Name: a rat model of orthotopic liver transplantation using a novel magnetic anastomosis technique for suprahepatic vena cava reconstruction
Uploaded: 2018-03-19T12:30:00
Description: Watch this Scientific Journal Video about A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction at JoVE.com

This work was supported by grants from the Ministry of Education Innovation Team Development Program of China (No. IRT16R57), National Natural Science Foundation of China (No. 81470896), and a research fund for Young Talent Recruiting Plans of Xi'an Jiaotong University (RW).

The protocol was carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals and was approved by the Committee on the Ethics of Animal Experiments of Xi&#39;an Jiaotong University, Xi&#39;an, Shaanxi Province, China.
NOTE: None of the procedures in this protocol was performed under a surgical microscope.
1. Preoperative Preparation
<ol>
	<li>The Design and Shape of the Magnetic Rings (Figure 1)
	<ol>
		<li...

<ol>
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Many clinical progresses in liver transplantation can be attributed to animal studies. Rat OLT is a widely used and well accepted model in the research of organ preservation, transplant immunology, physiology and pathology. However, it is also a very complicated procedure and requires advanced microsurgical skill. Despite many improvements in the procedure of rat OLT, the SHVC anastomosis remained a challenging step. In this article, we describe a novel magnetic anastomosis technique for SHVC reconstruction in rat OLT. T...

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research1,2. The first rat OLT was described by Lee et al. in 19733. In that model, all the vessels were reconstructed by the hand-suture technique. The hand-suture technique requires advanced microsurgical skills, which significantly limits its utilization. Since then, various modifications to the original protocol of rat OLT have been reported. Among them, the cuff technique for anastomosis of the portal vein (PV) and infrahepatic vena cava (IHVC) reported by Kamada et al. in 1979 is considered a major improvement to this model, as it significantly simplified the reconstruction procedures4. However, due to the short anterior wall of the recipients&#39; 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 OLT5,6,7.
The magnetic anastomosis technique (i.e., magnamosis) is a method of connecting two vessels or other tubular structures using the attractive force between two magnets8,9,10,11. The magnetic force gradually compresses and remodels the tissue into a strong, sutureless anastomosis12,13. This compression anastomosis has been proven to be effective in humans14,15. We have designed a pair of magnetic rings specifically for the anastomosis of the SHVC in rats. Our recent study has shown that the magnetic anastomosis technique is superior to the hand-suture technique for SHVC reconstruction in rat OLT16. The purpose of this article is to provide a detailed, step-by-step protocol for SHVC reconstruction in rats using the novel magnetic anastomosis technique.

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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.

After approximately 10 attempts, the magnetic anastomosis technique for SHVC reconstruction was successfully mastered by a researcher who had no prior microsurgical training. The reconstruction of the SHVC took less than 2 min. The anhepatic phase for the recipient rats was approximately 10 min. No thrombosis, bleeding or angiostegnosis was observed at the SHVC anastomotic site at 1, 5 and 30 days after reperfusion. The inferior vena cavography was performed at 2 weeks after transplantati...

Watch this Scientific Journal Video about A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction at JoVE.com

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

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 ...

