Name: organ ischemia-reperfusion injury by simulating hemodynamic changes in rat liver transplant model
Uploaded: 2021-03-06T13:00:00.000+00:00
Duration: 10 min 7 s
Description: Watch this Scientific Journal Video about Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model at JoVE.com

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>

<ol>
	<li>Dar, W. A., Sullivan, E., Byon, J. S., Eltzschig, H., Ju, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischaemia+reperfusion+injury+in+liver+transplantation:+Cellular+and+molecular+mechanisms.">Ischaemia reperfusion injury in liver transplantation: Cellular and molecular mechanisms.</a> Liver International. 39 (5), 788-801 (2019).</li><li>Qiao, P. F., Yao, L., Zhang, X. C., Li, G. D., Wu, D. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+shock+pretreatment+improves+stem+cell+repair+following+ischemia-reperfusion+injury+via+autophagy.">Heat shock pretreatment improves stem cell repair following ischemia-reperfusion injury via autophagy.</a> World Journal of Gastroenterology. 21 (45), 12822-12834 (2015).</li><li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+YAP+attenuates+hepatic+damage+and+fibrosis+in+liver+ischemia-reperfusion+injury.">Activation of YAP attenuates hepatic damage and fibrosis in liver ischemia-reperfusion injury.</a> Journal of Hepatology. 71 (4), 719-730 (2019).</li><li>Hirao, H., Dery, K. J., Kageyama, S., Nakamura, K., Kupiec-Weglinski, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heme+Oxygenase-1+in+liver+transplant+ischemia-reperfusion+injury:+From+bench-to-bedside.">Heme Oxygenase-1 in liver transplant ischemia-reperfusion injury: From bench-to-bedside.</a> Free Radical Biology and Medicine. 157, 75-82 (2020).</li><li>Moti&#241;o, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+role+of+hepatocyte+cyclooxygenase-2+expression+against+liver+ischemia-reperfusion+injury+in+mice.">Protective role of hepatocyte cyclooxygenase-2 expression against liver ischemia-reperfusion injury in mice.</a> Hepatology. 70 (2), 650-665 (2019).</li><li>Guo, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+search+for+a+magic+bullet+to+fight+multiple+organ+failure+secondary+to+ischemia/reperfusion+injury+and+abdominal+compartment+syndrome.">The search for a magic bullet to fight multiple organ failure secondary to ischemia/reperfusion injury and abdominal compartment syndrome.</a> Journal of Surgical Research. 184 (2), 792-793 (2013).</li><li>Elham, M., Mahmoudi, M., Nassiri-Toosi, M., Baghfalaki, T., Zeraati, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post+liver+transplantation+survival+and+related+prognostic+factors+among+adult+recipients+in+tehran+liver+transplant+center;+2002-2019.">Post liver transplantation survival and related prognostic factors among adult recipients in tehran liver transplant center; 2002-2019.</a> Archives of Iranian Medicine. 1 (23), 326-334 (2020).</li><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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+and+clinical+significance+of+hyperfibrinolysis+during+living+donor+liver+transplantation.">Incidence and clinical significance of hyperfibrinolysis during living donor liver transplantation.</a> Blood Coagulation and Fibrinolysis. 29 (3), 322-326 (2018).</li><li>Czig&#225;ny, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+research+practice+in+rat+orthotopic+and+partial+orthotopic+liver+transplantation:+a+review,+recommendation,+and+publication+guide.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+liver+transplantation+in+the+rat.+Technique+using+cuff+for+portal+vein+anastomosis+and+biliary+drainage.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+orthotopic+liver+transplantation+in+the+rat+and+some+studies+of+the+immunologic+responses+to+fully+allogeneic+liver+grafts.">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><li>Miyata, M., Fischer, J. H., Fuhs, M., Isselhard, W., Kasai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+orthotopic+liver+transplantation+in+the+rat.