear to be nucleoli when examined at higher power. Staining with ethidium bromide (Figure 2f) shows the nuclei of the nerve bodies in the ganglion, the Schwann cell nuclei, and a host of nuclei of the cells present in the surrounding mesenchyme. The nuclei of the Schwann cells appear orange-yellow because of the color mixing in the composite image of this triple-stained tissue.

Thus, by about the middle of the first trimester the airways are covered with smooth muscle and enveloped by a rich network of developing ganglia and nerve trunks. These trunks contain a population of Schwann cells.

Fetal Lung, 16 to 18 wk Gestation

The innervation and ASM were examined in the segmental airways of lobes from fetal lungs at 16 to 18 wk gestation. The airways were gently separated from the pulmonary vessels and parenchyma. Figure 3 shows the configuration of these airways from a lobe at 16 wk gestation. At the proximal end (arrow), the airway was opened and the bronchial wall imaged from both the adventitial and mucosal surface. Figure 4a shows a representative area of the adventitia where large nerve trunks (45- to 72-µm diameter), bronchial vessels (chiefly arterioles), and mucous glands are present. The -actin stains not only the airway and vascular smooth muscle but also the myoepithelial cells of mucous glands (17). Fine nerves to the acini of the mucous glands can just be detected (arrows) and appear varicosed when viewed at higher power. This projection (Figure 4a) is 120 µm in depth and comprises 31 optical sections. The bundles of ASM, which can be seen faintly, lie at the bottom of the projected volumetric data set. The specimen was mounted on a custom-made double-sided slide and imaged from both the adventitial and luminal aspects. Figure 4b was imaged from the luminal surface to a depth of 145 µm (35 optical sections), where bundles of nerves, separated by ~ 200 µm, run parallel with the long axis of the airway. This distance corresponds to the separation of the mucosal folds that could be seen under the dissecting microscope.

View larger version (101K): [in this window] [in a new window]   Figure 3.   Video image of a segmental bronchus and subsegmental branches from a lobe of a 16-wk-gestation human fetal lung oriented to show the luminal opening of the bronchus (arrow).

View larger version (70K): [in this window] [in a new window]   Figure 4.   The wall of a proximal part of the segmental bronchus (see Figure 3) stained for nerves with PGP 9.5 and smooth muscle with -actin, imaged from both the adventitial (a) and the mucosal (b) surfaces. (a) A representative single field of the adventitial surface showing large nerve trunks with ganglia (green), bronchial vessels (red), and mucous glands (red). These glands contain myoepithelial cells that stain for -actin and are innervated by fine nerve fibers (arrows). Some ASM bundles can be seen but most are obscured by the wall structures and adhering parenchyma. (b) A view of the mucosa from the luminal surface where bundles of intersecting nerves run parallel with the long axis of the airway, with bundles of the ASM lying underneath. A prominent bronchial vessel is visible that is located on the adventitial side of the smooth muscle (localized by confocal sectioning).

A large montage of the nerve tracts in the segmental and subsegmental airways of a lobe from an 18-wk fetal lung is shown in Figure 5a. It measures 11 mm in overall length and 2.2 mm in external diameter at the proximal end, reducing to a mean diameter of 200 ± 11 µm (n = 15) at the distal ends, spanning seven branchings. On the adventitial surface of the more distal airways, smaller trunks, nerve bundles, and interconnected ganglia were present, with a network of finer nerves and small ganglia lying over the ASM. Compared with the 53- to 58-d gestation lungs, the nerve trunks and associated nerve bundles are now more defined and compact, with larger ganglia located proximally in the more mature airways. In general, at least one and more often two major trunks run the length of these airways, with branches giving rise to a plexus of smaller nerves connected by small ganglia lying closer to the ASM. The continuity of the main nerves could be followed to the extremities of the bronchial tree. A large continuous nerve trunk in this segment measured ~ 45 µm at the proximal end, progressively decreasing to ~ 20 µm diameter at the airway extremities. Similar observations were made in the lung of the 16-wk-old fetus described previously. At higher power (not shown), the network of small-diameter fibers that branched from the larger nerves to overlie the smooth muscle was similar to that seen in fetal pigs at the end of the first trimester (6). The thin, varicosed fibers lying on the smooth muscle are not yet aligned with the orientation of the smooth-muscle cells, but connect between larger nerve bundles without an apparent orientation.

