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The microanatomical alterations of the surface of lung Clara cells were studied during secretion. Stimulation of Clara cells was induced in rats by chronic inflammation caused by a single intratracheal instillation of bleomycin performed 2.5 months before the animals were killed. Bleomycin treatment resulted in marked stimulation of secretion by the bronchiolar Clara cells, 31% of the Clara cells from the treated rats showing signs of active secretion whereas only 6.3% of Clara cells in control rats presented similar features. High-resolution views of lung airways were obtained by scanning electron microscopy of critical point dried tissue samples. The surface of Clara cells underwent several modifications associated with the secretory events. These alterations followed 3 major phases: (1) formation of a smooth apical dome made up of a large volume of cytoplasm; (2) progressive narrowing of this dome-like body at its base with the formation of a cap-like structure; (3) in toto release of the cytoplasmic cap-like body. In favourable views, thin pedicles linking the cap-like bodies to the remaining cytoplasm of the Clara cell were detected. In other instances, release of the cap-like body occurred without the previous formation of stalks. Secretion of intracellular granules was observed in some cells before severance of the cap-like body. It is concluded that: (1) the cap-like bodies are not artifactual features of Clara cells; (2) Clara cell secretion is both apocrine and merocrine, the former predominating; (3) chronic inflammation is associated with an increased formation and release of the secretory cap-like bodies by Clara cells.

The lymphatic microvessels of the deep lung tissue were studied in corrosion casts, which were observed by scanning electron microscopy (SEM) after injection of a methacrylate resin (Mercox) through the trachea of CD-1 mice. We found that the deep lymphatics of the murine lung were composed of two interconnecting networks: a poorly developed capillary system located at the interacinar region, and a rich plexiform complex surrounding the bronchus. Lymphatic capillaries did not penetrate the alveolar area, thus leaving most of the lung parenchyma devoid of direct access to lymphatic drainage. Lung lymphatic vessels showed a small luminal surface and a low density of endothelial nuclei. Pulmonary lymphatic capillaries often formed star-like anastomoses. The structural features of pulmonary lymphatics, including their three-dimensional organization, were distinctly separate from those of the blood microvasculature of the lung.

We have investigated the topography of particle-laden macrophages in the pulmonary tissue of CD-1 mice after intratracheal instillation of a suspension of 250 micrograms of calcium tungstate. The mice were sacrificed 1, 3, 7 and 14 days after the particle deposition. Lung fragments were studied by scanning electron microscopy (SEM) coupled with X-ray microanalysis that allowed in situ elemental identification of tungsten in the lungs. Tungsten-positive macrophages were distinctly located in the lungs of mice sacrificed at 1-3 days when compared with samples from mice killed 7-14 days after the calcium tungstate instillation. At 1-3 days, the tungsten-carrying macrophages were accumulated near the terminal bronchioles whereas they were seen predominantly in the alveolar ducts and sacs in the 7- to 14-day groups of mice. This suggests that during pulmonary inflammation there is a redistribution of the particle-containing macrophages throughout the deep lung tissue. In high-magnification SEM views, we observed that the tungsten-positive macrophages presented numerous surface microvilli. Tungsten-laden phagocytes were detected in interalveolar fenestrae, at the so-called Kohn pores. This finding documents that the Kohn pores may be used by inflammatory cells as a pathway for the migration of phagocytes in between adjacent alveolar sacs.

Tungsten has been implicated as a cause of a severe form of pneumoconiosis in humans, the so-called "hard metal" lung disease. We have investigated the effect of intratracheal instillation of a powder of calcium tungstate on the pulmonary tissue of CD-1 mice. The tungsten-induced alterations were studied using 3 microanatomical methods: cytologic study of exudates obtained by bronchoalveolar lavage (BAL); histologic examination of paraffin-embedded sections of the lung; and scanning electron microscopic (SEM) examination of lung samples using x-ray microanalysis to detect tungsten in situ. The animals were sacrificed 1, 3, 7, 14 and 21 days after a single intratracheal instillation of 250 micrograms calcium tungstate particles suspended in 100 microliters of saline. We found that the metal particles induced a marked inflammatory response in the bronchoalveolar space characterized by a biphasic attraction of leukocytes with cellular peaks observed at day 1 and 14. More than 50% of the BAL macrophages showed ingested tungsten. In the lung parenchyma, the inflammatory infiltrates were predominantly located at the periphery of the bronchiolar walls. From 7 days on after the tungsten deposition, large inflammatory exudates were seen invading focal areas of the alveolar domain of the lung. SEM views revealed that the tungsten particles could be inside alveolar macrophages, in cells making up the alveolar wall, or inside periacinar lymphatics. Our data document that tungsten particles cause a marked inflammatory response in the lung tissue and that the leukocyte exudates may invade alveolar areas of the lung.(ABSTRACT TRUNCATED AT 250 WORDS)

