A new method for pulmonary edema research: Scanning electron microscopy of frozen-hydrated edematous lung

We present an integrative method for studying alveolar fluid accumulation during pulmonary edema using scanning electron microscopy (SEM) of frozen-hydrated lung. We induced edema in dogs by fluid over-loading the vascular system while monitoring vascular and airway pressures. Terminally, we freeze-fixed the lungs in the open thorax while controlling airway pressure. Using a cryosample preparation chamber attached to a scanning electron microscope equipped with a low temperature stage, we in vacuo freeze-fractured the lung, gold coated the fractured surface and used secondary electron emission scanning electron microscopy to examine the frozen-hydrated lung with scanning electron micrographs of freeze-dried and airway fixed, critical point dried lung. We discuss the advantages of frozen-hydrated scanning lung preservation for pulmonary edema research, especially in regard to quantifying alveolar fluid distribution by stereo photogrammetry, and suggest areas of future research to improve the method.     

Three-dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multiphoton microscopy

The organization of collagen during fibrotic processes is poorly characterized because of the lack of appropriate methodologies. Here we show that multimodal multiphoton microscopy provides novel insights into lung fibrosis. We characterize normal and fibrotic pulmonary tissue in the bleomycin model, and show that second-harmonic generation by fibrillar collagen reveals the micrometer-scale three-dimensional spatial distribution of the fibrosis. We find that combined two-photon excited fluorescence and second-harmonic imaging of unstained lung tissue allows separating the inflammatory and fibrotic steps in this pathology, underlining characteristic features of fibroblastic foci in human Idiopathic Pulmonary Fibrosis samples. Finally, we propose phenomenological scores of lung fibrosis and we show that they unambiguously sort out control and treated mice, with a better sensitivity and reproducibility in the subpleural region. These results should be readily generalized to other organs, as an accurate method to assess extracellular matrix remodeling during fibrosis.     

Lung development: AT1 and AT2 property

The human respiratory system consists of the upper and the lower respiratory tracts. Anatomically, the lower respiratory tract consists of the trachea, bronchi, bronchioles (terminal bronchioles and respiratory bronchioles), alveolar duct, alveolar duct sacs, and alveoli. Alveoli are composed of two epithelial cell types, cuboidal alveolar type 2 (AT2) cells that secrete surfactant to prevent alveolar collapse and function as stem cells to regenerate alveolar type 1 (AT1) cells during damage repair, and squamous AT1 cells that cover most of the surface area of the alveoli and mediate gas exchange. Previous studies mainly focused on AT2 cells; this review summarizes the current studies on lung development and property of AT1 cells.     

Improved preservation of phospholipid-rich multilamellar bodies in conventionally embedded mammalian lung tissue—an electron spectroscopic study

Different conventional methods of tissue processing were studied to determine the extent to which phospholipid-rich multilamellar bodies of pulmonary alveolar epithelial type II cells of the pig were preserved. Prolonged treatment with half-saturated aqueous uranyl acetate yielded excellent results on the stabilization of the multilamellar substructure, irrespective of whether glutaraldehyde-paraformaldehyde or glutaraldehydetannic acid was used as a primary fixative. The lamellar periodicities were observed to be 5·5–6·1 nm. Differences in the phosphorus distribution among several types of lipid bodies of alveolar epithelial type II cells were studied by means of electron spectroscopic imaging and electron energy-loss spectroscopy. Multilamellar bodies gave phosphorus signals which were significantly higher than those obtained from granular regions of composite bodies, whereas homogeneous bodies gave phosphorus signals which were even lower than those obtained from mitochondria, endoplasmic reticulum membranes or ribosomes.     

Morphological aspects of particle uptake by lung phagocytes

Macrophages residing on the inner epithelial surfaces of airways and alveoli are the only lung phagocytes exposed directly to the environment. Their phagocytic and microbicidal activities are essential for maintaining this organ in a clean and sterile state. The morphology of these phagocytes can be investigated in situ only after implementing special techniques, which involve intravascular triple-perfusion of aqueous fixatives or instillation of nonpolar ones. Such studies have revealed the engulfment of particles by these cells to be rapid, the process being essentially complete within a day. Particles are entrapped within phagosomes and the host cells eventually transported out of the lungs by mucociliary action, macrophages with higher loads being more rapidly eliminated than those with lower ones. Very small particles or those persisting on the epithelial surfaces may be taken up by the eponymous cells. Translocation of particles into the underlying connective tissue and their subsequent phagocytosis by interstitial macrophages prolongs their retention time in the lungs. The still poorly studied pleural macrophages might be involved in cell-mediated immune responses within the pleural space. Intravascular pulmonary macrophages figure largely in the phagocytosis of circulating particles. The role played by dendritic cells in particle uptake by the lungs is not well understood. Airway and alveolar macrophages are the primary phagocytes of the lung. In nonoverload situations and for particles >1 microm, a small proportion of those recruited suffices to remove material from the epithelial surface before other phagocytes, with an apparently greater immunological potential, gain access to it.     

