Experimental animal research plays a pivotal role in the development of clinical transplantation practice. The porcine orthotopic liver transplantation model (OLTx) closely resembles human conditions and is frequently used in clinically oriented research. The following protocol contains all information for a reliable porcine OLTx model using an active porto-caval-jugular shunt.
The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation waiting list die without receiving an organ transplant or are delisted due to disease progression. Even after a successful transplantation, rejection and side effects of immunosuppression remain major concerns for graft survival and patient morbidity.
Experimental animal research has been essential to the success of liver transplantation and still plays a pivotal role in the development of clinical transplantation practice. In particular, the porcine orthotopic liver transplantation model (OLTx) is optimal for clinically oriented research for its close resemblance to human size, anatomy, and physiology.
Decompression of intestinal congestion during the anhepatic phase of porcine OLTx is important to guarantee reliable animal survival. The use of an active porto-caval-jugular shunt achieves excellent intestinal decompression. The system can be used for short-term as well as long-term survival experiments. The following protocol contains all technical information for a stable and reproducible liver transplantation model in pigs including post-operative animal care.
原位肝移植(OLTx)是治疗终末期肝病或晚期肝癌的唯一的治疗选择。在过去的25年中,考生在轮候名单上的人数逐渐增加,现在远远超过可移植的数量。在大多数移植的地区,病人轮候肝脏移植20〜30%死亡,而不会收到器官移植或摘牌是由于疾病的进展。策略,以增加供体库,因此,可移植的数量,都迫切需要。扩展标准器官分配,长时间保存移植和诱导免疫耐受仍然代表主要的临床挑战1-3。因此,实验OLTx研究是枢转,以优化临床OLTx实践。
猪OLTx是类似于人类OLTx在许多方面,包括升行之有效的实验模型艾弗大小,解剖学,生理学4-6。因此,它已成为一个标准的实验方法在研究领域,如外科手术技术,生理学,免疫学,保存,和缺血再灌注损伤。许多接枝采购,收件人肝切除,和,特别是,血管重建,技术在文献5已经描述。合适的技术的选择,根据研究人员的偏好和技术能力而变化。
与此相反的人的情况下,在无肝期内脏拥塞表示在猪OLTx一个重要问题。随后肠缺血和充血性血管损伤可引起严重的血液动力学不稳定,危及猪存活,因此,在实验7-9中的成功。因此,足够的肠减压是强制性的,尤其是在缺乏技术精制而成的实验设置。
我们荷兰国际集团积极波尔图腔静脉,颈静脉分流术无肝期的持续时间是一个可靠的选择,以避免肠道堵塞。该系统可用于早期再灌注的实验以及长期生存的情景。下面的协议中包含的所有信息建立一个稳定和可重复的肝移植模型的猪,包括供肝采购,受体手术切除包括和终端到终端的血管重建技术,和术后护理。
实验猪OLTx是没有临床情况的重症监护资源研究设定一个具有挑战性的过程。可能的并发症包括血流动力学不稳定,出血,器官缺血,低体温,和代谢,以及呼吸,代偿失调。对于任何研究组中,外科技术5以及猪麻醉14,15的足够程序训练是强制性的,以实现具有代表性和可再现的结果。
许多技术微妙之处已经在文献中进行了描述,特别是关于血管重建阶段5。上述OLTx协议提供了一个静脉-替换模型类似于人OLTx所需的信息。所提供的结果证明可靠的动物生存和移植物复苏都HBD和DCD模式。该协议适用于移植再灌注EXPE用短期生存场景riments,例如,以及在长期生存车型如公差研究。
猪OLTx的一个很大的障碍就是静脉和门静脉交叉夹紧相对耐受性差。在无肝期内脏充血引起静脉高压和毛细血管损伤甚至器官再灌注后7可能导致重大肠缺血和血液动力学不稳定的不可逆性休克的地步。由于腔静脉完全嵌入在肝实质中,静脉保背驮式程序是不可行的。下腔静脉在静脉重建阶段完全闭塞损害猪的血流动力学稳定。虽然几个报告表明,猪OLTx可以小于25分钟16,17总静脉,门静脉闭塞期间完成,血管重建时的门-静脉-颈静脉旁路技术是更安全和更实际的选择7- 9,18。在日Ë作者的经验,一个被动的波尔图,颈静脉旁路不是最佳的在无肝期,保持猪血流动力学稳定。旁路模式,包括肝下静脉和门静脉活跃的减压,使得肝上下腔静脉和门静脉吻合甚至延长阻断时间的平静重建阶段,由于不可预见的并发症。相反,以前的报告7,脾切除术是不是强制性的,当门户旁路导管被删除。无论脾动脉和静脉被关闭大约一半沿脾的长度留一半近端充分灌注。像并发症出血或空气栓塞,由于旁路断线都是可以避免通过确保旁路小心放置和固定正常。