+Cuff+technique+for+three+vascular+anastomoses.">A simple method for orthotopic liver transplantation in the rat. Cuff technique for three vascular anastomoses.</a> Transplantation. 30 (5), 335-338 (1980).</li><li>Kamada, N. A., Calne, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+experience+with+five+hundred+thirty+liver+transplants+in+the+rat.">Surgical experience with five hundred thirty liver transplants in the rat.</a> Surgery. 93 (1), 64-69 (1983).</li><li>Yang, L. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rat+model+of+orthotopic+liver+transplantation+using+a+novel+magnetic+anastomosis+technique+for+suprahepatic+vena+cava+reconstruction.">A rat model of orthotopic liver transplantation using a novel magnetic anastomosis technique for suprahepatic vena cava reconstruction.</a> Journal of Visualized Experiments. 19 (133), e56933 (2018).</li><li>Liu, L. X., He, C., Huang, T., Gu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+new+technique+for+reconstruction+of+hepatic+artery+during+liver+transplantation+in+sprague-dawley+rat.">Development of a new technique for reconstruction of hepatic artery during liver transplantation in sprague-dawley rat.</a> PLoS One. 10 (12), 0145662 (2015).</li><li>Paller, M. S., Hoidal, J. R., Ferris, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+free+radicals+in+ischemic+acute+renal+failure+In+the+rat.">Oxygen free radicals in ischemic acute renal failure In the rat.</a> Journal of Clinical Investigation. 74 (4), 1156-1164 (1984).</li><li>Schmidt, J., Lewandrowsi, K., Warshaw, A. L., Compton, C. C., Rattner, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphometric+characteristics+and+homogeneity+of+a+new+model+of+acute+pancreatitis+in+the+rat.">Morphometric characteristics and homogeneity of a new model of acute pancreatitis in the rat.</a> International Journal of Pancreatology. 12 (1), 41-51 (1992).</li><li>Chui, C. J., McArdle, A. H., Brown, R., Scott, H. J., Gurd, F. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+mucosal+lesion+in+low-flow+states.+I.+A+morphological,+hemodynamic,+and+metabolic+reappraisal.">Intestinal mucosal lesion in low-flow states. I. A morphological, hemodynamic, and metabolic reappraisal.</a> Archives of Surgery. 101 (4), 478-483 (1970).</li><li>Kozian, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-lung+ventilation+induces+hyperfusion+and+alveolar+damage+in+the+ventilated+lung:an+experimental+study.">One-lung ventilation induces hyperfusion and alveolar damage in the ventilated lung:an experimental study.</a> British Journal of Anaesthesia. 100 (4), 549-559 (2008).</li><li>Shimada, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heavy+water+(D2O)+containing+preservation+solution+reduces+hepatic+cold+preservation+and+reperfusion+injury+in+an+isolated+perfused+rat+liver+(IPRL)+model.">Heavy water (D2O) containing preservation solution reduces hepatic cold preservation and reperfusion injury in an isolated perfused rat liver (IPRL) model.</a> Journal of Clinical Medicine. 8 (11), 1818 (2019).</li><li>Nakamura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sirtuin+1+attenuates+inflammation+and+hepatocellular+damage+in+liver+transplant+ischemia/reperfusion:+from+mouse+to+human.">Sirtuin 1 attenuates inflammation and hepatocellular damage in liver transplant ischemia/reperfusion: from mouse to human.</a> Liver Transplantation. 23 (10), 1282-1293 (2017).</li><li>Blaire, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+Considerations+of+Hilar+Cholangiocarcinoma.">Surgical Considerations of Hilar Cholangiocarcinoma.</a> Surgical Oncology Clinics of North America. 28 (4), 601-617 (2019).</li></ol>

