无静脉-静脉旁路术的猪肝移植作为扩展标准供体模型
Summary
在该协议中，描述了在植入过程中不使用静脉 – 静脉旁路的情况下对供体器官静态冷藏20小时后猪原位肝移植的模型。该方法使用简化的手术技术，最大限度地减少无肝期，并采用复杂的容量和血管加压药管理。
Abstract
肝移植被认为是治疗各种致命肝脏疾病的金标准。然而，慢性移植失败、器官供体持续短缺以及边缘移植物使用增加等未解决的问题要求改进当前的概念，例如器官机器灌注的实施。为了评估移植物修复和调节的新方法，需要转化模型。关于与人类的解剖学和生理学相似性以及异种移植领域的最新进展，猪已成为移植模型中使用的主要大型动物物种。在1965年由Garnier等人首次引入猪原位肝移植模型后，在过去的60年中已经发表了一些修改。
由于特定的解剖学特征，在无肝期进行静脉-静脉搭桥被认为是减少导致血流动力学不稳定和围手术期死亡率的肠充血和缺血的必要条件。然而，旁路的实施增加了程序的技术和后勤复杂性。此外，相关并发症，如空气栓塞、出血和需要同时进行脾切除术，以前已有报道。
在该协议中，我们描述了不使用静脉 – 静脉旁路术的猪原位肝移植模型。静态冷藏20小时后对供体肝脏的植入 – 模拟扩展标准供体条件 – 表明这种简化的方法可以在没有显着血流动力学改变或术中死亡率的情况下进行，并且定期摄取肝功能（由胆汁产生和肝脏特异性CYP1A2代谢定义）。优化的手术技术以及复杂的麻醉容量和血管加压药管理确保了这种方法的成功。
该模型应该对专注于术后即刻病程、缺血再灌注损伤、相关免疫机制和扩展标准供体器官修复的工作组特别感兴趣。
Introduction
肝移植仍然是导致急性或慢性肝衰竭的各种不同疾病中生存的唯一机会。自1963年Thomas E. Starzl首次在人类中成功应用以来，肝移植的概念已发展成为全球应用的可靠治疗选择，这主要是由于对免疫系统的理解的进步，现代免疫抑制的发展以及围手术期护理和手术技术的优化1，2.然而，人口老龄化和对器官需求的增加导致献血者短缺，过去几十年中，来自扩展标准捐献者的边际移植物的使用增加，并出现了新的挑战。器官机器灌注的引入和广泛实施被认为为移植物修复和调节开辟了一系列可能性，并有助于缓解器官短缺和降低等待名单死亡率3，4，5，6。
为了评估这些概念及其在体内的效果，需要转化移植模型7。1983年，Kamada等人在大鼠中引入了一种有效的原位肝移植模型，该模型已被全球工作组广泛修改和应用8，9，10，11。小鼠原位肝移植模型在技术上要求更高，但在免疫可转移性方面也更有价值，并于1991年由钱等人首次报道12。尽管在可用性、动物福利和成本方面具有优势，但啮齿动物模型在临床环境中的适用性有限7。因此，需要大型动物模型。
近年来，猪由于其与人类的解剖和生理相似性，已成为用于转化研究的主要动物物种。此外，目前在异种移植领域的进展可能会进一步增加猪作为研究对象的重要性13，14。
Garnier等早在1965年就描述了猪肝移植模型15。几位作者，包括1967年的Calne等人和1971年的Chalsrey等人，随后报告了修改，最终导致了随后几十年内猪肝移植的安全可行的实验性概念16，17，18，19，20，21。
最近，不同的工作组使用猪原位肝移植技术提供了有关肝移植当前问题的数据，几乎总是包括主动或被动静脉静脉，即Porto-caval，旁路19，22。其原因是由于肠相对较大且门腔或腔腔分流较少（例如，缺乏无肝期），对下腔静脉和门静脉钳夹的物种特异性不耐受23。在人类受者中应用下腔静脉保留移植技术作为替代方案是不可行的，因为猪下腔静脉被肝组织包裹23。
然而，静脉-静脉旁路的使用进一步增加了已经要求苛刻的外科手术中的技术和后勤复杂性，因此可能会阻止工作组完全尝试实施该模型。除了搭桥术的直接生理和免疫学影响外，一些作者还指出了分流放置期间的显着发病率，例如失血或空气栓塞，以及需要同时进行脾切除术，这可能会影响植入后的短期和长期结果24，25。
以下协议描述了供体器官静态冷藏20小时后猪原位肝移植的简单技术，代表在植入过程中不使用静脉 – 静脉旁路的扩展标准供体条件，包括供体肝脏采购，背台准备，受体肝切除术和麻醉术前和术中管理。
该模型对于专注于术后即刻病程、缺血再灌注损伤、扩展标准供体器官的修复以及相关免疫机制的外科工作组应特别感兴趣。
Protocol
这项研究是在下萨克森州消费者保护和食品安全管理局（Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit [LAVES]; 19/3146）批准后在汉诺威医学院动物科学实验室进行的。 1. 供体肝脏采购 注意：肝脏供体是雌性家猪（Sus scrofa homeus），年龄为4-5个月大，平均体重约为50公斤，在手术前已经在动物研究机构隔离了至少10天。 <…
Representative Results
该方案中提出的技术在整个手术过程中的血流动力学稳定性和动物存活率以及术后移植物功能方面提供了可靠且可重复的结果。 最近，我们将该模型应用于缺血再灌注损伤的研究和治疗干预，以减轻术后立即的不利影响。取回和静态冷藏20小时后，以所述方式植入肝脏移植物（平均重量为983.38g）。在门静脉再灌注并以规定的时间间隔采样血液和胆汁以及肝脏和胆管组织采样?…
Discussion
最近的技术发展，如机器灌注的引入，有可能彻底改变肝移植领域。为了将移植物修复或修饰概念转化为临床环境，大型动物的可重复移植模型是不可避免的。
在猪原位肝移植最初引入后，在过去的五十年中，几位作者一直致力于改进这些技术。所报道的手术方法之间的差异通常很小，涉及血管和胆道吻合、麻醉和围手术期管理。尽管如此，与目前临床肝移植的情况相反，?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢Britta Trautewig，Corinna Löbbert，Astrid Dinkel和Ingrid Meder的勤奋和承诺。此外，作者感谢Tom Figiel制作图片材料。
Materials
| Abdominal retractor | No Company Name available | No Catalog Number available | |
| Aortic clamp, straight | Firma Martin | No Catalog Number available | |
| Arterial Blood Sampler Aspirator (safePICOAspirator) 1.5 mL | Radiometer Medical ApS | 956-622 | |
| Atropine (Atropinsulfat 0.5 mg/1 mL) | B.Braun | 648037 | |
