| Objective To develop mini-porcine models of hepatic steatosis; discuss the preservation, resuscitation and evaluation effect of NMP method for the donated liver from DCD porcine.Methods The first part was to set up animal models of mini-porcine with hepatic steatosis. 10 Bama mini-porcines were selected and then randomly divided into two groups: control group with 5 pigs and model(steatosis liver) group with 5 pigs(1 died during the procession). The control group was fed regularly; the model group was fed with formulated diet consisting of regular feeds(50%), fat-rich feeds(lard50%), alcoholic beverage and tetracycline. Gastric nutrient catheter was created in pigs for feeding lard and alcoholic beverage. Blood sample from auricular veins was collected at the beginning of feeding and on each ending day of the 2nd week, 4th week and 6th week to test the serum biochemical items(AST,ALT,GGT,TG,CHO). At the end of the 6th week, liver biopsy was performed through a abdominal incision. The criteria of developing a hepatic steatosis model were significant increase of blood lipid contents(TG, CHO) and seroenzyme levels, as well as pathological manifestation of multifocal inflammatory cell infiltration and hepatic steatosis. The second part: after DCD steatosis liver model was developed and warm ischemic time was kept for 30 mins, part(5cm×5cm) of liver was removed and then placed in UW preservation solution(UW group) and the rest of liver was perfused in normothermic perfusion machine(NMP group). Perfusion solution was sampled at different time points(1h, 2h, 3h, 4h, 5h, 6h) and AST, ALT, GGT, TG and CHO were examined. Bile production was recorded per hour during perfusion; pathological analysis was inspected for each group before perfusion and at 2h, 4h, 6h during perfusion; liver homogenate(AST, ALT, GGT, TG, and CHO) were examined. Comparison was conducted for biochemical variables and pathology at different time points between two groups.Result The first part:1. One pig died on the first day after gastrostomy.2. Compare the biochemical variable between model group and control group There was no significant difference after being fed 0 week and 2 weeks as to various biochemical indexes(P>0.05); Compared with controls, the model group had significantly higher serum AST,ALT,GGT and TG levels(P<0.001)after they were fed 4 weeks, but CHO did not reach statistical significance(P=0.056); The model group showed significant difference compared with the control group as to various biochemical indexes when they were fed 6 weeks(P<0.001).3. Biopsy of liver tissue Pathology of liver tissue from control group: clear structure of hepatic lobule, proper arrangement of hepatic cords, round and big cell nucleus which were in the central, no lipid droplets and inflammatory cell infiltration, few blood cells in central vein and hepatic sinusoid. Pathological expression of liver tissue from model group(at 6 weeks of feeding): vague structure of hepatic lobule, many various size of lipid droplet vacuole, central venous congestion and hepatic sinusoid congestion, inflammatory cell infiltration, extrusion of hepatic sinusoid which resulted in narrowing space.Second part:1. Comparison of indicators in perfusion solution among different perfusion time points in NMP group Level of AST and ALT had significantly increased after 2hrs’ perfusion compared with the beginning(P<0.05). The concentrations of ALT and AST at other time points were not significantly different from the beginning(P>0.05). In addition, levels of CHO and TG at 5h and 6h were significantly different from the beginning(P<0.05).2. Comparison of indicators of liver homogenate between NMP group and UW group at the end of preserving time Compare with UW group,level of AST,ALT and GGT of liver homogenate had significantly decreased in NMP group(t,P:AST:2.827,0.0311;ALT:3.524,0.0073;GGT:3.010,0.0224).However,there was no significant diffference between the two groups as to level of CHO and TG.(t,P:CHO:2.315,0.0947;TG:2.620,0.0511); intra-group comparison:compared with the beginning of preserving,the level of ALT,CHO and TG had significantly decreased in NMP group after preserving 6h(P<0.05),but level of AST and CHO did not reach statistical significance(P>0.05);level of GGT had significantly increased(P<0.05)but the rest did not reach statistical significance(P>0.05)in UW group.3. Comparison of bile production among different perfusion time points in NMP After 2hrs’ perfusion, biliary drainage volume was about 3.5ml/h, and the character of bile was thick. Between 2h and 4h, the volume was about 4ml/h which was the most and the color was clear. During the last 2hrs, the volume decreased to 2ml/h, but still golden and clear.4. Comparison of biopsy results between NMP group and UW group among different preserving time pointsIn UW group, hepatic sinus congestion increased in certain level,and there was no difference about the degree of steatosis in the process of cold storage for 6 h; In NMP group, central vein and hepatic sinus showed extensive congestion after preserving 2h,then gradually reduce, also the degree of steatosis reduce gradually.Conclusion From this experiment, it could be concluded that formulated diet consisting of fat-rich fees, alcoholic beverage and tetracycline could be used for rapid developing fatty –liver models in mini-porcine. NMP method could relieve the degree of hepatic lipid accumulation, prevent hepatocyte damge with the extention of preserving time, promote the condition of marginal liver donor, which could help to enlarge the donor pool, and contribute to ease the issue of organ shortage. During NMP process, real time pre-operation evaluation of donor liver could be done, which could optimize the donor liver selection, and subsequently improve the prognosis of recipients. It was predicted that with prolonged perfusion time, the advantage of NMP would be more obvious compared with SCS. |
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