肝分型与门静脉结扎大鼠分期肝切除 (ALPPS) 手术模型的建立
Summary
提出了联合肝分型和门静脉结扎术 (ALPPS) 诱导肝脏快速肥大切除交界性切除肝肿瘤的方法。该模型可以阐明参与快速肥大的机制, 并允许对促进或阻止再生加速的药物进行检测。
Abstract
最近的临床数据支持对原发性和转移性肝肿瘤采取积极的手术方法。对于某些适应症, 如结直肠癌肝转移, 肝切除后留下的肝组织数量已成为大、多肝肿瘤可切除性的主要限制因素。为了避免 post-hepatectomy 肝衰竭的严重并发症, 需要少量的功能性组织, 发病率和死亡率都很高。在肝外科手术中, 以门静脉栓塞的形式, 或以门静脉结扎术前几周的形式, 在肝脏切除术前, 诱导肝脏的生长变得更加成熟。最近, 肝脏再生是更广泛和快速的, 当实质横断被增加到门静脉结扎在第一阶段然后, 在仅一个星期等待之后, 切除在第二个阶段执行 (关联肝脏分割和门静脉结扎分期切除 = ALPPS)。ALPPS 在世界范围内迅速普及, 但因其围手术期死亡率高而备受诟病。这一程序引起的加速和广泛增长的机制还没有得到很好的理解。建立了动物模型, 探讨了 ALPPS 加速肝再生的生理和分子机制。该协议提出了一个大鼠模型, 允许机械探索加速再生。
Introduction
肝脏残余物的大小限制了肝脏肿瘤的可切除性。1一般来说, 当少于25% 的肝脏组织被留下时, 患者由于缺乏新陈代谢的作用为整个有机体 (“太小为大小综合症状), 增加死亡的风险从急性肝功能衰竭。2此 post-hepatectomy 肝衰竭是肝切除术后最具破坏性的并发症。因此, 临床医生试图通过操纵门静脉的流动, 在肝切除前诱导肝再生。3发现, 一旦门静脉被阻塞, 门静脉流的剩余部分开始以较慢的速度生长, 从而可以增加到60% 的大小。4手术结扎5或介入性门静脉闭塞均已临床建立。4肝脏体积和功能的增加是可靠的, 但肝门闭塞后肝脏的生长率只有 1/5, 与部分肝切除后残肝的生长情况比较。6
肝脏生长所需的时间是几周到几个月, 即使肝脏可以在切除后以更快的速度再生。因此, 肝脏是唯一的器官, 生长回正常功能后, 删除的一部分。7一个新的程序, 诱导肝脏再生的速度与部分 hepactectomy 后, 由一组医生发现, 增加横断之间的闭塞和 non-occluded 部分肝脏诱导肝肥大与肝切除后相同的生长速率, 但在切除前。9该过程在未来一周内启动快速肥大的80% 的肝脏残余物, 允许切除广泛的, 主要不能切除的, 肝脏肿瘤在一周内。这一过程被称为 “结合肝分割和门静脉结扎术分期切除 = ALPPS”, 并迅速风靡世界各地。10多个报告支持新技术实现的边缘切除肝肿瘤可切除性的扩展,11而复杂的外科手术也因其高并发症率而受到批评。12,13
自 ALPPS 于2012年出版以来, 一直试图发展一种啮齿动物和大型动物模型, 以使其能够更好地鉴定和了解这些机制, 并测试药物对动物肝脏组织的不同生长速率。开发的第一个动物模型是老鼠模型。在该模型中, 左、右正中叶实质横断后快速肥大, 右中叶加速再生。14稍后在鼠标中引入了不同的模型。在这个模型中, 左外侧叶切除, 门静脉分支到肝脏的每一个肺叶, 除了左中位叶被捆绑。15同时, 还描述了猪 ALPPS 的大型动物模型。16
为研究肝门静脉血流变化和压力、肝脏组织灌注和氧合等生理机制, 大鼠模型优于小鼠 ALPPS 模型。老鼠模型的另一个优点是, 在大鼠模型中, 没有必要切除左外侧叶,15可能会污染肝切除与 ALPPS 的影响。大鼠模型的对比并不能降低肝细胞的质量。猪的模型使用右后叶作为生长中的叶, 但猪肝是高度叶的。因此, 在右后、右前叶之间的已经薄的组织桥上建立一个横断平面是很困难的。相比之下, 大鼠的中位叶由两部分组成, 分别由门静脉提供, 并且在两者之间使用显微外科技术可以很容易地形成实质横断平面。小动物计算机断层扫描 (CT) 和/或磁共振成像 (MRI) 的可用性使得门静脉结扎与门静脉结扎和附加横断之间的容积增长非常精确, 这一点很重要以验证任何快速肝肥大模型。
这里介绍的协议描述了用于容积验证的手术技术和程序, 以及门静脉结扎和门静脉结扎术后慢、快速肥大模型的生理特性,分别在大鼠。
Protocol
本议定书的所有实验均由瑞士苏黎世的兽医当局批准 (编号 60/2014)。此外, 所有实验步骤都严格遵照瑞士医学科学院 (sam) 的《动物实验指南》和欧洲实验动物科学协会联合会 (FELASA) 的指导方针进行。. 1. 畜牧, 手术室设备和器械, 麻醉 在标准无病原体条件下, 在12/12 小时的光照/暗循环下, 在通风笼中保持雄性大鼠体重250-300 克。在22±1° c 的环境温度下, 让动物免费获得食…
Representative Results
两种不同的手术方法门静脉结扎 (PVL) 和 PVL 与横断 (PVL + T) 导致明显不同的生长动力学。PVL 在3天内会导致适度的体积增大, 而在 PVL + T 中可以看到更大的右中位叶 (RML) (图 5)。这可以通过每日容量来验证。RML 的体积大约在3天内的 PVL 倍, 而它在 PVL + T.17 流量测量在门静脉显露一个稳定的?…
Discussion
本议定书提出了一个动物模型的 ALPPS 与其快速肥大诱导的 PVL + T, 大约双打增加3天内的体积比单独 PVL。17右中肝叶被用作生长中的肝叶的模型, 因为中间的肝叶是由两个独立的门静脉向其左侧和右侧提供的一个连续的实质质量, 如图 1所示, 最近已发布的工作。17与其他报表相比, 该模型具有一些优点。从解剖学上来说, 选择中叶最能代表人?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者没有致谢。
