Vascular Hyperpermeability: Methods and Protocols (Methods in Molecular Biology, 2711)

✍ Scribed by Binu Tharakan (editor)

• Publisher: Humana
• Year: 2023
• Tongue: English
• Leaves: 266
• Category: Library

✦ Synopsis

This volume focuses on the latest research methods and techniques used by researchers to study vascular permeability/hyperpermeability in a science laboratory setting. The chapters in this book cover topics such as determination of solute permeability of microvascular endothelial cell monolayers in vitro; evaluation of barrier integrity used in a two-layered microfluidic device mimicking the blood-brain barrier; isolation and culture of human umbilical vein endothelial cells; measurement of blood-brain barrier hyperpermeability using Evans Blue extravasation assay; lymphatic vascular permeability determined from direct measurements of solute flux; intravital imaging of leukocyte-endothelial interaction by intravital multiphoton microscopy; and evaluation of tight junction integrity in brain endothelial cells using confocal microscopy. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Thorough and cutting-edge, Vascular Hyperpermeability: Methods and Protocols is a valuable resource that will aid novice researchers with creating new and affordable research projects, and for expert researchers to initiate new strategies and collaborations in their ongoing programs.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Determination of Solute Permeability of Microvascular Endothelial Cell Monolayers In Vitro
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Endothelial Cell Monolayers for Study
3.2 Assay Variation 1: Transwell Membranes
3.3 Assay Variation 2: Snapwell Membranes
4 Notes
References
Chapter 2: Evaluation of Vascular Permeability in Inflamed Vessels of the Cremaster Muscle in Live Mice
1 Introduction
2 Materials
2.1 Imaging Chamber Preparation
2.2 Blood Vessel Labeling
2.3 Instruments
3 Method
3.1 Cremaster Muscle Exposure Surgery
3.2 Two-Photon Intravital Microscopy
3.3 Vascular Permeability Measurement
4 Notes
References
Chapter 3: Lymphatic Vascular Permeability Determined from Direct Measurements of Solute Flux
1 Introduction
2 Materials
2.1 Krebs Buffer and Fluorescent Tracers
2.2 Forceps and Spring Scissors
2.3 Dissection Chamber, Recessed into Table
2.4 Glass Micropipette Fabrication
2.5 Syringes for Filling Micropipettes and Controlling Pressure
2.6 Micropipette Mounting Stage and Pump
2.7 Dissecting Microscope and Light Source
2.8 Inverted Fluorescence Microscope
2.9 Pressure Control System and Peristaltic Pump
3 Methods
3.1 Locate and Remove Mesenteric Lymphatic Vessel
3.2 Dissect the Vessel from Connective Tissues
3.3 Set Up the Micropipettes on the Mounting Stage
3.4 Cannulate the Vessel
3.5 Move the Cannulation Chamber to the Microscope
3.6 Conduct the Experiment
3.7 Cleaning of Equipment
4 Notes
References
Chapter 4: Evaluation of Mesenteric Microvascular Hyperpermeability Following Hemorrhagic Shock Using Intravital Microscopy
1 Introduction
2 Materials
2.1 Anesthesia Induction
2.2 Operative Equipment
2.3 Shock Induction
2.4 Vascular Permeability Monitoring
3 Methods
3.1 Anesthesia
3.2 Preparation
3.3 Jugular Vein
3.4 Carotid Artery
3.5 Femoral Artery
3.6 Mesentery
3.7 Preshock
3.8 Shock
3.9 Intravital Microscopy
4 Notes
References
Chapter 5: Determination of Endothelial Barrier Resistance by Electric Cell-Substrate Impedance Sensing (ECIS) System
1 Introduction
1.1 Working Principle
1.2 Prerequisite Considerations and Optimization
2 Materials
2.1 Instrument Requirement
2.2 Buffers and Solutions
3 Methods
3.1 Instrument Setup
3.2 Calibration with Test Array (To Locate Respective Buttons on the Software, See Fig.3)
3.3 Coating of Array Wells
3.4 Seeding of the Cells
3.5 Medium Change and Thrombin Treatment
3.6 Data Analysis
3.7 Data Modeling for Determination of Rb and α
4 Notes
References
Chapter 6: Time-Lapse Observation of Cell Dynamics During Angiogenesis Using the Rat Mesentery Culture Model
1 Introduction
2 Materials
2.1 Animals
2.2 Solutions and Reagents
2.3 Surgical Tools
2.4 Other Materials
3 Methods
3.1 Preparation of Surgical Tools and Space
3.2 Preparation of the Rat
3.3 Mesenteric Tissue Exteriorization
3.4 Harvesting Mesentery Tissue
3.5 Mounting the Tissues for Culture
3.6 Imaging and Observation of Microvascular Networks
3.7 Post-imaging Immunohistochemistry
3.8 Representative Observations
4 Notes
References
Chapter 7: Evaluation of Barrier Integrity Using a Two-Layered Microfluidic Device Mimicking the Blood-Brain Barrier
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
3 Methods
3.1 Cell Culture
3.2 Microfluidic Chip Preparation
3.3 Seeding hBMECs, Astrocytes, and Pericytes to the Chips
3.4 Measuring BBB Integrity
3.4.1 Permeability Assay
3.4.2 Immunofluorescence Microscopy
4 Notes
References
Chapter 8: Intravital Imaging of Leukocyte-Endothelial Interaction in Hindlimb Ischemia/Reperfusion Injury by Intravital Multi...
