This work is supported by the SFB 824 subprojects C6 and C7 (DFG Sonderforschungsbereich 824), German Research Foundation, Bonn, Germany.

Note: The following steps have been performed in accordance with the guidelines of our institution and the local government of Bavaria, Germany. All attempts to reproduce this protocol must be made in adherence with the local guidelines for the humane treatment of animals, as dictated by local animal care and use committee. For example, many institutes require that sterile gloves are worn during rodent surgery.&#160;Furthermore, work with viruses must be performed according to local regulations, taking care for personal and environmental safety. Care must be taken to properly dispose of contaminated waste and to clean instruments and workspace (i.e., by autoclaving and cleaning with an appropriate antiviral disinfectant according to the manufacturer&#39;s instructions).
1. Preparations Before Beginning Surgery
<ol>
	<li>Sterilize surgical instruments (i.e., by autoclaving).</li>
	<li>Dilute the virus to the appropriate concentration, which will vary depending on the toxicity of the specific virus being used and the susceptibility of the rat strain. 
	Note: For example, when using vesicular stomatitis virus in male Buffalo rats, we typically inject 107 pfu in a 1 ml volume, which is the maximum tolerated dose in that model.
	<ol>
		<li>Prefill the virus (using aseptic technique) &#160;in 1 ml syringes, and attach 30 G needles, taking care to eliminate air from the syringe and needle.</li>
	</ol>
	</li>
	<li>Prepare the surgical space with all materials and equipment required, and spray the area with ethanol to disinfect. Fill a small receptacle with sterile saline solution (0.9% NaCl) for moistening gauze swabs and cotton-tipped applicators.</li>
</ol>
2. Preparation of the Rat
<ol>
	<li>Administer an analgesic (as specified by the local animal care and use committee), at the appropriate dose 30 min prior to beginning the surgical procedure. For example, administer a high dose of metamizole (100-200 mg/kg).</li>
	<li>Anesthetize the rat using inhalation (isoflurane) or injectable (i.e., a mixture of Medetomidine (0.15 mg/kg), Midazolam (2 mg/kg), and Fentanyl (0.005 mg/kg), also known as MMF) anesthesia. Administer isolflurane through a vaporizer at 1-3% and adjust as necessary to maintain normal respiration and heart rate.
	<ol>
		<li>Before proceeding, ensure that the rat is fully sedated and unable to feel pain by pinching the foot pad and observing for a reaction.</li>
		<li>Apply a veterinary eye ointment to prevent dryness during anesthesia.</li>
	</ol>
	</li>
	<li>Shave the abdominal area from the sternum down to just above the genitals using a small veterinary hair clipper. Take care to achieve a close shave without injuring the skin.</li>
	<li>Fix the rat to the surgical bed using tape on the fore- and hindlimbs, and, if using inhalation anesthesia, place the snout into a suitably-sized nose cone. Place a heating pad underneath the rat to maintain body temperature. Disinfect the abdominal area using&#160;a surgical grade disinfectant, followed by an alcohol rinse and application of an antiseptic paint.&#160;</li>
</ol>
3. Laparotomy
<ol>
	<li>Using a small curved scalpel, such as #15, make a small vertical incision in the skin layer directly along the midline. Extend the incision up to the xyphoid process. Next, incise the muscle layer by gently lifting up with a pair of Adson forceps to make a scalpel incision, and then extend the incision up to the xyphoid process with surgical scissors. Take care not to puncture the organs of the abdominal cavity or to damage the small vessels on either side of the xyphoid process.</li>
	<li>Place a retractor across the abdomen to spread the muscle and skin and expose the entire abdominal area.</li>
	<li>Moisten two 7.5 x 7.5 cm2 gauze swabs with sterile&#160;physiological saline and spread one across the top of the surgical opening and one across the bottom, as shown in Figure 2A.</li>
</ol>
4. Isolation of the Hepatic Artery
<ol>
	<li>Using two moistened cotton applicator swabs moistened with sterile physiological saline, gently lift the intestines and cecum&#160;out of the abdominal cavity, and place them on the lower gauze swab. Fold the gauze over to keep the organs moist and to hold them out of the way.</li>
	<li>Using two moistened cotton applicator swabs, gently lift up the left lateral liver lobe, which will be the most predominant lobe in view at this stage. Flip the lobe upwards, and, using a small pair of spring scissors, cut through the fibrous membrane on the underside of the lobe, allowing the lobe to be completely flipped upwards and rest on top of the previously placed gauze swab. Fold the gauze swab over the lobe to hold it in place.</li>
