Novel three-dimensional analysis tool for vascular trees indicates complete micro-networks, not single capillaries, as the angiogenic endpoint in mice overexpressing human VEGF(165) in the brain.

To adequately supply tissues with oxygen and nutrients, the formation of functional vascular networks requires generation of normal, healthy vessels and their arrangement into an effective network architecture. While our knowledge about the development of single vessels significantly increased during the last years, mechanisms responsible for network formation are still poorly understood. This is probably due to the lack of suitable methods for quantification of structural properties of microvascular networks. Previously we showed that cerebral blood flow is not increased in mice exhibiting a 2- to 3-fold higher density of normal and perfused capillaries as a result of transgenic overexpression of the human vascular endothelial growth factor (VEGF(165)). Here we used vascular corrosion casting and hierarchical micro-computed tomography combined with a new network analysis tool to characterize the vascular architecture in gray and white matter of these mice. Our results indicate that VEGF overexpression leads to formation of additional micro-networks connected to higher order vessels rather than insertion of individual capillaries into the existing vessel structure. This implies that the smallest "angiogenic quantum", i.e. the final, stable result of angiogenesis and subsequent remodeling, is not a single microvessel, but a complete micro-network. In conclusion, high-resolution 3D imaging combined with network analysis can substantially improve our understanding of vascular architecture, beneficial for the development of therapeutic angiogenesis as a clinical tool for applications such as wound healing or treatment of ischemic diseases.

Hierarchical microimaging for multiscale analysis of large vascular networks.

There is a wide range of diseases and normal physiological processes that are associated with alterations of the vascular system in organs. Ex vivo imaging of large vascular networks became feasible with recent developments in microcomputed tomography (microCT). Current methods permit to visualize only limited numbers of physically excised regions of interests (ROIs) from larger samples. We developed a method based on modified vascular corrosion casting (VCC), scanning electron microscopy (SEM), and desktop and synchrotron radiation microCT (SRmicroCT) technologies to image vasculature at increasing levels of resolution, also referred to as hierarchical imaging. This novel approach allows nondestructive 3D visualization and quantification of large microvascular networks, while retaining a precise anatomical context for ROIs scanned at very high resolution. Scans of entire mouse brain VCCs were performed at 16-microm resolution with a desktop microCT system. Custom-made navigation software with a ROI selection tool enabled the identification of anatomical brain structures and precise placement of multiple ROIs. These were then scanned at 1.4-microm voxel size using SRmicroCT and a local tomography setup. A framework was developed for fast sample positioning, precise selection of ROIs, and sequential high-throughput scanning of a large numbers of brain VCCs. Despite the use of local tomography, exceptional image quality was achieved with SRmicroCT. This method enables qualitative and quantitative assessment of vasculature at unprecedented resolution and volume with relatively high throughput, opening new possibilities to study vessel architecture and vascular alterations in models of disease.