Left Lung Orthotopic Transplantation in a Juvenile Porcine Model for ESLP

Summary

This protocol describes a juvenile porcine model of orthotopic left lung allotransplantation designed for use with ESLP research. Focus is made on anesthetic and surgical techniques, as well as critical steps and troubleshooting.

Abstract

Lung transplantation is the gold-standard treatment for end-stage lung disease, with over 4,600 lung transplantations performed worldwide annually. However, lung transplantation is limited by a shortage of available donor organs. As such, there is high waitlist mortality. Ex situ lung perfusion (ESLP) has increased donor lung utilization rates in some centers by 15%-20%. ESLP has been applied as a method to assess and recondition marginal donor lungs and has demonstrated acceptable short- and long-term outcomes following transplantation of extended criteria donor (ECD) lungs. Large animal (in vivo) transplantation models are required to validate ongoing in vitro research findings. Anatomic and physiologic differences between humans and pigs pose significant technical and anesthetic challenges. An easily reproducible transplant model would permit the in vivo validation of current ESLP strategies and the preclinical evaluation of various interventions designed to improve donor lung function. This protocol describes a porcine model of orthotopic left lung allotransplantation. This includes anesthetic and surgical techniques, a customized surgical checklist, troubleshooting, modifications, and the benefits and limitations of the approach.

Introduction

Lung transplantation is the preeminent long-term treatment for end-stage lung disease. Over 4,600 lung transplantations are performed worldwide annually¹. However, lung transplantation currently has significant limitations. For one, the necessity for organs continues to eclipse available donors. Despite rates of lung transplantation increasing every year since 2012 due to the combined effects of more candidates being listed for transplant, an increase in the number of donors, and improved use of recovered organs, the transplant waitlist mortality has not decreased significantly². Organ quality concerns represent another major limitation, with reported organ utilization rates as low as 20%-30%^3,4,5. Finally, the trends in the post-operative outcomes of lung transplantation are less than satisfactory, with long-term graft and patient outcomes still lagging that of other solid organ transplantations².

An emerging technology, ex situ lung perfusion (ESLP), has the potential to mitigate these limitations. ESLP has been increasingly applied as a method to assess and recondition marginal donor lungs and has demonstrated acceptable short- and long-term outcomes following transplantation of extended criteria donor (ECD) lungs^6,7,8,9,10. Consequently, ESLP has increased utilization rates in some centers by 15%-20%^{6,7,8,9,10,11}.

Proper ESLP research requires the in vivo validation of in vitro findings; however, there is limited literature on porcine lung transplantation models for ESLP^12,13,14,15. Furthermore, available literature provides inadequate details regarding anesthetic management of Yorkshire pigs for lung transplantation, which can be highly unstable hemodynamically^12,13,14,15. Establishing an easily reproducible model would permit the in vivo validation of current ESLP strategies and the preclinical evaluation of various interventions to reduce lung ischemia-reperfusion injury. The objective of the present study is to describe a porcine model of orthotopic left lung allotransplantation for use with ESLP. The protocol includes descriptions of the anesthetic and surgical techniques, a custom surgical checklist, and details regarding the troubleshooting experience and protocol modifications. The limitations and benefits of the left lung porcine transplantation model have also been discussed in this work. This manuscript does not outline the retrieval process of porcine lungs in 35-50 kg Yorkshire pigs, nor does it cover the establishment and termination of ESLP. This protocol exclusively addresses the recipient transplantation operation.

Protocol

All the procedures were performed in compliance with the guidelines of the Canadian Council on Animal Care and the guide for the care and use of laboratory animals. The protocols were approved by the institutional animal care committee of the University of Alberta. This protocol has been applied in female juvenile Yorkshire pigs between 35-50 kg. Pigs are pathogen-free, food-grade specimens. They are purchased from the Swine Research and Technology Centre in Edmonton, AB, Canada (https://srtc.ualberta.ca). All individuals involved in ESLP procedures had received proper biosafety training.

1. Pre-surgical preparations and anesthesia

NOTE: Pigs are fasted overnight prior to surgery for a maximum duration of 12 h.

