Ex Vivo Artifacts and Histopathologic Pitfalls in the Lung

Context Surgical and pathologic handling of lung physically affects lung tissue. This leads to artifacts that alter the morphologic appearance of pulmonary parenchyma. Objective —To describe and illustrate mechanisms of ex vivo artifacts that may lead to diagnostic pitfalls. Design In this study 4 mechanisms of ex vivo artifacts and corresponding diagnostic pitfalls are described and illustrated. Results —The 4 patterns of artifacts are: (1) surgical collapse, due to the removal of air and blood from pulmonary resections; (2) ex vivo contraction of bronchial and bronchiolar smooth muscle; (3) clamping edema of open lung biopsies; and (4) spreading of tissue fragments and individual cells through a knife surface. Morphologic pitfalls include diagnostic patterns of adenocarcinoma, asthma, constrictive bronchiolitis, and lymphedema. Conclusion Four patterns of pulmonary ex vivo artifacts are important to recognize in order to avoid morphologic misinterpretations.

Download Full-text

Multidimensional immunolabeling and 4D time-lapse imaging of vital ex vivo lung tissue

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00061.2015 ◽

2015 ◽

Vol 309 (4) ◽

pp. L323-L332 ◽

Cited By ~ 13

Author(s):

Gerald Burgstaller ◽

Sarah Vierkotten ◽

Michael Lindner ◽

Melanie Königshoff ◽

Oliver Eickelberg

Keyword(s):

Smooth Muscle ◽

Lung Tissue ◽

Ex Vivo ◽

Smooth Muscle Actin ◽

Focal Adhesions ◽

Cellular Tissue ◽

Muscle Actin ◽

Human Lung Tissue

During the last decades, the study of cell behavior was largely accomplished in uncoated or extracellular matrix (ECM)-coated plastic dishes. To date, considerable cell biological efforts have tried to model in vitro the natural microenvironment found in vivo. For the lung, explants cultured ex vivo as lung tissue cultures (LTCs) provide a three-dimensional (3D) tissue model containing all cells in their natural microenvironment. Techniques for assessing the dynamic live interaction between ECM and cellular tissue components, however, are still missing. Here, we describe specific multidimensional immunolabeling of living 3D-LTCs, derived from healthy and fibrotic mouse lungs, as well as patient-derived 3D-LTCs, and concomitant real-time four-dimensional multichannel imaging thereof. This approach allowed the evaluation of dynamic interactions between mesenchymal cells and macrophages with their ECM. Furthermore, fibroblasts transiently expressing focal adhesions markers incorporated into the 3D-LTCs, paving new ways for studying the dynamic interaction between cellular adhesions and their natural-derived ECM. A novel protein transfer technology (FuseIt/Ibidi) shuttled fluorescently labeled α-smooth muscle actin antibodies into the native cells of living 3D-LTCs, enabling live monitoring of α-smooth muscle actin-positive stress fibers in native tissue myofibroblasts residing in fibrotic lesions of 3D-LTCs. Finally, this technique can be applied to healthy and diseased human lung tissue, as well as to adherent cells in conventional two-dimensional cell culture. This novel method will provide valuable new insights into the dynamics of ECM (patho)biology, studying in detail the interaction between ECM and cellular tissue components in their natural microenvironment.

Download Full-text

Combination of histochemical analyses and micro-MRI reveals regional changes of the murine cervix in preparation for labor

Scientific Reports ◽

10.1038/s41598-021-84036-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Antara Chatterjee ◽

Rojan Saghian ◽

Anna Dorogin ◽

Lindsay S. Cahill ◽

John G. Sled ◽

...