View larger version (123K): [in this window] [in a new window]   View larger version (64K): [in this window] [in a new window]   Figure 5.   (a) A composite montage showing the innervation of the adventitial surface of the bronchial tree of a segmental bronchus and its branches from a human fetal lung, 18 wk gestation. The tissue was stained for PGP 9.5 and -actin to show both nerves and smooth muscle. Nerve trunks extend to the most distal airways. Ganglia are present along the trunks and at the divisions of nerve bundles. The inset shows a higher-power projection of a ganglion at the junction of several nerve trunks (arrow). (b) A higher-power view of the straight region on the right-hand side of the montage in a showing the disposition of the nerves, ganglia, ASM, and bronchial arteries. PGP 9.5 (green) stained a plexus of fine nerves containing many small ganglia. -Actin (red) stained both ASM and arterioles of the bronchial circulation that accompany the larger nerve trunks, with some less-distinct vessels branching off to overlie the ASM.

Figure 5b shows a higher-power view of the straight segment of the right branch of the montage (Figure 5a). The bundles of ASM cells are regularly arranged around the circumference of the wall and lie essentially parallel to one another and perpendicular to the airway. The density of the ganglia averaged 70/mm² in this region and their size appeared to be proportional to the diameter of the nerves interconnecting with them. The diameter of the large, spherically shaped ganglia, which lay 20 to 30 µm from the smooth muscle, did not exceed 100 µm. There were many smaller ganglia lying closer to the muscle, some of which were less than 20 µm in diameter. Brightly staining bronchial arterioles ran along the path of the nerve trunks and frequently followed them as they divided. Smaller arterioles branched off to supply the ganglia. Other vessels, less distinct against the brightly staining ASM, branched off to overlie the ASM bundles. Blood vessels were more difficult to detect when the airway diameter decreased below 400 µm diameter because the fluorescent signal from these small vessels was much less intense than that of the large expanse of ASM. However, they could be revealed by examining only those optical sections lying above the ASM, where they appeared as fine, branching arterioles ranging from 10 to 30 µm in diameter.

The large ganglion near the right margin of the main airway was imaged at higher power (Figure 5a, arrow and inset), and optical sections at increasing depth were examined. Figure 6 shows an optical section through this ganglion and individual neurons with cell diameters of ~ 22 µm. Prominent nucleoli were present and appeared as black holes of 2 to 3 µm diameter within the nuclear region of the neurons. The ganglion was estimated to contain approximately 50 nerve bodies. The unstained cell profiles are attributed to glial cells and blood vessels. Schwann cells were seen as unstained, spindle-shaped cell profiles in the nerve trunks (see also Figures 2d and 2e).

View larger version (139K): [in this window] [in a new window]   Figure 6.   A single optical section of the ganglion shown in Figure 5 cut at a depth of 10.5 µm from its surface showing the neurons, some with prominent nucleoli. Some neurons show higher immunoreactivity for PGP 9.5 in the nuclear region. Between the neurons, unstained regions are observed that correspond in size and shape to satellite cells often seen in ganglia. The nerve trunks exhibit unstained cell profiles of Schwann cells (see Figure 2d).

A well-developed pulmonary arterial bed (Figure 7a) was present in the lobes of the lung at 16 wk gestation. These pulmonary arteries accompanied the subsegmental airways (see Figure 3) and comprised a branching structure of smooth-walled vessels. After separating the pulmonary arterial tree from the airways, it was used as a whole mount, stained for vascular smooth muscle and nerves, and imaged with the confocal microscope. Figure 7b shows the distal region of this arterial tree. The outer region of the vessel walls was strongly stained by the -actin, and in the most distal vessels a thin wall with hollow lumen was seen. A brightly staining plexus of fine nerves (< 17-µm diameter), with small ganglia-like swellings present at intervals along the length of the nerve bundles and at their divisions, was observed. In the larger vessels (~ 250-µm diameter), these nerves lay in the surrounding extracellular matrix at a distance of up to ~ 75 µm from the wall. In the most distal vessels (< 60-µm diameter) the nerve fibers lay closer, at a distance of ~ 16 µm. The small ganglia consisted of less than 10 neurons and gave rise to thin bundles of fibers, some of which could be followed to the vascular smooth muscle (Figure 7c).

