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.

Liver ischemia/reperfusion injury, a setting in which the functional mass is reduced and the role of PDE5 inhibitor

Liver ischemia reperfusion is induced during surgical procedures like liver transplantation and resection. Multiple mechanisms have been postulated to liver damage following liver ischemia reperfusion injury, such as oxidative stress and inflammatory reactions. The present study declares the possible mechanism of tadalafil, toward modulating the inflammatory response. Forty-eight rats were divided into 4 groups as follows; Sham group subjected to midline laparotomy only. Tadalafil group administered Tadalafil 10 mg/kg intraperitoneal 45 min before sham operation. I/R (Ischemiareperfusion) group, rats undergo 60 min of hepatic ischemia followed by 60 min of reperfusion. Tadalafil + I/R group rats undergo a similar pattern of I/ R after the treatment with Tadalafil 10 mg/kg, 45 min before ischemia. At the end of the reperfusion, the blood samples were collected for estimation of biochemical markers including liver enzymes using colorimetric assay method and serum: TNF-α (tumor necrosis factor-α), IL-6 (interleukin 6) levels, ICAM- 1 (Intercellular Adhesion Molecule-1) were measured. Tissues were evaluated by semiquantitative and morphometrical approaches. Tadalafil succeeded in restoring normal levels of liver enzymes and ameliorating the oxidative stress as evidenced by decreasing MDA and restoring reduced glutathione levels in liver tissue homogenate. Also, Tadalafil exhibits anti-inflammatory effects, as it significantly decreased the levels of TNF-α, IL6 and ICAM-1. The findings are supported by BCL-2, TNF-α immunomarkers. It is concluded that modulation of the inflammatory response might be one of the mechanisms of Tadalafil-mediated hepatoprotection, so it is recommended as an adjuvant therapy in liver surgery

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Novel H-shunt Venovenous Bypass for Liver Transplantation in Cynomolgus Macaques.

Cynomolgus monkeys are often used in preclinical transplantation research. Performing liver transplantation in cynomolgus monkeys is challenging because they poorly tolerate portal vein clamping during the anhepatic phase. Finding an alternative to portal vein clamping is necessary before preclinical liver transplant models can be performed with reliable outcomes. We used 3 different techniques to perform 5 liver transplants in male cynomolgus macaques (weight, 7.4-10.8 kg; mismatched for MHC I and II; matched for ABO). In procedure A, we clamped the portal vein briefly, as in human transplants, as well as the superior mesentery artery to minimize congestion at the expense of temporary ischemia (n = 2). In procedure B, we performed a temporary portocaval shunt with extracorporeal venovenous bypass (n = 1). For procedure C, we developed an H-shunt system (modified portocaval shunt) with extracorporeal bypass (n = 2). Postoperative immunosuppression comprised cyclosporine A, mycophenolate mofetil, and steroids. Recipients in procedure A developed hemodynamic instability and were euthanized within 2 d. The recipient that underwent procedure B was euthanized within 11 d due to inferior vena caval thrombosis. The H-shunt in procedure C led to minimal PV congestion during the anhepatic phase, and both recipients reached the 21-d survival endpoint with good graft function. Our novel H-shunt bypass system resulted in successful liver transplantation in cynomolgus macaques, with long-term posttransplant survival possible. This technical innovation makes possible the use of cynomolgus monkeys for preclinical liver transplant tolerance models.

Liver transplantation in the mouse: Insights into liver immunobiology, tissue injury, and allograft tolerance.

