An Optimized Evans Blue Protocol to Assess Vascular Leak in the Mouse

Summary

In this article, an economical, optimized, and simple protocol is described which uses the Evans blue dye method for assessing plasma extravasation in the organs of FVBN mice that can be adapted for use in other strains, species, and other organs or tissues.

Abstract

Vascular leak, or plasma extravasation, has a number of causes, and may be a serious consequence or symptom of an inflammatory response. This study may ultimately lead to new knowledge concerning the causes of or new ways to inhibit or treat plasma extravasation. It is important that researchers have the proper tools, including the best methods available, for studying plasma extravasation. In this article, we describe a protocol, using the Evans blue dye method, for assessing plasma extravasation in the organs of FVBN mice. This protocol is intentionally simple, to as great a degree as possible, but provides high quality data. Evans blue dye has been chosen primarily because it is easy for the average laboratory to use. We have used this protocol to provide evidence and support for the hypothesis that the enzyme neprilysin may protect the vasculature against plasma extravasation. However, this protocol may be experimentally used and easily adapted for use in other strains of mice or in other species, in many different organs or tissues, for studies which may involve other factors that are important in understanding, preventing, or treating plasma extravasation. This protocol has been extensively optimized and modified from existing protocols, and combines reliability, ease of use, economy, and general availability of materials and equipment, making this protocol superior for the average laboratory to use in quantifying plasma extravasation from organs.

Introduction

Vascular leak in the organs refers to extravasation, or leakage of blood plasma through gaps produced in the endothelium of post capillary venules in the organs. This plasma extravasation or increased vascular permeability, which may arise from some type of an inflammatory response, may have grave consequences. Thus, it is important that this phenomenon, its causes, modulators, and consequences, are studied and understood, and likewise, that investigators have good tools and protocols with which to study them. The endothelial gaps may be produced via a number of stimuli, but usually are produced by the action of peptide neurotransmitters and/or tachykinins on the endo....

Protocol

All applicable international, national, and/or institutional guidelines for the care and use of animals (mice) were followed in the experiments described in this manuscript.

This method uses FVBN adult mice, aged 16 - 20 weeks, found to be optimal for the purposes of this study. Day 1 includes steps 1 - 5 and Day 2 includes steps 6 - 7 (Figure 1).

1. Equipment Preparation

1. Secure an adequate supply of sterile, disposable syringes and needles, if ketamine/xylazine is used as the anesthetic (as recommended). If isoflurane is used as the anesthetic, check the oxygen tank and ....

Results

In Figure 1, a schematic of the procedure is shown, which has been found to result in the most reliable and consistent substance P-induced plasma extravasation values from organs of FVBN mice. This procedure usually takes two days of work, separated by at least 48 h of waiting time. It is possible to spread it out even more, if this is done consistently for all experiments to be compared. For example, after the organs are isolated on Day 1, the organs can be .......

