ObjectiveTo analyze the death causes of postoperative early-stage after orthotopic liver transplantation (OLT) in rats, and to provide appropriate treatment strategies. MethodsThree hundreds of rat OLT models were performed by modified Kamada two-cuff technique. Operation time of each stage during OLT and postoperative survival time of rats were recorded and analyzed. According to survival time, the rats were divide into 4 groups:intraoperative death group (rats died during operation), < 6 hours group, 6-24 hours group, and > 24 hours group. Then comparison of operation time of each stage during OLT in rats of 4 groups was performed, and reasons of death during 24 hours after OLT were analyzed. ResultsOf the 300 OLT models, 37 rats died during operation (12.33%), 51 rats died within 6 hours after operation (17.00%), 76 rats died during 6-24 hours after operation (25.33%), and 136 rats survived longer than 24 hours (45.34%). The most common death causes of < 6 hours group were as follows:lose too much blood during the operation (27.45%, 14/51), postoperative bleeding (27.45%, 14/51), and vascular embolization (15.69%, 8/51). However, the most common death causes of 6-24 hours group were as follows:angiostenosis (27.63%, 21/76), postoperative bleeding (21.05%, 16/76), and pulmonary edema (19.74%, 15/76). There were significant differences in the cold ischemia time and anhepatic phase among the 4 groups (P < 0.05). The cold ischemia time and anhepatic phase of intraoperative death group were longer than those of other 3 groups (P < 0.05), in addition, the cold ischemia time of > 24 hours group was shorter than those of other 3 groups (P < 0.05). ConclusionsThere are many reasons leading to the early death after OLT. The long time of anhepatic phase and the cold ischemia time, intraoperative and postoperative bleeding, thrombosis, angiostenosis, and pulmonary edema are key factors for the improvement of prognosis in rats after OLT operation. Improvements of the reasons above are helpful to improve the successful rate of modeling and quality of OLT rats.
Objective To summarize the diagnosis and treatment progress of borderline resectable pancreatic ductal adenocarcinoma (BR-PDAC) in recent years. Methods Through the retrieval of relevant literatures, the progress in the diagnosis and treatment of BR-PDAC in recent years were reviewed, to summarize the current status of definition, management, and outcome of BR-PDAC. Results Pancreatic surgery had significantly changed during the past years and resection approaches had been extended beyond standard procedures, including vascular and multivisceral resections. Consequently, BR-PDAC, which had recently been defined by the International Study Group for Pancreatic Surgery (ISGPS), had become a controversial issue with regard to its management in terms of upfront resection vs. neoadjuvant treatment and sequential resection. The key point was preoperative diagnostic accuracy to define the resectability of BR-PDAC and radical tumor resection followed by neoadjuvant treatment. Conclusion Surgery followed by neoadjuvant treatment is the only treatment option for BR-PDAC with the chance of long-term survival.
Objective To analyze the effects of different perfusion and different superior hepatic vena cava anastomosis methods on the establishment of rat orthotopic liver transplantation model. Methods Eighty SD rats were randomly grouped with donor group and recipient group with 40 rats in each group. Ten rats in the donor liver acquisition group received abdominal aorta infusion set drip irrigation (1 drop/s); 10 rats received abdominal aorta micro-pump perfusion method (6 mL/min); and then received recipient liver transplantation (corresponding to 10 rats in each group). The donor liver perfusion time and donor liver acquisition time of rats in the 2 groups were collected. HE staining was performed on liver tissues after perfusion and 24 h after liver transplantation. Ten rats in the recipient liver transplantation group received continuous anastomosis, and 10 rats received tension-reducing half-needle anastomosis. We collected the anastomosis time of superior and inferior hepatic vena cava, the time of anhepatic stage and the incidence of postoperative complications of the recipient. Results Contrasted with abdominal aorta infusion group, the perfusion time and acquisition time of donor liver were shorter in the abdominal aorta micro-pump perfusion group (P＜0.05). HE staining showed that the morphology of hepatocytes, portal vein and bile duct in the abdominal aorta micro-pump perfusion group did not change obviously, only a few lymphocytes infiltrated. Contrasted with continuous anastomosis group, the anastomosis time, anhepatic stage of the superior hepatic vena cava, incidences of postoperative anastomotic bleeding and incomplete perfusion of donor liver in the reduced tension half-needle anastomosis group were shorter or lower (P＜0.05). Conclusions Compared with abdominal aorta infusion set drip method, the quality of donor liver was improved by abdominal aorta micropump perfusion. Compared with continuous anastomosis method, the tension-reducing half-needle anastomosis can shorten the suture time of superior hepatic vena cava and anhepatic stage, and the incidence of anastomotic bleeding was reduced.