Materials
| Isoflurane, 250ml bottles | Attane, Piramal, Mumbai, India | LDNI 22098 | Standard vet. equipment |
| Tec-3 Isofluorane Vaporizer | Ohmeda, GE-Healthcare, Chicago, IL | not available anymore | Standard vet. equipment |
| Buprenorphine (Temgesic) | Indivior, Baar, Switzerland | 7680419310353 | GTIN-number |
| Vitamine A ointment | Bausch&Lomp, Zug, Switzerland | 7680223980247 | GTIN-number |
| Atropine sulfate 0.5mg/ml | Sintetica SA, Mendrisio, Switzerland | 7680565330045 | GTIN-number |
| Microsurgery microscope | Olympus, Tokio, Japan | SZX10 | Standard vet. equipment |
| Betadine | Mundipharma, Basel, Switzerland | 7680342821377 | GTIN-number |
| Sponges | Carl Roth GmbH, Karlsruhe, Germany | NK83.1 | Mini-sponges |
| Abdominal Wall retractors | N/A | N/A | Self-made from paper clips and Q-Tips |
| 3-0 silk | Ethicon, Sommerville, NJ | K872H | Standard surgical |
| Scissors | World precision instruments (WPI), Sarasota, FL | 503371 | Standard microsurgical |
| Adson forceps | World precision instruments (WPI), Sarasota, FL | 501244-G | Standard microsurgical |
| Fine tips microforceps | World precision instruments (WPI), Sarasota, FL | 501976 | Tips need to be polished regularly |
| Curved fine tips microforceps | World precision instruments (WPI), Sarasota, FL | 504513 | Essential to go around the portal vein branches |
| 6-0 LOOK black braided silk | Surgical Specalities Corporation, Wyomissing, PA | SP114 | Spool, precut prior to the procedure |
| 2-0 silk sutures | Ethicon, Sommerville, NJ | K833 | Standard surgical |
| 5-0 maxon sutures | Covidien, Dublin, Ireland | 6608-21 | Standard surgical |
| Bipolar microforceps | Sutter, Freiburg, Germany | 780148SGS | Essential for parenchymal transection |
| Q-tips small | Carl Roth GmbH, Karlsruhe, Germany | EH11.1 | Standard surgical |
| Q-tips big | Carl Roth GmbH, Karlsruhe, Germany | XL54.1 | Standard surgical |
| G30 needle | Terumo, Tokyo, Japan | NN-3013R | Standard anesthesia equipment |
| 2mm volume flow probe | Transonic Systems, Ithaca, NY | MA-2PS | Smallest available probe for HAT-311 flow meter |
| Transonic flow meter | Transonic Systems, Ithaca, NY | HAT-311 Transsonic flow QC meter | One of the first generation flow flow meters for surgery |
