Assessment of Blood–Brain Barrier Breakdown - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 388

BBB. We describe a semiquantitative method to assess microscopi-
cally the extravasation of the EB to the parenchyma.

1. Evans-blue (EB) (2% in saline)
2. Paraformaldehyde (4% in PBS)

3.1.1. Solutions

1. Fluorescence microscope (Zeiss, Axioskop 2 plus)
2. Camera (e.g., Cool Snap, Photometrics)
3. Vibrotome

3.1.2. Instruments

1. Animals are injected intravenously (2.4 ml/kg) or intraperito-
neally (5 ml) with EB.
2. At a fi xed time after injection (>30 min is recommended if EB
is injected intravenously or >60 min for intraperitoneally) rats
are transcardially perfused with 4% paraformaldehyde fi xation
solution.
3. The brain is quickly removed and sliced (ca. 500

3.1.3. Method

μ m thick)

using a vibrotome.
4. Sections are incubated in 4% paraformaldehyde solution for
24 h, followed by incubation in store-solution (PFA 0.02% in
PBS). Thin slices (~10-40 m) are prepared and assessed under

fl uorescence microscope (562 nm) for extravascular leakage of
the fl uorescence injected dye.
5. Tissue concentrations of the fl uorescent albumin-EB complex
are measured by microscopically reading color intensity in low-
power fi eld cortical imaging (10-20×) using home-made
Mathlab Scripts.

μ

3.2. Spectrometric
Measurements of
Evans Blue-Albumin
Complex in Brain
Homogenates

In this method, the fl uorescent intensity of the EB-albumin com-
plex is measured in a tissue homogenate following a peripheral
injection of EB (as in Sect. 2.1 ).

1. Evans-blue (EB) (2% in saline)
2. 0.9% Saline
3. 0.1 M Phosphate buffer containing 1% SDS (Carl Roth Gmbh,
Karlsruhe, Germany)

3.2.1. Solutions

1. Homogenizer
2. Centrifuge (Eppendorf Scientifi c)
3. Spectrophotometer (Eppendorf Biometer)

3.2.2. Instruments

1. Animals are injected intravenously (2.4 ml/kg BW) or intrap-
eritoneally (5 ml BW) with EB.

3.2.3. Method