	<li>Continue with the next lobe (the anterior caudate), which is now the most anterior lobe in view. With the aid of a dissecting microscope and fine forceps, dissect the thin membrane surrounding the lobe in order to release it and allow it to be freely flipped upwards. Place the lobe underneath the gauze swab together with the left lateral lobe.</li>
	<li>Observe the posterior caudate lobe, which will now be visible. Repeat step 4.3 to completely dissect the posterior caudate lobe and flip it upwards and inside the gauze together with the previously placed liver lobes.</li>
	<li>Just beneath the original position of the caudate lobe, find the common hepatic artery, which will now be visible and easily recognized by its strong pulsation. Following the artery to the anatomical right, observe the thick membranous material obscuring the gastroduodenal and proper hepatic arteries from view. Using fine forceps, lift up this material and gently tear it, taking care to avoid any small vessels in this area. Use cotton applicator swabs to stop any bleeding which might occur.
	<ol>
		<li>Locate the arterial branches, which will be clearly visible with the aid of a dissecting microscope when the membranes are properly dissected.</li>
	</ol>
	</li>
	<li>Using two fine, atraumatic forceps, carefully dissect the common hepatic artery, gastroduodenal artery, and the proper hepatic artery such that they are freed from the surrounding membranes (Figure 2B).</li>
</ol>
5. Preparation of the Artery for Injection
<ol>
	<li>Pass a 7-0 Prolene suture underneath the gastroduodenal artery, and use a micro-needle-holder to tie a permanent ligature at the distal end, just above the bifurcation (see Figure 1 for proper placement). Allow approximately 2 inches of suture material remaining on the ends, and clamp them with a needle holder to pull the artery taught, as in Figure 2C.</li>
	<li>Place a small clamp on the common hepatic artery to temporarily block blood flow (Figure 2C). 
	Note: This is important to prevent massive bleeding once the needle is removed from the artery. Furthermore, the blockage of arterial blood flow will allow for slower virus administration and better transduction efficiency.
	<ol>
		<li>Important: Work quickly to limit the amount of time that blood flow is stopped - perform the injection and subsequent ligation of the artery within 5 min of clamping the common hepatic artery.</li>
	</ol>
	</li>
	<li>If possible, increase the magnification on the dissecting microscope, such that the gastroduodenal artery is in sharp focus and appears large enough to accurately insert the 30 G needle. Place the needle of the injection syringe into the lumen of the gastroduodenal artery and slowly push on the plunger. 
	Note: Proper placement of the needle will result in the injected solution flowing directly through the proper hepatic artery and towards the liver.
	<ol>
		<li>Perform the injection very slowly to maximize transduction efficiency.</li>
	</ol>
	</li>
	<li>Once the entire injection volume has been administered, slowly retract the needle from the artery. 
	Note: There should be no bleeding from the injection site. In the case that bleeding does occur, it indicates that the clamp is not properly placed and should be quickly adjusted.</li>
</ol>
6. Closing
<ol>
	<li>Pass a second 7-0 Prolene suture beneath the gastroduodenal artery, and tie a second permanent ligature above the injection site.</li>
	<li>Remove the clamp from the common hepatic artery. Confirm that blood flow through the proper hepatic artery towards the liver has been restored.
	<ol>
		<li>Cut the loose ends of the ligatures.</li>
	</ol>
	</li>
	<li>Confirm that there is no bleeding before returning the intestines and liver to the abdominal cavity. Take care that the appendix is returned in its original orientation.</li>
	<li>Suture the muscle and skin in separate layers using 4-0 vicryl suture material with a curved needle. Place continuous sutures approximately 1 mm apart and pull tightly.
	<ol>
		<li>During the suturing phase, administer an analgesic such as buprenorphine (0.05 mg/kg).</li>
	</ol>
	</li>
	<li>Remove rat from anesthesia. If isoflurane has been used, the rat should regain consciousness within several minutes. If MMF has been used as an injectable anesthesia, antagonize it with a subcutaneous injection of Atipamezole (0.75 mg/kg), Flumazenil (0.2 mg/kg), and Naloxone (9.12 mg/kg), which will reverse the effects of the anesthesia and allow the animals to regain consciousness within 20 min. Keep rats warm with an infrared warming lamp during the initial wakening phase.
	<ol>
		<li>Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency.</li>
		<li>Do not return an animal that has undergone surgery to the company of other animals until fully recovered.</li>
	</ol>
	</li>
	<li>Monitor rats post-operatively for pathological signs of distress, and follow-up with analgesics as required by the local animal care and use committee. For example, buprenorphine can be administered at 0.05 mg/kg every 12 hr.</li>
</ol>

