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

这项工作得到了美国国立卫生研究院 T32AI 106704-01A1 和俄亥俄州立大学器官移植、灌注、工程和再生的 t 鲁道夫·弗莱切基金的支持。

所有程序都是按照《机构动物保育指南》和国家研究理事会《人道护理和使用实验动物手册》 (IACUC) 进行的, 并经俄亥俄州立大学 IACUC 委员会批准。
1. 初步设置
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结合以下方法制备灌注液:86 毫升25% 白蛋白, 184 毫升的威廉姆斯的培养基, 30 毫升青霉素/链霉素 (10 的 u/毫升青霉素和0.01 毫克/毫升链霉素), 胰岛素 (50 u/升), 肝素 (0.01 u/毫升), l-谷氨酰胺 (0.292 克/升), 和?...

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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. 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移植的同种异体肝有很大的不足, 在反应中, 捐献者的标准已经扩大了1、2、3、4、5。由于捐助者短缺, NEVLP 已被引入作为评估和修改器官功能6,7的方法。我们设计了一种 NEVLP 的大鼠模型。此外, 我们使用这个模型来证明它的一个重?...

在肝移植的等待名单上有14578名患者, 每年约有7000人进行移植手术,1,2。针对这一严重的捐助者短缺问题, 已扩大了肝捐献者的标准;这些通常被称为边际器官或延长的标准捐赠者, 并预期在移植后的表现不如同种异体标准标准, 具有较高的原发性移植功能障碍和延迟移植功能3, 4,5,6。结果, NEVLP 作为评价和修改器官功能6,7的方法被引入。我们设计了一个大鼠 NEVLP 模型, 并用该模型证明了它的一个重要的潜在应用--对肝脏灌流液的新型分子添加剂的检测。
NEVLP 已经被评估在鼠 (鼠) 和猪模型, 以及在被丢弃的人体器官6,8,9。NEVLP 第一次人体试验的结果最近也发表在10。虽然低温机灌注已明显成为肾脏保存的标准, 但肝机灌注的温度仍有争议。与低温和 subnormothermic 灌注相比, NEVLP 有许多建议的优点。这包括减少保存伤害, 恢复正常的器官功能在生理条件下, 能力评估器官的性能, 并作为一个平台的器官修复, 重塑和修改7,11, 12,13,14,15,16,17。
大量的研究已经完成使用猪 NEVLP 模型。尽管这些模型在考虑使用废弃人体器官或人体临床试验模型时比较便宜, 但与我们的小动物 NEVLP 模型相比, 它们是非常昂贵的。每个实验成本的一个重要组成部分是灌流液。我们能够完成4小时的灌注与300毫升的灌流液在相对较低的成本。此外, 小动物的成本, 包括大鼠是非常低的成本相比, 猪。
与其他模型的 NEVLP 在大鼠, 这里提出的模型是相对简单的实施和具有广泛的应用范围。在图 1中可以看到灌注电路。灌流液开始在灌流液水库 (1), 这是一个水套容器。灌流液是由一个滚子泵 (2) 从水库中拉出, 并推入 windkessel (3), 然后是肺 (4)。肺为逆流气体和灌流液流提供最大气体交换。灌流液然后进入加热线圈 (5) 内的灌注室, 以确保它是在生理温度, 和气泡陷阱 (6), 以防止空气气泡灌注有前器官 (7) 和后器官 (8) 样本端口, 这使得灌流液采样。灌流液然后通过门静脉插管进入肝脏。门静脉套管附在压力监测器上, 用于图表数据收集软件上的值。灌流液然后通过静脉插管退出肝脏, 并流入压力均衡器块 (9)。最后, 将灌流液从压力块中拉回, 通过滚子泵, 倒入储层中。该模型包括对门静脉进行连续灌注, 并将脉动流留到肝动脉和透析用在其他一些模型中, 每一个都需要一个单独的和额外的电路, 但以前已经证明不需要9,13。
为了探索新的治疗分子在灌流液中的加入, 我们选择了酶过氧化氢。过氧化氢酶是一种内源性 ROS 清除剂, 是细胞内部防御机制的一部分, 以减轻 ROS18的影响。过氧化氢酶在肝缺血再灌注损伤中的表达增加19。实验性地添加过氧化氢酶已被证明可以减少缺血再灌注眼, 脑, 肺20,21,22,23,24。聚乙二醇化已被证明的目标过氧化氢酶的内皮和帮助过氧化氢酶吸收到内皮细胞25。在减少肝缺血再灌注损伤方面, 对 PEG 进行系统的管理, 疗效有限;然而, 我们假设, 添加 PEG 猫到一个孤立的器官灌注电路将导致改善结果26,27,28。在这里, 我们添加 PEG 猫到我们的基地灌流液, 并表明其能力减轻肝脏保护伤害。

<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

移植的同种异体肝有很大的不足, 并在反应中扩大了供体的标准。因此, 常温体外肝灌注 (NEVLP) 作为评价和改变器官功能的一种方法。NEVLP 与低温和 subnormothermic 灌注相比有许多优点, 包括减少保存损伤、生理条件下正常脏器功能恢复、器官性能评估, 以及作为器官修复的平台。、改建和改装。对小鼠和猪 NEVLP 模型进行了描述。我们展示了一个大鼠模型的 NEVLP, 并使用该模型显示其重要的应用之一-使用的治疗分子添加到肝灌流液。过氧化氢酶是一种内源活性氧 (ROS) 清除剂, 已被证明可以减少缺血再灌注在眼, 脑, 肺。聚乙二醇化已被证明是以过氧化氢酶为靶向内皮细胞。在这里, 我们增加了 pegylated 过氧化氢酶 (PEG 猫) 的基础灌流液, 并证明了它的能力减轻肝脏保护伤害。我们的啮齿动物 NEVLP 模型的好处是它是便宜的与更大的动物模型比较。这项研究的一个局限是它目前不包括灌注后肝移植。因此, 不能肯定地预测器官移植后的功能。然而, 大鼠肝移植模型的建立是很好的, 可以与该模型结合使用。最后, 我们展示了一个廉价的, 简单的, 易于复制的 NEVLP 模型使用大鼠。该模型的应用包括测试新的 perfusates 和灌流液添加剂, 用于器官评估的测试软件, 以及用于修复器官的实验。

每组三只老鼠的样本大小被使用。ALT 测量了 0, 30, 60, 90, 120, 150, 180, 210, 和240分钟的灌注。我们使用学生的t检验, 以比较的结果之间的基础灌流液和基地灌流液加上 PEG 猫组在每个时间点。在比较基灌流液和基灌流液加上 peg-猫组, 有显着少 (p < 0.05) ALT 在基灌流液加上 PEG 猫组在 150, 180, 210 和240分钟 (图 14A)。
<p class="jove_content" fo:keep-toge...

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

体外常温肝灌注的小动物模型

肝脏捐献者数量严重不足, 肝脏捐献者的标准已经扩大。常温体外肝灌注 (NEVLP) 已发展为评价和修饰器官功能。本研究展示了 NEVLP 的大鼠模型, 并对 pegylated 过氧化氢酶的能力进行了试验, 以减轻肝脏保存损伤。

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A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

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