Measurements of vascular remodeling by confocal microscopy

The introduction of myographs was crucial for the study of structure and function of resistance arteries. The ability to support and maintain small blood vessels paved the way for true microscopic studies of the vascular cells. However, even after decades of study, we still do not know very much about the "normal" arrangement of smooth muscle cells in the vascular wall and how their distribution affects function. It was clearly time for the next technological step forward. We have shown here how the combination of myography and confocal microscopy creates a platform for the study of vascular structure at the cellular level and in 3D. In addition, the possibility of using live myograph-mounted vessels in combination with LSCM opens a new field of research to assess vascular remodeling from a physiological point of view and to study vascular function at a level not achieved by any other method at present. Now that the hardware is in place it is time to concentrate on the software and improve the methods of analysis. We have used 2D analysis of 3D data sets to describe differences in vascular structure and, at the same time, developed methods to semiautomate the process. The success of the 3D methods will ultimately depend on the reliability and accuracy of the analysis routines. There are still problems to overcome en route to finding a complete solution. However, we believe that the search for a robust fully (or semi-) automated method of 3D analysis will be more than worthwhile. We have defined vascular remodeling to include any changes in cellular arrangement or morphology. However, on a more subtle level, changes in receptors, enzymes, and proteins leading to altered functionality could also be regarded as remodeling. In that respect it may be interesting to map the distribution of the many receptors, channels, and proteins that regulate vascular growth, death, and function. Currently, there is a growing list of fluorescent ligands and antibodies that can be used in conjunction with confocal microscopy. It is possible that multiple stains could be used and imaged at different wavelengths with a view to constructing full 3D models of various structures and their colocalization. It is our belief that the confocal approach will prove to be a major tool in unraveling the complexities of cell-cell interactions and arrangements and will allow a better understanding of the process of vascular remodeling and function.