Gene expression profile in rat small intestinal allografts after cold preservation/reperfusion

AIM: To determine the changes of gene expression profile in small intestinal allografts in rats after cold preservation/ reperfusion, and to identify the genes relevant to cold preservation/reperfusion injury. METHODS: Heterotopic segmental small bowel transplantation was performed in six rats with a sham operation and they were used as controls. Total RNA was extracted from the allografts (experimental group) and normal intestines (control group) 1 h after cold preservation/ reperfusion, and then purified to mRNA, which was then reversely transcribed to cDNA, and labeled with fluorescent Cy5-dUTP and Cy3-dUTP to prepare hybridization probes. The mixed probes were hybridized to the cDNA microarray. After high-stringent washing, the fluorescent signals on cDNA microarray chip were scanned and analyzed. RESULTS: Among the 4 096 target genes, 82 differentially expressed genes were identified between the two groups. There were 18 novel genes, 33 expression sequence tags, and 31 previously reported genes. The selected genes may be divided into four classes: genes modulating cellular adhesion, genes regulating cellular energy, glucose and protein metabolism, early response genes and other genes. CONCLUSION: A total of 82 genes that may be relevant to cold preservation/reperfusion injury in small intestinal allografts are identified. Abnormal adhesion between polymorphonuclears and endothelia and failure in energy, glucose and protein metabolism of the grafts may contribute to preservation/reperfusion injury. The functions of the novel genes identified in our study need to be clarified further.

Adenosine 5'triphosphate transport and accumulation during the cold preservation of rat hepatocytes in University of Wisconsin solution

AIM: We used isolated hepatocytes to investigate how different concentrations of ATP in the University of Wisconsin (UW) solution affected both cellular ATP content and cell viability during the cold storage and the rewarming step. The mechanism involved in ATP transport and accumulation in hypothermia was also determined. METHODS: The cells were preserved up to 72 h in different conditions: UW solution without ATP (a-group), UW+5 mmol/L ATP (b-group), and UW+10 mmol/L ATP (c-group). The ATP content and the cell viability (LDH release) were determined during the cold storage and the rewarming step. In the groups a and c, the respiratory function of the cells at rewarming was studied. In addition, the cell volume of hepatocytes and the mechanism involved in ATP transport and accumulation were assessed. The extracellular degradation of exogenous nucleotides during transport experiments was investigated by a HPLC technique. RESULTS: After three days of cold storage a loss of cellular ATP content was observed in hepatocytes preserved either without nucleotides (a-group) or with 5 mmol/L ATP (b-group). In contrast, 10 mmol/L ATP (c-group) was able to maintain a normal ATP cellular content, with only a 6% diminution after 72 h of cold storage. The respiratory function was significantly different in hepatocytes preserved with 10 mmol/L ATP than without ATP. No significant change was detected For the three groups in cellular volume during the cold storage. We also report that the time course accumulation of [3H]-ATP by cold stored hepatocytes is a rapid process that is completed after 180 s with linear dependence on the extracellular ATP concentration (linear fitting results in a slope of 0.5624±0.1179 mmol/L ATP intracell/mmcl/L ATP extracell). CONCLUSION: Our results show that, during hypothermic storage in UW solution, hepatocytes are permeable to ATP by a diffusive mechanism. Also, we found that it is ATP the main extracellular nucleotide available for transport and it is not the breakdown products.

Effect of ulinastatin donor-pretreatment on liver graft during cold preservation in rats

Background Donor-pretreatment with ulinastatin may influence the liver graft during cold preservation. The aim of this research was to determine whether pretreatment of donor liver with Ulinastatin can attenuate cold preservation injury,and to explore the mechanism by which Ulinastatin affects the donor liver graft.Methods One hundred and forty-four Wistar rats were divided into the Ulinastatin treatment group （T group） pretreated with Ulinastatin 50 000 U/kg and control group （C group） treated with 0.9% normal saline via peritoneal injection prior to the anesthetization. After the abdominal cavity was opened and perfused with cold Ringer＇s lactate solution, the liver was harvested. The harvested liver was preserved in cold Ringer＇s lactate solution for 0, 2, 6, 24 hours, at which time the liver tissue was sampled for determination of dry weight and wet weight, Na＋-K＋-ATPase and Ca2＋-ATPase activity, lactic acid dehydrogenase （LDH） activity, lactic acid and malondialdehyde levels. Light microscopy and electron microscopy were used to observe liver morphology. The liver cold-preservation solution was taken for measurement of aspartate aminotransferase （AST） and alanine transaminase （ALT） levels. Correlation between ATPase activity and lactic acid level was analyzed by SPSS 13.0 for Windows.Results The morphology in the T group had improved cell boundaries vs. The C group at each time point. Dry weight to wet weight in the T group was lower than in the C group at 6 hours （P 〈0.05）, but the difference was not significant at 24 hours. ALT levels in the T group were lower than that in the C group at 6 hours （P 〈0.05） and 24 hours （P 〈0.01）. AST levels in the T group were lower than those in the C group at 2 hours （P〈0.05）, 6 hours （P 〈0.01） and 24 hours （P 〈0.01）.Na＋-K＋-ATPase activity in the T group was higher than in the C group and the mean difference between two groups was significant at 0 hour （P 〈0.05） and 2 hours （P 〈0.05）. Ca2＋-ATPase activity in the T group was higher than in the C group with the mean difference between two groups significant at 2 hours （P 〈0.05）. The T group had increased lactic acid levels at 0 hour （P 〈0.01） and 2 hours （P 〈0.05） compared with the C group, but there was no influence on the LDH activity at the same time. There were no obvious differences in the levels of malondialdehyde between the two groups at any time point. A linear correlation between Na＋-K＋-ATPase activity and lactic acid levels （r=0.295, P 〈0.05） was found.Conclusions Donor-pretreatment with ulinastatin may protect the cells in a liver graft from ischemia injury during cold preservation; the mechanism may be due to its promotion for cell glycolysis and its preservation of ATPase activity.

