Tiam Mana Saffari

74 CHAPTER 4 sutures. The ligation was placed as proximal as possible and distal to any large hepatic bifurcations, depending on the anatomic variation. The aorta was dissected from the vena cava distally using cotton tip applicators. This was performed approximately 1 cm proximal to the iliac bifurcation. A loose 5-0 Vicryl suture was placed under the aorta. To facilitate the passage of contrast, a 24 Gauge catheter (Jelco IV Catheter Radiopaque, Smiths Medical International, UK) connected to an IV tubing system, was introduced in the aorta just distally to the proximally placed grip sutures, while keeping the aorta on tension by slightly pulling the grip sutures (Figure 1). After the catheter was fixated with the previously placed suture around the aorta distally, 2-3 ml of saline (NaCl 0.9%) was infused through the tubing system to evaluate the patency of the aorta. The needle was removed off the cannula, while maintaining the cannula in the artery. Care was taken that the tip of the cannula would still be proximal to the iliac bifurcation, so that the contrast would reach both limbs. A yellow-colored (MV-122) Microfil® compound (MV 8ml, diluent 15 ml, and curing agent 1.2 ml, Flow Tech, Inc., Carver, MA, USA) in a 50 cc syringe was connected to the tubing system to be infused in the aorta. While putting pressure on the insertion site using gauze, the perfusion was performed with constant perfusion of approximately 100 mmHg. The perfusion was continued until the syringe was empty and yellow nailbeds on either paw were observed. After the perfusion was completed, a clamp was placed on the cannula to prevent leakage of the infused contrast. The rat was kept at room temperature while the agents cured for at least 90 minutes. Collection and clearing of samples After the vascular bed was perfused and the contrast cured, the sciatic nerve was exposed and harvested extending to approximately 3 mm on either side of the anastomoses. The contralateral nerve samples were harvested as non-operated, control samples (N=12). The 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 opacifiedmicrovascular structures that were filled with contrast.