Name: Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model (Video) | JoVE Journal
Uploaded: 2021-03-06T13:00:00.000+00:00
Duration: 10 min 7 s
Description: Explore a rat liver transplant model simulating hemodynamic changes to study ischemia-reperfusion injury. This method enables investigation of anhepatic phase effects on extrahepatic organs and survival outcomes.

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5 minutes

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&#39;s parents for their continuous support and encouragement. The work was supported by Ningbo Natural Science Foundation (2014A610248).

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 &#177; 2&#176;C and humidity of 50 &#177; 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
<ol>
	<li>After weighing, anesthetize the rats with isoflurane (5%) using an animal anesthesia machine.</li>
	<li>After 1-2 minutes, gently clamp the toes of the rat with tweezers. If the rat does not respond after pinching, it has entered a state of anesthesia. Use vet ointment on the eyes to prevent dryness. Use animal heating lamps to keep the rats&#39; body temperature at 37-38 &#176;C.</li>
	<li>Following abdominal disinfection (povidone iodine solution), fix the rat on the animal dissection table. Make a median incision of 3 cm below the xiphoid process using forceps and scissors.</li>
	<li>Open the abdominal cavity, expose the liver using a retractor, and mobilize the hepatogastric ligament. Use cotton swabs to flip the middle lobe of the liver gently and turn it upward to expose the porta hepatis. Identify the CBD, PV, and HA.</li>
	<li>Push the small intestine toward the left lower abdominal cavity using cotton swabs, cover it with wet gauze, and move the intrahepatic vena cava to the right renal vein.</li>
	<li>Isolate the portal vein, hepatic artery, and the inferior vena cava above the right renal vein with an intraocular lens and forceps marked with 3-0 silk thread, each with a slip knot.</li>
	<li>Cut open the left and right lower extremity skin and expose the femoral vein using ophthalmic forceps. Slowly inject low molecular weight heparin 625 IU/kg through the femoral vein to heparinize the whole body.</li>
	<li>Ligate the portal vein, hepatic artery, and inferior vena cava above the right renal vein with No. 3-0 sutures, lasting 45 minutes (Figure 1). Replace the small intestine in the abdominal cavity and cover it with gauze. Reduce inhalation anesthesia during these periods.</li>
	<li>After 45 minutes, release the portal vein, hepatic artery, and the inferior vena cava above the right renal vein.</li>
	<li>Suture the muscle and skin, layer by layer, and terminate the inhalational anesthesia. Provide postoperative analgesia using subcutaneous morphine of 5 mg/kg every 4 hours.</li>
	<li>Observe the rat until it is awake and feed under a temperature of 25 &#177; 2 &#176;C and humidity of 50 &#177; 10%. Animal heating lamps are necessary.</li>
</ol>

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<li>Kim, E. H., Ko, J. S., Gwak, M. S., Lee, S. K., Kim, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Incidence+and+clinical+significance+of+hyperfibrinolysis+during+living+donor+liver+transplantation%5BTitle%5D)+AND+%22Blood+Coagulation+and+Fibrinolysis%22%5BJournal%5D)">Incidence and clinical significance of hyperfibrinolysis during living donor liver transplantation.</a> Blood Coagulation and Fibrinolysis. 29 (3), 322-326 (2018).</li>
<li>Czigány, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improving+research+practice+in+rat+orthotopic+and+partial+orthotopic+liver+transplantation%3A+a+review%2C+recommendation%2C+and+publication+guide%5BTitle%5D)+AND+%22European+Surgical+Research%22%5BJournal%5D)">Improving research practice in rat orthotopic and partial orthotopic liver transplantation: a review, recommendation, and publication guide.</a> European Surgical Research. 55 (1-2), 119-138 (2015).</li>
<li>Kamada, N., Calne, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Orthotopic+liver+transplantation+in+the+rat.+Technique+using+cuff+for+portal+vein+anastomosis+and+biliary+drainage%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Orthotopic liver transplantation in the rat. Technique using cuff for portal vein anastomosis and biliary drainage.</a> Transplantation. 28 (1), 47-50 (1979).</li>
<li>Zimmermann, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Techniques+for+orthotopic+liver+transplantation+in+the+rat+and+some+studies+of+the+immunologic+responses+to+fully+allogeneic+liver+grafts%5BTitle%5D)+AND+%22Transplantation+Proceedings%22%5BJournal%5D)">Techniques for orthotopic liver transplantation in the rat and some studies of the immunologic responses to fully allogeneic liver grafts.</a> Transplantation Proceedings. 11 (1), 571-577 (1979).</li>
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The authors of this manuscript have no conflicts of interest to disclose.

