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Method Article

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

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DOI:

10.3791/57541

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June 27th, 2018

 ,  ,  ,  ,  ,  ,  ,  ,  ,  , 
1Collaboration for Organ Perfusion, Protection, Engineering and Regeneration (COPPER) Lab, Division of Transplant, Department of Surgery, Comprehensive Transplant Center, Ohio State University Wexner Medical Center, 2Department of Surgery, Division of Transplant, Ohio State University Wexner Medical Center, 3Department of Surgery, Division of CardioThoracic Surgery, Ohio State University Wexner Medical Center, 4Department of Medicine, Ohio State University Wexner Medical Center

In This Article

Summary

There is a significant liver donor shortage, and criteria for liver donors have been expanded. Normothermic ex vivo liver perfusion (NEVLP) has been developed to evaluate and modify organ function. This study demonstrates a rat model of NEVLP and tests the ability of pegylated-catalase, to mitigate liver preservation injury.

Abstract

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, normothermic ex vivo liver perfusion (NEVLP) has been introduced as a method to evaluate and modify organ function. NEVLP has many advantages in comparison to hypothermic and subnormothermic perfusion including reduced preservation injury, restoration of normal organ function under physiologic conditions, assessment of organ performance, and as a platform for organ repair, remodeling, and modification. Both murine and porcine NEVLP models have been described. We demonstrate a rat model of NEVLP and use this model to show one of its important applications — the use of a therapeutic molecule added to liver perfusate. Catalase is an endogenous reactive oxygen species (ROS) scavenger and has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung. Pegylation has been shown to target catalase to the endothelium. Here, we added pegylated-catalase (PEG-CAT) to the base perfusate and demonstrated its ability to mitigate liver preservation injury. An advantage of our rodent NEVLP model is that it is inexpensive in comparison to larger animal models. A limitation of this study is that it does not currently include post-perfusion liver transplantation. Therefore, prediction of the function of the organ post-transplantation cannot be made with certainty. However, the rat liver transplant model is well established and certainly could be used in conjunction with this model. In conclusion, we have demonstrated an inexpensive, simple, easily replicable NEVLP model using rats. Applications of this model can include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and experiments designed to repair organs.

Introduction

There are 14,578 patients on the waiting list for liver transplantation and approximately 7,000 transplants are performed per year1,2. In response to this significant donor shortage, the criteria for liver donors have expanded; these are often referred to as marginal organs or extended criteria donors and are expected to perform less well after transplantation than standard criteria allografts, with higher rates of primary graft dysfunction and delayed graft function3,4,5,6. As a result, NEVLP has been introduced as a method to evaluate and modify organ function6,7. We have designed a rat model of NEVLP and used this model to demonstrate one of its important potential applications – the testing of novel molecule additives to liver perfusate.

NEVLP has been evaluated in both murine (rat) and porcine models, as well as in discarded human organs6,8,9. The results of the first human trials of NEVLP have also recently been published10. Although hypothermic machine perfusion has clearly become the standard for kidney preservation, the temperature at which liver machine perfusion should occur is still controversial. NEVLP has many proposed advantages in comparison to hypothermic and subnormothermic perfusion. These include reduced preservation injury, restoration of normal organ function under physiologic conditions, the ability to assess organ performance, and as a platform for organ repair, remodeling, and modification7,11,12,13,14,15,16,17.

A significant number of studies have been completed using porcine NEVLP models. Although these models are comparatively inexpensive when considering models using discarded human organs or human clinical trials, they are very expensive when compared to our small animal NEVLP model. A significant component of the per experiment cost is the perfusate. We are able to complete a 4 h perfusion with 300 mL of perfusate at a relatively low cost. Additionally, the cost of small animals including rats is very low in comparison to the cost of pigs.

In comparison to other models of NEVLP in the rat, the model presented here is relatively simple to implement and has a broad range of applications. The perfusion circuit can be seen in Figure 1. The perfusate starts in the perfusate reservoir (1), which is a water jacketed container. Perfusate is pulled from the reservoir by a roller pump (2) and pushed into a windkessel (3) and then the oxygenator (4). The oxygenator is set for countercurrent gas and perfusate flow to provide maximum gas exchange. The perfusate then proceeds to a heating coil (5) inside the perfusion chamber to ensure it is at physiologic temperature, and a bubble trap (6) to prevent perfusion of air bubbles There are pre-organ (7) and post-organ (8) sample ports, which allow the perfusate to be sampled. The perfusate then enters the liver through the portal vein cannula. The portal vein cannula is attached to a pressure monitor that charts the values on the data collection software. The perfusate then exits the liver through the IVC cannula and flows into the pressure equalizer block (9). Finally, the perfusate is pulled from the pressure block back through the roller pump and emptied into the reservoir. This model includes continuous perfusion to the portal vein and leaves out the pulsatile flow to the hepatic artery and dialysis used in some other models, each of which requires a separate and additional circuit, but have previously been demonstrated to not be required9,13.

