THE ANATOMICAL RECORD 243:63-70 (1995) Three-Dimensional Organization of the Elastic Fibres in the Rat Lung CARLOS ALBERT0 GONCALVES, MARIA HELENA FIGUEIREDO, AND VASCO ANTONIO BAIROS Znstitute of Histology and Embryology, Faculty of Medicine, University of Coimbra, Coimbra Codex, Portugal ABSTRACT Background: The elastic framework of the distal lung has been studied by light microscopy (LM) and transmission electron microscopy (TEM). The preservation of the elastic fibres, for the three-dimensional observation in their relative positions, is difficult because they lack support when the normal methods of tissue processing are used. The goal of the present study was to understand the three-dimensional ultrastructure and organization of the elastic fibres of the lung preserved in their relative positions. Methods: A combination of intravascular resin injection and formic acid digestion was used. The resin cast of the microvasculature acted as a scaffold to preserve the in vivo arrangement of the elastic fibres that are, otherwise, easily collapsible. Scanning electron microscopy (SEM) samples were further processed for TEM in order to confirm that the fibres were indeed components of the elastic system. Results: SEM demonstrated a fine framework of elastic fibres, representing remnants of the alveolar walls, with the casted capillaries interwoven with the network of elastin. Each individual elastic fibre is composed of a small bundle of discrete fibrils. Some of these fibrils emerge from the fibre and join other fibres, producing an anastomosing appearance. Several elastic fibres link the walls of the intrapulmonary conducting airways, the vessels walls and the alveolar network, thus establishing an interrelated and interlaced framework. Conclusions: The method we have applied to visualize the elastic fibres of the lung is a unique approach to define the spatial organization of the pulmonary elastic fibres. We have demonstrated here the close relationship between the elastic fibres and the capillaries of the septa1 alveoli. The arrangement of the interwoven network of elastin and its relationship with the capillaries offers the structural setting for the distending capacity of the alveolar wall. o 1995 Wiley-Liss, Inc. Key words: Scanning electron microscopy, Corrosion casts, Lung, Elastic fibres, Elastin The dynamics of the respiratory function requires the existence of a well-developed elastic system in the lung. The major role of the elastic components of the lung is in the recoil of the organ as a unit. During the process of the lung development the genesis of elastic fibres is essential to the process of alveolization (Das, 1980; Bairos, 1986; Foster and Curtiss, 1990). Elastic fibres are present in the interalveolar septa, in the walls of the pulmonary conducting airways, and in the lung vessles, as it has been demonstrated by light microscopy (LM) (Loosli and Potter, 1959) and transmission electron microscopy (TEM) (Collet and Des Biens, 1974). Scanning electron microscopy (SEM) studies on isolated elastin from bovine ligamentum nucae were first published by Gotte et al. (1972). SEM studies were also 0 1995 WILEY-LISS, INC performed by Kadar (1977), before and after enzyme treatment of elastin purified by different methods using the bovine ligamentum nuchae and the human aorta as the main source of the fibres. A first SEM report on the lung alveolar elastic network was published by Wang and Ying (1977), showing disruption of coiled ends of elastic fibrils at the margins of the alveoli. Our goal in this study was to investigate the spatial Received November 1, 1994; accepted March 6, 1995. Address reprint requests to Dr. Vasco Antdnio Bairos, Institute of Histology and Embryology, Faculty of Medicine, University of Coimbra, 3049 Coimbra Codex, Portugal. 64 GONCALVES ET AL. organization of the rat lung elastic system, with minimal disruption or coiling of the fibres. For that we developed a combination of intravascular resin injection and formic acid treatment that was introduced by Imayama and Bravernan (1988) to study the superficial fascia of the rat. A major technical advantage of the casting of the microvasculature was to work as a scaffold to preserve the in vivo arrangement of the easily collapsible elastic tissue. The casting procedure uses perfusion fixation and injection of resin prior to digestive treatment, thus avoiding the deformation of the resin framework that occurs when the elastic fibres are isolated using autoclaving processes (Tsuji et al., 1979; Crissman and Guilford, 1984). We used here SEM to study the blocks of resin casts with the preserved elastic fibres. We also employed TEM of the same samples to confirm the elastic nature of the fibres. Correlative observations by LM and TEM were performed after the cytochemical detection of the elastic fibres. MATERIAL AND METHODS Animals Male Wistar rats, 6 weeks of age and weighing 200250 g, were obtained from the animal house of the Gulbenkian Foundation Institute of Science (Oeiras, Portugal). The rats were anaesthetized with sodium pentobarbital (30 mg/kg, intraperitoneally), and perfusion was performed through a cannula inserted into the right ventricle. Transmission Electron Microscopy (TEM) Complementary morphological studies were performed using LM and TEM. The animals were fixed by perfusion with a 4% formaldehyde in 0.1 M phosphatebuffered solution (pH 7.4) containing 0.5%glutaraldehyde and the lung pieces were then immersed in the same fixative for 2 hours a t 4°C. The material for the TEM study was postfixed for 1 hour