Femke Mathot

Chapter 5 84 Sample collection After the vascular bedwas perfused successfully and the contrast had cured, the sciatic nerve was exposed and harvested extending to approximately 3 mm on either side of the anastomoses. Nerve samples were collected in phosphate buffered saline (PBS) and cleared for five days by immersion in graded series of ethyl alcohol as follows: the samples were first placed in 25% ethyl alcohol and at successive 24-48 hour intervals the concentration was raised to 50%, 75%, 95% and 100%. As the final step, the samples were immersed in methyl salicylate. If tissue had not cleared, a second clearing starting from 95% ethyl alcohol stage was performed to repeat the final steps of the clearing procedure. This procedure allowed clearing of all structures, with exception of the opacified microvascular structures that were filled with contrast. Micro CT for calculating the vascular volume After clearing had taken place, the samples were scanned in a SkyScan 1276 micro CT (Bruker Corporation, Billerica, MA, USA) at 40 kV voltage, 200 µA current and 10 µm resolution to calculate vascular volume. Three samples were scanned at a time, taking approximately 26 minutes per scan with frame averaging set at three in order to reduce noise. Three-dimensional images of the samples were reconstructed using Hierarchical InstaRecon software (NRecon, 1.7.4.2., InstaRecon, 2.0.4.0. InstaRecon). This software 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 65mmMacro 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 nerve area in the graft were measured in a blinded fashion. For each image, a vessel/nerve area ratio was calculated and expressed in percentages. 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