The authors acknowledge funding by the German Ministry of Education and Research (BMBF) via the systems biology network "Virtual Liver", grant numbers 0315743 (ExMI), 0315765 (UK Jena), 0315769 (MEVIS).The authors also thank Frank Schubert for technical support.

Procedures involving animal subjects have been approved by Th&#252;ringer Landesamt f&#252;r Verbraucherschutz Abteilung Tiergesundheit und Tierschutz, Germany. Because the portal venous system was visualized separately from the hepatic venous system, separate animals were needed for the different vascular trees.
1. Reagents Preparation
<ol>
	<li>Heparin-saline solution
	<ol>
		<li>Add 0.1 ml heparin into 10 ml saline (5 IU/ml).</li>
	</ol>
	</li>
	<li>Silicone compound mixture
	<ol>
		<li>Add 2 ml MV-120 in one 5 ml tube. Dilute MV-120 by adding 3 ml MV diluent resulting in a 40% solution.</li>
	</ol>
	</li>
</ol>
2. Portal Venous System Silicone Injection
<ol>
	<li>Laparotomy
	<ol>
		<li>Place the mouse in the anesthesia induction chamber and anaesthetize it with 2% isoflurane and 0.3 L/min oxygen.</li>
		<li>Fix the anaesthetized mouse on the operating table using tape with continuous inhalation of 2% isoflurane and 0.3 L/min oxygen. Check the toe-pinch withdrawal reflex of the mouse and start operation if the reflex is absent.</li>
		<li>Make a transverse incision on the abdomen using scissors for the skin layer and an electric coagulator for the muscle layer. Move out the intestines to the left side using cotton tips and cover the intestines with saline soaked gauze.</li>
	</ol>
	</li>
	<li>Catheter insertion
	<ol>
		<li>Dissect portal vein under the microscope using micro-forceps. Place one 6-0 silk suture underneath the extrahepatic portal vein, in approximately 1 mm distance to its bifurcation and tie it loosely for later use.</li>
		<li>Inject prepared heparin-saline solution via penile vein (male) or inferior vena cava (female) for systemic heparinization for 5 min.</li>
		<li>Insert the 26-gauge (26 G) catheter with needle into portal vein and fix it with a clamp</li>
	</ol>
	</li>
	<li>Heparin-saline solution and silicone compound perfusion
	<ol>
		<li>Load heparin-saline solution in 5 ml syringe and turn on perfusion device. Fill the catheter fully with heparin-saline solution to avoid air bubbles.</li>
		<li>Connect extension tube to the catheter and fix them tightly. Start heparin-saline perfusion with a perfusion rate of 0.4 ml/min.</li>
		<li>Ligate pre-placed 6-0 silk suture for double fixing the catheter and blocking blood flow from splenic vein and mesenteric vein. Euthanatize the mouse by exsanguination via perfusion under anesthesia.</li>
		<li>Rinse the liver using saline during the whole perfusion procedure in order to keep it moist.</li>
		<li>Add 0.1 ml curing agent into the MV-120 tube. Change heparin-saline syringe to silicone syringe.</li>
		<li>Start silicone perfusion with a perfusion rate of 0.2 ml/min for approximately 1 min to prefill the catheter system and to fill the portal vein system. Stop silicone perfusion when the vessels on the surface turn blue.</li>
	</ol>
	</li>
	<li>Sampling
	<ol>
		<li>Keep the liver in-situ until the silicone is fully polymerized after approximately 15 to 30 min. Dissect ligaments connecting liver and adjacent organs with care to keep liver intact. Explant the liver and put it into formalin for fixation.</li>
	</ol>
	</li>
</ol>
3. Hepatic Venous System Silicone Injection
<ol>
	<li>Perform laparotomy as performed in step 2.1 and expose operative field fully.</li>
	<li>Catheter insertion
	<ol>
		<li>Dissect portal vein under the microscope using micro-forceps. Place one 6-0 silk suture underneath the extrahepatic portal vein, in approximately 1 mm distance to its bifurcation and tie it loosely for later use.</li>
		<li>Inject prepared heparin-saline solution via penile vein (male) or inferior vena cava (female) for systemic heparinization for 5 min.</li>
		<li>Insert one 26 G catheter (catheter 1) with needle into portal vein and fix it with a clamp. Insert another 26 G catheter (catheter 2) with needle into inferior vena cava and fix it with a clamp.</li>
		<li>Ligate the branches of inferior vena cava (including left and right renal veins) and its distal end using 6-0 synthetic, monofilament, nonabsorbable polypropylene suture.</li>
	</ol>
	</li>
	<li>Heparin-saline solution and silicone compound perfusion
	<ol>
		<li>Load heparin-saline solution in 5 ml syringe and turn on perfusion device. Fill catheter 1 completely with heparin-saline solution to avoid air bubbles. Connect extension tube to catheter 1 and fix it tightly. Start heparin-saline perfusion at a rate of 0.4 ml/min.</li>
		<li>Ligate pre-placed 6-0 silk suture for double fixing the catheter and blocking blood flow from splenic vein and mesenteric vein. Euthanatize the mouse by exsanguination via perfusion under anesthesia.</li>