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Diffusion Tensor ◽

Ex Vivo ◽

Muscle Cells ◽

Pregnant Mouse ◽

Collagen Fibers ◽

Muscular Layer ◽

Tissue Organization ◽

Picrosirius Red

AbstractThe cervix is responsible for maintaining pregnancy, and its timely remodeling is essential for the proper delivery of a baby. Cervical insufficiency, or “weakness”, may lead to preterm birth, which causes infant morbidities and mortalities worldwide. We used a mouse model of pregnancy and term labor, to examine the cervical structure by histology (Masson Trichome and Picrosirius Red staining), immunohistochemistry (Hyaluronic Acid Binding Protein/HABP), and ex-vivo MRI (T2-weighted and diffusion tensor imaging), focusing on two regions of the cervix (i.e., endocervix and ectocervix). Our results show that mouse endocervix has a higher proportion of smooth muscle cells and collagen fibers per area, with more compact tissue structure, than the ectocervix. With advanced gestation, endocervical changes, indicative of impending delivery, are manifested in fewer smooth muscle cells, expansion of the extracellular space, and lower presence of collagen fibers. MRI detected three distinctive zones in pregnant mouse endocervix: (1) inner collagenous layer, (2) middle circular muscular layer, and (3) outer longitudinal muscular layer. Diffusion MRI images detected changes in tissue organization as gestation progressed suggesting the potential application of this technique to non-invasively monitor cervical changes that precede the onset of labor in women at risk for preterm delivery.

Download Full-text

NTPDase1 Modulates Smooth Muscle Contraction in Mice Bladder by Regulating Nucleotide Receptor Activation Distinctly in Male and Female

Biomolecules ◽

10.3390/biom11020147 ◽

2021 ◽

Vol 11 (2) ◽

pp. 147

Author(s):

Romuald Brice Babou Kammoe ◽

Gilles Kauffenstein ◽

Julie Pelletier ◽

Bernard Robaye ◽

Jean Sévigny

Keyword(s):

Smooth Muscle ◽

Ex Vivo ◽

Smooth Muscle Contraction ◽

Receptor Activation ◽

Nucleoside Triphosphate ◽

Nucleotide Receptors ◽

Nerve Terminals ◽

Wild Type ◽

Nucleoside Triphosphate Diphosphohydrolase ◽

Bladder Contraction

Nucleotides released by smooth muscle cells (SMCs) and by innervating nerve terminals activate specific P2 receptors and modulate bladder contraction. We hypothesized that cell surface enzymes regulate SMC contraction in mice bladder by controlling the concentration of nucleotides. We showed by immunohistochemistry, enzymatic histochemistry, and biochemical activities that nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) and ecto-5′-nucleotidase were the major ectonucleotidases expressed by SMCs in the bladder. RT-qPCR revealed that, among the nucleotide receptors, there was higher expression of P2X1, P2Y1, and P2Y6 receptors. Ex vivo, nucleotides induced a more potent contraction of bladder strips isolated from NTPDase1 deficient (Entpd1−/−) mice compared to wild type controls. The strongest responses were obtained with uridine 5′-triphosphate (UTP) and uridine 5′-diphosphate (UDP), suggesting the involvement of P2Y6 receptors, which was confirmed with P2ry6−/− bladder strips. Interestingly, this response was reduced in female bladders. Our results also suggest the participation of P2X1, P2Y2 and/or P2Y4, and P2Y12 in these contractions. A reduced response to the thromboxane analogue U46619 was also observed in wild type, Entpd1−/−, and P2ry6−/− female bladders showing another difference due to sex. In summary, NTPDase1 modulates the activation of nucleotide receptors in mouse bladder SMCs, and contractions induced by P2Y6 receptor activation were weaker in female bladders.

Download Full-text

Reduced biomechanical models for precision-cut lung-slice stretching experiments

Journal of Mathematical Biology ◽

10.1007/s00285-021-01578-2 ◽

2021 ◽

Vol 82 (5) ◽

Author(s):

Hannah J. Pybus ◽

Amanda L. Tatler ◽

Lowell T. Edgar ◽

Reuben D. O’Dea ◽

Bindi S. Brook

Keyword(s):

