We thank P. Chu for performing H&#38;E and histology; and C. Wang and A. McCarty for helpful discussions. Research was supported through funding from the Virginia and D. K. Ludwig Fund for Cancer Research; the National Heart, Lung, and Blood Institute (R01HL058770 and U01HL099999); and the California Institute for Regenerative Medicine (RC1 00354) grants to I.W. Y.R. was supported by the Human Frontier Science Program Career Development Award (CDA00017), the German Research Foundation (RI 2787/1), the Siebel Stem Cell Institute, and the Thomas and Stacey Siebel Foundation (1119368-104-GHBJI). J.M.T. was supported by the NIH (T32GM007365), the National Research Service Award (1F30DK108561), and the Paul and Daisy Soros Fellowship for New Americans.

All animal experiments were carried out in strict accordance with the guidelines set forth by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) and Stanford University&#39;s Administrative Panel on Laboratory Animal Care (APLAC), (Protocol number #10266) and in the United States, or the European Animal Welfare Act, Directive 2010/63/EU. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Government of Bavaria, Germany, and received the permission No: 55.2-1-54-2532-150-2015.
1. Animal Preparation
<ol>
	<li>Prepare an empty cage with appropriate bedding on a heating pad.</li>
	<li>Prior to touching animals with gloves, rub the mother&#39;s bedding onto the gloves.</li>
	<li>Remove all pups from their mother and place into the empty cage. Remove some of the mother&#39;s bedding and place it in the empty cage with the pups.</li>
	<li>Place the mother in a separate, clean, dry cage away from the surgical field.</li>
	<li>Don new, clean sterile gloves prior to surgical field preparation.&#160;</li>
</ol>
2. Surgical Field Preparation
<ol>
	<li>Using a 10-mL pipette, add 10 mL of phosphate buffered saline (PBS) to a 10-cm Petri dish.</li>
	<li>Pipette 2 mL of betadine or equivalent anti-septic solution in the 10-cm Petri dish.</li>
	<li>Place a dissecting scope on the surgical field. Turn on dissecting scope lamp and adjust the level of light to the surgeon&#39;s comfort. A pup can be placed in the surgical field underneath the dissecting scope lamp. Adequate light can be confirmed by the absence of shadows on the ventral surface of the pup.</li>
	<li>Prepare the isoflurane anesthesia chamber (see Table of Materials) with a nose cone. The chamber should be cleaned without evidence of urine or feces. Place the nose cone and associated tubing under the dissecting scope in the surgical field. Divert the flow of oxygen and isoflurane solely to the nose cone.</li>
	<li>Prepare a post-operative recovery area with a heating pad set at approximately 37 &#176;C. The post-operative recovery should consist of 4 pieces of gauze placed on top of the heating pad. If possible, rub gauze pads in the mother&#39;s bedding, feces, and urine to preserve the mother&#39;s scent. 
	NOTE: It is ill-advised to use a heat lamp, as this makes it difficult to control the temperature. Elevated temperatures will result in the death of neonatal mice.</li>
	<li>Prepare and sterilize all surgical instruments with an autoclave. The tools needed include: micro-dissecting scissors, micro-dissecting forceps, gauze, hemostat, and any 6-0 monofilament non-absorbable suture. Surgical instruments can be resterilzed with any glass bead sterilizer.&#160;</li>
</ol>
3. Partial Lobular Hepatectomy
<ol>
	<li>Anesthetize the pup by placing it in the nose cone on its back and gently taping its feet and hands in place. The pup should be receiving 5% isoflurane in oxygen.
	<ol>
		<li>Allow the pup to sit for 5 min or until adequately anesthetized, which can be verified by a toe pinch test.</li>
		<li>Inject 5 mg/kg of carprofen subcutaneously prior to incision. 
		NOTE: The entire surgery should take no longer than 30 min. Poorer outcomes may be observed in neonates that are under general anesthesia for over 30 min. Take precautions to minimize the length of surgery through thorough field preparation and wetting of the skin prior to closure to minimize suture induced skin tears.</li>
	</ol>
	</li>
	<li>Gently clean off the posterior abdominal wall with a small gauze pad wet with betadine. Allow the betadine to dry for 1 min.</li>
	<li>Make a left mid-clavicular 0.5 cm incision immediately below the rib cage with the micro-dissecting scissors and forceps. Gently separate the skin using forceps and make a second deeper incision into the peritoneal cavity (See&#160;Figure 1C, left and center)</li>
