The mammalian lung´s structural design is optimized to serve its main function: gas exchange. It takes place in the alveolar region (parenchyma) where air and blood are brought in close proximity ...over a large surface. Air reaches the alveolar lumen via a conducting airway tree. Blood flows in a capillary network embedded in inter-alveolar septa. The barrier between air and blood consists of a continuous alveolar epithelium (a mosaic of type I and type II alveolar epithelial cells), a continuous capillary endothelium and the connective tissue layer in-between. By virtue of its respiratory movements, the lung has to withstand mechanical challenges throughout life. Alveoli must be protected from over-distension as well as from collapse by inherent stabilizing factors. The mechanical stability of the parenchyma is ensured by two components: a connective tissue fiber network and the surfactant system. The connective tissue fibers form a continuous tensegrity (tension + integrity) backbone consisting of axial, peripheral and septal fibers. Surfactant (surface active agent) is the secretory product of type II alveolar epithelial cells and covers the alveolar epithelium as a biophysically active thin and continuous film. Here, we briefly review the structural components relevant for gas exchange. Then we describe our current understanding of how these components function under normal conditions and how lung injury results in dysfunction of alveolar micromechanics finally leading to lung fibrosis.
Design-based stereology provides efficient methods to obtain valuable quantitative information of the respiratory tract in various diseases. However, the choice of the most relevant parameters in a ...specific disease setting has to be deduced from the present pathobiological knowledge. Often it is difficult to express the pathological alterations by interpretable parameters in terms of volume, surface area, length, or number. In the second part of this companion review article, we analyze the present pathophysiological knowledge about acute lung injury, diffuse parenchymal lung diseases, emphysema, pulmonary hypertension, and asthma to come up with recommendations for the disease-specific application of stereological principles for obtaining relevant parameters. Worked examples with illustrative images are used to demonstrate the work flow, estimation procedure, and calculation and to facilitate the practical performance of equivalent analyses.
A brief update on lung stereology OCHS, MATTHIAS
Journal of microscopy (Oxford),
June 2006, Letnik:
222, Številka:
3
Journal Article
Recenzirano
Lung stereology has a long and successful tradition. From mice to men, the application of new stereological methods at several levels (alveoli, parenchymal cells, organelles, proteins) has led to new ...insights into normal lung architecture, parenchymal remodelling in emphysema‐like pathology, alveolar type II cell hyperplasia and hypertrophy and intracellular surfactant alterations as well as distribution of surfactant proteins. The Euler number of the network of alveolar openings, estimated using physical disectors at the light microscopic level, is an unbiased and direct estimate of alveolar number. Surfactant‐producing alveolar type II cells can be counted and sampled for local size estimation with physical disectors at a high magnification light microscopic level. The number of their surfactant storage organelles, lamellar bodies, can be estimated using physical disectors at the EM level. By immunoelectron microscopy, surfactant protein distribution can be analysed with the relative labelling index. Together with the well‐established classical stereological methods, these design‐based methods now allow for a complete quantitative phenotype analysis in lung development and disease, including the structural characterization of gene‐manipulated mice, at the light and electron microscopic level.
Preservation of original tissue dimensions is an essential prerequisite for morphometric studies. Shrinkage occurring during tissue processing for histology may severely influence the appearance of ...structures seen under the microscope and stereological calculations. Therefore, shrinkage has to be avoided so that estimates obtained by application of unbiased stereology are indeed unbiased. The present study investigates the alterations of tissue dimensions of mouse lung samples during processing for histology. Different fixatives as well as embedding protocols are considered. Mouse lungs were fixed by instillation of either 4% formalin or a mixture of 1.5% glutaraldehyde/1.5% formaldehyde. Tissue blocks were sampled according to principles of stereology for embedding in paraffin, glycol methacrylate without treatment with osmium tetroxide and uranyl acetate, and glycol methacrylate including treatment with osmium tetroxide and uranyl acetate before dehydration. Shrinkage was investigated by stereological measurements of dimensional changes of tissue cut faces. Results show a shrinkage of the cut face areas of roughly 40% per lung during paraffin embedding, 30% during "simple" glycol methacrylate embedding, and <3% during osmium tetroxide/uranyl acetate/glycol methacrylate embedding. Furthermore, the superiority of the glutaraldehyde-containing fixative regarding shrinkage is demonstrated. In conclusion, the use of a glutaraldehyde-containing fixative and embedding in glycol methacrylate with previous treatment of the samples with osmium tetroxide and uranyl acetate before dehydration is recommended for stereological studies of the mouse lung.
