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. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+genomics+and+spatial+transcriptomics:+Discovery+of+novel+cell+states+and+cellular+interactions+in+liver+physiology+and+disease+biology.">Single-cell genomics and spatial transcriptomics: Discovery of novel cell states and cellular interactions in liver physiology and disease biology.</a> J Hepatol. 73 (5), 1219-1230 (2020).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 ...

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460 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

Cecal Ligation Puncture Procedure

Human sepsis is characterized by a set of systemic reactions in response to intensive and massive infection that failed to be locally contained by the host. Currently, sepsis ranks among the top ten causes of mortality in the USA intensive care units 1. During sepsis there are two established haemodynamic phases that may overlap. The initial phase (hyperdynamic) is defined as a massive production of proinflammatory cytokines and reactive oxygen species by macrophages and neutrophils that affects vascular permeability (leading to hypotension), cardiac function and induces metabolic changes culminating in tissue necrosis and organ failure. Consequently, the most common cause of mortality is acute kidney injury. The second phase (hypodynamic) is an anti-inflammatory process involving altered monocyte antigen presentation, decreased lymphocyte proliferation and function and increased apoptosis. This state known as immunosuppression or immune depression sharply increases the risk of nocosomial infections and ultimately, death. The mechanisms of these pathophysiological processes are not well characterized. Because both phases of sepsis may cause irreversible and irreparable damage, it is essential to determine the immunological and physiological status of the patient. This is the main reason why many therapeutic drugs have failed. The same drug given at different stages of sepsis may be therapeutic or otherwise harmful or have no effect 2,3. To understand sepsis at various levels it is crucial to have a suitable and comprehensive animal model that reproduces the clinical course of the disease. It is important to characterize the pathophysiological mechanisms occurring during sepsis and control the model conditions for testing potential therapeutic agents. 
To study the etiology of human sepsis researchers have developed different animal models. The most widely used clinical model is cecal ligation and puncture (CLP). The CLP model consists of the perforation of the cecum allowing the release of fecal material into the peritoneal cavity to generate an exacerbated immune response induced by polymicrobial infection. This model fulfills the human condition that is clinically relevant. As in humans, mice that undergo CLP with fluid resuscitation show the first (early) hyperdynamic phase that in time progresses to the second (late) hypodynamic phase. In addition, the cytokine profile is similar to that seen in human sepsis where there is increased lymphocyte apoptosis (reviewed in 4,5). Due to the multiple and overlapping mechanisms involved in sepsis, researchers need a suitable sepsis model of controlled severity in order to obtain consistent and reproducible results.

The mouse model of cecal ligation and puncture as a valuable tool for the study of human sepsis.

Human sepsis is characterized by a set of systemic reactions in response to intensive and massive infection that failed to be locally contained by the ...

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

In organs, the correct architecture of vascular and ductal structures is indispensable for proper physiological function, and the formation and maintenance of these structures is a highly regulated process. The analysis of these complex, 3-dimensional structures has greatly depended on either 2-dimensional examination in section or on dye injection studies. These techniques, however, are not able to provide a complete and quantifiable representation of the ductal or vascular structures they are intended to elucidate. Alternatively, the nature of 3-dimensional plastic resin casts generates a permanent snapshot of the system and is a novel and widely useful technique for visualizing and quantifying 3-dimensional structures and networks.
A crucial advantage of the resin casting system is the ability to determine the intact and connected, or communicating, structure of a blood vessel or duct. The structure of vascular and ductal networks are crucial for organ function, and this technique has the potential to aid study of vascular and ductal networks in several ways. Resin casting may be used to analyze normal morphology and functional architecture of a luminal structure, identify developmental morphogenetic changes, and uncover morphological differences in tissue architecture between normal and disease states. Previous work has utilized resin casting to study, for example, architectural and functional defects within the mouse intrahepatic bile duct system that were not reflected in 2-dimensional analysis of the structure1,2, alterations in brain vasculature of a Alzheimer's disease mouse model3, portal vein abnormalities in portal hypertensive and cirrhotic mice4, developmental steps in rat lymphatic maturation between immature and adult lungs5, immediate microvascular changes in the rat liver, pancreas, and kidney in response in to chemical injury6.
Here we present a method of generating a 3-dimensional resin cast of a mouse vascular or ductal network, focusing specifically on the portal vein and intrahepatic bile duct. These casts can be visualized by clearing or macerating the tissue and can then be analyzed. This technique can be applied to virtually any vascular or ductal system and would be directly applicable to any study inquiring into the development, function, maintenance, or injury of a 3-dimensional ductal or vascular structure.

A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating architecture.

In  organs, the correct architecture of vascular and ductal structures is  indispensable for proper physiological function, and the formation and  ...

