Imaging the Vascular Wall With Confocal Microscopy

Abstract

Blood vessels are capable of continuous structural changes in response to varying conditions and functional demands. In physiological situations, these structural modifications -known as “vascular remodelling”- are adaptive and aimed to maintain a relative constant shear and wall stress. Vascular remodelling is also common to several pathological conditions, such as hypertension and atherosclerosis, where the structural changes are no longer adaptive and contribute to the progression of the disease. Vascular remodelling is a dynamic process involving cell growth, death, as well as extracellular matrix synthesis and degradation. However, knowledge of vascular remodelling has been largely based on a long-standing, static view from two-dimensional histology and the 3-dimensional (3-D) organization of the different types of vascular cells and their relationship with the extracellular matrix and innervation has not been thoroughly explored. In addition, important questions related to the active process of vascular remodelling, such as which cells undergo proliferation, apoptosis, phenotypic change or migration, are still unsolved. An integrated view of the interrelationships of the different elements of the arterial wall is made possible by fluorescence Laser Scanning Confocal microscopy (LSCM). Confocal microscopes allow obtaining serial optical sections of relatively thick specimens without the need to cut them as with conventional histology. With the aid of image analysis software, these serial sections can be further reconstructed to obtain 3-D images, where the structures of interest are localized and quantified. LSCM can be combined with pressure myography to obtain simultaneously information on vascular function and 3-D structure at near-to-physiological conditions. The vessel is firstly pressurized at physiological pressure to study vascular function and mechanics. Thereafter, it is fixed and stained with the fluorescent dyes of the structures of interest. The fixed vessel is mounted intact on a slide provided with a small well to avoid 3-D distortion and it is visualized with a LSCM. The vascular wall is scanned from the adventitial to the endothelial layer and serial optical sections are acquired with the microscope. These serial images can be processed with an image analysis software that allows for reconstruction of 3-D models and for quantification of the stained structures. There are a vast number of fluorescent compounds useful to study vessel structure. For example, fluorescent dyes that bind with DNA, such as propidium iodide, DAPI or Hoescht 3332, are useful to identify the different types of vascular cells – adventitial, smooth muscle and endothelial cells- by the shape and orientation of their nuclei. With the aid of morphometric methods, which involve segmentation and object extraction, it is possible to identify and quantify cell type, number, shape, orientation and density in the different layers of the vascular wall. The use of fluorescent antibodies and kits allows locating, within an intact vascular wall, the distribution of nerves, specific enzymes and extracellular matrix elements and to study their relationship with the vascular cells. LSCM enables to scan and process relatively large amounts of tissue in short time. This makes possible to locate rather infrequent events in the vascular wall, such as cell apoptosis or proliferation, cells undergoing phenotypic change or migration. LSCM is not only useful to image vascular wall structure, but also to visualize and quantify by the intensity of fluorescence, the generation of vascular cell products, such as nitric oxide or superoxide anion. In conclusion, confocal microscopy and image analysis software permits to image vascular wall structure and function and to assess the active process of vascular remodelling in physiological and pathological situations