1 Introduction
1.1 Why Use Multiphoton Excitation in Intravital Microscopy?
1.2 Considerations for the Microscope Setup
1.3 Why Use the Tibialis Anterior Muscle for the Evaluation of Leukocyte-Endothelial Interaction?
2 Materials
2.1 Preparation of the Tracer: Labeling Bovine Serum Albumin with Texas Red
2.2 Animals
2.3 Supplies
2.4 Optional: For Heart Rate Monitoring
2.5 Surgical Instruments
2.6 Microscope Setup for Two-Photon Intravital Microscopy of I/R Injured Muscle
3 Methods
3.1 Tail Vein Cannulation for Intravenous Injections
3.2 Tail Artery Cannulation for Monitoring of Blood Parameters (Optional)
3.3 Surgical Procedure
3.4 Choosing Good Regions of Interest and the Right Vessel Diameter
3.5 Digital Image and Video Analysis
4 Notes
References
Chapter 9: Studying Angiogenesis Using Matrigel In Vitro and In Vivo
1 Introduction
2 Materials
2.1 Cell Culture
2.2 2D and 3D Angiogenesis
2.3 Ex Vivo Sprouting Angiogenesis
2.4 Matrigel Plug Assay Materials
3 Methods
3.1 2D Angiogenesis/Tube Formation Assay
3.2 3D Angiogenesis/Tube Formation Assay
3.3 Ex Vivo Sprouting Angiogenesis Assay
3.4 Matrigel Plug Assay
4 Notes
References
Chapter 10: Determination of Blood-Brain Barrier Hyperpermeability Using Intravital Microscopy
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
2.3 Supplies
2.4 Working Solutions
3 Methods
3.1 Anesthetizing the Animals
3.2 Intravenous Injection via Tail Vein
3.3 Surgical Procedures
3.4 Traumatic Brain Injury
3.5 Intravital Microscopy (IVM)
3.6 Imaging Procedure
4 Notes
References
Chapter 11: Imaging and Analysis of the Dynamics of Filamentous Actin Structures in Live Endothelial Cells
1 Introduction
2 Materials
2.1 Transfection of Endothelial Cells with GFP-Actin (Nucleofector Method)
2.2 Setting Up the Chamber System
2.3 Acquisition of Data
2.4 Data Analysis
3 Methods
3.1 Transfection of Endothelial Cells with GFP-Actin (Nucleofector Method)
3.2 Setting Up the Chamber System
3.3 Acquisition of Data
3.4 Analysis of Imaging Data
4 Notes
References
Chapter 12: Isolation and Culture of Human Umbilical Vein Endothelial Cells (HUVECs)
1 Introduction
2 Materials
2.1 Reagents for Isolation and Culture
2.2 Reagents for Characterization and Validation
2.3 Instrument and Other Materials
2.4 Preparation of Reagents and Solutions
2.4.1 Sample Collection Buffer
2.4.2 Fibronectin Solution for Coating
2.4.3 Collagenase Solution
2.4.4 Endothelial Cell Culture Media
2.4.5 Paraformaldehyde for Cell Fixation
2.4.6 Blocking Solution for Immunofluorescence
3 Methods
3.1 Umbilical Cord Collection and Processing
3.2 Isolation and Culture of HUVECs
3.3 Trypsinization and Subculture of HUVECs
3.4 Characterization and Validation of HUVECs
3.4.1 Morphological Validation
3.4.2 Immunofluorescence Staining for Endothelial Markers
3.4.3 Dil-Acetylated LDL Uptake Assay and Lectin Staining
3.4.4 Tube Formation Assay
3.5 Troubleshooting
4 Notes
References
Chapter 13: Microvascular Endothelial Glycocalyx Surface Layer Visualization and Quantification
1 Introduction
2 Materials
2.1 Vascular Cannulation and IVM Preparation
2.2 Glycocalyx Visualization with BSI-Lectin and Dye-Exclusion Assay
3 Methods
3.1 Blood Vessel Cannulation
3.2 IVM Preparation
3.3 IVM: FITC-Lectin
3.4 FITC-Lectin Image Analysis
3.5 IVM: Dye-Exclusion Assay
3.6 Dye-Exclusion Assay Image Analysis
4 Notes
References
Chapter 14: Measurement of Blood-Brain Barrier Hyperpermeability Using Evans Blue Extravasation Assay
1 Introduction
2 Materials
2.1 Reagents and Working Solutions
2.2 Equipment
2.3 Supplies
3 Methods
