Identification of Foreign Particles in Human Tissues Using Raman Microscopy.

The goal of this study was to precisely and unambiguously identify foreign particles in human tissues using a combination of polarized light microscopy and Raman microscopy, which provides chemical composition and microstructural characterization of complex materials with submicrometer spatial resolution. This identification for patient care and research has been traditionally studied using polarized light microscopy, electron microscopy with X-ray analysis, and electron diffraction, all with some limitations. We designed a model system of stained and unstained cells that contained birefringent talc particles and systematically investigated the influence of slide and coverslip materials, laser wavelengths, and mounting media on the Raman spectra obtained. Hematoxylin and eosin stained slides did not produce useful results because of fluorescence interference from the stains. Unstained cell samples prepared with standard slides and coverslips produce high quality Raman spectra when excited at 532 nm; the spectra are uniquely assigned to talc. We also obtain high quality Raman spectra specific for talc in unstained tissue samples (pleural tissue following talc pleurodesis and ovarian tissue following long-term perineal talc exposure). Raman microscopy is sufficiently sensitive and compositionally selective to identify particles as small as one micrometer in diameter. Raman spectra have been catalogued for thousands of substances, which suggests that this approach is likely to be successful in identifying other particles of interest in tissues, potentially making Raman microscopy a powerful new tool in pathology.

Malignant mesothelioma with fungating masses and multiple sites involvement.

Malignant mesothelioma (MM) is a rare tumor associated with asbestos exposure. It typically presents as thickening or nodularity of the pleura, although it can also originate from other sites consisting of mesothelia and have manifestations other than thickening or nodularity. Several studies have implied that these different manifestations are associated with a different tumor biology. We report the case of a 54-year-old man with multiple fungating masses diagnosed as MM on histological examination.

Friction and morphology of pleural mesothelia.

To verify the hypothesis that by enmeshing lubricants, microvilli reduce the coefficient of kinetic friction (µ) of pleural mesothelium, µ was measured during reciprocating sliding of rabbit's visceral against parietal pleura before and after addition of hyaluronan, and related to the morphological features of the microvillar network. Because no relation was found between µ or µ changes after hyaluronan and microvillar characteristics, the latter are not determinants of the frictional forces which oppose sliding of normal mesothelial surfaces under physiological conditions, nor of the effects of hyaluronan. Addition of hyaluronan increased µ slightly but significantly in normal specimens, probably by altering the physiological mix of lubricants, but decreased µ of damaged mesothelia, suggesting protective, anti-abrasion properties. Indeed, while sliding of an injured against a normal pleura heavily damaged the latter and increased µ when Ringer was interposed between the surfaces, both effects were limited or prevented when hyaluronan was interposed between the injured and normal pleura before onset of sliding.

Use of back-scatter electron signals to visualise cell/nanowires interactions in vitro and in vivo; frustrated phagocytosis of long fibres in macrophages and compartmentalisation in mesothelial cells in vivo.

BACKGROUND: Frustrated phagocytosis has been stated as an important factor in the initiation of an inflammatory response after fibre exposure. The length of fibrous structures has been linked to the potential of fibres to induce adverse health effects for at least 40 years. However, we only recently reported for the first time the threshold length for fibre-induced inflammation in the pleural space and we implicated frustrated phagocytosis in the pro-inflammatory effects of long fibres. This study extends the examination of the threshold value for frustrated phagocytosis using well-defined length classes of silver nanowires (AgNW) ranging from 3-28 µm and describes in detail the morphology of frustrated phagocytosis using a novel technique and also describes compartmentalisation of fibres in the pleural space. METHODS: A novel technique, backscatter scanning electron microscopy (BSE) was used to study frustrated phagocytosis since it provides high-contrast detection of nanowires, allowing clear discrimination between the nanofibres and other cellular features. A human monocyte-derived macrophage cell line THP-1 was used to investigate cell-nanowire interaction in vitro and the parietal pleura, the site of fibre retention after inhalation exposure was chosen to visualise the cell- fibre interaction in vivo after direct pleural installation of AgNWs. RESULTS: The length cut-off value for frustrated phagocytosis differs in vitro and in vivo. While in vitro frustrated phagocytosis could be observed with fibres≥14 µm, in vivo studies showed incomplete uptake at a fibre length of ≥10 µm. Recently we showed that inflammation in the pleural space after intrapleural injection of the same nanofibre panel occurs at a length of ≥5 µm. This onset of inflammation does not correlate with the onset of frustrated phagocytosis as shown in this study, leading to the conclusion that intermediate length fibres fully enclosed within macrophages as well as frustrated phagocytosis are associated with a pro-inflammatory state in the pleural space. We further showed that fibres compartmentalise in the mesothelial cells at the parietal pleura as well as in inflammatory cells in the pleural space. CONCLUSION: BSE is a useful way to clearly distinguish between fibres that are, or are not, membrane-bounded. Using this method we were able to show differences in the threshold length at which frustrated phagocytosis occurred between in vitro and in vivo models. Visualising nanowires in the pleura demonstrated at least 2 compartments--in leukocyte aggregations and in the mesothelium--which may have consequences for long term pathology in the pleural space including mesothelioma.