Ready to migrate? Reading cellular signs of migration in an epithelial to mesenchymal transition model

The epithelial to mesenchymal transition (EMT) is a cellular program that drives de-differentiation of cells in both physiological and pathological processes. One of the characteristics of cells describing an EMT is the (re)acquisition of a motility capacity that allows them to migrate through the original tissue as well as to other sites in the organism. The molecular mechanisms that control the EMT are rapidly emerging and here we add to the idea that the adaptation required for cells to commit to the EMT includes adjustments of the translation machinery and metabolic pathways to cope with a high demand of extracellular components.     

Effect of indirubin‐3‐monoxime against lung cancer as evaluated by histological and transmission electron microscopic studies

The aim of this study is to evaluate the antitumor effect of indirubin‐3‐monoxime and its mode of action in benzo(α)pyrene [B(α)P] induced lung cancer in A/J mice. Light microscopic examination of lung sections of [B(α)P] induced lung cancer mice revealed the presence of adenocarcinoma characterized by extensive proliferation of alveolar epithelium and loss of alveolar spaces. The control lung tissue showed a normal architecture with clear alveolar spaces. Interestingly the indirubin‐3‐monoxime treated groups showed the reduced adenocarcinoma with appearance of alveolar spaces. Transmission Electron Microscopic (TEM) studies of lung sections of [B(α)P] induced lung cancer mice showed the presence of phaemorphic cells with dense granules and increased mitochondria. The lung sections of mice treated with indirubin‐3‐monoxime showed the presence of shrunken, fragmented, and condensed nuclei implying apoptosis. The effects were dose dependent and prominent in 10 mg/kg/5 d/week groups suggesting the therapeutic role of indirubin analogue against this deadly human malignancy. Here, our results indicate that indirubin‐3‐monoxime brings antitumor effect against [B(α)P] induced lung cancer by its apoptotic action in A/J mice. Microsc. Res. Tech. 73:1053–1058, 2010. © 2010 Wiley‐Liss, Inc.     

Curcumin protects against hepatic and renal injuries mediated by inducible nitric oxide synthase during selenium‐induced toxicity in Wistar rats

The aim of this study is to evaluate the effect of curcumin in protecting against selenium‐induced toxicity in liver and kidney of Wistar rats. Light microscopy evaluation of selenium alone administered rats showed liver to be infiltrated with mononuclear cells, vacuolation, necrosis, and pronounced degeneration. Control liver sections showed a regular morphology of parenchymal cells with intact hepatocytes and sinusoids. Kidney from selenium alone administered rats showed vacuolar degeneration changes in the epithelial cells, cellular proliferation with fibrosis, thickening of capillary walls, and glomerular tuft atrophy. Such changes were also observed in rats administered with selenium and curcumin simultaneously and rats administered first with selenium and then curcumin 24 h later. Interestingly, such degenerative changes observed in liver and kidney induced by selenium were not seen in rats that were administered with curcumin first and selenium 24 h later. This clearly suggests the protective nature of curcumin against selenium toxicity. To understand the probable mechanism of action of curcumin, we analyzed inducible nitric oxide synthase (iNOS) expression by immunohistochemistry, and the results showed an increased iNOS expression in selenium‐alone induced liver and kidney. Such high iNOS levels were inhibited in liver and kidney of rats pretreated with curcumin and then with selenium 24 h later. Based on the histological results, it can be concluded that curcumin functions as a protective agent against selenium‐induced toxicity in liver as well as kidney, and this action is probably by the regulatory role of curcumin on iNOS expression. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Long-term monitoring of live cell proliferation in presence of PVP-Hypericin: a new strategy using ms pulses of LED and the fluorescent dye CFSE

During fluorescent live cell imaging it is critical to keep excitation light dose as low as possible, especially in the presence of photosensitizer drugs, which generate free radicals upon photobleaching. During fluorescent imaging, stress by excitation and free radicals induces serious cell damages that may arrest the cell cycle. This limits the usefulness of the technique for drug discovery, when prolonged live cell imaging is necessary. This paper presents a strategy to provide gentle experimental conditions for dynamic monitoring of the proliferation of human lung epithelial carcinoma cells (A549) in the presence of the photosensitizer Polyvinylpyrrolidone-Hypericin. The distinctive strategy of this paper is based on the stringent environmental control and optimizing the excitation light dose by (i) using a low-power pulsed blue light-emitting diode with short pulse duration of 1.29 ms and (ii) adding a nontoxic fluorescent dye called carboxyfluorescein-diacetate-succinimidyl-ester (CFSE) to improve the fluorescence signals. To demonstrate the usefulness of the strategy, fluorescence signals and proliferation of dual-marked cells, during 5-h fluorescence imaging under pulsed excitation, were compared with those kept under continuous excitation and nonmarked reference cells. The results demonstrated 3% cell division and 2% apoptosis due to pulsed excitation compared to no division and 85% apoptosis under the continuous irradiation. Therefore, our strategy allows live cell imaging to be performed over longer time scales than with conventional continuous excitation.     

