Femke Mathot

5 Novel methods for evaluation of vascularization of nerves 83 surrounding the thoracic aorta and vena cava was cleaned using cotton tip applicators taking care not to harm the vascular structures. The thoracic aorta and vena cava were ligated proximally with a 5-0 Vicryl suture (5-0 Vicryl Rapide, Ethicon Inc., Sommerville, NJ, USA) which was kept long to act as grip 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. Figure 1. Schematic drawing of the insertion of the catheter into the rat aorta. Long sutures indicate the grip sutures that have ligated the aorta and vena cava proximally. Short cut suture serves to hold catheter in place while injecting the Microfil® contrast compound into both common iliac arteries of the rat. (with permission of the Mayo Foundation, Copyright Mayo Foundation 2019)