Determination of a Safe Vascular Clamping Method for Liver Surgery

Abstract

To determine the safest method of hepatic vascular clamping associated with the least ischemiareperfusion injury of the liver during liver surgery. Setting: University laboratories. Subjects: Sixty-five adult male Wistar rats. Methods: The hilar area of the left lateral and median lobes of rat liver was clamped for 10 minutes (group 1), 15 minutes (group 2), or 20 minutes (group 3) followed by 5 minutes of reperfusion. The procedure was repeated for a total period of ischemia of 60 minutes in each group. Control rats underwent laparotomy without vascular clamping. In addition to histological examination, we determined calpain µ activity, a marker of liver injury, by Western blotting using specific antibodies against the intermediate (activated) and proactivated forms of calpain µ. Measurements were performed at the end of ischemia and after 2 hours of reperfusion. We also determined the degradation of talin, an intracellular substrate of calpain µ, by Western blotting. Results: The level of adenosine triphosphate and energy charge at 2 hours after reperfusion did not change after ischemia-reperfusion irrespective of the duration of ischemic cycle. After 60 minutes of intermittent ischemia followed by 2 hours of reperfusion, cell membrane bleb formation, calpain µ activation, and talin degradation were detected in groups 2 and 3 but not in group 1. Conclusion: The safest method of hepatic vascular clamping that produces a minimum or no ischemiareperfusion injury is 60 minutes of 6 cycles of 10minute vascular clamping interrupted by 5 minutes of reperfusion.

Protective effect of prednisolone on ischemia-induced liver injury in rats

“…Since cell membrane bleb formation is an irreversible phenomenon leading to cell necrosis, the role of calpain activation in cell membrane blebbing through the proteolysis of cytoskeletal proteins [15] is considered to be particularly important. Furthermore, we have previously reported that talin and α-actinin were degraded simultaneously with calpain μ activation in oxidative stress-induced hepatocyte injur y, and that all these events were suppressed by a specific calpain inhibitor, calpeptin [10][11][12][13][14] . Thus, the beneficial effects of prednisolone on hepatic ischemia-reperfusion injury may be at least due to inhibition of calpain μ activation.…”

“…These changes were associated with improved overall liver function as reflected by lowering of serum AST and ALT concentrations, relative to the control. Calpain μ is a major Ca 2+ -dependent cytosolic protease so far described [13,14] and is activated following increased [Ca 2+ ] i in hepatocyte injur y. Since cell membrane bleb formation is an irreversible phenomenon leading to cell necrosis, the role of calpain activation in cell membrane blebbing through the proteolysis of cytoskeletal proteins [15] is considered to be particularly important.…”

“…Partial (68%) hepatic ischemia was induced by clamping the branches of the portal vein, and hepatic artery feeding the left lateral and median lobes, the branches of the right lateral (24%) and caudate (8%) lobes were not clamped [13,14] . In this condition, intestinal congestion or other complications did not occur throughout the experiment (Figure 3).…”

“…The preparation and characteristics of an antibody that specifically recognizes the N-terminal peptide of intermediate (activated) (NH 2 -AQVQKQC-COOH) for m of calpain μ (78 kDa) have been described previously [13,14] .…”

“…Although the molecular mechanisms of bleb formation are unknown at present, Ca 2+ -dependent disruption of the cytoskeleton is considered to play an important role in the blebbing of plasma membrane [8,9] . Calpain μ, a Ca 2+ -sensitive for m of Ca 2+ -activated neutral protease (EC 3,4,22,17), has been shown to degrade various cytoskeletal proteins such as talin, α-actinin and filamin [10][11][12][13][14] .…”