Effect of Peroxiredoxin 1 on the biological function of airway epithelial cells and epithelial-mesenchymal transition

Peroxiredoxin 1 (PRDX1) participates in tumor cell proliferation, apoptosis, migration, invasion, and the epithelial-to-mesenchymal transition (EMT). This study aimed to investigate the effect of PRDX1 on the EMT of airway epithelial cells stimulated with lipopolysaccharide (LPS) and transforming growth factor-beta 1 (TGF-β1). PRDX1 overexpression significantly increased the proliferation and migration of human bronchial epithelial (BEAS-2B) cells, reduced cell apoptosis (p < 0.01), and induced EMT and collagen deposition by upregulating the expression of the matrix metallopeptidase (MMP)2, MMP9, α-smooth muscle actin (α-SMA), N-cadherin, vimentin and twist proteins and inhibiting E-cadherin expression (p < 0.05). PRDX1 overexpression promoted TGF-β1-mediated inhibition of cell proliferation and migration and significantly enhanced the TGF-β1-induced EMT and collagen synthesis (p < 0.05). Knockdown of PRDX1 inhibited cell proliferation, migration, EMT, and collagen synthesis (p < 0.01), reversed LPS-mediated inhibition of cell proliferation and migration, and significantly suppressed LPS-induced EMT and collagen synthesis (p < 0.01). The result indicating that PRDX1 may be involved in LPS/TGF-1-induced EMT and collagen synthesis in human bronchial epithelial cells.     

Electron micropscopic appearances of rapidly frozen lung.

Analysis of pulmonary structure-function relationships by microscopy requires that the lung be fixed under carefully controlled physiological conditions, since lung structure is extremely responsive to the relationship between airway and vascular pressures. Unfortunately, standard post-mortem fixation techniques leave some doubt as to the exact relationship between these pressures during fixation. This problem can be circumvented by stabilizing lung structure by rapid freezing under carefully controlled physiologic conditions. Using ethylene glycol in a freeze substitution technique we have developed procedures which yield a degree of preservation compatible with the high degree of resolution of the electron microscope. These can be used to obtain a more detailed understanding of pulmonary structure-function relationships under well-defined physiological conditions.     

Mesenchymal stem cell-derived exosomes as new remedy for the treatment of inflammatory eye diseases

Detrimental immune response has a crucially important role in the development and progression of inflammatory eye diseases. Inflammatory mediators and proteolytic enzymes released by activated immune cells induce serious injury of corneal epithelial cells and retinal ganglion cell which may result in the vision loss. Mesenchymal stem cells (MSCs) are regulatory cells which produce various immunosuppressive factors that modulate phenotype and function of inflammatory immune cells. However, several safety issues, including undesired differentiation and emboli formation, limit clinical use of MSCs. MSC-derived exosomes (MSC-Exos) are nano-sized extracellular vesicles which contain all MSC-derived immunoregulatory factors. Intraocular administration of MSC-Exos efficiently attenuated eye inflammation and significantly improved visual acuity in experimental animals without causing any severe side effects. As cell-free product, MSC-Exos addressed all safety issues related to the transplantation of MSCs. Therefore, MSC-Exos could be considered as potentially new remedy for the treatment of inflammatory eye diseases which efficacy should be explored in up-coming clinical trials.     

Pharmacotherapeutics and molecular docking studies of alpha-synuclein modulators as promising therapeutics for Parkinson’s disease

Parkinson’s disease (PD) is an age-related neurodegenerative ailment that affects dopamine-producing neurons in a specific area of the brain called the substantia nigra of the ventral midbrain. It is clinically characterized by movement disorder and marked with unusual synaptic protein alpha-synuclein accumulation in the brain. To date, only a few Food and Drug Administration (FDA) approved drugs are available on the market for the treatment of PD. Nonetheless, these drugs show parasympathomimetic related adverse events and remarkably higher toxicity; hence, it is important to find more efficacious molecules to treat PD. In our study, We chosen 22 natural compounds as inhibitors that potentially block the alpha-synuclein clump—the pathological hallmark of PD—and provide new avenues for its treatment. Most of these molecules exhibited good pharmacokinetic behaviors, making them decisively favorable drug candidates to cure PD. Molecular docking studies were performed to investigate the binding interactions between natural compounds and alpha-synuclein as anti-Parkinson drug targets. Among the examined compounds, curcumin and piperine emerged as promising phytochemicals with the highest binding affinity, key residual stable bindings and showed a good inhibitory features. Thus, the present study indicates that curcumin and piperine hold the potential to be developed as treatment options against PD. Experimental validations are needed for insights into their mechanism of action and potential clinical application.     