Objective To explore the pathological features of rejection reaction and whether it accord with antibody-mediated rejection (AMR) in the liver transplantation model of allo-sensitized rat. Methods Twelve male Lewis rats as the recipient, 250–290 g; 6 male Brown Norway (BN) rats as the donor, 250–280 g. Twelve Lewis recipient rats were randomly divided into 4 groups by random number method (n=3): Lewis control group (LC group, without any treatment), direct transplantation group (T group, livers from BN rats were directly transplanted into Lewis rats), sensitized group (S group, spleen lymphocytes from BN rats were injected into Lewis rats), and sensitized transplantation group (TS group, splenic lymphocytes from BN rats were injected into Lewis rats for 2 weeks before liver transplantation). On the 14th day after liver transplantation, 3–4 mL of recipient non-lethal blood was collected to detect serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TB), alkaline phosphatase (ALP) and creatinine (CRE) levels, and detect the expression of donor-specific alloantibody (DSA) and complement C4d in recipients. Hematoxylin-eosin (HE) staining and Masson staining were used to evaluate the morphological indexes of rat liver tissue, and CK-19, C4d and CD20 immunohistochemical staining methods were used to evaluate the degree of liver rejection and rejection activity index (RAI) score was performed. ResultsCompared with the T group, the serum AST, TB, and ALP levels, as well as the positive rates of DSA (IgG1, IgG2a, IgG2b, IgG2c) and C4d expression in Lewis rats in the TS group increased. Compared with the LC group, rats in the T group showed partial bile duct edema and lymphocyte infiltration, but no obvious damage of capillary structure was observed. Compared with the T group, a large number of lymphocytes or monocytes were infiltrated and capillaries were severely damaged in the anterior bile duct of rats in the TS group. The RAI and C4d scores of the TS group were higher than those of the T group. Conclusions More severe acute rejection and liver dysfunction occurred after liver transplantation in sensitized rats, and the acute rejection in sensitized rats was consistent with the characteristics of AMR. However, due to the small sample size in this study, further exploration of AMR model remains to be done.
Objective To study the mechanism of immune hyporesponsiveness of allograft rejection induced by transfusion nonpufsed allopeptide syngeneic immature dendritic cell (imDC) generated from recipient bone marrow progenitors and to explore a possible strategy for liver allograft protection in clinic. Methods Forty experimental rats were randomly divided into 4 group: control group, cyclosporine A (CsA) group, mature DC (mDC) group and imDC group. In control group, Wistar rats only received liver transplantation. In CsA group, Wistar rats underwent liver transplantation plus CsA treatment 〔10 mg/（kg·d)〕. In mDC group, recipient-derived mDC 1×106 were infused intravenously through the penile vein to Wistar rats. In imDC group, ImDC with the dose of 1×106 were injected into Wistar rats via the dorsum vein of penile. In each group, five recipients were killed on the 10th day after transplantation, the other five recipients were left to observe survival time. The levels of ALT, AST, TBIL, IL-2, IFN-γ, IL-4 and IL-10 were detected. The acute rejection and the expression of FasL/Fas in the grafts were detected by HE and immunohistochemical staining. Western blot was used to detect Scurfin protein expression of CD4+ CD25+ T cells. Results The median survival time of the liver allografts in CsA group and imDC group were significantly longer than that in control group and mDC group ( P ＜ 0.05). The levels of ALT and TBIL in control group and mDC group were significantly higher than those in CsA group and imDC group ( P ＜ 0.05). Compared with CsA group and imDC group, the levels of IL-2 and IFN-γ were higher but the levels of IL-4 and IL-10 were lower in control group and mDC group ( P ＜ 0.01). Slightly or no rejection reaction was found in CsA group and imDC group ( P ＜ 0.05). The Scurfin protein expressions of CD4+ CD25+ T cells of imDC group were significantly higher than those of other three groups. Conclusion Application of nonpufsed allopeptide syngeneic recipient-derived imDC is an effective way to induce immune hyporesponsiveness by blocking indirect recognition in rat liver transplantation model. Survival span is significantly prolonged by its protective effect. The mechanism of immune hyporesponsiveness induced by imDC transfusion might be involved in some aspects: T cell apoptosis, immune deviation of Thl/Th2 cytokine net and inhibition of T lymphocytes responsiveness by regulatory T cells.