A short primer on lung stereology Ochs, Matthias; Schipke, Julia
Respiratory research,
11/2021, Letnik:
22, Številka:
1
Journal Article
Recenzirano
Odprti dostop
The intention of this short primer is to raise your appetite for proper quantitative assessment of lung micro-structure. The method of choice for obtaining such data is stereology. Rooted in ...stochastic geometry, stereology provides simple and efficient tools to obtain quantitative three-dimensional information based on measurements on nearly two-dimensional microscopic sections. In this primer, the basic concepts of stereology and its application to the lung are introduced step by step along the workflow of a stereological study. The integration of stereology in your laboratory work will help to improve its quality. In a broader context, stereology may also be seen as a contribution to good scientific practice.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
COVID-19-induced “acute respiratory distress syndrome” (ARDS) is associated with prolonged respiratory failure and high mortality, but the mechanistic basis of lung injury remains incompletely ...understood. Here, we analyze pulmonary immune responses and lung pathology in two cohorts of patients with COVID-19 ARDS using functional single-cell genomics, immunohistology, and electron microscopy. We describe an accumulation of CD163-expressing monocyte-derived macrophages that acquired a profibrotic transcriptional phenotype during COVID-19 ARDS. Gene set enrichment and computational data integration revealed a significant similarity between COVID-19-associated macrophages and profibrotic macrophage populations identified in idiopathic pulmonary fibrosis. COVID-19 ARDS was associated with clinical, radiographic, histopathological, and ultrastructural hallmarks of pulmonary fibrosis. Exposure of human monocytes to SARS-CoV-2, but not influenza A virus or viral RNA analogs, was sufficient to induce a similar profibrotic phenotype in vitro. In conclusion, we demonstrate that SARS-CoV-2 triggers profibrotic macrophage responses and pronounced fibroproliferative ARDS.
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•Monocyte-derived macrophages accumulate in the lung in COVID-19 ARDS•Macrophages in COVID-19 express genes associated with profibrotic functions•Patients with severe COVID-19 ARDS display hallmarks of pulmonary fibrosis•SARS-CoV-2 induces a profibrotic transcriptome and proteome profile in macrophages
SARS-CoV-2 infection, but not influenza A, triggers immunological and pathological changes in the lung that are hallmarks of pulmonary fibrosis. A subset of CD163+ macrophages are found to drive this fibroproliferative acute respiratory distress.
The activation of NLRP3 inflammasome in macrophages has been proven to play a crucial role in the development of cardiovascular diseases. THP-1 monocytes can be differentiated to macrophages by ...incubation with phorbol-12-myristate 13-acetate (PMA), providing a suitable model for
studies. However, PMA has been shown to have effects on the levels of IL-1β, the main mediator of NLRP3 inflammasome, while the effects on the other mediators of the inflammasome have not been reported before.
THP-1 monocytes were incubated without (THP-1), with 5ng/ml PMA for 48h (PMA48h) or with 5ng/ml PMA for 48h plus 24h in fresh medium (PMArest). Morphological changes and the expression of macrophage surface markers (CD14, CD11b, CD36 and CD204) were evaluated by flow cytometry. Changes in intracellular levels of inflammasome components (NLRP3, ASC, pro-caspase-1, pro-IL1β) were analyzed by western blot and release of mature IL-1β in cell supernatant was analyzed by ELISA. ASC speck formation was determined by immunofluorescence.
After 48h incubation with PMA or subsequent rest in fresh medium, cells became adherent, and the differential expression of CD36, CD11b, CD14 and CD204 compared to THP-1 cells confirmed that PMArest resemble macrophages from a molecular point of view. Changes in the levels were detected in PMA48h group for all the NLRP3-related proteins, with increase of NLRP3 and pro-IL-1β and secretion of mature IL-1β. In PMArest, no pro-IL-1β and lower amounts of mature IL-1β were detected. No ASC speck was found in PMA treated groups, but the addition of a second stimulus to PMArest resulted in ASC speck formation, together with IL-1β production, confirming the responsiveness of the model.
Differentiation of THP-1 with 5ng/ml PMA followed by 24h resting period provides a model that morphologically and molecularly resembles macrophages. However, even at low concentrations, PMA induces production of IL-1β. The 24h rest period provides for down-regulation of pro-IL-1β in PMArest group, without affecting its ability to respond to a second stimulus through activation of inflammasome.
Aim
Mixed-methods approaches promise a deep understanding of psychotherapeutic processes. This study uses qualitative and quantitative data from daily diary entries and daily self-assessments during ...inpatient treatment. The aim of the study is to get an insight into the similarities and differences between both types of data and how they represent self-organized pattern transitions in psychotherapy. While a complete correlation of results is not expected, we anticipate observing amplifying and subsidiary patterns from both perspectives.
Materials and methods
Daily, five MDD patients wrote diaries and completed self-assessments using the Therapy Process Questionnaire, a questionnaire for monitoring the change dynamics of psychotherapy. The data were collected using the Synergetic Navigation System, an online tool for real-time monitoring. Diary entries of the patients described their experiences in everyday life. The qualitative text analysis was conducted using Mixed Grounded Theory, which provided categories representing the patients’ ongoing experiences of transformation and stagnation. The time series data was analyzed using the dynamic complexity algorithm and the pattern transition detection algorithm. Results from qualitative and quantitative analyses were combined and compared. Following the process of data triangulation, the leading perspective came from the theory of self-organization. In addition to presenting the overall results for all five patients, we delve into two specific case examples in greater detail.
Results
Specific and highly diversified diary entries of 5 patients were classified into the categories of perceived pattern stability, noticing improvement, broadening the perspective, critical instability, and experiencing moments of Kairos. Patients reported problems not only related to their disorder (e.g., lack of energy and hopelessness) but also to phases and steps of change, which could be related to the theory of self-organization (e.g., problem attractors, critical fluctuations, pattern transitions, and Kairos). Qualitative and quantitative analysis provide important supplementary results without being redundant or identical.
Conclusion
Data triangulation allows for a comprehensive and multi-perspective understanding of therapeutic change dynamics. The different topics expressed in the diary entries especially help to follow micro-psychological processes, which are far from being a simple reaction to interventions. The way patients experience themselves being in stability or instability and stagnation or transformation is surprisingly close to the general features of self-organizing processes in complex systems.
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