Optimizing the mouse orthotopic liver transplantation model: Learning curve, technical enhancements, and keys to success

Liver transplantation (LT) is currently the only effective treatment option for end-stage liver disease. The importance of animal models in transplantation is widely recognized among researchers. Because of the well-characterized mouse genome and the greater diversity and availability of both genetically modified animals and research reagents, mouse orthotopic LT (MOLT) has become an ideal model for the investigation of liver biology, tissue injury, regulation of alloimmunity and tolerance induction, and the pathogenesis of specific liver diseases. However, due to its complicated and technically demanding procedure, the model has merely been used by only a few research groups in the world for years. For a new learner, training lasting at least a couple of months or even years is required. Most of the investigators have emphasized the importance of elaborate techniques and dedicated instruments in establishing a MOLT model, but some details are often neglected. The nontechnical details are also significant, especially for researchers who have little experience in mouse microsurgery. Here, we review and summarize the crucial technical and nontechnical details in establishing the model of MOLT based on scientific articles and our experience in six aspects: animal selection, anesthesia, perioperative management, organ procurement, back-table preparation, and implantation surgery. We aim to enable research groups to shorten the learning curve and implement the mouse LT procedure with high technical success.

Mouse orthotopic liver transplantation: Challenges, achievements, and applications.

Key Points in Establishing a Model of Mouse Liver Transplantation

Translational research using animal models is essential for addressing the clinical issues in organ transplantations. Although mouse orthotopic liver transplantation (LT) is a useful tool for investigating the mechanisms of liver regeneration, ischemia-reperfusion injury, and immune responses following LT, technical challenges in this procedure restrict its feasibility. The key aspects for successful mouse orthotopic LT are vascular reconstruction in recipients, including the suprahepatic inferior vena cava (SHIVC), portal vein (PV), and infrahepatic inferior vena cava (IHIVC). In addition to the technical difficulties in reconstructing these vessels, the time limitation of the anhepatic phase to within 20 min makes this procedure more challenging. Although a cuff technique can be used for PV reconstruction, a safe and quick suture technique is required to reconstruct the SHIVC during the anhepatic phase. For IHIVC reconstruction, while both cuff and suture techniques are applicable, the suture technique leads to a shorter operation time in donor surgery and back-table preparation and can maintain a larger vascular lumen than the cuff technique. We herein provide guidelines for SHIVC and IHIVC reconstruction using a suture technique to facilitate mouse orthotopic LT.

Lymph node (LN) metastasis is one of the most common mechanisms underlying the spread of solid cancers. Mouse models of orthotopic tumor transplantation have been used to elucidate the metastatic mechanisms. Previous studies have mainly focused on metastasis in orthotopic models, and the correlation between histological findings of infiltrated cancer cells and tumor microenvironment has not been fully explored. Cancer-associated fibroblasts (CAFs) play a key role in metastasis, especially via secreting Wnt2, which plays a functional role in cancer progression. Here, we investigated the correlation between the incidence of LN metastases and histological findings in an orthotopic mouse model using 22 colorectal cancer cell lines. Lymphatic invasion was significantly associated with LN metastasis. Immunohistochemistry revealed that podoplanin and αSMA were highly expressed in the cancer stroma, thus suggesting that CAFs were also induced in the orthotopic implantation mouse model. Furthermore, Wnt2 positivity was mainly observed in the cancer stroma, and Wnt2 expression was significantly associated with the incidence of LN metastasis. Our results indicate that the orthotopic mouse model recapitulates cancer infiltration and the tumor microenvironment, including CAFs. The contribution of Wnt2 toward LN metastasis was also validated in an in vivo model. Thus, our findings provide new insights into the functional roles of CAFs and Wnt2 in metastasis.

