Femke Mathot

Chapter 6 98 anesthetized in an isoflurane induction chamber. Once asleep, the rats were euthanized with an overdose of pentobarbital. Vascular preservation A longitudinal incision over the abdomen exposed the abdominal organs. The aorta and inferior vena cava were dissected carefully. A catheter was placed and fixed inside the aorta over which 10 cc of saline was administered to flush the vasculature. Yellow Microfil® (Flow Tech inc., Carver, Massachusetts, USA) was administered by aortic infusion until both toenail matrixes were colored, which required at least 19 mL of Microfil®. 15 After 90 minutes of curing, the sciatic nerves were dissected and cleared in 5 days by immersing them in graded series of ethyl alcohol (25% - 50% - 75% - 95%) and a final day in methyl salicylate. Vascular surface area To optimize the accuracy of the measured level of revascularization, the vascular surface area and the vascular volume were obtained. The nerve samples were stretched by suturing both ends onto a solid holder. While in a petri dish with methyl salicylate, detailed pictures of the nerve were obtained with a Canon 5D Mark III camera, a Canon 65 mm Macro lens and a Canon Twin Lite Macro strobe light source. Polarized light was used to reduce reflections and a 1:1 magnification was used to ensure consistency of the pictures. To correct for the 3D structure of the nerve sample and the surface area that alters depending on the angle of observation, two pictures of each nerve sample were obtained 180 degrees rotated (i.e. front and back of nerve). The solid holder provided a clear distinction between the front and the back of the nerve sample. The pictures were blinded before analysis. Using Image Pro Plus Software, the total nerve area and the total vessel area were marked and measured and a vessel/nerve area ratio was calculated of each picture taken. The ratios of the two pictures (i.e. front and back of the nerve) were averaged. The obtained photographs also enabled the subjective assessment of the alignment of the preserved vasculature. 42 Vascular volume With a micro-CT (Inveon Multiple Modality PET/CT scanner, Siemens Medical Solutions USA, Inc., Knoxville, TN), a CT scan was obtained from the nerve samples with an effective pixel size of 9.91 m m. As the microfil was more radiopaque than the rest of the nerve, the volume of the total nerve and the volume of the vasculature could be measured with Analyze 12.0 software (AnalyzeDirect, Inc., Overland Park, KS, USA). Eventually, vessel/nerve volume ratios were calculated for each scan. 42 Correlations The correlation between the vascular surface area and volume measurements was obtained to determine whether the methods are complementary to each other. Statistical analysis The outcome measures were analyzed with one-way ANOVA, with Bonferroni correction for post-hoc multiple comparisons. Correlations were analyzed with the Pearson’s correlation test.