		<li>Rinse the liver using saline during the whole perfusion procedure in order to keep it moist. Add 0.1 ml curing agent into the MV-120 tube. Exchange heparin-saline syringe with silicone syringe.</li>
		<li>Place a clamp on the suprahepatic inferior vena cava to obstruct the outflow of the liver.</li>
		<li>Connect the extension tube to catheter 2 and start silicone perfusion with a perfusion rate of 0.2 ml/min for approximately 2 min to reach an objective hepatic vascular volume as reported. Stop silicone perfusion when the vessels on the surface turn blue.</li>
	</ol>
	</li>
	<li>Sampling
	<ol>
		<li>Dissect hepatic ligaments avoiding any injury to the liver. Explant the liver and put it into formalin for fixation.</li>
	</ol>
	</li>
</ol>
4. Micro-CT (&#181;CT) Scanning
To scan the explanted liver sample using &#181;CT, the following steps are needed.
<ol>
	<li>Take the liver sample out of the fixation solution. Place the liver onto the &#181;CT bed. Put the &#181;CT bed with the liver sample into the &#181;CT.</li>
	<li>Acquire topogram before starting the scan. Use one subscan for the small liver sample and two subscans for large samples.</li>
	<li>Select a &#181;CT protocol with a high resolution (e.g., HQD-6565-390-90). This protocol acquires 720 projections with 1,032 x 1,012 pixels during one full rotation with the scanning time of 90 sec per sub-scan. Start the &#181;CT scan.</li>
</ol>
5. Histological Serial Sections
<ol>
	<li>Embed the liver specimen in paraffin as a whole after &#181;CT scanning. Cut whole paraffin sample into 4&#181;m sections, resulting in a series of 2,000 to 2,500 sections.</li>
	<li>Stain sections with appropriate staining technique to visualize molecular events of interest such as Ki-67 as proliferation marker and HMGB1 as a marker of ischemic damage. Determine sequence of staining in respect to scientific question.</li>
	<li>Use a whole slide scanner to digitalize the stained sections.</li>
	<li>Perform 3D-reconstruction of vascular tree(s) (already feasible) and visualize 3D-distribution of molecular events in respect to vascular tree (research in progress).</li>
</ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+parameter+values+for+physiologically+based+pharmacokinetic+models.">Physiological parameter values for physiologically based pharmacokinetic models.</a> Toxicol.Ind.Health. 13 (4), 407-484 (1997).</li><li>Dai, D., 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=Elastase-Induced+Intracranial+Dolichoectasia+Model+in+Mice.">Elastase-Induced Intracranial Dolichoectasia Model in Mice.</a> Neurosurgery. , (2015).</li><li>Ding, B. S., 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=Inductive+angiocrine+signals+from+sinusoidal+endothelium+are+required+for+liver+regeneration.">Inductive angiocrine signals from sinusoidal endothelium are required for liver regeneration.</a> Nature. 468 (7321), 310-315 (2010).</li><li>Downey, C. 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=Quantitative+ex-vivo+micro-computed+tomographic+imaging+of+blood+vessels+and+necrotic+regions+within+tumors.">Quantitative ex-vivo micro-computed tomographic imaging of blood vessels and necrotic regions within tumors.</a> PLoS.One. 7 (7), 41685 (2012).</li><li>Ehling, 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=CCL2-dependent+infiltrating+macrophages+promote+angiogenesis+in+progressive+liver+fibrosis.">CCL2-dependent infiltrating macrophages promote angiogenesis in progressive liver fibrosis.</a> Gut. , (2014).</li><li>Ehling, 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=Micro-CT+imaging+of+tumor+angiogenesis:+quantitative+measures+describing+micromorphology+and+vascularization.">Micro-CT imaging of tumor angiogenesis: quantitative measures describing micromorphology and vascularization.</a> Am.J.Pathol. 184 (2), 431-441 (2014).</li><li>Ghanavati, S., Yu, L. 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L., 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=Radiomorphometric+quantitative+analysis+of+vasculature+utilizing+micro-computed+tomography+and+vessel+perfusion+in+the+murine+mandible.">Radiomorphometric quantitative analysis of vasculature utilizing micro-computed tomography and vessel perfusion in the murine mandible.</a> Craniomaxillofac.Trauma Reconstr. 5 (4), 223-230 (2012).</li><li>Melloul, E., 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=Small+animal+magnetic+resonance+imaging:+an+efficient+tool+to+assess+liver+volume+and+intrahepatic+vascular+anatomy.">Small animal magnetic resonance imaging: an efficient tool to assess liver volume and intrahepatic vascular anatomy.</a> J.Surg.Res. 187 (2), 458-465 (2014).</li><li>Schwier, M., Bohler, T., Hahn, H. 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The authors declare that they have no competing financial interests.