| ExiTron nano 12,000 | Miltenyi Biotech, Bergisch Gladbach, Germany | 130-095-698 | Nanomoloecular contrast medium that opacifies liver and spleen |
| G26 intravenous catheter | Becton Dickinson, Franklin Lakes, NJ | 391349 | Standard anesthesia equipment |
| Quantum FX MicroCT | Perkin Elmer, Waltham, MA | N/A | Standard small animal CT scanner at the institute of physiology, University of Zürich |
| OsiriX 8.0 | Pixmeo Sarl, Geneva, Switzerland | N/A | Public domain software : www.pixmeo.com |
Referências
- She, W. H., Chok, K. Strategies to increase the resectability of hepatocellular carcinoma. World J Hepatol. 7 (18), 2147-2154 (2015).
- Vauthey, J. N., et al. Standardized measurement of the future liver remnant prior to extended liver resection: methodology and clinical associations. Surgery. 127 (5), 512-519 (2000).
- Kinoshita, H., et al. Preoperative portal vein embolization for hepatocellular carcinoma. World J Surg. 10 (5), 803-808 (1986).
- van Lienden, K. P., et al. Portal Vein Embolization Before Liver Resection: A Systematic Review. Cardiovasc Intervent Radiol. , (2012).
- Kianmanesh, R., et al. Right portal vein ligation: a new planned two-step all-surgical approach for complete resection of primary gastrointestinal tumors with multiple bilateral liver metastases. J Am Coll Surg. 197 (1), 164-170 (2003).
- Nadalin, S., et al. Volumetric and functional recovery of the liver after right hepatectomy for living donation. Liver Transpl. 10 (8), 1024-1029 (2004).
- Michalopoulos, G. K., DeFrances, M. C. Liver regeneration. Science. 276 (5309), 60-66 (1997).
- Fulop, A., et al. Alterations in hepatic lobar function in regenerating rat liver. J Surg Res. 197 (2), 307-317 (2015).
- Schnitzbauer, A. A., et al. Right portal vein ligation combined with in situ splitting induces rapid left lateral liver lobe hypertrophy enabling 2-staged extended right hepatic resection in small-for-size settings. Ann Surg. 255 (3), 405-414 (2012).
- de Santibanes, E., Clavien, P. A. Playing Play-Doh to prevent postoperative liver failure: the "ALPPS" approach. Ann Surg. 255 (3), 415-417 (2012).
- Schadde, E., et al. Monosegment ALPPS hepatectomy: extending resectability by rapid hypertrophy. Surgery. 157 (4), 676-689 (2015).
- Dokmak, S., Belghiti, J. Which limits to the "ALPPS" approach?. Ann Surg. 256 (3), e6 (2012).
