The protocol received ethical approval from the Animal Care Committee, Toronto general research institute. Animals received humane care in compliance with the National Society of Medical Research and Guide for the care of laboratory animals, National Institute of Health (NIH), Ontario, Canada. For this study, 15-week-old unrelated Yorkshire male swine, weighing 40-50 kg were used.
NOTE: The entire protocol of the study is divided into the following major steps: (i) Organ retrieval and back-table preparation; (ii) Recipient total pancreatectomy and (iii) Graft implantation. The entire surgery of donor and recipient is done in 1 day.
1. Donor organ retrieval and back-table preparation
NOTE: The method for organ retrieval has been described in a separate protocol6. However, the protocol, in brief, is described here along with some additional take-home points specific to the surgical technique (surgical tips relevant to donor operation).
<ol>
	<li>In brief, anesthetize the donor pig, and secure the airway and central venous catheter (described below in the recipient pancreatectomy section). Incise and expose the viscera. Perform aortocaval dissection and mobilization till the bifurcation into iliac vessels.</li>
	<li>Bowel handling: Porcine small bowel is long and tortuous, and the large bowel is mostly distended. Make sure to reposition the bowel loops in their anatomical position after every step of donor operation to ensure adequate perfusion and avoid the possibility of mesenteric torsion.</li>
	<li>Hilar dissection: Ligate and divide the arterial branches and bile duct to expose the portal vein. Dissect the hilum (hepato-duodenal ligament) as high/distal as possible to avoid the risk of injuring the small vascular channels that perfuse the superior aspect of the pancreato-duodenal region. Start skeletonizing the vessels in a right to left sequence, till the anterior surface of the portal vein lies bare.</li>
	<li>Aorto-caval dissection: Expose the supra-hepatic infra-diaphragmatic part of the aorta by dividing the diaphragmatic crura. Avoid dissection of the inferior vena cava around the pancreato-duodenal grove. Use the renal arteries on both sides as the marked upper limit of dissection.</li>
	<li>Expose the pancreas by opening the lesser sac. Ligate and divide the renal arteries. Cannulate the infrarenal aorta and connect with the University of Wisconsin (UW) solution for flushing.</li>
	<li>Ligate the supra-hepatic aorta and clamp. Flush with 1.5 L-2 L of UW solution to ensure adequate flushing of the bowel and pancreas (evident by the visual appearance of pallor). During in situ flushing of the organ, reposition the small bowel and large bowel in sequence to the midline ensuring no mesenteric twist. This step ensures uniform flushing of the bowel and pancreas with the UW (University of Wisconsin) solution and is important to remember.</li>
	<li>Incise the portal vein and infrahepatic vena cava simultaneously after starting the flush to let out the venous effluent. Cover the abdomen with ice to ensure cold perfusion. Dissect the organ (pancreatoduodenal graft with parts of retroperitoneum, psoas, spleen, and adrenal) en masse.</li>
	<li>Cold dissection: Start mobilization of the graft from lateral to medial direction, from the tail of the pancreas to the junction between the head and the corpus, by sharp dissection of the fascia between the pancreas and large bowel. While performing this step, hold the pancreas by its retroperitoneal covering with forceps and ask the assistant to give counter traction on the large bowel toward the foot.</li>
	<li>Mesenteric clamping: After dividing the thin tissue around the corpus of the pancreas, duodenojejunal loop, and large bowel, ask the assistant to loop around the mesenteric pedicle and pull the large bowel loop caudally. This will ensure delineation of the mesentery and safe placement of the clamp to divide the mesenteric major vessels in sequence without injuring the pancreatic parenchyma.</li>
	<li>Complete extra-peritoneal dissection of the graft: After dividing the mesenteric vessels, start retrieving the graft by dissection from medial to lateral in a counterclockwise direction, dividing the extrapancreatic tissues, including the pararenal fascia, adrenal gland, cuff of IVC, psoas muscle, and diaphragmatic crus. Ensure an adequate plane away from the aorta to avoid injuring the superior mesenteric and coeliac arteries.</li>
	<li>Perform back table preparation on ice. Store the organ in an ice box (cold ischemia time of 5 h).</li>
</ol>
2. Recipient pancreatectomy
<ol>
	<li>Pre-operative preparation
	<ol>
		<li>Fast the animal for at least 6 h before the stipulated induction time. Administer an injection containing midazolam (0.15 mg/kg), atropine (0.04 mg/kg), and ketamine (25 mg/kg) subcutaneously (SC), 15 min before transporting the animal to the operating room (OR). Administer buprenorphine 0.3 mg sustained-release injection SC 15 min prior to transport to the OR.</li>
		<li>Positioning: Place the animal supine on the operating table and harness the forelimbs to ensure a stable position during the surgery.</li>
		<li>Airway and induction: Ventilate the animal using a bag and mask with 3%-5% isoflurane and 2-3 L oxygen per min, while connecting the pulse oximeter probe and monitoring the heart rate and O2 saturation.</li>