1. Administer intramuscular injections of ketamine (20 mg/kg) and atropine (0.05 mg/kg) as premedication for the recipient pig in the operating room.
2. Place the pig supine on a heated operating table to maintain normothermia and proceed with mask induction.
3. Titrate oxygen flow rate according to animal weight and the anesthetic system.
   NOTE: Oxygen flow should be 20-40 mL/kg.
4. Administer isoflurane at 4%-5% and reduce to 3% after 1-2 min.
5. Evaluate the depth of anesthesia, ensure the pig has no withdrawal reflex in response to a noxious stimulus. Repeat every 5 min.
   NOTE: If a pain response is present, increase the percentage of isoflurane administration until the appropriate depth of anesthesia is achieved. See step 10 of this section for further details on maintenance analgesia with ketamine and hydromorphone. No paralytics are administered. This allows for assessment of a withdrawal reflex. A nose pinch is used as a noxious stimulus.
6. Intubate the pig once the correct depth of anesthesia is confirmed. Use a custom 10 inch, flat blade laryngoscope and size 9 or 10 endotracheal tubes for pigs 40-50 kg.
7. Place a pulse oximeter probe on the tongue (preferred) or ear and target an oxygen saturation above 90%.
   NOTE: Temperature is monitored via a nasal probe. A heating pad is used to maintain normothermia.
8. To maintain the anesthesia, adjust oxygen flow (20-40 mL/kg) and inhalant gas rate (1%-3%).
9. Keep the ventilator settings at a respiratory rate of 12-30 breaths/min, TV of 6-10 mL/kg, PEEP of 5 cm H₂O, and Peak Pressure of 20 cm H₂O.
   NOTE: A standard ICU style positive pressure ventilator is used to create a closed system for anesthesia and ventilation. Vitals are continuously monitored and recorded at 15 min intervals. ABG’s are drawn every 15-60 min, depending on the stability of the animal. Although TVs are targeted as high as 10 mL/kg, 6-8 mL/kg are achieved. Figure 1 provides a schematic overview of the negative pressure ventilation (NPV)-ESLP for the transplant protocol applied in the lab.
10. Shave, wash and aseptically prepare the incision site using povidone iodine.
    ​NOTE: Following sedation with Ketamine/Atropine, the analgesic regime involves administering 3mg/kg Ketamine IV q 1 h (range 1-3 mg/kg depending on patient parameters) and Hydromorphone 0.05 mg/kg IM q 2 h via a peripherally inserted IV line in an ear vein. Any longer duration between doses results in breakthrough pain response, such as elevated heart rate and abnormal breathing patterns / abdominal muscle movement.

2. Insertion of central venous and arterial lines

1. Insert a central line for fluid and heparin administration.
   NOTE: Total IV fluid administration is calculated to 1 mL/kg/h, and fluid boluses are administered PRN to maintain a MAP >60 mmHg. Central line is also used to administer steroids, antibiotics, vasopressors, and inotropes. See Figure 2A for line positioning.
   1. Prep the skin using a povidone iodine prep solution and allow to dry completely. Use electrocautery to make a 5-8 cm midline incision centered over the trachea and extend cranially from the sternal notch.
   2. Divide the skin and subcutaneous fat using cautery.
   3. Divide the midline plane between the strap muscles, and then divide the connective tissue layers to identify the left or right carotid intravascular bundle lateral to the trachea.
   4. Obtain proximal and distal control of the jugular vein using silk ties (size 2-0) as vessel loops.
   5. Tie the cranial encircling tie and retract upwards on the proximal tie to control blood flow.
   6. Make a small incision in the vein using Metzenbaum scissors (see Table of Materials) to accommodate a two-port, 7 Fr central line (~1/3 the vessel's circumference).
   7. Simultaneously, release the tension on the proximal vessel loop, cannulate the vein, and then tie down to secure the cannula in the vein at a depth of 10 cm.
   8. Flush the line with heparin, connect to an IV line of 0.9% normal saline, and administer fluid if the pig is intravascularly depleted from dehydration.
      NOTE: Heparin lock any unused ports.
   9. Administer 500 mg of methylprednisone and 1 g of cefazolin IV.
2. Follow the same techniques to cannulate the common carotid artery using a 7 Fr arterial line for accurate blood pressure management.