Objective To explore improvement of orthotopic liver transplantation model in rhesus monkey. Methods Healthy rhesus monkeys were chosen to perform orthotopic liver transplantation for 10 cases. The model was established by drawing on a variety of animal model methods, and the portal vein cuff method was used to establish stable model of orthotopic liver transplantation in rhesus monkeys. Results Ten orthotopic liver transplantation models in rhesus were performed, and the achievement ratio of operation was 10/10. The time of donor hepatectomy and donor preparation was (20±5) min and (30±7) min, respectively. The operation time of recipient and anhepatic phase were (180±35) min and (17±4) min, respectively. After 24 h of operation 9 cases survived, one case died of intra-abdominal hemorrhage after 9 h of operation. After 72 h of operation 8 cases survived, and one case died of upper gastrointestinal bleeding after 38 h of operation. After one week of operation 5 cases survived, and 3 cases died of rejection after 9, 11, and 11 d of operation, respectively. The longest survival time was 32 d, but all of them also died of rejection. No portal vein thrombosis and biliary complications were found in all recipients.Conclusion The improved rhesus monkey model of orthotopic liver transplantation is easy to perform with high achievement ratio of operation. It is an ideal animal model for pre-clinical studies of liver transplantation.
Objective To explore the methods of hepatic artery reconst ruction with iliac arterial interpositiongraf t in orthotopic liver t ransplantation (OL T) and influential factor of relevant complications postoperatively.Methods Analyzed ret rospectively 8 OL T , the hepatic artery reconst ruction with arterial inflow based on recipientinf rarenal aorta using donor iliac artery graf t tunneled through the t ransverse mesocolon and pancreas. Results Thetime required for hepatic artery reconst ruction with iliac arterial interposition graf t was 52 - 126 minutes. Amongthe 8 patient s , 2 patient s developed postoperative bililary t ract complications , 1 with biliary fistula , 1 with int rahepatic biloma , the others were recovered smoothly and liver function returned to normal about one week af ter livert ransplantation. No complications of hepatic artery were observed. Conclusion Iliac arterial interpositional graft is aneffective and reliable method of revascularization in liver transplantation when the use of hepatic artery is not possible.
Objective To observe the dynamic histopathologic changes of acute rejection in rat orthotopic liver transplantation (OLT) model after tacrolimus discontinued and provide some prediction and evaluation data for clinical acute rejection after liver transplantation. Methods Kamada two-cuff technique was used to establish 60 rat OLT model， and male DA rats, male Lewis rats were used as donors and recipients respectively. Therapeutic amount of tacrolimus (0.05 mg/kg, twice per day, continued for 8 d, 1 d before operation and 7 d after operation, intragastric administrated) was administrated to recipients, then continuously half dose was decreased every day beginning from day 8 after operation and tacrolimus administration was stopped on day 13. Liver tissues were collected on day 7, 14, 21, and 28 after liver transplantation. Histopathologic changes and rejection activity index (RAI) of liver tissues were observed, survival time of recipients was calculated. Results Owing to protection effects of tacrolimus, liver tissues displayed no significant histopathologic changes of acute rejection in 7 d after OLT, while typical acute rejection histopathologic changes began to be observed on day 14 after OLT due to tacrolimus discontinuation. On day 14, 21, and 28, RAI were 3.7±0.9, 6.3±0.9, and 8.1±0.7 respectively. Survival time of recipients was (20.85±0.71) d with a median of 21 d. Conclusion Acute rejection could be induced in rat OLT model after tacrolimus discontinuation, and data collected from this model shows some extent of predictive value and assessment value for clinical liver acute rejection.
ObjectiveTo clone full-length cDNA of rat galectin-9 and construct recombinant adenovirus granule containing rat galectin-9 gene. MethodsThe galectin-9 gene was amplified by RT-PCR from rat liver tissue and inserted orientationally into plasmid pDC316-GFP digested by restriction endonucleases NotⅠ and HindⅢ. The recombinant pDC316-GFP-galectin-9 shuttle plasmid was identified by PCR, restriction endonuclease digestion and sequencing, and then co-transfected with rescue plasmid pBHGlox△E1.3Cre into HEK-293 cells by liposome reagent. Recombinant adenovirus vector containing rat galectin-9 gene (Ad5-galectin-9) was generated by sitespecific recombination and confirmed by PCR, and then Ad5-galectin-9 was propagated in HEK-293 cells and purified. The infectious titer of viral stock was determined by TCID50 assay. ResultsConstruction of pDC316-GFP-galectin-9 shuttle plasmid was confirmed to be correct by PCR, restriction endonuclease digestion and sequencing. Construction of recombinant adenovirus Ad5-galectin-9 was confirmed to be correct by PCR. The infective titer of Ad5-galectin-9 was 1.4×109 U/ml. ConclusionRecombinant adenovirus vector containing rat galectin-9 gene (Ad5-galectin-9) is successfully constructed, which provides the foundation of further research on the function of galectin-9 gene.