O116* Aims: The aim of the study is to probe into the protective mechanism of the combination therapy of FTY720 and fat-derived hormone adiponetin in a rat liver transplantation model using small-for-size fatty graft. Methods: A rat orthotopic liver transplantation model using 60% of liver grafts was applied. Fatty Zucker rats (240-280g) with 30% - 40% liver steatosis were used as donors. Lean Zucker rats (260-300g) were as recipients. In the FTY720 treatment group (Group 1), FTY720 (1mg/kg, IV) was administrated (1) at 20 minutes before graft harvesting in donor, (2) immediately before liver out in recipient and (3) immediately after reperfusion in recipient. In the adiponectin treatment group (Group 2), adiponectin protein from mammaline cells was given at 48 and 24 hours before graft harvesting in donor, and day1 and day 2 post-transplantation in recipient at the dose of 1.5mg/Kg by IV injection. In the combination therapy group (Group 3), adiponectin (1.5mg/kg, IV) was given at 48 and 24 hours before graft harvesting in donor, and day1 and day 2 post-transplantation in recipient in addition to FTY720. 7-day survival was compared among the treatment groups and control group (without any treatment). Liver tissues were sampled at 6, 24 and 48 hours after reperfusion for morphological examination (EM), apoptotic nuclei staining (TUNEL), energy metabolism (ATP) and intragraft gene detection for cell survival (Akt), apoptosis (Caspase) and MAPK signaling pathways by Western blot and immunostaining. Blood samples were analyzed for liver function. Fatty droplet and fat metablism were also compared. Results: FTY720 significantly improved 7-day graft survival rate to 62.5% (5/8) from 0% (0/0) of control group. Adiponectin treatment also increased 7-day survival rate remarkbly (50%, 5/10). Combined with adiponectin and FTY720, it reached to 80% (8/10). The liver steatosis by red oil staining was attenuated significantly from 30-40% to less than 10-20% at day2 post-transplantation in Group 2 and Group3. Combination therapy significantly reduced serum ALT and AST levels at 6, 24 and 48 hours after liver transplantation compared to the control or single treatment. FTY720 or adiponectin decreased serum ALT and AST only at 24 hours after transplantation compared to the control group. Combination therapy significantly increased the intragraft ATP levels compared to control group and single treatment groups. The cell survival signaling Akt pathway was significantly activated at ser473 both in FTY720 and combination groups by phosphorylation of GSK-3, Bad and FKHR. The cleaved Caspase 3, 7 and 9 were down regulated accompanied with less apoptotic nuclei. MAPK pathway was down regulated more significantly in combination groups. Hepatic ultrastructure was well maintained in combination treatment group compared to the control group with disruption of hepatic sinusoids. Conclusions: Combination therapy of FTY720 plus adiponectin rescued small-for-size fatty liver graft from injury in liver transplantation.

Because of the wide availability of genetically modified animals, mouse orthotopic liver transplantation is often preferred over rat liver transplantation. We present a simplified mouse liver transplantation technique and compare transplantation outcomes with versus without hepatic artery anastomosis. Instruments for liver implantation were designed and printed with a 3-dimensional (3D) printer. The suprahepatic vena cava anastomosis was performed with a 10-0 running suture. The vena porta and infrahepatic vena cava were joined on extraluminal cuffs, using the 3D-printed device for spatial alignment and stabilization. The hepatic artery was reconstructed in half of the recipients using intraluminal stents. Liver function tests (3, 7, and 28 days) and histology (7 and 28 days) were assessed after transplantation. We performed 22 consecutive syngeneic C57BL/6 mouse orthotopic liver transplantations. The median portal clamping time was 12.5 ± 1.5 minutes. The survival rate at 4 weeks was 100% for both arterialized and nonarterialized recipients (n = 7, 4 recipients of each group being killed for early histology at day 7). Liver function tests at 3, 7, and 28 days were similar between arterialized versus nonarterialized groups. Liver parenchyma demonstrated only irrelevant abnormalities in both groups. The proposed device allows for a shorter clamping time compared with the published literature. Using this technique, the artery does not need to be anastomosed, with no impact on graft and recipient outcomes. The device is available for 3D printing. Liver Transplantation 22 1688-1696 2016 AASLD.