in a solution of 1%osmium tetroxide in 0.1 M cacodylate buffer and embedded in an epoxy resin. Grids with thin sections were rinsed in 70% ethanol solution and immersed for 1 hour in 0.15% orcein in 70% ethanol containing 0.6 ml of concentrated HC1 for each 100 ml of the solution (Nakamura et al., 1977). The solution was ultrafiltrated before use. After staining, each grid was washed three times in 70% ethanol and in ultra-pure water, and counterstained with uranyl acetatellead citrate. For LM, lung samples were paraffin-embedded by standard methods and 3 pm sections were stained using the Veroheff technique for elastic fibres. The same samples previously submitted to the SEM study were embedded in an epoxy resin and thin sections (with and without the orcein staining) were studied by TEM, in order to confirm the presence of the elastic fibres and the surface morphology observed under the SEM. RESULTS The epithelial cells started to peel off from the alveolar surface 2 days after the beginning of the extraction procedure; at day 3, collagen fibres had a swollen appearance, while a lot of cellular debris could be seen attached to the vascular resin casts. Scanning Electron Microscopy (SEM) After 6 days of extraction procedure, the elastic fiThe trachea was cannulated and the lungs instilled bres were the only remaining fibrous structure, due to with an aldehyde mixture of 4% formaldehyde, 0.5% the hot hydrolitic action of the formic acid at 88% and glutaraldehyde in a 0.1M phosphate buffer, pH 7.4, confirmed by TEM observations of the same material with a pressure corresponding to a 20-cm high column. as presented in Figure 11. Then we selected day 6 for The pulmonary vascular system was flushed all the observations. The elastic fibres maintained through the cannula inserted into the right ventricle their three-dimensional arrangement due to the miwith heparinized saline serum (350 I.U. heparid10 ml crovascular support of the vascular cast, thus allowing solution) at 37°C for 2 minutes and the aorta was cut to a spatial view by SEM (Fig. 1). exsanguinate the animal. The lungs were perfusionThe blood alveolar vessels and their organization folfixed using the same cannula with 4% formaldehyde in lowed the architectural structure of the alveoli, which 0.1M phosphate buffer, pH 7.4, for 3-5 minutes, fol- is recognizable in normal lungs by its characteristic lowed by the injection of a methacrylate resin (Mer- repetitive, basketlike morphology (Ohtani, 1980; Peso cox@ C1-2B, blue color; Japan Vilene Co., Tokyo, Ja- et al., 1994). This aspect could be recognized in our pan), until the surface of the lung turned blue. samples (Figs. 1and 4). The cast vessels were the main After the resin had polymerised for 15 minutes, the structure that sustained the elastic network. lungs were removed and immersed in the same fixative A fine framework of elastic fibres representing remat 4°C for 4 days. After fixation, pieces of lung tissue nants of the alveolar walls, with capillaries interwoven measuring 3 x 3 x 3 mm were collected using a sharp with the network of elastin, was clearly observed (Figs. scalpel. These tissue blocks were then incubated in 2-4). 88% formic acid solution a t 45°C and daily processed Interestingly, some elastic fibres linked the walls of from day 2 to day 8. large vessels with the alveolar elastic network, thus After the different times of incubation, the samples establishing an interrelated and interlaced framework were washed in 0.002 N HC1 for 5 hours and then im- (Fig. 5). mersed in 2% tannic acid solution for 30 minutes. The At the boundary of three or more confluent alveoli, specimens were subsequently rinsed in water, post- large bundles of elastic fibres, 1-3 pm in diameter, were fixed for 1 hour in 2% osmium tetroxide in water, de- seen connected by a fine network of fibrils. They often hydrated in acetone, critical point dried, sputter-coated formed a triangular cribriform veil with the small with gold, and examined in a JEOL T330 scanning fibrils running in different directions and establishing electron microscope (SEMI. anastomoses among themselves (Figs. 4 and 6). The ELASTIC FIBRES IN THE RAT LUNG Fig. 1. Low magnification scanning electron micrograph of the elastic framework of the rat lung supported by a microvascular cast. Elastic fibres maintain the alveolar walls configuration. C, capillary. Bar = 100 pm. Fig. 2. Scanning electron micrograph of an alveolar duct showing the interweaving of capillary vessels with elastic fibres. Bar = 50 pm. 65 66 GONCALVES ET AL. Fig. 3.Elastic fibres running through the walls of different alveoli, establishing a n interlaced network. C , capillary. Bar = 10 km. Fig. 4. Scanning electron micrograph of the basket-like aspect of the alveoli, whose walls are limited by the resin cast of the capillaries and the elastic fibres. C, capillary. Bar = 10 km. ELASTIC FIBRES IN THE RAT LUNG Fig. 5. Scanning electron micrograph of elastic fibres (arrows) linking a vascular wall with some neighbouring alveoli. C, capillary; V, vessel. Bar = 10 pm. Fig. 6. A triangular cribriform veil of elastic fibrils at the boundary of three or more contiguous alveoli. = 10 Fm. Bar Fig. 7. Light micrograph of a Veroeff stained paraffin section of lung tissue, showing a similar aspect to the triangular cribriform veil seen in the scanning electron micrograph of Figure 6 . Bar = 100 pm. 67 68 GONCALVES ET AL. Fig. 8. Scanning electron micrograph of a bundle of anastomosing elastic fibrils around a capillary vessel. Bar = 5 km. Fig. 9. Successive branching of an elastic