To explore the addition of a novel therapeutic molecule to the perfusate, we chose the enzyme catalase. Catalase is an endogenous ROS scavenger that is part of the cells internal defense mechanism to mitigate the effects of ROS18. Catalase expression is increased in hepatic ischemia reperfusion injury19. Experimental addition of catalase has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung20,21,22,23,24. Pegylation has been shown to target catalase to the endothelium and aid in catalase uptake into endothelial cells25. PEG-CAT has been administered systemically with limited efficacy in reducing hepatic ischemia-reperfusion injury; however, we hypothesized that adding PEG-CAT to an isolated organ perfusion circuit would lead to improved results26,27,28. Here, we add PEG-CAT to our base perfusate and demonstrate its ability mitigate liver preservation injury.

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Protocol

All procedures were performed according to the guidelines of the Institutional Animal Care and National Research Council’s Guide for the Humane Care and Use of Laboratory Animals (IACUC) and has undergone approval by the Ohio State University IACUC committee.

1. Initial Set-up

  1. Prepare the perfusion solution by combining the following: 86 mL of 25% albumin, 184 mL of Williams’ media, 30 mL of penicillin/streptomycin (10 U/mL penicillin and 0.01 mg/mL streptomycin), insulin (50 U/L), heparin (0.01 U/mL), L-glutamine (0.292 g/L), and hydrocortisone (0.010 g/L) to a total volume of 300 mL. For the base perfusate and PEG-CAT group, add 625 U/mL of PEG-CAT.
    1. Buffer the perfusate solution using tris(hydroxymethyl)aminomethane (THAM) to pH 7.4. Use an arterial blood gas machine to confirm the perfusate pH.
  2. Set up the circuit (Figure 1).
    1. Turn on the water bath warmer and set it to 37 °C. Allow the organ chamber to warm up.
    2. Pour the mixed and buffered perfusate into the reservoir and start circulation.
      NOTE: The perfusate mentioned in this step was prepared in step 1.1.1.
    3. Turn on the oxygenating gas (95% oxygen and 5% carbon dioxide) to the flow counter through the in-line oxygenator.
    4. Turn on the data collection software and click “start” to record for the duration of the experiment.
  3. Set up the surgical microscope and the operating room (Figure 2).
    1. Turn on all equipment including the warming board, electrocautery, and the anesthesia and vital signs (heart rate and oxygen saturation) monitoring equipment.
      NOTE: The microscope settings will vary based on the microscope used and can be adjusted to the user’s comfort.
    2. Fill the 10 mL anesthesia syringe with 10 mL of liquid isoflurane for inhalation (molecular weight 184.5 g/mol) and place in the anesthesia unit.
    3. Position a 0.5 mL syringe of heparin (50 U), the surgical instruments, 4-0 and 7-0 silk suture, sterile cotton swabs, and 4 cm x 4 cm non-woven gauze sponges appropriately (Figure 2).
  4. Prepare the isoflurane chamber.

2. Induction of Anesthesia

  1. Wear the following personal protective equipment (PPE): surgical mask, surgical gloves, disposable gown.
  2. Weigh the rat.
    NOTE: We use Sprague-Dawley rats between 250–350 g.
  3. Turn on the oxygen compressor and isoflurane. Place the rat, after being weighed, into the isoflurane chamber and secure the lid. Induce anesthesia using 6% isoflurane delivered with 2 L/min of oxygen.
    NOTE: The exact isoflurane dose used will depend on the specific anesthesia system being used.
  4. Use electronic clippers to clip the animal’s abdominal hair.
  5. Replace the animal in the isoflurane chamber.
  6. Turn on the anesthesia unit located in the operating room.
  7. Remove the rat from the isoflurane chamber when anesthesia is fully induced. Confirm the depth of anesthesia using a toe pinch.

