All experiments in this protocol were approved by the Veterinary Authorities of the Jiangxi Provincial People&#39;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. The animals were provided ad libitum access to food and water at a controlled ambient temperature of 22 &#177; 1 &#176;C. The surgery was performed in a dedicated microsurgical room using a surgical microscope (Figure 1). The mice in the sham group were only operated by laparotomy, but no operation was performed on the liver. In the PVL group, part of the portal vein branches was ligated, and in the ALPPS group, the middle liver lobe was transected on the basis of portal vein ligation (PVL).
<ol>
	<li>Subject the mice to a 4-6 h&#160;preoperative fasting period. 
	NOTE: Fasting is required because eating causes gastric bulge in mice, it results in poor surgical field and is prone to accidental damage to the gastric wall.</li>
	<li>Arrange sterile surgical instruments, including microforceps, scissors, abdominal retractors, and pre-prepared 8-0 portal vein ligature sutures, on a side table. Soak intraoperative materials comprising sterile gauze and cotton balls in 37 &#176;C saline solution.</li>
	<li>For induction, expose the animals to 5% v/v isoflurane for 30 s, followed by maintenance anesthesia using 3 vol% isoflurane until achieving a deep anesthetic state. Examine the toe pinch reflex to ascertain the level of anesthesia. Subcutaneously inject medetomidine at a dose of 5 mg/kg during the induction phase.</li>
	<li>Apply ointment to the animals&#39; eyes and inject 1 mL of lactated Ringer&#39;s solution mixed with 5% glucose subcutaneously, dividing the dose equally between the lower abdomen on each side.</li>
	<li>Meticulously shave the abdominal region using small animal hair clippers, encompassing a 2 cm lateral area from the xiphoid process to the genital region.</li>
	<li>Thoroughly disinfect the surgical site 3 times&#160;with alternating rounds of povidone-iodine and alcohol.</li>
	<li>Make a length of 2.5-3.0 cm midline incision on the skin using a surgical scalpel, subsequently utilizing surgical scissors to delicately open the abdominal cavity.</li>
	<li>Employ the abdominal retractor to expand the incision, facilitating the gentle traction of the duodenum and a segment of the small intestine using sterile, moist cotton swabs. Cover these structures with the pre-prepared sterile gauze, simultaneously exposing the hepatic segment of the portal vein.</li>
</ol>
2. Portal vein ligation (PVL)
NOTE: Ensure that all actions are gentle throughout the whole procedure. Use moistened cotton balls when manipulating liver tissue.
<ol>
	<li>Peel off the peritoneum and intestinal tract and make sure the portal vein is fully exposed.</li>
	<li>In the course of exposing the main portal vein, proceed by encircling its branches using slow, deliberate forward-pushing and spreading movements.</li>
	<li>Sequentially identify the various branches of the portal vein in the following order: (1) the right posterior branch, (2) the combined left lateral and left median branches, and (3) the caudate branch (Figure 2A).</li>
</ol>
3. Dissection of the right posterior branch of portal vein branches
<ol>
	<li>Upon traversing the hepatic hilum via the main portal vein, observe a conspicuous branch of the right posterior lobe that has become visible.</li>
	<li>Use moistened cotton balls as a fulling agent and separate the liver&#39;s middle lobe and the right posterior lobe to avoid potential injury.</li>
	<li>Be sure to dissect along the Glisson&#39;s capsule (the peritoneum that extends over the surface of the liver) and pull a 1.5 cm silk to tie around portal vein branches.</li>
	<li>Ligate the right posterior branch of the portal vein with 8-0 silk suture. A reliable indicator of successful portal vein ligation is visible pallor of the liver.</li>
</ol>
4. Dissection of the left lateral and left medial branches of the portal vein
<ol>
	<li>The left lateral lobe (LLL) and left medial lobe (LML) branches share a common main portal vein. Be sure to dissect any potential interstice between the LLL and the caudate lobe. 
