Orthotopic Porcine Kidney Transplantation: A Technique to Transplant Graft Kidney in Porcine Model

프로토콜

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Basic techniques and common procedures

1. Use female German landrace pigs (or comparable) for this protocol. Figure 1 depicts the summary of the described experimental protocol. Deliver the animals to the research facility 14 days before the surgery for acclimatization and house them in a temperature- and humidity-controlled barrier environment with a 12 h light and dark cycle (Figure 2). Fast the animals overnight before surgery.
2. Premedicate by an initial intramuscular injection of azaperone (4 mg/kg) and atropine (0.1 mg/kg), followed by an injection of ketamine (15 mg/kg) 10 min later.
3. After premedication, weigh the animal and transfer it directly from the housing facility to the central OR facility anesthesia preparation room.
4. Cannulate one of the large ear veins using an 18 G peripheral venous catheter. Monitor the animal by a standard ECG and pulse oximetry.
5. Initiate the anesthesia with propofol (3 mg/kg).
6. Expose the vocal cord with a laryngoscope and insert a 7.5 mm endotracheal tube. The cuff is blocked with air according to the manufacturer's recommendations.
7. Insert an oro-gastric drainage tube to remove fluid and air from the stomach.
8. Insert a urinary catheter via the urethra.
9. Subsequently, trim the skin in the area of the surgical incision.
10. Apply eye ointment to prevent drying of the cornea during surgery.
11. After orotracheal intubation, maintain anesthesia with isoflurane (final expiratory 1.45-2.0 Vol.%) and fentanyl (3-7.5 μg/kg/h).
12. Ensure active intraoperative temperature control of the animal by a heating pad and using warmed air. Insert a rectal probe to monitor body temperature (target temperature 36.5 °C - 37.5 °C).
13. Administer antibiotic prophylaxis using cefuroxime (35 mg/kg i.v.). Infuse Ringer solution at 4 mL/kg/h and increase to 8 mL/kg/h after skin incision. Administer a prophylactic dose of pantoprazole (40 mg i.v.) over the ear vein access.
14. Perform all surgical procedures under sterile conditions according to the general principles of surgical asepsis and antisepsis. Disinfect the surgical field with povidone-iodine solution and cover with surgical drapes.

