Preservation Of Rat Liver In Newly Developed Shanghai Multiple-Organ Preservation Solution

Chapter 1 The optimal molecular weight and concentration of PEG added to the newly developed SMO solutionObjective: To evaluate the optimal molecular weight and concentration of PEG added to the newly developed SMO solution.Methods: Livers from SD rats were flushed and then preserved at 4°C for 24h in SMO solution with different molecular weight and concentration of PEG (no PEG, 1g/L PEG20, 10g/L PEG20, 30g/L PEG20, 1g/L PEG35, 10g/L PEG35 and 30g/L PEG35). At the end of the preservation, histological examination of hepatic tissues was performed. Then human venous blood was mixed with normal saline, UW solution, SMO solutions with different molecular weight and concentration of PEG. The dilution ratios of blood with preservation solution were 5:1 and 1:1. Sedimentation rate was measured by an automatic ESR analyzer. Human RBC aggregability and blood viscosity was investigated with an automatic hemorheological analyzer. Light microscopy was used to evaluate morphological characters of the RBC aggregates.Results: After cold preservation for 24h, SMO solution with PEG35 (1g/L) showed the best preservation effect among the tested solution. PEG20 (1 and 10g/L) and PEG35 (1g/L) had little effect on RBC aggregation, while PEG20 (30g/L) and PEG35 (10 and 30g/L) significantly increased the RBC sedimentation and blood viscosity, and had a hyperaggregating effect on RBC.Conclusion: Low concentration PEG35 (1g/L) would be the optimal choice added to the SMO solution.Chapter 2 Establishment of the isolated perfused rat liver model and its modificationObjective: To set up the isolated perfused rat liver model, provide a reliable study platform for the development of organ preservation solution.Methods: We modified the isolated perfused system described previously in references and establish CZ - 1 isolated perfused rat liver system. Then the effectiveness of the isolated perfused system was tested. Livers were harvested after the cannulation of the portal vein and bile duct. Then Livers were connected via the portal vein to a recirculating perfusion system for 120min. The reperfusion solution was Krebs-Henseleit solution at a constant temperature of 37°C and a flow rate of 20ml/min. After 120min reperfusion, bile production was evaluated. Routine HE staining examination of hepatic tissues was also performed.Results: The CZ - 1 isolated perfused rat liver system was cost-effective and reliable to use. It was easy to run. The bile volume collected from the isolated repefused liver were 0.248±0.094 ul/min·g (liver) and was not significantly different from that reported by references. Hepatic tissues in reperfusion group were also morphologically normal.Conclusion: The isolated perfused rat liver model closely mimics physiologic conditions and is the ideal model for investigation of organ preservation solution.Chapter 3 Effects of SMO solution in rat liver preservationObjective: To compare the protective effect of SMO solution with that of UW solution during cold preservation and normothermic reperfusion.Methods: SD rats were divided into four groups according to different preservation solution: control group (without cold preservation), lactated Ringers group (negative control), UW group and SMO group. Preservation injury after cold storage for 24, 48 and 72h was assessed microscopically. Meanwhile the pH value and osmotic pressure of the preservation solutions was measured. The change of the liver weight was also assessed. After 24h cold storage of rat liver in different preservation solutions, the isolated perfused rat liver model was applied to reperfuse the liver for 120min normothermically with Krebs-Henseleit solution. Reperfusion injury was analyzed in the isolated perfused liver by enzyme (AST, ALT and LDH) release and bile production. Routine HE staining and electron microscopic examination of hepatic tissues were performed. NF-κB, ICAM-1 and Caspase-3 expressions were assessed by immunohistochemical analysis. Hepatocellular apoptosis was also assessed Results: After preserving for 24h, histology showed no difference between the SMO and UW group. But after 48h cold preservation, UW solution provided rat livers with better protection against cold ischemia injury. During cold preservation, pH value of the SMO and UW solution decreased with preservation time, and the buffering capacity of both solutions was similar. The osmotic pressure of the both preservation solutions increased with preservation time, but they were higher in UW solution. Prolonged cold preaervation time resulted in an increase in liver weight with SMO-preserved livers and a decrease in weight with UW-preserved livers. After 24h cold storage, transaminase level in the both groups showed no difference. After reperfusion for 120min, ALT level was lower in SMO group, but higher AST and LDH levels were found in SMO group than in UW group. The amount of Bile product after reperfusion for 120min in UW group was more than that in SMO group. Morphological changes in SMO group were significiently worse than that in UW group. Histological results showed more necrotic regions in livers preserved in SMO solution. Electron microscopy revealed that ultrastructural impairments were similar in both groups. No significant differences were found in NF-κB and ICAM-1 expression between SMO group and UW group. The percentage of apoptotic cells and the expression patterns of apoptosis-related-genes were similar in livers preserved in SMO and UW solutions. The variables in both groups were better than those of livers preserved in Ringers solution.Conclusion: The short-term cold preservation effect of SMO solution is similar with that of UW solution. However, SMO solution is inferior to UW solution in protecting the liver from cold ischemia-reperfusion injury.