Contrasting the vascular tree by silicone injection and &#181;CT scanning has been introduced in tumor models and neurological disease models frequently to study the angiogenic progression5,7,8,10. Improvements in methodology of silicone injection were made in the present study for visualizing and quantifying vascular growth after partial hepatectomy in mice.
There are a number of critical steps needing attention to achieve good perfusion quality. First of all, systemic heparinizati...

Liver regeneration is often determined by measuring the increase of liver weight and volume and by assessing the hepatocyte proliferation rate16. However, liver regeneration is not only inducing parenchymal regeneration but also vascular regeneration6. Therefore, vascular growth should be further investigated with respect to its role in the progression of liver regeneration. Visualization of the hepatic vascular system is critical to advancing our understanding of vascular regeneration. Numerous indirect methods have been developed to study the underlying molecular mechanisms of hepatic vascular regeneration. Traditionally, detection of cytokines (vascular endothelial growth factor, VEGF)14, chemokines and their receptors (CXCR4/CXCR7/CXCL12)4 have been the mainstay for studying vascular regeneration. However, a 3D model together with quantitative analysis of the vasculature would add critical anatomic information to gain a better understanding of the important relationship between hepatic parenchymal and vascular regeneration.
To visualize the hepatic vascular system, which requires contrasting the vascular trees, mice were injected with a radiopaque silicone rubber contrast agent directly into the portal or hepatic venous vascular tree. After polymerization of the silicone and explantation of the organ, the liver samples were subjected to &#181;CT scanning using a CT scanner. The scans resulted in voxel image representations of the silicone-injection specimens9.
For quality control, the vascular system was first visualized in 3D using preclinical software. Segmentation was performed by setting a threshold between the soft tissue intensity and the vessel intensity. The resulting vessel mask was visualized using surface rendering. The software also allowed for manual determination of two parameters of vascular growth: maximal vessel length and radius.
A preclinical software was then used for 3D reconstruction of vascular trees and subsequent calculation of the supplying or draining vascular territories13. In addition, this software automatically determined certain parameters of vascular growth, such as the total length of all visible vascular structures also known as the total edge length or total vessel volume.
The silicone perfusion procedure was performed in naive mice and in mice that underwent 70% partial hepatectomy (PH). Livers were collected at different observation time points after resection for analyzing vascular and parenchymal liver regeneration using the aforementioned visualization and quantification technique.
The main goals of this film are to: (1) demonstrate the delicate injection-technique required to achieve optimal contrasting and (2) show the potential benefit resulting from a detailed analysis of the resulting specimen using &#181;CT and histological serial sections. After watching this film, the reader should have a better understanding of how to inject silicone compound into a specific vascular system and of the usefulness and applicability of the technique.

