Name: a small animal model of ex vivo normothermic liver perfusion
Uploaded: 2018-06-27T14:00:00
Description: Watch this Scientific Journal Video about A Small Animal Model of Ex Vivo Normothermic Liver Perfusion at JoVE.com

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.

All procedures were performed according to the guidelines of the Institutional Animal Care and National Research Council&#8217;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
<ol>
	<li>Prepare the perfusion solution by combining the following: 86 mL of 25% albumin, 184 mL of Williams&#8217; media, 30 mL of penicillin/streptomycin (10 U/mL penicillin and 0.01 mg/mL streptomycin...

<ol>
	<li>. National Data. Overall by Organ. Current U.S. Waiting List. Based on OPTN data as of October 19, 2017 Available from: <a target="_blank" href="https://optn.transplant.hrsa.gov/data/view-data-reports/national-data/">https://optn.transplant.hrsa.gov/data/view-data-reports/national-data/</a> (2017)</li><li>. National Data, Transplants by Donor Type, U.S. Transplants Performed January 1, 1988 - December 31, 2016, For Organ = Liver Available from: <a target="_blank" href="https://optn.transplant.hrsa.gov/data/view-data-reports/national-data/">https://optn.transplant.hrsa.gov/data/view-data-reports/national-data/</a> (2017)</li><li>Nemes, B., 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=Extended+criteria+donors+in+liver+transplantation+Part+I:+reviewing+the+impact+of+determining+factors.">Extended criteria donors in liver transplantation Part I: reviewing the impact of determining factors.</a> Expert Rev Gastroenterol Hepatol. 10 (7), 827-839 (2016).</li><li>Nemes, B., 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=Extended-criteria+donors+in+liver+transplantation+Part+II:+reviewing+the+impact+of+extended-criteria+donors+on+the+complications+and+outcomes+of+liver+transplantation.">Extended-criteria donors in liver transplantation Part II: reviewing the impact of extended-criteria donors on the complications and outcomes of liver transplantation.</a> Expert Rev Gastroenterol Hepatol. 10 (7), 841-859 (2016).</li><li>Pezzati, D., Ghinolfi, D., De Simone, P., Balzano, E., Filipponi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+to+optimize+the+use+of+marginal+donors+in+liver+transplantation.">Strategies to optimize the use of marginal donors in liver transplantation.</a> World J Hepatol. 7 (26), 2636-2647 (2015).</li><li>Marecki, H., 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=Liver+ex+situ+machine+perfusion+preservation:+A+review+of+the+methodology+and+results+of+large+animal+studies+and+clinical+trials.">Liver ex situ machine perfusion preservation: A review of the methodology and results of large animal studies and clinical trials.</a> Liver Transpl. 23 (5), 679-695 (2017).</li><li>Barbas, A. S., Knechtle, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+Donor+Pool+With+Normothermic+Ex+Vivo+Liver+Perfusion:+The+Future+Is+Now.">Expanding the Donor Pool With Normothermic Ex Vivo Liver Perfusion: The Future Is Now.</a> Am J Transplant. 16 (11), 3075-3076 (2016).</li><li>Dries, 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=Ex+vivo+normothermic+machine+perfusion+and+viability+testing+of+discarded+human+donor+livers.">Ex vivo normothermic machine perfusion and viability testing of discarded human donor livers.</a> Am J Transplant. 13 (5), 1327-1335 (2013).</li><li>Westerkamp, A. C., 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=End-ischemic+machine+perfusion+reduces+bile+duct+injury+in+donation+after+circulatory+death+rat+donor+livers+independent+of+the+machine+perfusion+temperature.">End-ischemic machine perfusion reduces bile duct injury in donation after circulatory death rat donor livers independent of the machine perfusion temperature.</a> Liver Transpl. 21 (10), 1300-1311 (2015).</li><li>Selzner, M., 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=Normothermic+ex+vivo+liver+perfusion+using+steen+solution+as+perfusate+for+human+liver+transplantation:+First+North+American+results.">Normothermic ex vivo liver perfusion using steen solution as perfusate for human liver transplantation: First North American results.</a> Liver Transpl. 22 (11), 1501-1508 (2016).