3. Left lung procurement

1. Position the pig in a right lateral decubitus position.
2. Perform a left anterolateral thoracotomy (Figure 2).
   1. Prep the skin using a povidone iodine prep solution and allow to dry completely. Mark the thoracotomy incision (20 cm) using the following landmarks: use palpation to identify the tip of the left scapula; likewise, identify the xiphoid process inferior to the sternum with palpation. Connect the two as shown in Figure 2B.
   2. Inject a total of 10 mL of 0.25% bupivacaine into the incisional line and two rib spaces above and below the incision.
   3. Use electrocautery to dissect the skin, subcutaneous layers, and muscle layers. The latissimus dorsi must be divided. Identify the rib immediately below the incision and cauterize on top of the rib to expose the intercostal muscles while avoiding the intercostal neurovascular bundle.
   4. Use a mosquito hemostat to puncture the intercostal muscles immediately above the rib, and then feel inside the chest for adhesions using a finger. Push the lung away using a Yankauer suction or finger (see Table of Materials) as you cauterize along the top edge of the rib to extend the thoracotomy.
      1. Extend the thoracotomy anteriorly until 1 inch away from the sternum. Extend the thoracotomy posteriorly to the paraspinal muscles.
   5. Insert a Cooley sternal retractor (see Table of Materials) to open the thoracotomy wide (10 cm) (Figure 2C). Retract the lung to expose the left hemi-azygous vein (Figure 2D).
   6. Circumferentially dissect the left hemiazygos vein using Metzenbaum scissors and a fine Lauer. Encircle the vessel with silk ties, and then ligate and transect it (Figure 2E). Keep a silk tie on the proximal stump for added control.
      NOTE: Lauer is a right angle clamp or a celiac clamp used for tissue dissection.
   7. Dissect out the left pulmonary artery (PA) and left pulmonary veins (PV). Encircle the veins in silk ties for control (Figure 2F).
      NOTE: The superior PVs are very small and are suture ligated at their branch points or common trunk, depending on the individual anatomy. The left mainstem bronchus is deep to the PA and LA (left atrium), so occasionally, it cannot be dissected easily until the artery and veins have been clamped and transected (Figure 2G).
   8. Administer 5000 units of heparin IV 5 min before clamping the PA.
      NOTE: Heparin 5000 units IV is also administered 5 min before unclamping the PA. For every hour after that, 1000 units of IV heparin is administered.
   9. Clamp the PA (DeBakey cross-clamp), left inferior pulmonary vein (Satinsky clamp), and the left bronchus (Spoon Potts clamp) individually (see Table of Materials). Decrease tidal volumes to 5 mL/kg once the left bronchus is clamped.
   10. Transect the PA, left inferior pulmonary vein, and the left bronchus. Leave at least 0.5 cm of tissue cuff to sew. Divide the left inferior pulmonary ligament and remove the left lung.
       NOTE: The left lung can be discarded or kept for control histology.

4. Termination of ESLP, division of left lung, and flushing with electrolyte solution

1. Clamp the ventilation tubing at maximal inspiration, terminate perfusion and ventilation, and disconnect the lungs from the ESLP device.
2. Weigh the lungs to determine the amount of edema formation.
   NOTE: Edema is tissue swelling due to the accumulation of excess fluid.
3. Take a tissue biopsy of the accessory lobe, divide it into three equal pieces, and place one piece into each of the following: optimum cutting temperature (OCT) gel, formalin, and snap freeze in liquid nitrogen.
   NOTE: This step is typically followed in the author's lab. The samples are then stored for future analysis: OCT and snap-frozen samples are kept in a -80 °C freezer, and formalin-stored samples are placed in a properly sealed container and stored in 4 °C refrigerators. Details of the specific ESLP protocol and tissue analysis are published elsewhere¹⁶.
4. Divide the left donor lung from the right lung. Leave 1 cm of donor PA, 1 cm of donor bronchus, and adequate donor LA cuff (~0.5 cm circumferentially) to sew to the recipient LA (Figure 2H). Leave the left inferior PV and left superior PVs in continuity with the donor LA wall to facilitate later anastomoses.
5. Weigh the left lung.
6. Cannulate the donor left PA using a drop sucker connected to an IV line and flush 500 mL of extracellular, low potassium, dextran-based electrolyte preservation solution antegrade through the lung vasculature. Secure the cannula in the PA with a silk tie during the flush, and release when the flush is complete.
   ​NOTE: The steps mentioned pertain to the specific ESLP device utilized for this work and may not be directly applicable to other devices.

