Biology Reference
In-Depth Information
BBB Opening
As positive control, potent BBB opening can be achieved using a
direct perfusion of the cortical surface with ACSF containing 1-2 mM
sodium deoxycholate (Sigma-Aldrich, see refs. ( 26, 30, 31 )). Below,
we show the effect of high (K + ) solution to imitate the effect of
delayed hemolysis of blood products within the subarachnoid space
as in the case of intracerebral hemorrhage (see refs. ( 32, 33 )).
1. Baseline signal images are obtained for 3-5 s, followed by intra-
venously injection of BBB non-permeable fl uorescent dye.
2. Full resolution (658 × 496) images of cortical surface vessels
are obtained at 20-30 images/s before, during, and after injec-
tion of the tracer (total of ca. 25 s).
3. Additional fl orescence images can be taken for longer periods
at a lower sampling rate (1 image/s) to follow late changes in
tracer behavior.
4. Depending on the tracer half life in blood, injections can be
repeated.
5. To compare vessels permeability to different molecules, animal
can also be injected with other BBB non-permeable tracer (see
Fig. 1e ).
6. Image analysis is performed off line.
Real-Time Fluorescence
Imaging
For detailed description of the analysis methods, see ref. ( 26 ) and
see Fig. 1 .
Image Analysis
1. Preprocessing can include image resizing (to reduce memory
usage and computation time) using 2D bicubic interpolation
( 34 ) and subpixel image registration to overcome artifacts due
to small movements. This is performed by applying a single
step discrete Fourier transform ( 35 ) to each image. To increase
the signal-to-noise ratio, each frame is registered according to
a moving average of several preceding images.
2. Signal intensity changes over time and space are then
analyzed.
3. Physiologically measured parameters: baseline, time to incline ,
incline (averaged slope) , time to maximal intensity , max imal
intensity , and decline (averaged slope) . Maps are performed for
the parameter of interest (e.g., Fig. 1c ).
4. Cluster analysis is performed on the measured parameters to
separate between compartments with different signal kinetics—
usually allowing a good separation of arterioles, venous, and
extravascular tissue. To overcome small changes in signal inten-
sity in the extravascular compartment under normal conditions
(probably due to nonspecifi c effect from underlying vessels), a
correction to a “near zero” change based on the intravascular
signal is optional.
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