Assessment of Blood–Brain Barrier Breakdown - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 384

2. Dynamic In Vivo
Imaging Method
for Measuring
Regional Blood
Flow Changes and
BBB Permeability
Changes

This method is based on in vivo real-time fl uorescence imaging of
small cortical vessels followed by quantitative image analysis. The
advantages of the method include high spatial and temporal reso-
lution, allowing the study of distinct anatomically and functionally
defi ned vessels (i.e., arterioles and venules) while maintaining a
high sensitivity for detecting changes in vessels permeability and
blood fl ow ( 26 ). In addition, the method allows following dynamic
changes in regional blood fl ow and vessels permeability in the liv-
ing brain. Limitations of the method are its invasiveness and its
limited use to anesthetized animals. Another disadvantage is that
to this end, the method has been tested only for surface vessels. It
should be pointed that although surface pial vessels appear to share
many morphological and functional features with deeper intracere-
bral vessels, there are some important differences (e.g., the distri-
bution of the TJs between ECs ( 27 )). While we could never detect
any apparent difference in permeability between pial and deep ves-
sels in response to experimental manipulations (using established
histological methods for detection leakage of proteins in fi xed tis-
sue), the future use of other imaging techniques (e.g., two-photon
microscopy, see refs. ( 5, 28 )) may allow the implementation of the
new analysis methods to deeper cortical vessels. Figure 1 shows a
typical experiment demonstrating the effect of elevated extracellu-
lar K + on vessels' diameter, regional cerebral blood fl ow, and BBB
permeability.

2.1. Main
Considerations

Choosing the Fluorescent tracer . The method is based on fast imag-
ing of signal intensity following the peripheral injection of a fl uo-
rescent molecule (tracer). Thus, the injected tracer should be
nontoxic and under normal conditions not permeable through the
BBB. To allow repeated imaging, the ideal tracer should have a
short half life in the circulation, preferentially secreted by the kid-
neys. We use small molecules, including Lucifer Yellow (LY,
FW = 521.58 Da) and Na-Fluorescein (NF, MW = 376 Da)—both
showing a relatively short half time (<10 min) within the circula-
tion. In an alternative approach, large molecules with a long serum
half life are injected and permeability changes are monitored dur-
ing treatment (e.g., FITC-labeled albumin, MW = 66 kDa); how-
ever, in such experiments repeated injections cannot be performed.
The use of tracers with different properties (e.g., molecular weight,
charge) allows a more detailed characterization of permeability
changes to specifi c agents, including brain to blood clearance under
healthy and pathological conditions (see below).