	<li>Gently apply lateral pressure from both sides of the abdomen using the back, blunt ends of the micro-dissecting scissors and forceps to force the apex of the left lobe out of the peritoneal cavity. The left apex of the left lobe should be easily visualized (Figure 1C, right).</li>
	<li>From the apex, amputate and weigh the amount of tissue to be removed.
	<ol>
		<li>Using the micro-dissection scissors, gently amputate the desired amount of tissue from the apex of the left lobe.</li>
		<li>Place the amputated tissue into a 1.5-mL tube filled with PBS. Weigh the amputated tissue using an analytical balance (see Table of Materials). 
		NOTE: Place a piece of gauze or paper on the balance and tare it. Then place the amputated tissue on the gauze or paper and measure its mass.</li>
		<li>Fix the amputated area in 2% paraformaldehyde at room temperature for 1 h and place in optimal cutting temperature (OCT) compound over dry ice for frozen section analysis. Analyze frozen sections by cutting 7-10 &#181;m sections using any standard cryostat.</li>
	</ol>
	</li>
	<li>Using a rolled piece of gauze, gently replace the left lobe into the peritoneal cavity. Leave the gauze in the cavity until the bleeding stops.</li>
	<li>Remove the gauze and wet the surgical site and the surrounding area with gauze soaked with PBS.</li>
	<li>Close the surgical site with 6-0 monofilament, non-absorbable sutures with a running stitch. The peritoneum and skin should be closed separately. 
	NOTE: Gently wetting the skin with gauze soaked with PBS may minimize suture induced tears.</li>
	<li>Gently but thoroughly clean the pup with gauze soaked with PBS and ensure no blood or betadine remains. Roll the soaked gauze pad and gently scrub over the wound sign to clean off any blood or betadine. 
	NOTE: This is especially important as the mother may cannibalize the pups if they are cleaned inadequately.</li>
	<li>Remove the pup from the nose cone and place it on the recovery area that includes the gauze pads exposed to the mother&#39;s feces and urine. Once the pup recovers, replace the pup in the empty cage with the mother&#39;s bedding. 
	NOTE: Do not use a heat lamp. The use of a heat lamp can cause the neonate to overheat.</li>
	<li>Repeat the procedure on the desired number of pups. Although all pups can be used, it is generally advisable to leave a few pups not operated on to be replaced together with the operated pups.</li>
	<li>Replace all pups simultaneously with their mother&#39;s bedding in the mother&#39;s cage.</li>
</ol>
4. Recovery and Analysis
<ol>
	<li>Follow up on the mice daily.
	<ol>
		<li>Check that the wound remains closed and remove any dead pups, if present.</li>
		<li>If the wound reopens, prepare the surgical site and recovery area as previously described, and repeat steps 3.8 to 3.12.</li>
		<li>Inject 5 mg/kg of carpofen subcutaneously 24 and 48 h following the procedure.</li>
		<li>Follow Pups for 56 days or more.</li>
	</ol>
	</li>
	<li>Euthanize the animals after the desired amount of post-operative days by carbon dioxide (CO2) exposure and cervical dislocation.
	<ol>
		<li>Place mice in the induction / euthanasia chamber and turn on the CO2 until mice stop breathing.</li>
		<li>Ensure euthanasia by cervical dislocation. Push down on the dorsal neck of the mouse using the fore-finger and thumb and using the other hand, pull down on tail.</li>
	</ol>
	</li>
	<li>Remove the entire liver en bloc and weigh it.
	<ol>
		<li>Carefully separate each lobe and weigh separately. 
		NOTE: Remove the liver by careful dissection of the diaphragm and hepatic and portal vessels. Separate the lobes from each other by carefully dissecting each lobe at its proximal attachment.</li>
	</ol>
	</li>
	<li>Determine the extent of regeneration by comparing the mass of the amputated left lobe to the mass of the whole liver. An uninjured left lobe is approximately 30% of the whole liver.</li>
</ol>

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The authors have no disclosures.

Acute hepatic injury has traditionally been studied using chemical (acetaminophen, ethanol, carbon tetrachloride), or surgical models (70% partial hepatectomy). The regenerative response after 70% partial hepatectomy has been characterized to involve global hepatocyte hypertrophy and multiple rounds of cell division4,5. To stop hemorrhaging, however, this model is limited, as the major vessels must be ligated leaving an abnormal environment for regeneration. Many...

