Investigation of blood-brain barrier permeability by means of computerized image analysis.

Intravital fluorescence microscopy has been widely used to study changes of blood-brain barrier (BBB) permeability in vivo. However, quantification of tracer extravasation by counting leaky spots provides only a rough estimate of permeability increase. We have therefore developed a new method for measurement of tracer extravasation from cerebral blood vessels by combining image analysis techniques with intravital fluorescence microscopy. This method is based on generation of subtraction images after shading and displacement correction. Subtraction images are further processed to eliminate noise and diminish artefacts due to vessel distortion and/or dilatation. The "degree of extravasation" (E(f) value) which is then calculated takes into account the number and size of leaky spots as well as their intensities. Examples are shown to illustrate that pure vasodilatation does not increase E(f) as long as the BBB-permeability is not disturbed. This newly developed method may prove helpful for comparison of tracer extravasation under different experimental conditions.