A Serial Study Of Cold/Warm Ischemia Reperfusion Injury On Death Pattern And Repair Factors Of Hepatocyte In Rats

Objective: To find out the mechanisms of cell death in I/R injury, we made a serial study of cold/warm ischemia reperfusion injury on death pattern and repair factors of hepatocyte in rats based on such new theory.Method: A serial study had been made. Models of warm ischemia/reperfusion of clamping partial liver vein and hepatic artery, cold ischemia/reperfusion of orthotopic liver transplantation were made. Relative factors of energy capacity, cell channel, repair of I/R injury had been investigated. The number of cells including normal, apoptosis, oncosis and necrosis were measured. The change of hepatocytes energy capacity, Proliferating cell nuclear antigen (PCNA); the expression ofhypoxia-inducible factor-1α(HIF-1α ), aquaporin-8(AQP-8), and caspase 3 at Oh,1h,12h and 72h following reperfusion, at 1h of ischemia were observed. At the same time, the pathologic change of liver was also observed. We also investigated the activity of inon channel such as Na+-K+ATPase,Ca2+-Mg2+ATPase during the whole process. The methods of immunohistochemistry, image analysis, flow cytometer and immunoelectron microscopic method had been used.Results: In groups of warm ischemia, ATP reduced immediately and oncosis was the dominant death model of hepatocytes during the ischemia phase. After reperfusion, ATP increased and the death model of hepatocytes conversed into apoptosis. While ATP reduced slowly and apoptosis was the dominant death model of hepatocytes during the ischemia phase in cold ischemia groups. The expression of caspase-3 was laid in summit at 12 hours following warm ischemia while there was little change in group of cold ischemia. To aspect of liver pathology, tissue damage during phrase of warm reperfusion was severity, infiltrated by flame cells . We found that the activity of Na+-K+ATPase decreased immediately, and the activity of Ca2+- ATPase Mg2+-ATPase decreased even dramatically. On the contrast, the activities of all ion channel measured decreased slightly and increased to normal faster following cold ischemia.The total expression of AQP8, the main AQP in liver, changed slightly following cold/warm ischemia. But the expression of AQP8 can be observed mostly in cell membrane following warm ischemia, while the expression of AQP-8 following cold ischemia was similar to that of control group. Through the methods of colloidal gold immunoelectron microscopic, the particles of AQP-8 can be found in the membrane of cell, mitochondria and Endoplasmic reticulum. The level of PCNA increased following cold/warm ischemia, and the level of cold ischemia group was lower than that of warm ischemia. At the same time, the peak of proliferation still increased after the number of apoptosis cells laid on the summit. In groups of warm ischemia, the expression of HIF-1α increased 1hour after ischemia, and laid in summit 12 of reperfusion. Then, it decreased at 72 hours of reperfusion, while still higher than that of control. In this group, the expressions of HIF-1α were strengthened in region I of liver acinus while weak in region and . In group of cold ischemia, the expressions of HIF-1α were lower, but up-regulated at 72 hour of reperfusion. Conclusion: The reduce energy capacity caused by ischemia and anoxia is the trigger factor which leads to the death of cell. The patterns of death can be change-over with each other by the effect of energy capacity. It would be useful to reduse the ischemia / reperfusion (R/I) injury and prlong the time of of clamping partial liver vein and hepatic artery, if the energy was treatment before operation or during operation. In other way, we find that nevertheless from microcosmic aspect as cell death or macroscopic aspect as R/I injury, water is the most important mediate. For example, oncosis is the passive death mediate by water while apoptosis is the initiative death of losing water. The substrate of energy capacity lost as soon as the water is deprived from the cell. The cell must be dead without water (reperfusion) in time or obtaining too much harm...