An in vivo Assay to Test Blood Vessel Permeability

Summary

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

Abstract

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

Introduction

Formation and maintenance of selective permeable barriers are essential for proper organ development and performance ^1,2. Endothelial cells line the blood vessel lumen and form a semi-permeable barrier that is essential in the selective transport between blood and the interstitial space of all organs. An adequate permeability barrier is maintained through tight cell-to-cell junctions that are strictly controlled by growth factors, cytokines and other stress related molecules ³. Disruption of the endothelial cell barrier can result in increased permeability and vascular leakage. These effects are seen in various disease states and the understandin....

Protocol

1. Intravenous Injection of Evans Blue in the Mouse Lateral Tail Vein

1. Prepare a 0.5% sterile solution of Evans blue in PBS. If necessary, filter-sterilize the solution to remove any particulate matter that has not dissolved.
2. Aspirate 200 μl Evans Blue solution into a syringe. Avoid all air bubbles that might have escaped into the syringe.
3. Place mice that are 8-12 weeks old into a restraint device so that the animal is not freely mobile but its tail can be handled.
4. Place the mouse restraint device on its side so the lateral tail vein is easily visible and are facing upward.
5. Hold onto ....

Results

We used an in vivo permeability assay to test vessel leakage in mice 8-12 weeks old. This test is useful in comparing the relative vascular permeability between animals of different genetic background or in a single strain of mice subjected to treatments that affect the vasculature. Our results show that a strain of genetically modified mice we created in our lab has a more permeable endothelium compared to wild-type mice. These changes are evident at macroscopic level in many organs (Figure 1).......

Discussion

Vascular permeability is a critical marker for blood vessel status. Increased vascular permeability has been shown to be present in several systemic diseases, including diabetes, hypertension, and autoimmune diseases ^6,7,8. Increased vascular permeability has been shown to be mediated by shear stress, growth factors like vascular endothelial growth factor and fibroblast growth factor, inflammatory mediators like serotonin, histamine and bradykinin ⁹. Extravasation of water and small molecules is tho.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
REAGENT
EVANS BLUE	SIGMA	E2129
FORMAMIDE	INVITROGEN	15515-026
PBS			0.2M Phosphate 1.5M NaCl pH 7.4
EQUIPMENT
SPECTROPHOTOMETER	EPPENDORF	952000006
MOUSE RESTRAINT DEVICE	HARVARD APPARATUS	340012
SYRINGE	BD	309659
NEEDLES	BD	305106	The gauge of the needle depends on the size of the animal.
BALANCE	DENVER INSTRUMENT	TP-64