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....

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 hepatic IRI is associated with 10% of early organ failures and may increase the incidence of acute and chronic rejection4,5. In addition, hepatic IRI may also induce multiple organ dysfunction syndromes or systemic inflammatory response syndrome, with high mortality6. Patients with extrahepatic organ involvement tend to stay longer in the hospital, spend more money, and have a worse prognosis7. The development of complications is closely related to the length of the anhepatic phase of liver transplantation8.
Orthotopic liver transplantation (OLT) in rats was first reported by the American professor Lee in 1973. The experimental operation simulated the steps of clinical liver transplantation and the anastomosis of blood vessels and the common bile duct (CBD) using the suture method. The procedure is difficult and time-consuming with a low rate of success9. In 1979, Kamada et al. made a significant improvement to OLT in rats by creatively using the &#39;two-cuff method&#39; for anastomosis of the portal vein to control the anhepatic phase within 26 minutes10. In the same year, Zimmermann proposed the &#39;single biliary stent method.&#39; On the basis of Lee&#39;s work, Zimmermann used polyethylene tubes to directly anastomose the CBD of the donor and recipient, simplified the reconstruction of CBD, and preserved the function of the sphincter, and this method became the standard for biliary reconstruction of OLT models11. In 1980, Miyata et al. proposed the &#39;three-cuff method&#39; where the portal vein (PV), suprahepatic vena cava (SVC), and intrahepatic vena cava (IVC) were anastomosed by the cuff method. However, there is a risk of distortion of the cannula with this method, which can lead to the obstruction of inferior vena cava reflux12. In 1983, the &#39;two-cuff method&#39; was proposed using the cuff method for anastomosis of the PV and IVC, but adopting the suture method for the SVC13. This method was adopted by scholars globally to establish OLT models. Since then, the cuff anastomosis steps have been improved to shorten the anhepatic phase and improve the survival rate of the rats14. Similarly, improved methods are used in clinical practice to shorten the anhepatic phase15. However, basic research into IRI after liver transplantation has shown that the survival rate is inversely related to the degree of injury to extrahepatic organs. Therefore, further research is required, and a simple and reproducible animal model is needed to simulate IRI after liver transplantation.
Based on the definition of the anhepatic phase, we simulated the hemodynamic changes in liver transplantation resulting in IRI of extrahepatic organs in rats. Herein, we provide a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into IRI after liver transplantation.