</li><li>Whitson, B. A., Black, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+assessment+and+repair+centers:+The+future+of+transplantation+is+near.">Organ assessment and repair centers: The future of transplantation is near.</a> World J Transplant. 4 (2), 40-42 (2014).</li><li>Tolboom, H., 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=Subnormothermic+machine+perfusion+at+both+20&#176;C+and+30&#176;C+recovers+ischemic+rat+livers+for+successful+transplantation.">Subnormothermic machine perfusion at both 20&#176;C and 30&#176;C recovers ischemic rat livers for successful transplantation.</a> J Surg Res. 175 (1), 149-156 (2012).</li><li>Nagrath, D., 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=Metabolic+preconditioning+of+donor+organs:+defatting+fatty+livers+by+normothermic+perfusion+ex+vivo.">Metabolic preconditioning of donor organs: defatting fatty livers by normothermic perfusion ex vivo.</a> Metab Eng. 11 (4-5), 274-283 (2009).</li><li>Boehnert, M. U., 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=Normothermic+acellular+ex+vivo+liver+perfusion+reduces+liver+and+bile+duct+injury+of+pig+livers+retrieved+after+cardiac+death.">Normothermic acellular ex vivo liver perfusion reduces liver and bile duct injury of pig livers retrieved after cardiac death.</a> Am J Transplant. 13 (6), 1441-1449 (2013).</li><li>Sch&#246;n, M. R., 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=Liver+transplantation+after+organ+preservation+with+normothermic+extracorporeal+perfusion.">Liver transplantation after organ preservation with normothermic extracorporeal perfusion.</a> Ann Surg. 233 (1), 114-123 (2001).</li><li>Reddy, 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=Non-heart-beating+donor+porcine+livers:+the+adverse+effect+of+cooling.">Non-heart-beating donor porcine livers: the adverse effect of cooling.</a> Liver Transpl. 11 (1), 35-38 (2005).</li><li>Banan, B., 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=Novel+strategy+to+decrease+reperfusion+injuries+and+improve+function+of+cold-preserved+livers+using+normothermic+ex+vivo+liver+perfusion+machine.">Novel strategy to decrease reperfusion injuries and improve function of cold-preserved livers using normothermic ex vivo liver perfusion machine.</a> Liver Transpl. 22 (3), 333-343 (2016).</li><li>Held, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> An Introduction to Reactive Oxygen Species: Measurement of ROS in Cells. , 1-14 (2012).</li><li>Chen, C. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethylene+glycol-conjugated+superoxide+dismutase+in+focal+cerebral+ischemia-reperfusion.">Polyethylene glycol-conjugated superoxide dismutase in focal cerebral ischemia-reperfusion.</a> Am J Physiol. 265 (1 Pt 2), H252-H256 (1993).</li><li>I&#351;lekel, S., I&#351;lekel, H., G&#252;ner, G., Ozdamar, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+superoxide+dismutase,+glutathione+peroxidase+and+catalase+activities+in+experimental+cerebral+ischemia-reperfusion.">Alterations in superoxide dismutase, glutathione peroxidase and catalase activities in experimental cerebral ischemia-reperfusion.</a> Res Exp Med (Berl). 199 (3), 167-176 (1999).</li><li>Li, G., Chen, Y., Saari, J. T., Kang, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalase-overexpressing+transgenic+mouse+heart+is+resistant+to+ischemia-reperfusion+injury.">Catalase-overexpressing transgenic mouse heart is resistant to ischemia-reperfusion injury.</a> Am J Physiol. 273 (3 Pt 2), H1090-H1095 (1997).</li><li>Nowak, K., 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=Immunotargeting+of+catalase+to+lung+endothelium+via+anti-angiotensin-converting+enzyme+antibodies+attenuates+ischemia-reperfusion+injury+of+the+lung+in+vivo.">Immunotargeting of catalase to lung endothelium via anti-angiotensin-converting enzyme antibodies attenuates ischemia-reperfusion injury of the lung in vivo.</a> Am J Physiol Lung Cell Mol Physiol. 293 (1), L162-L169 (2007).</li><li>Beckman, J. 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=Superoxide+dismutase+and+catalase+conjugated+to+polyethylene+glycol+increases+endothelial+enzyme+activity+and+oxidant+resistance.">Superoxide dismutase and catalase conjugated to polyethylene glycol increases endothelial enzyme activity and oxidant resistance.</a> J Biol Chem. 263 (14), 6884-6892 (1988).