To investigate the effect of different ischemic preconditioning (IPC) modes on ischemia-reperfusion (IR) injury of skeletal muscle in rats.

Forty male SD rats were selected to construct the model of IR injury of skeletal muscle by clamping one side of the femoral artery. In the IPC mode, the right femoral artery was clipped for 10 minutes, and reperfusion ensued for 10 minutes after the release of artery clamp. Such preconditioning procedure was repeated 3 times. The rats were divided into conventional IR group, IPC immediate group, IPC24-hour group, IPC48-hour group and sham operation group according to the random number table method, with 8 rats in each group.In conventional IR group, the right femoral artery was clipped for 3 hours and then the artery clamp was released to allow reperfusion for 3 hours. In IPC immediate group, the same treatment as conventional IR group was performed immediately after preconditioning. In IPC24-hour group, the rats received skin suturing after preconditioning and then fed in a cage for 24 hours before the same treatment as conventional IR group. In IPC48-hour group, the rats received skin suturing after preconditioning and then fed in a cage for 48 hours before the same treatment as conventional IR group. In sham operation group, the right femoral artery was bluntly separated but not clipped. At the end of reperfusion, the tibialis anterior muscle tissue, gastrocnemius muscle tissue and serum were collected. The ratio of wet weight to dry weight (W/D) of the tibialis anterior muscle tissue was calculated to evaluate tissue edema. After HE staining, histopathological changes of gastrocnemius muscle were observed and the injury severity of gastrocnemius tissue was scored. Serum levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and malondialdehyde (MDA) were determined by ELISA. Expressions of hypoxia receptor (EGLN1) and hypoxia-inducible factor-1α (HIF-1α) in the gastrocnemius tissue were detected by real-time fluorescence quantitative PCR (qRT-PCR).

The degree of tissue edema was reduced under different IPC modes when compared with conventional IR group (all P<0.01). The W/D in conventional IR group, IPC immediate group, IPC24-hour group and IPC48-hour group were 6.05±0.19, 5.70±0.12, 5.25±0.13 and 5.50±0.08, higher than 3.80±0.08 in sham operation group (all P<0.01). IPC24-hour group had the least degree of edema when compared with IPC immediate group and IPC48-hour group (P<0.05 or 0.01). The muscle fibers in sham operation group were neatly arranged with clear structure, and the muscle fibers in other groups showed different degrees of injury and inflammatory infiltration. The total scores for tissue injury severity in IPC immediate group, IPC24-hour group and IPC48-hour group were (8.15±0.15)points, (6.15±0.38)points and (6.90±0.19)points, lower than (9.60±0.50)points in conventional IR group and higher than (0.16±0.16)points in sham operation group (all P<0.01). The total scores for tissue injury severity in IPC24-hour group and IPC48-hour group were not significantly different (P>0.05), but both decreased when compared with IPC immediate group (P<0.05 or 0.01). ELISA showed that the levels of TNF-α, IL-1β and MDA under different IPC modes were higher than those in sham operation group (all P<0.01), and the order from high to low was conventional IR group, IPC immediate group, IPC48-hour group, IPC24-hour group and sham operation group. Levels of IL-1β, TNF-α and MDA in IPC24-hour group were decreased when compared with IPC immediate group and IPC48-hour group (all P<0.05). qRT-PCR showed that the expression of EGLN1 mRNA from high to low was conventional IR group, IPC48-hour group, sham operation group, IPC immediate group and IPC24-hour group and that the expression of EGLN1 mRNA in IPC24-hour group decreased when compared with conventional IR group, IPC immediate group and IPC48-hour group (all P<0.01). The expression of HIF-1α mRNA increased under different IPC modes when compared with sham operation group (all P<0.01), and the order from high to low was IPC24-hour group, IPC immediate group, IPC48-hour group, conventional IR group and sham operation group. The expression of HIF-1α mRNA in IPC24-hour group increased when compared with conventional IR group, IPC immediate group and IPC48-hour group (P<0.05 or 0.01), but there was no significant difference between IPC immediate group and IPC48-hour group (P>0.05).

Different ischemic preconditioning modes can reduce IR injury of skeletal muscle in rats by reducing tissue edema, inflammatory symptoms and oxidative stress response, among which reperfusion 24 hours after IPC has the best effect on IR injury. EGLN1 and HIF-1α may be involved in IPC to alleviate IR injury of skeletal muscle in rats.

Ischemic preconditioning; Reperfusion injury; Muscle, skeletal; Inflammation