The ability to regenerate an organ, or to restore form and function, has been thought to be mostly lost over evolutionary time. The regenerative potential of the adult mammalian liver after acute chemical or physical injury has been found to involve the mobilization of all remaining hepatocytes resulting in waves of hypertrophy and few rounds of cell division, resulting in a functional but architecturally different organ1,2,3,4,5. Recently, studies have begun to characterize the regenerative response of neonatal mammalian organs to injury within the first week of life6,7,8. These studies have shown that when injured during neonatal development, certain mammalian organs respond with morphological regeneration instead of compensatory growth or fibrosis7,8.
Recent studies have shown that regeneration of both global structure and function occurs during the early neonatal period6,7,8. Established liver injury protocols involve chemical injury or administration of ethanol9,10,11, acetaminophen12,13,14,15, carbon tetrachloride16,17,18,19, 70% partial hepatectomy4,20,21, or removal of the left and median lobes. Chemical administration leads to hepatocyte cell death, but often leaves micro- and macro-structures intact. Morphologic regeneration cannot be readily studied in this context, as the overall hepatic architecture was not obliterated. The 70% partial hepatectomy involves suture ligation of the major vessels, which is necessary to stop bleeding, but leaves a non-physiologic environment with permanent disruption of vasculature. Furthermore, this method has only been used on adult rodents, and its application to neonates is technically extremely difficult. With this in mind, we developed a method in which 20-30% of the apex of the left lobe is removed in a newborn P0 mouse (Figure 1A-1B). This method is surgically conservative, minimally invasive, not technically challenging, and leads to gross loss of morphology without the ligation of vasculature, leaving room for regeneration to occur. The resulting step-by-step protocol, described below, allows for any researcher to perform a partial lobular hepatectomy on neonatal mice in order to study mammalian neonatal regeneration in the early stages of post-natal life. This method also has clear applications to comparative studies in regenerative medicine and stem cell biology, as it can be used in the liver during later stages of life.
The most common acute liver injury studies are chemically-induced damage, adult liver amputation, or 70% partial hepatectomy. Chemical damage often involves intravenous, intraperitoneal or oral administration of acetaminophen, carbon tetrachloride, or ethanol, and is a relatively easy and non-invasive injury model. As previously discussed, chemical damage results in hepatocyte cell death, but often leaves stroma and parenchyma structures intact, making it difficult to make claims about morphologic regeneration. Chemical damage often centers on hepatic vessels, making it a useful technique to study site and cell-specific injury, but also makes it difficult to interrogate, at the whole organ level, other populations that may be situated further from vessels and that may contribute to regeneration. Despite these limitations, chemical damage still remains a useful and highly physiologically relevant injury model.
Adult 70% partial hepatectomy involves the removal of the left and median lobes following ligation of hepatic vasculature. The response to hepatectomy has been well characterized: the amputated liver 14 days post 70% partial hepatectomy develops a grossly different architecture from that of the original undamaged lobe, as the hepatocytes of the remaining right and caudate lobes undergo hypertrophy and a few rounds of cell division4,5. This makes up lost mass and function, but fails to regenerate the two amputated lobes, and therefore does not replace gross morphology. As a result, the injury response to 70% partial hepatectomy is useful to study compensatory growth mechanisms with limited regeneration.
Here, we fully describe a protocol for a neonatal partial lobular hepatectomy. The procedure involves appropriate animal selection and preparation, surgical field preparation, surgery, and recovery. Optimization and adaptation of each of these steps may be required for different applications of the protocol.
We have extensively performed and optimized this protocol on wild type C57BL/6J pups (JAX 000664), however, to study different cell populations and mechanisms of regeneration, we also used various transgenic animals including mice harboring various Cre and CreERT2 transgenes and/or knock-ins (Axin2CreERT2 JAX 018867, and Sox9CreERT2 JAX 018829) in combination with fluorescent reporters, such as the Rainbow and mTmG systems (R26VT2/GK3, R26mT/mG)22,23. We found no need to change this methodology for different mouse strains, as no differences in survival outcomes or regenerative potential were observed.
In addition to using different animal strains, we also performed partial lobular hepatectomies on neonatal mice treated with small molecules, such as 4-hydroxy-tamoxifen and 5-ethynyl-2&#8242;-deoxyuridine (EdU). Dimethyl sulfoxide (DMSO) and ethanol were used as solvents, as it was found that corn oil was a significant cause of morbidity. We otherwise found that intraperitoneal administration of small molecules did not affect survival or regenerative outcomes. We predict that this protocol will be adapted for use with other small molecules to interrogate various aspects of regeneration.
Neonatal mouse surgeries can be technically challenging and may require special expertise in animal handling and microscopic dissection. Animal husbandry expertise is necessary to avoid maternal cannibalism following surgery and during the immediate recovery period.

