Time course of post-ischemic superoxide generation in venous effluent from reperfused rabbit hindlimbs

In order to clarify the early time course of the generation of O2- produced from reperfused extremities after ischemia, the authors directly quantified the O2- of venous effluent from reperfused hindlimbs following various time periods of warm ischemia, using a rabbit reperfusion model. Using a total of 30 rabbit hindlimbs, the hindlimb-reperfusion model was created by temporarily cutting both soft-tissue and bony structures, exclusive of femoral vessels, around the thighs, and clamping the vessels with a microvascular clip. The various vascular pedicle clamping times at normothermia (25 degrees C) were used for three animal groups: 2-hr (n = 10), 4-hr (n = 10), and 6-hr (n = 10). For sham-operated controls (n = 7), the same surgical procedures with no clamping were performed. Venous effluent blood samplings from the femoral vein at the proximal side of the clamping point were collected before reperfusion, and from 5 min to 60 min after reperfusion at 5-min intervals in each animal, and peripheral blood was sampled to quantify O2- by a chemiluminescence (CLN) method using a derivative of luciferin (CLA). The time course of the production of O2- during reperfusion of all three experimental groups and controls showed polyphasic patterns with two or three peaks, but no significant changes throughout reperfusion. The time course of superoxide production after reperfusion in the 2-hr, 4-hr, and 6-hr groups showed significant differences (p < 0.05) in repeated measures by ANOVA, compared with controls. Superoxide production in the 6-hr group was greater than controls at relatively late times of reperfusion (20 min, 30 min, and 40 to 55 min after reperfusion). In the present study, it was found that the time course of superoxide production in ischemia-induced reperfused limbs showed a late polyphasic pattern without a significant initial peak, especially in the 6-hr ischemia group. In addition, the amount of superoxide production correlated with ischemic time period to some extent.