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

Abstract

Objective Ischemia-reperfusion (IR) injury is a significant problem in the management of patients with acute limb ischemia (ALI). Despite rapid restoration of blood flow following 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 is 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 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. Hematoxylin and eosin stained sections of hind limb skeletal muscle tissue were examined for fiber injury. For immunohistochemistry, mouse monoclonal anti-histone H2A/H2B/DNA complex antibody to detect NETs and rabbit polyclonal anti-myeloperoxidase (MPO) antibody were used to identify infiltrating cells containing MPO. Muscle ATP levels, nuclear NF-κB activity, IκBα, poly (ADP-ribose) polymerase (PARP) activity and iNOS expression were measured. Systemic levels of KC, MCP-1 and VEGF in the serum samples were also examined. Results IR injury in the hind limb of wild type mice demonstrated significant levels of muscle fiber injury, decreased energy substrates, increased NF-κB activation, decreased I-κBα levels, increased iNOS expression and increased PARP activity levels when compared to the TLR4 knockout mice samples. Additionally, there was marked decrease in the level of neutrophil and monocyte infiltration in the TLR4 mutant mice, which corresponded to similar levels of decreased NETs detection in the interstitial space and in microvascular thrombi. 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. 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.

Sensitization of Group III and IV Muscle Afferents in the Mouse After Ischemia and Reperfusion Injury

“…Both prolonged and transient ischemia induce some muscle tissue damage relative to these uninjured controls, but prolonged ischemia (BAO) appeared to affect the forepaw muscles to a slightly greater degree as evidenced by more extensive muscle fiber degeneration and muscle atrophy compared to the I/R condition. Although both ischemic injuries do not severely alter the muscles, our results are consistent with other reports of muscle tissue integrity during these types of artery occlusions 8,19,47,49 . To then confirm that our occlusion surgeries were not causing direct injury to the sensory neurons, we also analyzed ATF3 staining (a marker of neuronal injury) in the C8 DRGs in mice that underwent BAO or I/R.…”

Proteasome Inhibition Diminishes the Formation of Neutrophil Extracellular Traps and Prevents the Death of Cardiomyocytes in Coculture with Activated Neutrophils during Anoxia-Reoxygenation

“…Savchenko et al [28] demonstrated that extracellular chromatin released through NETosis exacerbates myocardial ischemic-reperfusion injury. Oklu et al [29] demonstrated that hind-limb ischemia-reperfusion injury is reduced in Toll-like receptor-4 mutant mice that are characterized by decreased NET formation.…”

Cell death, clearance and immunity in the skeletal muscle

“…Finally, the generation of neutrophil extracellular traps (NETs), an event strictly associated with the activation of autophagy pathway in inflammatory leukocytes 48 and modulated by the recognition of apoptotic cells, 49 has been described in the muscle upon ischemia-reperfusion injury. 50 Further studies will evaluate the relevance of NET contribution to muscle remodeling during autoimmune diseases and in homeostatic conditions.…”