Use of Evans Blue to monitor Blood Brain Barrier Permeability

June 29, 2015

Mass General Hospital
Department of Neurosurgery
Postdoctoral Fellow

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Company:

Sigma-Aldrich

Product Name:

Evans Blue

Catalog Number:

E2129 SIGMA

We wanted to analyse blood brain barrier permeability in a neurologically diseased mouse model. There are many ways to analyse this but we chose a simple but powerful method using the Evans Blue [EB] Dye. Since EB binds to albumin, a high molecular weight protein it cannot cross the blood brain barrier if the barrier is structurally and functionally intact. However if there are innate problems with the barrier, the dye would show up in the parenchyma of the brain.

Experimental Design and Results Summary

Application

Evaluate blood brain barrier permeability

Starting Material

Powder

Protocol Overview

Twenty-one day old wild type and symptomatic diseased Canavan mice (n=3) were injected with Evans blue (EB) (4 ml/kg). Control mice were injected with saline or EB combined with mannitol pretreatment (3 ml/100 g) since mannitol reversibly opens the BBB. 30 minutes later, mice were perfused with PBS, and their brains homogenized. The supernatant was measured for absorbance at 610 nm to check the presence of EB using known concentrations as standard.

Tips

Inject the animals with the Evans Blue solution (4 mg/kg) and wait until the extremities turn blue indicating that the dye has entered the bloodstream. It is important to sacrifice the animal within an hour so as to avoid clearance from the system physiologically. After sacrifice the animal must be perfused to remove the dye from the blood vessels. the brain (and a control organ like liver) should then be homogenized and the supernatant used for measuring absorbance in a spectrophotometer We used water to dissolve the powder to make a 1mg/ml solution. It was extremely easy to dissolve and filter sterilize too. I would recommend filter sterilizing if made in PBS or physiological saline. The solution made in water (but not salt solution like saline or PBS) can be autoclaved.

Results Summary

Our results showed that the BBB is not disrupted at the young age of Canavan mice

Additional Notes

None

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Summary

The Good

It was extremely easy to dissolve and filter sterilize too. I would recommend filter sterilizing if made in PBS or physiological saline.

The Bad

If the solution is made in salt solution like saline or PBS, it cannot be autoclaved but can be filter sterilized

The Bottom Line

It is an extremely useful and cheap product to perform an important assay. I highly recommend its use

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