scholarly journals Sensing of Vascular Permeability in Inflamed Vessel of Live Animal

2018 ◽  
Vol 2018 ◽  
pp. 1-6 ◽  
Author(s):  
Sang A Park ◽  
Soi Jeong ◽  
Young Ho Choe ◽  
Young-Min Hyun
Keyword(s):  
Vascular Permeability ◽  
Real Time ◽  
Intravital Microscopy ◽  
Early Stage ◽  
Neutrophil Migration ◽  
Cremaster Muscle ◽  
Live Animal ◽  
Two Photon ◽  
Inflammatory State ◽  
Permeability Increase

Increase in vascular permeability is a conclusive response in the progress of inflammation. Under controlled conditions, leukocytes are known to migrate across the vascular barriers to the sites of inflammation without severe vascular rupture. However, when inflammatory state becomes excessive, the leakage of blood components may occur and can be lethal. Basically, vascular permeability can be analyzed based on the intensity of blood outflow. To evaluate the amount and rate of leakage in live mice, we performed cremaster muscle exteriorization to visualize blood flow and neutrophil migration. Using two-photon intravital microscopy of the exteriorized cremaster muscle venules, we found that vascular barrier function is transiently and locally disrupted in the early stage of inflammatory condition induced by N-formylmethionyl-leucyl-phenylalanine (fMLP). Measurement of the concentration of intravenously (i.v.) injected Texas Red dextran inside and outside the vessels resulted in clear visualization of real-time increases in transient and local vascular permeability increase in real-time manner. We successfully demonstrated repeated leakage from a target site on a blood vessel in association with increasing severity of inflammation. Therefore, compared to other methods, two-photon intravital microscopy more accurately visualizes and quantifies vascular permeability even in a small part of blood vessels in live animals in real time.

Endothelial LSP1 is involved in endothelial dome formation, minimizing vascular permeability changes during neutrophil transmigration in vivo

Blood ◽  
2011 ◽  
Vol 117 (3) ◽  
pp. 942-952 ◽  
Author(s):  
Björn Petri ◽  
Jaswinder Kaur ◽  
Elizabeth M. Long ◽  
Hang Li ◽  
Sean A. Parsons ◽  
...  
Keyword(s):  
Vascular Permeability ◽  
Neutrophil Migration ◽  
Endothelial Activation ◽  
Specific Protein ◽  
Actin Binding ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Factor Α

Abstract The endothelium actively participates in neutrophil migration out of the vasculature via dynamic, cytoskeleton-dependent rearrangements leading to the formation of transmigratory cups in vitro, and to domes that completely surround the leukocyte in vivo. Leukocyte-specific protein 1 (LSP1), an F-actin–binding protein recently shown to be in the endothelium, is critical for effective transmigration, although the mechanism has remained elusive. Herein we show that endothelial LSP1 is expressed in the nucleus and cytosol of resting endothelial cells and associates with the cytoskeleton upon endothelial activation. Two-photon microscopy revealed that endothelial LSP1 was crucial for the formation of endothelial domes in vivo in response to neutrophil chemokine keratinocyte-derived chemokine (KC) as well as in response to endogenously produced chemokines stimulated by cytokines (tumor necrosis factor α [TNFα] or interleukin-1β [IL-1β]). Endothelial domes were significantly reduced in Lsp1−/− compared with wild-type (WT) mice. Lsp1−/− animals not only showed impaired neutrophil emigration after KC and TNFα stimulation, but also had disproportionate increases in vascular permeability. We demonstrate that endothelial LSP1 is recruited to the cytoskeleton in inflammation and plays an important role in forming endothelial domes thereby regulating neutrophil transendothelial migration. The permeability data may underscore the physiologic relevance of domes and the role for LSP1 in endothelial barrier integrity.

