Tiam Mana Saffari

71 OBJECTIVE EVALUATION OF ANGIOGENESIS USING MICRO CT AND PHOTOGRAPHY 4 INTRODUCTION It has been postulated that blood supply affects nerve regeneration 1-7 , as it is reported that vascular endothelial cells directly guide the regeneration of peripheral nerve axons 7 . Vascularization (angiogenesis) can be enhanced through growth factors 8 , in particular angiopoietin-1 (Ang-1) an important angiogenic factor that promotes vascular stabilization 9 and vascular endothelial growth factor (VEGF) which enhances intraneural angiogenesis 2 . To quantify blood vessels in nerve grafts or conduits, previous studies have focused on immunohistochemical staining 10-12 . Unfortunately, the amount of (neo)angiogenesis was not evaluated in these studies. Histomorphometric analyses are used to describe angiogenesis, but are limited in the ability to identify the three-dimensional interconnectivity of the vasculature in serial histological sections 13,14 and focuses on representing superficial blood flow 15 . Connectivity defines the maximal number of branches that may be cut without separating the structure 15 . Insight in the connectivity of the vascular tree may contribute to crucial description of neovascularization patterns. Therefore, two- dimensional measures of vasculature deliver incomplete information 13 . Three- dimensional reconstruction of blood vessels in sciatic nerves of the rat has been technically difficult because the average diameter of the small endoneurial vessels in the rat is 8.8 mm 16 . Micro-computed tomography (micro CT) facilitates the visualization of contrast enhanced microvessels and could separate vessels from surrounding tissues, such as bone, fat tissue or nerve 17 . It provides three-dimensional volume imaging with spatial resolution at the micrometer scale and is applied in many fields, for instance in tumor visualization, cardiovascular plaque imaging 15,17 and evaluation of surgical angiogenesis in a bone allotransplantation models 18-20 . As technology continues evolving, modern micro CT systems are more commonly available and are capable of generating very small voxels with short scan acquisition times allowing both ex vivo and in vivo scanning 15 . Moreover, due to voxels as small as 1μm, the vascular system in rat and even mice could now be visualized 13,14,21,22 . As perfusion with a radiopaque contrast agent is the only requirement to delineate the