2. Contralateral nephrectomy and orthotopic kidney auto-transplantation

1. During the recipient operation, adapt premedication and initial anesthesia to the restricted renal metabolism and avoid the use of ketamine. Induction is performed with propofol (3-5 mg/kg i.v.), midazolam (0,25 mg/kg i.v.), and atropine (0.1 mg/kg i.m.). Thereafter, the preoperative preparation is identical to the procedures described in section 1.
2. Maintain anesthesia with isoflurane (final expiratory 1.45-2.0 Vol.%) and fentanyl (3 - 7,5 μg/kg/h) and propofol (2 - 4 mg/kg/h).
3. Check and continuously monitor ECG, pulse oximetry, rectal temperature and the function of the telemetry transponder implanted in the right flank.
   NOTE: Strict anesthesia and blood pressure control is of crucial importance during the implantation procedure.
4. In rare cases where the arterial blood pressure signal registered over the telemetry transponder is not satisfactory due to the supine position of the animal, place a further arterial catheter into the right femoral artery using percutaneous puncture and the Seldinger technique.
5. Following sterile draping, reopen the median laparotomy and expose the surgical field using the abdominal retractor. The colon and small bowel are placed to the left side of the abdomen to expose the intact right kidney.
6. Dissect the contra-lateral (right) kidney and its vessels from the surrounding tissue. Dissect the right renal vein and renal artery in the direction of the kidney hilum to ensure sufficient vessel length for anastomosis.
7. Five minutes before vascular clamping, inject natrium-heparin intravenously (100 I.U./kg).
8. Clamp the right renal artery and the right renal vein using vascular clamps. The right kidney is removed. The vessels are checked for integrity before starting the anastomoses.
9. Place the preserved graft kidney into the abdomen and start the venous and arterial anastomoses.
10. From this point onwards, keep the mean arterial pressure over 80-90 mm Hg to ensure good early perfusion of the kidney graft following reperfusion. Achieve this partially by adequate volume management and partially by the administration of norepinephrine (0.1 - 1.0 μg/kg/min as a continuous infusion using the mean arterial blood pressure and heart rate for monitoring the efficiency).
11. Perform end-to-end anastomosis of the renal vein:
    1. After placing two corner stitches using 5-0 polypropylene, suture the back wall in a continuous fashion.
    2. Tie the cranial corner stitch and tie it together with the thread used for the back wall.
    3. After finishing the back wall, use the cranial corner stitch to suture the front wall in a cranio-caudal direction. Flush the vein with a heparinized saline solution (100 I.U./mL). Tie the caudal corner stitch.
       NOTE: In case of a size mismatch between the donor and recipient sides, a small growth-factor can be used to ensure a wide and sufficient anastomosis. There are many possible variations of the porcine renal vein branches. In the case of complex venous anatomy, a modified anastomosis approach is necessary (see Figure 3).
12. Perform the end-to-end anastomosis of the renal artery:
    1. Use a 6-0 polypropylene cranial corner stitch to perform the arterial anastomosis. Placing a further caudal, supporting corner stitch which is later removed, is optional.
    2. Suture the back wall in a continuous fashion using the parachute technique. After arriving at the caudal corner remove the second corner stitch (if applicable).
    3. Suture the front wall with the other end of the double-armed 6-0 polypropylene suture. Flush the artery with a heparinized saline solution (100 I.U./mL). Tie the two threads at the caudal corner.
13. Record the time needed for performing both anastomoses with a target warm ischemia time of <40 min.
14. Reperfuse the kidney by opening the venous vascular clamp and subsequently the arterial clamp. Check for significant bleeding.
15. If no significant bleeding from the anastomoses is observed, unwrap the kidney graft and pour warm normal saline solution in the abdomen covering the reperfused graft.
16. Reposition the graft, if needed, to ensure homogeneous reperfusion and avoid congestion.
17. Administer papaverine topically to the outside of the renal artery and the arterial anastomosis (5 mL undiluted).
18. After reperfusion, infuse 250 mL of 20% glucose solution to induce osmotic dieresis followed by the administration of a single dose of 80 mg of furosemide.
    NOTE: Following this, initial urine production may be observed.
19. To ensure urinary drainage, pass a 12 French pediatric urine catheter through the abdominal wall of the right flank of the animal, retroperitoneally.
20. Secure the catheter in the ureter using ligatures (2-0 polyglactin) and block the catheter with 2 mL saline. Further sutures are used to adapt and secure the ureter to the peritoneum of the abdominal wall (2-0 polypropylene). The catheter is also secured to the skin with at least two single knot sutures (2-0 polypropylene).
21. Close the peritoneal layer over the kidney to prevent dislocation of the kidney graft and kinking of the vascular anastomoses (3-0 polyglactin).
22. Close the abdomen in a similar 4-layer fashion as described earlier for the graft retrieval.
23. Following abdominal closure, maintain normothermia on the OR table.
    NOTE: Mean arterial blood pressure should be maintained over 80 mm Hg until the animal is awake and is in a prone position. Following abdominal closure, use color Doppler ultrasound to ensure adequate arterial and venous perfusion of the kidney graft (Figure 4). Monitor the animal closely until it is fully awake and can walk and drink spontaneously. The animals are given 1 L of Ringer solution during the recovery phase. Subsequently, return the animal to its box in the housing facility.

결과

Figure 1: Study flowchart and protocol.

Abbreviations used: POD-postoperative day; ECG-electrocardiography.

Figure 2: Animal housing facility with real-time and continuous telemetry monitoring of up to 6 animals.