<table><tbody><tr><td>PERFUSOR&amp;reg; VI</td><td>B.BRAUN</td><td>87 222/0</td><td></td></tr><tr><td>Pipetus&amp;reg;-akku</td><td>Hirschmann</td><td>9907200</td><td></td></tr><tr><td>Pipets</td><td>Greiner</td><td>606180</td><td></td></tr><tr><td>micro scissors</td><td>Fine Science Tools (F&amp;middot;S&amp;middot;L)</td><td>No. 14058-09</td><td></td></tr><tr><td>micro serrefine</td><td>Fine Science Tools (F&amp;middot;S&amp;middot;L)</td><td>No.18055-05</td><td></td></tr><tr><td>Micro clamps applicator</td><td>Fine Science Tools (F&amp;middot;S&amp;middot;L)</td><td>No. 18057-14</td><td></td></tr><tr><td>Straight micro forceps</td><td>Fine Science Tools (F&amp;middot;S&amp;middot;L)</td><td>No. 00632-11</td><td></td></tr><tr><td>Curved micro forceps</td><td>Fine Science Tools (F&amp;middot;S&amp;middot;L)</td><td>No. 00649-11</td><td></td></tr><tr><td>needle-holder</td><td>Fine Science Tools (F&amp;middot;S&amp;middot;L)</td><td>No. 12061-01</td><td></td></tr><tr><td>1 ml syringe</td><td>B.Braun</td><td>9161406V</td><td></td></tr><tr><td>5 ml syringe</td><td>B.Braun</td><td>4606051V</td><td></td></tr><tr><td>extension and connection lines</td><td>B.Braun</td><td>4256000</td><td>30 cm, inner &amp;oslash; 1.2 mm</td></tr><tr><td>6-0 silk (Perma-Hand Seide)</td><td>Ethicon</td><td>639H</td><td></td></tr><tr><td>6-0 prolene</td><td>Ethicon</td><td>8711H</td><td></td></tr><tr><td>Microfil&amp;reg; MV diluent</td><td>FLOW TECH, INC</td><td></td><td></td></tr><tr><td>Microfil&amp;reg; MV - 120</td><td>FLOW TECH, INC</td><td>MV - 120 (blue)</td><td></td></tr><tr><td>MV curing agent</td><td>FLOW TECH, INC</td><td></td><td></td></tr><tr><td>Heparin 2500 I.E./5 ml</td><td>Rotexmedica</td><td>ETI3L318-15</td><td></td></tr><tr><td>Saline</td><td>Fresenius Kabi Deutschland GmbH</td><td>E15117/D DE</td><td></td></tr><tr><td>Imalytics Preclinical software</td><td>Experimental Molecular Imaging, RWTH Aachen University, Germany</td><td></td><td></td></tr><tr><td>HepaVision</td><td>Fraunhofer MEVIS, Bremen, Germany</td><td></td><td></td></tr><tr><td>NanoZoomer 2.0-HT&amp;nbsp;Digital slide scanner</td><td>Hamamatsu Electronic Press, Japan&amp;nbsp;</td><td>C9600</td><td></td></tr><tr><td>Tomoscope Duo CT&amp;nbsp;</td><td>CT Imaging GmbH, Erlangen, Germany</td><td>TomoScope&amp;reg;&amp;nbsp;Synergy</td><td></td></tr></tbody></table>

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.

Quality Criteria
The quality of silicone injection can be judged with the naked eye during the procedure. The small vessels on liver surface fill gradually with the blue compound. If the normal vascular structure was observed on the liver surface, the silicone rubber injection quality was good. If the perfusion volume was insufficient, the small vessels on the liver surface were not fully filled. In contrast, ov...

