Procurement of Parathyroid Glands from Living Donor Pigs and Ex Vivo Identification

Summary

This protocol presents a rapid and non-lethal method for procuring parathyroid glands from pigs. The procedure consists of three main steps: porcine preparation with anesthesia, unilateral thymus retrieval, and ex vivo identification and extraction of the intrathymic parathyroid gland.

Abstract

Hypoparathyroidism, often resulting from surgical complications, severely disrupts calcium homeostasis, leading to disabling symptoms. Current treatments offer limited relief, highlighting the need for alternative therapies like parathyroid transplantation. This study introduces a simple method for living-donor parathyroid procurement from pigs, with potential applications in transplantation research, especially for the field of xenotransplantation. The procedure involves three key steps: porcine preparation with anesthesia, unilateral thymus retrieval, and ex vivo identification and extraction of the intrathymic parathyroid gland. The protocol utilizes Landrace pigs, with procedures ensuring ethical treatment and recovery post-operation. We successfully extracted parathyroid glands, confirming their viability and integrity through subsequent analysis. PTH levels in tissue supernatant exceeded 1,000 pg/mL, and immunofluorescence staining distinguished parathyroid glands from thymic tissue, validating the procedure's reliability. This protocol offers a rapid, non-lethal, and ethically sound method for parathyroid gland procurement from pigs, addressing anatomical differences between pigs and humans. It offers a sustainable approach with significant implications for hypoparathyroidism research and xenotransplantation.

Introduction

This protocol presents a rapid and non-lethal method for parathyroid gland procurement from pigs. Hypoparathyroidism, characterized by insufficient parathormone secretion, critically disrupts calcium homeostasis¹. This condition, predominantly a surgical complication following parathyroidectomy or thyroidectomy, manifests through symptoms like asthenia, chronic pain, tachycardia, seizures, and even end-stage renal disease². Occasionally, it arises from rare autoimmune or genetic disorders. Current treatments, mainly calcium and vitamin D oral supplementation or recombinant parathormone, provide only partial relief from systemic symptoms like fatigue and depression^3,4. The growing necessity for parathyroid allotransplantation as a potential remedy for severe hypoparathyroidism underscores the necessity for the establishment of uniform transplant protocols^5,6,7,8,9. However, preclinical studies on parathyroid transplantation remain limited, and there is a lack of standardized animal models. Furthermore, the recent boost in the xenotransplantation field^10,11, increases the interest in clinical applications of xenotransplantation, especially in the field of endocrine cell therapy^12,13. However, porcine models of parathyroid xenotransplantation are challenging due to the complexity of identifying parathyroid tissue in pigs. There is, thus, a need to provide a reproducible, sustainable, and ethical technique to retrieve parathyroid glands from pigs to further develop parathyroid xenotransplantation.

Unlike human beings, who typically have four parathyroid glands located behind the thyroid, pigs possess, for most, two parathyroid glands, each located in the cranial part of the thymus. Previous studies that aimed to locate parathyroid glands in pigs either relied on lethal methods for ex vivo identification or fluorophore-based techniques, which may be difficult to implement in xenotransplantation^14,15,16. We propose here a simple and non-lethal technique, suitable for xenotransplantation, based on two key steps: porcine thymus retrieval and ex vivo identification of the parathyroid gland using transillumination. This technique might be useful for research on parathyroid physiology, as well as transplantation.

Protocol

All animal studies were approved by the animal ethical committee of the University of Lille (referenced #20491-20190219161313).

1. Porcine anesthesia

1. Administer buprenorphine (0.015 mg/kg, intramuscularly) 30 min before the first incision to provide preoperative analgesia.
2. Perform an intramuscular injection of ketamine (50 mg/kg) and xylazine (3 mg/kg) to induce sedation. Administer 5% isoflurane via inhalation for 3 to 5 min to deepen anesthesia.
3. Assess the depth of anesthesia by checking the eyelid closure reflex and the swallowing reflex. Intubate the pig and position it supine on the operating table. Reduce isoflurane concentration to 2% via inhalation for anesthesia maintenance.
4. Continuously monitor the heart rate and oxygen saturation using a portable multi-parameter veterinary monitor to ensure optimal anesthetic depth and physiological stability throughout the procedure.

2. Exposition of the area of interest

1. Shave and meticulously clean the cervical region, extending from the submandibular area to the sternum.
2. Apply antiseptic soap, followed by chlorhexidine, to ensure a sterile field. Thoroughly dry the entire area.
3. Position sterile drapes to isolate the anterior cervical region for surgical intervention.

3. Porcine thymus exposure

1. Perform a 10 cm vertical incision along the white cervical line using a scalpel. Carefully dissect the area with Metzenbaum scissors to separate the prethyroid muscles, creating a clear surgical field.
2. Place a self-retaining retractor between the omohyoid muscle and the thyroid cartilage to facilitate dissection of the connective tissue and expose the thymus.
   NOTE: Avoid excessive pressure on the trachea to prevent injury.
3. 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.
   NOTE: To prevent injury, use electrocoagulation to achieve hemostasis.