	NOTE: Ligation of the LLL and LML branches of the portal vein is the most difficult in this surgical model because the microforceps must traverse the space around the portal vein and avoid liver injury.</li>
	<li>Introduce the microforceps through the entrance in step 4.1 and move the hand slowly, as even the slightest resistance hints at the potential proximity to the liver or blood vessels.</li>
	<li>When resistance is felt, adjust the microforceps slightly to move forward and through the surface of Glisson&#39;s capsule of the right medial lobe (RML) (Figure 3C).</li>
	<li>Employ the prepared 8-0 silk suture to tie around the portal vein of the LLL and LML branches. Look for a striking ischemic demarcation within the liver middle lobe, while the left lobe assumes pallor, upon ligation of the portal vein branches.</li>
</ol>
5. Dissection of the caudate branch of the portal vein branches
<ol>
	<li>Dissect the caudate lobe and use a cotton swab to fill the potential space between the main portal vein and the caudate lobe.</li>
	<li>Peel off the peritoneum of the caudate lobes to visualize the portal vein branches of the lobe.</li>
	<li>Use Stitch-up suture rather than tie-around to ligate the caudate branch, ensuring that the needle path adequately wraps the caudate branch of the portal vein. 
	NOTE: The difficulty of ligating the caudate branch of the portal vein is because the caudate lobe portal vein encapsulates the liver tissue and is close to the main portal vein. A needle penetration depth of 1 mm is recommended.</li>
</ol>
6. Liver transection and cholecystectomy
<ol>
	<li>Observe a distinct demarcation line in the middle liver lobe and perform the transection along this line (Figure 2B).</li>
	<li>Use an electrocautery pen to create a 0.5 mm precut line along the demarcation line.</li>
	<li>Use micro forceps and an electrocautery pen to cut liver tissue, applying cotton swabs to stop bleeding. 
	NOTE: The difficulty in the transection is to master the depth. The goal is to split the liver as close to the vena cava as possible. Intraoperative bleeding is difficult to avoid and requires repetitive practice to master the depth of transection. Hot burning, cotton swabs, and gelatin sponges can be used to assist in hemostasis.</li>
	<li>Remove the gallbladder last as this will provide a good pulling point during the whole operation.</li>
	<li>Employ a cotton swab to apply controlled pressure for hemostasis, and if hemorrhage persists, the inferior vena cava is injured and the effect of suture repair will be poor; euthanize the animal without hesitation.</li>
</ol>
7. Final steps of surgery
<ol>
	<li>Ensure meticulous placement of the small bowel within the abdominal cavity prior to closure.</li>
	<li>Securely close the peritoneum and abdominal wall using 6-0 absorbable sutures and the skin with 4-0 absorbable sutures.</li>
	<li>Disinfect the skin surface again to ensure optimal cleanliness.</li>
	<li>Subcutaneously administer 1 mL of saline solution and fentanyl 0.02 mg/kg plus meloxicam 4 mg/kg at the incision site to alleviate postoperative pain and address any supplementary intraoperative fluid loss.</li>
	<li>Place the animal on a warmed surface to facilitate natural awakening, and monitor the blood supply to their extremities. Subsequently, house them individually in cages for continued observation as they regain consciousness.</li>
	<li>Subcutaneously inject medetomidine at a dose of 4 mg/kg upon recovery, continuing for 24 h postoperatively.</li>
</ol>
8. Intraoperative measurement of liver regeneration and liver function
<ol>
	<li>Euthanize the mice and collect liver as well as blood samples at intervals of 1, 2, 3, and 7 days post surgery.</li>
	<li>Perform HE staining and liver function test and examine serum inflammatory markers, thereby providing additional validation for the impact of the model construction.</li>
	<li>Document the daily variations in the body weight and FLR/BW (body weight) ratio of the mice over a span of 1 week following the surgical procedure.</li>
</ol>

