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

The overall goal of this protocol is to assess and relatively quantitate plasma extravasation or leakage in the organs of mice or other small animal species using a modified Evans Blue Dye method. This method can help answer key questions in the vascular field such as, which endogenous or exogenous factors modify plasma extravasation in a specific species or organ of interest? The main advantage of this technique is that although catheterization requires a modest degree of surgical competence, the method provides durable venous access and is reproducible from mouse to mouse.

Generally, individuals new to this method may struggle because of the modest surgical difficulty of the catheter placement. After weighing a 16 to 20-week-old adult FVB NJ mouse, confirm a lack of response to toe pinch and shave the ventral neck area of the animal. Place the mouse in the supine position on a preheated pad and tape all four paws to the surgical surface.

Apply ointment to the animal's eyes to prevent drying. Make a one centimeter incision in the right ventral neck over the jugular vein. Apply one or two drops of 1-2%lidocaine to the incision area for pain management and to promote vasodilation.

Isolate the right internal jugular vein via blunt dissection. Tie the vein off with a 4-0 suture and use a hemostat to gently retract the rostral end of the vessel. Using fine scissors, cut a hole about three millimeters caudal to the suture and approximately halfway through the diameter of the vein.

Next, insert a PV1 polyvinyl catheter into the jugular vein through the hole and thread the catheter approximately 1.5 centimeters toward the caudal end of the vessel. Use a new 4-0 suture to tie the catheter securely within the caudal section of the vessel and tie the rostral part of the vessel to the outside of the catheter with the loose ends of the suture originally used to tie off the rostral jugular vein. Tack the skin loosely back together around the catheter with another piece of 4-0 suture to prevent dehydration, loss of body heat and tissue desiccation.

Then, connect the catheter to a syringe containing heparinized saline and flush the catheter with the saline solution. To assess extravasation and leakage from the organ of interest, first inject 50 microliters of Evans Blue solution into the jugular vein catheter, followed by a small volume of heparinized saline to flush the line. Two minutes later, inject 100 microliters Substance P into the catheter, followed by a second flush of heparinized saline.

Eighteen minutes after Substance P injection, open the chest cavity of the animal and gravity perfuse the heart and blood vessels with 50 milliliters of 50 millimolar sodium citrate solution to remove the excess Evans Blue from within the blood vessels. Harvest the organs and tissues of interest and remove any residual contents for each tissue as necessary. Rinse the organs in room temperature PBS, blot the organs with a lab wipe and cut each organ in half.

Obtain the wet weights of each organ half on a balance and dry 1/2 of each organ on a piece of foil in a drying oven at 150 degrees Celsius for 48 hours. Then, place the other half of each organ in up to 200 microliters of formamide in a microfuge tube for 48 to 72 hours for Evans Blue Dye extraction. Neprilysin knockout mice demonstrate an increased Substance P-induced plasma extravasation from the urinary bladder compared to wild type animals suggesting a role for Neprilysin in the negative regulation of Substance P-induced plasma extravasation from the bladder.

Doxycycline treatment of mice transgenic for the over expression of Neprilysin for one week to activate Neprilysin over expression indicates an additional role for this enzyme in duodenal extravasation as doxycycline-treated transgenic animals exhibit a significantly reduced Substance P-induced plasma extravasation from the duodenum compared to all three groups of animals that is not observed after doxycycline treatment of wild type or knockout animals. Once mastered, the catheterization, injection, and organ isolation steps can be completed in 45 to 55 minutes if they are performed properly. Allow an additional two to three days for the remainder of the protocol.

When first learning this technique, it's important to repeat the procedure in at least 10-20 practice animals before performing an experiment. After watching this video, you should have a good understanding of how to assess and to relatively quantitate plasma extravasation or leakage in the organs of mice or other small animal species using this modified Evans Blue Dye method.