Electron-microscopic imaging of endothoracic fascia in the thoracic paravertebral space in rats.

BACKGROUND AND OBJECTIVES: Anesthesia and analgesia with paravertebral block are reportedly variable. Existence of an endothoracic fascia has been proposed as one of the possible mechanisms leading to variability. We undertook an electron-microscopy imaging study to investigate the endothoracic fascia in the thoracic paravertebral space (TPS) in rats. METHODS: Male Wistar rats were studied in accordance with the principles of laboratory animal care. After the rats were euthanized in a CO2 chamber, the thoracic paravertebral tissues were removed en bloc and cut into consecutive transverse sections of approximately 3 mm. Stereomicroscopy and electron-microscopy assessments were performed by 2 independent observers. RESULTS: The endothoracic fascia was consistently identified in all specimens. The fascia was located between the parietal pleura and the innermost intercostal muscles or ribs. Its thickness ranged from 15 to 27 µm (mean, 20 ± 3 µm). The endothoracic fascia divided the TPS in 2 compartments: one, extrapleural and anterolateral (EPC); another, subendothoracic and posteromedial (SETC). The spinal nerves with their ganglia were found within SETC, whereas the sympathetic ganglia were consistently located within the EPC. CONCLUSIONS: The endothoracic fascia in rats appears to divide the TPS into EPC and SETC. These anatomic characteristics may have implications in thoracic paravertebral blockade.

Microscopic anatomy of the lower respiratory tract of the grey short-tailed opossum (Monodelphis domestica).

The respiratory tracts of seven grey short-tailed opossums were histologically examined. Six opossums were prepared by perfusion with buffered formalin. Opossum seven was perfused with gluteraldehyde. Samples taken from the respiratory passages and lungs of specimens 1-6 were stained with haematoxylin and eosin. A mixture of methylene and azure blue was used for specimen 7. The trachea and right and left principal bronchi are lined with a pseudostratified ciliated columnar epithelium with occasional goblet cells. The secondary and tertiary bronchi and the primary and secondary bronchioles are lined by a simple ciliated columnar epithelium. The terminal bronchioles and a portion of the respiratory bronchioles are lined by a simple ciliated cuboidal epithelium. The terminal portion of the respiratory bronchioles and the alveolar ducts are lined with simple squamous epithelium. Alveoli are lined by type I and II pneumocytes. Tracheal glands are present in the tela submucosa. The fibromusculocartilaginous tunic of the trachea consists of c-shaped cartilage rings and the trachealis muscle. A lamina muscularis mucosa begins in the intrapulmonary portion of the principal bronchus and continues into the respiratory bronchioles. Bronchial glands are present in the propria submucosa and tela submucosa of the principal bronchi. The musculocartilaginous tunic is localized to the extrapulmonary portion of the principal bronchus. The bronchial cartilages are irregular shaped plates and limited to the extrapulmonary portion of the principal bronchus. The visceral pleura is a simple squamous mesothelium covering the outer surface of the lung.

Length-dependent retention of carbon nanotubes in the pleural space of mice initiates sustained inflammation and progressive fibrosis on the parietal pleura.

The fibrous shape of carbon nanotubes (CNTs) raises concern that they may pose an asbestos-like inhalation hazard, leading to the development of diseases, especially mesothelioma. Direct instillation of long and short CNTs into the pleural cavity, the site of mesothelioma development, produced asbestos-like length-dependent responses. The response to long CNTs and long asbestos was characterized by acute inflammation, leading to progressive fibrosis on the parietal pleura, where stomata of strictly defined size limit the egress of long, but not short, fibers. This was confirmed by demonstrating clearance of short, but not long, CNT and nickel nanowires and by visualizing the migration of short CNTs from the pleural space by single-photon emission computed tomographic imaging. Our data confirm the hypothesis that, although a proportion of all deposited particles passes through the pleura, the pathogenicity of long CNTs and other fibers arises as a result of length-dependent retention at the stomata on the parietal pleura.

