Pentobarbital

Pentobarbital, salts, and buffers were purchased from Sigma-Aldrich (St. Louis, MO, USA) Pentobarbital was dissolved in ND96 electrophysiology buffer (see below) and pH adjusted to 7.5 on the day of use.

Male SD rats (aged 6–8 weeks) were randomly divided into two groups: the CKD group (n = 5) and sham group (n = 5), All animals were obtained from the Shanghai Model Organisms Center, Inc. (Shanghai, China). All animal experiments were approved by the Ethic Committee of Shanghai Tenth People’s Hospital, School of Medicine, Tongji University (2016-33; Shanghai, China). In brief, rats were housed under constant environmental conditions with a 12-h light–dark cycle. Standard rat chow and water were provided ad libitum before and after surgery (Hsu et al., 2015 (link)). A two-step 5/6 nephrectomy was carried out to induce CKD as previously described (Hanifa et al., 2019 (link); Uchiyama et al., 2016 (link)). Rats were anesthetized by peritoneal injection of pentobarbital (30–50 mg/kg) (Merck KGaA, Darmstadt, Germany) and right kidney was then exposed through a flank incision and ∼2/3 of the right kidney were removed. One week later, the left kidney was exposed and the upper and lower poles were resected through a flank incision. For sham rats, each flank of the kidney underwent sham surgery by incising the flanks, exposing the kidney and suturing each flank. Eight weeks after the second step of nephrectomy, rats were sacrificed and blood samples were obtained by cardiac puncture and urine was collected in individual metabolic cages.

After implantation of the lung and endometrium tissue grafts, the macroscopic appearance of the skinfold chamber preparation was controlled daily. In 15 mice with a total of 34 analysable lung transplants and 12 endometrium grafts (7 mice with 2–4 lung grafts, 8 mice with 1–2 lung grafts and 1–2 endometrium grafts), intravital fluorescence microscopic analyses were performed on days 0 (day of transplantation), 3, 6, 10 and 14 after transplantation. Functional capillary density was measured within three areas of interest per graft and observation time point. Microvessel diameters and microhemodynamic parameters were determined by analysing 10 microvessels per region of interest. Microvessels were selected randomly inasmuch as those microvessels were chosen, which crossed a vertical line drawn over the center of the video screen. At the end of the in vivo experiments, i.e. day 14 after transplantation of lung and endometrium grafts, the animals were euthanized with an overdose of pentobarbital (Merck, Darmstadt, Germany), and the dorsal skinfold chamber preparations were processed for haematoxylin and eosin staining and immunohistochemistry.

General anesthesia was administered to Lewis rats (12–16-weeks old, 350–430 g; Charles River Laboratories Japan) by the inhalation of 1.0–2.5% isoflurane and injection of pentobarbital (10 mg/kg intraperitoneal, Merck & Co., Inc.) An incision was made in the midline neck of the rats. The cervical trachea was exposed from the cricoid ring to the suprasternal notch and dissected from the esophagus. Three cartilage rings of the cervical tracheal segment were resected. We switched to intubation in the operative field. Anastomoses were performed at the proximal and distal ends of the tracheal analogue graft having 3 rings. The rats were allowed to recover gradually after the discontinuation of the isoflurane delivery. Analgesia (buprenorphine 0.017 mg/kg/day, subcutaneous) was administered regularly for the first five postoperative days. After the predetermined periods of implantation (1 week, 1 month, or 8 months), the rats were sacrificed by injecting sodium pentobarbital (200 mg/kg), and the implants with the surrounding tracheas were harvested.