在长期的生存OLTx实验,胆管吻合术被认为是一个薄弱点,由于其并发症发生率高19。胆道组织是很脆弱的,NEEDS特殊照顾,当被处理。许多不同的吻合技术已经描述5,19。端至端吻合术在技术上是容易的,并以最少的并发症19相关联。连续缝合与非切割针包括胆管周围结缔组织的大段似乎要优于间断缝合。胆管下放置不必要的紧张,当单针间断缝合打结是。这可能会导致组织撕裂和连续胆汁泄漏。缝合材料 – 可吸收或不可吸收的 – 通常是不重要的,因为它的寿命有限,直到猪被终止。在过去的数个月的长期生存模型,可吸收缝合线 – 就像人类OLTx – 是可取的。
具体必须注意与术后随访。充足的营养和流体供应,有可靠的疼痛缓解协议,和一个适当的免疫抑制制度是强制性的。对于长期实验中,免疫抑制显得尤为重要。相对于其他哺乳类动物,猪表明OLTx 20,21后一个令人惊讶的低免疫排斥反应率。圆形细胞浸润是在移植后的第二周最大,减少自发即使没有免疫抑制。拒绝是很少死亡猪OLTx 22后的原因。然而,即使使用免疫抑制协议涉及这里提到给予类固醇四和钙调磷酸酶抑制剂口服,移植物排斥是由OLTx之后开始在约4天转氨酶轻度增加表示,并通过表观门户字段圆形细胞浸润证实。钙调磷酸酶抑制剂可以给任何婆23,24或25,26 IV;这两种方法都有缺点。即使口服应用助剂,实际量到达胃肠道仍然难以捉摸。另一方面,持续静脉输注猪的笔有积极的动物是困难的。因此,四应用必须进行以大丸剂,这导致在高药物浓度的峰值随着潜在的毒性作用。然而,应用这两种方法出现,以允许长期生存。
类似于临床上,手术后应激性溃疡的预防建议。手术后出血从消化性溃疡是一种常见的问题,并可能与一个肝功能损害27。经过这两个团OLTx消化道出血少数情况下,作者开始定期预防与泮托拉唑,并没有遇到任何胃肠道出血至今。
严格维持在无菌条件术,可比的条件在临床手术室,以及随之而来的预防性抗生素,降低感染并发症的风险。
总之,本品Provides实用信息在研究环境建立猪OLTx程序。足够的奉献精神,实践和团队合作是为了降低学习期间,产生可靠的结果,并降低成本,研究动物的数量重要。
The authors have nothing to disclose.
The study was supported by research grants from the Roche Organ Transplant Research Foundation (ROTRF) and Astellas. Markus Selzner was supported by an ASTS Career Development Award. Matthias Knaak was supported by the Astellas Research Scholarship. We thank Uwe Mummenhoff and the Birmingham family for their generous support.
Atropine Sulphate 15mg/30mL | Rafter 8 Products | 238481 | |
Buprenorphine 0.3mg/mL | RB Pharmaceuticals LDT | N/A | |
Cefazolin 1g | Pharmaceutical Partners of Canada Inc. | 2237138 | |
Cyclosporin Oral Solution 5000mg/50mL | Novartis Pharmaceuticals Canada Inc. | 2150697 | |
Fentanyl Citrate 0.25mg/5mL | Sandoz Canada Inc. | 2240434 | |
Heparin 10,000iU/10mL | Leo Pharma A/S | 453811 | |
Isoflurane 99.9%, 250mL | Pharmaceutical Partners of Canada Inc. | 2231929 | |
Ketamine Hydrochloride 5000mg/50mL | Bimeda-MTC Animal Health Inc. | 612316 | |
Lactated Ringer’s + 5% Dextrose, 0.5L | Baxter Corporation | 61131 | |
Lacteted Ringer’s, 1L | Baxter Corporation | 61085 | |
Metronidazole 500mg/100mL | Baxter Corporation | 870420 | |
Midazolame 50mg/10mL | Pharmaceutical Partners of Canada Inc. | 2242905 | |
Pantoprazole 40mg | Sandoz Canada Inc. | 2306727 | |
Potassium Chloride 40mEq/20mL | Hospira Healthcare Corporation | 37869 | |
Propofol 1000mg/100mL | Pharmascience Inc. | 2244379 | |
Protamine Sulfate 50mg/5mL | Pharmaceutical Partners of Canada Inc. | 2139537 | |
Saline 0.9%, 1L | Baxter Corporation | 60208 | |
Sodium Bicarbonate 50 mEq/50mL | Hospira Healthcare Corporation | 261998 | |
Solu-Medrol 500mg | Pfizer Canada Inc. | 2367963 | |
Tranexamic Acid 1000mg/10mL | Pfizer Canada Inc. | 2064413 | |
University of Wisconsin Solution, SPS-1 | Organ Recovery Systms | SPS-1 | |
Xylocaine Endotracheal 10mg/50mL | AstraZeneca | 2003767 | |