The surgically demanding mouse orthotopic liver transplant model was first described in 1991. It has proved to be a powerful research tool for the investigation of liver biology, tissue injury, the regulation of alloimmunity and tolerance induction, and the pathogenesis of specific liver diseases. Liver transplantation in mice has unique advantages over transplantation of the liver in larger species, such as the rat or pig, because the mouse genome is well characterized and there is much greater availability of both genetically modified animals and research reagents. Liver transplant experiments using various transgenic or gene knockout mice have provided valuable mechanistic insights into the immunobiology and pathobiology of the liver and the regulation of graft rejection and tolerance over the past 25 years. The molecular pathways identified in the regulation of tissue injury and promotion of liver transplant tolerance provide new potential targets for therapeutic intervention to control adverse inflammatory responses/immune-mediated events in the hepatic environment and systemically. In conclusion, orthotopic liver transplantation in the mouse is a valuable model for gaining improved insights into liver biology, immunopathology, and allograft tolerance that may result in therapeutic innovation in the liver and in the treatment of other diseases.

Manufacturing devices and instruments for easier rat liver transplantation.

Orthotopic rat liver transplantation is a popular model, which has been shown in a recent JoVE paper with the use of the "quick-linker" device. This technique allows for easier venous cuff-anatomoses after a reasonable learning curve. The device is composed of two handles, which are carved out from scalpel blades, one approximator, which is obtained by modifying Kocher's forceps, and cuffs designed from fine-bore polyethylene tubing. The whole process can be performed at a low-cost using common laboratory material. The present report provides a step-by-step protocol for the design of the required pieces and includes stencils.

Propuesta de un programa de formación usando un modelo experimental para el manejo anestésico del trasplante hepático / Proposed experimental model to provide training in the management of anesthesia in liver transplantation

Objetivos: Proponemos y evaluamos un programa de entrenamiento para anestesiólogos, en modelo porcino, para aumentar la calidad del manejo anestésico durante el trasplante hepático. Material y método: Cinco anestesiólogos, facultativos especialistas de área, sin experiencia previa en el manejo anestésico del trasplante hepático que no trabajan en centros donde se realiza la cirugía de trasplante hepático, realizaron el programa de entrenamiento y evaluación de manera voluntaria, anestesiando cinco animales cada uno. La cirugía se realizó bajo anestesia total balanceada, realizando monitorización hemodinámica, ventilatoria, determinación bioquímica, gasometrías arteriales y coagulación. Para evaluar a los alumnos se utilizaron criterios publicados previamente, considerando su habilidad para mantener al paciente estable. Fueron evaluados en los tiempos T1 (basal), T2-15 min fase preanhepática, T3-final fase preanhepática, T4-15 min fase anhepática, T5-final fase anhepática, T6-15 min fase postanhepática, T7-final fase postanhepática. Los alumnos completaron una encuesta al finalizar el programa para valorar su utilidad. Los cambios en la puntuación de los criterios de calidad fueron analizados utilizando ANOVA. Resultados: Durante la fase postimplantación, los cambios metabólicos (acidosis) y cardiacos (hipotensión y bradicardia) fueron las alteraciones más críticas a las que tuvieron que hacer frente los alumnos. Se evidenció un incremento significativo en las habilidades de los alumnos para hacer frente a los cambios hemodinámicos y metabólicos (p < 0,05). Todos los alumnos expresaron su preferencia por la realización de un entrenamiento previo en modelo porcino. Conclusiones: Utilizando este modelo de entrenamiento, los cambios hemodinámicos y fisiológicos que acontecen durante el trasplante, fueron entendidos y manejados. En nuestra opinión, la realización de un programa de formación en modelo porcino permite al anestesiólogo adquirir experiencia y habilidad para el manejo anestésico del trasplante hepático(AU)