Studying Liver Regeneration of ALLPS in Animal Models for Staged Hepatectomy

<ol>
	<li>Michalopoulos, G. K., Bhushan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+regeneration:+Biological+and+pathological+mechanisms+and+implications.">Liver regeneration: Biological and pathological mechanisms and implications.</a> Nat Rev Gastroenterol Hepatol. 18 (1), 40-55 (2021).</li><li>Chapelle, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+the+future+remnant+liver+function+is+a+better+tool+to+pr+edict+post-hepatectomy+liver+failure+than+platelet-based+liver+scores.">Estimation of the future remnant liver function is a better tool to pr edict post-hepatectomy liver failure than platelet-based liver scores.</a> Eur J Surg Oncol. 43 (12), 2277-2284 (2017).</li><li>Isfordink, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Portal+vein+ligation+versus+portal+vein+embolization+for+induction+of+hypertrophy+of+the+future+liver+remnant:+A+systematic+review+and+meta-analysis.">Portal vein ligation versus portal vein embolization for induction of hypertrophy of the future liver remnant: A systematic review and meta-analysis.</a> Surg Oncol. 26 (3), 257-267 (2017).</li><li>Linecker, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+adjustment+in+alpps+is+associated+with+a+dramatic+decrease+in+ear+ly+mortality+and+morbidity.">Risk adjustment in alpps is associated with a dramatic decrease in ear ly mortality and morbidity.</a> Ann Surg. 266 (5), 779-786 (2017).</li><li>Darnis, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subtotal+hepatectomy+in+swine+for+studying+small-for-size+syndrome+and+portal+inflow+modulation:+Is+it+reliable.">Subtotal hepatectomy in swine for studying small-for-size syndrome and portal inflow modulation: Is it reliable.</a> HPB (Oxford). 17 (10), 881-888 (2015).</li><li>Casellas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inbred+mouse+strains+and+genetic+stability:+A+review.">Inbred mouse strains and genetic stability: A review.</a> Animal. 5 (1), 1-7 (2011).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+associating+liver+partition+and+portal+vein+ligation+for+staged+hepatectomy+from+2012+to+2021:+A+bibliometric+analysis.+Review.">Evolution of associating liver partition and portal vein ligation for staged hepatectomy from 2012 to 2021: A bibliometric analysis. Review.</a> Int J Surg. 103, 106648 (2022).</li><li>Parola, M., Pinzani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+fibrosis:+Pathophysiology,+pathogenetic+targets+and+clinical+iss+ues.">Liver fibrosis: Pathophysiology, pathogenetic targets and clinical iss ues.</a> Mol Aspects Med. 65, 37-55 (2019).</li><li>Kikuchi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+associating+liver+partition+and+portal+vein+occlusion+for+st+aged+hepatectomy+on+tumor+growth+in+a+mouse+model+of+liver+metastasis.">Impact of associating liver partition and portal vein occlusion for st aged hepatectomy on tumor growth in a mouse model of liver metastasis.</a> Eur J Surg Oncol. 44 (1), 130-138 (2018).</li><li>Schlegel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpps:+From+human+to+mice+highlighting+accelerated+and+novel+mechanism+s+of+liver+regeneration.">Alpps: From human to mice highlighting accelerated and novel mechanism s of liver regeneration.</a> Ann Surg. 260 (5), 839-846 (2014).</li><li>Dili, A., Lebrun, V., Bertrand, C., Leclercq, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associating+liver+partition+and+portal+vein+ligation+for+staged+hepate+ctomy:+Establishment+of+an+animal+model+with+insufficient+liver+remnan+t.">Associating liver partition and portal vein ligation for staged hepate ctomy: Establishment of an animal model with insufficient liver remnan t.</a> Lab Invest. 99 (5), 698-707 (2019).</li><li>Schadde, E., Hertl, M., Breitenstein, S., Beck-Schimmer, B., Schl&#228;pfer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+model+of+the+associating+liver+partition+and+portal+vein+ligation+for+staged+hepatectomy+(alpps)+procedure.">Rat model of the associating liver partition and portal vein ligation for staged hepatectomy (alpps) procedure.</a> J Vis Exp. (126), e55895 (2017).</li><li>Allaire, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+frontiers+in+liver+resection+for+hepatocellular+carcinoma.">New frontiers in liver resection for hepatocellular carcinoma.</a> JHEP Rep. 2 (4), 100134 (2020).</li><li>Memi, F., Ntokou, A., Papangeli, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crispr/cas9+gene-editing:+Research+technologies,+clinical+applications+and+ethical+considerations.">Crispr/cas9 gene-editing: Research technologies, clinical applications and ethical considerations.</a> Semin Perinatol. 42 (8), 487-500 (2018).</li><li>Saviano, A., Henderson, N. C., Baumert, T. 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The authors have no conflicts of interest to declare.

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&#39;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 ...

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 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 models9,10. Creating a standardized murine model under controlled conditions could advance our understanding of the rapid liver regeneration observed after ALPPS procedures.

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

a mouse model of the associating liver partition and portal vein ligation for staged hepatectomy procedure aided by microscopy

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.

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...

Read this Scientific Article Protocol about A Mouse Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy Procedure Aided by Microscopy at JoVE.com

Author Spotlight: Advancing Liver Regeneration Research through ALPPS Mouse Model

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.