		<li>After adequate relaxation, confirmed by relaxation of the jaws and stable oxygen saturation and heart rate, ask the assistant to hold open the mouth with adequate traction on the relaxed upper and lower jaws of the animal, and visualize the vocal cords using the laryngoscope. Spray 2% lidocaine to relax the vocal cords to prevent spasms induced by intubation (the porcine vocal cords are highly vascular and fragile!).</li>
		<li>Intubate using a 7 mm endotracheal tube and inflate the cuff using 3-5 mL of air. Ensure the position of the tube using the end-tidal CO2 (ETCO2) probe and connect the tube to the ventilator, with 14-16 breaths per min to achieve a tidal volume of 10-15 mL/kg. Lower the isoflurane to 2.5% as a maintenance dose of inhalational anesthesia.</li>
		<li>Intravenous line: Aseptically prepare the surgical site with betadine and place a sterile drape. Then, identify the landmark to place the central venous catheter, which is the centroid of the triangle formed between the mastoid process, acromion process, and head of the clavicle. Use a 16 G needle to puncture the vein; after ensuring free flow of venous blood, apparent by the color and lack of pulsatility, introduce a guidewire using the Seldinger technique.</li>
		<li>Dilate the tract using the dilator provided. Replace the guidewire and dilator with an 8.5 x 10 Fr catheter and secure in place by suturing with a 0-0 silk suture cutting needle. Connect the IV line to the intravenous antibiotics containing cefazolin (1 gm) and metronidazole (500 mg) followed by intravenous anesthesia done by propofol infusion at the rate of 10 mL/h.</li>
		<li>Invasive blood pressure monitoring: After aseptically preparing the surgical site with betadine, place a sterile drape and make an incision parallel to the trachea 2 cm from the midline. Dissect the fibers of sternomastoid muscle and paratracheal fascia by blunt dissection using Lahey&#39;s right-angled forceps and hemostatic forceps.</li>
		<li>Identify the jugular vein lateral to the fibers of the strap muscles and dissect along the muscle compartmentalizing it from the carotid artery medially using blunt dissection as described above. Identify the artery by its pulsation, texture (cord-like), and perivascular plexus. Mobilize the vessel and sling it using two silk 2-0 ties.</li>
		<li>Puncture the vessel with a 16 G angio-catheter with a beveled tip pointing upward and withdraw the inner stylet once inside the vessel. Slide the outer catheter sheath using the Seldinger technique and connect the outlet to the arterial blood pressure (BP) measuring system. Prior to connecting the invasive BP monitor, calibrate the reading to zero to ensure accurate recording and secure the catheter in place using the silk ties across.</li>
		<li>Temperature monitoring and warming: Place the temperature recording probe orally. Cover the animal with the Bair-hugger heating duvet and ensure a temperature of 37-38 &#176;C. Lubricate the eyes with a neutral eye lubricant gel. Paint and drape the surgical field</li>
	</ol>
	</li>
	<li>Surgical procedure 
	NOTE: The skin is aseptically prepared for all surgical procedures using betadine scrubs followed by the placement of sterile drapes.
	<ol>
		<li>Incision and exposure: Make a midline incision from the xiphisternum to the symphysis pubis using the pure cutting mode of Bovie&#39;s electrocautery. Ensure to keep lateral to the urethra in the lower part of the incision beyond the phallus to avoid injury. Place the self-retaining abdominal wall retractor taking care not to injure the spleen and liver, to optimize exposure of the surgical field.</li>
		<li>Mobilization of the head of the pancreas: With the assistant providing counter-traction to the pancreato-duodenal grove, start by dissecting along the avascular fascial plane between the pancreatic parenchyma (duodenal lobe) and the infra-hepatic IVC.</li>
		<li>Pancreatic ring mobilization: The porcine pancreas forms a ring around the mesenterico-portal axis (connecting lobe). Dissect along the fascia on either aspect of the portal vein (PV) to ensure complete mobilization of the connecting lobe from the overlying PV. The safest way is to start the dissection along either side of the portal vein and separate the tissues circumferentially in a clockwise or anticlockwise direction.</li>
		<li>Pancreatic tail mobilization: Next, identify the junction between the pancreatic and pararenal tissues (seen as a white line) and start dissecting the tail off the underlying splenic vein staying close to the parenchyma. Ligate and divide the small venous tributaries to the parenchyma, taking care not to injure the splenic vein using silk 3/0 ties.</li>
		<li>Pancreatic corpus mobilization: Dissect along the thin layer of tissue separating the parenchyma from the large bowel and the stomach, taking care to preserve the thin vascular arcade along the infra-pyloric region.</li>
		<li>Pancreato-duodenal mobilization: Next, separate the parenchyma from the C loop of the duodenum by sharp dissection in the pancreato-duodenal grove by traction-counter traction, taking care to preserve the thin vascular arcade along the C loop of the duodenum.</li>
		<li>Division of the pancreatic duct: Identify the pancreatic duct, ligate with silk 2-0 ties and divide keeping around 3-5 mm stump of the duct on the duodenal C loop, staying away from the duodenal vascular arcade (in close relation to the duct).</li>
