A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

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 year^1,2. 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 function^3,4,5,6. As a res....

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 ....

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.......

Discussion

There is a significant shortage of liver allografts available for transplantation and in response donor criteria have been expanded^1,2,3,4,5. As a result of the donor shortage, NEVLP has been introduced as a method to evaluate and modify organ function^6,7. We have designed a rat model of NEVLP. Further.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Perfusate
8% Albumin	CLS Behring, King of Prussia, PA	0053-7680-32
Williams Media	Sigma Aldrich, St. Louis, MO	W1878
Penicillin/Streptomycin	Sigma Aldrich, St. Louis, MO	P4333
Insulin	Eli Lilly, Indianapolis, IL	0002-8215-91
Heparin	Fresnius Lab, Lake Zurich, IL	C504701
L-glutamine	Sigma Aldrich, St. Louis, MO	G3126
Hydrocortisone	Sigma Aldrich, St. Louis, MO	H0888
THAM	Hospira, Inc,	0409-1593-04
Polyethylene Glycol - Catalase	Sigma Aldrich	S9549 SIGMA
Personal Protective Equipment
Surgical Mask	Generic	N/A
Protective Gown	Generic	N/A
Surgical Gloves	Generic	N/A
Liver Procurement
Sprague-Dawley Rat	Harlan Sprague Dawley Inc.	250 -350 grams
Surgical Microscope	Leica	M500-N w/ OHS
Charcoal Canisters	Kent Scientific	SOMNO-2001-8
Isoflurane	Piramal Healthcare	N/A
Pressure-Lok Precision Analytical Syringe	Valco Instruments Co, Inc.	SOMNO-10ML
Electrosurgical Unit	Macan	MV-7A
Warming Pad	Braintree Scientific	HHP2
SomnoSuite Small Animal Anesthesia System	Kent Scientific	SS-MVG-Module
PhysioSuite	Kent Scientific	PS-MSTAT-RT
Isoflurane chamber	Kent Scientific	SOMNO-0530LG
SurgiVet	Isotec	CDS 9000 Tabletop
Oxygen	Praxair	98015
Rib retractors	Kent Scientific	INS600240
GenieTouch	Kent Scientific	GenieTouch
Normal Saline	Baxter	NDC 0338-0048-04
4x4 Non-Woven Sponges	Criterion	104-2411
Sterile Q-Tips	Henry Schein Animal Health	1009175
U-100 27 Gauge Insulin Syringe	Terumo	22-272328
5mL Syringe	BD	REF 309603
4-0 Braided Silk Suture	Deknatel, Inc.	198737LP
7-0 Braided Silk Suture	Teleflex Medical	REF 103-S
16 gauge Catheters	BBraun Introcan Safety	4252586-02
14 gauge Catheters	BBraun Introcan Safety	4251717-02
Bile Duct Cannular Tubing	Altec	01-96-1727
Liver Perfusion Circuit Components
Water Bath Warmer	Lauda Ecoline Staredition	E103
Data Collection Software	ADInstruments	Labchart 7
Liver Perfusion Circuit	Harvard Apparatus	73-2901
Membrane Oxygenator	Mediac SPA	M03069
Roller Pump	Ismatec	ISM827B
Gas (95% oxygen and 5% carbon dioxide)	Praxair	98015
Organ Chamber	Harvard Apparatus	ILP-2
1.8 mL Arcticle Cryogenic Tube	USA Scientific	1418-7410
Mucasol	Sigma-Aldrich	Z637181
Microsurgical Instruments
Small Scissors	Roboz	RS-5610
Large Scissors	S&T	SAA-15
Forceps - Large Angled	S&T	JFCL-7
Forceps - Small Angled	S&T	FRAS-15 RM-8
Clip Applier	ROBOZ	RS-5440
Scissors - non micro	FST 14958-11	14958-11
Forceps - Straight Tip	S&T	FRS-15 RM8TC
Large Microsurgical Clip	Fine Scientific Tools	18055-01
Small Microsurgical Clip	Fine Scientific Tools	18055-01
Small Microsurgical Clip	Fine Scientific Tools	18055-02
Small Microsurgical Clip	Fine Scientific Tools	18055-03
Small Mosquito Clamps	Generic	N/A
Post-Experiment Analysis
Alanine Aminotransferase (ALT) Activity Colorimetric/Fluorometric Assay Kit	BioVision	K752
Adenosine Triphosphate (ATP) Colorimetric/Fluorometric Assay Kit	BioVision	K354
Glutathione Assay Kit	Cayman Chemical	703002
Lipid Peroxidation (MDA) Assay Kit	Abcam	ab118970
Caspase-Glo 3/7 Assay Systems	Promega	G8090
POLARstar OMEGA Microplate Reader	BMG LABTECH	N/A