| Backhaus clamp | Bernshausen | BF432 | |
| Bipolar forceps, 23 cm | SUTTER | 780222 SG | |
| Bowl 5 L, 6 L, 9 L | Chiru-Instrumente | 35-114327 | |
| Braunol Braunoderm | B.Braun | 3881059 | |
| Bulldog clamp | Aesculap | No Catalog Number available | |
| Button canula | Krauth + Timmermann GmbH | 1464LL1B | |
| Calcium gluconate (2.25 mmol/10 mL (10%)) | B.Braun | 2353745 | |
| Cell Saver (Autotransfusion Reservoir) | Fresenius Kabi AG | 9108471 | |
| Central venous catheter 7Fr., 3 Lumina, 30 cm 0.81 mm | Arrow | AD-24703 | |
| Clamp | INOX | B-17845 / BH110 / B-481 | |
| Clamp | Aesculap | AN909R | |
| Clamp, 260 mm | Fehling Instruments GMbH &Co.KG | ZAU-2 | |
| Clip Forceps, medium | Ethicon | LC207 | |
| Clip forceps, small | Ethicon | LC107 | |
| CPDA-1 solution | Fresenius Kabi AG | 41SD09AA00 | |
| Custodiol (Histidin-Tryptophan-Ketogluterat-Solution) | Dr.Franz Köhler Chemie GmbH | 2125921 | |
| Dissecting scissors | LAWTON 05-0641 | No Catalog Number available | |
| Dissecting scissors, 180 mm | Metzenbaum | BC606R | |
| Endotracheal tube 8.0 mm | Covetrus | 800764 | |
| Epinephrine (Adrenalin 1:1000) | InfectoPharm | 9508734 | |
| Falcon Tubes 50ml | Greiner | 227 261 L | |
| Femoralis clamp | Ulrich | No Catalog Number available | |
| Fentanyl 0.1mg | PanPharma | 00483 | |
| Forceps, anatomical | Martin | 12-100-20 | |
| Forceps, anatomical, 250 mm | Aesculap | BD052R | |
| Forceps, anatomical, 250 mm | Aesculap | BD032R | |
| Forceps, anatomical, 250 mm | Aesculap | BD240R | |
| Forceps, surgical | Bernshausen | BD 671 | |
| Forceps, surgical | INOX | B-1357 | |
| G40 solution | Serag Wiessner | 10755AAF | |
| Gelafundin ISO solution 40 mg/mL | B. Braun | 210257641 | |
| Guidewire with marker | Arrow | 14F21E0236 | |
| Haemostatic gauze ("Tabotamp" 5 x 7.5 cm) | Ethicon | 474273 | |
| Heparin sodium 25,000IE | Ratiopharm | W08208A | |
| Hico-Aquatherm 60 | Hospitalwerk | No Catalog Number available | |
| Infusion Set Intrafix | B.Braun | 4062981 L | |
| Intrafix SafeSet 180 cm | B.Braun | 4063000 | |
| Introcan Safety, 18 G | B.Braun | 4251679-01 | |
| Isofluran CP | CP-Pharma | No Catalog Number available | |
| Large-bore venous catheter, 7Fr. | Edwards Lifesciences | I301F7 | |
| Ligaclip, medium | Ethicon | LT200 | |
| Ligaclip, small | Ethicon | LT100 | |
| Material scissors | Martin | 11-285-23 | |
| Methylprednisolone (Urbason solubile forte 250 mg) | Sanofi | 7823704 | |
| Monopolar ERBE ICC 300 | Fa. Erbe | No Catalog Number available | |
| NaCl solution (0.9%) | Baxter | 1533 | |
| Needle holder | Aesculap | BM36 | |
| Needle holder | Aesculap | BM035R | |
| Needle holder | Aesculap | BM 67 | |
| Neutral electrode | Erbe Elektromedizin GmbH Tübingen | 21191 – 060 | |
| Norepinephrine (Sinora) | Sintetica GmbH | 04150124745717 | |
| Omniflush Sterile Filed 10 mL | B.Braun | 3133335 | |