Objetive: To propose and evaluate the use of a porcine model for training in how to manage and improve the quality of anesthesia during liver transplantation. Material and Methods: Five trained anesthesiologists who had no previous experience in managing anesthesia during liver transplantation and who did not work in hospitals where the procedure was performed volunteered for the training course and evaluated it. Each trainee anesthetized 5 animals. Surgery was performed under total balanced anesthesia with monitoring of hemodynamics, ventilation, biochemistry, arterial blood gases, and coagulation. Previously set criteria were used to evaluate the trainees' skill in maintaining patient stability. Their work was assessed 7 times: at baseline, 15 minutes into the preanhepatic phase, at the end of the preanhepatic stage, 15 minutes into the anhepatic phase, at the end of the anhepatic phase, 15 minutes into the postanhepatic phase, and at the end of the postanhepatic phase. After completing the course, the trainees filled in a questionnaire to evaluate its usefulness. Analysis of variance was applied to score changes in anesthetic quality criteria. Results: After implantation, changes in metabolic (acidosis) and cardiac (hypotension and bradycardia) status were the most critical abnormalities the trainees faced. Their skill in coping with hemodynamic and metabolic changes improved significantly (P<.05). All participants expressed a preference for receiving training in a porcine model before providing anesthesia in this surgical setting. Conclusions: Under this training model, the anesthesiologists understood and were able to manage the hemodynamic and physiologic changes that develop during a liver transplant procedure. We believe that training using a porcine model allows an anesthesiologist to acquire experience and skill in this setting(AU)

Liver transplantation in swine without venovenous bypass.

INTRODUCTION: A model of orthotopic liver transplantation in swine was developed to investigate an advanced reperfusion approach. Thereby, we consciously disclaim otherwise commonly practiced venovenous bypass during the recipient operation. MATERIAL AND METHODS: Ten liver transplantations were performed according to the described technique without using venovenous bypass. In each swine the observation period was 48 h. RESULTS: All transplantations were carried out after a median cold ischemic time of 307.5 min (295-340); the median warm ischemic time in these cases was 25 min (20-32). Eight of 10 swine survived 48 h after the operation. CONCLUSION: Orthotopic liver transplantations in the recipient swine are feasible even without using venovenous bypass.

Intraoperative vital and haemodynamic monitoring using an integrated multiple-channel monitor in rats.

The aim of this study is to give a hands-on description of the successful monitoring procedure established for extended liver resections and liver transplantations in rats and to provide the typical range of data as obtained before and after a hepatobiliary surgical procedure (right median hepatic vein [RMHV] ligation) in healthy male Lewis rats. All manipulations were performed in anaesthetized (3% isoflurane in O(2) 1 L/min) healthy male Lewis rats (250-350 g) with an integrated multiple-channel intraoperative monitor (Powerlab system) using a series of sensors for data acquisition. Vital parameters (body temperature, electrocardiogram, respiratory rate and heart rate), haemodynamic parameters (mean arterial blood pressure [MAP] and central venous pressure) and liver perfusion parameters (inferior hepatic venous pressure, portal vein pressure [PVP], blood flow of portal vein and inferior hepatic cava) were monitored. Catheters were placed in microsurgical technique after careful exposure guided by anatomical landmarks. Vascular incisions were closed with interrupted sutures. Complete instrumentation of animals was performed within 1 h. No specific complications occurred. Vital and haemodynamic parameters such as MAP (94 +/- 16.2 mmHg) or portal pressure (9.6 +/- 1.34 mmHg) were in the same range as known for humans (MAP = 100 mmHg, portal pressure = 5-10 mmHg), whereas parameters dependent on the size of the body or organ such as flow rates (portal blood flow = 16.2 +/- 6 mL/min) were obviously different compared with those of humans (portal blood flow = 800 mL/min). In conclusion, the normal range for vital, haemodynamic and liver perfusion parameters was reported as reference values to allow quality control for future surgical hepatobiliary research projects. As the procedure can be easily learned, the extensive intraoperative monitoring can be used routinely.

Oral buprenorphine and aspirin analgesia in rats undergoing liver transplantation.