Discussion

As discussed above, the study of plasma extravasation may ultimately lead to new knowledge concerning the causes of or new ways to inhibit or treat plasma extravasation. The successful use of the plasma extravasation protocol (above), using Evans blue dye, has been demonstrated in the current manuscript. Although the data shown back the hypothesis that NEP may protect the vasculature against plasma extravasation, this is a secondary goal presently, with the primary goal being to present an optimized protocol which may be.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
isoflurane	Vet One	200-070	inhaled anesthetic
ketamine	Vet One	200-055	injectable anesthetic
xylazine	Lloyd Laboratories	139-236	injectable anesthetic
syringes (10,3 & 1 cc)	Becton Dickinson	309604, 309657, 309659
needles (20G1,23G1 & 26G1/2)	Becton Dickinson	305178, 305193, 305111
isoflurane induction chamber	VetEquip	941443	1 Liter
nosecone breathing circuits	VetEquip	RC2	Rodent Circuit Controller 2
oxygen tank	Airgas	UN 1072	100% medical
heating pad	CWE Inc.	TC-1000	temperature controller
rectal temperature probe	CWE Inc.	10-09012	mouse
balance (for rodents)	Ohaus	CS 2000
surgical tools-scissors	Fine Science tools	15000-00	Vannas Spring scissors 3mm straight blade (cutting vessels)
surgical tools-forceps	Fine Science tools	11151-10	Graefe extra fine forceps (isolating mouse vessels)
surgical tools-hemostats	Fine Science tools	13009-12	Halstead-mosquito hemostats (blunt dissect, hold tissue)
surgical tools -suture drivers	Fine Science tools	12502-12	Olsen-Hegar suture drivers (suturing)
surgical tools-forceps	Fine Science tools	11627-12	Adson-Brown alligator forceps (tissue grasping suturing, rat)
surgical tools-scissors	Fine Science tools	14110-15	Mayo tough cut scissors 15 cm (surgery, dissection, bones, rat)
surgical tools-forceps	Fine Science tools	18025-10	suture tying forceps (used for Millar cath)
surgical tools-scissors	Fine Science tools	14078-10	Lexer Baby scissors straight (surgery, mouse)
surgical tools-forceps	Fine Science tools	11254-20	Dumont #5 fine-tip forceps (rat vessels, dissection)
surgical tools-scissors	Fine Science tools	14082-09	Dissector scissors 12 mm (surgery, rat mouse)
surgical tools-forceps	Fine Science tools	11051-10	10 cm Graefe forceps (tissue grasping, rat mouse)
surgical tools-forceps	Fine Science tools	11251-35	Dumont 5/45 forceps (introducer for vessels)
surgical tools-retractors	Fine Science tools	17012-11	Weitlaner retractors 2/3 tooth (rat surgical)
surgical tools-forceps	Fine Science tools	11294-00	Dumont #4 forceps (vessel isolation rats, mice)
surgical tools-forceps	Fine Science tools	11297-00	Dumont #7 forceps (tissue grasping, dissection)
surgical tools-scissors	Fine Science tools	14058-11	tough cut iris scissors (mouse dissection, bones)
surgical tools-forceps	Fine Science tools	11009-13	serrated, curved Semken forceps (tissue grasping, mouse rat)
surgical tools-hemostats	Fine Science tools	13003-10	Hartman curved hemostats (blunt dissect, hold tissue)
surgical tools-forceps	Fine Science tools	11006-12	Adson serrated forceps (tissue grasping)
clippers	Oster	A5
tape	Fisherbrand	159015G
artificial tear ointment	Akorn Inc	13985-600-03
lidocaine	Hospira	0409-4277-01	2% injectable
polyvinyl catheters	Tygon	PV-1
Evans blue	Sigma Aldrich	E2129
Substance P	Bachem	H-1890
heparin	Sagent Pharmaceuticals	25201-400-10	1000 U/ml
saline solution	Hospira	0409-7138-09	0.9% sodium chloride
phenobarbital	Vortech	0298-9373-68
sodium citrate	Fisher Scientific	BP327-1
PBS	Sigma Aldrich	P4417-50TAB
Kimwipes for blotting	Fisher Scientific	06-666A
formamide	Sigma Aldrich	47670
microbalance	Denver Instrument	APX-60
microfuge tubes	Fisher Scientific	07-200-534
polystyrene 96 well plate	Becton Dickenson	351172
absorbance plate reader	BioTek	Synergy 2
polyacrylamide gels	Bio-Rad	3450014
protein molecular weight standard	Bio-Rad	1610374
Protran supported nitrocellulose	Amersham (GE)	10600015
gel box	Bio-Rad	1658005
Tris	Fisher Scientific	BP152-1
Tween20	Sigma Aldrich	P-1379
sodium chloride	Fisher Scientific	S271-1
primary NEP polyclonal antibody	R & D Systems	AF1182
doxycycline chow	Teklad (HARLAN)	TD.130750
FVB/NJ wild type mice	Jackson	001800
secondary antibody (goat anti-rabbit)	ZyMed	81-6120
ECL solution-Western Lightening Plus	PerkinElmer	NEL104001EA
film	Pierce	34091