Femke Mathot

Chapter 5 88 the smallest angiogenic vessels to allow complete study of the vascular network. 15 The injected Microfil® does not directly interact with the histology but could influence the proportion of axon and nerve areas during the analysis. Therefore, we would suggest harvesting other tissue prior to Microfil® injection to secure reliable histologic analysis. There are a few considerations associated with these techniques. Although micro CT systems become more commonly available with improving quality 15 , costs could add up and it is still conceivable that micro CT devices with small effective voxel size are not available for all researchers. In this case, solely the described conventional photography strategy could be used, as it is cost-effective, simple to perform and correlates with micro volume measurements. However, the limitation of conventional photography is that it does not describe vascularization in space and lacks detailed information. As only two sides of the nerve sample are measured and the surface area could not be corrected for the thickness of the nerve (i.e. depth of the obtained photo), representation of the amount of angiogenesis using vascular surface area may be questionable. The difference between various groups of nerve samples, however, could be described with conventional photography. The use of Microfil® to fill the complete vascular bed including the smallest arterial and venous branches allows visualization of the desired vascular system but limits any other histological examination in the same samples, as processing is different. The micro CT has the advantage of precisely measuring vessel volume in relation to total measured nerve volume. Also, clearing of samples is not necessary as the lead pigments in Microfil® provide high contrast compared to background tissue to acquire complete high resolution 3D images of the vessels. 17 Our results indicate the significant correlation between the vascular volume and the vascular surface area measurements demonstrating that the methods could be used either complementary or separately, depending on the goals of the study. These methods will allow us to advance angiogenesis related research by improving the tools for studying and understanding vascular development and the mechanisms of neoangiogenesis. CONCLUSION This study provides accurate objective analysis of the newly formed vascular network of the sciatic nerve. The use of the micro CT and conventional photography provides many modalities for vascular exploration, allowing the exploration of the structure and organization of blood vessels. These imaging methods are easily reproducible and could be extrapolated to any other desired target organ ex vivo in small animals to investigate the vascular network.