		<li>Removal of the specimen: Complete the last part of the mobilization by dissecting the parenchyma from the junction between the connecting lobe, duodenum, and colon on either side of the portal vein. Extract the specimen en-masse (Figure 1) by dividing the connecting lobe on the anterior aspect of the PV. Division of the parenchyma is required to extract the specimen out because of its circumferential location around the PV.</li>
		<li>Ensure hemostasis along the areas of dissection and inspect the duodenum for any injury and vascular congestion (Figure 2).</li>
	</ol>
	</li>
	<li>Implantation of the pancreato-duodenal graft
	<ol>
		<li>Preparation of the graft: Prepare the graft PV and proximal aortic end by trimming the edges for anastomosis. Place the prepared graft in the organ bag with UW solution on an ice bath. Take core needle biopsies from the pancreatic tail -store in formalin, snap freeze, and extract RNA at a later stage. Suture the biopsy site using Prolene 6-0 in a figure of 8 fashion.</li>
		<li>Aorto-caval dissection: Begin by identifying the right ureter and dissect along the fascia separating the ureter from the retroperitoneal covering over the inferior vena cava. Dissect along the lateral border of the aorta to expose it from the underlying psoas muscle. Next, dissect along the interaortocaval grove to separate the aorta from the IVC.</li>
		<li>Clamp test: Ensure adequate mobilization of the aorta and IVC by a trial of Satinsky&#39;s vascular clamp placement. Switch position with the first assistant and prepare the surgical field by retracting the bowel loops on the left side to expose the major vessels for anastomosis. Place the trial clamps on IVC and aorta to re-assess an adequate mobilization of both the vessels for anastomosis.</li>
		<li>Venous anastomosis: After placing the vascular side-biting clamp in the IVC, make a small opening in the anterior wall of the vessel and extend it to an adequate size (matched with the graft vessel) cranially and caudally using Pott&#39;s scissors. Flush the lumen with heparinized saline using the flexible sheath of an IV cannula.</li>
		<li>Using Prolene 6-0 double-needle, make corner sutures on the IVC (inside-out) and graft portal vein (inside-out). Secure the caudal corner suture and run one of the needles along the posterior wall of the graft and recipient vein in a continuous fashion. Secure the anterior wall in a similar (outside-in continuous) fashion and secure the knot in the center of the anterior wall after flushing the lumen of the anastomosis with heparinized saline.</li>
		<li>De-clamping the vein: Place a Bulldog&#39;s vascular clamp on the graft portal vein, away from the anastomosis, and slowly release the side-biting clamp from the recipient IVC. Check for venous refilling and any major bleed from the anastomosis.</li>
		<li>Arterial anastomosis: Place the side-biting clamp on the aorta taking care not to injure the lumbar branches along the posteromedial wall of the vessel. Instruct the anesthetist to inject heparin (100 U/kg BW) and methylprednisolone (500 mg) through the central venous catheter.</li>
		<li>Make an opening on the anterior wall of the vessel and extend it cranially and caudally taking care not to dissect along the vessel wall. Flush the lumen with heparinized saline and using Prolene 6-0 double-needle, suture the proximal end of the graft aorta to the recipient aortic opening in a continuous fashion by the Parachute technique. Flush the lumen with heparinized saline before tying the final knot on the anterior wall of the vessel.</li>
		<li>Reperfusion: Take care of the following two aspects while performing this step. First, the hemodynamic alterations where a drastic fall in the mean BP occurs. Monitor the invasive BP minute to minute and perform a dose titration of the vasopressor (norepinephrine infusion), and fluid preload to keep the target mean BP between 45-50 mmHg.</li>
		<li>Second, maintain the hemostasis. After removal of the bulldog clamp from the vein, assess for any major bleed from the parenchyma, para-aortic region, and peri-portal area; then, release the aortic clamp and check for bleeding from the arterial anastomotic site. Secure the bleeding points with ligatures and hemostatic suturing. Ask the anesthetist to inject a vial of tranexamic acid through the central venous catheter. 
		NOTE: An important rule is to minimize aggressive handling of the pancreas during this phase of reperfusion (to avoid graft oedema and hematoma).</li>
		<li>Bowel anastomosis: After ensuring no mesenteric torsion, isolate a loop of jejunum, 40-50 cm away from the duodenojeunal junction, and bring it close to the graft. Anastomose the graft duodenum to the recipient jejunum in a side-to-side continuous fashion using Polydioxanone 4-0 suture, taking care to ensure an adequate luminal diameter of 1.5-2 cm.</li>
		<li>Hemostasis and post-reperfusion biopsy: Ensure hemostasis around the graft and at the sites of anastomosis. Take three core needle biopsies from the pancreatic tail1 h after reperfusion-store in formalin, snap freeze, and extract RNA at a later stage. Suture the biopsy site with Prolene 6-0 in a figure of 8 fashion.</li>
		<li>Abdominal wall closure: After assessing for retained mops and securing hemostasis, suture the rectus abdominis in a continuous fashion using Polydioxanone 0 needle taking care in the lower midline to keep away from the urethra. Close the skin using silk 0 suture in a continuous fashion. 