<ol>
	<li>Murray, C. J., Lopez, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence-based+health+policy+-+lessons+from+the+Global+Burden+of+Disease+Study.">Evidence-based health policy - lessons from the Global Burden of Disease Study.</a> Science. 274, 740-743 (1996).</li><li>Parkin, D. M., Bray, F., Ferlay, J., Pisani, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+world+cancer+burden:+Globocan+2000.">Estimating the world cancer burden: Globocan 2000.</a> Int J Cancer. 94, 153-156 (2001).</li><li>Bruix, J., Sala, M., Llovet, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoembolization+for+hepatocellular+carcinoma.">Chemoembolization for hepatocellular carcinoma.</a> Gastroenterology. 127, 179-188 (2004).</li><li>Miraglia, R., Pietrosi, G., Maruzzelli, L., Petridis, I., Caruso, S., Marrone, G., Mamone, G., Vizzini, G., Luca, A., Gridelli, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+transcatheter+embolization/chemoembolization+(TAE/TACE)+for+the+treatment+of+single+hepatocellular+carcinoma.">Efficacy of transcatheter embolization/chemoembolization (TAE/TACE) for the treatment of single hepatocellular carcinoma.</a> World J Gastroenterol. 13, 2952-2955 (2007).</li><li>Breedis, C., Young, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood+supply+of+neoplasms+in+the+liver.">The blood supply of neoplasms in the liver.</a> Am J Pathol. 30, 969-977 (1954).</li><li>Nakashima, T., Kojiro, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathologic+characteristics+of+hepatocellular+carcinoma.">Pathologic characteristics of hepatocellular carcinoma.</a> Semin Liver Dis. 6, 259-266 (1986).</li><li>Heo, 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=Randomized+dose-finding+clinical+trial+of+oncolytic+immunotherapeutic+vaccinia+JX-594+in+liver+cancer.">Randomized dose-finding clinical trial of oncolytic immunotherapeutic vaccinia JX-594 in liver cancer.</a> Nat Med. 19, 329-336 (2013).</li><li>Altomonte, J., Marozin, S., Schmid, R. M., Ebert, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+newcastle+disease+virus+as+an+improved+oncolytic+agent+against+hepatocellular+carcinoma.">Engineered newcastle disease virus as an improved oncolytic agent against hepatocellular carcinoma.</a> Mol Ther. 18, 275-284 (2010).</li><li>Shinozaki, K., Ebert, O., Kournioti, C., Tai, Y. S., Woo, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncolysis+of+multifocal+hepatocellular+carcinoma+in+the+rat+liver+by+hepatic+artery+infusion+of+vesicular+stomatitis+virus.">Oncolysis of multifocal hepatocellular carcinoma in the rat liver by hepatic artery infusion of vesicular stomatitis virus.</a> Mol Ther. 9, 368-376 (2004).</li><li>Shinozaki, K., Ebert, O., Woo, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eradication+of+advanced+hepatocellular+carcinoma+in+rats+via+repeated+hepatic+arterial+infusions+of+recombinant+VSV.">Eradication of advanced hepatocellular carcinoma in rats via repeated hepatic arterial infusions of recombinant VSV.</a> Hepatology. 41, 196-203 (2005).</li><li>Altomonte, 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=Synergistic+antitumor+effects+of+transarterial+viroembolization+for+multifocal+hepatocellular+carcinoma+in+rats.">Synergistic antitumor effects of transarterial viroembolization for multifocal hepatocellular carcinoma in rats.</a> Hepatology. 48, 1864-1873 (2008).</li></ol>

The authors have nothing to disclose.