- Aloia, T. A., Vauthey, J. N. Associating liver partition and portal vein ligation for staged hepatectomy (ALPPS): what is gained and what is lost?. Ann Surg. 256 (3), e9 (2012).
- Yao, L., et al. Establishment of a rat model of portal vein ligation combined with in situ splitting. PLoS One. 9 (8), e105511 (2014).
- Schlegel, A., et al. ALPPS: from human to mice highlighting accelerated and novel mechanisms of liver regeneration. Ann Surg. 260 (5), 839-846 (2014).
- Croome, K. P., et al. Characterization of a porcine model for associating liver partition and portal vein ligation for a staged hepatectomy. HPB (Oxford). 17 (12), 1130-1136 (2015).
- Schadde, E., et al. Hypoxia of the growing liver accelerates regeneration. Surgery. 161 (3), 666-679 (2017).
- Moris, D., et al. Mechanistic insights of rapid liver regeneration after associating liver partition and portal vein ligation for stage hepatectomy. World J Gastroenterol. 22 (33), 7613-7624 (2016).
- Garcia-Perez, R., et al. Associated Liver Partition and Portal Vein Ligation (ALPPS) vs Selective Portal Vein Ligation (PVL) for Staged Hepatectomy in a Rat Model. Similar Regenerative Response?. PLoS One. 10 (12), e0144096 (2015).
- Shi, H., et al. A preliminary study of ALPPS procedure in a rat model. Sci Rep. 5, 17567 (2015).
- Almau Trenard, H. M., et al. Development of an experimental model of portal vein ligation associated with parenchymal transection (ALPPS) in rats. Cir Esp. 92 (10), 676-681 (2014).
- Dhar, D. K., Mohammad, G. H., Vyas, S., Broering, D. C., Malago, M. A novel rat model of liver regeneration: possible role of cytokine induced neutrophil chemoattractant-1 in augmented liver regeneration. Ann Surg Innov Res. 9, 11 (2015).
- Wei, W., et al. Establishment of a rat model: Associating liver partition with portal vein ligation for staged hepatectomy. Surgery. 159 (5), 1299-1307 (2016).
- Tschuor, C., et al. Salvage parenchymal liver transection for patients with insufficient volume increase after portal vein occlusion – an extension of the ALPPS approach. Eur J Surg Oncol. 39 (11), 1230-1235 (2013).
- Schadde, E., et al. Early survival and safety of ALPPS: first report of the International ALPPS Registry. Ann Surg. 260 (5), 829-836 (2014).
- Harnoss, J. M., et al. Prolyl Hydroxylase Inhibition Enhances Liver Regeneration Without Induction of Tumor Growth. Ann Surg. , (2016).
- Olthof, P. B., et al. Comparable liver function and volume increase after portal vein embolization in rabbits and humans. Surgery. 161 (3), 658-665 (2017).
- Olthof, P. B., van Gulik, T. M., Bennink, R. J. Optimal use of hepatobiliary scintigraphy before liver resection. HPB (Oxford). 18 (10), 870 (2016).
- Lau, L., Christophi, C., Muralidharan, V. Intraoperative functional liver remnant assessment with indocyanine green clearance: another toehold for climbing the "ALPPS". Ann Surg. 261 (2), e43-e45 (2015).
- Cieslak, K. P., et al. Assessment of Liver Function Using (99m)Tc-Mebrofenin Hepatobiliary Scintigraphy in ALPPS (Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy). Case Rep Gastroenterol. 9 (3), 353-360 (2015).
- Truant, S., et al. Drop of Total Liver Function in the Interstages of the New Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy Technique: Analysis of the "Auxiliary Liver" by HIDA Scintigraphy. Ann Surg. 263 (3), e33-e34 (2016).
Citar este artigo
Schadde, E., Hertl, M., Breitenstein, S., Beck-Schimmer, B., Schläpfer, M. Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) Procedure. J. Vis. Exp. (126), e55895, doi:10.3791/55895 (2017).