A simple stereologic method for analysis of cerebral cortical microvessels using image analysis

Previous methods for determining morphological features of vascular networks in cerebral cortex were subject to arbitrary variation and bias. Unbiased estimates of vessel number, volume, surface area and length can be obtained using stereology but these techniques tend to be tedious and time-consuming. Stereologic protocols generally require micrographs that have to be analyzed manually for intersections of vessels on grid points or lines. In this report, we provide a simpler and more precise method for measuring morphological features of cerebral cortical microvessels. Images of microvessels in 1 microm toluidine blue stained sections were captured using a popular image analysis software package. Luminal surfaces of endothelial cells were automatically traced using commonly available features; the two-dimensional data of vessels (diameter, area, perimeter and number of vessels) were automatically computed and transferred to a spreadsheet. Three-dimensional features were then determined using basic stereologic equations. The method eliminates the need for manual measurements and is particularly time- and cost-effective for quantitative studies where numerous images have to be evaluated.