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. Thus, we used inhalation anesthesia in subsequent trials for rapid onset of action and rapid exclusion of in vitro characteristics. The switch to inhalation anesthesia significantly improved the tolerance time and the postoperative survival of rats. Investigators should pay attention to the breathing and heart rates of the rat to prevent overdosing the anesthetic. Biosystems can be used to monitor the heart rate and blood pressure. We also observed the impact of surgical suture thickness on the ligation of blood vessels. Although lines smaller than 3-0 could perfectly ligate the blood vessels, they were difficult to loosen and may lead to the rupture of blood vessels. On the contrary, lines larger than 3-0 may result in incomplete vascular occlusion, which prevents hemodynamic changes. These material problems will be improved upon in future experiments. There are some limitations to our protocol. Heat lamps are not recommended for temperature maintenance due to their potential for overheating; alternate heating suggestions, such as recirculating water blankets, are recommended for the animal&#39;s benefit.
There are many reasons for distal organ injury after OLT. First, injury can be caused by cold preservation of the donor liver in vitro20. Second, IRI can occur and cause tissue damage when blood supply returns to tissue (reperfusion) after a long period of ischemia. Ischemia is the leading cause of injury, and reperfusion is the process where the injury occurs. After simultaneously blocking the IVC and PV during the anhepatic phase, a large amount of blood stasis occurred in the lower limbs and internal organs. The effective circulating volume (ECV) decreased sharply, and the MAP decreased. However, due to vagus nerve stimulation, there was no compensatory increase in heart rate in rats. In this experiment, we found that rats underwent significant hemodynamic changes within 5 minutes of ligation and vessel release, which met the definition of the ischemia-reperfusion syndrome.
Ischemia occurred in some tissues outside the liver. After the anhepatic phase, ECV increased. MAP returned to normal after the IVC and PV were unblocked, with injury occurring outside the liver after reperfusion. Furthermore, IRI of the donor&#39;s liver produced inflammatory mediators (TNF-&#945;, interleukin-1, interleukin-6, interleukin-8) that attacked the distal organs21. In this experiment, the hemodynamics during the anhepatic phase were simulated, which caused the passive congestion of IVC and kidney, damage to the gastrointestinal barrier, bacterial translocation, ischemia of the organs (e.g., lung, heart, pancreas, kidney, etc.) where the SVC is located, and IRI to the extrahepatic organs.
The pathological findings showed that the peak of ischemic injury and the recovery time were different in each organ. Although cold storage and damage caused by immune factors could not be simulated in this study, anhepatic IRI can be replicated and compared with other animal models to research extrahepatic organ injury. Our model and OLT model can be compared and observed to provide a basis for the research on extrahepatic organ injury. Furthermore, our model is similar to some clinical liver operations, such as that for Hilar cholangiocarcinoma. Hilar cholangiocarcinoma is a malignant tumor that frequently invades the PV or the IVC and often requires PV clamping during surgery22. Hepatic portal reconstruction was performed; when the tumor invades the IVC, intraoperative clamping of the IVC is also required, and the resulting hemodynamic changes are consistent with our model.
To summarize, our model in rats is easy-to-use and straightforward, without microsurgery, and provides the basis for fundamental research into IRI of extrahepatic organs after hepatic ischemia.

