Ultrastructural Changes in Rat Anterior Tibial and Soleus Muscles Following Graded Periods of Ischemia and Reperfusion

Paik, Doo Jin; Ahn, Dong Choon; Hwang, Kyu Sung; Kim, Hey Joo; Park, Cheol Hong; Chung, Ho Sam; Jo, Geun Yeol

Abstract

Skeletal muscles are known to have tolerance to ischemia, but a prolonged ischemia can cause damage to muscular tissues. The ischemia-reperfusion injury results from the oxygen free radicals released by leucocytes and formed by the reaction of hypoxanthine and xanthine oxidase. Superoxide dismutase (SOD), one of major antioxidant enzymes ocurring in the various tissues of the body metabolizes or scarvanges the oxygen free radicals. Although many studies reported difference in tolerance to ischemia and reperfusion between white and red muscles, some other investigators failed in finding such difference. The present study was performed to examine effects of graded periods of ischemia and reperfusion on the cellular ultrastructure and activity of SOD in white and red muscles. The Sprague-Dawley rats (200~250 g) were used as experimental animals. Under pentobarbital (50 mg/kg IP) anesthesia, incision was made on lower abdomen and left common iliac artery was occluded by means of a vascular clamp for 2, 4 and 6 hour (hrs). Thereafter, the superficial portion of mid-belly of anterior tibial muscle and soleus muscles were excised at 0, 24 and 72 hrs after onset of reperfusion. The specimens were sectioned into slices, 2 mm in length, 1 mm in width and thickness. Some specimens were prepared for electron microscopic observation and others for determination of SOD activity by using antihuman Cu, Zn- and Mn-SOD antibodies. The results obtained were as follows. 1. In anterior tibial muscle, areas with loose electron-density and dilated cristae were observed in the mitochondria immediately after 2 hrs of ischemia, while widened intermyofibrillar spaces and dilated cisternae of sarcoplasmic reticulum were seen after 2 hrs and 24 hrs reperfusion. When subjected to 2 hrs ischemia and 72 hrs reperfusion, no significant change was found in the cellular ultrastructure. 2. In soleus muscle, electron density was loose in the matrix of mitochondria immediately after 2 hrs of ischemia, while cisternae of sarcoplasmic reticulum were dilalated after 2 hrs of ischemia and 24 hrs reperfusion. Following 2 hrs of ischemia and 72 hrs reperfusion, the electron microscopic findings were similar to those of normal rats. 3. The changes in cellular ultrastructure were more prominent in both the 4 hrs and 6 hrs ischemia groups, in which degree of ultrastructural changes were proportional to duration of reperfusion. 4. In anterior tibial muscle, trace or weak immunoreactivities of Cu, Zn- and Mn-SOD were seen, whereas trace immunoreactivity of Cu, Zn-SOD and trace or weak immunoreactivity of Mn-SOD were observed in soleus muscle. 5. The immunoreactivities of Cu, Zn- and Mn-SOD were not altered in 2 hrs ischemic and 72 hrs reperfused group, while they were increased slightly in 2 hrs ischemic and 24 hrs reperfused group. 6. In both muscles, the activity of SOD increased following 4 hrs or 6 hrs ischemia and 24 hrs or 72 hrs reperfusion. The changes in immunoreactivity of Mn-SOD were not different between two muscles, whereas immunoreactivity of Cu, Zn-SOD were higher in anterior tibial muscle. Consequently, it is suggested that significant ischemia reperfusion injuries are produced after 4~6 hrs ishemia followed by 24 hrs or 72 hrs reperfusion, that anterior tibial muscle is more susceptible to ischemic reperfusion injury and that the ischemic-reperfusion injury is closely related with activity of SOD.