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

Hepatectomy is widely regarded as the primary treatment for hepatic malignancies; yet, postoperative liver failure remains a major cause of perioperative ...

a-mouse-model-associating-liver-partition-portal-vein-ligation-for

Research

JoVE Journal

Medicine

502 Views.  The First Affiliated Hospital of Nanchang Medical College. 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.

Video: Author Spotlight: Advancing Liver Regeneration Research through ALPPS Mouse Model

Use of a Hanging-weight System for Liver Ischemia in Mice

Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and can result in liver failure which has a high mortality rate1-2. Therefore murine studies of hepatic ischemia have become an important field of research by providing the opportunity to utilize pharmacological and genetic studies3-9. Specifically, conditional mice with tissue specific deletion of a gene (cre, flox system) provide insights into the role of proteins in particular tissues10-13 . Because of the technical difficulty associated with manually clamping the portal triad in mice, we performed a systematic evaluation using a hanging-weight system for portal triad occlusion which has been previously described3. By using a hanging-weight system we place a suture around the left branch of the portal triad without causing any damage to the hepatic lobes, since also the finest clamps available can cause hepatic tissue damage because of the close location of liver tissue to the vessels. Furthermore, the right branch of the hepatic triad is still perfused thus no intestinal congestion occurs with this technique as blood flow to the right hepatic lobes is preserved. Furthermore, the portal triad is only manipulated once throughout the entire surgical procedure. As a result, procedures like pre-conditioning, with short times of ischemia and reperfusion, can be easily performed. Systematic evaluation of this model by performing different ischemia and reperfusion times revealed a close correlation of hepatic ischemia time with liver damage as measured by alanine (ALT) and aspartate (AST) aminotransferase serum levels3,9. Taken together, these studies confirm highly reproducible liver injury when using the hanging-weight system for hepatic ischemia and intermittent reperfusion. Thus, this technique might be useful for other investigators interested in liver ischemia studies in mice. Therefore the video clip provides a detailed step-by-step description of this technique.

We established a novel murine model of a hanging weight system for portal triad occlusion. This technique may be useful for future investigations of ischemia in murine hepatic models.

Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and ...

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

Cancer Research

Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse

Metabolic diseases such as diabetes, pre-diabetes, non-alcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH) are becoming increasingly common. Ex vivo liver perfusions allow for a comprehensive analysis of liver metabolism using nuclear magnetic resonance (NMR), in nutritional conditions that can be rigorously controlled. As in silico simulations remain a primarily theoretical means of assessing hormone actions and the effects of pharmaceutical intervention, the perfused liver remains one of the most valuable test beds for understanding hepatic metabolism. As these studies guide basic insights into hepatic physiology, results must be accurate and reproducible. The greatest factor in the reproducibility of ex vivo hepatic perfusion is the quality of surgery. Therefore, we have introduced an organized and streamlined method to perform ex vivo mouse liver perfusions in the context of in situ NMR experiments. We also describe a unique application and discuss common issues encountered in these studies. The overall purpose is to provide an uncomplicated guide to a technique we have refined over several years that we deem the golden standard for obtaining reproducible results in hepatic resections and perfusions in the context of in situ NMR experiments. The distance to the center of the field for the magnet as well as the inaccessibility of the tissue to intervention during the NMR experiment makes our methods novel.

Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse

The protocol describes a straightforward method of resectioning an intact mouse liver for metabolic studies through portal vein perfusion.

Metabolic diseases such as diabetes, pre-diabetes, non-alcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH) are becoming ...

Biochemistry

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (&#181;CT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees.
The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after &#181;CT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using &#181;CT and histological serial sections.

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) Procedure

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver metastases, the amount of liver tissue left behind after liver resection has become the main limiting factor of resectability of large or multiple liver tumors. A minimal amount of functional tissue is required to avoid the severe complication of post-hepatectomy liver failure, which has high morbidity and mortality. Inducing liver growth of the prospective remnant prior to resection has become more established in liver surgery, either in the form of portal vein embolization by interventional radiologists or in the form of portal vein ligation several weeks prior to resection. Recently, it was shown that liver regeneration is more extensive and rapid, when the parenchymal transection is added to portal vein ligation in a first stage and then, after only one week of waiting, resection performed in a second stage (Associating Liver Partition and Portal vein ligation for Staged hepatectomy = ALPPS). ALPPS has rapidly become popular across the world, but has been criticized for its high perioperative mortality. The mechanism of accelerated and extensive growth induced by this procedure has not been well understood. Animal models have been developed to explore both the physiological and molecular mechanisms of accelerated liver regeneration in ALPPS. This protocol presents a rat model that allows mechanistic exploration of accelerated regeneration.

