Time course of superoxide generation in reperfusion after arterial ischaemia and venous congestion

In order to identify differences in superoxide (O2−) production after reperfusion following arterial ischaemia and venous congestion, we directly quantified O2− in the venous effluent from reperfused hindlimbs in the rabbit. Using a total of 20 rabbit hindlimbs, we made hindlimb-reperfusion models by section of both soft tissue and bony structures exclusive of femoral vessels around the thighs and clamping of the artery alone (group A: n=10) or vein alone (group V: n=10) by microvascular clamp. A pilot study revealed that the critical time for venous congestion was 2 h. Accordingly, clamping was performed at 25°C (normothermia) for 2 h. Sham operations were performed in the control (n=7). Venous effluent blood samples from the femoral vein proximal to the clamping point were collected before reperfusion, and from 5 to 60 min after reperfusion at 5 min intervals. Effluent blood samples were used to quantify O2− using a chemiluminescence method using a derivative of luciferin. The time course of O2− production after reperfusion in group A and group V showed significant differences (P<0.05), compared with the controls. There was no significant difference in the time course of O2− production in group A and that in group V. In the early reperfusion phase, no difference in O2− production was observed after arterial ischaemia or venous congestion. In future studies, we will test the contributions of free radicals by setting longer ischaemic times, longer reperfusion times, and studying secondary ischaemia.