Femke Mathot

5 Novel methods for evaluation of vascularization of nerves 81 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 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 endoneural 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 imagingwith spatial resolution at themicrometer scale and is applied in many fields, for instance in tumor visualization, cardiovascular plaque imaging 15, 17 and evaluation of surgical angiogenesis in bone allotransplantation models. 18-20 As technology continues to evolve, 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 1 μm, 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 vascular tree, a closer-meshed network of smaller vessels, contrast enhanced micro CT can become a powerful tool in quantifying angiogenesis. 13 Information collected from conventional photography may complement the micro CT. Photographs could be analyzed by measuring the ratio of vessel area and total nerve area digitally. 23 More conventional methods such as manual vessel counts per sub-segment of the nerve, providing a vessel density per mm2, have also been described. 24 These techniques have never been verified and are questionably representative for the entire quantification of the nerve vasculature when used solely. However, with evolving technology, the quality of conventional photography has improved and there are numerous photo editing software programs available providing a cost-effective way to measure vessel and nerve surface areas. The purpose of this study was to describe three-dimensional visualization of the vascular