A Mouse Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy Procedure Aided by Microscopy

Summary

Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) has been shown to result in remarkable liver regeneration after several weeks post first-stage surgery. Animal models that have undergone ALPPS have been developed to explore potential liver regeneration ability and achieve favorable clinical outcomes.

Abstract

Hepatectomy is widely regarded as the primary treatment for hepatic malignancies; yet, postoperative liver failure remains a major cause of perioperative mortality, severely impacting patient outcomes. In a robust hepatic environment, the future liver remnant (FLR) must exceed 25%, and in cases of cirrhosis, this requirement increases to over 40%. The inadequacy of FLR is currently a major obstacle in the progression of hepatic surgery.

Traditional methods to enhance FLR hypertrophy mainly focus on portal vein embolization (PVE), but its effectiveness is considerably limited. In recent years, there have been numerous reports on a novel biphasic hepatectomy method involving hepatic partitioning and portal vein ligation, known as associating liver partition and portal vein ligation for staged hepatectomy (ALPPS). ALPPS surpasses PVE in efficiently and considerably inducing FLR hypertrophy. However, the detailed mechanisms driving ALPPS-facilitated hepatic regeneration are not fully understood. Thus, replicating ALPPS in animal models is crucial to thoroughly investigate the molecular mechanisms of hepatic regeneration, offering valuable theoretical and practical insights.

Introduction

The liver harbors formidable regenerative potential, swiftly proliferating and restoring metabolic demands within a mere 3 months following resection for diverse hepatic ailments¹. However, the imperative to ascertain the completeness of tumor margins necessitates the inevitability of expansive hepatic excision. Thus, ensuring an ample volume of the participatory hepatic milieu, known as the future liver remnant (FLR), assumes paramount importance². ALPPS has been a breakthrough technique in hepatic surgery over the past few decades, particularly tailored for patients with inadequate residual hepatic volume subsequent to tumor resection, heralded as one of the most auspicious breakthroughs in the realm of hepatic oncological surgery³.

Remarkable progress has been made in developing ALPPS animal models. An ideal model typically requires independent blood inflow (portal vein and hepatic artery) and outflow (hepatic vein) in the preserved hepatic lobe and a clear separation between the preserved and the to-be-resected hepatic lobes to prevent collateral circulation⁴. Although ALPPS stimulates rapid hepatic regeneration in the remaining liver tissue, the specific mechanisms of this process are still unclear.

Currently, ALPPS models are categorized into three types: large animal models (e.g., swine and sheep), medium-sized models (e.g., rabbits and rodents), and small models (e.g., mice)⁵. The use of mice, with their quick breeding, cost-effectiveness, and ease of genetic modification, is particularly effective for in-depth studies of hepatic regeneration mechanisms⁶. Moreover, the liver structure of mice, especially their middle hepatic vein, closely resembles that of humans, making them highly suitable for ALPPS model development.

It is important to note that most hepatocellular carcinoma patients in clinical practice have underlying liver conditions, unlike the healthy liver models typically used in studies⁷. Thus, using mice preconditioned with hepatic fibrosis or viral infections can more accurately simulate the surgical responses and postoperative liver regeneration seen in patients with various liver diseases⁸. This approach could reveal new therapeutic targets of clinical relevance.

So far, a few renowned research groups, such as those at the University of Zurich and the University of Tokyo, have successfully developed murine ALPPS models^9,10. Creating a standardized murine model under controlled conditions could advance our understanding of the rapid liver regeneration observed after ALPPS procedures.

Protocol

All experiments in this protocol were approved by the Veterinary Authorities of the Jiangxi Provincial People's Hospital (number 70/2022). Furthermore, all experimental steps were performed in strict compliance with the Institutional Animal Care and Use Committee. 1. Start of surgery NOTE: Male C57BL/6 mice weighing 20-22 g were housed in a well-ventilated cage, maintained in a standard pathogen-free environment with a 12 h/12 h light/dark cycle. …

Representative Results

In contrast to the PVL (protocol steps 2,3,4, and 5), the mice that were subjected to the ALPPS (steps 2,3,4,5, and 6) surgical intervention demonstrated a heightened proclivity towards cellular proliferation. Figure 2 delineates the ischemic demarcation of the middle liver lobe following portal vein ligation, along with the visual field during liver transection, which represents a key distinction between ALPPS and PVL. Notably, the levels of inflammatory mediators, including (Tumor Necrosis…

Discussion

This study presents a protocol for the initial phase of ALPPS surgery in mice, involving partial ligation of the portal vein and division of the median liver lobe. This method closely mimics the human ALPPS procedure, with the mouse liver's unique lobular structure and dual portal venous supply to the median lobe ensuring distinct blood flow post-surgery¹¹. It is noteworthy that the ALPPS procedure comprises two distinct surgical stages. In this study, we exclusively present the initial stage …

Materials

5% glucose injection (500 mL)	Shapuaisi pharma (http://www.zjspas.com/)	H20003666	Efficient, cheap,Easy to access
anaesthesia machine	RWD (www.rwdls.com)	R500	low price and valuable quality. It is suitable for operations with beginners
C57 BL/6	The Jackson Lab	22349-2023	Stability of strains
isoflurane	KCSW (kcsw.szqisoubao.com)	H19980141	durable, cost-effective
meloxicam	Boehringer-Ingelheim	H20020217	Durable and efficient
microforceps	maydeal	60018920	Durable and efficient
microinstrument	CH microsurgical instrument factory	HC-A804-1	durable, cost-effective
sodium lactate ringer	Shapuaisi pharma (http://www.zjspas.com/)	H20193277	Efficient, cheap, easy to access
suture line	CH microsurgical instrument factory	6-0/8-0	high performance-price ratio
topical antibodies	chenxin pharmacology (www.cisen-pharma.com)	H20020217	Effectively avoid incision infection