자료

Name	Company	Catalog Number	Comments
Anesthesia materials, drugs and medications
Atropine sulfate solution for injection, 100mg	Dr. Franz Köhler Chemie GmbH, Bensheim, Germany	1821288	parasympatholytic agent, premedication
Bepanthen ointment for eyes and nose	Bayer Vital AG, Leverkusen, Germany	1578675	eye ointment
BD Discardit II syringes, 2ml, 5ml, 10ml,20ml	Becton Dickinson GmbH, Heidelberg, Germany	300928, 309050,309110, 300296	syringes
BD Micolance 3 (20G yellow) Cannula	Becton Dickinson GmbH, Heidelberg, Germany	305888	venous catheter
BD Venflon Pro Safety (20G pink)	Becton Dickinson GmbH, Heidelberg, Germany	4491101	venous catheter
Buprenorphine (Buprenovet)	Bayer Vital AG, Leverkusen, Germany	794-996	analgesia
Cefuroxime 750mg, powder for preparing injection solution	FRESENIUS KABI Deutschland GmbH, Bad Homburg, Germany	J01DC02	antibiotics
Covidien Hi-Contour, Endotracheal Tube 7,5 with Cuffed Murphy Eye	Covidien Deutschland GmbH,Neustadt/Donau, Germany	COV-107-75E	endotracheal Tube
FENTANYL 0,5 mg Rotexmedica solution for injection Rotexmedica GmbH	Arzneimittelwerk, Trittau, Germany	4993593	opioide analgetic agent
Furosemide-ratiopharm 250 mg/25 ml solution for injection	Ratiopharm GmbH, Ulm, Germany	1479542	loop diuretics
Glucose 5% solution for infusion (500ml, 250ml)	B. Braun Deutschland GmbH & Co. KG, Melsungen, Germany	3705273, 03705422	infusion fluid
Glucose 20% solution for infusion	B. Braun Deutschland GmbH & Co. KG, Melsungen, Germany	4164483	osmotic diuresis
Heparin-Sodium 5000 I.E./ml	B. Braun Deutschland GmbH & Co. KG, Melsungen, Germany	15782698	anticoagulant
Isoflurane-Piramal (Isoflurane)	Piramal Critical Care Deutschland GmbH, Hallbergmoos, Germany	9714675	volatile anaesthetic agent
Ketamine (Ketamine hydrochloride) 10%	Medistar Arzneimittelvertrieb GmbH, Ascheberg, Germany	4230	general anaestetic agent
MIDAZOLAM 15mg/3ml	Rotexmedica GmbH, Arzneimittelwerk, Trittau, Germany	828093	hypnotica, sedative agent
NaCl 0,9% solution for infusion (500ml,1000ml)	B. Braun Deutschland GmbH & Co. KG, Melsungen, Germany	864671.8779	infusion fluid
Norepinephrine (Arterenol)	Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany	16180	increase in blood pressure
Pantoprazole 40mg/solution for injection	Laboratorios Normon,Madrid, Spain	11068	proton pump inhibitor
Paveron N 25mg/ml solution for injection (Papaverine Hydrochloride)	LINDEN Arzneimittel-Vertrieb-GmbH, Heuchelheim, Germany	2748990	spasmolytic agent for vasodilatation
Propofol 1% (10mg/ml) MCT Fresenius	FRESENIUS KABI Deutschland, GmbH, Bad Homburg, Germany	654210	general anaesthetic agent
Ringer solution	B. Braun Deutschland GmbH & Co. KG, Melsungen, Germany	1471411	infusion fluid
Urine catheter ruffle 12CH	Wirutec Rüsch Medical Vertriebs GmbH, Sulzbach, Germany	RÜSCH-180605-12	transurethral urine catheter
Surgical materials
Appose ULC Skin Stapler	Covidien Deutschland GmbH,Neustadt/Donau, Germany	8886803712	skin stapler
Feather Disposable Scapel (11)(21)	Feather, Japan	8902305.395	scapel
Prolene 2-0, blue monofil VISIBLACK, FS needle	Johnson & Johnson Medical GmbH- Ethicon Deutschland, Norderstedt, Germany	EH7038H	skin
Prolene 5-0 (simply angulated, C1 needle) blue monofil VISI-BLACK	Johnson & Johnson Medical GmbH- Ethicon Deutschland, Norderstedt, Germany	EH7227H	vascular
Prolene 5-0 (double armed, C1 needle) 60cm	Johnson & Johnson Medical GmbH- Ethicon Deutschland, Norderstedt, Germany	KBB5661H	vascular
