Tiam Mana Saffari

76 CHAPTER 4 was used to adjust the following parameters while reconstructing the images; Beam Hardening Correction (%) was set at 51, Ring Artifact Correction at 9, Smoothing at 1, Post alignment compensation and Histogram windows were manually adjusted for each scan. After obtaining reconstruction of the images, AnalyzePro software (AnalyzeDirect, Inc., Overland Park, KS, USA) was used to measure the volume of the vasculature and the volume of the total nerve. A vessel/nerve area ratio was calculated and expressed in percentages (vessel%). Photography for calculating the vascular surface area After micro CT scanning was completed, the nerve samples were stretched by suturing both nerve ends onto a solid holder. Detailed pictures of the samples were obtained using a Canon 5D Mark IV camera, (Manual Mode, ISO 200, 1/200th of a sec, f/16), a Canon MP-E 65mm Macro lens and a Canon MT-26-RT Twin Lite Macro strobe light source. During photography, samples were placed in a petri dish with methyl salicylate in order to obtain clean photographs allowing for the specimen to be separated from the background for better measurement. The petri dish was placed on a black background to achieve maximum contrast with the yellow vessels in the nerve samples. Polarized light was used to reduce reflections and a 1:1 magnification was used to ensure consistency of the pictures. To correct for the surface area that altered depending on the angle of the image, two pictures of each nerve sample were obtained; one of the front whereafter the holder was flipped and the picture of the other side was obtained. With NIS-Elements software (NIS-Elements BR 4.51.01), the total vessel area and the total vessel area in the graft were measured in a blinded fashion. For each image, a vessel/nerve area ratio was calculated. The ratios of the two images (front and back) were averaged per sample. Statistical analysis The vascular volume and the vascular surface area were analyzed and compared to the non-operated side (control). A nonpaired student t-test for comparisons between time points and between the two groups was used for statistical investigation. Correlations were analyzed using Pearson’s correlation test. Results were reported as the mean and standard error or the mean (SEM), and the level of significance was set at α ≤ 0.05.