Figure 1. Schematic diagram of acute hind limb ischemia reperfusion and treatment protocols.

C57BL6 male mice were subjected to 1.5 hours of unilateral hind limb tourniquet ischemia followed by 24 hours reperfusion. In the first set of experiments (diagram A), mice were subjected to IR and split into two groups. The first group received 50 μg of human recombinant DNase-I (Pulmozyme, n=8) intraperitoneally at 5 minutes before reperfusion and a second dose of 10μg intravenously at 10 minutes after reperfusion and a third dose of 50μg intraperitoneally at 12 hours after reperfusion. The second control group received similar volumes of carrier buffer alone at the same time points (n=8). In the second part of this study (diagram B), two separate groups of mice received daily injections of either rabbit anti-mouse neutrophil serum intraperitoneally for neutrophil depletion (n=8), or normal rabbit serum (n=7) for 48hrs two days prior to IR. Mice were then subjected to 1.5hrs unilateral ischemia IR injury and received additional rabbit anti-mouse serum or normal serum 1 minuute after the start of reperfusion. AT the end of 24 hours IR period, hind limb perfusion was documented with a laser Doppler imaging, then mice were euthanized and hind limb muscle tissues were harvested for analysis.