Prolene 6-0 (double armed, C1 needle) 60cm	Johnson & Johnson Medical GmbH- Ethicon Deutschland, Norderstedt, Germany	EH7228H	vascular
Sempermed derma PF Surgical Gloves Seril Gr. 7, 7.5, 8	Semperit investment Asia Pte Ltd, Singapore	4200782, 4200871, 4200894	surgical gloves
Sentinex® PRO Surgical Gowns Spunlace XL 150cm	Lohmann & Rauscher GmbH & Co. KG, Neuwied, Germany	19302	surgical gown
Telasorp Belly wipes (green 45x45cm)	PAUL HARTMANN AG, Heidenheim, Germany	4542437	abdominal towel
Pediatric urine catheter	Uromed Kurt Drews KG, Oststeinbeck, Germany	PZN 03280856	used for the uretero-cutaneus stoma
VICRYL- 0 MH Plus	Johnson & Johnson Medical GmbH- Ethicon Deutschland, Norderstedt, Germany	V324	fascial closure
VICRYL - 3-0, SH1 Plus needle, 75cm	Johnson & Johnson Medical GmbH- Ethicon Deutschland, Norderstedt, Germany	W9114	subcutaneous suture, peritoneal suture
VICRYL - 3-0, SH1 Plus needle, 75cm	Johnson & Johnson Medical GmbH- Ethicon Deutschland, Norderstedt, Germany	V780	subcutaneous suture, peritoneal suture
VICRYL - ligatures Sutupak purple braided, 3-0	Johnson & Johnson Medical GmbH- Ethicon Deutschland, Norderstedt, Germany	V1215E	threads for ligature
3M™ Standard Surgical Mask 1810F	3M Deutschland GmbH, Neuss, Germany	3M-ID 7000039767	surgical mask
Surgical instruments
Anatomical forceps Standard	ASANUS Medizintechnik GmbH, Tuttlingen, Germany	PZ0260	anatomical forceps
Atraumatic tweezers steel, De Bakey Tip 1,5mm 8"	ASANUS Medizintechnik GmbH, Tuttlingen, Germany	GF0840	anatomical atraumatic forceps
Bipolar forceps 16 cm straight, Branch 0,30 mm pointed, universal fit	Bühler Instrumente Medizintechnik GmbH,Tuttlingen, Germany	08/0016-A	biopolar forceps
Bulldog clamp atraumatic, curved, De bakey 78 mm, 3"	ASANUS Medizintechnik GmbH, Tuttlingen, Germany	GF0900	bulldog clamps
DE BAKEY-SATINSKY vascular clamp 215mm	ASANUS Medizintechnik GmbH, Tuttlingen, Germany	GF1661	vascular clamp
Dissecting scissors Mayo, 250 mm, 10"	ASANUS Medizintechnik GmbH, Tuttlingen, Germany	SC2232	Scissors for dissection
Dissecting scissors Metzenbaum-Fino, 260 mm, 101/4"	ASANUS Medizintechnik GmbH, Tuttlingen, Germany	SC2290	Scissors for dissection
Draeger CATO Anesthetic machine with PM8050 Monitor	Dräger, Drägerwerk AG & Co. KGaA, Lübeck, Germany	106782	Ventilation System
Fine Tweezers, ADSON 180 mm	ASANUS Medizintechnik GmbH, Tuttlingen, Germany	ADSONPZ0571	fine forceps
Gosset abdomenal wall spreader	CHIRU-INSTRUMENTE, Kaierstuhl, Germany	09-621512	abdominal retractor
Microsurgical/watermaker tweezers LINZ 150mm 6" Ergo round handle	ASANUS Medizintechnik GmbH, Tuttlingen, Germany	MN0087	fine microsurgical forceps
Surgical forceps Standard 5 3/4"	ASANUS Medizintechnik GmbH, Tuttlingen, Germany	PZ1260	surgical forceps
Surgical scissors standard pointed-blunt (thread/cloth scissors)175 mm, 7"	ASANUS Medizintechnik GmbH, Tuttlingen, Germany	SC1522	surgical scissors
Further material
Heating pad	Eickemeyer - Medizintechnik für Tierärzte KG, Tuttlingen, Germany	648050 MHP-E1220	maintain body temperature during surgery
Laryngoscope, customized	Wittex GmbH, Simbach, Germany	333222230	expose the vocal cord
Rectal temperature probe	Asmuth Medizintechnik, Minden, Germany	ASD-RA4	measure body temperature
Ultrasound device, Sonosite Edge-II	FUJIFILM SonoSite GmbH, Frankfurt, Germany	V21822	ultrasound and color Doppler
Urine bag 2000ml Volume	ASID BONZ GmbH, Herrenberg, Germany	2062578	disposable urine bag connected to the uretero-cutaneous fistula catheter