3. Procurement Procedure

  1. Prepare the 16 G portal cuff (Figure 3).
    1. Begin with a 16 G angiocatheter. Cut a 7 mm section of tubing. Determine the midpoint of the 7 mm section by measuring 3.5 mm. Incise at the midpoint and remove the anterior half of the tubing.
    2. Use a hemostat to crush this now flat portion. Use a lighter to melt the other end of the angiocatheter to create a lip. Do not place the tip directly into the flame or it will ignite.
  2. Prepare the bile duct cannula.
    1. Take a 27 G angiocath and cut off the injection port leaving only the catheter. Connect this to a 10 cm section of the 27 G cannular tubing.
  3. Position the rat with its nose in the anesthesia nose cone, and its four extremities immobilized. Monitor the vital signs by attaching the monitor to the left hind extremity. Perform a toe pinch to confirm appropriate depth of anesthesia. Continue anesthesia at 4% isoflurane (for animals that weigh >250 g).
  4. Spray the animal’s abdomen with 70% isopropyl alcohol. Allow to dry. Place a sterile drape over the animal.
  5. Make a midline incision from the xiphoid to the pubis using sharp scissors and extending through the skin (Figure 4). Gently enter the peritoneum and incise the muscle. Take care to avoid damaging the bladder in the inferior aspect of this incision and the liver in the superior aspect of this incision.
  6. Extend the incision laterally to the left and right to form a cross at the level of the inferior border of the liver.
  7. Turn the anesthesia down to 2% (for animals weighing >250 g).
  8. Retract the xiphoid process using a curved mosquito clamp and the ribs using rib retractors (Figure 5).
  9. Cut the falciform, phrenic, and gastrohepatic ligaments with sharp scissors.
  10. Locate and tie-off the phrenic vein with a 7-0 suture as close to its origin as possible to prevent leaking.
  11. Eviscerate the rat using a sterile moistened cotton tip applicator and wrap the bowel in gauze moistened with 0.9% normal saline. Take care not to stretch the vasculature of the small bowel.
  12. Dissect over the inferior vena cava (IVC) to remove excess tissue. Dissect behind the IVC just superior to the bifurcation and pass a loop of a 4-0 silk suture for later use (Figure 6).
  13. Retract the right kidney to provide exposure to the right adrenal vein. Retract the right lobe of the liver superiorly with gauze. Tie off the right adrenal vein with a 7-0 silk suture as close to the IVC as possible and cauterize across it distal to the tie (Figure 7).
  14. Carefully dissect out the splenic vein, tie it off using two 7-0 silk sutures and cut across it between the two sutures.
  15. Tie off and ligate additional veins using 7-0 silk suture for additional length on the portal vein, if needed.
    NOTE: There are sometimes small branches of the infrahepatic IVC between the right adrenal vein and the inferior liver.
  16. Dissect around the gastroduodenal artery, tie off the gastroduodenal artery with a 7-0 silk suture, and ligate the gastroduodenal artery.
  17. Dissect around the hepatic artery and then place a 7-0 silk suture tie around it (Figure 8).
  18. Dissect out the bile duct.
    1. Check the length of the bile duct. Tie off the bile duct at the distal end using a 7-0 silk suture. Place a loop of a 7-0 silk suture around the bile duct as proximally as possible.
    2. Cut a hole that is half the diameter of the duct with small scissors and place a 27 G catheter into the bile duct proximally. Tie the catheter in place using a roman sandal tie suture (Figure 9).
  19. Inject 0.5 mL of heparin (50 U) into the penile vein or IVC of the animal using a 27 G needle.
    NOTE: A 27 G insulin syringe can also be used instead.
  20. Clamp and tie off the IVC using the previously placed 4-0 silk suture.
  21. Tie off the hepatic artery using the previously placed 7-0 silk suture.
  22. Clamp off the portal vein using a microsurgical clip. Cannulate the portal vein using a 22 G angiocatheter. Flush the portal vein with 60 mL of cold 0.9% normal saline with 1 mL heparin (100 U) until the liver blanches (Figure 10).
    NOTE: If the liver does not blanch immediately it can be massaged with sterile cotton tip applicators.
  23. Expose the suprahepatic IVC and cut across it as high in the chest as possible.
  24. Perform a hepatectomy as follows. Cut around the diaphragm, cut the hepatic artery, cut the IVC, cut the portal vein, cut any additional ligaments, and take the liver out. Place the liver in ice cold 0.9% normal saline (Figure 11).
  25. Place a 16 G vascular cuff in the portal vein (Figure 12). Place the liver on the ex vivo normothermic liver perfusion circuit.

