Effects of Ischemic Preconditioning on Injury of Rat Skeletal Muscle During Use of Tourniquet

[Purpose] The use of a pneumatic tourniquet for an extended period of time in limb surgery can lead to apoptosis after reperfusion. While ischemic preconditioning (IPC) has a protective effect on reperfusion injury, the effect of age is unknown. This study thus examines the effects of IPC and age on reperfusion injury in the skeletal muscle. [Methods] A pneumatic tourniquet was applied on the hindlimb of rats aged 3–24 months with a tourniquet pressure of 300 mmHg. The IPC⊖ group had two 120-minute tourniquet applications with a 5-minute reperfusion period in between, followed by reperfusion. The IPC⊕ group underwent 5 minutes of tourniquet application and 5 minutes of reperfusion repeated 3 times. The gastrocnemius muscles were then excised 72 hours after reperfusion. The ratio of apoptotic cells was compared using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The expression of apoptosis inducers was examined by Western blot and immunostaining. Six-month-old rats were treated as in the experiment described above, and IPC⊖ and IPC⊕ groups were prepared. Gastrocnemius muscles were then harvested immediately after reperfusion and 24 hours and 72 hours after reperfusion. The temporal changes in VEGF and 8-OHdG expression levels were examined by immunostaining. [Results] Degenerated and atrophied myocytes were observed in the gastrocnemius muscles of 3-month-old to 12-month-old rats, whereas partial necrosis and inflammatory cell infiltration were observed in those of 24-month-old rats. Compared with the IPC⊖ group, the ratio of apoptotic cells decreased in the 6- and 12-month-old IPC⊕ rats. Immunostaining and Western blotting showed that Bax expression decreased in the 6-month-old IPC⊕ rats, whereas Bax and caspase-9 expression decreased in the 12-month-old IPC⊕ rats. [Conclusions] IPC⊕ suppresses apoptosis after the application of pneumatic tourniquet. However, apoptosis cannot be prevented when IPC⊕ is applied in elderly groups. Age-related changes in myocyte mitochondria may be involved in the apoptotic and necrotic process due to ischemia-reperfusion injury in elderly patients despite IPC.