Reduced murine hind limb ischemia-reperfusion injury in toll-like receptor mutant mice is associated with decreased nets

Purpose Mechanism of ischemia-reperfusion (IR) injury in peripheral vascular disease is unknown. The pattern recognition receptors, Toll-like receptor (TLR) family of proteins, are key players in IR associated with stroke and myocardial infarction. Our aim was to: 1) create a murine model of hind limb injury to evaluate the role of TLR4 in IR, and 2) determine whether inactive TLR4 led to a decrease in the detection of neutrophil extracellular traps (NETs), which is known to be highly thrombogenic and may mediate microvascular injury. Materials and Methods A calibrated tension tourniquet was applied to unilateral hind limb of wild type (WT) and TLR4 knockout mice (TLR4m) for 1.5 hours to induce ischemia and then immediately removed to initiate reperfusion. At the end of 48 hours of reperfusion, mice were sacrificed and hind limb tissue as well as serum specimens were collected for analysis. For immunohistochemistry, mouse monoclonal anti-histone H2A/H2B/DNA complex antibody to detect NETs, rabbit polyclonal anti-H2A and rabbit polyclonal anti-myeloperoxidase were used. Muscle ATP levels were determined using ATP-lite luminescence assay. Nuclear NF-κB activity was determined using a Trans-AM™ NF-κB p65 transcription factor assay. IκBα expression was quantitated using Western blot analysis. ELISA assay was performed to compare levels of KC, MCP-1 and VEGF in the serum samples isolated from both the WT and TLR4m groups. Results IR injury in the hind limb of WT demonstrated significant levels of muscle fiber injury, decreased energy substrates, increased NF-κB activation and decreased I-κBα levels when compared to the TLR4 knockout mice samples. There was marked decrease in the level of neutrophil and monocyte infiltration in the TLR4 mutant mice, marked decrease in the levels of NETs detection. In situ nuclease treatment of wild-type tissue sections significantly diminished the level of NETs immunostaining demonstrating the specificity of our antibody to detect NETs and suggesting a potential role for nuclease treatment in IR injury. Conclusion These results suggest a pivotal role for TLR4 in mediating hind limb IR injury and suggest that NETs may potentially exacerbate the extent of muscle fiber injury.