In situ analysis of ischaemia/reperfusion injury in rat liver studied in three different models

Animal models of liver ischaemia and reperfusion are frequently used to study the consequences on liver cells of transient oxygen deprivation. In 3 different rat models we studied ischaemia/reperfusion effects on liver cell membrane integrity, cytoplasmic enzyme proteins and enzyme activities by in situ histochemical techniques. In vivo ischaemia, as well as no-flow hypoxia, or N2-induced hypoxia in isolated perfused livers, reduced the activity of 5′-nucleotidase, a sensitive marker for plasma membrane damage in hepatocytes. As little as 2 minutes of reoxygenation in each model resulted in leakage of soluble enzymes from parenchymal and non-parenchymal liver cells, as shown by decreased protein level and activity of cytoplasmic enzymes. Whereas a multifocal decrease was observed after in vivo reperfusion, a decrease was found in all periportal and midzonal cells after blood-free reoxygenation. As judged by alkaline phosphatase activity and immunohistochemistry, an influx of inflammatory cells was not found in the in vivo model. Our findings indicate that reoxygenation itself, rather than restoration of flow, accounts for the loss of soluble enzymes from liver cells after a period of hypoxia. In situ detection of enzyme protein and activity proved useful for the examination of very early ischaemia/reperfusion effects on rat liver cells

Influence of acidosis and hypoxia on liver ischemia and reperfusion injury in an in vivo rat model

The contribution of acidosis to the development of reperfusion injury is controversial. In this study, we examined the effects of respiratory acidosis and hypoxia in a frequently used in vivo liver ischemia and reperfusion (I/R) injury rat model. Rats were anesthetized with intraperitoneal anesthetics and subjected to partial liver ischemia (70%) for 60 min and subsequent reperfusion for 90 min under the following conditions: 1) no acidosis and normoxia, maintained by controlled ventilation; 2) acidosis and normoxia, maintained by passive supply with oxygen; 3) no acidosis and hypoxia, maintained by bicarbonate administration without respiratory support; and 4) acidosis and hypoxia, i.e., without respiratory support or pH correction. Changes in plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured as parameters of hepatocellular injury, and bile secretion was monitored. AST and ALT levels were lowest in the ventilated rats and highest in the bicarbonate-treated rats. No differences in bile secretion were found between groups. Our results suggest that respiratory acidosis significantly enhanced liver I/R injury under normoxic conditions, whereas respiratory acidosis significantly reduced liver I/R injury under hypoxic condition

Decrease in core liver temperature with 10 degrees C by in situ hypothermic perfusion under total hepatic vascular exclusion reduces liver ischemia and reperfusion injury during partial hepatectomy in pigs

OBJECTIVE: We attempted to assess liver ischemia/reperfusion injury under a mild decrease in core liver temperature of 10 degrees C by in situ hypothermic perfusion during ischemia. METHODS: Liver ischemia was induced in pigs by total hepatic vascular exclusion with concomitant in situ perfusion with hypothermic (4 degrees C) Ringer-glucose (cold perfused group, core liver temperature maintained at 28 degrees C), with normothermic (38 degrees C) Ringer-glucose (warm perfused group) or without in situ perfusion (control group). RESULTS: In the cold perfused, warm perfused, and control groups, 24-hour survival was 5/5, 0/5, and 3/5, respectively. Hemodynamic parameters in the cold perfused group remained stable, whereas pigs in both other groups required circulatory support. Plasma AST and interleukin-6 levels were lower in the cold perfused group than in both other groups. Hepatocellular function was best preserved in the cold perfused group as indicated by complete recovery of bile production during reperfusion and no loss of indocyanine green clearance capacity. In both other groups, bile production and indocyanine green clearance capacity were reduced significantly. The hyaluronic acid uptake capacity of pigs in the cold perfused group or control group did not differ, indicating preserved sinusoidal endothelial cell function. Histopathologic injury scores during reperfusion were significantly lower in the cold perfused group when compared to both other groups. CONCLUSIONS: A mild decrease in core liver temperature of 10 degrees C by in situ hypothermic liver perfusion during ischemia protects the liver from ischemia/reperfusion injury. This protection appears to be related to cooling of the liver rather than to the washout of blood during perfusio

Preoperative assessment of postoperative remnant liver function using hepatobiliary scintigraphy

Hepatic resection is the therapy of choice for malignant and symptomatic benign hepatobiliary tumors. The concept of remnant liver volume (RLV) has been introduced and can be assessed with CT. However, inhomogeneous liver function distribution and a lack of correlation between morphologic hypertrophy and functional recovery fuelled the enthusiasm for functional imaging. The aim of the present study was to assess liver function reserve (LFR) and remnant liver function (RLF) before and after major liver surgery with hepatobiliary scintigraphy (HBS) and to compare scintigraphic results with volumetric CT data and indocyanine-green (ICG) clearance test results. Furthermore, HBS was used to assess functional recovery of liver function, and results were compared with volumetric data. Methods: Fifteen patients with a partial liver resection were included. HBS was performed before, 1 d after, and 3 mo after surgery. ICG clearance and CT were performed before and 3 mo after surgery. Liver function determined with HBS was compared with ICG and volumetric data. Results: Liver function determination using HBS was highly reproducible. A strong positive association (r = 0.84) was found between LFR determined with HBS and ICG clearance. Little or no association (r = 0.27) was found between CT volumetric analysis and corresponding ICG clearance. A strong positive association (r = 0.95) was found between the RLF determined preoperatively on HBS and the actually measured value postoperatively. A weak positive association (r = 0.61) was found between functional liver regeneration and liver volume regeneration in the 3 mo after partial liver resection. Conclusion: HBS offers a unique combination of functional liver uptake and excretion with the ability to assess the preoperative LFR and to estimate the RLF preoperatively. Determination of the RLF instead of the RLV might clarify some of the discrepancies observed in the literature between RLV and clinical outcome in patients with an inhomogeneous liver function. Finally, liver function regeneration can be monitored using HB