CD-1 mice were used to study the cellular kinetics of the inflammatory response of the pleural space to the injection of 250 micrograms of silica or of tungsten microparticles. The pleural exudates were collected by lavage of the serous cavity of mice that were sacrificed at 30 min and up to 7 days after the intrapleural instillation of the particles. The samples were studied by light and electron microscopy (transmission and scanning modes); the quantitative cellular kinetics of the inflammation was determined by leukocyte counting in exudates using cytocentrifuge preparations. The normal resident population of cells of CD-1 mice was made up of (2.47 +/- 0.37) x 10(6) cells. It consisted mostly of macrophage-like cells ((2.03 +/- 0.26) x 10(6) cells, 82% of total cells), some lymphocytes ((0.37 +/- 0.07) x 10(6) cells, 15% of total cells), a few mast cells and eosinophilic granulocytes (1-2% of total cells). The initial inflammatory reaction (30-60 min after injection) was characterized by a decrease in the number of cells harvested from the pleural space. This was followed by an intense recruitment of granulocytes and monocytes that resulted in a peak of intrapleural cells at 24 h ((16.8 +/- 4.0) x 10(6) cells induced by silica particles and (18.3 +/- 4.2) x 10(6) cells induced by tungsten particles). In tungsten-injected mice (but not in silica-treated animals) the enhancement in the number of intrapleural macrophages continued up to 72 h after particle injection. The highest percentage of macrophages with ingested tungsten (50% of total macrophages) was found early (6 h) and decreased thereafter; at day 7 it encompassed just 17% of the macrophages. Injection of any of the two particulates led to the disappearance of mast cells from the pleural space of mice. Silica particles attracted a high number of eosinophils to the pleural cavity of mice. Light and electron microscopy documented that pleural macrophages underwent striking morphological changes during the inflammatory response: the phagocytes showed marked increase in size and in number of surface processes, and their cytoplasm often contained large amounts of the injected particles and also of cellular debris. This study establishes the mouse as a reliable animal model to study the dynamics of the pleural space and it offers a precise definition of the cellular kinetics of inflammation in this serous cavity. The data indicate that the kinetics of experimental pleural inflammation induced by particulates may depend on the nature of the injected particles.

We instilled tungsten powder (CaWO4) into the pleural space of the dog and studied the kinetics and distribution of particle translocation from the pleural space to the thoracic lymph nodes over 1-7 days. We found that the transport of tungsten particles to regional lymph nodes was present at day 1, and reached its peak at day 3. In situ detection of tungsten by elemental particle analysis of lymph node sections by scanning electron microscopy complemented by light microscopy and X-ray analysis allowed precise mapping of the marker in the thoracic nodes. The first lymph nodes to become tungsten-laden was the parasternal group (day 1-3). From day 3 to 7 tungsten inclusions decreased in these parasternal nodes while moderately increasing in the remaining intrathoracic lymph nodes. Retrocardiac pleural folds containing numerous "milky spots" also accumulated prominent amounts of tungsten early after intrapleural injection of CaWO4. These data indicate that 1) particle translocation from the pleural space to regional lymph nodes is a rapid process and is first directed to the parasternal lymph nodal subgroup; 2) particle dissemination to virtually all other lymph nodes within the thorax follows thereafter; 3) retrocardiac pleural folds contribute to the clearance of particles from the pleural space.

We studied the anatomical features of the arterial vasculature of the lymphoid follicles (LF) of tracheobronchial lymph nodes in the dog. The microvascular organization of the lymph node was visualized after systemic perfusion of the canine arterial system with gelatinated colloidal carbon. We found that LF contained a substantial number of blood capillaries forming a vascular network with a density comparable to that of neighboring domains of the node. The density of the vascular supply was greater in the secondary than in the primary LF. The geometric arrangement of LF capillaries was also different in the two types of LF. These findings are in contrast with the current view that the LF is a vascular-poor domain of the lymph node. We also document the plasticity of the LF blood microvasculature that appears to proliferate in conjunction with lymphoblast turnover and immune reactivity characteristic of secondary LF.

