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 large 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 Nomega-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 myelo-peroxidase activity. At the end of the experiment, skeletal muscle necrosis was quantified using the nitroblue tetrazolium staining technique, and 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.