4. Ex Vivo Normothermic Liver Perfusion

NOTE: The perfusate used here was prepared in protocol step 1.1.1.

  1. Place the portal vein cannula into the cuffed portal vein (Figure 13).
  2. During the portal vein cannula placement, maintain flow of the perfusate through the circuit at 2 mL/min to start. Watch the monitor for any spikes in portal vein pressure; this may indicate the vessel has become occluded and repositioning of the cannula is needed.
  3. Suture in the IVC cannula for the return flow of the perfusate using a 7-0 silk suture.
  4. Once both cannulas are in place, begin turning up the flow at 1 mL/min until a physiological pressure in the range of 10–16 cmH2O is reached.
  5. Take a 1 mL sample from pre- and post-ports at 0, 30, 60, 90, 120, 150, 180, 210, and 240 min of perfusion. Divide the 1 mL sample into two 0.5 mL samples.
    NOTE: 0.5 mL of this will be used in protocol step 4.5.1, and 0.5 mL will be used in protocol step 4.5.2.
    1. Snap freeze 0.5 mL of this sample in cryogenic tubes in liquid nitrogen.
    2. Run an arterial blood gas analysis using the remaining 0.5 mL of perfusate.
    3. After running the blood gas analysis at each time point (0, 30, 60, 90, 120, 150, 180, 210, and 240 min) examine the pH levels and buffer the perfusate as needed to return to pH 7.4.
  6. At the conclusion of 4 h of perfusion, disconnect the liver from the perfusion circuit. Divide the liver into 0.5 g segments. Snap freeze the liver tissue in cryogenic tubes in liquid nitrogen.

5. Post-experiment Analysis

  1. Determine alanine aminotransferase (ALT) level in the perfusate at 0, 30, 60, 90, 120, 150, 180, 210, and 240 min using a commercial colorimetric assay kit.
    1. In brief, incubate the perfusate with the reaction mix reagents at 37 °C for 60 min. Measure the optical density values at 570 nm using a microplate reader.
  2. Homogenize 0.5 g of liver tissue with 100 µL of lysis buffer and analyze the tissue lysate for adenosine triphosphate (ATP), glutathione (GSH), and malondialdehyde (MDA).
    1. In brief, measure the ATP levels of the liver tissue samples using a commercial assay kit. Mix the sample with the reaction buffer and incubate at room temperature for 30 min. Measure the optical density at 570 nm using a microplate reader.
    2. Measure the GSH levels of the liver tissue samples using a commercial assay kit. Mix the tissue samples with the assay cocktail. Measure the optical density values at 405–414 nm.
    3. Measure the MDA levels of the liver tissue samples using a commercial assay kit. Mix the samples with TBA and heat to 95 °C for 60 min. Centrifuge the reactant and transfer the supernatant to a 96-well plate. Measure the optical density at 532 nm.
  3. Homogenize 0.5 g of liver tissue with 100 µL of lysis buffer and analyze the tissue lysate for relative caspase-3/7 activity using a commercial assay kit.
    1. Mix the tissue lysate with the caspase-3-7 reagent assay buffer and incubate at room temperature for 30 min.
    2. Measure the fluorescence level in each well using a microplate reader.
  4. Determine the level of apoptotic cells in the liver tissue samples using a commercial in situ death detection kit.
    1. Pre-treat the 0.5 g tissue sections with 10 U/mL Proteinase K for 10 min and then incubate with the reaction mixture at 37 °C for 60 min. Perform the analysis using a fluorescent microscope.

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Results

A sample size of three rats per group was used. ALT was measured at 0, 30, 60, 90, 120, 150, 180, 210, and 240 min of perfusion. We used Student's t-tests to compare results between the base perfusate and base perfusate plus PEG-CAT groups at each time point. In comparing the base perfusate and base perfusate plus PEG-CAT groups, there is significantly less (p <0.05) ALT in the base perfusate plus PEG-CAT group at 150, 180, 210, and 240 min (Figur...

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Discussion

There is a significant shortage of liver allografts available for transplantation and in response donor criteria have been expanded1,2,3,4,5. As a result of the donor shortage, NEVLP has been introduced as a method to evaluate and modify organ function6,7. We have designed a rat model of NEVLP. Further...