</li><li>Yabe, Y., Nishikawa, M., Tamada, A., Takakura, Y., Hashida, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+delivery+and+improved+therapeutic+potential+of+catalase+by+chemical+modification:+combination+with+superoxide+dismutase+derivatives.">Targeted delivery and improved therapeutic potential of catalase by chemical modification: combination with superoxide dismutase derivatives.</a> J Pharmacol Exp Ther. 289 (2), 1176-1184 (1999).</li><li>Yabe, 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=Prevention+of+neutrophil-mediated+hepatic+ischemia/reperfusion+injury+by+superoxide+dismutase+and+catalase+derivatives.">Prevention of neutrophil-mediated hepatic ischemia/reperfusion injury by superoxide dismutase and catalase derivatives.</a> J Pharmacol Exp Ther. 298 (3), 894-899 (2001).</li><li>Ushitora, M., 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=Prevention+of+hepatic+ischemia-reperfusion+injury+by+pre-administration+of+catalase-expressing+adenovirus+vectors.">Prevention of hepatic ischemia-reperfusion injury by pre-administration of catalase-expressing adenovirus vectors.</a> J Control Release. 142 (3), 431-437 (2010).</li><li>Kakizaki, 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=The+Effects+of+Short-Term+Subnormothermic+Perfusion+after+Cold+Preservation+on+Liver+Grafts+from+Donors+after+Cardiac+Death:+An+Ex+Vivo+Rat+Model.">The Effects of Short-Term Subnormothermic Perfusion after Cold Preservation on Liver Grafts from Donors after Cardiac Death: An Ex Vivo Rat Model.</a> Transplantation. , (2018).</li><li>Kumar, R., Chung, W. Y., Dennison, A. R., Garcea, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+Vivo+Porcine+Organ+Perfusion+Models+as+a+Suitable+Platform+for+Translational+Transplant+Research.">Ex Vivo Porcine Organ Perfusion Models as a Suitable Platform for Translational Transplant Research.</a> Artif Organs. , (2017).</li><li>Nativ, N. I., 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=Liver+defatting:+an+alternative+approach+to+enable+steatotic+liver+transplantation.">Liver defatting: an alternative approach to enable steatotic liver transplantation.</a> Am J Transplant. 12 (12), 3176-3183 (2012).</li><li>Yeung, J. C., 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=Ex+vivo+adenoviral+vector+gene+delivery+results+in+decreased+vector-associated+inflammation+pre-+and+post-lung+transplantation+in+the+pig.">Ex vivo adenoviral vector gene delivery results in decreased vector-associated inflammation pre- and post-lung transplantation in the pig.</a> Mol Ther. 20 (6), 1204-1211 (2012).</li><li>Goldaracena, N., 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=Inducing+Hepatitis+C+Virus+Resistance+After+Pig+Liver+Transplantation-A+Proof+of+Concept+of+Liver+Graft+Modification+Using+Warm+Ex+Vivo+Perfusion.">Inducing Hepatitis C Virus Resistance After Pig Liver Transplantation-A Proof of Concept of Liver Graft Modification Using Warm Ex Vivo Perfusion.</a> Am J Transplant. 17 (4), 970-978 (2017).</li><li>Van Raemdonck, D., Neyrinck, A., Rega, F., Devos, T., Pirenne, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Machine+perfusion+in+organ+transplantation:+a+tool+for+ex+vivo+graft+conditioning+with+mesenchymal+stem+cells?.">Machine perfusion in organ transplantation: a tool for ex vivo graft conditioning with mesenchymal stem cells?.</a> Curr Opin Organ Transplant. 18 (1), 24-33 (2013).</li><li>Pratschke, 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=Results+of+the+TOP+Study:+Prospectively+Randomized+Multicenter+Trial+of+an+Ex+Vivo+Tacrolimus+Rinse+Before+Transplantation+in+EDC+Livers.">Results of the TOP Study: Prospectively Randomized Multicenter Trial of an Ex Vivo Tacrolimus Rinse Before Transplantation in EDC Livers.</a> Transplant Direct. 2 (6), e76 (2016).</li><li>Pratschke, 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=Protocol+TOP-Study+(tacrolimus+organ+perfusion):+a+prospective+randomized+multicenter+trial+to+reduce+ischemia+reperfusion+injury+in+transplantation+of+marginal+liver+grafts+with+an+ex+vivo+tacrolimus+perfusion.">Protocol TOP-Study (tacrolimus organ perfusion): a prospective randomized multicenter trial to reduce ischemia reperfusion injury in transplantation of marginal liver grafts with an ex vivo tacrolimus perfusion.</a> Transplant Res. 2 (1), 3 (2013).</li><li>Nativ, N. 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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...