5. Left lung transplantation

1. Insert the donor lung into the recipient's chest, beginning with the lower lobe. Do not force the lung into place.
   NOTE: The lower ribcage may need to be lifted upwards to accommodate the donor lung by torquing on the sternal retractor. Ideally, the recipient is a few kilograms larger than the donor to facilitate a size match.
2. Perform the bronchial anastomosis first using 4-0 prolene on a TF needle (Figure 2I).
   NOTE: A running, end-to-end anastomosis works well. Trim any excess length from the two anastomotic ends before sewing to avoid kinking caused by redundant tissue.
3. Perform the LA anastomosis second with 6-0 prolene on BV-1 needles using a running, end-to-end anastomosis. Again, trim excess tissue to avoid kinking.
   NOTE: The LA is friable and benefits from the small BV-1 needle. Horizontal bites on the donor may be required to purchase adequate tissue and correct the size mismatched caused by sewing the donor IPV and SPV to the recipient IPV/LA opening.
4. Incorporate the donor SPVs into the inferior PV and LA anastomosis to allow left upper lung lobe venous drainage (Figure 2J).
   NOTE: The branch superior pulmonary veins (SPVs) are less than 0.5 cm in diameter. The common SPV trunk is variable in length and is not routinely present, making direct anastomosis between the donor and recipient SPVs a poor option.
5. Complete the PA anastomosis with 6-0 prolene on BV-1 needles using a running, end-to-end anastomosis. Again, trim excess tissue to avoid kinking.
6. Remove the bronchial clamp and increase TVs to target 10 mL/kg.
7. Confirm heparinization, administer a potassium shift (40 mg of furosemide, 10 units of insulin, 100 mL of 25% dextrose solution), open the PA clamp partially, de-air, and tie the PA suture. Completely release the PA clamp after 10 min.
8. Meanwhile, de-air the LA, tie the sutures, and remove the LA clamp.
9. Take a reperfusion blood gas from the central line and a reperfusion tissue biopsy from the left middle lobe.
   NOTE: To take a tissue biopsy, use a size 0-silk tie to encircle a 1 cm portion of the middle lobe apex, tie-down to ensnare the tissue, and then cut the isolated portion with Metzenbaum scissors. Divide the biopsy into three equal portions and manage as previously described.
10. Perform a left and right lung bronchoscopy to assess the bronchial anastomosis and to suction secretions. Insert a bronchoscope into the endotracheal tube using an adaptor connection.
    1. Connect the scope to suction. Advance the bronchoscope into the left bronchus. Inspect the bronchial anastomosis (Figure 2N). Advance the scope down the bronchioles and suction any fluid. Repeat on the right side.
       NOTE: Do not allow the oxygen saturation to fall below 90%. If saturations fall below this level, remove the scope and allow the pig a few minutes of uninterrupted ventilation to recover.
11. Insert a 20 Fr malleable chest tube (Figure 2L), close the thoracotomy in three layers (Figure 2M), and prone the pig as soon as the arterial blood gases (ABGs) are stable (Figure 2O).
12. Monitor the pig over 4 h in the prone position. Perform an ABG analysis every 30 min. Administer 1000 units of heparin every hour after reperfusion.
    1. Take a 10 mL blood sample every hour for centrifugation and enzyme-linked immunosorbent assay (ELISA) analysis of inflammatory markers¹⁶.
       ​NOTE: Centrifugation parameters are detailed later.

6. Isolated Left Lung Assessment

1. Position the pig supine and re-prep the sternum using povidone iodine prep solution. Perform a midline sternotomy for final isolated left lung assessment (Figure 2P).
2. Open the left pleura using Metzenbaum scissors and take a tissue biopsy from the left lower lobe as previously described (NOTE to step 5.9).
3. Open the accessory lobe pleura and dissect out the common vein using Metzenbaum scissors.
   NOTE: This will be clamped later on.
4. Take a blood sample from the LA anastomosis using a 21 G needle. Direct the needle toward the left pulmonary veins and away from the common left atrium or accessory lobe trunk.
5. Open the right pleura to create space for the right hilar clamps (see Table of Materials). Dissect the right inferior pulmonary ligament up to the hilum. Ensure that a clamp can be placed around the hilum superiorly, inferiorly, and anteriorly.
   NOTE: This ensures that the hilum is occluded, and all oxygenation is dependent on the left lung. The right lung will not ventilate at this time, which should be evident by a lack of inflation/deflation with ventilator respirations. The right lower lobe can be lifted out of the chest to accomplish this.
6. Clamp the accessory lobe vein using a DeBakey aortic cross-clamp (see Table of Materials) to occlude any accessory lobe drainage into the LA (Figure 2Q).
7. Clamp the right hilium and take the following serial blood samples from the left PV anastomosis with a 21 G needle directed toward the left lung: 0 min, 1 min, 2 min, 5 min, and 10 min after clamping.
   NOTE: Five samples are taken to monitor for any trend in partial pressure of oxygen (PaO₂) (Figure 2R). The PaO₂ should remain relatively stable to represent proper left lung function. Five samples also provide insurance of a quality assessment if there is an issue with clotting of any samples or a problem arises with ABG analysis.
8. Transect the anastomoses, and remove the left lung. Transect the IVC to expedite euthanasia under anesthesia via exsanguination.
   NOTE: Total anesthesia time for the recipient pig is 8 h.
9. Weigh the donor lung to assess for edema formation and inspect it for overall appearance. Inspect the PA, bronchus, and LA cuff for signs of clot or other pathology within the donor lung and the recipient mediastinum.
10. Run the final gas analyses, centrifuge the perfusate samples, and store the tissue biopsies as previously described (NOTE to step 4.3).
    NOTE: The centrifugation settings are: 112 x g, 9 acceleration, 9 deceleration, 4 °C, and 15 min duration.