Smooth Muscle ◽

Muscle Contraction ◽

Ex Vivo ◽

Finite Thickness ◽

Biomechanical Model ◽

Local Stress ◽

Smooth Muscle Contraction ◽

Biomechanical Models ◽

Cytokine Activation ◽

Asymptotic Reduction

AbstractPrecision-cut lung-slices (PCLS), in which viable airways embedded within lung parenchyma are stretched or induced to contract, are a widely used ex vivo assay to investigate bronchoconstriction and, more recently, mechanical activation of pro-remodelling cytokines in asthmatic airways. We develop a nonlinear fibre-reinforced biomechanical model accounting for smooth muscle contraction and extracellular matrix strain-stiffening. Through numerical simulation, we describe the stresses and contractile responses of an airway within a PCLS of finite thickness, exposing the importance of smooth muscle contraction on the local stress state within the airway. We then consider two simplifying limits of the model (a membrane representation and an asymptotic reduction in the thin-PCLS-limit), that permit analytical progress. Comparison against numerical solution of the full problem shows that the asymptotic reduction successfully captures the key elements of the full model behaviour. The more tractable reduced model that we develop is suitable to be employed in investigations to elucidate the time-dependent feedback mechanisms linking airway mechanics and cytokine activation in asthma.

Download Full-text

Myorelaxant Effect of the Dysphania ambrosioides Essential Oil on Sus scrofa domesticus Coronary Artery and Its Toxicity in the Drosophila melanogaster Model

Molecules ◽

10.3390/molecules26072041 ◽

2021 ◽

Vol 26 (7) ◽

pp. 2041

Author(s):

Luiz Jardelino de Lacerda Neto ◽

Andreza Guedes Barbosa Ramos ◽

Renata Evaristo Rodrigues da Silva ◽

Luís Pereira-de-Morais ◽

Fernanda Maria Silva ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Smooth Muscle ◽

Coronary Artery ◽

Essential Oil ◽

Coronary Arteries ◽

Sus Scrofa ◽

Ex Vivo ◽

Alternative Methods ◽

Major Constituent ◽

Sus Scrofa Domesticus

Purpose: Alternative methods for the use of animals in research have gained increasing importance, due to assessments evaluating the real need for their use and the development of legislation that regulates the subject. The principle of the 3R’s (replacement, reduction and refinement) has been an important reference, such that in vitro, ex vivo and cord replacement methods have achieved a prominent place in research. Methods: Therefore, due to successful results from studies developed with these methods, the present study aimed to evaluate the myorelaxant effect of the Dysphania ambrosioides essential oil (EODa) using a Sus scrofa domesticus coronary artery model, and the toxicity of both the Dysphania ambrosioides essential oil and its major constituent, α-terpinene, against Drosophila melanogaster in toxicity and negative geotaxis assays. Results: The EODa relaxed the smooth muscle of swine coronary arteries precontracted with K+ and 5-HT in assays using Sus scrofa domesticus coronary arteries. The toxicity results presented LC50 values of 1.546 mg/mL and 2.282 mg/mL for the EODa and α-terpinene, respectively, thus showing the EODa and α-terpinene presented toxicity to these dipterans, with the EODa being more toxic. Conclusions: Moreover, the results reveal the possibility of using the EODa in vascular disease studies since it promoted the relaxation of the Sus scrofa domesticus coronary smooth muscle.

Download Full-text

Diagnostic Open Lung Biopsy in the Critically Ill Child

PEDIATRICS ◽

10.1542/peds.52.4.605 ◽

1973 ◽

Vol 52 (4) ◽

pp. 605-608

Author(s):

Stacy A. Roback ◽

William H. Weintraub ◽

Mark Nesbit ◽

Panayiotis K. Spanos ◽

Barbara Burke ◽

...

Keyword(s):

Pulmonary Function ◽

Critically Ill ◽

Lung Tissue ◽

Lung Biopsy ◽

Open Lung Biopsy ◽

University Of Minnesota ◽

Biopsy Technique ◽

Open Lung ◽

Critically Ill Child ◽

The University

Forty-six open biopsies in 40 acutely ill children with rapidly decreasing pulmonary reserve were performed at the University of Minnesota Hospitals between January 1, 1970, and January 1, 1972. Tissue obtained was adequate in all patients and no serious complications ensued. Information obtained resulted in the change in treatment in 30 patients. This procedure is recommended over closed biopsy when the magnitude of the patient's illness and degree of pulmonary function do not allow acceptance of the risks known to occur with a closed biopsy technique and when histologic examination of lung tissue is required.