<table><tbody><tr><td>4% paraformaldehyde solution</td><td>Shanghai Macklin Biochemical Co.,Ltd</td><td>P804536</td><td></td></tr><tr><td>air drying oven</td><td>Shanghai Binglin Electronic Technology Co., Ltd.</td><td>BPG</td><td></td></tr><tr><td>Alanine aminotransferase (ALT)Kit</td><td>Elabscience Biotechnology Co.,Ltd</td><td>E-BC-K235-S</td><td></td></tr><tr><td>ammonia</td><td>Sinopharm Chemical Reagents Co. Ltd</td><td>10002118</td><td></td></tr><tr><td>amylase Kit</td><td>Elabscience Biotechnology Co.,Ltd</td><td>E-BC-K005-M</td><td></td></tr><tr><td>anhydrous ethanol</td><td>Sinopharm Chemical Reagents Co. Ltd</td><td>100092183</td><td></td></tr><tr><td>Animal anesthesia machine</td><td>Shenzhen Ruiwode Life Technology Co. Ltd</td><td>R640</td><td></td></tr><tr><td>aspartate aminotransferase (AST)kit</td><td>Rayto Life and Analytical Sciences Co., Ltd.</td><td>S03040</td><td></td></tr><tr><td>automatic biochemical analyzer.</td><td>SIEMENS AG FWB:SIE, NYSE:SI Co., Ltd.</td><td>2400</td><td></td></tr><tr><td>Biosystems (when nessary)</td><td>Chengdu Taimeng Electronics Co., Ltd.</td><td>BL-420F</td><td></td></tr><tr><td>Centrifuge</td><td>Baiyang Medical Instrument Co., Ltd.</td><td>BY-600A</td><td></td></tr><tr><td>cover glass</td><td>Jiangsu Shitai Experimental Equipment Co. Ltd</td><td>10212432C</td><td></td></tr><tr><td>creatinine Kit</td><td>Rayto Life and Analytical Sciences Co., Ltd.</td><td>S03076</td><td></td></tr><tr><td>dewatering machine</td><td>Hungary 3DHISTECH Co.,Ltd</td><td>Donatello Series 2</td><td></td></tr><tr><td>embedding machine</td><td>Hubei Xiaogan Kuohai Medical Technology Co., Ltd.</td><td>KH-BL1</td><td></td></tr><tr><td>frozen machine</td><td>Wuhan Junjie Electronics Co., Ltd</td><td>JB-L5</td><td></td></tr><tr><td>hematoxylin-eosin dye solution</td><td>Wuhan Saiwell Biotechnology Co., Ltd</td><td>G1005</td><td></td></tr><tr><td>high-efficiency paraffin wax</td><td>Shanghai huayong paraffin wax co., Ltd</td><td>Q/YSQN40-91</td><td></td></tr><tr><td>hydrochloric acid</td><td>Sinopharm Chemical Reagents Co. Ltd</td><td>10011018</td><td></td></tr><tr><td>intraocular lens (IOL)forceps</td><td>Guangzhou Guangmei Medical Equipment Co., Ltd.</td><td>JTZRN</td><td></td></tr><tr><td>Isoflurane</td><td>Shenzhen Ruiwode Life Technology Co. Ltd</td><td>&amp;mdash;</td><td></td></tr><tr><td>micro Scissors(when nessary)</td><td>Shanghai Surgical Instrument Factory</td><td>WA1010</td><td></td></tr><tr><td>needle holders</td><td>Shanghai Surgical Instrument Factory</td><td>J32010</td><td></td></tr><tr><td>neutral gum</td><td>Shanghai Huashen Healing Equipment Co.,Ltd.</td><td>&amp;mdash;</td><td></td></tr><tr><td>normal optical microscope</td><td>Nikon Instrument Shanghai Co., Ltd</td><td>Nikon Eclipse CI</td><td></td></tr><tr><td>ophthalmic forceps</td><td>Shanghai Surgical Instrument Factory</td><td>J3CO30</td><td>straight</td></tr><tr><td>ophthalmic forceps</td><td>Shanghai Surgical Instrument Factory</td><td>JD1060</td><td>bending</td></tr><tr><td>ophthalmic Scissors</td><td>Shanghai Surgical Instrument Factory</td><td>J1E0</td><td></td></tr><tr><td>pathological slicer</td><td>Shanghai Leica Instrument Co., Ltd</td><td>RM2016</td><td></td></tr><tr><td>pipettes</td><td>Dragon Laboratory Instruments Co., Ltd.</td><td>7010101008</td><td></td></tr><tr><td>retractors</td><td>Beijing Jinuotai Technology Development Co.,Ltd.</td><td>JNT-KXQ</td><td></td></tr><tr><td>scanner</td><td>Hungary 3DHISTECH Co.,Ltd</td><td>Pannoramic 250</td><td></td></tr><tr><td>slide</td><td>Wuhan Saiwell Biotechnology Co., Ltd</td><td>G6004</td><td></td></tr><tr><td>xylene</td><td>Sinopharm Chemical Reagents Co. Ltd</td><td>1330-20-7</td><td></td></tr></tbody></table>

organ ischemia-reperfusion injury by simulating hemodynamic changes in rat liver transplant model

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

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.

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. ...

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.

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...

organ-ischemia-reperfusion-injury-simulating-hemodynamic-changes-rat

Research

JoVE Journal

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Explore a rat liver transplant model simulating hemodynamic changes to study ischemia-reperfusion injury. This method enables investigation of anhepatic phase effects on extrahepatic organs and survival outcomes.

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

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