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9. Rolling fraction (RFx = RF/TF).
10. Sticking fraction (SFx = SF/RFx), number of rolling cells that
become fi rmly adherent.
11. Locomotion of adherent cells on the endothelium.
12. Transendothelial migration of adherent cells (TEM).
13. Rolling velocity/fast cell velocity.
The fi rst fi ve parameters mentioned above that can be assessed
by fl uorescence microscopy characterize the morphological aspects
and changes of the superfi cial macro- and microvasculature, pro-
viding the possibility to evaluate structural and functional changes
quantitatively, and therefore, giving further insight in course and
severity of the cerebral vasospasm in the observed blood vessels
(Fig.
3a
, b).
The second set of eight parameters documents leukocyte activ-
ity as well as leukocyte-endothelial interaction as a marker for
infl ammatory changes in the observed vessels or the surrounding
brain tissue (Fig.
3c
).
The following equipment and materials are required for intra-
vital fl uorescence microscopy:
14. A fl uorescence microscope equipped with a high pressure
mercury lamp with a set of different fi lters (ultraviolet,
340-380 nm; blue, 450-490 nm; and green, 520-570 nm)
(e.g. Zeiss, Oberkochen, Germany).
15. A set of different objectives (e.g. 3.2× and 10× long distance,
20× and 40× water immersion objectives).
16. Low-light, charge-coupled (CCD) microscope-mounted video
camera connected to a recording system for later off-line
analysis.
17. FITC-dextrane (2% w/v, MW 150,000, Sigma Chemical Co.,
St. Louis, MO).
18. Rhodamine 6G (0.2% w/v, MR 489, Serva Feinbiochemica
GmbH & Co, Heidelberg, Germany).
19. Computer-assisted image analysis system.
The following paragraph outlines the procedure of intravital
microscopy after chronic cranial window preparation:
20. Measurements are done under general anaesthesia.
21. All measurements have to be performed in a dark chamber to
eliminate fading of the fl uorescent dyes and to provide maximum
detection of the emitted fl uorophores.
22. Immobilization of the animal in the stereotaxic head frame
after reopening of the skin incision over the cranial window.
23. Cleansing by irrigation.
24. Intravenous application of 0.1 ml of 2% FITC-dextrane.
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