Download Full-text

Childhood Asthma: Update

Pediatrics in Review ◽

10.1542/pir.13.11.403 ◽

1992 ◽

Vol 13 (11) ◽

pp. 403-412

Author(s):

Gail G. Shapiro

Keyword(s):

Smooth Muscle ◽

Mast Cells ◽

Airway Inflammation ◽

Immunoglobulin E ◽

Bronchial Hyperresponsiveness ◽

Basement Membrane Thickening ◽

Infiltrating Cells ◽

Lung Biopsies ◽

Airways Reactivity ◽

Mast Cell Mediator Release

Definition and Pathophysiology Asthma is a reversible airways disease characterized by both smooth muscle hyperreactivity and airway inflammation. During the 1970s and early 1980s the focus was on smooth muscle constriction, and it was believed that better bronchodilators would greatly diminish our difficulties in controlling this condition. This, unfortunately, was not the case. The emphasis of therapy today has turned to airway inflammation. Lung biopsies from patients who have asthma show destruction of respiratory epithelium, basement membrane thickening, and inflammatory cellular infiltrate. Among the infiltrating cells are eosinophils, macrophages, and neutrophils that are called to the site of inflammation by the chemotactic products released by activated mast cells. Upon their arrival, these cells release their own products of inflammation, which amplify this immunologic response. A variety of neuropeptides also play a role, some serving to stabilize and others to destabilize the airway. One result of this airway inflammation is airways reactivity, also known as bronchial hyperresponsiveness. A common example of this scenario is the child who has allergic asthma and encounters a problematic allergen. This child has immunoglobulin E (IgE) to this allergen bound to mast cells in his or her airway. Upon exposure to the allergen, the binding of IgE and antigen triggers mast cell mediator release within minutes.

Download Full-text

MO583IN VITRO AND EX VIVO EXPERIMENTS OF VASCULAR CALCIFICATION

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfab086.0021 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

Jana Holmar ◽

Heidi Noels ◽

Joachim Jankowski ◽

Setareh Orth-Alampour

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Calcification ◽

Vascular Smooth Muscle Cells ◽

Incubation Time ◽

Fetal Calf Serum ◽

Ex Vivo ◽

Calf Serum

Abstract Background and Aims Vascular calcification (VC) is one major complication in patients with chronic kidney disease whereas a misbalance in calcium and phosphate metabolism plays a crucial role. The mechanisms underlying VC have not been entirely revealed to date. Therefore are the studies aiming at the identification and characterization of the mediators/uremic toxins involved in VC ongoing and highly relevant. However, currently many different protocols being used in the studies of vascular calcification processes. This complicates the comparison of study outcomes, composing systematic reviews, and meta-analyses. Moreover, the reproducibility of data is hampered, and the efficiency in calcification research through the lack of a standardized protocol is reduced. In this study, we developed a standardized operating protocol for in vitro and ex vivo approaches to aiming at the comparability of these studies. Method We analysed in vitro and ex vivo experimental conditions to study VC. Vascular smooth muscle cells (HAoSMCs) were used for in vitro experiments and aortas from Wistar rats were used for ex vivo experiments. The influence of the following conditions was studied in detail: • Phosphate and calcium concentrations in calcifying media. • Incubation time. • Fetal calf serum (FCS) concentration. The degree of calcification was estimated by quantification of calcium concentrations that were normalized to protein content (in vitro) or to the dry weight of the aortic ring (ex vivo). Additionally, the aortic rings were stained using the von Kossa method. Optimal conditions for investigating medial vascular calcification were detected and summarized in the step-by-step protocol. Results We were able to demonstrate that the degree and the location of VC in vascular smooth muscle cells and aortic rings were highly dependent on the phosphate and CaCl2 concentration in the medium as well as the incubation time. Furthermore, the VC was reduced upon increasing fetal calf serum concentration in the medium. An optimized protocol for studying vascular calcification in vitro and ex vivo was developed and validated. The final protocol (Figure 1) presented will help to standardize in vitro and ex vivo approaches to investigate the processes of vascular calcification. Conclusion In the current study, we developed and validated a standardized operating protocol for systematic in vitro and ex vivo analyses of medial calcification, which is essential for the comparability of the results of future studies.