The Use Of Two Photon Microscopy To Image Vaso-Occulsion In Pulmonary Microvessels Of Living Mice With Sickle Cell Disease

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 976-976
Author(s):  
Natacha Ralainirina ◽  
Ferron Lynn Sonderegger ◽  
Hong Pei ◽  
Grzegorz Chodaczek ◽  
Joel Linden
Keyword(s):  
Endothelial Cells ◽  
Sickle Cell Disease ◽  
Real Time ◽  
Sickle Cell ◽  
Intravital Microscopy ◽  
Blood Cells ◽  
White Blood Cells ◽  
Cell Disease ◽  
Alexa Fluor ◽  
Two Photon

Abstract Although sickle cell anemia is initiated by red cell pathology, it is accompanied by an inflammatory immune response involving platelets and white blood cells that contribute to vaso-occlusvie episodes including painful vaso-occlusive crises (pVOC) and acute chest syndrome (ACS). In order to better understand the cellular and molecular bases of vaso-occlusion we are in the process of developing procedures to image microvessels in the lung, liver and spleen of living mice by 2-photon microscopy, a procedure that is based on excitation of a fluorophore by two photons simultaneously. The two-photon technique utilizes infrared light that efficiently penetrates tissues up to 200 microns with low phototoxicity allowing time-lapse imaging. Two-photon intravital microscopy can be used to study the behavior of intravascular cells during vaso-occlusive events. Mice are prepared for lung intravital microscopy by the intraperitoneal injection of a mixture of ketamine and xylazine. Additional anesthesia is added during experimentation. The trachea is opened and the mouse intubated. The chest is opened to allow access to the left lobe of the lung through a window that is a few millimeters in diameter. PBS is applied to keep the lung moist. A custom built suction device is placed on the lung and covered with a cover glass at the same time pressure is exerted to seal the organ and the glass cover together. Throughout the procedure, the mouse is held at a temperature of 37°C. Once surgery is completed, a mixture of antibodies coupled to fluorophores is given by retro-orbital injection. In order to minimize photobleaching we used antibodies conjugated to Alexa Fluor 488, Alexa Fluor 555 or Alexa Fluor 647. We are able to visualize and quantify interactions between red blood cells, white blood cells, and endothelial cells as well as the expression of adhesion molecules on endothelial cells in real time. During pVOC triggered by hypoxia, cell adhesion of neutrophils, lymphocytes and monocytes to the endothelium is observed that is associated with an increase in endothelial expression of ICAM-1 and V-CAM. We label endothelial cells with anti-CD31, lymphocytes with anti-CD45, monocytes with anti-Ly6C and neutrophils with anti-Ly6G. Platelets are labeled with anti-CD41 or anti-CD62P, NK cells with anti-NKp46, and macrophages with anti-F4/80 and anti-CD1d. We are able to quantify cell shape, rolling, adhesion and movement. Our preliminary results demonstrate that it is possible in real time to image the sequence of events occurring during pulmonary vasoocclusion in sickle cell disease. In conclusion, intravital 2-photon microscopy holds great potential for enabling us to better understand inflammatory responses within the blood vessels of living SCD mice. Disclosures: No relevant conflicts of interest to declare.

Rational design of a ratiometric two-photon fluorescent probe for real-time visualization of apoptosis

2018 ◽  
Vol 54 (74) ◽  
pp. 10495-10498 ◽  
Author(s):  
Yinliang Huang ◽  
Qin Zhou ◽  
Yan Feng ◽  
Wan Zhang ◽  
Guoshun Fang ◽  
...  
Keyword(s):  
Real Time ◽  
Fluorescent Probe ◽  
Rational Design ◽  
Early Stage ◽  
Two Photon ◽  
Dual Color ◽  
Real Time Visualization ◽  
Color Visualization

A ratiometric two-photon fluorescent probe (Mito-DCHO) was rationally designed for real-time assessing and dual-color visualization of the early stage of apoptosis.

scholarly journals The Ability of Metabolomics to Discriminate Non-Small-Cell Lung Cancer Subtypes Depends on the Stage of the Disease and the Type of Material Studied