Appose ULC 35W skin stapler | Covidien Canada | 803712 | |
Maxon, 1 | Covidien Canada | 606173 | |
Sofsilk, 0 | Covidien Canada | S606 | |
Sofsilk, 2-0 | Covidien Canada | S405 | |
Sofsilk, 3-0 | Covidien Canada | S404 | |
Surgipro II, 4-0 | Covidien Canada | VP581X | |
Surgipro II, 5-0 | Covidien Canada | VP725X | |
Surgipro II, 6-0 | Covidien Canada | VP733X | |
Catheter i.v, 18 G | BD Canada | 381147 | |
Cook TPN catheter, 9.5Fr | Cook Medical Company | C-TPNS-9.5-90 | |
PSI Kit for sheath catheter, 8.5Fr | Arrow International | ASK-09803-UHN | |
Infusion Pump Line | Smith Medical ASD Inc. | 21-0442-25 | |
Liver Admin Set (flush line) | CardioMed Supplies Inc | 17175 | |
Mallinckrodt, Tracheal Tube, 6.5mm | Covidien Canada | 86449 | |
Med-Rx Suction Connecting Tube | Benlan Inc. | 70-8120 | |
Organ Bag | CardioMed Supplies Inc | 2990 | |
Suction Tip | Tyco Healthcare Group LP | 8888501023 | |
Valleylab, Cautery Pencil | Covidien Canada | E2515H | |
Valleylab, Patient Return Electrode | Covidien Canada | E7507 | |
Bypass Connector 3/8” x 1/4“ | Raumedic AG | 955083-001 | |
Bypass Connector 3/8” x 3/8” Luer Lock | Raumedic AG | 955163-001 | |
Bypass Connector Y 3/8” x 3/8” x 1/4” | Raumedic AG | 961360-002 | |
Bypass Tubing 1/4” x 1/16” | Raumedic AG | 039505-010 | |
Bypass Tubing 3/8” x 3/32” | Raumedic AG | 039535-005 | |
Rotaflow Cenrtifugal Pump | Maquet-Dynamed | HC 2821 | |
Stainless Steel Hose Clamp Ring, 5mm | Oetiker | 16700007 | |
Abdominal Retractor | Medite GmbH | N/A | |
De Bakey – Beck, Infrahepatic Cava Clamp | Aesculap Inc. | FB519R | |
Diethrich, Atraumaitc Clamp (Portal Vein) | Aesculap Inc. | FB525R | |
Gregory Bull Dog Clamp, curved | Aesculap Inc. | FB382R | |
Gregory Bull Dog Clamp, straight | Aesculap Inc. | FB381R | |
Potts – De Martel, Scissors | Aesculap Inc. | BC648R | |
Satinsky, Suprahepatic Cava Clamp | Aesculap Inc. | FB605R | |
Symetrical Tubing Clamp | Codman Instruments | 198010 | |
Anesthesia Machine, Optimax | Moduflex Anesthesia Equipment | SN5180 | |
Bypass Flow meter, HT 110 | Transonic Systems Inc. | HT110B11106 | |
Flow meter probe, H6XL | Transonic Systems Inc. | H6Xl689 | |
Heat Therapy Pump, T/Pump | Gaymar Industries Inc | TP500-G89D19 | |
Infusion Pump 3000 | SIMS Graseby LTD. | SN300050447 | |
Isoflurane Vapor 19.1 | Draeger Medical Canada Inc. | N/A | |
Monitor, Datex AS 3 | Instrumentarium Corp./ Hitachi | D-VHC14-23-02 | |
Rotaflow Centrifugal Drive Unit | Marquet-Dynamed | 952301 | |
Rotaflow Console | Marquet-Dynamed | 706035 | |
Temperature Therapy Pad | Gaymar Industries Inc | TP26E | |
Valleylab Force Tx | Valleylab Inc. | 216151480 | |
Ventilator, AV 800 | DRE Medical Equipment | 40800AVV | |
Warm Touch, Patient Warming System | Nellcor/ Covidien Canada | 5015300A |