Reduced hind limb ischemia-reperfusion injury in Toll-like receptor-4 mutant mice is associated with decreased neutrophil extracellular traps.

Objective: Ischemia-reperfusion (IR) injury is a significant problem in the management of patients with acute limb ischemia. Despite rapid restoration of blood flow after technically successful open and endovascular revascularization, complications secondary to IR injury continue to occur and limit clinical success. Our aim was to create a murine model of hind limb IR injury to examine the role of Toll-like receptor-4 (TLR4) and to determine whether inactive TLR4 led to a decrease in the detection of neutrophil extracellular traps (NETs), which are known to be highly thrombogenic and may mediate microvascular injury.

Methods: A calibrated tension tourniquet was applied to unilateral hind limb of wild-type (WT) and TLR4 receptor mutant (TLR4m) mice for 1.5 hours to induce ischemia and then removed to initiate reperfusion. At the end of 48 hours of reperfusion, mice were euthanized and hind limb tissue and serum specimens were collected for analysis. Hematoxylin and eosin-stained sections of hind limb skeletal muscle tissue were examined for fiber injury. For immunohistochemistry, mouse monoclonal antihistone H2A/H2B/DNA complex antibody to detect NETs and rabbit polyclonal antimyeloperoxidase antibody were used to identify infiltrating cells containing myeloperoxidase. Muscle adenosine triphosphate levels, nuclear factor (NF)-κB activity, the α-subunit of inhibitor of NF-κB light polypeptide gene enhancer, poly (adenosine diphosphate-ribose) polymerase activity, and inducible nitric oxide synthase expression were measured. Systemic levels of keratinocyte-derived chemokine, monocyte chemotactic protein-1, and vascular endothelial growth factor in the serum samples were also examined.

Results: IR injury in the hind limb of WT mice demonstrated significant levels of muscle fiber injury, decreased energy substrates, increased NF-κB activation, decreased levels of α-subunit of inhibitor of NF-κB light polypeptide gene enhancer, increased inducible nitric oxide synthase expression, and increased poly (adenosine diphosphate-ribose) polymerase activity levels compared with the TLR4m samples. Additionally, there was marked decrease in the level of neutrophil and monocyte infiltration in the TLR4m mice, which corresponded to similar levels of decreased NET detection in the interstitial space and in microvascular thrombi. In situ nuclease treatment of WT tissue sections significantly diminished the level of NET immunostaining, demonstrating the specificity of the antibody to detect NETs and suggesting a potential role for nuclease treatment in IR injury.

Conclusions: These results suggest a pivotal role for TLR4 in mediating hind limb IR injury and suggest that NETs may contribute to muscle fiber injury.