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3314
Author(s):  
Tomasz Kowalczyk ◽  
Joanna Kisluk ◽  
Karolina Pietrowska ◽  
Joanna Godzien ◽  
Miroslaw Kozlowski ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Early Stage ◽  
Chronic Obstructive ◽  
Liquid Chromatography Mass Spectrometry ◽  
Obstructive Pulmonary Disease ◽  
Cell Lung Carcinoma ◽  
Cancer Subtypes ◽  
Inflammatory State ◽  
Nsclc Patients

Identification of the NSCLC subtype at an early stage is still quite sophisticated. Metabolomics analysis of tissue and plasma of NSCLC patients may indicate new, and yet unknown, metabolic pathways active in the NSCLC. Our research characterized the metabolomics profile of tissue and plasma of patients with early and advanced NSCLC stage. Samples were subjected to thorough metabolomics analyses using liquid chromatography-mass spectrometry (LC-MS) technique. Tissue and/or plasma samples from 137 NSCLC patients were analyzed. Based on the early stage tissue analysis, more than 200 metabolites differentiating adenocarcinoma (ADC) and squamous cell lung carcinoma (SCC) subtypes as well as normal tissue, were identified. Most of the identified metabolites were amino acids, fatty acids, carnitines, lysoglycerophospholipids, sphingomyelins, plasmalogens and glycerophospholipids. Moreover, metabolites related to N-acyl ethanolamine (NAE) biosynthesis, namely glycerophospho (N-acyl) ethanolamines (GP-NAE), which discriminated early-stage SCC from ADC, have also been identified. On the other hand, the analysis of plasma of chronic obstructive pulmonary disease (COPD) and NSCLC patients allowed exclusion of the metabolites related to the inflammatory state in lungs and the identification of compounds (lysoglycerophospholipids, glycerophospholipids and sphingomyelins) truly characteristic to cancer. Our results, among already known, showed novel, thus far not described, metabolites discriminating NSCLC subtypes, especially in the early stage of cancer. Moreover, the presented results also indicated the activity of new metabolic pathways in NSCLC. Further investigations on the role of NAE biosynthesis pathways in the early stage of NSCLC may reveal new prognostic and diagnostic targets.

Cascade photocaging of diazeniumdiolate: a novel strategy for one and two photon triggered uncaging with real time reporting

2017 ◽  
Vol 53 (68) ◽  
pp. 9470-9473 ◽  
Author(s):  
Krishna Kalyani Behara ◽  
Y. Rajesh ◽  
Yarra Venkatesh ◽  
Bhaskar Rao Pinninti ◽  
Mahitosh Mandal ◽  
...  
Keyword(s):  
Real Time ◽  
Two Photon ◽  
New Strategy ◽  
Novel Strategy

We report a new strategy, viz. cascade photocaging, for protecting diethylamine diazeniumdiolate (O2-position), a light sensitive molecule.

scholarly journals Real‐time two‐photon confocal microscopy using a femtosecond, amplified Ti:sapphire system

1996 ◽  
Vol 181 (3) ◽  
pp. 253-259 ◽  
Author(s):  
G. J. BRAKENHOFF ◽  
J. SQUIER ◽  
T. NORRIS ◽  
A. C. BLITON ◽  
M. H. WADE ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Real Time ◽  
Two Photon

In vivo imaging of early stage apoptosis by measuring real-time caspase-3/7 activation

APOPTOSIS ◽  
2010 ◽  
Vol 16 (2) ◽  
pp. 198-207 ◽  
Author(s):  
Matteo Scabini ◽  
Fabio Stellari ◽  
Paolo Cappella ◽  
Sara Rizzitano ◽  
Gemma Texido ◽  
...  
Keyword(s):  
Real Time ◽  
In Vivo Imaging ◽  
Caspase 3 ◽  
Early Stage
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