Quantification of the pathological response and fate in the lung and pleura of chrysotile in combination with fine particles compared to amosite-asbestos following short-term inhalation exposure.

The marked difference in biopersistence and pathological response between chrysotile and amphibole asbestos has been well documented. This study is unique in that it has examined a commercial chrysotile product that was used as a joint compound. The pathological response was quantified in the lung and translocation of fibers to and pathological response in the pleural cavity determined. This paper presents the final results from the study. Rats were exposed by inhalation 6 h/day for 5 days to a well-defined fiber aerosol. Subgroups were examined through 1 year. The translocation to and pathological response in the pleura was examined by scanning electron microscopy and confocal microscopy (CM) using noninvasive methods. The number and size of fibers was quantified using transmission electron microscopy and CM. This is the first study to use such techniques to characterize fiber translocation to and the response of the pleural cavity. Amosite fibers were found to remain partly or fully imbedded in the interstitial space through 1 year and quickly produced granulomas (0 days) and interstitial fibrosis (28 days). Amosite fibers were observed penetrating the visceral pleural wall and were found on the parietal pleural within 7 days postexposure with a concomitant inflammatory response seen by 14 days. Pleural fibrin deposition, fibrosis, and adhesions were observed, similar to that reported in humans in response to amphibole asbestos. No cellular or inflammatory response was observed in the lung or the pleural cavity in response to the chrysotile and sanded particles (CSP) exposure. These results provide confirmation of the important differences between CSP and amphibole asbestos.

Influence of the softness of the parietal pleura on respiratory sliding mechanisms.

The pleural surfaces of the lung and chest wall slide against each other with low friction. Normal load support can be effected either by a combination of quasi-static fluid pressure and solid-solid contacts of relatively stiff asperities, or by shear-induced hydrodynamic pressures in the pleural fluid layer. To distinguish between these mechanisms, we measured surface topography and spatial distribution of stiffness of rat parietal pleura using atomic force microscopy. The topography of the pleural surface has unevenness at length scales smaller than the thickness of pleural fluid, similar to mesothelial cell diameters. The estimated maximum normal contact pressure that could be borne by asperities of the soft pleura is much less than that required to support a substantial difference between pleural fluid pressure and the pleural surface pressure. These results suggest that during sliding motion, unevenness of the pleural surface is smoothed by local hydrodynamic pressure, preventing any significant contribution of solid-solid contacts.

Anatomy of the pleura.

The pleura is a monolayer of mesothelial cells covering the lung and inner surface of the chest cavity, creating the pleural space. The mesothelial cells rest on a matrix of collagen, elastic fibers, blood vessels, and lymphatics, which allow the lung and chest to expand and contract, protected from friction by the pleural fluid and properties of the mesothelial cells. With a rich blood supply and lymphatic system just deep to the mesothelial layer, the pleura is a dynamic layer protecting the lung and pleural cavity from infection while transmitting the forces of respiration without damage to the underlying lung parenchyma.

The primary cilium of telocytes in the vasculature: electron microscope imaging.

Blood vessels are highly organized and complex structure, which are far more than simple tubes conducting the blood to almost any tissue of the body. The fine structure of the wall of blood vessels has been studied previously using the electron microscope, but the presence the telocytes associated with vasculature, a specific new cellular entity, has not been studied in depth. Interestingly, telocytes have been recently found in the epicardium, myocardium, endocardium, human term placenta, duodenal lamina propria and pleura. We show the presence of telocytes located on the extracellular matrix of blood vessels (arterioles, venules and capillaries) by immunohistochemistry and transmission electron microscopy. Also, we demonstrated the first evidence of a primary cilium in telocytes. Several functions have been proposed for these cells. Here, the telocyte-blood vessels cell proximity, the relationship between telocytes, exosomes and nervous trunks may have a special significance.

Telocytes in pleura: two- and three-dimensional imaging by transmission electron microscopy.