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy ALPPS Procedure

Inducing rapid liver hypertrophy using Associating Liver Partition and Portal vein ligation for a Staged hepatectomy (ALPPS) has been proposed for resection of borderline resectable liver tumors. This model may elucidate mechanisms involved in rapid hypertrophy and allows testing of drugs that promote or block the acceleration of regeneration.

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver ...

A Mouse Model of Chronic Liver Fibrosis for the Study of Biliary Atresia

Biliary atresia (BA) is a fatal disease involving obstructive jaundice, and it is the most common indication for liver transplantation in children. Due to the complex etiology and unknown pathogenesis, there are still no effective drug treatments. At present, the classic BA mouse model induced by rhesus rotavirus (RRV) is the most commonly used model for studying the pathogenesis of BA. This model is characterized by growth retardation, jaundice of the skin and mucosa, clay stools, and dark yellow urine. The histopathology shows severe liver inflammation and obstruction of the intrahepatic and extrahepatic bile ducts, which are similar to the symptoms of human BA. However, the livers of end-stage mice in this model lack fibrosis and cannot fully simulate the characteristics of liver fibrosis in clinical BA. The presented study developed a novel BA mouse model of chronic liver fibrosis by injecting 5-10 &#181;g of anti-Ly6G antibody four times, with gaps of 2 days after each injection. The results showed that some of the mice successfully formed chronic BA with typical fibrosis after the time lapse, meaning these mice represent a suitable animal model for the virus-induced liver fibrosis mechanistic study of BA and a platform for developing future BA treatments.

We established a mouse model of chronic liver fibrosis, which provides a suitable animal model for the virus-induced liver fibrosis mechanistic study of biliary atresia (BA) and a platform for future BA treatments.

Biliary atresia (BA) is a fatal disease involving obstructive jaundice, and it is the most common indication for liver transplantation in children. Due to ...

Immunology and Infection

3D Imaging of the Liver Extracellular Matrix in a Mouse Model of Non-Alcoholic Steatohepatitis

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can progress to fibrosis and eventually to cirrhosis, a significant risk factor for hepatocellular carcinoma. The extracellular matrix (ECM) provides structural support and maintains liver homeostasis via matricellular signals. Liver fibrosis results from an imbalance in the dynamic ECM remodeling process and is characterized by excessive accumulation of structural elements and associated changes in glycosaminoglycans. The typical fibrosis pattern of NASH is called &#34;chicken wire,&#34; which usually consists of zone 3 perisinusoidal/pericellular fibrosis, based on features observed by Masson&#39;s trichrome stain and Picrosirius Red stains. However, these traditional thin two-dimensional (2D) tissue slide-based imaging techniques cannot demonstrate the detailed three-dimensional (3D) ECM structural changes, limiting the understanding of the dynamic ECM remodeling in liver fibrosis.
The current work optimized a fast and efficient protocol to image the native ECM structure in the liver via decellularization to address the above challenges. Mice were fed either with chow or fast-food diet for 14 weeks. Decellularization was performed after in situ portal vein perfusion, and the two-photon microscopy techniques were applied to image and analyze changes in the native ECM. The 3D images of the normal and NASH livers were reconstituted and analyzed. Performing in situ perfusion decellularization and analyzing the scaffold by two-photon microscopy provided a practical and reliable platform to visualize the dynamic ECM remodeling in the liver.

The present protocol optimizes the liver in situ perfusion/decellularization and two-photon microscopy methods to establish a reliable platform to visualize the dynamics of extracellular matrix (ECM) remodeling during non-alcoholic steatohepatitis (NASH).

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can ...

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile duct proliferation. However, long-term experiments can lead to increased morbidity and mortality. In select mouse strains with underlying liver disease, meaningful comparisons can be made even with ligation of a single lobe of the liver, which can reduce animal losses and expenses. Here, we describe partial bile duct ligation (pBDL) in the mouse, in which only the left hepatic bile duct is ligated, causing biliary obstruction in the left lobe but not the remaining lobes. With careful microsurgical technique, pBDL experiments can be cost-effective, since the unligated lobe serves as an internal control to the ligated lobes, when subjected to the same conditions in the same animal. Unlike cBDL, a separate sham-operated control group is not necessary. pBDL is highly useful to directly compare localized versus systemic effects of cholestasis and other retained bile components. pBDL can also be repurposed as a novel method to investigate mechanisms related to medications and cell migration.