Cytocentrifugation of cell suspensions onto glass slides is a widely used procedure in contemporary cytology. We employed here scanning electron microscopy (SEM) to investigate putative morphological changes induced in cells submitted to cytocentrifugation. The fine structure of murine pleural exudate cells (macrophages mainly) processed by spinning was compared with that of similar cells treated without centrifugation (poly-L-lysine attachment of the cells to glass slides at 1 g). Cells of cytocentrifuged preparations showed a significant increase in diameter and smoothening of the cell surface as compared with the morphology of non-centrifuged cells. Cytocentrifugation also induced the formation of thin elongations coming out of the cellular outlines. The centrifugation-induced flattening of the pleural macrophages improved the detection of large intracellular inclusions (containing tungsten particles): these bodies were readily identified by secondary-electron imaging mode of SEM in cytospinned cells whereas their detection in non-centrifuged spherical cells required the use of the backscattered-electron imaging mode of SEM. We conclude that the cytocentrifugation methodology, on one hand, requires caution on the interpretation of the microanatomy of the cells and, on the other hand, the procedure may be an adequate method to improve the identification of large intracellular inclusions by routine (secondary-electron imaging mode) SEM.

Studies of pleural clearance of calcium tungstate particles were made in the dog. By using scanning electron microscopy and elemental microanalysis, we show that mesothelial cells of the visceral leaflet of the pleura are also involved in the clearance of particles present in the pleural space. The histological study of lung parenchyma shows many macrophages loaded with tungsten particles, and we conclude that this way may be an important pathway for the transmission of pathologic processes from the pleural space to the lung.

Scanning electron microscopy was used to investigate the submicroscopic organization of the blood vessels of dog lymph nodes (LN). The LN vessels were casted by systemic perfusion of the animals vasculature with two resins of distinct viscosity. This resulted in the retrieval of two types of LN vascular replicas that depicted either the arterial blood system alone (methacrylate casting) or both the arterial and venous blood systems (Mercox casting). We found that the dog LN showed a significantly higher density of arterial vessels in the cortex than in the medulla. In the cortical domain, a subcapsular layer stood out because of its rich content in arterial capillaries. The use of the high-resolution resin (mercox) resulted in excellent structural detail of the luminal surface of the LN vessels that allowed a clear distinction between arterioles and venules based on the geometrical pattern of the imprints left in the replicas by the nuclei of endothelial cells. At the cortex-medulla frontier, most arterioles showed narrowings of their lumen that suggested the existence of sphincters at this level. Our findings document that the microanatomical arrangement of blood vessels in the LN of the dog is different from that of LN from other mammals studied so far, in particular from rodents where vascular-poor microdomains have been reported in the LN cortex. The arteriolar sphincters that we detected at the innermost zone of the cortex may represent the structural counterpart of the physiological modulation of the blood supply of the cortex exerted by arterial branches coming from the hilus.

The organization of the arterial vessels of dog lymph nodes (LN) was studied using methods of visualization of the vasculature by systemic injection of different tracers (colloidal carbon, Micropaque resin and methylmethacrylate) followed by observation of the samples by light microscopy (after clearing of the thick sections of LN) or scanning electron microscopy (corrosion casts). LN from all of the three groups of nodes studied (tracheobronchial, paratracheal and popliteal) showed an extensive network of arterial vessels encircling the capsule of the organ. We found that branches of these capsular arteries penetrated deeply into the cortical domain of LN. The capsule-originating vessels appeared to have a significant participation in the blood supply of the LN parenchyma at the cortical domains of the organs. Our findings are in contrast with current views on the angiology of the LN that consider that virtually all of the arterial capillaries of the LN parenchyma come from hilar arteries. We propose, therefore, that important segments of the LN cortex receive their blood supply from capsular arteries rather than from hilar vessels.