View larger version (85K): [in this window] [in a new window]   View larger version (115K): [in this window] [in a new window]   Figure 7.   (a) Videomicrograph of a pulmonary arterial tree from a lobe of a 16-wk human fetal lung. The large-diameter central region of the artery is located adjacent to the segmental bronchus shown in Figure 3. (b) A low-power confocal image of a distal region of the pulmonary vascular bed stained for nerves with PGP 9.5 and for the vascular smooth muscle with -actin. The fine nerve bundles are supported by an extracellular matrix. Small ganglia are present along the length of the nerves and at branching points. (c) A higher-power view of the ganglia with a few fine fibers distributing to the arterial wall.

The Postnatal Lung

The airways of postnatal lungs were also examined to allow for a more complete view of the development of the airway innervation. Lungs from two infants who died of SIDS, at ages 5 and 10 mo, respectively, were studied. Figure 8 shows a representative field of a distal airway (1-mm diameter) from the 5-mo-old infant, stained for nerves (Figure 8a) and for smooth muscle (Figure 8b). The field shown was chosen because the rich vascular supply that obscured most of the airway wall was sparse enough here to allow the ASM to be imaged. The composite image showing both nerves and smooth muscle is presented in Figure 8c. Two nerve trunks, showing numerous folds, run along the longitudinal axis of the airway, with smaller nerve bundles and varicosed fibers aligned with the smooth-muscle cells. The loss of sharpness of the fine nerve fibers and the poor definition of the varicosities suggests that some autolysis had occurred during the 22 h that elapsed between death and fixation.

View larger version (100K): [in this window] [in a new window]   Figure 8.   Nerves and smooth muscle in a whole mount of a distal airway (1-mm diameter) from the left upper lung lobe of an infant that died of SIDS. The tissue was stained for nerves with PGP 9.5 (a) and ASM with -actin (b). c shows the composite image of nerves and smooth muscle. (a) Two nerve trunks (only part of the lower one can be seen at the bottom of the field) that show regular folding give rise to smaller nerve bundles and varicosed fibers. (b) Bundles of ASM cells that exhibit partial separation. A large blood vessel with several smaller branches crosses the field from the left-hand side. (c) This composite image shows that the general direction of the varicosed nerve fibers is along the long axis of the ASM bundles.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We have shown here, from early in gestation in the human fetal lung, that ASM covers the branching epithelial tubules that are destined to become the future bronchial tree. In turn, the smooth-muscle layer is ensheathed in a network of nerves and ganglia, and these two tissues persist as an integral part of the airways into postnatal life. This study commenced at 7.5 wk gestation, which corresponds to the end of the embryonic period and, in the lung, the early pseudoglandular stage. The airways of the developing bronchial tree are conventionally described as a layer of epithelial cells that are cuboidal at the growing tips, becoming columnar as the airways enlarge and the wall thickens; that is, maturation occurs centripetally (4, 18). Our findings show that the airway wall is a more complex structure than is generally appreciated at this early stage of development.

The ASM runs continuously from the trachea to the growing tips, where it ends at the base of the terminal sac, the locus of smooth-muscle differentiation from mesenchymal cells that occurs via epithelial-mesenchymal interaction (19, 20). The muscle cells form a cylindrical layer around the airway wall, pitched perpendicularly to its long axis; this orientation is maintained throughout the length of the airways to the base of the terminal sac, and throughout gestation into postnatal life. At the trachea the ASM is closely packed, is multilayered, and forms bundles typically seen in postnatal airways (M. P. Sparrow and H. W. Mitchell, unpublished data), indicative of its greater state of maturity; whereas more distally, the layer of ASM is one to two cells deep (2). The spontaneous narrowing and relaxation of these epithelial tubules in the first trimester in human (1) and in pig (2, 3) fetal lungs indicates that the smooth muscle is functional. It was not possible to ascertain whether smooth muscle is present around the epithelial tubules earlier than 53 d of gestation because of the lack of younger human tissue. However, in embryonic pig lungs of comparatively earlier gestational age, ASM is present in the trachea (M. Weichselbaum and M. P. Sparrow, unpublished data). Postnatally, in the distal airways of SIDS lungs, the ASM was present as bundles, many of which were partially separated from each other, a feature frequently seen in excised lungs inflated to 25 cm H₂O before fixation. This separation could be caused in part by lengthening of the airways due to inflation, and by the dissection procedure needed to separate the airways from the surrounding structures. The varicosed nerve fibers overlying the ASM were typically oriented along the muscle bundles, as seen in distal porcine airways (6).