Heterotopic Auxiliary Rat Liver Transplantation With Flow-regulated Portal Vein Arterialization in Acute Hepatic Failure

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the failing native liver has regenerated.1-3 The APOLT-method, the orthotopic implantation of auxiliary segments- averts most of the technical problems. However this method necessitates extensive resections of both the native liver and the graft.4 In 1998, Erhard developed the heterotopic auxiliary liver transplantation (HALT) utilizing portal vein arterialization (PVA) (Figure 1). This technique showed promising initial clinical results.5-6 We developed a HALT-technique with flow-regulated PVA in the rat to examine the influence of flow-regulated PVA on graft morphology and function (Figure 2).
A liver graft reduced to 30 % of its original size, was heterotopically implanted in the right renal region of the recipient after explantation of the right kidney.&#160; The infra-hepatic caval vein of the graft was anastomosed with the infrahepatic caval vein of the recipient. The arterialization of the donor&#8217;s portal vein was carried out via the recipient&#8217;s right renal artery with the stent technique. The blood-flow regulation of the arterialized portal vein was achieved with the use of a stent with an internal diameter of 0.3 mm. The celiac trunk of the graft was end-to-side anastomosed with the recipient&#8217;s aorta and the bile duct was implanted into the duodenum. A subtotal resection of the native liver was performed to induce acute hepatic failure. 7
In this manner 112 transplantations were performed. The perioperative survival rate was 90% and the 6-week survival rate was 80%. Six weeks after operation, the native liver regenerated, showing an increase in weight from 2.3&#177;0.8 g to 9.8&#177;1 g. At this time, the graft&#8217;s weight decreased from 3.3&#177;0.8 g to 2.3&#177;0.8 g.
We were able to obtain promising long-term results in terms of graft morphology and function. HALT with flow-regulated PVA reliably bridges acute hepatic failure until the native liver regenerates.

Auxiliary liver transplantation provides a temporary support in acute hepatic failure, until regeneration of the failing liver. The heterotopic auxiliary liver transplantation (HALT) with portal vein arterialization (PVA) renders sufficient liver function. We developed an analogous technique in the rat, to examine the influence of the portal vein arterialization on the morphology and function of the graft.

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the ...

A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation of the animal for surgery, opening the thoracic body wall, exposing the desired rib from the surrounding intercostal muscles, excising the desired section of rib without inducing a pneumothorax, and closing the incisions. Compared to the bones of the appendicular skeleton, the ribs are highly accessible. In addition, no internal or external fixator is necessary since the adjacent ribs provide a natural fixation. The surgery uses commercially available supplies, is straightforward to learn, and well-tolerated by the animal. The procedure can be carried out with or without removing the surrounding periosteum, and therefore the contribution of the periosteum to repair can be assessed. Results indicate that if the periosteum is retained, robust repair occurs in 1 - 2 months. We expect that use of this protocol will stimulate research into rib repair and that the findings will facilitate the development of new ways to stimulate bone repair in other locations around the body.

The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the aorta in mice using the sleeve method was short and easy averaging 20 min, permitting studies of iso/allo grafts. The following article describes the aortic transplantation procedure used in our laboratory. The mice were anesthetized with a mixture of 1.5% volume isoflurane and 100% oxygen through a face mask. At this point, the segment of the aorta between the renal arteries and its bifurcation was separated from the vena cava, freely prepared and clampedat the proximal and distal segments with a single silk suture. Prior to the removal of the aorta, a saline solution containing heparin was injected into the inferior vena cava. Then the aorta was cut between the clamps and a saline heparin solution was used to flush the lumen. The sleeve technique with monofilament sutures was used in order to transplant the abdominal aorta in the orthotopic position.

We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

Evaluating the Procedure for Performing Awake Cystometry in a Mouse Model

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among laboratories. The goal of this study was to describe the microsurgical procedure used to implant an intravesical tube and the experimental technique for recording urinary bladder pressure in an awake, freely moving mouse. In addition, experimental data is presented to show how surgery, as well as tubing type and size, affect lower urinary tract function and recording sensitivity. The effect of tube diameter on pressure recording was assessed in both polyethylene and polyurethane tubing with different internal diameters. Subsequently, the best performing tube from both materials was surgically implanted into the dome of the urinary bladder of male C57BL/6 mice. Twelve-hour, overnight micturition frequency was recorded in healthy, intact animals and animals 2, 3, 5, and 7 days post-surgery. At harvest, bladders were assessed for signs of swelling using gross observation and were subsequently processed for pathological analysis. The greatest extent of bladder swelling was observed on day 2 and 3, which correlated with behavioral voiding data showing significantly impaired bladder function. By day 5, bladder histology and voiding frequency had normalized. Based on the literature and evidence provided by our studies, we propose the following steps for in vivo recording of intravesical pressure and voided volume in an awake mouse: 1) Perform the surgery using an operating microscope and microsurgical tools, 2) Use polyethylene-10 tubing to minimize movement artifacts, and 3) Perform cystometry on post-operative day 5, when bladder swelling resolves.

This study describes the surgical procedures and experimental techniques for performing awake cystometry in a freely moving mouse. In addition, it provides experimental evidence to support its optimization and standardization.

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among ...

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 ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...