小鼠手术模型中扩展 78% 肝切除术
Summary
部分 2/3 （66%） 肝切除术的小鼠模型在文献中有很好的描述，但很少使用更扩展的肝切除术，模仿肝移植后的小尺寸综合征。我们描述了在小鼠模型中延长的 78% 肝切除术程序，该手术在健康小鼠中导致约 50% 的术后致死率。
Abstract
小鼠部分 2/3 肝切除术用于研究肝脏的再生能力，并探索肝切除术在许多疾病模型中的结果。在小鼠经典的 2/3 部分肝切除术中，五个肝叶中的两个，即左叶和正中叶，约占肝脏质量的 66%，被 整体 切除，预期术后存活率为 100%。更具侵略性的部分肝切除术在技术上更具挑战性，因此很少用于小鼠。我们小组开发了一种扩展肝切除术技术的小鼠模型，其中五个肝叶中的三个，包括左、正中和右上叶，分别切除约占肝脏总质量的 78%。在其他方面健康的小鼠中，这种延长的切除术会留下残余的肝脏，不能总是维持充分和及时的再生。由于暴发性肝衰竭，未能再生最终导致 1 周内术后 50% 的致死率。这种在小鼠中延长 78% 肝切除术的手术代表了一种独特的手术模型，用于研究小尺寸综合征和评估治疗策略，以改善肝移植或癌症延长肝切除术中的肝脏再生和结果。
Introduction
小鼠和大鼠手术肝切除模型于 1931 年首次描述，是用于研究肝脏再生分子基础的最常见实验模型。它们还可用于转化科学研究，以测试和制定策略，以改善延长肝切除术或次优肝移植物移植后的结果 1,2,3,4。小鼠部分肝切除术 （PH） 需要切除约 2/3 （66%） 的肝质量总量 （TLM），当在健康动物中进行时，该手术具有特殊的结果5。该过程持续时间短，由于小鼠肝脏解剖结构变化不大，因此易于复制，术后存活率通常接近 100%1。
部分 2/3 肝切除术包括左叶 （LL） 和正中叶 （ML） 切除术，可使残叶相对不受肺叶炎症或肝脏流入和流出限制的阻碍再生。相反，PH 后门静脉血流增加和随后对肝窦内皮细胞的剪切应力导致内皮一氧化氮合酶 （eNOS） 表达持续上调和随后的一氧化氮 （NO） 释放，这有助于肝细胞启动增殖和肝脏再生3。在疾病模型（如非酒精性脂肪性肝病）或特定遗传背景中，通常在 2/3 PH 后研究的结果包括急性肝衰竭的风险、肝脏再生能力的定性和定量测量，以及对压力或创伤性损伤的其他生物学反应 1,3。
然而，模仿功能性或解剖学上较小尺寸综合征的小鼠模型，因为它发生在癌症扩展肝切除术或边缘性（脂肪变性或长时间缺血时间）或部分（分裂或来自活体供体肝脏）肝移植物之后，仍有待充分建立。为了满足这一需求，需要更广泛的肝切除模型，这些模型超越了维持最小（和功能性）肝脏质量，以模拟小于大小的肝脏综合征以及与该综合征相关的高死亡率6,7。
小鼠肝脏解剖结构表现出最小的变异。小鼠肝脏由五个肺叶组成，每个肺叶占肝脏总质量的百分比：左叶（LL;34.4±1.9%）、中叶（ML;26.2±1.9%）、右上叶（也称为右上叶）叶（RUL;16.6±1.4%）、右下叶（也称为右下叶）（RLL;14.7±1.4%）和尾状叶（CL;8.1±1.0%）1，5.每个肺叶由一个门静脉三联征供血，包括肝动脉的一个分支、门静脉的一个分支和胆管5。从历史上看，有几种技术被描述为通过切除 LL 和 ML 来执行 2/3 PH。这些包括 1） 经典技术，该技术由每个切除叶的基部的单个结扎线组成;2）止血夹技术，使用钛夹应用于切除叶的基部;3）一种以血管为导向的薄壁组织保留技术，在钳夹近端使用穿刺缝合线;4） 一种以血管为导向的显微外科技术，即在肺叶切除术1 之前结扎门静脉和肝动脉分支。虽然每种技术都有相对的优点和缺点，但没有一种技术能产生更高的致死率1,8,9。
在这项研究中，我们提出了一种在小鼠中延长 78% PH 值的新方法。在该模型中，五个肝叶中的三个，包括LL，ML和RUL，使用结扎技术分别切除（图1）。该手术切除约占肝脏总质量的 78%（77.2 ± 5.2%）。我们选择单独切除 LL 和 ML，而不是像经典 PH 技术那样“整体”切除，这样可以最大限度地减少与这两个叶整体切除相关的并发症，例如肝上腔静脉狭窄和当单结扎线太靠近腔静脉时，剩余叶坏死的风险增加1， 10,11,12,13,14.在进入此过程的最后一步以删除 RUL 之前，这一点至关重要。这种在 8-12 周龄野生型 C57BL/6 小鼠中的广泛肝切除术在手术后 1 周内引起 50% 的致死率，原因是肝脏再生失败导致暴发性肝衰竭15,16。这种在延长 78% 肝切除术后致死率升高的小鼠模型适当地概括了小尺寸综合征的病理生理学，并能够开发和测试新的策略以改善结果。
Protocol
Representative Results
Discussion
为了成功进行延长的 78% 肝切除术，导致小鼠 50% 的致死率，精确切除每个肝叶至关重要。只有重复执行该程序，才能达到这种能力和精确度水平。培训曲线因操作员而异，但通常需要 3-6 个月的实践。切除少于 78% TLM 的肝切除术将产生更高的存活率，而切除超过 78% TLM 的肝切除术将导致更高的致死率。每个肺叶切除术都具有挑战性，尽管程度不同。
左肺叶是最容易可靠切除?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了 NIH R01 赠款的支持，DK063275和 CF HL086741。 PB 和 TA 是 NHLBI T32 培训赠款 HL007734 的 NRSA 奖学金的获得者。
Materials
| 2 x 2 Gauze | Covidien | 2146 | Surgery: dissection |
| 5-O Nylon Monofilament Suture | Oasis | 50-118-0631 | Surgery: Skin closure |
| 5-O Silk Suture | Fine Science Tools | 18020-50 | Surgery: liver lobe ligation |
| 5-O Vicryl Suture | Ethicon | NC9335902 | Surgery: Abdominal wall closure |
| Addson Forceps | Braintree Scientific | FC028 | Surgery: dissection |
| Alcohol Swabs (2) | BD | 326895 | Disinfectant |