The overall goal of this protocol is to introduce a rat orthotopical liver transplantation model using a magnetic anastomosis technique for suprahepatic vena cava reconstruction. This method can help answer key question in the liver transplantation model about suprahepatic vena cava reconstruction. The main advantage of this technique is that it reduces the anhepatic phase of liver transplantation and greatly improves the postoperative survival rate.First, shave the entire abdominal skin of the rat. Disinfect the corresponding skin with a povidone-iodine solution. Dissect the falciform ligament around the liver.Next, isolate and ligate the left inferior phrenic vein. Perform a V incision on the anterior wall. Insert the stent into the lumen of the bile duct.Make sure at least half of the stent lies outside the bile duct. Secure the stent with a 6-0 silk suture. Cut one end of the suture.Keep the other one to hold during later anastomosis. Isolate the infrahepatic vena cava down to the left renal vein level. Dissect it until the infrahepatic vena cava is skeletonized.Separate and ligate the right suprarenal vein. Separate and ligate the right renal vein. Heparinize the rat through the dorsal vein of the penis.Insert a 22-gauge catheter into the abdominal aorta below the left renal vein. Perform a thoracotomy. Clamp the thoracic aorta using a microforceps.Perfuse the donor liver through the catheter in the aorta. Transect the suprahepatic vena cava. In the meantime, cut the vena cava below the right renal veins to allow the perfusate flowing out of the liver.Dissect the connective tissue surrounding the portal vein. Ligate and divide the pyloric vein. Transect the portal vein at the level of the splenic vein.Transect the infrahepatic vena cava at the level of the left renal vein. Liver graft preparations are performed in four degree Celsius saline bath. Insert the infrahepatic vena cava into its cuff, with the cuff extension pointing towards to the liver.Evert the distal end of the infrahepatic vena cava over the cuff body. Secure the edge with a 6-0 silk suture. Repeat the above steps on the portal vein to attach a cuff to the donor's portal vein.Pull the diaphragm through the forceps, and trim the suprahepatic vena cava. Pass the suprahepatic vena cava through the magnetic ring. Evert the distal end of the suprahepatic vena cava over the ring.And secure the ring with a 6-0 silk suture. Then, remove the excess diaphragm around the liver. Place a 6-0 silk suture, and ligate the common bile duct.Isolate the common bile duct from the first hepatic portal. Ligate and divide the right adrenal vein. Place a strap in the suprahepatic vena cava of the liver.Clamp the infrahepatic vena cava just above the right renal vein with microvessel clips. Clamp the portal vein at the level of pyloric vein. Inject two milliliter normal saline through the portal vein slowly to flush the blood out of the liver.Drag down the liver by pulling the previous placed strap. Clamp the suprahepatic vena cava and part of the diaphragm with a Satinsky clamp. Excise the liver by transecting the suprahepatic vena cava just above the liver.Transect the infrahepatic vena cava close to the parenchyma and the portal vein at the porta hepatis. Insert the recipient's remaining suprahepatic vena cava into a magnetic ring. Fix the magnetic ring on a Satinsky clamp by magnetic attraction.Evert the suprahepatic vena cava over the magnetic ring. Place the donor liver graft orthotopically, and fill the lumen with normal saline solution, coupling the magnetic rings to fulfill the vessel construction. Reconstruct the portal vein.Fill the lumen of the portal vein with normal saline. Insert the cuff body of the donor's portal vein into the recipient's portal vein. And secure that with a circumferential 6-0 silk ligature.Remove the Satinsky clamp from the suprahepatic vena cava. Incise a V on the anterior wall of the common bile duct. Insert the donor stent into the lumen of the recipient bile duct.And fix it with a 6-0 silk circumferential suture. Pull the sutures on the two bile ducts together, and tie the sutures to bring the bile ducts close to each other. The angiography two weeks POD showed a patent blood flow of the suprahepatic vena cava anastomosis.The post-transplant survival rate was up to 85%to day 30 POD. The magnetic anastomosis technique makes easy and fast suprahepatic vena cava reconstruction in rats possible. After watching this video, without advanced microsurgical skills, can produce reliable and reproducible results using this rat model of liver transplantation.

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Research

JoVE Journal

Medicine

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

Electrolytic Inferior Vena Cava Model (EIM) of Venous Thrombosis

Animal models serve a vital role in deep venous thrombosis (DVT) research in order to study thrombus formation, thrombus resolution and to test potential therapeutic compounds (1). New compounds to be utilized in the treatment and prevention of DVT are currently being developed. The delivery of potential therapeutic antagonist compounds to an affected thrombosed vein has been problematic. In the context of therapeutic applications, a model that uses partial stasis and consistently generates thrombi within a major vein has been recently established. The Electrolytic Inferior vena cava Model (EIM) is mouse model of DVT that permits thrombus formation in the presence of continuous blood flow. This model allows therapeutic agents to be in contact with the thrombus in a dynamic fashion, and is more sensitive than other models of DVT (1). In addition, this thrombosis model closely simulates clinical situations of thrombus formation and is ideal to study venous endothelial cell activation, leukocyte migration, venous thrombogenesis, and to test therapeutic applications (1). The EIM model is technically simple, easily reproducible, creates consistent thrombi sizes and allows for a large sample (i.e. thrombus and vein wall) which is required for analytical purposes.

Electrolytic Inferior Vena Cava Model EIM of Venous Thrombosis

The electrolytic induction of endothelial activation to the internal surface of the Inferior Vena Cava results in venous type thrombus formation due to endothelial activation and partial blood stasis, two components of Virchow's triad.

Animal models serve a vital role in deep venous thrombosis (DVT) research in order to study thrombus formation, thrombus resolution and to test potential ...

Mouse Complete Stasis Model of Inferior Vena Cava Thrombosis

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE). In the United States (U.S.), the high morbidity and mortality rates make VTE a serious health concern 1-2. After heart disease and stroke, VTE is the third most common vascular disease 3. In the U.S. alone, there is an estimated 900,000 people affected each year, with 300,000 deaths occurring annually 3. A reliable in vivo animal model to study the mechanisms of this disease is necessary.
The advantages of using the mouse complete stasis model of inferior vena cava thrombosis are several. The mouse model allows for the administration of very small volumes of limited availability test agents, reducing costs dramatically. Most promising is the potential for mice with gene knockouts that allow specific inflammatory and coagulation factor functions to be delineated. Current molecular assays allow for the quantitation of vein wall, thrombus, whole blood, and plasma for assays. However, a major concern involving this model is the operative size constraints and the friability of the vessels. Also, due to the small IVC sample weight (mean 0.005 grams) it is necessary to increase animal numbers for accurate statistical analysis for tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent (ELISA), zymography, vein wall and thrombus cellular analysis, and whole blood and plasma assays 4-8.
The major disadvantage with the stasis model is that the lack of blood flow inhibits the maximal effect of administered systemic therapeutic agents on the thrombus and vein wall.

