Tiam Mana Saffari

219 MICRO CT ANALYSIS AFTER STEM CELL DELIVERY AND ANGIOGENESIS 10 Outcome measurements Preserved vasculature in the nerve segments was quantified using a SkyScan 1276 micro CT (Bruker Corporation, Billerica, MA, USA) at 40kV voltage, 200 µA current and 10 µm resolution, as previously described 12 . Three-dimensional images of the samples were reconstructed using Hierarchical InstaRecon software (NRecon, 1.7.4.2., InstaRecon, 2.0.4.0. InstaRecon) and AnalyzePro software (AnalyzeDirect, Inc.,Overland Park, KS) was used to segment blood vessels. The volume of the vasculature was expressed as a percentage of the total nerve volume (vessel%). Evaluation of blood vessel morphology For all transplanted nerve groups and controls, a vasculature tree skeleton was generated using an ERODE/DILATE program (written by the Department of Physiology and Biomedical Engineering at Mayo Clinic, Rochester, MN) 19 . This program allowed for optimal morphological information of the entire vasculature network including measurement of the following morphological parameters; volume and percentage of vessels at different diameters. Using this program, the volume of vessels within a selected lumen diameter range was estimated. Specifically, the distance between the two most distant lying voxels within these vessels was determined to describe vessel thicknesses. The average of the local voxel thicknesses within the structure yields the vessel thickness (lumen diameter in µm). As the cubic voxel side dimension was 10 µm, the nerve microvessels were always derived in 20 µm increments. Due to a finite imaging field, some vessels could not be visually connected and were excluded from the data. The volume (in mm 3 ) and percentage (in %) of the averaged vessel counts were calculated for each group to depict the vascular distribution. Analysis Vascular volume results were analyzed and compared to ten non-operated sciatic nerve (control) samples, randomly selected from two rats of each experimental group. The nerve samples were included in the study if they underwent successful preservation of vasculature, defined by observation of yellow nailbeds on either paw. Investigators could not be blinded to the experimental groups during perfusion of Microfil®, but the micro CT was performed by an independent technician to provide