| Buprenorphine Extended Release Formulation | Zoopharm | N/A | Analgesia |
| Cordless Trimmer | Braintree Scientific | CLP-9868-14 | Shaving |
| Curved Forceps | Braintree Scientific | FC0038 | Surgery: dissection |
| Hemostat | Braintree Scientific | FC79-1 | Surgery: dissection |
| Isoflurane Inhalant Anesthetic | Patterson Veterinary | RXISO-250 | General Anesthesia |
| Magnet Fixator (2-slot) (2) | Braintree Scientific | ACD-001 | Surgery: to hold small retractors |
| Magnet Fixator (4-slot) | Braintree Scientific | ACD-002 | Surgery: to hold small retractors |
| Microscissors | Braintree Scientific | SC-MI 151 | Surgery: dissection |
| Operating tray | Braintree Scientific | ACD-0014 | Surgery: for establishment of surgical field |
| Povidone Iodine 10% Swabstick (2) | Medline | MDS093901ZZ | Disinfectant |
| Scalpel (15-blade) | Aspen Surgical Products | 371615 | Surgery: dissection |
| Sharp Scissors (Curved) | Braintree Scientific | SC-T-406 | Surgery: dissection |
| Sharp Scissors (Straight) | Braintree Scientific | SC-T-405 | Surgery: dissection |
| Small Cotton-Tipped Applicators | Fisher Scientific | 23-400-118 | Surgery: dissection |
| Tissue Forceps (Straight x2) | Braintree Scientific | FC1001 | Surgery: dissection |
| Warming Pad (18" x 26") | Stryker | TP 700 | Warming |
| Warming Pad Pump | Stryker | TP 700 | Warming |
| Wire Handle Retractor (2) | Braintree Scientific | ACD-005 | Surgery: to facilitate exposure of peritoneal cavity |
| Xenotec Isoflurane Small Animal Anesthesia System | Braintree Scientific | EZ-108SA | General Anesthesia: Contains Isoflurane vaborizer & console, Induction chamber, Regulator/Hose, Facemask (M) |
References
- Martins, P. N., Theruvath, T. P., Neuhaus, P. Rodent models of partial hepatectomies. Liver Int. 28 (1), 3-11 (2008).
- Higgins, G., Anderson, R. Experimental pathology of the liver I. Restoration of the liver of the white rat following partial surgical removal. Arch Pathol. 12, 186-202 (1931).
- Koniaris, L. G., McKillop, I. H., Schwartz, S. I., Zimmers, T. A. Liver regeneration. J Am Coll Surg. 197 (4), 634-659 (2003).
- Fausto, N., Campbell, J. S., Riehle, K. J. Liver regeneration. Hepatology. 43 (2), S45-S53 (2006).
- Inderbitzin, D., et al. Magnetic resonance imaging provides accurate and precise volume determination of the regenerating mouse liver. J Gastrointest Surg. 8 (7), 806-811 (2004).
- Clavien, P. A., et al. What is critical for liver surgery and partial liver transplantation: size or quality. Hepatology. 52 (2), 715-729 (2010).
- Dahm, F., Georgiev, P., Clavien, P. A. Small-for-size syndrome after partial liver transplantation: definition, mechanisms of disease and clinical implications. Am J Transplant. 5 (11), 2605-2610 (2005).