		NOTE: Ideally, monofilament sutures (Nylon) are recommended; however, since this&#160;is a 3-day survival model, silk is acceptable in these&#160;experimental models.</li>
		<li>Strapping the central venous catheter: Create a subcutaneous tunnel on the neck and secure the central venous catheter by burying it under the tunnel and suturing the overlying skin with a silk 0-0 cutting needle.</li>
		<li>Removal of the arterial catheter: After ensuring a stable mean BP (40-50 mmHg) and an improving trend of pH and lactate levels on the blood gas analysis, remove the arterial catheter and ligate the vessel to secure hemostasis in the neck. Close the overlying skin with silk 0 suture in a continuous fashion.</li>
		<li>Position the animal and extubation: Under all necessary precautions, turn the animal sternal on a transporting cart. Observe for the O2 saturation and reversal of anesthesia (movement of limbs, spontaneous breathing efforts, SpO2 &#62;95% off O2, and ventilator support) and extubate the animal. Transport to the pen and position the animal sternal and monitor the hemodynamic status till stable.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64203/64203fig01.jpg" /> 
Figure 1: Pancreatectomy specimen resected en masse. Note the ring of pancreatic tissue surrounding the portal vein in vivo. <a href="https://www.jove.com/files/ftp_upload/64203/64203fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64203/64203fig02.jpg" /> 
Figure 2: Image of the duodenum. The C loop of the duodenum with a preserved vascular arcade (arrowhead) compared with the surrounding jejunal loop and assessed for congestion. <a href="https://www.jove.com/files/ftp_upload/64203/64203fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<ol>
	<li>Gruessner, R. W., Gruessner, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+current+state+of+pancreas+transplantation.">The current state of pancreas transplantation.</a> Nature Reviews. Endocrinology. 9 (9), 555-562 (2013).</li><li>Redfield, R. R., Rickels, M. R., Naji, A., Odorico, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreas+transplantation+in+the+modern+era.">Pancreas transplantation in the modern era.</a> Gastroenterology Clinics of North America. 45 (1), 145-166 (2016).</li><li>Canadian Institute for Health Information. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annual+Statistics+on+Organ+Replacement+in+Canada:+Dialysis,+Tranplantation+and+Donation,+2009+to+2018.">Annual Statistics on Organ Replacement in Canada: Dialysis, Tranplantation and Donation, 2009 to 2018.</a> Canadian Institute for Health Information. , (2019).</li><li>Prudhomme, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex-situ+perfusion+of+pancreas+for+whole-organ+transplantation:+Is+it+safe+and+feasible?A+systematic+review.">Ex-situ perfusion of pancreas for whole-organ transplantation: Is it safe and feasible?A systematic review.</a> Journal of Diabetes Science and Technology. 14 (1), 120-134 (2020).</li><li>Ferrer, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pig+pancreas+anatomy:+Implications+for+pancreas+procurement,+preservation,+and+islet+isolation.">Pig pancreas anatomy: Implications for pancreas procurement, preservation, and islet isolation.</a> Transplantation. 86 (11), 1503-1510 (2008).</li><li>Parmentier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normothermic+ex+vivo+pancreas+perfusion+for+the+preservation+of+pancreas+allografts+before+transplantation.">Normothermic ex vivo pancreas perfusion for the preservation of pancreas allografts before transplantation.</a> Journal of Visualized Experiments. , (2022).</li><li>Chaib, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+pancreatectomy:+Porcine+model+for+inducing+diabetes+-+Anatomical+assessment+and+surgical+aspects.">Total pancreatectomy: Porcine model for inducing diabetes - Anatomical assessment and surgical aspects.</a> European Surgical Research. 46 (1), 52-55 (2011).</li><li>Prudhomme, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+pancreatectomy+and+pancreatic+allotransplant+in+a+porcine+experimental+model.">Total pancreatectomy and pancreatic allotransplant in a porcine experimental model.</a> Experimental and Clinical Transplantation. 18 (3), 353-358 (2022).</li><li>Grussner, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptozotocin-induced+diabetes+mellitus+in+pigs.">Streptozotocin-induced diabetes mellitus in pigs.</a> Hormone and Metabolic Research. 25 (4), 199-203 (1993).</li><li>Mazilescu, L. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normothermic+ex+situ+pancreas+perfusion+for+the+preservation+of+porcine+pancreas+grafts.">Normothermic ex situ pancreas perfusion for the preservation of porcine pancreas grafts.</a> American Journal of Transplantation. 22 (5), 1339-1349 (2022).</li><li>Kumar, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+normothermic+porcine+pancreas:+A+physiological+model+for+preservation+and+transplant+study.">Ex vivo normothermic porcine pancreas: A physiological model for preservation and transplant study.</a> International Journal of Surgery. 54, 206-215 (2018).</li><li>Kaths, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normothermic+ex+vivo+kidney+perfusion+for+the+preservation+of+kidney+grafts+prior+to+transplantation.">Normothermic ex vivo kidney perfusion for the preservation of kidney grafts prior to transplantation.</a> Journal of Visualized Experiments. (101), e52909 (2015).</li></ol>

