Effects of Ischemic Preconditioning, Adenosine and Pinacidil on the Expression of Cu,Zn- and Mn-SOD mRNA in the Rectus Femoris Muscle of the Rat after Ischemia and Timely Reperfusion

Paik, Doo Jin; Yoo, Young Mi; Hwang, Kyu Sung; Ahn, Dong Choon; Chung, Ho Sam; Kang, Bong Kyun

Abstract

A brief episode of ischemia and reperfusion termed 'ischemic preconditioning' has been established as rendering muscle tolerance to damage during a subsequent prolonged ischemia. The effects of ischemic preconditioning in the cardiac muscle are related to the stimulation of adenosine A1 receptor and the opening of KATP channel. The effect and mechanisms of ischemic preconditioning in the skeletal muscle are not known clearly. The superoxide radical injures the skeletal muscle during the ischemia and reperfusion. There are two types of SOD, which metabolizes the superoxide radicals to H2O2 and O2, in the cell. One of them is Cu, Zn-SOD in the cytoplasm and the other is Mn-SOD in the mitochondria. The activities of SOD are increased against the formation of superoxide radical during the reperfusion. The author performed the present study to investigate the effect and the mechanisms of ischemic preconditioning by measuring the expression of SOD mRNA on timely reperfused ischemic muscles. The healthy Sprague-Dawley rats weighing from 300 g to 350 g were used as experimental animals. Under pentobarbital (50 mg/kg) anesthesia, lower abdominal incision was done and left common iliac artery was occluded by vascular clamp for 2 hours. Rectus femoris muscles were obtained respectively at 3, 6, 12, 24 and 72 hours after reperfusion. The ischemic preconditioning group underwent three episodes of 5 minute occlusion and 5 minute reperfusion of common iliac artery followed by 2 hours of ischemia and timely reperfusion. Adenosine (50 microgram/kg) or pinacidil (1 mg/kg) was administered intravenously before ischemia. 8-cyclopentyl-1, 3-dipropylxanthine (15 mg/kg) or glibenclamide (0.5 mg/kg) was administered intravenously before ischemic preconditioning. Paraffin sections with 4 micrometer thickness in all groups were obtained. The expression of Cu, Zn- and Mn-SOD mRNA was observed by use of in situ hybridization. The results obtained were as follows. 1. The expression of SOD mRNA was seen only in small muscle fibers of the rectus femoris muscle of the rat. 2. Weak expressions of Cu, Zn- and Mn-SOD mRNA were observed in the normal control rat. 3. After 2 hours of ischemia, moderate expression of Cu, Zn-SOD mRNA was observed until 72 hours of reperfusion. Weak or moderate expression of Mn-SOD mRNA at 3 hours and 6 hours of reperfusion, weak or trace expression at 12 hours of reperfusion, moderate expression at 24 hours of reperfusion and weak or moderate expression at 72 hours of reperfusion were observed. 4. After ischemic preconditioning, moderate expressions of Cu, Zn-SOD mRNA were seen in the groups of 3, 6, 12 and 24 hours of reperfusion. Moderate expressions of Mn-SOD mRNA were seen in the group of 0, 3, 6 and 12 hours of reperfusion and strong expression was seen in the group of 24 hours of reperfusion after ischemic preconditioning. 5. After 2 hours of ischemia with ischemic preconditoining, moderate expressions of Cu, Zn-SOD mRNA were seen in the groups of 0, 3, 6, 12, 24 hours of reperfusion. Moderate expressions of Mn-SOD mRNA were observed in the groups of 0, 3, 6, and 12 hours of reperfusion and moderate or strong expression was seen in the group of 24 hours of reperfusion. 6. After 2 hours of ischemia with the pretreatment of adenosine, moderate expressions of Cu, Zn-SOD mRNA were seen in the group of 0, 3, 6, 12 and 24 hours of reperfusion. Moderate expression of Mn-SOD mRNA in the groups and 3 hours of reperfusion, strong expression in the group of 6 and 12 hours of reperfusion and moderate expression in the group of 24 hours of reperfusion were seen. 7. After 2 hours of ischemia with the pretreatment of pinacidil, moderate expressions of Cu, Zn-SOD mRNA were seen in the groups of 0, 3, 6 and 12 hours of reperfusion and those of Mn-SOD mRNA were seen in the groups of 3, 6, 12 and 24 hours of reperfusion. 8. After 2 hours of ischemia with ischemic preconditioning and the pretreatment of 8-cyclopentyl-1, 3- dipropylxanthine, moderate expression of Cu, Zn-SOD mRNA were observed in the groups of 0, 3, 6, and 12 hours of reperfusion and those of Mn-SOD were seen in the groups of 6, 12 and 72 hours of reperfusion. 9. After 2 hours of ischemia with ischemic preconditioning and the pretreatment of glibenclamide, moderate expressions of Cu, Zn- and Mn-SOD mRNA were seen in all groups of reperfusion. Consequently, these results suggest that the expression of Cu, Zn and Mn-SOD mRNA increases during 2 hours ischemia and reperfusion with or without ischemic preconditioning. The effects of ischemic preconditioning are closely related to the stimulation of adenosine A1 receptor and KATP channel.