静脉大鼠部分肝移植的体内血管定向70% 肝切除术
1Department of Surgery,The First Affiliated Hospital of Wenzhou Medical University, 2Reproductive Center,The First Affiliated Hospital of Wenzhou Medical University, 3Department of Transplantation,The First Affiliated Hospital of Wenzhou Medical University, 4Department of Surgery,Perelman School of Medicine at the University of Pennsylvania
Summary
本文提出了一种静脉大鼠部分肝移植的协议。具体地说, 70% 例肝切除术是通过使用一种最新的血管性肝切除技术在体内进行的。肝动脉以端对侧的方式重建。对袖套技术进行了改进, 缩短了 infrahepatic 腔静脉的吻合时间。
Abstract
在临床上发展了肝移植和活体肝供肝移植, 以更有效地利用肝脏器官。为更好地了解这些手术过程的机制, 建立了大鼠部分肝移植 (PLTx) 模型进行相关的外科研究。由于大鼠 PLTx 模型的复杂性, 需要一个具有详细描述的协议。此前发表的一篇文章报告了一项协议, 其中前体肝切除术用于实现50% 大鼠 PLTx。与此协议相比, 我们引入了一个 PLTx体内70% 肝切除的重新静脉。以血管为导向的肝切除术被纳入大鼠 PLTx, 以改进显微外科手术。肝实质切除前, 左外侧叶和正中叶的门静脉和肝动脉分别进行解剖和结扎, 从而降低残肝残端出血的几率。此外, 还介绍了肝动脉与肝动脉扩张的端到端血管吻合术, 以重新 arterialize 肝脏动脉。采用端到端血管吻合技术, 扩大了吻合口的直径, 降低了手缝的难度, 保持了较高的吻合通畅率。此外, infrahepatic 腔静脉的袖套吻合略有改变。在袖状吻合术期间, 在接受者的腔静脉周围有一节环性肝实质, 以维持血管腔的三维形状。这部分肝实质在完成吻合术后被切除。经此修改, 省略了放置缝合线的步骤, 从而进一步缩短了袖套吻合时间。采用本协议对大鼠 PLTx, 在显微外科手术后实现了低肝酶水平、完整的肝小叶结构和高存活率。
Introduction
目前, 捐赠肝脏的数量和等待捐献肝脏的病人数量之间存在很大的差异。肝脏器官的短缺是一个全球性的问题。为了扩大捐献者的水池, 分裂肝移植 (LTx) 和活体捐献者 LTx 被开发用于使用部分肝脏作为移植物1。
为进一步研究部分肝移植 (PLTx) 的机制, 建立了相关动物模型2,3,4,5。在大鼠 PLTx 中, 肝叶切除体内和前体, 以模拟活体捐献者 LTx 的条件, 并分别在人类体内分裂 LTx。在可视化实验杂志上发表的一篇论文提出了一个详细的协议, 涉及50% 只老鼠 PLTx 使用前体肝切除5。然而, 在可视化文献中还没有报道一只老鼠 PLTx 与体内肝切除术有关。
除了体内和前体hepatectomies 之间的差异外, 执行肝切除术本身也在确定 PLTx 的结果方面起着重要作用. 目前, 在许多外科研究中使用大鼠 PTLx模型, 肝叶切除后, 放置一个简单的结扎在肝瓣的椎弓根2,3,6,7,8,9。然而, 在切除前放置一个简单的结扎不适合所有肝叶, 因为不同的肝叶有不同的形状和大小。在正中裂片的基础上进行简单结扎, 有很高的风险导致腔静脉收缩, 这可能最终影响部分肝移植的流出10,11。因此, 在大鼠 PTLx 的研究中, 需要以大鼠肝脏解剖知识为基础的更新肝切除技术。
在本研究中所述的协议中, 将最新的以血管为导向的70% 肝切除术纳入大鼠 PLTx 的程序中。在肝实质切除前分别对左外侧叶 (微光) 和正中叶 (ML) 的门静脉和肝动脉进行解剖和分切。然后用穿刺缝线结扎了微光和 ML 的肝静脉。通过使用单独的结扎和多重穿刺缝合, 而不是简单的结扎, 毫升的残余树桩能够扩散到腔静脉。因此, 避免了手术结扎引起的 infrahepatic 腔静脉的收缩。另外, 在切除肝实质之前, 单独结扎对微光和 ML 血供的阻断减少了残肝残端的出血率, 从而最大限度地减少了血液损失对实验的影响11。
对于 microsurgeons, 由于该血管直径极小, 重建肝移植肝动脉 (PHA) 是一项重大的挑战。虽然在 LTx 中重新动脉是否真的是必要的问题仍在辩论中12,13,14, 已经提出了许多重建肝动脉的显微外科技术14.本文介绍了一种新的肝移植动脉技术, 它以端到端的方式将普通肝动脉 (CHA) 与扩大后的医用血管吻合。使用这种端对端技术, 两个肝动脉连接到一个更大的直径吻合。吻合口直径越大, 缝合越容易, 吻合通畅的改善越大。
Protocol
所有的程序都遵循了温州医科大学动物政策和福利委员会批准的《啮齿动物外科指南》 15。 1. 动物 用雄性 Lewis 老鼠称250-350 克作为捐献者和受体。 2. 操作环境 在手术显微镜下执行所有显微外科手术, 放大率从7X 到20X 不等。手术前要消毒所有外科器械。注意: 操作室和操作表清洁, 但不消毒。 <p class="jove_titl…
Representative Results
共完成31例自体静脉大鼠 PLTx 的应用本协议。所有收件人都存活到观察时间结束为止。患者的体重在术后一天 (POD) 4 开始恢复。在 POD 6 (图 8) 之后, 体重的斜率接近正常的 Lewis 鼠, 这间接地暗示了接收方的恢复。组织学上, 接受者观察到胆管的轻微增生, 接受者的肝小叶结构完好。没有发现坏死或明显的正弦扩张 (图 9)。血清丙氨…
Discussion
大鼠 PLTx 是一种复杂的显微外科手术, 其培训计划成本高且持续时间为20。大鼠 PLTx 协议的复杂性使得研究人员无法使用这种动物模型。与全尺寸大鼠 LTx 相比, 大鼠 PLTx 的 microsurgeon 不仅与移植过程的挑战有关, 而且与小动物肝脏切除的挑战有关。因此, 详细描述整个过程的可视化显微外科协议是建立这一复杂模型的必要条件。Nagai et所报告的先前可视化的文章提出了一项50%…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究获得中国国家自然科学基金 (81501382 号赠款) 和浙江省自然科学基金 (批准号:) 的支持。LQ13H100003 和 LQ16H100002)。
Materials
| Surgical microscope | Möller-Wedel, Germany | 654359 | |
| Light source | OSRAM (China) Lighting Co., Ltd | 64627 | |
| Isoflurane Vaporizer | MIDMARK Corporation | VIP3000 | |
