Visualisation of morphological changes in living intact human microvessels using confocal microscopy

Conventional techniques to visualise microvascular structure often involve fixed tissue slices that provide two-dimensional images. A previous study using diffusive labelling of fresh, dissected tissue samples with fluorescently-tagged endothelial markers demonstrated the possibility of examining the three-dimensional architecture of the microvasculature using confocal microscopy. The present study extends the use of this quick and simple method of diffusive labelling to examine the possibility of repeatedly measuring changes in the morphology of intact microvessel in response to pharmacological stimuli. Initially, three-dimensional surface-rendered images of the same microvessel derived from the placenta and subcutaneous biopsies demonstrated morphological and topological changes in response to temperature and increasing potassium changes of physiological salt solutions, respectively. Furthermore, a dose-response study was performed with subcutaneous microvessels using the potent vasodilator, adrenomedullin. Analysis of a series of z-stack, superimposed to form a single maximum brightness image, demonstrated an inverse dose-response relationship, with responses to increasing adrenomedullin concentrations (10(-12) to 10(-8) M). In vessels that had constricted in response to noradrenaline (diameters: 22.4 to 58.0 microm), physiological concentrations of 10(-12) M increased vessel diameter by 108% above baseline conditions. Control treatment using physiological salt solution did not demonstrate any changes. The technique described suggest that diffusive labelling with vascular endothelial markers such as ulex europeaus agglutinin I in live tissue samples may be used in conjunction with confocal microscopy to demonstrate heterogeneous morphological and topological changes in intact segments of the microvasculature.