Determining Blood-Brain Barrier Disruption Using Fluorescent Dyes in an EAE Mouse Model

Start with a solution containing green fluorescent dye bound to a large dextran chain. Then, add a solution of red dye bound to a small dextran chain.

Mix the dyes thoroughly and take into a syringe.

Inject this dye mixture intravenously into an anesthetized mouse through the retro-orbital sinus, a vein behind the eye.

This mouse suffers from experimental autoimmune encephalomyelitis or EAE.

In this condition, the blood-brain barrier is disturbed with weakened tight junctions between the endothelial cells of the brain's blood vessels with gaps.

Allow the mouse to fully awake and regain motility, enabling the dyes to circulate.

The red dye with the small dextran chain easily diffuses through the small gaps into the brain tissue, while the green dye bound to the large dextran chain diffuses if the damage is severe.

The amount of each fluorescent dye in the brain tissue correlates with the extent of the blood-brain barrier disruption.

For preparing dextran stock solutions, dissolve 10 milligrams of three kilodalton Dextran Texas Red in 500 microliters of 0.9% sodium chloride solution, and 5 milligrams of 10 kilodalton Dextran AlexaFluor 488 in 250 microliters of 0.9% sodium chloride solution.

For the dextran working solution, just before injection, pipette 55 microliters of 10 kilodalton Dextran AlexaFluor 488 stock solution onto a piece of sealing film, then, add 55 microliters of three kilodalton Dextran Texas Red stock solution. Mix and collect 100 microliters into a disposable fine syringe.

Next, place an anesthetized mouse in a lateral position on the table. Intravenously inject 100 microliters of fluorescent tracers into the mouse before the mouse wakes up. Let the tracer circulate for 15 minutes.