The authors have no conflicts of interest to disclose.

The current protocol has been performed to demonstrate the technique and the feasibility of pancreatectomy and pancreas allotransplantation in porcine models. The animals were observed for a period of 3 days after transplantation to demonstrate the reliability of the technique of pancreatectomy and the allotransplantation. All animals were monitored and nursed for 3 days after the surgery using a standardized animal care protocol of antibiotics, fluids, analgesics, supplemental nutrition, and immunosuppressants (i.e., cy...

Over the past several decades, there has been significant progress in perioperative management strategies and surgical techniques leading to the evolution of pancreas transplantation into one of the most promising strategies for the treatment of diabetes mellitus with end-stage renal disease (usually in conjunction with kidney transplantation)1. However, complications such as graft pancreatitis, ischemia-reperfusion injury, and vascular thrombosis remain the biggest challenges to overcome to ensure successful outcomes, more so in the more damaged extended criteria grafts2. In addition, pancreas grafts are the most commonly discarded grafts from procurement and have the lowest utilization rates (9%) for any organ3. Therefore, machine perfusion aims to provide an optimum homeostatic milieu to the pancreas graft with the goal of increasing graft utilization rate, similar to what has been achieved in liver, kidney, and lung transplantation4. The porcine pancreatic anatomy is complex in terms of its lobular architecture (comprising three lobes), its extensions all around the mesenterico-portal axis, its mesenteric vascular variations (in 40%-50%), and its delicate vascular channels along the C loop of the duodenum5. These anatomical attributes contribute to a challenging dissection in both retrieval of the pancreato-duodenal graft and recipient pancreatectomy to induce an iatrogenic apancreatic diabetes state, i.e., surgically induced state of diabetes mellitus with a fasting glucose level above 8 mmol/L. Based on these features, porcine pancreatectomy with transplantation provides the closest possible replication of the technique that could be performed in humans as a definitive treatment against end-stage diabetes mellitus. The present article aims at covering the following aspects: (i) outline of the peri-operative porcine care during recipient pancreatectomy and pancreas graft implantation; (ii) technical step by step details of the recipient pancreatectomy and implantation of the pancreato-duodenal graft and (iii) tips and tricks of donor and recipient pancreatic operation in porcine models to minimize graft and recipient injury.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Belzer UW Cold storage solution</td>
			<td>Bridge to life Ltd (Columbia, SC, USA)</td>
			<td>4055</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium gluconate (10%)</td>
			<td>Fresenius Kabi Canada Ltd (Toronto, ON)</td>
			<td>C360019</td>
			<td></td>
		</tr>
		<tr>
			<td>Composelect (blood collection bags)</td>
			<td>Fresenius Kabi Canada Ltd (Toronto, ON)</td>
			<td>PQ31555</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin (10000 IU/10 ml)</td>
			<td>Fresenius Kabi Canada Ltd (Toronto, ON)</td>
			<td>C504710</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactated Ringer&#39;s</td>
			<td>Baxter (Mississauga, ON, Canada)</td>
			<td>JB2324</td>
			<td></td>
		</tr>
		<tr>
			<td>Percutaneous Sheath Introducer Set with Integral Hemostasis Valve/side Port for use with 7-7.5 Fr Catheters&#160;</td>
			<td>Arrow International LLC</td>
			<td>SI-09880</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (8.4%)</td>
			<td>Fresenius Kabi Canada Ltd (Toronto, ON)</td>
			<td>C908950</td>
			<td></td>
		</tr>
		<tr>
			<td>Solu-Medrol</td>
			<td>Pfizer Canada Inc.&#160;</td>
			<td>52246-14-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical retreival and transplant instrument set</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

