Exercise-induced hyperemia unmasks regional blood flow deficit in experimental hindlimb ischemia

Many experimental models of hindlimb ischemia are characterized by spontaneous and rapid normalization of resting muscle blood flow (BF) rates which complicates the long-term evaluation of angiogenic therapies to reverse limb ischemia. We tested the hypothesis that peroneal nerve stimulation in an ischemic hindlimb would increase the oxygen (O(2)) demand and BF rate, thereby unmasking a severe blood flow deficit that is not apparent at rest.

Ischemia was induced in adult rats by ligation of the left common iliac, femoral arteries, and their branches. Peroneal nerves were stimulated to allow measurement of exercise-induced regional BF rates with fluorescent microspheres. Hemodynamics were monitored. Fluorescent microspheres were injected before and after 5 min of nerve stimulation 3, 10, and 24 days postischemia. The tibialis anterior (TA) and gastrocnemius (GC) muscles and skin were harvested and weighed, and fluorescence was measured. BF rate was calculated as milliters per minute per gram of tissue and compared to normal muscle and skin of unoperated control rats. In order to determine the accuracy of BF rate measurements in ischemic muscle when <400 microspheres was delivered per specimen, 3 rats were studied by simultaneous injection of 4 x 10(5) blue and 1 x 10(5) yellow-green fluorescent microspheres. The correlation coefficient between the number of different colored microspheres delivered was measured.

The ischemia caused atrophy of the TA and GC muscles. The mean muscle mass of the ischemic TA and GC as a percentage of total body weight decreased over time vs control [TA 0.13 +/- 0.05% vs 0.25 +/- 0.03%, P < 0.05; GC 0.51 +/- 0.27% vs 0.70 +/- 0.07%, P = 0.07 at 24 days (24D)]. Despite clinical evidence of severe hindlimb ischemia in experimental groups, i.e., pressure sores, muscle atrophy, and weakness, resting BF rates were not significantly different from those of control. The BF rate of the TA was of 0.11 ml/min/g after 3D of ischemia, 0.14 ml/min/g after 10D, and 0.13 ml/min/g after 24D. The mean BF rate in normal muscle of unoperated controls was 0.16 ml/min/g (P > 0.05). However, the exercise-induced hyperemia in the skeletal muscle was significantly blunted in all of the ischemic groups. The unoperated control TA had a greater than 10-fold increase in BF to 1.95 ml/min/g in response to exercise while the ischemic TA had no increase in BF at 3D, 2-fold increase at 10D, and a 5-fold increase at 24D. Parallel findings were noted in the GC muscles. There was no significant difference in the BF rate in the skin. The accuracy of this microsphere technique in measuring very low BF rates found in ischemic muscle was supported by the significant correlation coefficient (r = 0.99) comparing two quantities of microspheres injected simultaneously.

Despite clinical signs of severe hindlimb ischemia, resting BF rates in the ischemic groups were not significantly decreased. Peroneal nerve stimulation resulted in up to 10-fold increase in BF rate and unmasked a severe deficit in vascular reserve in the ischemic groups. Resting BF rate is not always an accurate reflection of the flow deficit in models of critical limb ischemia, and this model of exercise-induced hindlimb hyperemia may allow better long-term evaluation of angiogenic therapies designed to reverse critical limb ischemia.