Computerised image analysis in conjunction with fluorescence microscopy for the study of blood-brain barrier permeability in vivo

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Abstract
The present paper describes a new method using computerised image analysis techniques for quantification of tracer extravasation over the blood-brain barrier as studied by intravital fluorescence microscopy. Cats were equipped with an open cranial window and continuously infused with fluorescein isothiocyanate-labelled dextran (FITC-dextran, mol. wt. 70 000) to maintain a steady plasma concentration. Several cortical fields were recorded in each experiment and the images stored on video tape for off-line analysis. This procedure, which largely eliminates the superficial pial vasculature and allows extraction of the extravasation areas, consists of the following steps: (1) averaging of images, (2) software shading correction based on the original images for compensation of optical non-uniformity, (3) correction of displacement artefacts, (4) intensity adjustment, (5) generation of subtraction images by subtracting the first image of a series from the subsequent ones, (6) median filtering and thresholding, (7) a length recognition algorithm, and (8) elimination of small areas. Compared to the previously described method, step (2) has been newly developed and steps (4) and (8) added to enhance sensitivity for detecting tracer extravasation. The degree of extravasation in a cortical field at a given time point [E(f) value] was calculated as the mean intensity of the remaining pixels. TheE(f) is a quantitative value computed by a fully automatised procedure which takes into account the number, as well as the size and intensity, of extravasation areas in a given cortical field. TheE(f) values obtained at different times in a series of experiments were averaged to give theE(I) value. TheE(I) value did not alter when hypercapnia was employed to induce pure vasodilatation. On the other hand it increased dramatically, indicating tracer extravasation, during topical application of high concentrations of adenosine (10−5–10−3 M). The new computerised image analysis procedure may therefore be suitable for measuring quantitatively tracer extravasation over the blood-brain barrier in vivo under different experimental conditions. It may also be applicable to study changes of vascular permeability in peripheral vascular beds.