Although direct intratumoral injection is undoubtedly the simplest method to result in efficient tumor transduction of a single tumor nodule, hepatic arterial infusion represents an ideal administration route to target multifocal, orthotopic HCC. This method has proven to be both safe and effective for treating HCC in immune competent rats with oncolytic viruses. Furthermore, since HCC patients are routinely treated by transarterial application of chemoembolization, the method described here is readily translatable to ...

Hepatocellular carcinoma (HCC) is the fifth most prevalent cancer worldwide, and the third leading cause of cancer-related death, making it a significant health concern1,2. For patients who are not eligible for tumor resection, or those awaiting liver transplantation, locoregional therapy involving transarterial embolization (TAE) or transarterial chemoembolization (TACE) are applied as standard palliative care3,4. These therapies exploit the unique feature of dual blood supply in the liver whereby tumors are fed almost exclusively by hepatic arterial blood flow, while the surrounding liver receives the majority of its blood supply from the portal vein5,6.
Due to the extremely limited efficacies of established therapies for HCC, oncolytic viruses have emerged as promising alternative therapeutics. JX-594, recently renamed Pexa-Vec, is a thymidine kinase-deleted vaccinia vector, armed with granulocyte-macrophage colony-stimulating factor (GM-CSF), which has completed phase II clinical trial for HCC7. More recently, a recombinant vesicular stomatitis virus vector (VSV) expressing human interferon-beta has entered a phase I clinical trial for sorafenib-refractory HCC (NCT01628640). As oncolytic viruses move closer to obtaining approval for clinical application for HCC in patients, the need for an effective administration route to target multifocal disease is evident. While systemic delivery is largely ineffective due to inefficient tumor transduction, intratumoral applications could limit the efficacy of the therapy to the injected tumor, leaving uninjectable microscopic lesions susceptible to disease progression.
We have established a method of isolating the hepatic artery in rats to administer oncolytic virus therapy in a locoregional manner to target orthotopic HCC. We have demonstrated that this administration route results in safe and effective transduction of multifocal HCC nodules, resulting in significant survival prolongation in immune competent rats8-10. Here, we describe the method of accessing, dissecting, and injecting into the hepatic artery in rats. A scheme of the procedure is shown in Figure 1 (previously published9)

<table><tbody><tr><td>Veterinary clippers</td><td>Aesculap</td><td>GT415</td><td>Small, cordless trimmer ideal for removing fur from surgical area</td></tr><tr><td>Stereomicroscope</td><td>Zeiss</td><td>Stemi SV6</td><td></td></tr><tr><td>30 G Needles</td><td>Braun</td><td>4656300</td><td>30 G x &amp;frac12;&amp;rdquo;</td></tr><tr><td>1 ml syringes</td><td>Braun</td><td>9161406V</td><td>Tuberculin syringe</td></tr><tr><td>Disposable scalpel</td><td>Feather</td><td>2975#15</td><td>#15 blade&amp;nbsp;</td></tr><tr><td>Standard surgical scissors</td><td>Fine Science Tools</td><td>14001-13&amp;nbsp;</td><td>Sharp/blunt, for opening skin and muscle</td></tr><tr><td>Adson forcep</td><td>Fine Science Tools</td><td>1101-12</td><td>With teeth, for grasping skin and muscle</td></tr><tr><td>Alm retractor</td><td>Fine Science Tools</td><td>17008-07</td><td>With blunt teeth, for spreading abdominal cavity open during surgery</td></tr><tr><td>Gauze swabs</td><td>Lohmann &amp;amp; Rauscher</td><td>18504</td><td>7.5 cm x 7.5 cm, should be autoclaved prior to use</td></tr><tr><td>Cotton-tipped applicator swabs</td><td>Lohmann &amp;amp; Rauscher</td><td>11970</td><td>Sterile</td></tr><tr><td>Fine-tipped foreceps</td><td>Fine Science Tools</td><td>11063-07</td><td>0.4 mm, angled tip, for dissecting hepatic artery</td></tr><tr><td>Vannas spring scissors</td><td>Fine Science Tools</td><td>91500-09</td><td>For delicate cutting</td></tr><tr><td>Micro-needle holder</td><td>Fine Science Tools</td><td>12076-12</td><td>For ligating gastroduodenal artery</td></tr><tr><td>Needle holder</td><td>Fine Science Tools</td><td>12005-15</td><td>Tungsten carbide jaws</td></tr><tr><td>7-0 Prolene sutures</td><td>Ethicon</td><td>8648H</td><td>Polypropylene suture with curved needle, for ligating gastroduodenal artery</td></tr><tr><td>4-0 Vicryl sutures</td><td>Ethicon</td><td>V3040H</td><td>With curved needle attached</td></tr><tr><td>Infrared warming lamp</td><td>Beurer</td><td>IL11</td><td>For maintaining body temperature post-operatively</td></tr></tbody></table>

transarterial administration of oncolytic viruses for locoregional therapy of orthotopic hcc in rats