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 &#8211; 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.

<table>
	<tbody>
		<tr>
			<td>Perfusate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>8% Albumin</td>
			<td>CLS Behring, King of Prussia, PA</td>
			<td>0053-7680-32</td>
			<td></td>
		</tr>
		<tr>
			<td>Williams Media</td>
			<td>Sigma Aldrich, St. Louis, MO</td>
			<td>W1878</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma Aldrich, St. Louis, MO</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Eli Lilly, Indianapolis, IL</td>
			<td>0002-8215-91</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Fresnius Lab, Lake Zurich, IL</td>
			<td>C504701</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Sigma Aldrich, St. Louis, MO</td>
			<td>G3126</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma Aldrich, St. Louis, MO</td>
			<td>H0888</td>
			<td></td>
		</tr>
		<tr>
			<td>THAM</td>
			<td>Hospira, Inc,</td>
			<td>0409-1593-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene Glycol - Catalase</td>
			<td>Sigma Aldrich</td>
			<td>S9549 SIGMA</td>
			<td></td>
		</tr>
		<tr>
			<td>Personal Protective Equipment </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Mask</td>
			<td>Generic</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Protective Gown</td>
			<td>Generic</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Gloves</td>
			<td>Generic</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Liver Procurement</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague-Dawley Rat</td>
			<td>Harlan Sprague Dawley Inc.</td>
			<td>250 -350 grams</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Microscope</td>
			<td>Leica</td>
			<td>M500-N w/ OHS</td>
			<td></td>
		</tr>
		<tr>
			<td>Charcoal Canisters</td>
			<td>Kent Scientific</td>
			<td>SOMNO-2001-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal Healthcare</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure-Lok Precision Analytical Syringe&#160;</td>
			<td>Valco Instruments Co, Inc.</td>
			<td>SOMNO-10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrosurgical Unit</td>
			<td>Macan</td>
			<td>MV-7A</td>
			<td></td>
		</tr>
		<tr>
			<td>Warming Pad</td>
			<td>Braintree Scientific</td>
			<td>HHP2</td>
			<td></td>
		</tr>
		<tr>
			<td>SomnoSuite Small Animal Anesthesia System</td>
			<td>Kent Scientific</td>
			<td>SS-MVG-Module</td>
			<td></td>
		</tr>
		<tr>
			<td>PhysioSuite</td>
			<td>Kent Scientific</td>
			<td>PS-MSTAT-RT</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane chamber</td>
			<td>Kent Scientific</td>
			<td>SOMNO-0530LG</td>
			<td></td>
		</tr>
		<tr>
			<td>SurgiVet</td>
			<td>Isotec</td>
			<td>CDS 9000 Tabletop</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen</td>
			<td>Praxair</td>
			<td>98015</td>
			<td></td>
		</tr>
		<tr>
			<td>Rib retractors</td>
			<td>Kent Scientific</td>
			<td>INS600240</td>
			<td></td>
		</tr>
		<tr>
			<td>GenieTouch</td>
			<td>Kent Scientific</td>
			<td>GenieTouch</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Saline</td>
			<td>Baxter</td>
			<td>NDC 0338-0048-04</td>
			<td></td>
		</tr>
		<tr>
			<td>4x4 Non-Woven Sponges</td>
			<td>Criterion</td>
			<td>104-2411</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Q-Tips</td>
			<td>Henry Schein Animal Health</td>
			<td>1009175</td>
			<td></td>
		</tr>
		<tr>
			<td>U-100 27 Gauge Insulin Syringe</td>
			<td>Terumo</td>
			<td>22-272328</td>
			<td></td>
		</tr>
		<tr>
			<td>5mL Syringe</td>
			<td>BD</td>
			<td>REF 309603</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 Braided Silk Suture</td>
			<td>Deknatel, Inc.</td>
			<td>198737LP</td>
			<td></td>
		</tr>
		<tr>
			<td>7-0 Braided Silk Suture</td>
			<td>Teleflex Medical</td>
