Expression Pattern of Nitric Oxide Synthases in Rat Skeletal Muscle after Cyclic Episodes of Short Ischemia and Reperfusion

Seo, Youn Kyoung; Jeon, Su Kyoung; Park, Hyun Joo; Lee, Sang Wan; Koh, Bong Cheol; Lee, Duk Ku; Chang, Nam Jin; Paik, Doo Jin; Lee, Wan Hee

Abstract

The ischemic preconditioning was initially identified as a protective maneuver induced by brief periods of ischemia followed by reperfusion. Although ischemic preconditioning can reduce ischemic injury of heart, skeletal muscle and neuronal tissue, it's protective mechanism remains elusive. Recently, several investigations suggest the associations of nitric oxide with protection from ischemic injury. Nitric oxide synthesized by a member of nitric oxide synthase (NOS) family has been known to increase or decrease the ischemic injury. The purpose of this study was to observe the expression patterns of NOS 1, NOS 2 and NOS 3 in the rat skeletal muscle after cyclic episodes of short ischemia and reperfusion. Nine and thirty-five weeks-old male Sprague-Dawley rats were divided into control and cyclic short ischemia and reperfusion groups. The experimental group was further divided into 3 groups based on cycles of short ischemia and reperfusion. For cyclic short ischemia and reperfusion, left commom iliac artery was occluded 3, 6 and 10 times for 5 minutes ischemia followed by 5 minutes reperfusion using rodent vascular clamps. The animals were sacrificed at hours 0, 3, 6, 24 and 72 after reperfusion and the left rectus femoris muscles were removed. The expression profiles and distribution of NOS 1, NOS 2 and NOS 3 were examined with immunohistochemical staining. The results were as follows; In the cyclic of short ischemia and reperfusion groups, the mortality was increased with increasing of cyclic episodes at 72 hours after reperfusion, and aging. In the control group, NOS 1, NOS 2 and NOS 3 immunoreactivities showed no differenes with aging. In the 9 weeks-old rats, NOS 1 immunoreactivities were observed moderate at 24 hours after 6 times of short ischemia and reperfusion, and moderate and strong at 24 hours after 10 times of short ischemia and reperfusion. In the 35 weeks-old rats, NOS 1 immunoreactivities were observed trace or mild at 24 hours after 6 and 10 times of short ischemia and reperfusion. At 3 hours after 3 times of short ischemia and reperfusion, NOS 2 immunoreactivities were observed moderate or strong, and trace in the 9 and 35 weeks-old rats, respectively. At 3 hours after 10 times of short ischemia and reperfusion, NOS 3 immunoreactivities were observed mild or moderate, and trace or negative in the 9 and 35 weeks-old rats, respectively. In summary, the expression profile of NOS 1, NOS 2 and NOS 3 were observed differently with increasing episodes of short ischemia and reperfusion. The alteration was the most prominent in NOS 3 than in NOS 1 and NOS 2. These results suggest that the alteration of NOS 3 known to protect tissue against ischemic injury may be associated with increasing mortality after multiple episodes of short ischemia and reperfusion.