- Hori, T., et al. Simple and reproducible hepatectomy in the mouse using the clip technique. World J Gastroenterol. 18 (22), 2767-2774 (2012).
- Kamali, C., et al. Extended liver resection in mice: state of the art and pitfalls-a systematic review. Eur J Med Res. 26 (1), 6 (2021).
- Mitchell, C., Willenbring, H. A reproducible and well-tolerated method for 2/3 partial hepatectomy in mice. Nat Protoc. 3 (7), 1167-1170 (2008).
- Borowiak, M., et al. Met provides essential signals for liver regeneration. Proc Natl Acad Sci U S A. 101 (29), 10608-10613 (2004).
- Boyce, S., Harrison, D. A detailed methodology of partial hepatectomy in the mouse. Lab Anim (NY). 37 (11), 529-532 (2008).
- Greene, A. K., Puder, M. Partial hepatectomy in the mouse: technique and perioperative management. J Invest Surg. 16 (2), 99-102 (2003).
- Mitchell, C., Willenbring, H. Erratum: A reproducible and well-tolerated method for 2/3 partial hepatectomy in mice. Nat Protoc. 9 (6), 1532 (2014).
- Studer, P., et al. Significant lethality following liver resection in A20 heterozygous knockout mice uncovers a key role for A20 in liver regeneration. Cell Death Differ. 22 (12), 2068-2077 (2015).
- Longo, C. R., et al. A20 protects mice from lethal radical hepatectomy by promoting hepatocyte proliferation via a p21waf1-dependent mechanism. Hepatology. 42 (1), 156-164 (2005).
- Michalopoulos, G. K., DeFrances, M. C. Liver regeneration. Science. 276 (5309), 60-66 (1997).
- Diehl, A. M., Rai, R. M. Liver regeneration. 3. Regulation of signal transduction during liver regeneration. FASEB J. 10 (2), 215-227 (1996).
- . A comparison of selected organ weights and clinical pathology parameters in male and female CD-1 and CByB6F1 hybrid mice 12-14 weeks in age Available from: https://www.criver.com/sites/default/files/resources/doc_a/AComparisonofSelectedOrganWeightsandClinicalPathologyParametersinMaleandFemaleCD-1andCByB6F1HybridMice12-14WeeksinAge.pdf (2023)
- CD-1® IGS mouse. Charles River Laboratories Available from: https://www.criver.com/products-services/find-model/cd-1r-igs-mouse?region=3611 (2023)
- C57BL/6J mouse organ weight. The Jackson Laboratory Available from: https://www.jax.org/de/-/media/jaxweb/files/jax-mice-and-services/b6j-data-summary.xlsx (2023)
- Inderbitzin, D., et al. Regenerative capacity of individual liver lobes in the microsurgical mouse model. Microsurgery. 26 (6), 465-469 (2006).
- Zhou, X., et al. L-carnitine promotes liver regeneration after hepatectomy by enhancing lipid metabolism. J Transl Med. 21 (1), 487 (2023).
- Linecker, M., et al. Omega-3 fatty acids protect fatty and lean mouse livers after major hepatectomy. Ann Surg. 266 (2), 324-332 (2017).
- Haber, B. A., et al. High levels of glucose-6-phosphatase gene and protein expression reflect an adaptive response in proliferating liver and diabetes. J Clin Invest. 95 (2), 832-841 (1995).
- Rickenbacher, A., et al. Arguments against toxic effects of chemotherapy on liver injury and regeneration in an experimental model of partial hepatectomy. Liver Int. 31 (3), 313-321 (2011).
- Aravinthan, A. D., et al. The impact of preexisting and post-transplant diabetes mellitus on outcomes following liver transplantation. Transplantation. 103 (12), 2523-2530 (2019).
- Gonzalez, H. D., Liu, Z. W., Cashman, S., Fusai, G. K. Small for size syndrome following living donor and split liver transplantation. World J Gastrointest Surg. 2 (12), 389-394 (2010).
- Mahmud, N., et al. Risk prediction models for post-operative mortality in patients with cirrhosis. Hepatology. 73 (1), 204-218 (2021).
- Kooby, D. A., et al. Impact of steatosis on perioperative outcome following hepatic resection. J Gastrointest Surg. 7 (8), 1034-1044 (2003).
- Ma, K., et al. A mesenchymal-epithelial transition factor-agonistic antibody accelerates cirrhotic liver regeneration and improves mouse survival following partial hepatectomy. Liver Transpl. 28 (5), 782-793 (2022).
- Hori, T., et al. Simple and sure methodology for massive hepatectomy in the mouse. Ann Gastroenterol. 24 (4), 307-318 (2011).
- Ramsey, H. E., et al. A20 protects mice from lethal liver ischemia/reperfusion injury by increasing peroxisome proliferator-activated receptor-alpha expression. Liver Transpl. 15 (11), 1613-1621 (2009).
- Arvelo, M. B., et al. A20 protects mice from D-galactosamine/lipopolysaccharide acute toxic lethal hepatitis. Hepatology. 35 (3), 535-543 (2002).
