Tiam Mana Saffari

113 THE LOCAL MICROENVIRONMENT OF NERVE ALLOGRAFTS AFTER ANGIOGENESIS 6 minutes at 7,500g in 4°C, the supernatant was removed, the RNA was resuspended in 15 µl RNAse free water and RNA concentrations were measured using a NanoDropTM 2000/2000c Spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). Complementary DNA (cDNA) was generated using reverse transcriptase and obtained in a concentration of 10 ng/µl. cDNAwas amplified with a CFX384 real time PCR system (Bio-Rad Laboratories, Inc., CA, USA) using SYBR Green detection. Genes evaluated Genes were selected based on their involvement in nerve regeneration and neovascularization, as well as other biological properties and functions 7,15-17 (Table 2). Results were normalized to the mRNA for the general cytoskeletal protein β-actin gene ( Actb ) because this reference gene remains relatively invariable among many tissues and cell types 18 . The differences in gene expression levels were quantified using the comparative delta crossover threshold (2 -ΔΔ CT ) method 19,20 . Primer sequences per gene are provided in the Appendix. Immunohistochemistry To visualize nerve fibers and vascularization, nerve grafts obtained from four rats of each group were used for immunohistochemical staining at 12 and 16 weeks after reconstruction. Nerve grafts were harvested, fixed in 10% formalin under agitation for 48 hours, stored in 70% ETOH and embedded in paraffin. Cross-sectional sections (5 µm) from the mid-distal portion of the graft were cut and stained for protein gene product 9.5 (PGP9.5) a pan-neuronal marker (diluted 1:500, Agilent, USA), which stains both myelinated and unmyelinated nerve fibers 21 , S100B, a common marker of neural tissue (diluted 1:5000, Agilent, USA) and CD34 (diluted 1:4000, Abcam, USA), staining vascular endothelial cells and extensively expressed on blood vessels 22 . A qualitative assessment of the number of blood vessels and nerve fibers in the nerve sections was performed by four blinded observers, using an established scoring system 23 . Statistical analysis Results of qPCR measurements were analyzed for each gene using analysis of variance (ANOVA) corrected with Bonferroni post-hoc testing. Cytokine blood levels were