4. Porcine thymus retrieval

1. Once released, extract the cranial part of the thymus, which typically measures 5 - 8 cm.
2. Rinse the extracted thymus in isotonic saline solution to facilitate the identification of the parathyroid gland.
   NOTE: This step is crucial for enhancing visualization and differentiation of the parathyroid gland's distinct texture

5. Identification of the intrathymic parathyroid gland ex vivo

1. Gently flatten the thymic tissue to facilitate parathyroid gland identification.
   NOTE: The parathyroid gland exhibits a distinctive texture, particularly in comparison to the surrounding tissue. The fibrotic capsule of the parathyroid gland is firmer and of a similar size to a pea.
2. Use a light source to identify the parathyroid gland via transillumination.
   NOTE: The vasculature surrounding the parathyroid gland should be clearly visible, aiding in its differentiation.
3. Carefully excise the capsule and extract the entire parathyroid gland. Using Debakey forceps for delicate handling and Metzenbaum scissors for precise dissection. Isolate the gland from the surrounding thymic tissue.
4. Place the isolated parathyroid gland in cold storage solution and maintain it at 4 °C to preserve its viability for subsequent procedures.
5. Embed the tissue in paraffin for further processing.

6. Surgical closure and awakening

1. 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.
2. Gradually wean the pig off anesthesia. Upon regaining consciousness, monitor the pig for immediate postoperative complications.
3. Administer analgesics and antibiotics as per postoperative protocol (buprenorphine 0.03 à 0.05 mg/kg and Clamoxyl 15 mg/kg for 48 h). Return the pigs to their housing facility for recovery under veterinary supervision.

7. Immunostaining protocol

1. Perform deparaffinization and dehydration from paraffin-embedded samples: xylene for 5 min, xylene for 10 min, 100% ethanol for 5 min, 90% ethanol for 7 min 30, 70% ethanol for 7 min 30, 50% ethanol for 5 min, distilled water for 5 min and phosphate-buffer-saline (PBS) for 5 min.
2. Perform antigen retrieval by microwaving the samples in citrate buffer pH 6, at 650 W for 9 min. Allow the samples to cool down in the buffer for 20 min. Rinse three times with PBS for 5 min each.
3. Incubate the samples, encircled with a Dako-pen, at room temperature for 20 min with protein-block solution to block non-specific binding.
4. Incubate the samples overnight at 4°C with the primary antibody (PTH Antibody (H-7), Mouse IgG-α-human, sc-398856, 1:100 ) diluted in 1% Bovine Serum Albumin (BSA). Wash twice with PBS for 5 min each.
5. Incubate for 1 h and 30 min at room temperature with the secondary antibody (Alexa Fluor 488, goat-α-Mouse, A11001, 1:500) diluted in 1% bovine serum albumin. Wash twice with PBS and once with double distilled water for 5 min.
6. Mount the slides with DAPI solution and store the slides at 4 °C.

Results

This study's primary outcomes include the successful extraction of parathyroid glands from Landrace pigs and subsequent analysis to confirm their viability and integrity. The results are summarized as follows. Identification of parathyroid tissue was confirmed intraoperatively via transillumination (Figure 1A). After extraction, two small samples of the tissue were collected (approximately 10% each). One sample was minced in a dish-plate in 1 mL of DMEM-F12 culture medium. After min...

Discussion

This protocol presents a rapid and non-lethal method for parathyroid gland procurement in pigs. The key step within the protocol involves localizing the porcine thymus to ensure complete retrieval for further ex vivo investigation. Once the thymus is extracted, the parathyroid gland can be easily identified using transillumination.

One of the main challenges of this technique is the need to avoid relying on human surgical habits associated with parathyroidectomy, as our protocol circu...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Equipment
22G Short Peripheral Catheter	Smiths Medical	5060
Basic set II, crepe	Hartmann	9389253
Bupaq (buprénorphine)	Virbac
Clippers Favorita II	Aesculap Schermaschinen	GT104
Disposable Scalpels	Swann-Morton	0510
Electrode spatula	Erbe	21191-002
Electrosurgery Generator BE VIO 200p	Erbe	10140-400
Euroklav 23 VS+	MELAG	10056
Gravity infusion 3-way valve with mobile Luer Lock tip	Care Fusion	A64-3V
Handle with two buttons for electrodes Slim-Line	Erbe	20190-065
Isoflurane Funnel Fill Vaporizer	Supera Anesthesia Innovations	VAP3000
Isorane (isoflurane)	Axience
Ketamidor (kétamine)	Axience
Light Ceiling Double	RIMSA	PENTA81+30E
Medisorb Carbon Dioxide Absorbent	Vyaire	8570043
Mersutures 0	Ethicon	F2520
Microtome	LEICA RM2255
Miller Blade	Opticlar	600.020
Oxygen Concentrator	DeVilbiss	525KS
Paxman (xylazine)	Virbac
Portable Multi-Parameter Veterinary Monitor	Mindray	IPM 10
Reusable Laryngoscope Handles	Opticlar	600.080
Small Animal Anesthesia Ventilator	Midmark	91806001
Sodium Chloride Injection Viaflo	Baxter
Surgical Instruments	Kohler medizintechnik
Surgical Table	Vetinox	TA700005
Tracheal Tube	Smiths Medical	100/179/070
Vicryl 2-0	Ethicon	JV453
VMS	Midmark	91800003
Materials and Reagents
DMEM F-12 medium	ThermoFischer	11320033
Parathormone antibody	SantaCruz	sc-398856
Rapid intraoperative pth assay	Beckman Coulter	A16972