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

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&#39; 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. Eight survived for 14 days in the I45 group, and only 2 survived in the I60 group. These results suggest that the rats could tolerate the anhepatic phase for a maximum of 45 minutes (Table 1).
Effects of vascular ligation on circulation in rats 
During the experiment, Biosystems recorded the heart rate and blood pressure (right internal carotid artery intubation) before and after the anhepatic phase. We found that the heart rate and mean arterial pressure (MAP) of rats changed dramatically after vascular ligation (Figure 2).
Effects on extrahepatic organs 
Hepatic ischemia congestion and edema were found in the intestines, gastric varices, and splenomegaly after ligation. Eighty rats were randomly divided into 8 groups for ischemia for 45 minutes (T0), reperfusion for 6 hours (T6), 12 hours (T12), 24 hours (T24), 48 hours (T48), 72 hours (T72), 7 days (D7), and 14 days (D14). After the rats were sacrificed, tissue from the kidney, pancreas, small intestine, heart, and lung were taken and stained with hematoxylin-eosin (HE). The whole staining process consists of five steps: dewaxing, staining, dehydration, transparency, and sealing. Except for the heart, pathological scores were assigned as previously described16,17,18,19.
The time until maximum injury to the extrahepatic organs varied; it was 6-24 hours after the operation for the pancreas and 24-48 hours for the lungs. The intestinal tract and kidney were most severely injured after 45 minutes of ischemia. There was no obvious abnormality of the intestinal mucosa 24 hours after the operation, and the kidneys recovered after 48 hours. After reperfusion, local myocardial cell necrosis, cell fragmentation and dissolution, inflammatory cell infiltration, and local vasodilation and congestion were found in the heart by 24-48 hours after the operation (Figure 3).
Lungs 
Neutrophil infiltration was found in the lung tissue after ischemia. With the increase of reperfusion time(T0,T6), mucus of the bronchial lumen could also be seen in the lung tissue. Inflammatory cell infiltration occurred in the alveolar wall, which became severely thickened. Alveolar collapse and disappearance of the alveolar cavity could also be found in some tissue. There was no significant alveolar edema or capillary congestion in the alveolar walls. They were most severely injured 24-48 hours after the operation, with some rats showing dyspnea and other manifestations 7 days post-operation. HE staining results suggested lymphadenitis in the airway, mild inflammatory cell infiltration in the alveolar wall, and local hemorrhage (Figure 3A, Figure 4).
Kidneys 
A small amount of eosinophilic substance was found in renal tubules after ischemia at phase T0, but no inflammatory cell infiltration and other abnormalities were seen. However, swollen renal tubular epithelial cells, porous or vacuolated cytoplasm, necrotic cells in few lumens, karyopyknosis, fragmentation, brush border loss, and tube-shaped acidic group in many lumens were seen 6-48 hours after the operation. In addition, a small number of renal tubular epithelial cells were seen with granular degeneration, and porous and lightly stained cytoplasm seen 48 hours after the operation. Significant interstitial telangiectasia, yet no severe inflammatory cell infiltration, was found (Figure 3B, Figure 5).
Small intestine 
The small intestine was most severely injured after ischemia (T0). There was severe inflammatory cell infiltration, mucosa epithelium shedding, and telangiectasia. With the increase of reperfusion time, the injury healed quickly. The mucosal epithelium was restored completely 24 hours after the operation, and only mild inflammatory cell infiltration was seen (Figure 3C, Figure 6).
Pancreas 
Severe inflammatory cells infiltrated around the pancreatic tissue at phase T0. However, the pancreatic lesions were not uniform. Six out of 10 had pancreatic necrosis and inflammatory infiltration 24 hours after surgery, and the other 4 had no apparent abnormalities. Twenty-four hours after the operation, in addition to the infiltration of inflammatory cells, there was edema, widening of interlobular space, hemorrhage, necrosis of a small number of acinar cells, unclear demarcation of the cells, nuclear fragmentation and dissolution, and mild inflammatory cell infiltration in the visual field. Then, the inflammation disappeared slowly (Figure 3D, Figure 7 ).
Heart 
By phase T0, cardiac myocytes were arranged regularly with clear demarcation, normal cell morphology, local interstitial congestion, and mild brown-yellow pigment deposition. Moreover, inflammatory cell infiltration was observed in myocardial interstitial and perivascular regions. With the increase in reperfusion time, local myocardial cell necrosis, cell fragmentation and dissolution, inflammatory cell infiltration, local vasodilation, and congestion were found in tissues 24-48 hours after the operation. Ventricular dilatation, porous structure, increased myocardial interstitium, and mild inflammatory cell infiltration were seen in some specimens. After 48 hours, local cardiomyocytes disappeared and were replaced by a small amount of fibrous connective tissue with mild inflammatory cell infiltration. By then, no other obvious abnormalities were seen (Figure 8).
