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

我们要感谢广西医科大学第二附属医院的李文涛博士和吴继华博士提出的有益建议。作者要感谢我们的队友们的有益评论和讨论。作者还要感谢JoVE的匿名评论者和编辑们的评论。特别感谢袁医生的父母们的不断支持和鼓励。这项工作得到了宁波市自然科学基金委员会（2014A610248）的支持。

动物伦理委员会批准了广西医科大学（No20190920）的实验。所有动物均由广西医科大学动物实验中心提供。我们使用SPF雄性斯普拉格道利大鼠（200-250克，10-12周），保持在室温下25±2°C和湿度50±10%。喂食在手术前24小时停止：然而，提供了水。
注：一名操作员无需显微外科或手术显微镜即可执行所有操作。
1. 操作
<ol><li>称量后，用异氟化物麻醉大鼠 （5%?...

<ol>
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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. 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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. 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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>

大鼠OLT是研究肝移植、IRI、移植排斥、免疫耐受性、移植病理学和药理学、同质移植和异种移植器官保存的理想模型。目前，它已广泛应用于肝移植的实验研究。
在试验性研究中，我们首先对五巴比妥钠进行腹膜内麻醉，发现这导致术后死亡率高，对血动性变化的耐受性短。因此，我们在随后的试验中使用吸入麻醉剂，以快速开始行动和快速排除 体外 特征。转向吸入...

缺血性输液损伤（IRI）是肝脏移植后常见的并发症。肝 IRI 是一个病理过程，涉及缺血介导细胞损伤和肝脏再输液异常恶化。肝ARI和局部与生俱来的免疫反应可分为冷热ARI，根据临床环境的差异1。热 IRI 是由干细胞损伤引起的，通常由肝脏移植、休克和创伤2引起。冷ARI是由内皮细胞和外周循环3引起的肝移植并发症。临床报告显示，肝IRI与10%的早期器官衰竭有关，并可能增加急性和慢性排斥的发病率4，5。此外，肝ARI也可能诱发多种器官功能障碍综合征或全身炎症反应综合征，死亡率高6.肝外器官介入的患者往往在医院停留更长的时间，花更多的钱，并有一个更差的预后7。并发症的发展与肝移植8的肝移植阶段的长度密切相关。
1973年，美国李教授首次报道了大鼠骨科肝移植（OLT）。实验操作采用缝合法模拟临床肝移植步骤、血管和普通胆管（CBD）的解剖步骤。这个过程是困难和耗时的，成功率低9。1979年，Kamada等人创造性地使用"双袖法"对门户静脉进行解剖，在26分钟内控制肝炎期，从而大大改善了大鼠的OLT。同年，齐默尔曼提出了"单胆汁支架法"。在李安工作的基础上，齐默尔曼利用聚乙烯管直接对捐赠人和接受者的CBD进行解剖，简化了CBD的重建，保留了括约肌的功能，使之成为OLT模型11的胆汁重建标准。1980年，宫田等人提出了"三袖法"，即门户静脉（PV）、超肝静脉卡瓦（SVC）和内皮静脉卡瓦（IVC）被袖口法麻醉。然而，有扭曲的管子与这种方法的风险，这可能导致阻碍劣质的静脉曲道倒流12。1983年，"双袖法"被提出使用袖口法进行光伏和IVC的解剖，但采用SVC13的缝合方法。这种方法被全球学者采用，以建立OLT模型。此后，袖口解剖步骤得到改善，缩短了肝病期，提高了14只大鼠的存活率。同样，改进的方法用于临床实践，以缩短肝阶段15。然而，肝移植后对IRI的基础研究表明，存活率与肝外器官的损伤程度成反比。因此，需要进一步的研究，需要一个简单和可重复的动物模型来模拟肝移植后的IRI。
基于肝相的定义，我们模拟了肝移植的造血变化，导致大鼠肝外器官的IRI。在此，我们提供了如何建立大鼠肝相（肝缺血）动物模型的详细描述，以促进肝移植后对IRI的基础研究。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>&#8212;</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>&#8212;</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

大鼠的正畸肝移植 （OLT） 是一种经过验证的动物模型，用于术前、术中和术后研究，包括肝外器官的缺血-输液损伤 （IRI）。此模型需要大量的实验和设备。肝相的持续时间与移植后开发 IRI 的时间密切相关。在这个实验中，我们利用血动力学的变化诱导大鼠的肝外器官损伤，并确定了最大耐受时间。对于不同的器官来说，直到最严重的器官损伤的时间各不相同。这种方法可以很容易地复制，也可用于研究肝移植后肝外器官的IRI。

老鼠对肝缺血的耐受性 在这个动物模型中，在操作过程中血管被放出的部位显示在图1中。大鼠被随机分成5组进行缺血15分钟（I15组）、30分钟（I30组）、45分钟（I45组）、60分钟（I60组）和假组，每组10只大鼠。手术后14天观察到每组的存活率。所有老鼠在I15组、I30组和假组存活下来。在I45组，有8人存活了14天，在I60组中只有2人幸存下来。这些结果表明，大鼠?...