The mouse complete stasis model of inferior vena cava thrombosis yields quantifiable amounts of vein wall tissue and thrombus. It has proven useful for evaluating interactions between the vein wall and the occlusive thrombus and in assessing the progression from acute to chronic inflammation.

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE).  In the United States (U.S.), the high morbidity and ...

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 ...

Technique of Porcine Liver Procurement and Orthotopic Transplantation using an Active Porto-Caval Shunt

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation waiting list die without receiving an organ transplant or are delisted due to disease progression. Even after a successful transplantation, rejection and side effects of immunosuppression remain major concerns for graft survival and patient morbidity. 
Experimental animal research has been essential to the success of liver transplantation and still plays a pivotal role in the development of clinical transplantation practice. In particular, the porcine orthotopic liver transplantation model (OLTx) is optimal for clinically oriented research for its close resemblance to human size, anatomy, and physiology. 
Decompression of intestinal congestion during the anhepatic phase of porcine OLTx is important to guarantee reliable animal survival. The use of an active porto-caval-jugular shunt achieves excellent intestinal decompression. The system can be used for short-term as well as long-term survival experiments. The following protocol contains all technical information for a stable and reproducible liver transplantation model in pigs including post-operative animal care.

Experimental animal research plays a pivotal role in the development of clinical transplantation practice. The porcine orthotopic liver transplantation model (OLTx) closely resembles human conditions and is frequently used in clinically oriented research. The following protocol contains all information for a reliable porcine OLTx model using an active porto-caval-jugular shunt.

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation ...

Orthotopic Rat Kidney Transplantation: A Novel and Simplified Surgical Approach

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of renal replacement therapy. Over the past few decades, the rat kidney transplantation model has been used to study the immunological phenomena of rejection and tolerance. This model has become an indispensable tool to test new immunomodulatory pharmaceuticals and regimens prior to proceeding with expensive preclinical large animal studies.
This protocol provides a detailed overview of how to reliably perform orthotopic kidney transplantation in rats. This protocol includes three distinctive steps that increase the probability of success: perfusion of the donor kidney by flushing through the portal vein and the use of a cuff system to anastomose the renal veins and ureters, thereby decreasing cold and warm ischemia times. Using this technique, we have achieved survival rates beyond 6 months with normal serum creatinine in animals with syngeneic or tolerant kidney transplants. Depending on the aim of the study, this model can be modified by pre- or posttransplant treatments to study the acute, chronic, cellular, or antibody-mediated rejection. It is a reproducible, reliable, and cost-effective animal model to study different aspects of kidney transplantation.

The purpose of this manuscript and protocol is to explain and demonstrate in detail the surgical procedure of orthotopic kidney transplantation in rats. This method is simplified to achieve the correct perfusion of the donor kidney and shorten the reperfusion time by using the venous and ureteral cuff anastomosis technique.

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of ...

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 ...

A Rat Orthotopic Renal Transplantation Model for Renal Allograft Rejection

Renal allograft rejection limits the long-term survival of patients after renal transplantation. Rat orthotopic renal transplantation is an essential model to investigate the mechanism of renal allograft rejection in pre-clinical studies and could aid in the development of novel approaches to improve the long-term survival of renal allografts. Donor kidney implantation in rat orthotopic renal transplantation is commonly performed by end-to-side anastomosis to recipients' aorta and inferior vena cava. In this model, the donor's kidney was implanted using end-to-end anastomosis to the recipients' renal artery and renal vein. The donor's ureter was anastomosed to the recipient's bladder in an end-to-side 'tunnel' method. This model contributes to better healing of ureter-bladder anastomosis and increases the recipients' survival by avoiding interference with blood supply and venous reflux of the lower body. This model can be used to investigate the mechanisms of acute and chronic immune and pathologic rejection of renal allografts. Here, the study describes the detailed protocols of this orthotopic renal transplantation between rats.

The rat orthotopic renal transplantation model contributes to investigating the mechanism of renal allograft rejection. The current model increases the recipients' survival without interference with blood supply and venous reflux of the lower body using an end-to-end anastomosis of kidney implantation and an end-to-side "tunnel" method of ureter-bladder anastomosis.

Renal allograft rejection limits the long-term survival of patients after renal transplantation. Rat orthotopic renal transplantation is an essential ...

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 ...