<table><tbody><tr><td>&lt;strong&gt;Animals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mother with litter of day 0 neonatal pups (any strain)</td><td></td><td></td><td></td></tr><tr><td>Surrogate mother and surrogate litter (optional)</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Standard Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Phosphate Buffered Serum (PBS)</td><td></td><td></td><td></td></tr><tr><td>Providine-iodine or equivalent antiseptic solution</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Surgical Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dissecting microscope</td><td>Zeiss</td><td>ZEMSDV4L MFR # 435421-9901-000</td><td></td></tr><tr><td>3mm straight spring micro scissors</td><td>Vannas</td><td>72932-01</td><td></td></tr><tr><td>5SF Forceps</td><td>Dumont</td><td>11252-00</td><td></td></tr><tr><td>Straight Kelly forceps</td><td>Grainger</td><td>17-050G</td><td></td></tr><tr><td>Heating pad</td><td>Sunbeam</td><td>000771-810-000</td><td></td></tr><tr><td>Isoflurane</td><td>Abbott Labs</td><td>0044-5260-05</td><td></td></tr><tr><td>Rodent Anesthesia System</td><td>Kent Scientific</td><td>1205S</td><td></td></tr><tr><td>Gauze, 10.16 x 10.16cm</td><td>Fisher Scientific</td><td>13-761-52</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Standard Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1.5ml microcentrifuge tube</td><td>Eppendorf</td><td>22363204</td><td></td></tr><tr><td>6-0 monocryl sutures</td><td>Ethicon</td><td>MCP489G</td><td></td></tr><tr><td>Petri dish</td><td>Fisher Scientific</td><td>S35839</td><td></td></tr><tr><td>Pipet-Aid, Plain, 110V</td><td>Drummond</td><td>4-000-110</td><td></td></tr><tr><td>Mettler Toledo NewClassic ME Analytical Balances</td><td>Fisher Scientific</td><td>01-912-402</td><td></td></tr><tr><td>Low Cost Induction Chamber</td><td>Kent Scientific</td><td>SOMNO-0730</td><td></td></tr></tbody></table>

partial lobular hepatectomy: a surgical model for morphologic liver regeneration

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult liver regeneration after 70% partial hepatectomy results in hepatocyte hypertrophy with some replication in remaining lobes with restoration of metabolic activity, but with permanent loss of the injured lobe&#39;s morphology and architecture. Here, we detail a new surgical method in the neonate that leaves a physiologic environment conducive to regeneration. This model involves amputation of the left lobe apex and a subsequent conservative management regimen, and lacks the necessity for ligation of major liver vessels or chemical injury, leaving a physiologic environment where regeneration may occur. We extend this protocol to amputations on juvenile (P7-14) mice, during which the injured liver transitions from organ regeneration to compensatory growth by hypertrophy. The presented, brief 30 min protocol provides a framework to study the mechanisms of regeneration, its age-associated decline in mammals, and the characterization of putative hepatic stem or progenitors.

Figure 1A details a general timeline of the neonatal partial lobular hepatectomy (schematic in Figure 1B), and the expected length of time to wait until regeneration is observed. Subtle regeneration of the left lobe can be observed 7-14 days post surgery. Full regeneration was often observed after 56 days post surgery. Mice should show no signs of physiologic abnormalities after surgery.
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Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration

Here, we present a new method for partial resection of the left hepatic lobe in neonatal (day 0) mice. This new protocol is suitable for studying acute liver injury and injury response in the neonatal setting.

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult ...

partial-lobular-hepatectomy-surgical-model-for-morphologic-liver

Research

JoVE Journal

Developmental Biology

12.1K Views.  Stanford University School of Medicine. Hier presenteren we een nieuwe methode voor gedeeltelijke resectie van de linker hepatische kwab bij neonatale (dag 0) muizen. Dit nieuwe protocol is geschikt voor de studie van acute lever schade en letsel reactie in de neonatale instelling.