The objective of this study was to establish effective postoperative analgesia for Dark Agouti rats undergoing liver transplantation with minimal additional stress due to handling and no adverse effect on transplant outcome. Oral administration of buprenorphine (0.5 mg/kg/dose) or aspirin (100 mg/kg/dose) in raspberry-flavoured gelatine were compared to controls receiving no treatment or plain gelatine. The drugs were presented five times: immediately on recovery from anaesthesia and at 12 h intervals thereafter. All rats underwent right nephrectomy and replacement of their liver by an arterialized liver isograft preserved optimally for 24 h. All groups had reversible hepatic damage, lost weight and demonstrated severely reduced dark cycle activity after surgery. Neither treatment appeared to ameliorate the loss of body weight that probably reflected hepatic insufficiency during the first week as well as pain and surgical stress. In the second week, when liver function was 'normal', rats began to regain weight at the pre-transplant rate. Aspirin treatment significantly increased activity during the first and second dark cycles after surgery, whereas buprenorphine significantly increased activity during the second dark cycle only. Neither drug had any apparent adverse effects on the rats or on graft function. Postoperative oral administration of aspirin should be incorporated into future programmes of liver transplantation in rodents. More effective treatment in the immediate postoperative period may require oral administration of analgesia prior to surgery or a single subcutaneous injection of an analgesic agent on completion of surgery in addition to postoperative oral administration of aspirin.

Prospects for xenotransplantation of the liver.

A severe shortage of human livers for allotransplantation has sparked interest in the potential use of animals in lieu of humans as a source of livers, that is xenotransplantation. Xenotransplantation might also provide a means by which recurrence of hepatitis might be averted. Among the types of xenografts that might be undertaken are extracorporeal "xenoperfusion" or perfusion of devices containing xenogeneic hepatocytes, auxiliary liver transplants, bridge liver transplants, and hepatocyte transplants. The hurdles to xenotransplantation of the liver include the immune response of the recipient against the graft, incompatibility of the graft with complex physiologic and biochemical systems of the recipient, and the possibility of transferring infectious agent from the graft to the recipient. Recent progress in characterizing and overcoming these hurdles has encouraged some optimism regarding the ultimate application of xenotransplantation for the treatment of human disease.

Division of donor liver for successful split-liver transplantation in pigs.

Split Liver Transplantation (SLT) is an attractive method to solve the problem of a shortage of liver grafts. A through knowledge of the anatomy of the porcine liver vessels and bile duct is essential in performing the experimental SLT. This study was undertaken to decide the split line for successful SLT in pigs by examining the main branching patterns both vessels and bile duct in 30 porcine livers macroscopically and angiographically. The hepatic arterial branching patterns were divided into three types and bile duct patterns into two types. There was no exception in branching patterns of the portal vein and the hepatic vein. We conclude it is desirable that the donor liver should be divided into two grafts between the left medial lobe and quadrate lobe.

An improved model for rat liver transplantation including arterial reconstruction and simplified microvascular suture techniques.

For experimental liver transplantation in the rat, the models that have been used most frequently do not include reconstruction of the arterial blood supply to the liver. In these procedures, specially developed cuff anastomoses rather than the conventional microvascular suture technique are used almost exclusively in the recipient operation, so that the anhepatic time is minimized. In this study the technical details of an improved rat model for orthotopic liver transplantation are described. During the donor operation in this experimental method, the liver is prepared with an arterial pedicle that includes the abdominal segment of the aorta, permitting perfusion in situ of the portal vein as well as the hepatic artery. The transplantation of the excised donor organ into the recipient site is carried out with simplified microvascular suture techniques and includes reconstruction of the arterial supply to the liver. Anastomosis of the bile duct is accomplished by choledocho-choledochostomy with a splint technique and supplemental suturing. For the entire procedure, magnifying glasses with 2- to 2.5-fold magnification are sufficient. When this technique has been mastered, the average duration of the anhepatic phase is about 20 min, well below the critical 30-min limit for survival of the experimental animals. As proficiency increased, the perioperative mortality was reduced to 9.2% (n = 130). With the combination of portal and arterial in situ flushing during the donor operation and the rearterialization of the transplant during the recipient operation, the clinical conditions can be approximated more closely than is possible when the transplanted rat liver is supplied only by the portal vein. Use of microvascular suture techniques, without cuff anastomoses, reduces the need for ex situ handling of the donor organ.