Superoxide anion release (O₂^-) after ischemia and reperfusion

Neutrophils have been implicated as mediators of the reperfusion injury following ischemia. In order to measure neutrophil activation, O₂^- was determined after 2 hr of ischemia followed by 1 hr of reperfusion (no clinical reperfusion injury) and 3 hr of ischemia followed by 1 hr of reperfusion (significant clinical reperfusion injury). Using New Zealand white rabbits, baseline blood samples were drawn from an ear artery. The right iliac and femoral arteries were exposed and clamped. Just prior to clamp release, blood was obtained from the right iliac vein (ischemia). After 1 hr of reperfusion, blood was again taken from the right iliac vein (reperfusion). Neutrophils were isolated from the blood samples. O₂^- was determined by the reduction of cytochrome e using a spectrophotometer. In the 2-hr group, results (expressed as μmole O₂^-/min/2 × 10⁶ cells) were: baseline, 0.337 ± 0.025; ischemia, 0.512 ± 0.039;* and reperfusion, 0.634 ± 0.064*. (*P < .05 as compared to baseline). In the 3-hr group, results were: baseline, 0.391 ± 0.038; ischemia, 0.413 ± 0.051; and reperfusion, 0.258 ± 0.043** (**P < 0.05 as compared to 2 hr reperfusion). A significant increase in O₂^- was seen after 2 hr of ischemia followed by 1 hr of reperfusion. However, little O₂^- increase was seen after 3 hr of ischemia and a significant O₂^- decrease was seen after 1 hr of reperfusion. We conclude: (1) Neutrophil O₂ is stimulated early in ischemia followed by reperfusion; (2) after reperfusion injury occurs (3 hr), neutrophils have been activated and O₂^- can no longer be stimulated; and (3) O₂^- in this model may be involved in the clinical reperfusion injury seen.