All animals were housed in the specific pathogen-free facility (Tongji Medical College, Huazhong University

of Science and Technology, Wuhan, China) and had access to water and food ad libitum. All the studies were performed PCI-32765 purchase in compliance with the Principles of Laboratory animal care (NIH publication Vol 25, No. 28 revised 1996) and the Tongji Medical College Animal Care and Use Committee Guidelines. Tracheas from Balb/c mice were implanted into Balb/c mice (syngeneic, n = 45) or C57BL/6 mice (allogeneic, n = 45). Each donor tracheal graft was evenly divided into three segments, and then simultaneously implanted into orthotopic, intra-omental and subcutaneous sites of each recipient. Grafts (15 syngeneic or 15 allogeneic grafts from each transplant site) were harvested on Days 14, 21 and 28 after transplantation for histologic and immunohistologic analyses. The donors were euthanized by intraperitoneally injecting pentobarbital (80 mg/kg). A midline cervical

incision was performed Vemurafenib to expose the entire trachea. The trachea below the cricoid cartilage distal to the bifurcation was dissected, harvested, and then it was flushed and preserved with cold sterile saline at 4 °C. Prior to implantation, the full-length trachea (approximately 12 cartilage rings) was divided into three segments of 4 cartilage rings, which were then randomly transplanted into various sites respectively. The recipients were anesthetized by intraperitoneally injecting pentobarbital (50 mg/kg). Initially, a short midline cervical incision was performed to visualize the entire laryngotracheal complex. The recipient trachea was carefully dissected, and then transected at the third intercartilage below the epiglottis while spontaneous breathing was maintained. One of the 4-ring donor tracheal segments was implanted end-to-end, starting with the distal anastomosis using 9-0 Prolene suture (Ethicon). The cervical incision was closed in layers with continuous 7-0 Vicryl suture (Ethicon). Subsequently, the recipient mouse underwent Ergoloid a midline laparotomy followed by exposure of the greater omentum. The second tracheal segment

was wrapped and fixed into the greater omentum using 9-0 Prolene, and then the abdominal wall was closed in layers with continuous 7-0 Vicryl suture. Finally, a small incision was made in the dorsal suprascapular area of the recipient mice. A subcutaneous pouch was made with blunt dissection, and then the third tracheal segment was placed into it. The skin was closed with interrupted 7-0 Vicryl suture. The operative procedures were performed with the assistance of a surgical microscope (× 10 magnification) in a sterile fashion. All recipient animals received no immunosuppression. The grafts were harvested from CO2 euthanized recipient mice on Day 14, 21, and 28 after transplantation for histologic and immunohistochemical analyses.