Here, we present partial bile duct ligation as a surgical model of liver injury and regeneration in rodents.

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile ...

Biology

Mouse Extended Hepatectomy Model for Liver Regeneration Studies

Extended 78&#37; Hepatectomy in a Mouse Surgical Model

Partial 2/3 hepatectomy in mice is used in research to study the liver's regenerative capacity and explore outcomes of liver resection in a number of disease models. In the classical partial 2/3 hepatectomy in mice, two of the five liver lobes, namely the left and median lobes representing approximately 66% of the liver mass, are resected en bloc with an expected postoperative survival of 100%. More aggressive partial hepatectomies are technically more challenging and hence, have seldom been used in mice. Our group has developed a mouse model of an extended hepatectomy technique in which three of the five liver lobes, including the left, median, and right upper lobes, are resected separately to remove approximately 78% of the total liver mass. This extended resection, in otherwise healthy mice, leaves a remnant liver that cannot always sustain adequate and timely regeneration. Failure to regenerate ultimately results in 50% postoperative lethality within 1 week due to fulminant hepatic failure. This procedure of extended 78% hepatectomy in mice represents a unique surgical model for the study of small-for-size syndrome and the evaluation of therapeutic strategies to improve liver regeneration and outcomes in the setting of liver transplantation or extended liver resection for cancer.

Author Spotlight: Evaluating Therapeutic Strategies to Enhance Liver Regeneration

The mouse model of partial 2/3 (66%) hepatectomy is well described in the literature, but more extended hepatectomies mimicking small-for-size syndrome after liver transplantation have seldom been used. We describe an extended 78% hepatectomy procedure in a mouse model that results in approximately 50% postoperative lethality in healthy mice.

Partial 2/3 hepatectomy in mice is used in research to study the liver's regenerative capacity and explore outcomes of liver resection in a number of ...

Imaging Fixture Supported Mouse Liver Staging for Multiphoton Microscopy Based Vascular Visualization: A Stabilization Device-assisted Imaging Technique to Obtain Multi-dimensional Images of Blood Vessels in Mouse Liver

After confirming a lack of righting reflex in an anesthetized 8-week-old male C57BL/6 mouse, wipe the mouse&#39;s tail with 75% alcohol and use a 1-microliter syringe equipped with a 30-gauge needle to inject 10 milligrams per milliliter of freshly prepared Rhodamine B isothiocyanate-dextran in 100 microliters of Rhodamine B isothiocyanate-dextran into the caudal tail vein.
When all of the solution has been delivered, use a cotton swab to apply pressure to the puncture site and use gauze wet with sterile water to soak the abdominal fur. Use a razor to shave the abdomen, making strokes in the direction of the fur, and place the mouse onto a 37 degrees Celsius, alcohol-disinfected heating pad.
To fix the mouse liver with a body organ imaging frame, first, place a clean 5-millimeter diameter suction cup in a fixed position and wipe the heating pad and suction cup with 75% alcohol. Connect the suction cup to the vacuum pump hose and turn on the pump.
On a 75% alcohol-sterilized table, use surgical scissors to cut 2 centimeters of skin from the lower sternal border of the mouse and expose the liver. Place the mouse and the heating pad onto the holder base of the body frame and adjust the organ imaging fixture so that the suction cup can hold the liver. Then, use a negative pressure of 30 to 35 kilopascals for suction so that the liver attaches to the cup.
To set up the multiphoton laser scanning microscope, turn on the microscope and select the 60x objective. Fix the frame and the mouse under the objective and add a drop of normal saline to the cup large enough to cover the lens. Adjust the objective so the lens just touches the normal saline and turn on the laser software.

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

Summary

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Extended 78% Hepatectomy in a Mouse Surgical Model

Imaging Fixture Supported Mouse Liver Staging for Multiphoton Microscopy Based Vascular Visualization: A Stabilization Device-assisted Imaging Technique to Obtain Multi-dimensional Images of Blood Vessels in Mouse Liver