Effect of SP-C on surface potential distribution in pulmonary surfactant: Atomic force microscopy and Kelvin probe force microscopy study

The air–lung interface is covered by a molecular film of pulmonary surfactant (PS). The major function of the film is to reduce the surface tension of the lung's air–liquid interface, providing stability to the alveolar structure and reducing the work of breathing. Earlier we have shown that function of bovine lipid extract surfactant (BLES) is related to the specific molecular architecture of surfactant films. Defined molecular arrangement of the lipids and proteins of the surfactant film also give rise to a local highly variable electrical surface potential of the interface. In this work we investigated a simple model of artificial lung surfactant consisting of DPPC, eggPG, and surfactant protein C (SP-C).     

一种慢性病危险因素的二值控制及应用

高血压、糖尿病、冠心病、脑卒中、慢性阻塞性肺病(COPD)等是严重威胁人类健康的慢性病,其控制的最佳途径是控制导致慢性病发生的危险因素.根据个体人的实际身体状态,用控制理论的方法设计个体人危险因素控制策略,以实现个体化预防医学的目的.设人体系统的身体状态分为健康状态、高危状态和疾病状态,判断指标是血压、血糖、心率、血脂、体重指数、呼气容积肺活量比等,判断限值来自于各种疾病的中国防治指南.危险因素的状态控制系统包括人体、比较器、二值控制策略、标准等所构成的反馈控制,并设计RFCS软件帮助医师进行危险因素控制.通过19例志愿者的应用表明这个方法对社区的慢性病防治是有效的.     

An alkali digestion method to expose connective tissue fibers: A scanning electron microscopy study of rat lung

Alkali digestion has been used to remove cellular elements of tissues thus exposing the underlying connective tissue framework. We studied the action of this severe alkali treatment on the delicate tissues of rat lung. The lungs of male Sprague-Dawley rats were perfused with saline to remove blood and then inflated by fixation through the airways at 20 cm pressure. Sections of lung 2 × 5 × 5 mm were immersed in 2.5 M NaOH at 25°C for 6 h, 16 h, 24 h, 48 h, and 72 h. The alkali was changed daily. Tissues were washed to neutral with water (24 h), treated with tannic acid (1%, 3h), post-fixed with osmic acid (1%, 3 h) and processed for SEM. At 6 h, epithelial cells started to peel off the alveolar surface. At 16 h the digestion process was well advanced. At 48 h the cells were completely removed revealing the lattice network of connective tissue fibers within the alveolar surface. The method allows the complete removal of cellular elements of the lung while retaining the very fine 3D structure of the connective tissue matrix.     

山区铁路钢轨磨损情况调查与防治技术试验研究

介绍了对实际线路钢轨现状调查的结果:随着我国五次铁路提速,铁路的超负荷运行,加上路线等原因导致铁路钢轨磨损严重．通过制定相应的抗磨损措施并进行实践操作,在对比分析基础上,得出机车合理轮缘喷脂间距能有效减缓钢轨鱼鳞伤损的发生和发展,提高钢轨防断能力,在此基础上提出适合我国山区铁路的抗磨损方法和保养措施．结论对我国铁路钢轨的养护具有参考价值．     

High-resolution X-ray refraction imaging of rat lung and histological correlations

This study was performed to observe microstructures of the rat lung, using a synchrotron radiation beam and to compare findings with histological observations. X-ray refraction images from ex-vivo ventilating rat lung were obtained with an 8 KeV monochromatic beam and 20-mum thick CsI(Tl) scintillation crystal. The visual image was magnified using a 20x microscope objective and captured using an analog CCD camera. Obtained images were compared with conventional light microscopic findings from the same tissue. Pulmonary microstructures, including alveolar ducts, alveolar sacs, alveoli, alveolar walls, and perialveolar capillary networks were clearly identified with spatial resolution of as much as 1.2 mum and had good correlation with conventional light microscopic findings. The shape of alveoli appeared more round in SR images than in the light microscopic images. The results suggest that X-ray microscopy study of the lung using synchrotron radiation demonstrates the potential for clinically relevant microstructure of lung tissue without sectioning and fixation.     

钻孔灌注桩常见施工质量问题及防治措施

钻孔灌注桩是各类房屋及民用建筑及桥梁基础的最重要组成部份,并在建筑中得了广泛应用.因此对它的施工质量提出了严格的要求,并针对经常出现的问题提出了切实可行的防治措施,以确保建筑质量.