To investigate the protective effect of bone marrow mesenchymal stem cells (BMMSC) combined with normothermic mechanical perfusion (NMP) on biliary epithelial cells (BEC) in rats receiving donation after cardiac death (DCD) donor liver transplantation. The BMMSC were isolated from male Sprague-Dawley (SD) rats aged 2-3 weeks and weighing 40-60 g, and then cultured, identified and expanded to the third generation in vitro. Male SD rats aged 6-8 weeks and weighing 200-220 g were divided into sham-operated group (Sham group), static cold storage (SCS group), simple NMP group (NMP group) and BMMSC combined with NMP group (BMMSC+NMP group) by random number table method with 44 rats in each group. The DCD donor liver transplantation models in rats were reproduced with 30-minute warm ischemic time. While the rats in Sham group merely received perihepatic ligaments-separation, which did not affect their liver blood supply, and then their incisions were sutured after 30 minutes. The DCD donor grafts in SCS group were preserved in the University of Wisconsin (UW) cold storage solution for 4 hours. While the DCD donor grafts in the NMP group and the BMMSC+NMP group were perfused with the DMEM/F12-based culture solution or combined with BMMSC for 4 hours through the established ex vivo NMP system. The orthotopic liver transplantation model was reproduced, and the survival rate of the recipients was observed at 0, 1, 7 and 14 days after liver transplantation. The biochemical liver function of rats in different groups was determined at each time point after operation. The morphological changes in bile ducts of liver grafts were observed by hematoxylin-eosin (HE) staining, and the expression of cytokeratin 19 (CK19) was determined qualitatively by immunohistochemistry and quantitatively by Western Blot after protein extraction from BEC in liver samples. The morphology, differentiation function and phenotypic identification of BMMSC confirmed that the stem cells used in this experiment were standard BMMSC. The survival rates of rats in the NMP group and the BMMSC+NMP group were significantly higher than that in the SCS group at 0, 1, 7 and 14 days after operation. The increase was more significant in the BMMSC+NMP group, with 100% on postoperative day (POD) 0, and the 14-day survival rate was still significantly higher than that in the SCS group and the NMP group [80.0% (16/20) vs. 20.0% (4/20), 70.0% (14/20), both P < 0.05]. As the time after liver transplantation prolonged, the liver function parameters of rats in the SCS group were deteriorated gradually, which reached the peak at 1-7 days after operation. The damage of biliary tissue increased gradually under the microscope, and the injury was most serious on POD 7 in the SCS group, showing a lot of balloon-like changes in hepatocytes, with obvious bile duct dilatation accompanied by large area inflammatory cell infiltration. Immunohistochemistry and Western Blot showed that the expression of CK19 in BEC cytoplasm was decreased gradually in the SCS group, reached the lowest on POD 7, and then gradually increased. The BMMSC+NMP group and the NMP group were significantly better than the SCS group in terms of liver function, pathological injury of biliary tract and CK19 expression in BEC, and the improvement was more significant in the BMMSC+NMP group. These results suggested that the protective effects of BMMSC combined with NMP on BEC was significantly better than that of the SCS and NMP. Preservation of rat DCD donor liver by BMMSC combined with NMP can reduce the BEC injury after liver transplantation significantly, thus improving both the prognosis and the survival rate after transplantation.

CD47 blockade alleviates acute rejection of allogeneic mouse liver transplantation by reducing ischemia/reperfusion injury

Bile Duct Stent and Strain Selection Influences Long-Term Survival of Mouse Orthotopic Liver Transplantation

Spectrum of chronic lung allograft pathology in a mouse minor-mismatched orthotopic lung transplant model.