surgical tips and tricks for performing porcine pancreas transplantation

Despite the promising results of pancreas transplantation in type 1 diabetes mellitus and metabolic syndrome, the biggest concern around this state-of-the-art technique remains the paucity of organs deemed fit for transplantation. High intravascular resistance, delicate intraparenchymal capillary framework, and complex lobular anatomy around the mesenteric vasculature are what make this organ more susceptible to injury and less tolerant to trivial trauma compared to organs such as the liver and kidney. Meticulous surgical dissection and judicious tissue handling form the cornerstone of the entire exercise of pancreas transplantation. Owing to morphological similarity between the anatomy of the porcine pancreas to the surrounding mesenteric vessels and the organs when compared to the human anatomy, demonstration of the technique in the porcine model could help to most accurately extrapolate this to a human setting. The present article aims to outline the essential surgical tips and tricks that need to be followed, in order to ensure a higher success rate of transplantation of this highly susceptible organ in a porcine 3-day survival model.

The peri-operative and post-operative, up to 3-day, biochemical parameters from the five pancreas transplant survival models are summarized below (numbered as PTX 1 to 5 in chronology). Of the five total pancreas transplants, all fared well during the 3-day survival period, as evident by their general well-being and the pancreatic injury and endocrine function tests. The results depicted below are representative of the experience of a 3-day survival model of recipient pancreatectomy followed by pancreas allotransplantati...

Watch this Scientific Journal Video about Surgical Tips and Tricks for Performing Porcine Pancreas Transplantation at JoVE.com

Surgical Tips and Tricks for Performing Porcine Pancreas Transplantation

The video article summarizes the technique of pancreatectomy and pancreas allotransplantation in a porcine 3-day survival model with a step-by-step description of the method and emphasis on the surgical tips and tricks to deal with the precarious and delicate porcine visceral anatomy.

Despite the promising results of pancreas transplantation in type 1 diabetes mellitus and metabolic syndrome, the biggest concern around this ...

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1.6K Views.  University Health Network, Toronto general hospital. The video article summarizes the technique of pancreatectomy and pancreas allotransplantation in a porcine 3-day survival model with a step-by-step description of the method and emphasis on the surgical tips and tricks to deal with the precarious and delicate porcine visceral anatomy.

