Assessment of Blood–Brain Barrier Breakdown - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 389

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 saline solution (ca. 200 ml/kg BW).
3. The rat is decapitated, and the brain is quickly removed.
Regions of interest are dissected, transferred to liquid nitrogen
and kept in −20°C for analysis.
4. Tissue is dissolved in the phosphate-SDS buffer solution
(10 l/mg tissue).

5. Homogenize and centrifuge (12,000 × g ; 5 min) to precipitate
confounding cell debris.
6. Spectrophotometrical measurements of the albumin-EB com-
plex concentrations at 595 nm wavelength.

μ

4. Other Methods

Immunostaining for serum proteins in brain tissue —After fi xation,
direct staining of brain slices with species-specifi c antibodies against
blood proteins, such as IgG ( 40 ) or albumin ( 41 ). Positive staining
indicates BBB disruption. The method is specifi c and under nor-
mal conditions leakage is small. However, the drawback of this
approach is that it gives rather a yes/no answer whether there was
an abnormal diffusion of the identifi ed protein during the time
between injection and brain removal.
Brain's biomarkers in serum —an alternative approach for dynamic/
continuous measure of BBB leakage is the detection of brain's
derived proteins in the serum. For example, astrocytic-specifi c pro-
teins are synthesized and may be released next to capillaries, but
owing to the negligible transendothelial permeability, signifi cant
extravasation into the serum only occurs when the BBB is breached.
The astrocytic protein S100B has been identifi ed as a potential
biomarker in serum for BBB breakdown ( 42-45 ). Its short half life
in serum (~30 min, see ref. ( 46 )) has an advantage as it refl ects
recent leakage. However, other factors—including changes in S100
levels due to upregulation following injury, increased secretion
from activated astrocytes or release from injured or dying cells—
may affect S100B serum levels.
Transendothelial electrical resistance —The BBB has a low paracel-
lular permeability to ions and thus a high electrical resistance, in
the order of 10-25 k

× cm 2 ( 47 ). An alternative method to study
BBB permeability is by measuring the transendothelial electrical
resistance ( 48, 49 ). The method is based on the infi nite leaky cable
theory in which current is injected through a microelectrode tip
inside a blood vessel causes voltage displacement which decays with
distance from the current source ( 48 ). The fall in voltage is due in
part to leakage of current across the vessel wall which is defi ned by

Ω