			<td>REF 103-S</td>
			<td></td>
		</tr>
		<tr>
			<td>16 gauge Catheters</td>
			<td>BBraun Introcan Safety</td>
			<td>4252586-02</td>
			<td></td>
		</tr>
		<tr>
			<td>14 gauge Catheters</td>
			<td>BBraun Introcan Safety</td>
			<td>4251717-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Bile Duct Cannular Tubing</td>
			<td>Altec</td>
			<td>01-96-1727&#160;&#160;&#160;&#160;&#160; &#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Liver Perfusion Circuit Components </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water Bath Warmer</td>
			<td>Lauda Ecoline Staredition</td>
			<td>E103</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Collection Software</td>
			<td>ADInstruments&#160;</td>
			<td>Labchart 7</td>
			<td></td>
		</tr>
		<tr>
			<td>Liver Perfusion Circuit</td>
			<td>Harvard Apparatus</td>
			<td>73-2901</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane Oxygenator</td>
			<td>Mediac SPA</td>
			<td>M03069</td>
			<td></td>
		</tr>
		<tr>
			<td>Roller Pump</td>
			<td>Ismatec</td>
			<td>ISM827B</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas (95% oxygen and 5% carbon dioxide)</td>
			<td>Praxair</td>
			<td>98015</td>
			<td></td>
		</tr>
		<tr>
			<td>Organ Chamber</td>
			<td>Harvard Apparatus</td>
			<td>ILP-2</td>
			<td></td>
		</tr>
		<tr>
			<td>1.8 mL Arcticle Cryogenic Tube</td>
			<td>USA Scientific</td>
			<td>1418-7410</td>
			<td></td>
		</tr>
		<tr>
			<td>Mucasol</td>
			<td>Sigma-Aldrich</td>
			<td>Z637181</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsurgical Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small Scissors</td>
			<td>Roboz</td>
			<td>RS-5610</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Scissors</td>
			<td>S&#38;T</td>
			<td>SAA-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps - Large Angled</td>
			<td>S&#38;T</td>
			<td>JFCL-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps - Small Angled</td>
			<td>S&#38;T</td>
			<td>FRAS-15 RM-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Clip Applier</td>
			<td>ROBOZ</td>
			<td>RS-5440</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors - non micro</td>
			<td>FST 14958-11</td>
			<td>14958-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps - Straight Tip</td>
			<td>S&#38;T</td>
			<td>FRS-15 RM8TC</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Microsurgical Clip</td>
			<td>Fine Scientific Tools</td>
			<td>18055-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Microsurgical Clip</td>
			<td>Fine Scientific Tools</td>
			<td>18055-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Microsurgical Clip</td>
			<td>Fine Scientific Tools</td>
			<td>18055-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Microsurgical Clip</td>
			<td>Fine Scientific Tools</td>
			<td>18055-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Mosquito Clamps</td>
			<td>Generic</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Post-Experiment Analysis </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alanine Aminotransferase (ALT) Activity Colorimetric/Fluorometric Assay Kit</td>
			<td>BioVision</td>
			<td>K752</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine Triphosphate (ATP) Colorimetric/Fluorometric Assay Kit</td>
			<td>BioVision</td>
			<td>K354</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione Assay Kit</td>
			<td>Cayman Chemical</td>
			<td>703002</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipid Peroxidation (MDA) Assay Kit</td>
			<td>Abcam</td>
			<td>ab118970</td>
			<td></td>
		</tr>
		<tr>
			<td>Caspase-Glo 3/7 Assay Systems</td>
			<td>Promega</td>
			<td>G8090</td>
			<td></td>
		</tr>
		<tr>
			<td>POLARstar OMEGA Microplate Reader</td>
			<td>BMG LABTECH</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

