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
1Department of Intensive Care Unit,The Second Affiliated Hospital of Guangxi Medical University, 2Department of hepatobiliary surgery,The Affiliated Ningbo Medical Center Lihuili Hospital of Medical School of Ningbo University, 3Department of transplantation,The Second Affiliated Hospital of Guangxi Medical University, 4Digital Technology and Engineering college,Ningbo University of Finance & Economics
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 称量后，用异氟化物麻醉大鼠 （5%?…
Representative Results
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?…
Discussion
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…
Declarações
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 4% paraformaldehyde solution | Shanghai Macklin Biochemical Co.,Ltd | P804536 | |
| air drying oven | Shanghai Binglin Electronic Technology Co., Ltd. | BPG | |
| Alanine aminotransferase (ALT)Kit | Elabscience Biotechnology Co.,Ltd | E-BC-K235-S | |
| ammonia | Sinopharm Chemical Reagents Co. Ltd | 10002118 | |
| amylase Kit | Elabscience Biotechnology Co.,Ltd | E-BC-K005-M | |
| anhydrous ethanol | Sinopharm Chemical Reagents Co. Ltd | 100092183 | |
| Animal anesthesia machine | Shenzhen Ruiwode Life Technology Co. Ltd | R640 | |
| aspartate aminotransferase (AST)kit | Rayto Life and Analytical Sciences Co., Ltd. | S03040 | |
| automatic biochemical analyzer. | SIEMENS AG FWB:SIE, NYSE:SI Co., Ltd. | 2400 | |
| Biosystems (when nessary) | Chengdu Taimeng Electronics Co., Ltd. | BL-420F | |
| Centrifuge | Baiyang Medical Instrument Co., Ltd. | BY-600A | |
| cover glass | Jiangsu Shitai Experimental Equipment Co. Ltd | 10212432C | |
| creatinine Kit | Rayto Life and Analytical Sciences Co., Ltd. | S03076 | |
| dewatering machine | Hungary 3DHISTECH Co.,Ltd | Donatello Series 2 | |
| embedding machine | Hubei Xiaogan Kuohai Medical Technology Co., Ltd. | KH-BL1 | |
| frozen machine | Wuhan Junjie Electronics Co., Ltd | JB-L5 | |
| hematoxylin-eosin dye solution | Wuhan Saiwell Biotechnology Co., Ltd | G1005 | |
| high-efficiency paraffin wax | Shanghai huayong paraffin wax co., Ltd | Q/YSQN40-91 | |
| hydrochloric acid | Sinopharm Chemical Reagents Co. Ltd | 10011018 | |
| intraocular lens (IOL)forceps | Guangzhou Guangmei Medical Equipment Co., Ltd. | JTZRN | |
| Isoflurane | Shenzhen Ruiwode Life Technology Co. Ltd | — | |
| micro Scissors(when nessary) | Shanghai Surgical Instrument Factory | WA1010 | |
| needle holders | Shanghai Surgical Instrument Factory | J32010 | |
| neutral gum | Shanghai Huashen Healing Equipment Co.,Ltd. | — | |
| normal optical microscope | Nikon Instrument Shanghai Co., Ltd | Nikon Eclipse CI | |
| ophthalmic forceps | Shanghai Surgical Instrument Factory | J3CO30 | straight |
| ophthalmic forceps | Shanghai Surgical Instrument Factory | JD1060 | bending |
| ophthalmic Scissors | Shanghai Surgical Instrument Factory | J1E0 | |
| pathological slicer | Shanghai Leica Instrument Co., Ltd | RM2016 | |
| pipettes | Dragon Laboratory Instruments Co., Ltd. | 7010101008 | |
| retractors | Beijing Jinuotai Technology Development Co.,Ltd. | JNT-KXQ | |
| scanner | Hungary 3DHISTECH Co.,Ltd | Pannoramic 250 | |
| slide | Wuhan Saiwell Biotechnology Co., Ltd | G6004 | |
| xylene | Sinopharm Chemical Reagents Co. Ltd | 1330-20-7 |
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Citar este artigo
Yuan, Y., Chen, M., Huang, J., Tian, Y., Qin, K., Yuan, Z., Wang, W., Wu, Z., Tian, X., Zhang, Y. Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model. J. Vis. Exp. (169), e61779, doi:10.3791/61779 (2021).