Effect of in situ hypothermic perfusion on intrahepatic pO(2) and reactive oxygen species formation after partial hepatectomy under total hepatic vascular exclusion in pigs

Aim: This study examined attenuation of ischemia and reperfusion (I/R) induced liver injury during liver resections by hypothermic perfusion of the liver under total hepatic vascular exclusion (THVE). Method: Reactive oxygen species (ROS) formation, microcirculatory integrity and endothelial cell damage were investigated. Left hemihepatectomy (LHX) was performed without in situ perfusion (control-LHX, n = 5) or with concomitant in situ perfusion with hypothermic (4 degreesC) Ringer-glucose (cold-LHX, n = 5) or normothermic (38 degreesC) Ringer-glucose (warm-LHX, n = 5). Glutathione (GSH) and malondialdehyde (MDA) concentrations, tissue pO(2) levels and hyaluronic acid (HA) uptake capacity were determined. Results: After cold, warm and control-LHX, 24 h survival was 5/5, 0/5 and 3/5, respectively. GSH levels were best preserved after cold-LHX during reperfusion. MDA levels increased in all groups without significant differences between the groups during reperfusion. Tissue pO(2) levels increased after cold-LHX whereas after warm-LHX and control-LHX, pO(2) levels decreased during reperfusion. HA uptake capacity remained normal after cold-LHX. After warm-LHX and control-LHX, HA uptake capacity decreased after 6 h of reperfusion but recovered after 24 h of reperfusion in the control-LHX group. Conclusion: Moderate hypothermic perfusion protects the liver from I/R injury during LHX under THVE. This protective effect depended on maintenance of liver microcirculation rather than a reduction in ROS formatio

Factor VIII expression in liver disease

Liver disease is associated with markedly elevated plasma factorVlll (FVIII) levels,whereas the synthesis of many other coagulation factors and proteins is reduced. In order to define the mechanism of FVIII increase, we have determined the expression levels of FVIII, both at mRNA and protein level, in patients with liver disease who underwent partial liver resection. In addition, the expression of von Willebrand factor (VWF) and low density lipoprotein receptor-related protein (LRP), proteins known for their ability to modulate FVIII plasma levels, were examined. Tissue samples for RNA extraction were obtained from 4 patients with cirrhosis, 9 patients with liver failure without cirrhosis and 6 patients with liver metastasis of a colon or rectum carcinoma (control group). In patients with liver cirrhosis hepatic FVIII and LRP mRNA levels were significantly lower than controls (p less than or equal to 0.010), while VWF mRNA was significantly higher (p less than or equal to 0.050). Immunohistochemical analysis revealed that cellular VWF protein distribution was also increased in cirrhotic livers compared to liver tissue from patients with non-cirrhotic liver disease. In cirrhotic tissue enlarged portal veins appeared to overgrow FVIII producing sinusoidal endothelial cells. Similarly, the number of LRP-producing cells appeared to be lower in cirrhotic tissue than in controls. The plasma concentration of both FVIII and VWF was significantly higher in patients with cirrhosis than control subjects (p = 0.038 and 0.010 respectively). These results demonstrate that elevated plasma FVIII levels in liver cirrhosis are associated with increased hepatic biosynthesis of VWF and decreased expression of LRP, rather than increased FVIII synthesi

Preoperative assessment of liver function: a comparison of 99mTc-Mebrofenin scintigraphy with indocyanine green clearance test

BACKGROUND/AIMS: The indocyanine green (ICG) clearance test is the most frequently used test for preoperative assessment of liver parenchymal function but has its limitations. The aim of this study was to investigate the correlation between ICG clearance test and the liver uptake of 99-Technetium-labelled (99mTc)-Mebrofenin (99mTc-Mebrofenin) as measured with hepatobiliary scintigraphy. METHODS: Fifty-four patients were diagnosed as hepatocellular carcinoma (n=9), hilar tumours (n=20) and 25 patients with non-parenchymal tumours including colorectal metastasis (n=15) and miscellaneous tumours (n=10). One day prior to operation, hepatobiliary 99mTc-Mebrofenin scintigraphy was performed after intravenous injection of 85 MBq and the 15-min clearance rate of ICG (ICG-C15) was measured. RESULTS: The mean ICG-C15 was 86.86+/-1.19% (SEM). The mean 99mTc-Mebrofenin uptake rate was 12.87+/-0.52%/min. A significant correlation was obtained between 99mTc-Mebrofenin uptake rate by scintigraphy and ICG-C15 (r=0.73, P <0.0001). The mean clearance capacity of the right liver segments (79.83+/-1.63, range 47.75-95.97%) was larger than that of the left segments (20.24+/-1.55, range 6.51-52.51%). CONCLUSION: 99mTc-Mebrofenin uptake rate as assessed by scintigraphy is an efficient method for determining liver function and correlates well with ICG clearance. At the same time, 99mTc-Mebrofenin scintigraphy provides information of segmental functional liver tissue, which is of additional use when planning liver resectio