a small animal model of ex vivo normothermic liver perfusion

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 &#8212; 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.

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&#39;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 &#60;0.05) ALT in the base perfusate plus PEG-CAT group at 150, 180, 210, and 240 min (...

Watch this Scientific Journal Video about A Small Animal Model of Ex Vivo Normothermic Liver Perfusion at JoVE.com

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

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.

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

This method can help answer key questions in the field of transplantation and organ preservation. Applications of this method include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and performing experiments designed to repair organs. The main advantage of this technique is that the normothermic ex vivo liver perfusion model presented here is simple, easily replicable, low cost, and has a broad range of applications.Start by preparing the 16 gauge portal cuff. Cut a five millimeter section of tubing, and determine the midpoint of the section by measuring 2.5 millimeters. Incise at the midpoint and remove the anterior half of the tubing.Use a hemostat to crush this now flat portion. Use a lighter to melt the other end of the angiocatheter to create a lip. Next, prepare the bile duct canula by cutting the injection port from a 27 gauge angiocatheter, leaving only the catheter.Connect this to a 10 centimeter section of 27 gauge cannula tubing. Position an anesthetized rat with its nose in the anesthesia nose cone and its four extremities immobilized. Monitor vital signs by attaching the monitor to the left hind extremity.Perform a toe pinch to confirm appropriate depth of anesthesia. Spray the animal's abdomen with 70%isopropyl alcohol and allow it to dry. Place a sterile drape over the animal.Then, after making a midline incision from the xiphoid to the pubis using sharp scissors, gently enter the peritoneum and incise the muscle. Extend the incision laterally to the left and right to form a cross at the level of the inferior border of the liver. At this point, turn the isoflurane anesthesia down to 2%Retract the xiphoid process using a curved mosquito clamp and the ribs using rib retractors.Then use sharp scissors to cut the falciform, phrenic, and gastrohepatic ligaments. Locate and tie off the phrenic vein with a 7-0 suture as close to its origin as possible to prevent leaking. Next, use a sterile moistened cotton-tip applicator to eviscerate the rat.Wrap the bowel in gauze moistened with 0.9%normal saline, taking care not to stretch the vasculature. Then dissect over the inferior vena cava to remove excess tissue. Dissect behind the IVC just superior to the bifurcation and pass a loop of a 4-O silk suture for later use.Next, retract the right kidney to provide exposure to the right adrenal vein and 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. Then carefully dissect out the splenic vein.Tie if off using two 7-0 silk sutures and cut across it between the two sutures. After dissecting around the gastroduodenal artery, tie off the gastroduodenal artery with a 7-0 silk suture and ligate. Then dissect around the hepatic artery and loosely place a 7-0 silk suture tie around it.Next, dissect out the bile duct. Then check the length of the bile duct, tie it off at the distal end, and place a loop of suture around the bile duct and proximally as possible. Use small scissors to cut a hole that is half the diameter of the duct, and place a 27 gauge catheter into the bile duct proximally.Tie the catheter in place using a roman sandal tie suture. Next, use a 27 gauge needle to inject 0.5 milliliters of heparin into the penial vein, or IVC. Then clamp and tie off the IVC using the previously placed 4-0 silk suture and tie off the hepatic artery using the previously placed 7-0 silk suture.Now, use a microsurgical clip to clamp off the portal vein. Cannulate the portal vein using a 22 gauge angiocatheter and flush with 60 milliliters of cold 0.9%normal saline containing 100 units of heparin until the liver blanches. After flushing the liver, expose the suprahepatic IVC and cut across it as high in the chest as possible.Perform the hepatectomy by cutting around the diaphragm and then cutting the hepatic artery, the IVC, the portal vein, and any additional ligaments. Lift out the liver and place an ice-cold 0.9%normal saline. Lastly, place the 16 gauge vascular cuff in the portal vein and connect the liver to the ex vivo normothermic liver perfusion circuit.Before beginning each perfusion, visual inspection of the circuit should be performed to identify and damage or build-up on circuit components or tubing. If there is build-up of bacteria or other substances on the circuit, parts should be replaced or cleaned. Start by flowing the perfusate at two milliliters per minute.Watch the monitor for any spikes in portal vein pressure, as this may indicate the vessel has become occluded and necessitate repositioning of the cannula. Insert the protal vein cannula into the cuffed portal vein for the return flow of the perfusate and suture in using a 7-0 silk suture. Once both cannulas are in place, begin turning up the flow of the perfusate by one milliliter per minute until the physiological pressure in the range of 10 to 16 centimeters of water is reached.Remove one milliliter samples from the pre-port and post-port during the perfusion. Divide each one milliliter sample into two 0.5 milliliter samples. Snap freeze one of the 0.5 milliliter aliquots in cryogenic tubes in liquid nitrogen and run an arterial blood gas analysis using the remaining 0.5 milliliters of perfusate.Then, measure the pH and buffer the perfusate as needed to return to pH 7.4. After four hours of perfusion, disconnect the liver from the perfusion circuit. Divide the liver into 0.5 gram segments, transfer to cryogenic tubes, and snap freeze in liquid nitrogen.ALT was measured at zero, 30, 60, 90, 120, 150, 180, 210, and 240 minutes of perfusion. There is significantly less ALT in the base perfusate plus PEG-CAT group compared to base perfusate, which is shown in black at 150, 180, 210, and 240 minutes. Tissue ATP was maintained in the base perfusate plus PEG-CAT group in comparison to the base perfusate alone group.Tissue MDA production was significantly higher in the base perfusate group than in the base perfusate plus PEG-CAT group. Total GSH was maintained in the base perfusate plus PEG-CAT in comparison to the base perfusate alone group. Each proposed application of normothermic ex vivo liver perfusion will need to be methodically tested in animal models prior to testing in discarded human organs and then in human livers.The model presented here is ideal as it is easily replicable, eliminates extraneous tests, and is low-cost. After watching this video, you should have a good understanding of how to execute an inexpensive, easily replicable, normothermic ex vivo liver perfusion model using rats.