fibre with the emergent fibrils joining other major elastic fibres, giving an anastomosing aspect. Bar = 5 km. 69 ELASTIC FIBRES IN THE RAT LUNG Fig. 10. Transmission electron micrograph of the normal histological structure of the tip of a n alveolar septum. The elastic fibres are close to an interstitial cell of the lung. E, elastic fibres; I, interstitial cell. Bar = 1 pm. surface of the elastic fibres represent the gold particles of the gold coating used for the scanning electron microscopic observation. The interface of the two resins, Mercox and epoxy is indicated by a black line of gold particles (arrows) from the gold coating process. E, elastic fibres; Er, epoxy resin; Mr, Mercox resin. Bar = 0.1 pm. Fig. 11. Transmission electron micrograph of a sample previously observed by scanning electron microscopy. The fine black dots on the anastomosis of the fine fibres defines a delicate network with a mesh of round holes. These structures are frequently observed in the wall of various contiguous alveoli. This aspect was also noticeable in the images obtained by LM with the conventional Veroeff staining method for elastin (Fig. 7). Closeup views of SEM revealed that the elastic fibres were made up of small bundles of fine fibrils, approximately 100 nm in diameter (Figs. 8 and 9). Some of these fibrils, sprouting from the fibre, established a direct connection with other fibres, thus producing an anastomosing appearance (Fig. 8). The number of fibrils form the main reason for increased or decreased diameters observed in different elastic fibres; some of the fibres were reduced in size because of repetitive branching along its major axes with the emerging fibrils joining other fibres (Fig. 9). The fabrillar substructure of the elastic fibres was clearly perceptible even in large bundles, where sets of parallel fibrils could be seen a t the surface (Fig. 9). TEM study showed that elastic fibres of the alveolar walls, mainly composed by the amorphous component, are localized at the tip of the septa, near the connective intersticial lung cells (Fig. 10). After SEM observation, the same samples were processed for TEM. The thin sections showed a total absence of collagen fibres in every sample observed). The presence of elastic fibres and the MercoxB resin casts of alveolar vessels was observed wrapped in a contin- uous line of fine black dots, corresponding to the gold coating process used for the SEM study (Figs. 11). The interface between the acrilic resin representing the blood vessels and the epoxilic resin used in the embeding process is also represented by a fine, black line of gold particles from the coating procedure (Fig. 11). The microfibrillar component of the elastic fibres is not observable on the periphery of the amorphous component of the elastic fibres, probably due to its hydrolisis by the incubation in formic acid. DISCUSSION We have investigated here the three-dimensional arrangement of the elastic fibres at the level of the lung alveolar septa. The combination of resin vascular injection and formic acid corrosion proved to be an elective method to study the elastic network of the lung in its actual topography. The instillation of fixative through the trachea a t a pressure corresponding to a 20-cm high column (Gil, 1971) prevented the collapse of the lungs and allowed the fixation of the elastic fibres in their physiologic distensible state that is at the end of the inspiration. The artificial shrinkage of the elastic material fixed with aldehydes, which occurs with the critical point drying (Wasano and Yamamoto, 19831, could be substantially minimized by tannic acid (Imayma and Braverman, 1988). The scaffold provided by the resin vascular casts 70 GONCALVES ET AL. maintained the elastic fibres in its natural spatial arrangement, avoiding the curling and kinking that occurs when the elastic fibres lack support (Crissman, 1986). The unusually large spaces with disrupted or coiled ends of elastic fibrils described by Wang and Ying (1977) in the lung were not seen in the present work, because we had provided the elastic network with the supporting structure offered by the resin vascular cast. This greatly reduced the loss of elastic material during the process of corrosion. With the TEM studies carried out on the same samples used for the SEM investigation we could confirm that the wire-like structures stretched between the alveolar vessels are indeed elastic fibres. The methodology is a useful tool to investigate the spatial organization of the pulmonary elastic fibres under normal or pathological conditions. ACKNOWLEDGMENTS We thank Professor Nuno Grande (Department of Anatomy, Abel Salazar Institute for the Biomedical Sciences, University of Porto) for his support throughcyt this study. We are also grateful to Professor Artur Aguas (Department of Anatomy, Abel Salazar Institute for the Biomedical Sciences, University of Porto) for his critical review of the manuscript. LITERATURE CITED Bairos, V. 1986 Studies on the morphogenesis, histogenesis and histophysiology of the lung. Ph.D. Thesis, University of Coimbra, Portugal, pp. 227-233. Collet, A.J. and G. Des Biens 1974 Fine structure of myogenesis and elastogenesis in the developing rat lung. Anat. Rec., 1974:343359. Crissman, R.S. and W. Guilford 1984 The three-dimensional architecture of the elastic-fiber network in canine hepatic portal system. Am. J. Anat., 171:401-413. Crissman, R.S. 1986 SEM observations of the elastic network in canine femoral artery. Am. J . Anat., 175:481-492. 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