Protective effect of ischaemic preconditioning against ischaemia-induced reperfusion injury of skeletal muscle: how many preconditioning cycles are appropriate?

We investigated the efficacy of ischaemic preconditioning (IPC), consiting of repeated brief episodes of vascular occlusion followed by reperfusion, as protection against ischaemia-reperfusion injury of skeletal muscle, using a rat amputation-like model. Wistar rats underwent temporary amputation at the level of the femur, excluding the femoral vessels. The femoral artery and wein were clamped for 4h, using a micro-clamp, in the groups exposed to ischaemia. The rats were randomly divided into eight groups: a control (C) group (n=7) with non-amputated and non-ischaemic hind limbs; a sham control (SC) group (n=7) with amputated but non-ischaemic hind limbs; an ischaemia-reperfusion (IR) group (n=7) with amputated and ischaemic hind limbs; and five IPC groups (n=7 in each) with hind limbs that were subjected to 4h of ischaemia after one to five cycles of brief ischaemia and reperfusion for 10 min each, respectively. All rats were sacrificed 24 h after reperfusion. The, viability of the anterior tibial muscles was evaluated using nitroblue tetrazolium staining. The total viable area ratio (T-VAR) of the muscle tissue was calculated in each animal as follows: T-VAR=total viable area/total slice area × 100%. The T-VAR values of the eight groups were as follows: C group, 100%±0%; SC group, 100%±0%; IR group, 73.5%±1.7%; IPC1 group, 79.4%±6.5%; IPC2 group, 70.5%±6.2%; IPC3 group, 90.6%±2.8%; IPC4 group, 90.0%±1.6%; and IPC5 group, 87.8%±1.8%. The T-VARs in the IPC3, IPC4 and IPC5 groups were significantly higher (α<0.01) than those in the IR group. In contrast, there were no significant differences between the T-VARs of the IPC1 and IPC2 groups and those of the IR group. In conclusion, three to five cycles of IPC could protect skeletal muscle against ischaemia.