Assessment of Blood–Brain Barrier Breakdown - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 387

5. Signal intensity time onset, slope, maximal intensity, and dura-
tion refl ect the fl ow of the tracer within the compartment are
measured and followed during the experiment (Fig. 1d ).

3. Detection
of Protein Leakage
into the Brain

Evans Blue (tetrasodium diazo salt, EB)—is a small, BBB-
permeable molecule (MW = 960.81) which rapidly and selectively
binds to albumin (MW = ~66 kDa) when injected into a peripheral
vein, therefore confers to its properties of a protein tracer ( 36 ).
It has been thus traditionally used as a reliable tracer to detect
signifi cant BBB breakdown (( 37-39 ) and Fig. 2a ). In addition,
the complex EB-albumin exhibits a red-orange fl uorescence and
thus can be detected within the CNS using a standard fl uorescent
microscope and analysis techniques (( 31 ) and Fig. 2b-d ).
Traditionally, measuring leakage of EB-albumin complex is done
in the “steady state” condition on tissue removed from the experi-
mental animal. Although EB binds rapidly and stably to the protein
and the amount of free dye in serum is usually negligible, it is
important to note that EB injection into the brains' arterial circula-
tion (or left heart) may result in overestimation of BBB permeability
due to leakage of nonbound molecules. Here, we present two
alternative methods to measure EB-albumin leakage across the

3.1. Morphological
Examination of Evans
Blue-Albumin Complex
Extravasation into the
Brain

Fig. 2. Detection of EB-albumin complex within the brain: Rats are injected with the albumin-binding dye Evans blue,
perfused with PFA; the brain is then sliced to ca. 500 μ m thin sections ( a ). The treated region of the brain is showing BBB
breakdown ( blue ). Fluorescence histological sections show albumin-EB complex in red ; in the healthy cortex fl uorescent
staining is limited to blood vessels ( b - d control). In brain regions where the BBB is compromised clear extravascular stain-
ing can be seen, including uptake by cellular elements ( b - d , treated.).