Femke Mathot

Chapter 5 82 system in the rat sciatic nerve and to quantify angiogenesis of nerve reconstruction. The micro CT and conventional photography were used to objectively quantify vascular volume and vascular surface area, respectively, as measurements of angiogenesis in rat nerve. MATERIALS AND METHODS Animal experiments were approved by our Institutional Animal Care and Use Committee (IACUC A3348-18). For this study, male Lewis rats (Envigo, USA) were used, weighing between 250 – 300 grams. All animals were housed with ad libitum access to food and water, with a 12- hour light-dark cycle after surgery. Experimental design In 12 Lewis rats, unilateral 10 mm sciatic nerve gaps were repaired with ipsilateral reversed autologous nerve grafts to create a mismatch in the alignment of the nerve fibers. 25, 26 This group was considered the gold standard for nerve repair. Rats survived for either 12- or 16 weeks. At the time of the sacrifice, autograft nerves (N=6 per time point) and the contralateral nerve samples as untreated, control samples (N=6 per time point) were harvested. The nerve vasculature was preserved to obtain the vascular volume and vascular surface area measurements. Surgical procedure After anesthesia in an isoflurane chamber, rats were shaved, prepped and positioned in the nosecone to maintain anesthesia throughout the procedure. Preoperatively the following were administrated subcutaneously; infection prophylaxis provided by Enrofloxacin (Baytril, Bayer, Germany, 10mg/kg), 5 ml of NaCl 0.9% solution to prevent dehydration and Buprenorphine SR (Buprenorphine SR-LAB, ZooPharm pharmacy, 0.6mg/kg) for pain control. During surgery, body temperature was maintained at 37°C with a heating pad. The sciatic nerve was fully exposed proximally from the inferior margin of the piriformis muscle to approximately 5 mm distal to the bifurcation, under an operating microscope (Zeiss OpMi 6, Carl Zeiss Surgica, Oberkochen, Germany). A 10 mm segment of the sciatic nerve was excised by sharp transection with microsurgical scissors. The nerve graft was reversed and reconstructed with six 10-0 nylon (Ethilon, Ethicon Inc., Sommerville, NJ, USA), epineural interrupted sutures on either side of the anastomosis. Wounds were closed in layers, approximating muscle with two 5-0 absorbable interrupted sutures (5-0 Vicryl Rapide, Ethicon Inc., Sommerville, NJ, USA). The skin was closed subcutaneously, using the same suture. Postoperatively, the rats were kept warm with towels. The rats were observed weekly until completion of the experiment. Perfusion of contrast Twelve and 16 weeks postoperatively, rats were sacrificed. Access for aortic infusion catheter placement was achieved via the abdomen. A large midline incision was made in the abdomen to expose the aorta and vena cava. A small retractor was used to retract the digestive organs providing stable exposure of the aorta and vena cava throughout the experiment. The fat