Effects of hemodynamic changes on liver, kidney, pancreas, and heart serological indexes 
Serum was collected, and the levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST), creatinine, and amylase were detected by an automatic biochemical analyzer. All indicators peaked at 24-48 hours, unlike the pathological changes. Although these levels were normal 48 hours after the operation, pathological damage continued (Figure 9).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61779/61779fig01.jpg" /> 
Figure 1: Location of ligature: PV, HA, IVC upper of right renal vein. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61779/61779fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Group</td>
			<td>n</td>
			<td>survival at 24 h, n (%)</td>
			<td>survival at 7 d, n (%)</td>
			<td>survival at 14 d, n (%)</td>
		</tr>
		<tr>
			<td>sham</td>
			<td>10</td>
			<td>10</td>
			<td>&#8212;</td>
			<td>&#8212;</td>
		</tr>
		<tr>
			<td>I 15 min</td>
			<td>10</td>
			<td>10/10</td>
			<td>10 (100)</td>
			<td>10 (100)</td>
		</tr>
		<tr>
			<td>I 30 min</td>
			<td>10</td>
			<td>10/10</td>
			<td>10 (100)</td>
			<td>10 (100)</td>
		</tr>
		<tr>
			<td>I 45 min</td>
			<td>10</td>
			<td>8/10</td>
			<td>8/10 (80)</td>
			<td>8/10 (80)</td>
		</tr>
		<tr>
			<td>I 60 min</td>
			<td>10</td>
			<td>2/10</td>
			<td>2/10 (20)</td>
			<td>2/10 (20)</td>
		</tr>
	</tbody>
</table>
Table 1: Rats&#39; tolerance to liver ischemia
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61779/61779fig02.jpg" /> 
Figure 2: Hemodynamic changes in I45 min group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61779/61779fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61779/61779fig03.jpg" /> 
Figure 3:&#160;Scores of organ histology. (A) lung; (B) kidney; (C) intestine;&#160;(D) pancreas; *Statistically significant compared to the sham group (P&#160;&#60;&#160;0.05). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61779/61779fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61779/61779fig04.jpg" /> 
Figure 4: Pathological changes in the lungs after the operation. (A) Sham group; (B) Ischemia group (T0 group); (C) Reperfusion 6 hours (T6) group;&#160;(D) Reperfusion 12 hours (T12) group; (E) Reperfusion 12 hours (T12) group;&#160;(F) Reperfusion 24 hours (T24) group; (G) Reperfusion 48 hours (T48) group; (H) Reperfusion 7 days (D7) group; (I) Reperfusion 14 days (D14) group (scale 50 &#181;m). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61779/61779fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61779/61779fig05.jpg" /> 
Figure 5: Pathological changes in the kidneys after the operation.&#160;(A) Sham group; (B) Ischemia group (T0 group); (C) Reperfusion 6 hours (T6) group;&#160;(D) Reperfusion 12 hours (T12) group; (E) Reperfusion 12 hours (T12) group;&#160;(F) Reperfusion 24 hours (T24) group; (G) Reperfusion 48 hours (T48) group; (H) Reperfusion 7 days (D7) group; (I) Reperfusion 14 days (D14) group (scale 50 &#181;m). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61779/61779fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61779/61779fig06.jpg" /> 
Figure 6: Pathological changes in the small intestine after the operation. (A) Sham group; (B) Ischemia group (T0 group); (C) Reperfusion 6 hours (T6) group;&#160;(D) Reperfusion 12 hours (T12) group; (E) Reperfusion 12 hours (T12) group;&#160;(F) Reperfusion 24 hours (T24) group; (G) Reperfusion 48 hours (T48) group; (H) Reperfusion 7 days (D7) group; (I) Reperfusion 14 days (D14) group (scale 50 &#181;m). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61779/61779fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61779/61779fig07.jpg" /> 
Figure 7: Pathological changes in the pancreas after the operation. (A) Sham group; (B) Ischemia group (T0 group); (C) Reperfusion 6 hours (T6) group;&#160;(D) Reperfusion 12 hours (T12) group; (E) Reperfusion 12 hours (T12) group;&#160;(F) Reperfusion 24 hours (T24) group; (G) Reperfusion 48 hours (T48) group; (H) Reperfusion 7 days (D7) group; (I) Reperfusion 14 days (D14) group (A and D scale 50 &#181;m; BCEFGHI scale 50 &#181;m). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61779/61779fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61779/61779fig08.jpg" /> 
Figure 8:&#160;Pathological changes in the heart after the operation. (A) Sham group; (B) Ischemia group (T0 group); (C) Reperfusion 6 hours (T6) group;&#160;(D) Reperfusion 12 hours (T12) group; (E) Reperfusion 12 hours (T12) group;&#160;(F) Reperfusion 24 hours (T24) group; (G) Reperfusion 48 hours (T48) group; (H) Reperfusion 7 days (D7) group; (I) Reperfusion 14 days (D14) group (A scale 50 &#181;m; BCDEFGHI scale 100 &#181;m). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61779/61779fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/61779/61779fig09.jpg" /> 
Figure 9: Changes of ALT, AST, creatinine (Cr) and amylase in each group; *Statistically significant compared to the sham group (P&#60;0.05). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61779/61779fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model at JoVE.com