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

本文详细介绍了如何构建大鼠肝相（肝缺血）动物模型，促进肝移植后缺血-输液损伤的基础研究。

器官缺血-输液损伤模拟大鼠肝脏移植模型的血动力学变化

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

通过模拟大鼠肝脏移植模型的血动力学变化，器官缺血-输液损伤。缺血性输液损伤是肝移植后常见的应用。肝外器官介入的患者往往在医院停留的时间更长，花费更多，预后更差。并发症的发展与肝移植的肝移植阶段的长度密切相关。然而，对肝移植后缺血性输液损伤的基础研究表明，肝移植后的存活率与触觉外器官的损伤程度成反比。基于触觉相的定义，我们模拟了肝移植的造血性变化，导致触觉外器官缺血-再输液损伤。本文详细介绍了如何构建大鼠触觉阶段动物模型，对肝移植后缺血-输液损伤进行基础研究。袁博士负责所有视频动物操作。两个眼钳，直和弯曲，和眼间透镜，IOL钳子，眼科剪刀，一到两个缩回器，棉签，3-0手术缝合，iodoform纱布，缝合针，生物系统，必要时，和眼间透镜，IOL钳子用于分离血管，因为圆头。动物麻醉机、除水机、嵌入机、病理切片机、 冷冻机、组织切片机、空气干燥炉、神经光学显微镜、扫描仪、一般麻醉的苯丙胺、4%甲醛溶液、任何水性乙醇、二甲苯、高效石蜡、羟基苯丙胺染料溶液、盐酸、氨、滑动、盖玻璃、中性胶和垂死试剂以及消耗材料。移液器，离心机，自动生化分析仪，阿兰氨基转移酶，ALT套件，阿斯巴酸盐，氨基转移酶，AST套件，肌氨酸套件。称量后，大鼠被动物麻醉机用异氟化物麻醉。腹部消毒后，使用钳子和剪刀在xiphoid工艺下方三厘米处切口中位数。打开腹腔，使用缩管暴露肝脏，并调动肝胃韧带。使用棉签轻轻翻转肝脏的中叶，并向上转动以暴露端口习惯。识别胆管、入口静脉和肝动脉。使用棉交换将小肠向左下腹腔推，将劣质静脉卡瓦移到右肾静脉，用眼内透镜（IOL）将入口静脉、肝动脉和右肾静脉上方的劣质静脉卡瓦隔离，用3-0丝线标记，每个带滑结。切开左下肢的皮肤，用眼钳露出股骨静脉。慢慢地通过股骨静脉注射低分子量肝素625 IU/kg，使整个身体肝素化。用 3 - 0 号缝合的右肾静脉上方的入口静脉、肝动脉和劣质静脉卡瓦， 持续 45 分钟。更换腹腔中的小肠，用纱布盖住。在此期间，吸入麻醉减少。45分钟后，释放入口静脉、肝动脉和右肾静脉上方的劣质静脉卡瓦。一层一层地缝合肌肉和皮肤，终止吸入麻醉。每四小时使用5毫克/千克的皮下吗啡提供术后镇痛观察大鼠直到清醒，并在25加零下2摄氏度的温度下保持喂养，需要50加或负10%的湿度动物加热灯。在这个动物模型中，大鼠根据缺血随机分为五组15分钟，I 15组，30分钟，I 30组，45分钟，I 45组，60分钟，I 60，和假组，每组10只老鼠。手术后14天观察了每组的存活率。所有的老鼠都活在I15，我30和假组。在I 45组中，有8人存活了14天，在I 60组中只有两人幸存下来。结果表明，大鼠最多能承受45分钟的肝相。在实验中，生物系统用于记录心率和血压的变化，右内胡萝卜动脉在肝相前后。我们发现，心率和平均动脉压力，大鼠的MAP，在血管结结后发生了巨大的变化。韧带后，肠道的肝缺血、充血和水肿、胃变和脾脏明显。80只大鼠被随机分成8组，包括缺血45分钟、T0、输液6小时、T6、12小时、T12、24小时、T24、48小时、T48、72小时、T72、7天、T7、14天、T14。在老鼠被牺牲后，肾脏、胰腺、小肠、心脏和肺组织被取走并沾染了 HE。除了心脏，病理学分数分别根据参考给出。结果表明，肝外器官最大损伤的时间各不相同。胰腺手术后6至24小时，肺部手术24至48小时。肠道和肾脏在缺血45分钟后受了重伤。手术后24小时肠道粘膜没有明显异常，肾脏在48小时后恢复。至于心脏，随着输液后时间的流逝，局部心肌细胞嗜睡、细胞分裂和溶解、炎症细胞渗透、局部血管增生和充血在手术后24至48小时内被发现在组织内。血清被收集，通过自动生化分析仪检测到ALT、AST、肌氨酸和淀粉酶的水平。所有指标在24至48小时内达到峰值，与病理变化不同。虽然手术后48小时水平基本正常，但病理损伤仍在继续。综上所述，大鼠的模型简单易用，无需显微外科，为肝缺血后肝外器官缺血-输液损伤的基础研究提供了依据。

Research

JoVE Journal

Medicine

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

常温心脏骤停和心肺复苏术：缺血再灌注损伤的小鼠模型

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

科研

医学

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury

在原位横向腹直肌肌皮瓣：肌缺血再灌注损伤模型大鼠

Free tissue transfer is the gold standard of reconstructive surgery to repair complex defects not amenable to local options or those requiring composite ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

急性肾损伤和伤后纤维化小鼠缺血再灌注模型

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Technique of Porcine Liver Procurement and Orthotopic Transplantation using an Active Porto-Caval Shunt

使用Active波尔图腔静脉分流术猪肝脏采购和原位移植技术

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation ...

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

诱导和缺血再灌注损伤评估中的Langendorff灌注鼠心

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

Murine Model of Intestinal Ischemia-reperfusion Injury

肠缺血再灌注损伤的小鼠模型

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

视网膜缺血再灌注损伤的小鼠模型通过眼压升高

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...

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

直接节段性肝内交付使用大鼠肝门部夹具模型方法

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

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

体外常温肝灌注的小动物模型

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

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

大鼠心肌缺血再灌注后冠状动脉流动储备评价

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...