| Original Perfusorline 300 cm | B.Braun | 21E26E8SM3 | |
| Overhold clamp | INOX | BH 959 | |
| Overhold clamp | Ulrich | CL 2911 | |
| Pentobarbital sodium(Release 500 mg/mL) | WDT, Garbsen | 21217 | |
| Perfusers | B.Braun | 49-020-031 | |
| Perfusor Syringe 50 mL | B.Braun | 8728810F | |
| Petri dishes 92 x 17 mm | Nunc | 150350 | |
| Poole Suction Instrument Argyle flexibel | Covidien, Mansfield USA | 20C150FHX | |
| Potassium chloride (7.45%) | B.Braun | 4030539078276 | |
| Pressure measurement set | Codan pvb Medical GmbH | 957179 | |
| Propofol (1%) | CP-Pharma | No Catalog Number available | |
| S-Monovette 2.6 mL K3E | Sarstedt | 04.1901 | |
| S-Monovette 2.9 mL 9NC | Sarstedt | 04.1902 | |
| S-Monovette 7.5 mL Z-Gel | Sarstedt | 11602 | |
| Sartinski clamp | Aesculap | No Catalog Number available | |
| Scalpel No.11 | Feather Safety Razor Co.LTD | 02.001.40.011 | |
| Scissors | INOX | BC 746 | |
| Seldinger Arterial catheter | Arrow | SAC-00520 | |
| Sodium bicarbonate (8.4%) | B.Braun | 212768082 | |
| Sterilization Set ("ProSet Preparation Kit CVC") | B.Braun | 4899719 | |
| Sterofundin ISO solution | B.Braun | No Catalog Number available | |
| Suction | Dahlhausen | 07.068.25.301 | |
| Suction Aesculap Securat 80 | Aesculap | No Catalog Number available | |
| Suction catheter | ConvaTec | 5365049 | |
| Sultamicillin (Unacid: 2000 mg Ampicillin/1000 mg Sulbactam) | Pfizer | DL253102 | |
| Suprapubic urinary catheter, "bronchialis", 50 cm | ConvaTec | UK 1F02772 | |
| Suprasorb ("Toptex lite RK") | Lohmann & Rauscher | 31654 | |
| Suture Vicryl 3-0 | Ethicon | VCP 1218 H | |
| Suture Vicryl 4-0 | Ethicon | V392H | |
| Suture, Prolene 4-0 | Ethicon | 7588 H | |
| Suture, Prolene 5-0, double armed | Ethicon | 8890 H | |
| Suture, Prolene 5-0, single armed | Ethicon | 8720 H | |
| Suture, Prolene 6-0, double armed | Ethicon | 7230 H | |
| Suture, Prolene 6-0, single armed | Ethicon | EH 7406 H | |
| Suture, Prolene: blau 3-0 | Ethicon | EH 7499H | |
| Suture, Safil 2/0 | Aesculap | C 1038446 | |
| Suture, Terylene 0 | Serag Wiessner | 353784 | |
| Syringe 2 mL, 5 mL, 10 mL, 20 mL | B.Braun | 4606027V | |
| TransferSet "1D/X-double" steril 330 cm | Fresenius Kabi AG | 2877101 | |
| Ultrasound Butterfly IQ+ | Butterfly Network Inc. | 850-20014 | |
| Ventilator "Oxylog Dräger Fl" | Dräger Medical AG | No Catalog Number available | |
| Yankauer Suction | Medline | RA19GMD | |
| Zoletil 100 mg/mL (50 mg Zolazepam, 50 mg tiletamin) | Virbac | 794-861794861 |
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Citar este artigo
Beetz, O., Oldhafer, F., Weigle, C. A., Cammann, S., DeTemple, D., Sieg, L., Eismann, H., Palmaers, T., Vondran, F. W. R. Porcine Liver Transplantation Without Veno-Venous Bypass As an Extended Criteria Donor Model. J. Vis. Exp. (186), e64152, doi:10.3791/64152 (2022).