Tiam Mana Saffari

59 THE SUPERFICIAL INFERIOR EPIGASTRIC ARTERY FASCIA FLAP IN RATS 3 5B. The flap edges were trimmed to fit the defect (Figure 5C). Care was taken to ensure that there was no tension on the nerve anastomoses while positioning the flap under the reconstructed nerve. After ensuring that there was no pedicle compression in the subcutaneous tunnel, the vascular pedicle remained freely mobile with full ranging of the leg and that the nerve anastomoses were without tension, two loosely tied 10-0 nylon-sutures (10-0 Ethilon, Ethicon Inc., Sommerville, NJ, USA) were placed through the SIEF flap (Figure 5D). Wounds were closed in layers, with muscle approximated with 5-0 absorbable sutures (5-0 Vicryl Rapide, Ethicon Inc., Sommerville, NJ, USA), and the skin of the leg and the abdomen was closed subcutaneously using the same suture. Evaluation SIEF flap The viability of the SIEF flap was evaluated at sacrifice by 12 and 16 weeks. This was performed using the milking patency test 28 . The SIEA was found and the vessel was occluded with forceps distal to the flap. The other forceps was placed just distally to the first. The vessel would be milked a few millimeters away from the flap. Thereafter the proximal forceps would be released. Rapid filling fromproximal to distal would indicate that the artery was not occluded (1), if no filling occurred, the test would be scored a (0). Viability of the flap was also characterized by color of the flap and active bleeding at the edges of the flap. Immunohistochemistry At 12 weeks, immunohistochemical staining of the nerves was obtained to confirm revascularization of the nerve. After sacrifice, the nerve grafts were harvested and fixed in 10% formalin (Fisher Scientific, NH, USA) for 48 hours and then transferred to 70% Ethanol and stored at 4°C. After embedding in paraffin, serial sections (5µm) were obtained from distal parts of the nerve grafts. These sections were stained with hematoxylin and eosin (H&E) and primary antibody rabbit anti-rat CD34 (1:4000, Abcam, Cambridge, MA, USA) to visualize vascularization and fibrosis in the nerve graft. CD34 is a transmembrane phosphoglycoprotein and established as a marker of hematopoietic cell types, including vascular endothelial progenitors and extensively expressed on blood vessels 29 . Contralateral nerve samples were stained as control. Immunohistochemical digital photographs were taken at 40x magnification with a microscope (Nikon Eclipse 50i) equipped with a digital camera. Images were qualitatively assessed.