Watch this Scientific Journal Video about Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice at JoVE.com

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

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 ...

visualization-vascular-parenchymal-regeneration-after-70-partial

Research

JoVE Journal

Medicine

8.0K Views.  Jena University Hospital. 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 µCT and histological serial sections.

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

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.

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 ...

Developmental Biology

Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable during renal transplantation. Experimental models that accurately and reproducibly recapitulate renal IRI are crucial in dissecting the pathophysiology of AKI and the development of novel therapeutic agents. Presented here is a mouse model of renal IRI that results in reproducible AKI. This is achieved by a midline laparotomy approach for the surgery with one incision allowing both a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia of the left kidney. By careful monitoring of the clamp position and body temperature during the period of ischaemia this model achieves reproducible functional and structural injury. Mice sacrificed 24 hr&#160;following surgery demonstrate loss of renal function with elevation of the serum or plasma creatinine level as well as structural kidney damage with acute tubular necrosis evident. Renal function improves and the acute tissue injury resolves during the course of 7 days following renal IRI such that this model may be used to study renal regeneration. This model of renal IRI has been utilized to study the molecular and cellular pathophysiology of AKI as well as analysis of the subsequent renal regeneration.

The mouse model of renal ischaemia reperfusion injury described here comprises of a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia that results in acute kidney injury. This model uses a midline laparotomy approach with all steps performed via one incision.

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable ...

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

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 ...

Liver Transplantation in Mouse and Analyzing Allograft Patency

Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality images to be obtained of micro-vasculature and are high-fidelity tools in the field of organ transplantation. In this model of orthotopic liver transplantation (OLT) in mice, microCT affords the opportunity to evaluate allograft anastomosis in real time and has the added benefit of not having to sacrifice study animals. The choice of contrast, as well as image acquisition settings, create a high-definition image, which gives researchers invaluable information. This allows for evaluation of the technical aspects of the procedure as well as potentially evaluating different therapeutics over an extended duration of time. In this protocol, we detail an OLT model in mice in a stepwise fashion and finally describe a microCT protocol that can give high-quality images, which aid researchers in in-depth analysis of solid organ transplantation. We provide a step-by-step guide for liver transplantation in a mouse, as well as briefly discuss a protocol for evaluating the patency of the graft through microCT angiography.

Author Spotlight: Enhancing Transplantation Research Through MicroCT Angiography in Murine Models 

In this protocol, we discuss the implementation of a model of successful orthotopic liver transplantation (OLT) in mice. Additionally, adjuvants to further analyze allograft patency after successful OLT in a mouse are discussed as well, specifically utilizing microcomputed tomography (microCT) scans.

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality ...

Strategies for Efficient Vascular Anastomosis in Mouse Liver Transplantation

Vascular Reconstruction with the Cuff Technique in Mouse Orthotopic Liver Transplantation

Mouse orthotopic liver transplantation is an effective methodology for investigating the underlying mechanisms of liver ischemia and reperfusion injury. However, the technical challenges pose a barrier to utilizing this valuable experimental model and passing on these skills to the next generation. The most challenging aspect of this procedure is vascular reconstruction, including the portal vein (PV), infrahepatic inferior vena cava (IHIVC), and suprahepatic inferior vena cava. The use of plastic cuffs, rather than sutures, allows for smoother PV and IHIVC reconstruction. Vessels are reconstructed by attaching a cuff made from an intravenous catheter to the tip of the graft vessel and interposing the cuff into the recipient vessel. The two most crucial aspects are properly visualizing the inner lumen of the vessel and avoiding the use of excessive force. Our aim is to provide a technical overview of vascular reconstructions using the cuff technique in recipient surgery. These technical tips for the cuff technique are expected to help microsurgeons facilitate vascular reconstruction and advance their research.

Author Spotlight: Exploring Cold Ischemia and Warm Reperfusion Injury in Fatty Liver Transplantation

This protocol provides technical information for vessel reconstruction using the cuff technique in mouse orthotopic liver transplantation.

Mouse orthotopic liver transplantation is an effective methodology for investigating the underlying mechanisms of liver ischemia and reperfusion injury. ...

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 ...

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 ...

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

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