Download Full-text

Axial Stretch Increases Cell Proliferation in Arteries in Organ Culture

10.1115/imece2000-2516 ◽

2000 ◽

Author(s):

Hai-Chao Han ◽

Raymond P. Vito ◽

Kristin Michael ◽

David N. Ku

Keyword(s):

Cell Proliferation ◽

Smooth Muscle ◽

Organ Culture ◽

Vascular Function ◽

Culture System ◽

Carotid Arteries ◽

Ex Vivo ◽

Axial Stretch ◽

Organ Culture System ◽

Physiological Flow

Abstract To study the effect of axial stretch on vascular function and wall remodeling, porcine carotid arteries were cultured under conditions of physiological flow and elevated axial stretch in an ex vivo organ culture system. Smooth muscle cell proliferation was measured by bromodeoxyuridine index. Results showed that cell proliferation was significantly increased in the highly stretched arteries when compared to the normally stretched arteries. This may indicate the feasibility of stimulating new arterial growth by stretching natural arteries.

Download Full-text

Abstract 1710: Pharmacologic Profile of SCH 530348, a Novel Oral Antiplatelet Agent Selective for the Protease-Activated Receptor-1 (PAR-1)

Circulation ◽

10.1161/circ.118.suppl_18.s_374 ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Madhu Chintala ◽

Ho-Sam Ahn ◽

Carolyn Foster ◽

Jacqueline Agans ◽

George Boykow

Keyword(s):

Smooth Muscle ◽

Platelet Aggregation ◽

Thymidine Incorporation ◽

Human Platelet ◽

Ex Vivo ◽

Calcium Transients ◽

Thrombin Receptor ◽

Cynomolgus Monkeys ◽

Sch 530348 ◽

Atherothrombotic Disease

Antiplatelet agents are a cornerstone of therapy for atherothrombotic disease. However, despite the proven efficacy of agents targeting the thromboxane A2 (aspirin) and P2Y12 ADP (clopidogrel, prasugrel) platelet activation pathways, the residual risk for ischemic events remains considerable. Binding of thrombin to the platelet PAR-1 is an important platelet activation pathway not targeted by existing agents. Inhibition of PAR-1 may thus provide incremental clinical benefits over aspirin and ADP receptor antagonists. PAR-1 receptors expressed on endothelial cells, smooth muscle cells, monocytes and neutrophils have been reported to mediate the pro-inflammatory and chemotactic responses to thrombin. In the present study, we report pharmacologic characterization of SCH 530348, a novel thrombin receptor antagonist (TRA) selective for PAR-1, using in vitro assays with human platelet membranes and cultured human coronary artery smooth muscle cells (HCASMC), and ex vivo platelet aggregation assays in cynomolgus monkeys. The affinity of SCH 530348 for the PAR-1 receptor was determined in human platelet membranes. Functional studies involving calcium transients, thymidine incorporation, and receptor kinetics were performed in HCASMC. The oral antiplatelet effects of SCH 530348 in cynomolgus monkeys were evaluated in ex vivo platelet aggregation assays. SCH 530348 exhibited high affinity for PAR-1 receptor. SCH 530348 potently inhibited thrombin-stimulated calcium transients and thymidine incorporation in HCASMC, and displayed slow dissociation from PAR-1 receptor. In cynomolgus monkeys, SCH 530348 administered orally at doses ranging from 0.1 mg/kg to 3 mg/kg, provided rapid, complete, and sustained inhibition of thrombin receptor agonist peptide (TRAP)-induced ex vivo platelet aggregation for 24 hours. Significant inhibition of TRAP-induced platelet aggregation was maintained at 48 hours after dosing of SCH 530348. SCH 530348 is a highly selective, potent, and orally active PAR-1 antagonist. Inhibition of PAR-1 by SCH 530348 may translate into beneficial clinical effects in patients with atherothrombotic disease, and this hypothesis is currently being evaluated in 2 large trials.

Download Full-text