We have studied the subcellular distribution of ferritin in inflammatory macrophages present in regional lymph nodes from dogs subjected to a pulmonary inflammatory reaction. The inflammatory reaction was induced by intrabronchial instillation of calcium tungstate (CaWO4), a water-insoluble powder. Ferritin was identified by electron microscopy, and its electron density was enhanced by the use of a modified Perls method. From day 14 on after the CaWO4 deposition, tungsten-positive lymph node macrophages showed a massive accumulation of ferritin. Most of the ferritin was stored in membrane-bounded vesicles that showed heterogeneous concentrations of the protein. A significant complement of ferritin was also detected in the cytoplasmic ground substance of phagocytes. The cell surface of the ferritin-rich, tungsten-positive macrophages showed deep infoldings that encompassed small pockets of connective tissue fibers. These features were not observed in control samples or in lymph nodes from dogs subjected to CaWO4-induced inflammation for periods shorter than 1 week. Our data indicate that inflammatory macrophages greatly increase their content of ferritin macrophages greatly increased their content of ferritin and that ferritin is stored predominantly by a membrane-bounded vesicular compartment. This is in contrast with suggestions that the inflammation-induced increase in macrophage iron is restricted to the labile pool of iron and it does not involve the iron bound to ferritin molecules. Our observation of nodules of connective-tissue fibers in intimate topographical association with ferritin-rich macrophages may indicate that the increase in intracellular ferritin in the macrophage is in some way related to the secretion of factors by the phagocyte that will stimulate fibrillogenesis by neighboring fibroblats.

We sprayed a tungsten powder (CaWO4) into the airway of a single lobe (left apical) of the dog lung in order to study: (a) the kinetics of particle translocation from the bronchoalveolar lining to hilar lymph nodes, and (b) the sorting in lung lymph nodes of inhaled microcrystals. We found that the transport of the tungsten particles to the regional lymph node takes at least 24 hours and reaches its peak at day 7. In situ detection of tungsten by elemental particle analysis of lymph node sections by scanning electron microscopy allowed precise mapping of the marker in the node; the method was complemented by light microscopy and thin-section electron microscopy of the same nodes. Virtually all of the lymph node tungsten was located inside macrophages. The first tungsten-positive macrophages seen in the regional lymph nodes (day 1 to day 3) were restricted to the subcapsular space. This was followed by massive filling of the same sinus and of the narrow interfollicular areas by the particle-laden macrophages (day 3 to day 7). The even distribution of the tungsten-bearing phagocytes found in these anatomical regions of the node indicated that the subcapsular area in the dog was a continuous domain rather than the segmented region observed in nodes of common laboratory animals such as the rat. By day 7 after tungsten instillation, a moderate number of tungsten-positive macrophages was also detected in the paracortical region of the node. Finally, the presence of tungsten-bearing macrophages was extended to the outer lymph node medulla (day 7 to day 14); here, the macrophages were located in association with cords of plasmacytes and showed interdigitations with these lymphocytes. Only minimal amounts of tungsten were detected inside lymphoid follicles in association with dendritic cells. Some of the tungsten initially deposited in the airway of the apical left lung lobe was detected in contralateral hilar lymph nodes. We conclude that: (i) particle translocation from the alveolus to regional lymph nodes is a slow process that is mediated by pulmonary macrophages, in agreement with the findings of Harmsen et al Science 230:1277, 1985); (ii) in the lymph node, particle-bearing macrophages are sorted through narrow interfollicular sinuses into the outer medulla where they interact extensively with plasma cells; (iii) the migrating macrophages cannot penetrate the follicular domains of the node; minute quantities of exogenous particles may, nevertheless, be transferred from macrophages to follicular dendritic cells; and (iv) contralateral drainage may be a feature of the lymphatic system in the lung.

Left lobe lung biopsies were obtained in dogs allowed to breathe in atmospheres of coal or barium sulphate dust before ligature of the left pulmonary veins and at 15 min, 1, 5, 7, 15, and 30 days after ligature. All samples were prepared for transmission electron microscopy (TEM) and scanning electron microscopy (SEM); in addition, samples from the barium sulphate group were examined by electron diffraction analysis (EDA). Two main phagocytic cell types were identified. In addition lymphatic capillary-like structures were observed in the alveolar septum after ligature of the pulmonary veins.

The AA describe a hypoplastic right ventricular chamber of the heart obtained by autopsy and belonging to a man who died of traumatic lesions and paraplegia. This malformation is very rare and the authors attribute to it the small size of the patient who was below the average Portuguese male height.

The authors studied the anatomical structures of the pulmonary lymphatic vessels in the dog and in man using injection techniques and macro- and microangiographic and histological methods. The results show that injection in the basal lobes is easier than in the apical lobes. The dog, an animal with a thin pleura, has a very dense superficial lymphatic network, like man. The superficial and the deep lymphatic vessels are connected through the pleural septa.