UBC Theses and Dissertations
Quantitative changes in Factor II messenger RNA levels during ischemic/reperfusion injury in porcine liver Donnachie, Elizabeth Mary
When organs are harvested, stored and transplanted they are subjected to a period of ischemia followed by reperfusion. This process results in significant damage to the organ and the success of transplantation is frequently dictated by the magnitude of this insult. It is for this reason that a high priority has been given to studying the pathological mechanisms underlying this type of ischemic and reperfusion injury. Ischemic/reperfusion injury to the liver significantly decreases the ability of the organ to synthesize proteins. In liver transplant recipients a decrease from pre-operative values is seen in the levels of all plasma protein clotting factors. In particular, Factor II levels decrease to 36% of their pre-operative level. Studies in ischemic rat liver have indicated that during post-ischemic recovery, the translatable levels of mRNA that code for albumin are qualitatively altered. It is not known whether these changes are quantitative. For these reasons, we elected to quantitate the levels of Factor II mRNA in tissue and compare them with plasma levels of Factor II in a porcine model of warm hepatic ischemic/reperfusion injury. In the model we employed, hepatic ischemia was achieved by diverting the portal blood through a shunt to the right external jugular vein and by clamping the hepatic and gastroduodenal arteries. Reperfusion was initiated following 90 minutes of ischemia by removal of the shunt and clamps. Blood and tissue biopsy samples were collected prior to ischemia, following ischemia and at 90 minutes, 270 minutes, 1 day and 2 days of reperfusion. Tissue mRNA was extracted and quantitated relative to the total DNA content. The extraction efficiencies were monitored and corrected for by means of a synthesized internal standard developed for this study. The effect of ischemic/reperfusion injury on Factor II mRNA was assessed using a Factor II cDNA probe and "dot-blot" hybridization techniques. A quantitative method for the determination of porcine Factor II in plasma during ischemic/reperfusion injury was established using a synthetic chromogenic substrate. In addition, routine plasma measurements of liver function and Indocyanine Green clearance tests were performed. The changes seen in the routine plasma measurements performed were found to be similar to those of other investigators. Plasma AST (aspartate aminotransferase) levels rose significantly during the reperfusion phase indicating that hepatocellular damage had occured. Plasma glucose and lactate levels increased significantly during ischemia and returned to normal by 90 minutes of reperfusion. Plasma K⁺ levels decreased significantly during the early stages of reperfusion (15 minutes) and returned to normal by 90 minutes of reperfusion. In contrast to the changing plasma levels of lactate, AST, glucose and K⁺, bilirubin values did not vary throughout the operative procedure. The clearance of ICG decreased significantly during ischemia due to the decrease of blood flow to the liver. During reperfusion, the clearance of ICG was also decreased significantly, and it was concluded that this reduction was due to some degree of hepatocellular injury although differences in hepatic blood flow and perfusion cannot be ruled out. At one and two days of reperfusion, the ICG clearances returned to normal. Plasma Factor II levels decreased significantly during the ischemic phase. Concomitant with the decrease in plasma levels was trend in which there was an increase in the tissue levels of Factor II mRNA. However, during reperfusion, the tissue levels of Factor II mRNA decreased to control biopsy values. The decrease in the levels of Factor II mRNA may have occurred as the result of damage inflicted during the reperfusion phase, specifically the production of oxygen radicals. With continued reperfusion (two days postoperatively), the Factor II mRNA levels remained low in some of the animals studied; in others, the levels started to rise again. The plasma Factor II levels, however, remained low throughout. It is anticipated that these findings will further our understanding of the pathological mechanisms underlying ischemic/reperfusion injury.
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