Comparison Of Different Solutions In Preservation Of Liver From Non-heart-beating Rats

Objects:1. To modify the two-cuff technique of orthotopic liver transplantation (OLT) in rats and set up a stable model of OLT.2. To compare the effects of University of Wisconsin (UW) solution, Histidine-trytophan- ketoglutarate (HTK) solution and Celsior (CS) solution on preservation of livers from non-heart-beating rats.3. To explore the effect of recombinant human hepatocyte growth factor (rhHGF) on protection from warm ischemia and its possible mechanisms,Methods:The two-cuff technique of OLT in rats was modified and the hepatic arterial was reconstructed with micro-stent. The thoracic descending aorta and supradiagragmatic inferior vena cava were cross-clamped for 10 min to induce warm ischemia as a NHBD model. After flushed with the respective cold preservation solution, NHBD livers were stored at 4℃ for 8 h or 16 h. Then they were transplanted. The bile discharge, water volume of liver tissue, liver enzymes (ALT and AST), ATP content in liver tissue, and ET-1 level in serum at 1h, 3h, 6h time point after reperfusion were measured. Cytokines (TNF-α, IL-1, INF-γ, IL-4 and IL-10) level in the serum were measured with ELISA method. Histopathology examination was analyzed by hematoxylin and eosin staining or an electron microscope. RT-PCR method was used to detect the mRNA expressions of INF-γ, IL-4 and IL-10.Results:1. The 3-week survival rate of the model of OLT with arterial reconstruction through micro-stent in rats was 92. 1% and the artery recanalization rate was 91.4%.2. When NHBD livers were preserved for 8h, the bile discharge at 1h after reperfusion in the group HTK and CS was more than that in the group UW (HTK and CS vs UW, p<0.05) ; The water volumes of liver tissue at lh, 3h and 6h after reperfusion were no significant difference between the three different solutions (p>0.05) ; The ALT and AST leakages at lh, 3h and 6h after reperfusion in the group UW were significantly higher than those in the group HTK and CS (UW vs HTK and CS, p<0. 05) ; The ATP contents in liver tissue in the group UW at lh and 3h after reperfusion were significantly lower than those in the group HTK and CS (UW vs HTK and CS, p<0. 05), but there was no difference at 6h time point between the three solutions (p > 0. 05) ; The serum ET-1 levels at lh and 3h after reperfusion in the group HTK and CS were significantly lower than those in the group UW (p<0. 05) , but there was no difference at 6h time point between the three solutions (p> 0.05 ) ; There was significant difference at lh and 3h after reperfusion in the TNF-α and IL—1 level between the different solutions (p<0. 05) , but there was no difference at 6h time point(p > 0.05) . Histopathology examination showed a well-preserved hepatic architecture, milder portal congestion and milder inflammatory infiltration in the group HTK and CS as compared with the findings in the group UW; Compared with the group HTK and UW, the ultrastructure of the bile duct cell in the group CS showed milder swelling of mitochondria; Recipients showed a 25. 0%, 33. 3% and 58. 3% one-week survival rate in the group UW , HTK and CS, respectively( UW vs HTK and CS, p<0. 05) .3. When NHBD livers were preserved for 16h, there was no significant difference in bile discharge, water volume of liver tissue, serum ALT and AST level, ATP content of liver tissue, serum ET-1 level and serum cytokines (TNF-α and IL—1) level at the respective time point between any of the three solutions (p>0.05); Histopathology examination showed that there was no much difference in the tissue damage between livers stored in any of the three solutions; All recipients died within 3 days and there was no any difference between the three solutions(p >0. 05).4. The experimental group (CS solution supplemented with rhHGF) showed more bile discharge, less water volume of liver tissue, lower serum ALT and AST level and higher ATP content in liver tissue as compared with the class group (CS solution without rhHGF). Histopathology of liver specimens revealed significant sinusoidal prominence, severe swelling/necrosis and diffused vacuolization of hepatocytes with neutrophil infiltration, portal congestion in the class group, while these findings were milder in the experimental group. The apoptosis of hepatocytes in the experimental group apparently decreased as compared with that in the class group; The serum level and the mRNA expression in the liver tissue of INF-γ in the experimental group were lower than those in class group, respectively( p<0.05) ; The serum level and the mRNA expression in the liver tissue of IL-4 and IL-10 in the experimental group were higher than those in the class group, respectively ( p<0. 05) . Posttransplant 7-day survival rate was 0% (0/12) in the class group and 16. 67% (2/12) in the experimental group ( p<0. 05 ).Conclusions:1. The animal model of OLT with arterial reconstruction through micro-stent in rats was stable and reproducible.2. UW solution was less effective in preserving NHBD livers after 8h of cold storage as compared with CS and HTK solutions. And CS may have advantages in protecting the bile duct cell from reperfusion-ischemia injury.3. When the NHBD livers were preserved 16h, UW, HTK or CS all showed bad preservation effects. The NHBD livers had better be preserved no more than 16h no matter what kind of preservation solution was used.4. rhHGF enhanced the protective effect of CS solution on preservation of the NHBD livers.5. rhHGF can protect the NHBD livers from cold-warm ischemia injury through alleviating inflammation response, decreasing apoptosis of hepatocytes and directly or indirectly reversing the rate of the Th₁/Th₂ cytokines after reperfusion.