Neural tissue invested the epithelial tubules, forming a rich network of ganglia interconnected by nerve trunks at 7.5 to 8 wk gestation. These ganglia (or, rather, ganglia precursors) appeared as thickenings or swellings of the nerve trunks. The neural plexus covering the airway wall gave rise to smaller bundles that descended toward the ASM (Figure 2c). The large nerve trunks stained with PGP 9.5 did not appear smooth but exhibited many cellular profiles that were shown to be due to the Schwann cells, as revealed using the Schwann cell marker S-100. Some Schwann cell processes were also present in the ganglia. The neurons of the ganglia appeared spherical in shape, with a diameter of ~ 8.5 µm, and contained one or more nucleoli in their nuclei.

By 16 to 18 wk the nerve trunks were more compact and severalfold larger in diameter. Ganglia also increased in size and became more spherical in shape. Smaller nerve bundles divided into fine varicosed fibers overlying the surface of the smooth-muscle layer, as seen in the fetal pig lung (6). Direct contact of these fibers with the smooth-muscle cells cannot be established by means of confocal microscopy, but requires the resolving power of electron microscopy. With ongoing maturation, the orientation of the thin, varicosed fibers was increasingly aligned to the smooth-muscle cells. The arrangement of the nerve cells in the ganglia, with their axons pointing toward the center of the ganglion, gave the appearance of spherical neurons at the surface of the ganglion (Figure 5a, inset). However, in cross-sections (Figure 6), the less regular shape of the neurons became apparent and axon hillocks were identifiable.

In adult airways, ganglia are considered to be relatively few and primarily associated with the extrapulmonary airways, with occasional small ganglia present at bifurcations of intrapulmonary bronchi (21). We found a large number of ganglia in the segmental and subsegmental airways at the canalicular stage (16 wk onward), and we predict that this complement of ganglia persists into postnatal life and adulthood. The reported infrequency with which ganglia are detected in postnatal airways is likely to be methodological. The chance of finding them in cross-sections by conventional histology is low because of the great increase in airway surface area during growth that results in a substantial separation of the ganglia.

The function of the smooth muscle in the fetal lung in early gestation is proposed to provide tone to maintain the lung liquid at a positive pressure to stimulate lung growth (2, 3). This pressure has been estimated to be about 2.4 to 2.7 cm H₂O in third-trimester fetal sheep (22), 1.4 to 2.9 cm H₂O in fetal rabbits (M. P. Sparrow and G. Miserocchi, unpublished data), and 1.4 to 4.2 cm H₂O in cultured fetal mouse lungs (23). This force, generated by the ASM acting across the airway wall and adjacent parenchyma, may stimulate lung growth by mechanically induced expression of growth factors from early in gestation. Increasing the intraluminal pressure by obstructing the trachea in the late-term fetal sheep increases the production of insulin-like growth factor II and causes lung hyperplasia (24). The spontaneous narrowing of the airway wall of the fetal lung that has been demonstrated in isolated human and pig fetal lung (1, 2) would confer the additional advantage of a pulsatile stimulus that is more effective than a static one in stimulating lung growth in vitro (25, 26).

The function of the neural tissue, particularly in early gestation, is unlikely to be in neurotransmission but may contribute trophically to the development of the lung. Nerves are currently viewed as secreting trophic factors that influence the growth and survival of their developing target organs (27)ASM and mucous glands, in this instance. In turn, the target organs secrete neurotrophins that maintain survival of the nerves and may even influence the phenotypic expression of the neurotransmitters secreted during development (28). The close association of the neural plexus and the ASM throughout development of the lung makes a trophic role for the nerves seem possible.