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Research

JoVE Journal

Medicine

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for liver transplantation die without receiving an organ transplant or are delisted for disease progression. One strategy to increase the donor pool is the utilization of marginal grafts, such as fatty livers, grafts from older donors, or donation after cardiac death (DCD). The current preservation technique of cold static storage is only poorly tolerated by marginal livers resulting in significant organ damage. In addition, cold static organ storage does not allow graft assessment or repair prior to transplantation.
These shortcomings of cold static preservation have triggered an interest in warm perfused organ preservation to reduce cold ischemic injury, assess liver grafts during preservation, and explore the opportunity to repair marginal livers prior to transplantation. The optimal pressure and flow conditions, perfusion temperature, composition of the perfusion solution and the need for an oxygen carrier has been controversial in the past.
In spite of promising results in several animal studies, the complexity and the costs have prevented a broader clinical application so far. Recently, with enhanced technology and a better understanding of liver physiology during ex vivo perfusion the outcome of warm liver perfusion has improved and consistently good results can be achieved.
This paper will provide information about liver retrieval, storage techniques, and isolated liver perfusion in pigs. We will illustrate a) the requirements to ensure sufficient oxygen supply to the organ, b) technical considerations about the perfusion machine and the perfusion solution, and c) biochemical aspects of isolated organs.

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

Marginal grafts, such as fatty livers, grafts from older donors, or livers retrieved after cardiac death (DCD) tolerate conventional, cold static storage only poorly. We developed a novel model of subnormothermic ex vivo liver perfusion for preservation, assessment, and repair of marginal liver grafts prior to transplantation.

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for ...

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal hyperplasia (IH) that eventually leads to vessel occlusion and graft failure. Mechanical forces, particularly low shear stress and high wall tension, are thought to initiate and to sustain these cellular and molecular changes, but their exact contribution remains to be unraveled. To selectively evaluate the role of pressure and shear stress on the biology of IH, an ex&#160;vivo perfusion system (EVPS) was created to perfuse segments of human saphenous veins under arterial regimen (high shear stress and high pressure). Further technical innovations allowed the simultaneous perfusion of two segments from the same vein, one reinforced with an external mesh. Veins were harvested using a no-touch technique and immediately transferred to the laboratory for assembly in the EVPS. One segment of the freshly isolated vein was not perfused (control, day 0). The two others segments were perfused for up to 7 days, one being completely sheltered with a 4 mm (diameter) external mesh. The pressure, flow velocity, and pulse rate were continuously monitored and adjusted to mimic the hemodynamic conditions prevailing in the femoral artery. Upon completion of the perfusion, veins were dismounted and used for histological and molecular analysis. Under ex&#160;vivo conditions, high pressure perfusion (arterial, mean = 100 mm Hg) is sufficient to generate IH and remodeling of human veins. These alterations are reduced in the presence of an external polyester mesh.

