Use of a nitric oxide precursor to protect pig myocutaneous flaps from ischemia-reperfusion injury

Nitric oxide is a radical with vasodilating properties that protects tissues from neutrophil-mediated ischemia-reperfusion injury in the heart and intestine. Previous studies in our laboratory suggested that L-arginine, a nitric oxide precursor, can protect skin flaps from ischemia-reperfusion injury. In this study, we examined the effects of L-arginine on the survival of myocutaneous flaps in a lar-ge animal model and established whether this effect was mediated by nitric oxide and neutrophils. Two superiorly based 15 x 7.5 cm epigastric myocutaneous island flaps were dissected in 15 Yorkshire pigs weighing 45 to 50 kg. One of the flaps was subjected to 6 hours of arterial ischemia and then reperfused for 4 hours (ischemia-reperfusion flaps), whereas the other flap was used as a non-ischemic control (non-ischemia-reperfusion flaps). The flaps were divided into four groups: control non-ischemia-reperfusion flaps that received only saline (group I); ischemia-reperfusion flaps that were treated with saline (group II); and flaps treated with either L-arginine (group III) or N<(omega)over bar>-nitro-L-arginine methylester (L-NAME), a nitric oxide synthase competitive inhibitor, plus L-arginine in equimolar amounts (group IV). These drugs were administered as an intravenous bolus 10 minutes before the onset of reperfusion, followed by a 1-hour continuous intravenous infusion. Full-thickness muscle biopsies were taken at baseline, 3 and 6 hours of ischemia, and 1 and 4 hours of reperfusion. The biopsies were evaluated by counting neutrophils and measuring myeloperoxidase activity. At the end of the experiment, skeletal muscle necrosis was quantified using the nitroblue tetrazolium staining technique, anti a full-thickness biopsy of each flap was used for determination of water content. Statistical analysis was performed using analysis of variance and the Newman-Keuls test. Non-ischemia-reperfusion flaps showed no muscle necrosis. Ischemia-reperfusion flaps treated with saline had 68.7 +/- 9.1 percent necrosis, which was reduced to 21.9 +/- 13.6 percent with L-arginine (p < 0.05). L-NAME administered concomitantly with L-arginine demonstrated a necrosis rate similar to that of saline-treated ischemia-reperfusion flaps (61.0 +/- 17.6 percent). Neutrophil counts and myeloperoxidase activity after 4 hours of reperfusion were significantly higher in ischemia-reperfusion flaps treated with L-NAME and L-arginine as compared with the other three groups (p < 0.05). Flap water content increased significantly in ischemia-reperfusion flaps treated with saline and L-NAME plus L-arginine versus non-ischemia-reperfusion flaps (p < 0.02) and L-arginine-treated ischemia-reperfusion flaps (p < 0.05), There was no difference in flap water content between ischemia-reperfusion flaps treated with L-arginine and non-ischemia-reperfusion flaps. Administration of L-arginine before and during the initial hour of reperfusion significantly reduced the extent of flap necrosis, neutrophil accumulation, and edema due to ischemia-reperfusion injury in a large animal model. This protective effect is completely negated by the use of the nitric oxide synthase blocker L-NAME. The mechanism of action seems to be related to nitric oxide-mediated suppression of ischemia-reperfusion injury through neutrophil activity inhibition.