| Isoflurane | Baxter Healthcare Corporation | 40032609 | For Inhalation anesthesia |
| Normal Saline | Zhejiang Tianrui Pharmaceutical Co., Ltd. | 716092103 | |
| Povidone Iodine Solution | HANGZHOU MINSHENG PHARMACEUTICAL | H33021567 | For disinfection |
| Hemostatic forceps | Shanghai Medical Instruments(Group) Ltd | J31020 | |
| Surgical scissors | Shanghai Medical Instruments(Group) Ltd | JC2116 | |
| Needle-holder | Shanghai Medical Instruments(Group) Ltd | J32030 | |
| Dressing Forceps | Shanghai Medical Instruments(Group) Ltd | J42020 | |
| Mosquito hemostatic forceps | Shanghai Medical Instruments(Group) Ltd | W40340 | For liver resection; For retracting the stay sutures. |
| Micro-scissors | Shanghai Medical Instruments(Group) Ltd | WA1040 | |
| Micro needle-holder | Shanghai Medical Instruments(Group) Ltd | WA2060 | |
| Curved micro typing forceps | Shanghai Medical Instruments(Group) Ltd | WA3061 | |
| straight micro typing forceps | Shanghai Medical Instruments(Group) Ltd | WA3060 | |
| Micro hemostatic forceps | Shanghai Medical Instruments(Group) Ltd | WA3020 | |
| Bulldog clamp | Shanghai Medical Instruments(Group) Ltd | XEC350 | |
| Vessel clamps | Shanghai Medical Instruments(Group) Ltd | W40160 | |
| Micro Serrefine-curved | Fine Science Tools, Inc | 18055-05 | |
| Dacryosyrinx | Shi Zhuo medical instruments Co., Ltd. | 5# curve | Use as the curved needle for forcing out the air bubbles in the vascular lumen |
| Sterilized gauze swabs | Fuqing Health & Integral Medical | 20230R | |
| 5mL syringe | Zhejiang Yusheng Medical Instrument Co.,Ltd | 811932416 | |
| 18G needles | Shanghai Kindly Enterprise Development Group Co., Ltd | 01-014 | For fixing the upper abdomen wall |
| Intima II 22G intraveous catheter | Becton Dickinson Medical Devices (Shanghai) Co Ltd. | 383207y | For making biliary stent; For infusing perfusate |
| 7-0 polypropylene sutures | Ningbo Medical Needle Co., Ltd | 151026 | For SHVC anastomsis |
| 6-0 silk suture | Shanghai Pudong Jinhuan Medical Products Co., Ltd | 2650182 | For ligation |
| Culture dish (100mm) | Corning Incorporated | 430165 | used in back table for storage ice. The back table dish was placed on it. |
| Culture dish (30 mm) | Corning Incorporated | 3296 | As a vessel for organ preservation |
| Angiocath 12G intravenous catheter | Becton Dickinson Medical Devices (Shanghai) Co Ltd. | 382277 | For making a IHVC cuff |
| Angiocath 14G intravenous catheter | Becton Dickinson Medical Devices (Shanghai) Co Ltd. | 382269 | For making a PV cuff |
| Buprenorphine (hydrochloride) | Tianjin Institute of Pharmaceutical Research Pharmaceutical Co., Ltd | H12020275 | For analgesic |
| Cefuroxime sodium for injection | Esseti FarmaceuticiS.r.l | H20160013 | As an antibiotics |
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Cite This Article
Chen, X., Yu, R., Xu, Z., Zhang , Y., Liu, C., Chen, B., Jin, H. Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy. J. Vis. Exp. (134), e56392, doi:10.3791/56392 (2018).