Information about the ultrastructure of connective (interstitial) cells supporting the pleural mesothelium is scarce. Our aim has been to examine whether telocytes (TCs) are present in pleura, as in epicardium and mesentery. TCs are a distinct type of cell, characterized by specific prolongations named telopodes (Tp). We have used transmission electron microscopy (TEM) and electron tomography (ET) to determine whether ultrastructural diagnostic criteria accepted for TCs are fulfilled by any of the cell subpopulations existing in the sub-mesothelial layer in mouse and human pleura. TCs have been identified with TEM by their characteristic prolongations. Tp appear long and moniliform, because of the alternation of podomeres (thin segments of less than 0.2 µm) and podoms (small dilations accommodating caveolae, mitochondria, and endoplasmic reticulum). Tp ramifications follow a dichotomic pattern and establish specialized cell-to-cell junctional complexes. TCs, via their Tp, seem to form an interstitial network beneath the mesothelium, covering about two-thirds of the abluminal mesothelial layer. ET has revealed complex junctional structures and tight junctions connecting pleural TCs, and small vesicles at this level in Tp. Thus, pleural TCs share significant similarities with TCs described in other serosae. Whether TCs are a (major) player in mesothelial-cell-induced tissue repair remains to be established. Nevertheless, the extremely long thin Tp and complex junctional structures that they form and the release of vesicles (or exosomes) indicate the participation of TCs in long-distance homo- or heterocellular communication.

Asbestos, carbon nanotubes and the pleural mesothelium: a review of the hypothesis regarding the role of long fibre retention in the parietal pleura, inflammation and mesothelioma.

The unique hazard posed to the pleural mesothelium by asbestos has engendered concern in potential for a similar risk from high aspect ratio nanoparticles (HARN) such as carbon nanotubes. In the course of studying the potential impact of HARN on the pleura we have utilised the existing hypothesis regarding the role of the parietal pleura in the response to long fibres. This review seeks to synthesise our new data with multi-walled carbon nanotubes (CNT) with that hypothesis for the behaviour of long fibres in the lung and their retention in the parietal pleura leading to the initiation of inflammation and pleural pathology such as mesothelioma. We describe evidence that a fraction of all deposited particles reach the pleura and that a mechanism of particle clearance from the pleura exits, through stomata in the parietal pleura. We suggest that these stomata are the site of retention of long fibres which cannot negotiate them leading to inflammation and pleural pathology including mesothelioma. We cite thoracoscopic data to support the contention, as would be anticipated from the preceding, that the parietal pleura is the site of origin of pleural mesothelioma. This mechanism, if it finds support, has important implications for future research into the mesothelioma hazard from HARN and also for our current view of the origins of asbestos-initiated pleural mesothelioma and the common use of lung parenchymal asbestos fibre burden as a correlate of this tumour, which actually arises in the parietal pleura.

Inhaled carbon nanotubes reach the subpleural tissue in mice.

Carbon nanotubes are shaped like fibres and can stimulate inflammation at the surface of the peritoneum when injected into the abdominal cavity of mice, raising concerns that inhaled nanotubes may cause pleural fibrosis and/or mesothelioma. Here, we show that multiwalled carbon nanotubes reach the subpleura in mice after a single inhalation exposure of 30 mg m(-3) for 6 h. Nanotubes were embedded in the subpleural wall and within subpleural macrophages. Mononuclear cell aggregates on the pleural surface increased in number and size after 1 day and nanotube-containing macrophages were observed within these foci. Subpleural fibrosis unique to this form of nanotubes increased after 2 and 6 weeks following inhalation. None of these effects was seen in mice that inhaled carbon black nanoparticles or a lower dose of nanotubes (1 mg m(-3)). This work suggests that minimizing inhalation of nanotubes during handling is prudent until further long-term assessments are conducted.

[Asbestos fibre lung burden and exposure indices in asbestos-cement workers]. / Carico polmonare di fibre di asbesto e indici di esposizione cumulativa in lavoratori del cementoamianto.

BACKGROUND: In many previous studies, the asbestos fibres retained in the lung were regarded as a good index of cumulative occupational asbestos exposure. Twelve workers suffering from asbestos-related diseases and had been employed in an asbestos-cement factory operating from 1961 to 1994 underwent post mortem investigations in the course of a criminal law suit. OBJECTIVES: Samples of lung tissues were collected for electron microscopy analysis to measure the asbestos fibre burden of the lungs in workers with high exposure, and assess the possible correlation between asbestos fibre lung burden and the estimated levels of cumulative exposure. METHODS: Samples of lung parenchyma obtained from a consecutive series of 12 post-mortem examinations that were performed between 1994 and 2007and included 5 cases of malignant pleural mesothelioma, 4 lung cancers, 1 case of asbestosis and2 ofpleuralplagues, were collected, stored and analysed by SEM electron microscopy, according to the methods suggested by the current scientific literature. For each worker, all males, a detailed occupational history was reconstructed by means ofpersonal interviews; both the measurements of airborne asbestos fibresperformed by the factory in the 1970's and the duration of each single job in the plant were taken into account to estimate an individual cumulative exposure index. RESULTS: A wide variation of total asbestos fibre concentrations in the lung (1,320-118 million) was observed; in all 12 workers, the lung amphibole fibre burden exceeded 1,000,000 fibres per g/dry tissue, The highest values were detected in the mesothelioma cases, in which the mean fibre concentrations differed statistically (t=2.29, p=0.045) from the mean calculated for the other asbestos-related diseases; in 9 subjects only amphibole fibres were detected. There was a good correlation between total asbestos fibre concentration and cumulative exposure index (r=0.91, p<0.0001). CONCLUSION: This study, which was numerically the biggest ever performed in Italy for this category of workers, confirms a wide range of total asbestos fibre burden in heavily occupationally exposed workers and showed that of the asbestos-related diseases, the highest lung concentrations of asbestos fibres were reached in cases of mesothelioma. It was also observed that almost the entire lung burden consists of only amphibole fibres, all exceeding 1 million per gramme of dry tissue. This study tested a synthetic cumulative occupational exposure index, which appears to be well correlated to the level of exposure established by biological analysis.