Evaluation of IGL-1 preservation solution using an orthotopic liver transplantation model

AIM： To compare, in a pig the protective effect of UW liver transplantation model, with that of IGL-1, a highsodium preservation solution containing polyethylene glycol （PEG） as an oncotic supply. METHODS： All livers were harvested and grafted orthotopically according to standard techniques. The livers were washed out and preserved for 7 h in IGL-1 （n = 6） or in UW solution （n = 7） at 4℃. In a sham group （n = 4）, the livers underwent a 60-min warm ischemia at 37℃. The hepatocellular injury was assessed in organ preservation solution washed out from the graft at the end of ischemic storage （before revascularization）, and in serum 2 h after reperfusion and daily for up to 6 d. RESULTS： Livers preserved in IGL-1 solution released markedly less AST than that preserved in the UW solution before and after revascularization （P 〈 0.05）. Besides, the activity of creatine kinase-BB, a marker of sinusoidal lining cells injury, was higher in the UW group than in the IGL-1 group （P 〈 0.05）. Histological results showed less necrotic regions in livers preserved in IGL-1 solution; however, no difference was observed for inflammation. CONCLUSION： IGL-1 liquid effectively protects parenchymal and non-parenchymal cells against preservation-reperfusion injuries.

Performance of cold-preserved rat liver Microorgans as the biological component of a simplified prototype model of bioartificial liver

AIMTo develop a simplified bioartificial liver (BAL) device prototype, suitable to use freshly and preserved liver Microorgans (LMOs) as biological component. METHODSThe system consists of 140 capillary fibers through which goat blood is pumped. The evolution of hematocrit, plasma and extra-fiber fluid osmolality was evaluated without any biological component, to characterize the prototype. LMOs were cut and cold stored 48 h in BG35 and ViaSpan® solutions. Fresh LMOs were used as controls. After preservation, LMOs were loaded into the BAL and an ammonia overload was added. To assess LMOs viability and functionality, samples were taken to determine lactate dehydrogenase (LDH) release and ammonia detoxification capacity. RESULTSThe concentrations of ammonia and glucose, and the fluids osmolalities were matched after the first hour of perfusion, showing a proper exchange between blood and the biological compartment in the minibioreactor. After 120 min of perfusion, LMOs cold preserved in BG35 and ViaSpan® were able to detoxify 52.9% ± 6.5% and 53.6% ± 6.0%, respectively, of the initial ammonia overload. No significant differences were found with Controls (49.3% ± 8.8%, P ® cold preserved LMOs, respectively (n = 6, P CONCLUSIONThis prototype relied on a simple design and excellent performance. It’s a practical tool to evaluate the detoxification ability of LMOs subjected to different preservation protocols.

Risk factors of severe ischemic biliary complications after liver transplantation

BACKGROUND:Ischemia-related biliary tract complications remain high after orthotopic liver transplantation.Severe ischemic biliary complications often involve the hepatic duct bifurcation and left hepatic duct,resulting finally in obstructive jaundice.Prevention and management of such complications remain a challenge for transplant surgeons.METHODS:All 160 patients were followed up for at least 180 days after transplantation.One-way analysis of variance (ANOVA) and comparative univariate analysis were made using 3 groups (no complications;mild complications;severe complications),to analyze risk factors associated with biliary complications.Multiple logistic regression and linear regression analysis were used to analyze independent risk factors for severe ischemic biliary complications,after excluding other confounding factors.RESULTS:By ANOVA and comparative univariate analysis,the risk factors associated with biliary complications were preoperative bilirubin level (P=0.007) and T-tube stenting of the anastomosis (P=0.016).Multiple logistic regression analysis showed that the use of T-tube and preoperative serum bilirubin were not independent risk factors for severe ischemic biliary complications after orthotopic liver transplantation.Chi-square analysis indicated that in the incidence of severe ischemic biliary lesions,bile duct second warm ischemic time longer than 60 minutes was a significant risk factor.Linear regression demonstrated a negative correlation between cold preservation time and warm ischemia time.CONCLUSIONS:Preoperative serum bilirubin level and the use of T-tube stenting of the anastomosis were independent risk factors for biliary complications after liver transplantation,but not for severe ischemic biliary complications.The second warm ischemia time of bile duct longer than 60minutes and prolonged bile duct second warm ischemia time combined with cold preservation time were significant risk factors for severe ischemic biliary complications after liver transplantation with grafts from non-heart-beating donors.