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis. In recent years, oncolytic virotherapies have proven themselves to be potentially powerful tools to fight malignancy. Due to the unique dual blood supply in the liver, it is possible to apply therapies locally to orthotopic liver tumors, which are predominantly fed by arterial blood flow. We have previously demonstrated that hepatic arterial delivery of oncolytic viruses results in safe and efficient transduction efficiency of multifocal HCC lesions, resulting in significant prolongation of survival in immune competent rats. This procedure closely mimics the application of transarterial embolization in patients, which is the standard palliative care provided to many HCC patients. The ability to administer tumor therapies through the hepatic artery in rats allows for a highly sophisticated preclinical model for evaluating novel viral vectors under development. Here we describe the detailed protocol for microdissection of the hepatic artery for infusion of oncolytic virus vectors to treat orthotopic HCC.

With experience, a nearly 100% success rate (meaning that the artery was successfully dissected and injected, and the animal survived surgery) can be achieved. However, the health status of the rat prior to surgery (degree of tumor burden, underlying liver function, etc.) will obviously play a role in the outcome of the surgical intervention. Following surgery, rats can be expected to experience transient weight loss and a slight, transient elevation of liver enzymes, even if buf...

Watch this Scientific Journal Video about Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats at JoVE.com

Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats

Oncolytic virotherapies are under development as novel therapeutics for the treatment of hepatocellular carcinoma (HCC). Here we describe a method for locoregional therapy of HCC via hepatic arterial administration of oncolytic virus.

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis.  In recent years, oncolytic virotherapies have proven ...

transarterial-administration-oncolytic-viruses-for-locoregional

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JoVE Journal

Medicine

9.5K Views.  Klinikum rechts der Isar der TU München. Oncolytic virotherapies are under development as novel therapeutics for the treatment of hepatocellular carcinoma (HCC). Here we describe a method for locoregional therapy of HCC via hepatic arterial administration of oncolytic virus.

Video: Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors 

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

Orthotopic Liver Transplantation in Rats

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat liver transplantation in 1979 by Kamada et al.3, this model has remained the gold standard despite various proposed alternative techniques4. Nevertheless, its broader use is limited by its steep learning curve5.
In this video paper, we show a simple and easy-to-establish revision of Kamada's two-cuff technique. The suprahepatic vena cava anastomosis is performed manually with a running suture, and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick-linker cuff system6. Manufacturing the quick-linker kit is shown in a separate video paper.

We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat.

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

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 ...

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, play a crucial role in metastatic CRC progression and predict poor patient prognosis. However, there is a lack of satisfactory mouse models to study the crosstalk between metastatic cancer cells and CAFs. Here, we present a method to investigate how liver metastasis progression is regulated by the metastatic niche and possibly could be restrained by stroma-directed therapy. Portal vein injection of CRC organoids generated a desmoplastic reaction, which faithfully recapitulated the fibroblast-rich histology of human CRC liver metastases. This model was tissue-specific with a higher tumor burden in the liver when compared to an intra-splenic injection model, simplifying mouse survival analyses. By injecting luciferase-expressing tumor organoids, tumor growth kinetics could be monitored by in vivo imaging. Moreover, this preclinical model provides a useful platform to assess the efficacy of therapeutics targeting the tumor mesenchyme. We describe methods to examine whether adeno-associated virus-mediated delivery of a tumor-inhibiting stromal gene to hepatocytes could remodel the tumor microenvironment and improve mouse survival. This approach enables the development and assessment of novel therapeutic strategies to inhibit hepatic metastasis of CRC.

Portal vein injection of colorectal cancer (CRC) organoids generates stroma-rich liver metastasis. This mouse model of CRC hepatic metastasis represents a useful tool to study tumor-stroma interactions and develop novel stroma-directed therapeutics such as adeno-associated virus-mediated gene therapies.

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the ...

Cancer Research

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 ...