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mechanisms leading to the development of intimal hyperplasia (IH) and vein graft failure are still poorly understood. This study describes an ex&#160;vivo system to perfuse human veins under controlled flow and pressure. Furthermore the efficiency of external mesh reinforcement to limit the development of IH was evaluated.

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Ex Situ Normothermic Machine Perfusion of Donor Livers

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous perfusion of organs provides the opportunity to improve organ quality and allows ex situ viability assessment of donor livers prior to transplantation. This video article provides a step by step protocol for ex situ normothermic machine perfusion (37 &#176;C) of human donor livers using a device that provides a pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous perfusion of the portal vein. The perfusion fluid is oxygenated by two hollow fiber membrane oxygenators and the temperature can be regulated between 10 &#176;C and 37 &#176;C. During perfusion, the metabolic activity of the liver as well as the degree of injury can be assessed by biochemical analysis of samples taken from the perfusion fluid. Machine perfusion is a very promising tool to increase the number of livers that are suitable for transplantation.

Ex Situ Normothermic Machine Perfusion of Donor Livers

Here we present a protocol describing oxygenated ex situ machine perfusion of donor liver grafts. This article contains a step by step protocol to procure and prepare the liver graft for machine perfusion, prepare the perfusion fluid, prime the perfusion machine and perform oxygenated normothermic machine perfusion of the liver graft.

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to &#160;transplantation.

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

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 ...

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent hemorrhages at a later stage. The exact effects of plasma coagulation on liver tissue are only poorly examined. In our porcine model, the coagulation effects can be examined close to the clinical application. A combined laser Doppler flowmeter and spectrophotometer documents microcirculation changes during coagulation at 8 mm tissue depth noninvasively, providing quantifiable information about hemostasis beyond the subjective clinical impression. The temperature at coagulation site is assessed with an infrared thermometer prior and post coagulation and with a thermographic camera during coagulation, a measurement of the gas beam temperature is not possible due to the upper threshold of the devices. The depth of coagulation is measured microscopically on hematoxylin/eosin stained sections after calibration with an object micrometer and gives an exact information about the power setting-coagulation depth-relation. The sealing effect is examined on the bile ducts as it is not possible for a plasma coagulator to seal larger blood vessels. Burst pressure experiments are carried out on explanted organs to rule out blood pressure related effects.

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Here we present a protocol to experimentally assess plasma coagulation in liver tissue in vivo. In a porcine model, microcirculation is examined by laser Doppler, coagulation depth is measured histologically, temperature at coagulation site by infrared thermometer and thermographic camera, and duct sealing effect is documented by burst pressure experiments.

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation time and increases the risk of adverse post-transplantation outcomes. Moreover, the static nature of CS does not allow for organ evaluation or intervention during the preservation interval. Normothermic ex situ heart perfusion (ESHP) is a novel method for preservation of the donated heart that minimizes cold ischemia by providing oxygenated, nutrient-rich perfusate to the heart. ESHP has been shown to be non-inferior to CS in the preservation of standard-criteria donor hearts and has also facilitated the clinical transplantation of the hearts donated after the circulatory determination of death. Currently, the only available clinical ESHP device perfuses the heart in an unloaded, non-working state, limiting assessments of myocardial performance. Conversely, ESHP in working mode provides the opportunity for comprehensive evaluation of cardiac performance by assessment of functional and metabolic parameters under physiologic conditions. Moreover, earlier experimental studies have suggested that ESHP in working mode may result in improved functional preservation. Here, we describe the protocol for ex situ perfusion of the heart in a large mammal (porcine) model, which is reproducible for different animal models and heart sizes. The software program in this ESHP apparatus allows for real-time and automated control of the pump speed to maintain desired aortic and left atrial pressure and evaluates a variety of functional and electrophysiological parameters with minimal need for supervision/manipulation.

Normothermic ex situ heart perfusion (ESHP), preserves the heart in a beating, semi-physiologic state. When performed in a working mode, ESHP provides the opportunity to perform sophisticated assessments of donor heart function and organ viability. Here, we describe our method for myocardial performance evaluation during ESHP.

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...