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小鼠常母离 体 肝机灌注

Published: September 25, 2023
doi:
10.3791/65363
, , , ,
1Experimental Transplantation Surgery, Department of General, Visceral and Vascular Surgery,Jena University Hospital, 2Institute of Pathology,Klinikum Chemnitz GmbH, 3Department of Pathology, Faculty of Veterinary Medicine,Menoufia University

Summary

为小鼠肝脏创建了常温 离体 肝灌注（NEVLP）系统。该系统需要显微外科手术经验，但允许可重复的灌注结果。利用小鼠肝脏的能力有助于研究分子途径以鉴定新型灌注添加剂，并能够执行专注于器官修复的实验。

Abstract

该协议提出了使用小鼠肝脏的优化的无红细胞NEVLP系统。小鼠肝脏的 离 体保存是通过采用改良的插管和改编自传统商业 离体 灌注设备的技术来实现的。该系统用于评估灌注12 h后的保存结果。C57BL / 6J小鼠作为肝脏供体，通过插管门静脉（PV）和胆管（BD）排出肝脏，随后用温（37°C）肝素化盐水冲洗器官。然后，将移出的肝脏转移到灌注室并进行常温氧合机灌注（NEVLP）。每隔3小时收集入口和出口灌注液样品进行灌注分析。灌注完成后，获得肝脏样本进行组织学分析，并通过苏木精 – 伊红（HE）染色使用改良的铃木评分评估形态完整性。优化实验得出以下结果：（1）体重超过30g的小鼠由于其胆管（BD）的尺寸较大，被认为更适合实验。（2） 与聚丙烯插管相比，2 Fr（外径 = 0.66 mm）聚氨酯插管更适合插管门静脉 （PV）。这归因于聚氨酯材料增强了抓地力，从而减少了从身体转移到器官室过程中导管打滑。（3）对于胆管（BD）的插管，发现1Fr（外径= 0.33mm）聚氨酯套管比聚丙烯UT-03（外径= 0.30mm）套管更有效。通过这种优化的方案，小鼠肝脏成功保存了12小时，对组织学结构没有显着影响。苏木精-伊红 （HE） 染色显示肝脏形态结构保存完好，其特征是主要存活的肝细胞，具有清晰可见的细胞核和肝窦的轻度扩张。

Introduction

肝移植是终末期肝病患者的金标准治疗。令人遗憾的是，对捐献器官的需求超过了现有供应，导致严重短缺。2021 年，约有 24,936 名患者在等待肝移植的名单上，而只有 9,234 例移植成功进行1。肝移植物供需之间的巨大差异凸显了研究替代策略以扩大供体库和提高肝移植物可及性的迫切需要。扩大捐助者库的一种方法是使用边际捐助者2.边缘捐赠者包括高龄、中度或重度脂肪变性的人。尽管边缘器官移植可能会产生有利的结果，但总体结果仍然不理想。因此，目前正在制定旨在增强边际捐赠者功能的治疗策略3,4。

其中一种策略是利用机器灌注，特别是常温氧合机灌注，来改善这些边缘器官的功能5。然而，对常温氧合机灌注（NEVLP）有益作用的分子机制仍然了解有限。小鼠具有丰富的转基因菌株，是研究分子途径的宝贵模型。例如，自噬途径在减轻肝缺血再灌注损伤中的重要性已得到越来越多的认可6,7。肝缺血再灌注损伤中一条重要的分子途径是 miR-20b-5p/ATG7 途径8。目前，有许多ATG敲除和条件敲除小鼠品系可用，但没有相应的大鼠品系9。

基于这一背景，目的是为小鼠肝脏移植物生成小型化的NEVLP平台。该平台将有助于探索和评估旨在改善供体肝脏功能的潜在转基因策略。此外，该系统必须适合长期灌注，使肝脏能够 离体 治疗，通常称为“器官修复”。

考虑到小鼠肝脏灌注的相关 体外 数据有限，文献综述侧重于在大鼠中进行的研究。使用“正常体温肝灌注”、“体外 或 体外”和“大鼠”等关键词对 2010 年至 2022 年的文献进行了系统检索。该搜索旨在确定啮齿动物的最佳条件，使我们能够确定最合适的方法。

灌注系统由密封的水套玻璃缓冲储液罐、蠕动辊泵、氧合器、气泡阱、热交换器、风琴室和封闭循环管系统组成（图 1）。该系统确保使用专用的恒温机器精确保持 37 °C 的恒定灌注温度。蠕动辊泵驱动灌注液在整个回路中的流动。灌注回路在绝缘水套储液罐处启动。随后，灌注液被引导通过氧合器，氧合器从专用气瓶中接收95%氧气和5%二氧化碳的气体混合物。充氧后，灌注液通过气泡阱，其中任何被困住的气泡都通过蠕动泵重定向回储液器。剩余的灌注液流经热交换器并进入器官室，从那里返回储液器。