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor (CAR) T Cell Infusions in Mice

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy and radiotherapy. As many tumors lack efficacious treatments, there is a crucial need to develop more promising therapeutic options, such as immunotherapies; the use of chimeric antigen receptor (CAR) T cell therapy&#160;directed against CNS tumors is of particular interest. Cell surface targets such as B7-H3, IL13RA2, and the disialoganglioside GD2 are highly expressed on the surface of several pediatric and adult CNS tumors,&#160;raising the opportunity to use CAR T cell therapy against these and other surface targets. To evaluate the repeated locoregional delivery of CAR T cells in preclinical murine models, an indwelling catheter system that recapitulates indwelling catheters currently being used in human clinical trials was established. Unlike stereotactic delivery, the indwelling catheter system allows for repeated dosing without the use of multiple surgeries. This protocol describes the intratumoral placement of a fixed guide cannula that has been used to successfully test serial CAR T cell infusions in orthotopic murine models of pediatric brain tumors. Following orthotopic injection and engraftment of the tumor cells in mice, intratumoral placement of a fixed guide cannula is completed on a stereotactic apparatus and secured with screws and acrylic resin. Treatment cannulas are then inserted through the fixed guide cannula for repeated CAR T cell delivery. Stereotactic placement of the guide cannula can be adjusted to deliver CAR T cells directly into the lateral ventricle or other locations in the brain. This platform offers a reliable mechanism for the preclinical testing of repeated intracranial infusions of CAR T cells and other novel therapeutics for these devastating pediatric tumors.

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor CAR T Cell Infusions in Mice

Central nervous system (CNS) tumors are the leading cause of cancer-related death in children, and locoregional immune-based therapies are increasingly being tested for patients in clinical trials. This protocol describes methods for locoregional cannula implantation in mice for the preclinical evaluation of immunotherapeutic infusions targeting CNS tumors.

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy ...

Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact. For this strategy to be successful, recent studies have shown that involvement of the host immune system is essential. Therefore, oncolytic virotherapy should be evaluated within the context of an immunocompetent model. Furthermore, the study of antitumor therapies in tolerized animal models may better recapitulate results seen in clinical trials. Cotton rats, commonly used to study respiratory viruses, are an attractive model to study oncolytic virotherapy as syngeneic models of mammary carcinoma and osteosarcoma are well established. However, there is a lack of published information on the proper handling procedure for these highly excitable rodents. The handling and capture approach outlined minimizes animal stress to facilitate experimentation. This technique hinges upon the ability of the researcher to keep calm during handling and perform procedures in a timely fashion. Finally, we describe how to prepare cotton rat mammary tumor cells for consistent subcutaneous tumor formation, and how to perform intratumoral and intraperitoneal injections. These methods can be applied to a wide range of studies furthering the development of the cotton rat as a relevant pre-clinical model to study antitumor therapy.

Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model.

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact.  For this strategy to be ...

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. ...

A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep learning curve. The introduction of the cuff technique for anastomosis of the portal vein (PV) and infrahepatic vena cava (IHVC) has significantly simplified the transplant procedure in rats. However, due to the short anterior wall of the recipients' suprahepatic vena cava (SHVC), the cuff technique is very difficult to use for the reconstruction of the SHVC. Most researchers in this field still use the hand-suture technique for SHVC reconstruction, which makes it the bottleneck step in rat orthotopic liver transplantation. The magnetic anastomosis technique (i.e., magnamosis) is a method of connecting two vessels using the attractive force between two magnets. Our recent study has shown that the magnetic anastomosis technique is superior to the hand-suture technique for SHVC reconstruction in rats. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using the novel magnetic anastomosis technique. In this model, the reconstruction of the PV and IHVC was performed by the standard cuff technique, while the reconstruction of the bile duct (BD) was performed by a stent technique. The hepatic re-arterialization was not performed. The magnetic anastomosis technique made SHVC reconstruction much easier and significantly shortened the anphepatic phase. After a reasonable learning curve, even researchers without advanced microsurgical skills can produce reliable and reproducible results using this rat model of OLT.

The reconstruction of the suprahepatic vena cava (SHVC) remains a difficult step in rat orthotopic liver transplantation. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using a novel magnetic anastomosis technique.

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep ...

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver&#39;s common hepatic artery to the recipient liver&#39;s proper hepatic artery.

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...

Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats

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