[Mehdi] This video describes the ex vivo living donor parathyroid procurement in pigs, adapted for parathyroid xenotransplantation. Hypoparathyroidism, characterized by insufficient parathormone secretion, critically disrupts calcium homeostasis. Current treatments offer limited relief from systemic symptoms like fatigue and depression. Parathyroid allotransplantation is currently under the highlight, as well as xenotransplantation. The objectives of the study is to propose a simple and non-lethal technique adapted for xenotransplantation based on two key steps. One, porcine thymus retrieval. And two, identification of the parathyroidal gland ex-vivo using transillumination. The experiments were performed in the University of Lille large animal facility. All animal studies were approved by the animal ethical committee. Step one, porcine anesthesia. Administer buprenorphine 30 minutes before the first incision to provide preoperative analgesia. Perform an intramuscular injection of ketamine and xylazine to induce sedation. Administer 5% isoflurane via inhalation for three to five minutes to deepen anesthesia. Assess the depth of anesthesia by checking the eyelid closure reflex and swallowing reflex. Intubate the pig and position it supine on the operating table. Reduce isoflurane concentration to 2% via inhalation for anesthesia maintenance. Continuously monitor heart rate and oxygen saturation using a portable, multi-parameter veterinary monitor to ensure optimal anesthetic depths and physiological stability throughout the procedure. Step two, exposition of the area of interest. Shave and meticulously clean the cervical region, extending from the submandibular area to the sternum. Apply antiseptic soap followed by chlorhexidine to ensure a sterile field. Thoroughly dry the entire area. Position sterile drapes to isolate the anterior cervical region for surgical intervention. Step three, porcine thymus exposure. Perform a 10 centimeter vertical incision along the white cervical line using a scalpel. Carefully dissect the area with Metzenbaum scissors to separate the pre thyroid muscles, creating a clear surgical field. Place a self-retaining retractor between the omohyoid muscle and the thyroid cartilage to facilitate dissection of the connective tissue and expose the thymus. Gradually expose the cranial part of the thymus and carefully separate it from the carotid sheath, which contains the vagus nerve, internal jugular vein, and common carotid artery. Step four. Porcine thymus retrieval. Once released, extract the cranial part of the thymus, which typically measures five to eight centimeters. Rinse the extracted thymus in isotonic saline solution to facilitate the identification of the parathyroid gland. Step five. Identification of the intrathymic parathyroid gland ex vivo. Gently flatten the thymic tissue to facilitate parathyroid gland identification. Use a light source to identify the parathyroid gland via transillumination. Carefully excise the capsule and extract the entire parathyroid gland. Using the Babcock forceps for delicate handling, and Metzenbaum scissors for precise dissection, isolate the gland from the surrounding thymic tissue. Place the isolated parathyroid gland in UW Cold Storage Solution, and maintain it at four degrees to preserve its viability for subsequent procedures. Step six. Surgical closure and awakening. Following the retrieval of the parathyroid gland, close the surgical site in layers. Suture muscles and subcutaneous tissues using absorbable sutures. Close the skin with non-absorbable sutures. Gradually wean the pig off anesthesia. Upon regaining consciousness, monitor the pig for immediate postoperative complications. Administer analgesics and antibiotics as per postoperative protocol. Return the pig to its housing facility for recovery under veterinary supervision. Representative results. The thymus is excised and analyzed ex vivo to identify the parathyroid gland. This can be visible in panel A following the white arrow. Parathyroid glands are then identified by peritoneum staining using immunofluorescence, which can be visible in panel B. Finally, peritoneal levels are measured in the supernatant of small pieces from the gland minced, which can be seen in panel C. Conclusion. This protocol presents a rapid and non-lethal method for parathyroid gland procurement in pigs. Porcine thymus retrieval and the use of transillumination are keystones to identify porcine parathyroid glands ex vivo. The ability to procure and study parathyroid glands in a living model opens new avenues for research and therapy. The preservation of the animal post-procedure, an ethical imperative in modern research, are essential in the context of xenotransplantation.