Video: Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Medicine

Patient&#45;Derived Hepatocarcinoma Cell Organoids for Liver Regeneration Study

A Hepatocellular Cancer Patient&#45;Derived Organoid Xenograft Model to Investigate Impact of Liver Regeneration on Tumor Growth

Recurrence poses a notable challenge after hepatocellular carcinoma (HCC) treatment, impacting more than 70% of patients who undergo surgical resection. Recurrence stems from undetected micro-metastasis or de novo cancer, potentially triggered by postsurgical liver regeneration. Prior research employed HCC cell lines in orthotopic models to study the impact of liver regeneration, but their limited validity prompted the need for a more representative model. Here, we introduce a novel approach utilizing patient-derived HCC organoids to investigate the influence of liver regeneration on HCC.
Patient tumor tissues are processed to create tumor organoids, embedded in a three-dimensional basement membrane matrix, and cultured in a liver-specific medium. One million organoids are injected into the right superior lobe (RSL) of immunodeficient mice, confirming macroscopic tumor growth through sonography. The intervention group undergoes resection of the left lateral lobe (LLL) (30% of total liver volume) or additionally, the middle lobe (ML) (65% of total liver volume) to induce liver regeneration within the tumor site. The control group experiences re-laparotomy without liver tissue resection. After 2 weeks, both groups undergo tumor and normal tissue explantation.
In conclusion, this patient-derived HCC organoid model offers a robust platform to investigate the impact of liver regeneration post-cancer resection. Its multi-cellular composition, genetic diversity, and prolonged culture capabilities make it an invaluable tool for studying HCC recurrence mechanisms and potential interventions.

Organoids generated from patient tumors are orthotopically injected into the mouse liver. The resection of non-tumorous liver tissue leads to a regenerative environment in the liver tissue where the tumor is located.

Recurrence poses a notable challenge after hepatocellular carcinoma (HCC) treatment, impacting more than 70% of patients who undergo surgical resection. ...

Cancer Research

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Bioengineering

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 ...

Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological conditions. Partial hepatectomy also facilitates the increased engraftment and proliferation of transplanted cells by accelerating neovascularization and cell migration towards the liver. Here, we describe a simple protocol for performing 30% hepatectomy and transplantation of cells in the spleen of a non-obese diabetic/severe combined immunodeficient NOD.SCID (NOD.CB17-Prkdcscid/J) mouse. 
In this procedure, two small incisions are made. The first incision is to expose and resect the left lobe of the liver, and another small incision is made to expose the spleen for the intrasplenic transplantation of cells. This procedure does not require any specialized surgical skills, and it can be completed in 5-7 minutes with less stress and pain, faster recovery, and better survival. We have demonstrated the transplantation of hepatocytes isolated from a green fluorescent protein (GFP) expressing mouse (Transgenic C57BL/6-Tg (UBC-GFP) 30Scha/J), as well as hepatocyte like cells of human origin (NeoHep) in partially hepatectomized NOD.SCID mice.

We have described a protocol for performing partial hepatectomy (PHx) and cell transplantation via spleen in NOD.SCID (NOD.CB17-Prkdcscid/J) mice. In this protocol, an incision is made to expose and resect the left lobe of the liver followed by another incision for the intrasplenic transplantation of cells.

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological ...

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. ...

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream single-cell applications is challenging. A marked accumulation of lipids within regenerating hepatocytes is observed during the first 2 days of normal liver regeneration in mice. This so-called transient regeneration-associated steatosis (TRAS) is temporary but partially overlaps the major proliferative phase. Density-gradient purification is the backbone of most existing protocols for the isolation of primary hepatocytes. As gradient purification relies on the density and size of cells, it separates non-steatotic from steatotic hepatocyte populations. Therefore, fatty hepatocytes often are lost, yielding non-representative hepatocyte fractions. 
The presented protocol describes an easy and reliable method for the in vivo isolation of regenerating hepatocytes regardless of their lipid content. Hepatocytes from male C57BL/6 mice are isolated 24-48 h after hepatectomy by a classic two-step collagenase perfusion approach. A standard peristaltic pump drives the warmed solutions via the catheterized inferior vena cava into the remnant, using a retrograde perfusion technique with outflow through the portal vein. Hepatocytes are dissociated by collagenase for their release from the Glisson's capsule. After washing and careful centrifugation, the hepatocytes can be used for any downstream analyses. In conclusion, this paper describes a straightforward and reproducible technique for the isolation of a representative population of regenerating hepatocytes after partial hepatectomy in mice. The method may also aid the study of fatty liver disease.

Lipid-laden hepatocytes are inherent to liver regeneration but are usually lost upon density-gradient centrifugation. Here, we present an optimized cell isolation protocol that retains steatotic hepatocytes, yielding representative populations of regenerating hepatocytes after partial hepatectomy in mice.

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream ...

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

Studying Liver Regeneration of ALLPS in Animal Models for Staged Hepatectomy

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.

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.

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

Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration

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