Video: Surgical Tips and Tricks for Performing Porcine Pancreas Transplantation

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep learning curve. The introduction of the cuff technique for anastomosis of the portal vein (PV) and infrahepatic vena cava (IHVC) has significantly simplified the transplant procedure in rats. However, due to the short anterior wall of the recipients' suprahepatic vena cava (SHVC), the cuff technique is very difficult to use for the reconstruction of the SHVC. Most researchers in this field still use the hand-suture technique for SHVC reconstruction, which makes it the bottleneck step in rat orthotopic liver transplantation. The magnetic anastomosis technique (i.e., magnamosis) is a method of connecting two vessels using the attractive force between two magnets. Our recent study has shown that the magnetic anastomosis technique is superior to the hand-suture technique for SHVC reconstruction in rats. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using the novel magnetic anastomosis technique. In this model, the reconstruction of the PV and IHVC was performed by the standard cuff technique, while the reconstruction of the bile duct (BD) was performed by a stent technique. The hepatic re-arterialization was not performed. The magnetic anastomosis technique made SHVC reconstruction much easier and significantly shortened the anphepatic phase. After a reasonable learning curve, even researchers without advanced microsurgical skills can produce reliable and reproducible results using this rat model of OLT.

The reconstruction of the suprahepatic vena cava (SHVC) remains a difficult step in rat orthotopic liver transplantation. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using a novel magnetic anastomosis technique.

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep ...

A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart failure. Multiple small-animal models of heart transplantation have been used to study the acute and long-term effects of novel therapies. However, few are tested and demonstrated success in clinical trials. It is of critical importance to evaluate new therapies in a clinically relevant large-animal model for efficient and reliable translation of basic studies&#39;&#160;findings. Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and previously used to investigate novel cardioprotective strategies. This procedure focuses on acute ischemia-reperfusion injury and is a reliable method to investigate novel interventions which have been tested and validated in smaller experimental models, such as the murine model. We demonstrate its usefulness in assessing cardiac performance during the early post-transplantation period and other potential possibilities enabled by the model.

Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies.

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart ...

Orthotopic Rat Kidney Transplantation: A Novel and Simplified Surgical Approach

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of renal replacement therapy. Over the past few decades, the rat kidney transplantation model has been used to study the immunological phenomena of rejection and tolerance. This model has become an indispensable tool to test new immunomodulatory pharmaceuticals and regimens prior to proceeding with expensive preclinical large animal studies.
This protocol provides a detailed overview of how to reliably perform orthotopic kidney transplantation in rats. This protocol includes three distinctive steps that increase the probability of success: perfusion of the donor kidney by flushing through the portal vein and the use of a cuff system to anastomose the renal veins and ureters, thereby decreasing cold and warm ischemia times. Using this technique, we have achieved survival rates beyond 6 months with normal serum creatinine in animals with syngeneic or tolerant kidney transplants. Depending on the aim of the study, this model can be modified by pre- or posttransplant treatments to study the acute, chronic, cellular, or antibody-mediated rejection. It is a reproducible, reliable, and cost-effective animal model to study different aspects of kidney transplantation.

The purpose of this manuscript and protocol is to explain and demonstrate in detail the surgical procedure of orthotopic kidney transplantation in rats. This method is simplified to achieve the correct perfusion of the donor kidney and shorten the reperfusion time by using the venous and ureteral cuff anastomosis technique.

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of ...

Laparoscopic Radical Left Pancreatectomy for Pancreatic Cancer: Surgical Strategy and Technique Video

Radical resection margins, resection of Gerota&#8217;s (perirenal) fascia, and adequate lymph node dissection are crucial for an adequate oncological resection of left-sided pancreatic cancer. Several surgical techniques have been described in recent years, but few were specifically designed for minimally invasive approaches. This study describes and demonstrates a standardized and reproducible technique for an adequate oncological resection of pancreatic cancer: laparoscopic radical left pancreatectomy (LRLP).
A 61-year-old woman presented with an incidental finding of a 3 cm mass in the left pancreas suspect for malignancy. Imaging did not reveal distant metastases, central vascular involvement, or morbid obesity, hence the patient was suitable for LRLP. This study describes the main steps of LRLP for pancreatic cancer. First, the lesser sac is opened by transecting the gastrocolic ligament. The splenic flexure of the colon is mobilized and the inferior border of the pancreas including Gerota&#39;s fascia is dissected down to the inferior border of the spleen. The pancreas is tunneled and hung, including Gerota&#8217;s fascia with a vessel loop. At the pancreatic neck, a tunnel is created between the pancreas and the portal vein, likewise a vessel loop is passed. The pancreas is then transected using the graded compression technique with an endostapler. Both the splenic vein and artery are transected before completing the resection. The entire specimen is extracted in a retrieval bag via a small Pfannenstiel incision.
Duration of the surgery was 210 min with 250 mL blood loss. Pathology revealed a R0-resection (&#62;1 mm) of a well-to-moderately differentiated adenocarcinoma originating from an intraductal papillary mucinous neoplasm. A total of 15 tumor-negative lymph nodes were resected. This is a detailed description of LRLP for left-sided pancreatic cancer as is currently being used within the international, multicenter randomized DIPLOMA (Distal Pancreatectomy Minimally Invasive or Open for PDAC) trial.