Organization and developmental aspects of lymphatic vessels.

The lymphatic system plays important roles in maintaining tissue fluid homeostasis, immune surveillance of the body, and the taking up dietary fat and fat-soluble vitamins A, D, E and K. The lymphatic system is involved in many pathological conditions, including lymphedema, inflammatory diseases, and tumor dissemination. A clear understanding of the organization of the lymphatic vessels in normal conditions would be critically important to develop new treatments for diseases involving the lymphatic vascular system. Therefore, the present paper reviews the organization of the lymphatic vascular system of a variety of organs, including the thyroid gland, lung and pleura, small intestine, cecum and colon in the rat, the diaphragm in the rat, monkey, and human, Peyer's patches and the appendix in the rabbit, and human tonsils. Methods employed include scanning electron microscopy of lymphatic corrosion casts and tissues with or without treatment of alkali maceration technique, transmission electron microscopy of intact tissues, confocal microscopy in conjunction with immunohistochemistry to some lymphatic-specific markers (i.e., LYVE-1 and VEGFR-3), and light microscopy in conjunction with enzyme-histochemistry to 5'-nucleotidase. Some developmental aspects of the lymphatic vessels and lymphedema are also discussed.

Ten cases of feline mesothelioma: an immunohistochemical and ultrastructural study.

In the cat only 10 cases of mesothelioma, mainly of the peritoneum, have been previously reported. This paper describes a further 10 cases, eight pleural and two peritoneal, in males and females aged 1-17 years. Histologically, five tumours were epithelial, three fibrosarcomatous and two biphasic. Immunohistochemical markers used in human pathology for the identification of mesotheliomas include vimentin, cytokeratin (CK) AE1/AE3, HBME-1, CK 5/6, calretinin, thrombomodulin, carcinoembryonic antigen (CEA), CD15, E-cadherin and desmin. All 10 feline mesotheliomas were positive for vimentin and CK AE1/AE3, six were positive for HBME-1, two for CK5/6, three for CEA and four for E-cadherin. All were negative for desmin and calretinin. Antibodies to thrombomodulin and CD15 failed to cross-react with feline tissues. Electron microscopy, performed in four cases, revealed microvillar structures, desmosomes and intracytoplasmic lumina, confirming its value as a diagnostic tool. The study showed that mesothelial marker antibodies commonly used in human patients can be used for the diagnosis of feline mesothelioma, preferably as a panel of antibodies rather than only one.

Serosal membranes (pleura, pericardium, peritoneum). Normal structure, development and experimental pathology.

This monograph offers a comprehensive review of the present knowledge of the structure of the serosal coverings of the pleural, pericardial, and peritoneal cavities in humans and laboratory animals. The authors provide data from their own research--with transmission and scanning electron microscopy--on the structure of the main components of the serosal membranes: mesothelial cells, underlying basal lamina, and submesothelial connective tissue layer. Two main types of mesothelial cells (flat and cubic) are distinguished and their distribution on the parietal serosal sheets and on the visceral coverings of various organs is described. The openings between mesothelial cells (stomata) and their relations with lymphatic lacunae are described thoroughly. Special reference is made to the serosal accumulations of lymphoid tissue (milky spots). The transcellular and intercellular transport to and from serosal cavities is studied by means of horseradish peroxidase tracing experiments. The prenatal and postnatal developmental studies are focused on human and rat pleura. The alterations of serosal membranes after experimental hemothorax, pneumonectomy, and peritonitis caused by Pseudomonas aeruginosa application suggest the existence of early, reversible, and late, definite periods.