BackgroundThis study aimed to develop a nude mouse model of orthotopic liver transplantation of HCCLM3 human hepatocellular carcinoma (HCC) cell xenografts and the use of imaging and histology to evaluate tumor development and progression.Material/MethodsHCCLM3 cells were injected subcutaneously into 25 healthy male athymic BALB/c (nu/nu) nude mice. The tumors that developed were transplanted into the liver of a new set of nude mice. After four weeks and six weeks, the mice were imaged using ultrasound (US), software-assisted contrast-enhanced ultrasound (CEUS), fluorodeoxyglucose-positron emission tomography (FDG-PET). Histology was performed on the liver and liver tumors, and included immunohistochemistry for vascular endothelial growth factor (VEGF), CD31, CD34, and α-smooth muscle actin (α-SMA).ResultsThe success rate for orthotopic tumor transplantation in the mouse liver was 90% (18/20). Liver tumors measured 11.8±2.6 mm in diameter and 525.9±250.8 mm3 in volume on the sixth week. CEUS showed rapid wash-in and washout in the liver tumors, and PET showed low tumor cell metabolism. Bone metastases were present in 45% (9/20) of mice in the sixth week. Immunohistochemistry showed positive expression for VEGF, CD31, CD34, and α-SMA.ConclusionsThe nude mouse orthotopic liver transplantation model of human HCC was shown to be a reliable model that has the potential for future research on the pathogenesis and progression of HCC and studies on drug development.

BackgroundThe mouse model of arterialized orthotopic liver transplantation (AOLT) has played an important role in biomedical research. The available methods of sutured anastomosis for reconstruction of the hepatic artery are complicated, resulting in a high incidence of complications and failure. Therefore, we developed and evaluated a new model of AOLT in mice.Materials and methodsMale inbred C57BL/6 mice were used in this study. A continuous suture approach was applied to connect the suprahepatic inferior vena cava (SHVC). The portal vein and infrahepatic inferior vena cava (IHVC) were connected according to the "two-cuff" method. The common bile duct was connected by a biliary stent. We used the stent (G3 group) or aortic trunk (G2 group) to reconstruct the hepatic artery. The patency of the hepatic artery was verified by transecting the artery near the graft after one week. The survival rate of the recipients and serum alanine aminotransferase (ALT) levels, hepatic pathologic alterations, apoptosis and necrosis were observed at one week postoperatively.ResultsThe patency of the hepatic artery was verified in eight of ten mice in G3 and in six of ten mice in G2. The 7-day survival rate, extents of necrosis and apoptosis, and TGF-β levels were not significantly different among the three groups (P>0.05). However, the serum ALT levels and operation time were markedly lower in G3 compared with G2 or G1 (both P<0.05).ConclusionsReconstruction of the hepatic artery using a stent can be performed quickly with a high rate of patency. This model simplifies hepatic artery anastomosis and should be promoted in the field of biomedical research.

The risk of malignant tumor progression has been a concern associated with the use of anti-tumor necrosis factor-alpha monoclonal antibody (anti-TNFα mAb). On the contrary, recent observational studies have reported negatively on this risk and instead suggested that anti-TNFα mAb acts as a tumor suppressor in inflammatory carcinogenesis models and subcutaneous transplant models of colorectal cancer. However, no consensus has been established regarding the actual effects of anti-TNFα mAb on malignant tumors. Here, we aimed to evaluate, for the first time, the effect of anti-TNFα mAb on the tumor microenvironment in the absence of intestinal inflammation in a colorectal cancer orthotopic transplant mouse model suitable for tumor microenvironment assessment. The orthotopic transplantation model was developed by transplanting CT26 cells into the cecum of BALB/c mice. Changes in tumor size and weight were recorded 3 weeks after transplantation, and the tumor microenvironment was assessed via RNA sequencing and immunohistological staining. In the orthotopic transplant model, the administration of anti-TNFα mAb led to a reduction in colorectal cancer. The RNA sequencing analysis showed upregulation of immune-related pathways and apoptosis and suppression of stromal- and tumor growth-related pathways. Additionally, Gene Ontology analysis showed inhibition of angiogenesis. Immunohistochemical staining showed inhibition of tumor growth, increase in apoptosis, suppression of stromal response, suppression of angiogenesis, enhancement of tumor immunity, and reduction in the number of tumor-associated macrophages. Anti-TNFα mAb acts as an inhibitor of tumor progression in the tumor microenvironment of a colorectal cancer orthotopic transplant mouse model.

Impaired hepatic regeneration by ischemic preconditioning in a rat model of small-for-size liver transplantation