Results

All of the results are in the context of 4 h of reperfusion following 12 h of NPV-ESLP¹⁶. During lung explant, there are several clinical outcomes to anticipate (Figure 3). Typically, the pig will remain hemodynamically stable following a successful left lung explantation but may require a low dose infusion of phenylephrine (dose range: 2-10 mg/h) due to a vasodilatory response to surgery. Heart rate should target approximately 100-120 bpm, respiratory rate (RR) 8-30 ...

Discussion

Several critical surgical steps are involved in this protocol, and troubleshooting is needed to ensure successful transplantation and lung assessment. Juvenile porcine lungs are incredibly delicate compared to adult human lungs, so the operating surgeon must be cautious when handling porcine lungs. This is especially true after a 12 h run of ESLP as the organ will have taken on fluid volume and be susceptible to injury from excessive manipulation. Any undue pressure will cause atelectasis or trauma to the experimental lu...

Materials

Name	Company	Catalog Number	Comments
ABL 800 FLEX Blood Gas Analyzer	Radiometer	989-963
Adult-Pediatric Electrostatic Filter HME - Small	Covidien	352/5877
Allison Lung Retractor	Pilling	341679
Arterial Filter	SORIN GROUP	01706/03
Backhaus Towel Clamp	Pilling	454300
Bovine Serum Albumin	MP biomedicals	218057791
Biomedicus Pump	Maquet	BPX-80
Bronchoscope
Cable Ties – White 12”	HUASU International	HS4830001
Calcium Chloride	Fisher Scientific	C69-500G
Cooley Sternal Retractor	Pilling	341162
CUSHING Gutschdressing Forceps	Pilling	466200
Debakey-Metzenbaum Dissecting	Pilling	342202
Scissors	Pilling	342202
DeBakey Peripheral Vascular Clamp	Pilling	353535
Debakey Straight Vascular Tissue Forceps	Pilling	351808
D-glucose	Sigma-Aldrich	G5767-500G
Drop sucker
Endotracheal Tube 9.0mm CUFD	Mallinckrodt	9590E
Flow Transducer	BIO-PROBE	TX 40
Infusion Pump	Baxter	AS50
Inspire 7 M Hollow Fiber Membrane Oxygenator	SORIN GROUP	K190690
Intercept Tubing Connector 3/8" x 1/2"	Medtronic	6013
Intercept Tubing 1/4" x 1/16" x 8'	Medtronic	3108
Intercept Tubing 3/8" x 3/32" x 6'	Medtronic	3506
Laryngoscope	N/A	N/A	Custom-made with 10-inch blade
Metzenbaum Dissecting Scissors	Pilling	460420
Medical Carbon Dioxide Tank	Praxair	5823115
Medical Oxygen Tank	Praxair	2014408
Medical Nitrogen Tank	Praxair	NI M-K
Mosquito Clamp	Pilling	181816
Harken Auricle Clamp
Organ Chamber	Tevosol
PlasmaLyte A	Baxter	TB2544
Poole Suction Tube	Pilling	162212
Potassium Phosphate	Fischer Scientific	P285-500G
PERFADEX Plus	XVIVO	19811
Satinsky Clamp	Pilling	354002
Scale	TANITA	KD4063611
Silicon Support Membrane	Tevosol
Sodium Bicarbonate	Sigma-Aldrich	792519-1KG
Sodium Chloride 0.9%	Baxter	JB1324
Sorin XTRA Cell Saver	SORIN GROUP	75221
Sternal Saw	Stryker	6207
Surgical Electrocautery Device	Kls Martin	ME411
TruWave Pressure Transducer	Edwards	VSYPX272
Two-Lumen Central Venous Catheter 7fr X2	Arrowg+ard	CS-12702-E
Vorse Tubing Clamp	Pilling	351377
Willauer-Deaver Retractor	Pilling	341720
Yankauer Suction Tube	Pilling	162300
0 ETHIBOND Green 1X36" Endo Loop 0	ETHICON	D8573
0 PDS II CP-1 2x27”	ETHICON	Z467H
1 VICRYL MO-4 1x18”	ETHICON	J702D
2-0 SILK Black 12" x 18" Strands	ETHICON	SA77G
4-0 PROLENE Blue TF 1x24”	ETHICON	8204H
6-0 PROLENE Blue BV 2x30”	ETHICON	M8776
21-Gauge Needle