3.1 Anesthetizing the Animals
3.2 Intravenous Injection of Evans Blue
3.3 Surgical Procedures
3.4 Traumatic Brain Injury
3.5 Evans Blue Injection
3.6 Transcardial Perfusion and Tissue Collection
3.7 Evans Blue Dye Extraction
4 Notes
References
Chapter 15: Assessment of Endothelial Barrier Functions in Extra Embryonic Vasculature of Chick Embryo as an Alternative Model
1 Introduction
1.1 Limitations
2 Materials
2.1 Instrumentation
2.2 Fennel Seed Extract (FSE) Preparation
2.3 Texas Red Preparation
2.4 Cell Culture Medium
2.5 Materials Required for CAM Experiments
2.6 Paraformaldehyde Preparation
3 Methods
3.1 In Vivo Chorioallantoic Membrane Permeability Assay Using Texas Red
3.2 Creating Ischemic on Chick Vascular Bed
3.3 Global Hypoxia Induction on Chick Vascular Bed
3.4 Phalloidin Staining in Chick Vascular Bed
3.5 Histology of the Vessel Middle of the Bed (VMD)
4 Notes
References
Chapter 16: Measurement of Transendothelial Electrical Resistance in Blood-Brain Barrier Endothelial Cells
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 17: An In Vitro Bilayer Model of Human Primary Retinal Pigment Epithelial and Choroid Endothelial Cells for Permeabili...
1 Introduction
2 Materials
2.1 Cell Culture Media and Chemicals
2.1.1 Cell Culture Plasticware and Other Accessories
2.2 ELISA Kits
2.3 Immunofluorescence Antibodies, Chemicals, and Plastics
2.4 RT PCR Materials
3 Methods
3.1 Coating the Culture Dishes with Extracellular Matrix Material
3.2 Preparation of Cadaver Eye Tissues Prior to Cell Isolation
3.2.1 Isolation and Culturing of Retinal Pigment Epithelial Cells from Human Eyecups
3.2.2 Isolation of Choroid Microvascular Endothelial Cells from Human Eyecups
3.3 Characterization of Isolated Primary Culture
3.3.1 RNA Isolation
3.3.2 cDNA Conversion and Semiquantitative RT-PCR
3.3.3 Gel Run and Imaging
3.3.4 Immunofluorescence Imaging
3.3.5 Tube Formation Assay
3.4 Bilayer Culture on Transwell Inserts
3.5 ELISA for PEDF and VEGF
3.6 Paracellular Permeability Assay Using the Bilayer Model
4 Notes
References
Chapter 18: Quantifying Adhesion of Inflammatory Cells to the Endothelium In Vitro
1 Introduction
2 Materials
3 Methods
3.1 Gelatin Coating of 48-Well HUVEC Culture Plates
3.2 Growth and TNFα-Mediated Activation of HUVECs
3.3 Growth and Labeling of THP-1 Cells
3.4 Assay of the Adhesion of the THP-1 Cells to the Endothelial Cell Monolayer
4 Notes
References
Chapter 19: Determination of Tight Junction Integrity in Brain Endothelial Cells Based on Tight Junction Protein Expression
1 Introduction
2 Materials
3 Methods
3.1 RBMEC Cell Lysate Preparation
3.2 Western Blot Analysis of ZO-1
4 Notes
References
Chapter 20: Evaluation of Glycolysis and Mitochondrial Function in Endothelial Cells Using the Seahorse Analyzer
1 Introduction
1.1 Cell Mito Stress Test
1.2 Glycolytic Rate Assay
1.3 ATP Production Rate Assay
2 Materials
3 Methods
3.1 Preparation of Cells and Cartridges
3.2 Hydration of Sensor Cartridges
3.3 Assay Media Preparation
3.4 Mito Stress Test Instructions
3.4.1 Prepare Compound Stock Solutions and Working Solutions
3.4.2 Prepare Seahorse XFp Cell Culture Miniplate for Assay
3.4.3 Running the Assay
3.5 Glycolytic Rate Assay
3.6 ATP Rate Assay
3.7 Protein Assay for Normalization
3.8 Data Analysis
4 Troubleshooting
References
Chapter 21: Evaluation of Tight Junction Integrity in Brain Endothelial Cells Using Confocal Microscopy
1 Introduction
2 Materials
3 Methods
3.1 Endothelial Cell Seeding
3.2 ZO-1 Immunofluorescence and Rhodamine Phalloidin Labeling for f-actin
4 Notes
References
Index