Method Article

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

DOI:

10.3791/61779

⸱

March 6th, 2021

 ,  ,  ,  ,  ,  ,  ,  ,  , 
1Department of Intensive Care Unit, The Second Affiliated Hospital of Guangxi Medical University, 2Department of hepatobiliary surgery, The Affiliated Ningbo Medical Center Lihuili Hospital of Medical School of Ningbo University, 3Department of transplantation, The Second Affiliated Hospital of Guangxi Medical University, 4Digital Technology and Engineering college, Ningbo University of Finance & Economics

In This Article

Summary

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This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

Abstract

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Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

Introduction

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Ischemia-reperfusion injury (IRI) is a common complication after liver transplantation. Hepatic IRI is a pathological process involving ischemia-mediated cell damage and abnormal deterioration of liver reperfusion. Hepatic IRI and the local innate immune response can be divided into hot and cold IRI, according to differences in the clinical environment1. Hot IRI is induced by stem cell injury, usually as a result of liver transplantation, shock, and trauma2. Cold IRI is a complication of liver transplantation caused by endothelial cells and peripheral circulation3. Clinical reports have shown that....

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Protocol

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The Animal Ethics Committee approved the experiment of Guangxi Medical University (No20190920). All animals were supplied by the Animal Experiment Center of Guangxi Medical University. We used SPF male Sprague Dawley rats (200-250 g, 10-12 weeks), kept under the room temperature of 25 ± 2°C and humidity of 50 ± 10%. Feeding was stopped 24 hours before operation; however, water was provided.

NOTE: One operator can perform all operations without a microsurgery basis or surgical microscope.

1. Operation

  1. After weighing, anesthetize the rats with isoflurane (5%) using an animal anesthesia ma....

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Results

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Rats' tolerance to liver ischemia
In this animal model, the sites at which blood vessels were ligated during operating are shown in Figure 1. The rats were randomly divided into 5 groups for ischemia for 15 minutes (I15 group), 30 minutes (I30 group), 45 minutes (I45 group), 60 minutes (I60), and sham group, with 10 rats in each group. The survival rate of each group was observed 14 days after the operation. All rats survived in the I15 group, I30 group, and sham group. .......

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Discussion

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OLT in rats is an ideal model for studying organ preservation in liver transplantation, IRI, transplant rejection, immune tolerance, transplantation pathology and pharmacology, homotransplantation, and xenotransplantation. At present, it is widely used in the experimental research of liver transplantation.

During pilot studies we first administered pentobarbital sodium intraperitoneal anesthesia and found that this led to high postoperative mortality and short tolerance to hemodynamic changes........

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Disclosures

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The authors of this manuscript have no conflicts of interest to disclose.

Acknowledgements

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We would like to acknowledge the useful suggestions given by Dr. Wen-tao Li and Dr. Ji-hua Wu of the Second Affiliated Hospital of Guangxi Medical University. The authors would like to thank our team-mates for useful comments and discussions. The authors would also like to thank the anonymous reviewers and editors of JoVE for their comments. Special thanks should go to Dr Yuan's parents for their continuous support and encouragement. The work was supported by Ningbo Natural Science Foundation (2014A610248).

....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
4% paraformaldehyde solutionShanghai Macklin Biochemical Co.,LtdP804536
air drying ovenShanghai Binglin Electronic Technology Co., Ltd.BPG
Alanine aminotransferase (ALT)KitElabscience Biotechnology Co.,LtdE-BC-K235-S
ammoniaSinopharm Chemical Reagents Co. Ltd10002118
amylase KitElabscience Biotechnology Co.,LtdE-BC-K005-M
anhydrous ethanolSinopharm Chemical Reagents Co. Ltd100092183
Animal anesthesia machineShenzhen Ruiwode Life Technology Co. LtdR640
aspartate aminotransferase (AST)kitRayto Life and Analytical Sciences Co., Ltd.S03040
automatic biochemical analyzer.SIEMENS AG FWB:SIE, NYSE:SI Co., Ltd.2400
Biosystems (when nessary)Chengdu Taimeng Electronics Co., Ltd.BL-420F
CentrifugeBaiyang Medical Instrument Co., Ltd.BY-600A
cover glassJiangsu Shitai Experimental Equipment Co. Ltd10212432C
creatinine KitRayto Life and Analytical Sciences Co., Ltd.S03076
dewatering machineHungary 3DHISTECH Co.,LtdDonatello Series 2
embedding machineHubei Xiaogan Kuohai Medical Technology Co., Ltd.KH-BL1
frozen machineWuhan Junjie Electronics Co., LtdJB-L5
hematoxylin-eosin dye solutionWuhan Saiwell Biotechnology Co., LtdG1005
high-efficiency paraffin waxShanghai huayong paraffin wax co., LtdQ/YSQN40-91
hydrochloric acidSinopharm Chemical Reagents Co. Ltd10011018
intraocular lens (IOL)forcepsGuangzhou Guangmei Medical Equipment Co., Ltd.JTZRN
IsofluraneShenzhen Ruiwode Life Technology Co. Ltd—
micro Scissors(when nessary)Shanghai Surgical Instrument FactoryWA1010
needle holdersShanghai Surgical Instrument FactoryJ32010
neutral gumShanghai Huashen Healing Equipment Co.,Ltd.—
normal optical microscopeNikon Instrument Shanghai Co., LtdNikon Eclipse CI
ophthalmic forcepsShanghai Surgical Instrument FactoryJ3CO30straight
ophthalmic forcepsShanghai Surgical Instrument FactoryJD1060bending
ophthalmic ScissorsShanghai Surgical Instrument FactoryJ1E0
pathological slicerShanghai Leica Instrument Co., LtdRM2016
pipettesDragon Laboratory Instruments Co., Ltd.7010101008
retractorsBeijing Jinuotai Technology Development Co.,Ltd.JNT-KXQ
scannerHungary 3DHISTECH Co.,LtdPannoramic 250
slideWuhan Saiwell Biotechnology Co., LtdG6004
xyleneSinopharm Chemical Reagents Co. Ltd1330-20-7

References

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  1. Dar, W. A., Sullivan, E., Byon, J. S., Eltzschig, H., Ju, C. Ischaemia reperfusion injury in liver transplantation: Cellular and molecular mechanisms. Liver International. 39 (5), 788-801 (2019).
  2. Qiao, P. F., Yao, L., Zhang, X. C., Li, G. D., Wu, D. Q.

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Tags

Hemodynamic Changes SimulationAnhepatic Phase DurationExtrahepatic Organ DamageHistopathological AnalysisSerum Biochemical MarkersAnimal Model DevelopmentIschemia Tolerance TimeOrgan Injury Assessment

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