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

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

Nanyang Technological University

Research

JoVE Journal

Medicine

5.0K Views.  The Second Affiliated Hospital of Guangxi Medical University. Orgaanischemie-reperfusie letsel door het simuleren van hemodynamische veranderingen in rat lever transplantatie model. Ischemia-reperfusie letsel is een veel voorkomende toepassing na levertransplantatie. Patiënten met extrahepatische orgaanbetrokkenheid blijven meestal langer in het ziekenhuis, geven meer uit en hebben een slechtere prognose.De ontwikkeling van de complicatie hangt nauw samen met de lengte van de anhepatische fase van levertransplantatie. Fundamenteel onderzoek naar...

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

Orthotopic Liver Transplantation in Rats

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat liver transplantation in 1979 by Kamada et al.3, this model has remained the gold standard despite various proposed alternative techniques4. Nevertheless, its broader use is limited by its steep learning curve5.
In this video paper, we show a simple and easy-to-establish revision of Kamada's two-cuff technique. The suprahepatic vena cava anastomosis is performed manually with a running suture, and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick-linker cuff system6. Manufacturing the quick-linker kit is shown in a separate video paper.

We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat.

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver&#39;s common hepatic artery to the recipient liver&#39;s proper hepatic artery.

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...

Avoiding Ischemia Reperfusion Injury in Liver Transplantation

Currently, ex situ machine perfusion is a burgeoning technique that provides a better preservation method for donor organs than conventional static cold preservation (0&#8211;4 &#176;C). A continuous blood supply to organs using machine perfusion from procurement and preservation to implantation facilitates complete prevention of ischemia reperfusion injury and permits ex situ functional assessment of donor livers before transplantation. In this manuscript, we provide a step-by-step ischemia-free liver transplantation protocol in which an ex situ normothermic machine perfusion apparatus is used for pulsatile perfusion through the hepatic artery and continuous perfusion of the portal vein from human donor livers to recipients. In the perfusion period, biochemical analysis of the perfusate is conducted to assess the metabolic activity of the liver, and a liver biopsy is also performed to evaluate the degree of injury. Ischemia-free liver transplantation is a promising method to avoid ischemia-reperfusion injury and may potentially increase the donor pool for transplantation.

Presented here is a protocol to provide a step-by-step ischemia-free liver transplantation protocol under ex situ normothermic machine perfusion (37 &#176;C) of human livers from donors to recipients.

Currently, ex situ machine perfusion is a burgeoning technique that provides a better preservation method for donor organs than conventional static cold ...

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...

A Heterotopic Rat Heart Transplantation Model using Circulatory Death Donor Hearts

The objective of this protocol is to set up a rat heterotopic heart transplantation model with donation after circulatory death (DCD) donor hearts. There are two setups for this protocol: heart donor setup and recipient setup. In the heart donor setup, Sprague Dawley rats are anesthetized, endotracheally intubated, and ventilated. The right carotid artery is cannulated to deliver heparin and the paralytic agent vecuronium-bromide. The DCD process is initiated by terminating the ventilation. After 20 min, the heart is exposed and the aorta distal to the brachiocephalic branch is clamped. At 25 min from terminating the ventilator, ice-cold University of Wisconsin (UW) solution is perfused through the carotid catheter to flush the heart. The heart is procured by dividing the aorta, pulmonary artery, venae cavae, and pulmonary veins and stored in UW solution for implantation. In the recipient setup, the Lewis rat is anesthetized with isoflurane. Slow-release buprenorphine is administered subcutaneously to facilitate a smooth postoperative recovery. Through a midline abdominal incision, the infra-renal aorta and the inferior vena cava are isolated and clamped with an atraumatic vascular clamp. The donor heart aorta and pulmonary artery are sutured to the recipient abdominal aorta and vena cava, respectively, with a running 8-0 Prolene. The vascular clamp is removed to reperfuse the heart. The abdominal wall is closed and the rat is recovered. After a set interval (24 h to 2 weeks), the recipient rat is anesthetized, the transplanted heart is exposed, and a balloon-tip-catheter is inserted into the left ventricle via the apex to record developed pressure and dP/dt using a data acquisition system. The heart tissue is collected for histology, immunology, or molecular analysis. A successful DCD donor rat heart transplantation model will allow further studies on the cardioprotective approaches to improve heart transplantation outcomes from DCD donors.

Here, we describe the preparation and technical details of murine heterotopic heart transplantation utilizing a circulatory death donor heart.

The objective of this protocol is to set up a rat heterotopic heart transplantation model with donation after circulatory death (DCD) donor hearts. There ...

