Effect of DNase I treatment and neutrophil depletion on acute limb ischemia-reperfusion injury in mice

Abstract

Objective Extracellular traps-(ETs) consist of DNA-protein-complexes formed following tissue injury contributes to the inflammatory and thrombosis cascades thereby exacerbating injury. Exogenous DNase-1 has been suggested as a therapeutic strategy to limit injury in the brain and myocardium.. These studies were designed to evaluate the effects of exogenous DNase-I treatment on skeletal muscle injury following acute hind limb ischemia-reperfusion (IR) injury in mice, and to determine whether neutrophils were a major source of ETs in postischemic muscle tissue. Methods C57BL6 mice were subjected to 1.5 hrs tourniquet ischemia and 24 hrs reperfusion with and without human recombinant DNase-I treatment. A separate set of mice was subjected to neutrophil depletion, followed by the same intervals of IR. Laser Doppler imaging and tissue harvesting was done at 24 hrs for assessment of limb perfusion, muscle fiber injury, ATP, markers of inflammation, thrombosis, and ETs formation. Results DNase-I treatment significantly reduced ETs detection in post-ischemic muscle but did not alter skeletal muscle fiber injury, levels of pro-inflammatory molecules or ATP. DNase-I treatment did enhance postischemic hind limb perfusion, decreased infiltrating inflammatory cells and reduced the expression of Thrombin-Anti-Thrombin-III. Neutrophil depletion resulted in a significant yet small reduction in ETs in the post ischemic muscle. Neutrophil depletion did not alter skeletal muscle fiber injury, hind limb perfusion, or ATP levels. Conclusions These data suggest that neither DNase-I treatment nor Neutrophil depletion were protective against IR injury, even though both decreased ETs detection in skeletal muscle following IR. Neutrophils are not the only source of ETs following IR.

Neutrophil elastase plays a non‐redundant role in remodeling the venular basement membrane and neutrophil diapedesis post‐ischemia/reperfusion injury

“…The migration of neutrophils from the blood stream into inflamed tissues is a tightly regulated process essential for the rapid development of an effective immune response against injury and invading pathogens. This phenomenon, extensively studied during physiological responses in healthy tissues , is also a key element in the development of many acute and life‐threatening inflammatory pathologies , such as I/R injury . Several therapeutic strategies have been tested both in preclinical and clinical studies of inflammatory conditions to inhibit neutrophil recruitment and/or functions through the use of natural or synthetic (e.g.…”

“…These innate immune cells, whilst vital for host defense against pathogens, are implicated in the pathogenesis of many disorders due to their capacity to release a wide range of proinflammatory mediators , reactive oxygen species (ROS) and destructive proteolytic enzymes. Clear evidence for the involvement of neutrophils in I/R injury has been provided by both preclinical and clinical studies in which neutrophil depletion was protective against further tissue damage . Similarly, blocking neutrophil interaction with blood vessel walls within reperfused areas of ischemic tissues improves disease outcome in patients .…”

Therapeutic targeting of neutrophil exocytosis

“…16,91,[146][147][148][149][150] Inhibition of neutrophil recruitment reduces organ injury in many animal models. [151][152][153][154][155][156][157][158] Data suggest that release of granule cargo and ROS generation mediate neutrophil-dependent organ damage. 154,159,160 A role for ROS is supported by finding oxidant-modified proteins in organs damaged by ischemia-reperfusion injury.…”

Mouse neutrophils release extracellular traps in response to Naegleria fowleri

“…We suggest the future study should use immunized mice with total N fowleri extract plus cholera toxin and challenge it with a lethal dose of the amoeba, to obtain evidence whether the protection is a result of the interference by NETs entrapment of trophozoites or otherwise. We could either consider the proposal of Albadawi et al of using DNase‐I treatment through nasal cavity or considerate neutrophil depletion.…”