Intravital fluorescence microscopy for the study of blood-brain-barrier function

Abstract

Vascular diameters and blood-brain-barrier (BBB) function were investigated in the parietal cortex of cats using an open skull window technique and intravital fluorescence microscopy. The cortical surface was superfused with artificial cerebrospinal fluid containing: bradykinin (BK), Na+-arachidonate (AA), or xanthine-oxidase (XO). Na+-fluorescein (MW: 376), fluorescein-isothiocyanate-labelled (FITC) albumin (MW: 67 000), or FITC-dextran (MW: 19 400-62 000) were given intravenously as blood-brain-barrier indicators of different molecular size. In control experiments, the effect of continuous exposure of the preparation to the light source used for fluorescence excitation was studied. Dependent on the molecular size of the tracer and light intensity, continuous light exposure led to extravasation and disturbances of the microcirculation in small veins. When Na+-fluorescein was employed as barrier indicator at a magnification of X 40, at least 55 min of continuous illumination were required to induce extravasation. Brief, i.e. 0.5-15 s and discontinuous illumination for taking microphotographs amounting to less than 20 min in total did not induce extravasation in control experiments of 3.5 h. Opening of the blood-brain-barrier was studied during superfusion with a hypertonic solution (2000 mOsmol 1(-1)). The results obtained with bradykinin, Na+-arachidonate, or xanthine-oxidase indicate that opening of the barrier can occur independently from a corresponding vasodilating reaction. BK, or AA led to initial venular leakage as a result of an increase of selective, or global barrier permeability. On the other hand, XO did not induce extravasation, although cerebral vessels were markedly dilated. Taken together, the experimental model presented is suitable to simultaneously analyze dynamic changes of the permeability of cerebral vessels in-vivo with excellent spatial resolution and of the cerebral vasomotor behaviour under physiological and pathological conditions.