| The establishment of a mouse model of hepatic cold ischemia-reperfusion in situLiver transplantation is the most effective therapy for the patients with end-stage liver diseases. According to series reports, 8000 cases undergo liver transplantation all over the world per year, and the total number is increasing year by year. The ischemia-reperfusion injury in liver transplantation cannot be avoided and is closely associated with the occurrence of the primary graft non-function, dysfunction and stricture of bile duct. The complication caused by ischemia-reperfusion has become the main cause of retransplantation and death. Accompanied with the increasing number of liver transplantation and shortage of liver graft, this problem is becoming serious in the future, and the study of ischemia-reperfusion mechanism becomes a hot point in the transplant field.Presently, three methods are applied on the study of ischemia-reperfusion mechanism: (1) liver reperfusion with artificial or mechanical method in vivo or in vitro, (2) warm ischemia-reperfusion in vivo, (3) orthotopic liver transplantation. Each method has its virtue. In clinical, with the development of liver-harvest skill, warm ischemia time is shortened. The injury of cold ischemia is noticed and considered having close relationshipwith the graft dysfunction, acute rejection, survival rate of graft and recipient. In order to study conveniently, we established hepatic cold ischemia-reperfusion mouse model of orthotopic liver transplantation.Materials and methodsMale BALB/C mice, weighing 25 to 30g, aged from 8 to 12 weeks, were randomly divided into 3 groups: observation group, sham group and experiment group. All animals operated under intraperitoneal anaesthesia with 2% ketamine hydrochloride 0.2 ml in clean environment. Operation steps: after opening abdomen cavity, we dissociated infrahepatic vena cava, hepatic artery, pylorus vein and portal vein, and clamped the three formers. The portal vein was cannulated with a 30G puncture pin and the liver was perfused through portal vein with 4 C saline 1ml. After 30sec, micro-vascular clamp was used to occlude the portal vein at porta hepatis and Ringer's frazil covered liver surface. After 20min anhepatic phase, we declamped the vena cava, liver artery and pylorus vein. In the experiment group, we collected blood samples via vena cava at 1h, 3h, 6h and 24h after reperfusion, respectively, and executed the animal to acquire the liver tissue. We observed the animal survival rate, serum levels of ALT and AST and tissue pathological changes.ResultsThe mouse survived more than 7 days postoperatively was considered as live forever, and the 7-day survival rate was 83.3%. The operative time before and after reperfusion was (10.6+2.2)min and (5.1+1.3)min respectively. The bleeding volume was lower than 0.5ml. Two mice died, one of adhesive obstruction in small intestine and the other of unknown reason. Within reperfusion, the serum levels of ALT and AST increased obviously in 1 hour (317+70/L vs 80+4.2/L; 662+96/L vs 124+10.2/L P<0.05, respectively), reached a maximal level in 6 hours, and decreased dramatically in 24 hours. Histopathological results showed that the hepatic tissue injured remarkably in 6 hours of reperfusion as neutrophil infiltrating, hepatocyte swelling, hepatic cord arraying irregularly, certain quantity of apoptosis cells and secondary necrosis.Conclusion1. Clamping infrahepatic vena cava, hepatic artery and portal vein can maintain the liver no-blood state after perfusion during anhepatic phase.2. Mouse can serve as a good hepatic cold ischemia-reperfusion animal model of orthotopic liver transplantation. This model can well reflect the dynamic change of serum enzymology and histopathology after liver ischemia-reperfusion injury.
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