Oncologically safe left pancreatectomy requires radical resection (R0), Gerota&#8217;s (perirenal) fascia resection, and adequate lymph node dissection. This study describes the technical details of laparoscopic radical left pancreatectomy (LRLP), used in the first international multicenter randomized trial comparing minimally invasive with open left pancreatectomy for pancreatic cancer, the DIPLOMA trial.

Radical resection margins, resection of Gerota&#8217;s (perirenal) fascia, and adequate lymph node dissection are crucial for an adequate oncological ...

Orthotopic Kidney Auto-Transplantation in a Porcine Model Using 24 Hours Organ Preservation And Continuous Telemetry

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for kidney transplantation (KT). Even though, the use of allografts from extended criteria donors (ECD) could partially ease the shortage of organ donors, ECD organs carry a potentially higher risk for inferior outcomes and postoperative complications. Dynamic organ preservation techniques, modulation of ischemia-reperfusion and preservation injury, and allograft therapies are in the spotlight of scientific interest in an effort to improve allograft utilization and patient outcomes in KT.
Preclinical animal experiments are playing an essential role in translational research, especially in the medical device and drug development. The major advantage of the porcine orthotopic auto-transplantation model over ex vivo or small animal studies lies within the surgical-anatomical and physiological similarities to the clinical setting. This allows the investigation of new therapeutic methods and techniques and ensures a facilitated clinical translation of the findings. This protocol provides a comprehensive and problem-oriented description of the porcine orthotopic kidney auto-transplantation model, using a preservation time of 24 hours and telemetry monitoring. The combination of sophisticated surgical techniques with highly standardized and state-of-the-art methods of anesthesia, animal housing, perioperative follow up, and monitoring ensure the reproducibility and success of this model.

Large animal models play an essential role in preclinical transplantation research. Due to its similarities to the clinical setup, the porcine model of orthotopic kidney auto-transplantation described in this article provides an excellent in vivo setting for the testing of organ preservation techniques and therapeutic interventions.

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for ...

Robot-Assisted Kidney Transplantation

This paper describes robot-assisted kidney transplantation (RAKT) from a living donor. The robot is docked between the parted legs of the patient, placed in the supine Trendelenburg position. Kidney allografts are provided by a living donor. Before vascular anastomosis, the kidney allograft is prepared by inserting a double-J stent in the ureter, and the temperature for the anastomosis is lowered by wrapping it in an ice-packed gauze. A 12 mm or 8 mm port for the robotic camera and three 8 mm ports for robotic arms are placed. A peritoneal pouch is created for the kidney allograft by raising the peritoneal flaps on both sides over the psoas muscle before dissecting the iliac vessels and bladder. A 6 cm Pfannenstiel incision is made to insert the kidney into the peritoneal pouch, lateral to the right iliac vessels. 
After clamping the external iliac vein with Bulldogs clamps, a venotomy is performed, and the graft renal vein is anastomosed to the external iliac vein in an end-to-side continuous manner with a 6/0 polytetrafluoroethylene suture. After clamping the graft renal vein, the iliac vein is declamped. This is followed by clamping of the external iliac artery, arteriotomy, arterial anastomosis with a 6/0 polytetrafluoroethylene suture, clamping of the graft renal artery, and declamping of the external iliac artery. Reperfusion is then carried out, and ureteroneocystostomy is performed using the Lich-Gregoir technique. The peritoneum is closed at a few locations with polymer locking clips, and a closed-suction drain is placed through one of the working ports. After deflating the pneumoperitoneum, all incisions are closed.

This paper provides technical details for robot-assisted kidney transplantation from a living donor.

This paper describes robot-assisted kidney transplantation (RAKT) from a living donor. The robot is docked between the parted legs of the patient, placed ...