在这里，我们报告了我们为小鼠肝脏建立NEVLP的经验，并分享了使用不含氧载体的含氧培养基进行的试点实验的有希望的结果。

Protocol

动物实验是根据现行的德国动物福利法规和指南以及ARRIVE动物研究报告指南进行的。动物实验方案由德国图林根州Thüringer Landesamt für Verbraucherschutz批准（批准号：UKJ – 17 – 106）。 注意：将体重34±4g（平均值[SEM]的平均±标准误差）的雄性C57BL / 6J小鼠用作肝脏供体。它们在受控的环境条件下（50%湿度和18 – 23°C）下保持，并自由获取标准小鼠食物和水。在整个手术过程中，呼吸?…

Representative Results

建立外科手术本实验共使用17只动物：14只小鼠用于优化器官获取过程，包括门静脉（PV）和胆管（BD）的插管，而3只小鼠用于验证该程序（表1）。比较组织学结果（图3）以方便确定最佳灌注条件。 灌注液的选择本研究选择了以前使用的肝细胞培养基10,11。William’s E?…

Discussion

协议中的关键步骤
肝植的两个关键步骤是门静脉插管（PV）和随后的胆管插管（BD）。这些步骤对于确保成功的器官取回和随后的灌注或移植程序至关重要。

挑战和解决方案
PV插管存在三个挑战：血管壁的损伤，导管的移位和插入过程的实用性。PV血管壁的脆弱性质使其在插管过程中不小心处理，容易被刺穿和随后的出血。此外，PV的任何失血都?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

在撰写本文的整个过程中，我得到了大量的支持和帮助。我特别要感谢我的队友陈新培，感谢他在我手术过程中的出色合作和耐心支持。

Materials

0.5 ml Micro Tube PP Sarstedt 72699
1 Fr Rubber Cannula Vygon Sample Cannula
10 µL Micro Syringe Hamilton 701N
2 Fr Rubber Cannula Vygon Sample Cannula
24 G Butterfly Cannula Terumo SR+OF2419
26 G Butterfly Cannula Terumo SR+DU2619WX
30 G Hypodermic Needle Sterican 100246
50 ml Syringe Pump Braun 110356
6-0 Perma-Hand Seide Ethicon 639H
Arterial Clip Braun BH014R
Autoclavable Moist Chamber Hugo Sachs Elektronik 73-4733
Big Cotton Applicator  NOBA Verbandmittel Danz GmbH 974018
Bubble Trap Hugo-Sachs-Elektronik V83163
Buprenovet (0.3 mg / ml) Elanco /
CIDEX OPA solution (2 L) Cilag GmbH 20391
Electrosurgical Unit for Monopolar Cutting VIO® 50 C ERBE /
Fetal Bovine Serum(500 ml)  Sigma-Aldrich F7524-500ML
Gas Mixture (95 % oxygen & 5 % carbon dioxide) House Supply /
Heating Circulating Baths Harvard-Apparatus 75-0310
Heparin 5000 (I.E. /5 ml) Braun 1708.00.00
Hydrocortisone (100 mg / 2 ml) Pfizer 15427276
Insulin(100 IE / ml) Sigma I0516-5ML
Iris Scissors  Fine Science Instruments 15000-03
Isofluran (250 ml) Cp-Pharma 1214
Membrane Oxygenator Hugo Sachs Elektronik T18728
Microsurgery Microscope  Leica M60
Mouse Retractor Set  Carfil Quality 180000056
NanoZoomer 2.0 HT Hamamatsu /
Non-Woven Sponges  Kompressen 866110
Penicillin Streptomycin (1 mg / ml)  C.C.Pro Z-13-M
Perfusion Extension Tube (30 cm) Braun 4256000
Peristaltic Pump Harvard-Apparatus P-70
Petri Dishc 100×15 mm VWR® 391-0578
Povidon-Jod (Vet-Sep Spray) Livisto 799-416
Pressure Transducer Simulator UTAH Medical Products 650-950
Reusable Blood Pressure Transducers AD Instruments MLT-0380/D
S & T Vessel Cannulation Forceps Fine Science Instruments 00608-11
Small Cotton Applicator NOBA Verbandmittel Danz GmbH 974116
Straight Forceps 10 cm  Fine Science Instruments 00632-11
Suture Tying Forceps Fine Science Instruments 11063-07
Syringe 50ml Original Perfusor Braun 8728810F-06
UT – 03 Cannula Unique Medical, Japan /
Vannas Spring Scissors Fine Science Instruments 15018-10
Veterinary Saline (500 ml) WDT 18X1807
Water Jacketed Reservoir  2 L Harvard-Apparatus 73-3441
William's E Medium (500 ML) Thermofischer Scientific A1217601
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Tags

Keywords: NormothermicEx-vivoLiverMachine PerfusionMouseMolecular MechanismsGenetically Modified AnimalsTransgenic MiceThermostatic ControllerPeristaltic PumpOxygenatorBubble TrapPortal VeinSuprahepatic CavaBile DrainageSemi-sterile30G Needle26G CatheterHeparinized SalineOrgan ChamberRoller PumpHeat Exchanger
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Chen, H., Dirsch, O., Albadry, M., Ana, P. H., Dahmen, U. Normothermic Ex Vivo Liver Machine Perfusion in Mouse. J. Vis. Exp. (199), e65363, doi:10.3791/65363 (2023).
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