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Disclosures

All authors report that they have no relevant disclosures.

Acknowledgements

This work was supported by NIH T32AI 106704-01A1 and the T. Flesch Fund for Organ Transplantation, Perfusion, Engineering and Regeneration at The Ohio State University.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Perfusate
8% AlbuminCLS Behring, King of Prussia, PA0053-7680-32
Williams MediaSigma Aldrich, St. Louis, MOW1878
Penicillin/StreptomycinSigma Aldrich, St. Louis, MOP4333
InsulinEli Lilly, Indianapolis, IL0002-8215-91
HeparinFresnius Lab, Lake Zurich, ILC504701
L-glutamineSigma Aldrich, St. Louis, MOG3126
HydrocortisoneSigma Aldrich, St. Louis, MOH0888
THAMHospira, Inc,0409-1593-04
Polyethylene Glycol - CatalaseSigma AldrichS9549 SIGMA
Personal Protective Equipment
Surgical MaskGenericN/A
Protective GownGenericN/A
Surgical GlovesGenericN/A
Liver Procurement
Sprague-Dawley RatHarlan Sprague Dawley Inc.250 -350 grams
Surgical MicroscopeLeicaM500-N w/ OHS
Charcoal CanistersKent ScientificSOMNO-2001-8
IsofluranePiramal HealthcareN/A
Pressure-Lok Precision Analytical Syringe Valco Instruments Co, Inc.SOMNO-10ML
Electrosurgical UnitMacanMV-7A
Warming PadBraintree ScientificHHP2
SomnoSuite Small Animal Anesthesia SystemKent ScientificSS-MVG-Module
PhysioSuiteKent ScientificPS-MSTAT-RT
Isoflurane chamberKent ScientificSOMNO-0530LG
SurgiVetIsotecCDS 9000 Tabletop
OxygenPraxair98015
Rib retractorsKent ScientificINS600240
GenieTouchKent ScientificGenieTouch
Normal SalineBaxterNDC 0338-0048-04
4x4 Non-Woven SpongesCriterion104-2411
Sterile Q-TipsHenry Schein Animal Health1009175
U-100 27 Gauge Insulin SyringeTerumo22-272328
5mL SyringeBDREF 309603
4-0 Braided Silk SutureDeknatel, Inc.198737LP
7-0 Braided Silk SutureTeleflex MedicalREF 103-S
16 gauge CathetersBBraun Introcan Safety4252586-02
14 gauge CathetersBBraun Introcan Safety4251717-02
Bile Duct Cannular TubingAltec01-96-1727       
Liver Perfusion Circuit Components
Water Bath WarmerLauda Ecoline StareditionE103
Data Collection SoftwareADInstruments Labchart 7
Liver Perfusion CircuitHarvard Apparatus73-2901
Membrane OxygenatorMediac SPAM03069
Roller PumpIsmatecISM827B
Gas (95% oxygen and 5% carbon dioxide)Praxair98015
Organ ChamberHarvard ApparatusILP-2
1.8 mL Arcticle Cryogenic TubeUSA Scientific1418-7410
MucasolSigma-AldrichZ637181
Microsurgical Instruments
Small ScissorsRobozRS-5610
Large ScissorsS&TSAA-15
Forceps - Large AngledS&TJFCL-7
Forceps - Small AngledS&TFRAS-15 RM-8
Clip ApplierROBOZRS-5440
Scissors - non microFST 14958-1114958-11
Forceps - Straight TipS&TFRS-15 RM8TC
Large Microsurgical ClipFine Scientific Tools18055-01
Small Microsurgical ClipFine Scientific Tools18055-01
Small Microsurgical ClipFine Scientific Tools18055-02
Small Microsurgical ClipFine Scientific Tools18055-03
Small Mosquito ClampsGenericN/A
Post-Experiment Analysis
Alanine Aminotransferase (ALT) Activity Colorimetric/Fluorometric Assay KitBioVisionK752
Adenosine Triphosphate (ATP) Colorimetric/Fluorometric Assay KitBioVisionK354
Glutathione Assay KitCayman Chemical703002
Lipid Peroxidation (MDA) Assay KitAbcamab118970
Caspase-Glo 3/7 Assay SystemsPromegaG8090
POLARstar OMEGA Microplate ReaderBMG LABTECHN/A

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A Small Animal Model of Ex Vivo Normothermic Liver Perfusion
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