Effect of ischaemic exercise training of a normal limb on angiogenesis of a pathological ischaemic limb in rabbits

The present study was designed to test the hypothesis that local exercise of a normal limb can promote angiogenesis in a pathological ischaemic limb. New Zealand White rabbits underwent left femoral artery ligation (Lig) and electrode implantation on the right sciatic nerve. The rabbits were randomly assigned to four groups: (i) Lig-N group, which did not receive ES (electrical stimulation); (ii) Lig-High group, which received high-intensity ES (2.5 mA, 40Hz for 1 ms) on the right hindlimb; (iii) Lig-Low group, which received low-intensity ES (0.3 mA, 40Hz for 1 ms) on the right hindlimb; (iv) Double-Lig-High group, which underwent femoral artery ligation on both hindlimbs and received high-intensity ES (2.5 mA, 40Hz for 1 ms) on the right hindlimb. The ES procedure included 5 min of stimulation, followed by 5 min of rest, and was repeated eight times a day for 4 weeks. Collateral circulation was examined grossly by angiography, resting blood flow was measured using the microspheres technique, and capillary supply was evaluated by immunohistochemistry. VEGF (vascular endothelial growth factor) mRNA and protein were analysed by real-time RT (reverse transcription)–PCR and Western blotting respectively. Collateral blood flow in all of the major muscles of the left hindlimb in the Lig-High group was highest among the four groups (P<0.01). Capillary supply (P<0.001), VEGF mRNA (P<0.01) and VEGF protein (P<0.01) in the gastrocnemius muscle increased remarkably in the Lig-High group; no statistically significant difference was observed among the other three groups. In conclusion, angiogenesis associated with an up-regulation of VEGF expression in pathological ischaemic limb may be facilitated by 4 weeks of physiological ischaemic exercise training in a normal limb.