Porcine Liver Transplantation Without Veno-Venous Bypass As an Extended Criteria Donor Model

Liver transplantation is regarded as the gold standard for the treatment of a variety of fatal hepatic diseases. However, unsolved issues of chronic graft failure, ongoing organ donor shortages, and the increased use of marginal grafts call for the improvement of current concepts, such as the implementation of organ machine perfusion. In order to evaluate new methods of graft reconditioning and modulation, translational models are required. With respect to anatomical and physiological similarities to humans and recent progress in the field of xenotransplantation, pigs have become the main large animal species used in transplantation models. After the initial introduction of a porcine orthotopic liver transplant model by Garnier et al. in 1965, several modifications have been published over the past 60 years.
Due to specifies-specific anatomical traits, a veno-venous bypass during the anhepatic phase is regarded as a necessity to reduce intestinal congestion and ischemia resulting in hemodynamic instability and perioperative mortality. However, the implementation of a bypass increases the technical and logistical complexity of the procedure. Furthermore, associated complications such as air embolism, hemorrhage, and the need for a simultaneous splenectomy have been reported previously.
In this protocol, we describe a model of porcine orthotopic liver transplantation without the use of a veno-venous bypass. The engraftment of donor livers after static cold storage of 20 h - simulating extended criteria donor conditions - demonstrates that this simplified approach can be performed without significant hemodynamic alterations or intraoperative mortality and with regular uptake of liver function (as defined by bile production and liver-specific CYP1A2 metabolism). The success of this approach is ensured by an optimized surgical technique and a sophisticated anesthesiologic volume and vasopressor management.
This model should be of special interest for workgroups focusing on the immediate postoperative course, ischemia-reperfusion injury, associated immunological mechanisms, and the reconditioning of extended criteria donor organs.

In this protocol, a model of porcine orthotopic liver transplantation after static cold storage of donor organs for 20 h without the use of a veno-venous bypass during engraftment is described. The approach uses a simplified surgical technique with minimization of the anhepatic phase and sophisticated volume and vasopressor management.

Liver transplantation is regarded as the gold standard for the treatment of a variety of fatal hepatic diseases. However, unsolved issues of chronic graft ...

Liver Transplantation in Rats and Analyzing Allograft Patency

Heterotopic Auxiliary Whole Liver Rat Transplant Model Utilizing a Hepaticoureterostomy for Allograft Rejection Studies

Small animal transplant models are indispensable for organ tolerance studies investigating feasible therapeutic interventions in preclinical studies. Rat liver transplantation (LTx) protocols typically use an orthotopic model where the recipients&#39; native liver is removed and replaced with a donor liver. This technically demanding surgical procedure requires advanced micro-surgical skills and is further complicated by lengthy anhepatic and lower body ischemia times. This prompted the development of a less complicated heterotopic method that can be performed faster with no anhepatic or lower body ischemia time, reducing post-surgery stress for the recipient animal.
This heterotopic LTx protocol includes two main steps: excising the liver from the donor rat and transplanting the whole liver into the recipient rat. During the excision of the donor liver, the surgeon ligates the supra-hepatic vena cava (SHVC) and hepatic artery (HA). On the recipient side, the surgeon removes the left kidney and positions the donor liver with the portal vein (PV), infra-hepatic vena cava (IHVC), and bile duct facing the renal vessels. Further, the surgeon anastomoses the recipient&#39;s renal vein end to end with the IHVC of the liver and arterializes the PV with the renal artery using a stent. A hepaticoureterostomy is utilized for biliary drainage by anastomosing the bile duct to the recipient&#39;s ureter, permitting the discharge of bile via the bladder.
The average duration of the transplantation was 130 min, cold ischemia duration was around 35 min, and warm ischemia duration was less than 25 min. Hematoxylin and eosin histology of the auxiliary liver from syngeneic transplants showed normal hepatocyte structure with no significant parenchymal alterations 30 days post-transplant. In contrast, 8-day post-transplant allogeneic graft specimens demonstrated extensive lymphocytic infiltration with a Banff Schema rejection activity index score of 9. Therefore, this LTx method facilitates a low morbidity rejection model alternative to orthotopic LTx.

The rat heterotopic auxiliary liver transplant protocol described here offers a practical investigational tool for exploring mechanisms of hepatic allograft rejection. This model helps to alleviate the surgical hurdles and animal stress of orthotopic liver transplantation in rats.

Small animal transplant models are indispensable for organ tolerance studies investigating feasible therapeutic interventions in preclinical studies. Rat ...

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video