Intravital Microscopic Evaluation of the Effects of a CXCR2 Antagonist in a Model of Liver Ischemia Reperfusion Injury in Mice

Abstract

BackgroundIschemia–reperfusion (IR) is a major contributor to graft rejection after liver transplantation. During IR injury, an intense inflammatory process occurs in the liver. Neutrophils are considered central players in the events that lead to liver injury. CXC chemokines mediate hepatic inflammation following reperfusion. However, few studies have demonstrated in real-time the behavior of recruited neutrophils. We used confocal intravital microscopy (IVM) to image neutrophil migration in the liver and to analyze in real-time parameters of neutrophil recruitment in the inflamed tissue in animals treated or not with reparixin, an allosteric antagonist of CXCR1/2 receptors.Materials and methodsWT and LysM-eGFP mice treated with reparixin or saline were subjected to 60 min of ischemia followed by different times of reperfusion. Mice received Sytox orange intravenously to show necrotic DNA in IVM. The effect of reparixin on parameters of local and systemic reperfusion-induced injury was also investigated.ResultsIR induced liver injury and inflammation, as evidenced by high levels of alanine aminotransferase and myeloperoxidase activity, chemokine and cytokine production, and histological outcome. Treatment with reparixin significantly decreased neutrophil influx. Moreover, reparixin effectively suppressed the increase in serum concentrations of TNF-α, IL-6, and CCL3, and the reperfusion-associated tissue damage. The number of neutrophils in the liver increased between 6 and 24 h of reperfusion, whereas the distance traveled, velocity, neutrophil size and shape, and cluster formation reached a maximum 6 h after reperfusion and then decreased gradually. In vivo imaging revealed that reparixin significantly decreased neutrophil infiltration and movement and displacement of recruited cells. Moreover, neutrophils had a smaller size and less elongated shape in treated mice.ConclusionImaging of the liver by confocal IVM was successfully implemented to describe neutrophil behavior in vivo during liver injury by IR. Treatment with reparixin decreased not only the recruitment of neutrophils in tissues but also their activation state and capacity to migrate within the liver. CXCR1/2 antagonists may be a promising therapy for patients undergoing liver transplantation.

Mannan-binding lectin deficiency exacerbates sterile liver injury in mice through enhancing hepatic neutrophil recruitment

“…CXCL2 is also known as an essential mediator in sterile liver injury which could alter hepatic function and hepatotoxicity by regulating hepatic neutrophil accumulation . A recent study showed that treatment with reparixin, an antagonist of the CXCL2 receptor, decreased not only the recruitment of neutrophils in tissues but also their activation state in the liver which was associated with better liver function and histologic outcome in sterile liver injury . Our result showed reduced hepatic neutrophil infiltration and attenuated liver damage in CCl 4 ‐injected mice that received anti‐CXCL2 neutralizing antibody, in line with the previous reports, indicating that CXCL2 is an important mediator of sterile liver inflammation.…”

“…However, the hepatic macrophages in CCl 4 ‐treated MBL −/− mice displayed a higher level of CXCL2 compared with WT counterparts. It is noteworthy that CXCL2 is a potent chemotactic and activation factor of neutrophils and plays a critical role in neutrophil recruitment during acute inflammation . CXCL2 is also known as an essential mediator in sterile liver injury which could alter hepatic function and hepatotoxicity by regulating hepatic neutrophil accumulation .…”

The role of neutrophil extracellular traps and TLR signaling in skeletal muscle ischemia reperfusion injury

“…1,[5][6][7] Neutrophils comprise a substantial portion of the amplifier cell population in the initial immune response, and their involvement in IR injury is hypothesized to be, in part, due to the formation of neutrophil extracellular traps (NETs) containing web-like extrusions of nuclear DNA, citrullinated histones, and antimicrobial peptides released into the tissue by activated neutrophils. [8][9][10] NETs have been implicated in a wide variety of diseases, notably autoimmune diseases such as lupus [11][12][13] and rheumatoid arthritis. 14,15 Additionally, NETs have also been shown to form following traumatic injury both systemically and at the site of injury, [16][17][18][19][20][21] including in models of ischemic injury in vital organs.…”

Effects of positive acceleration (+Gz stress) on liver enzymes, energy metabolism, and liver histology in rats

“…Hepatic energy metabolism and an optimal intracellular environment rely on an adequate blood supply. Hepatocytes are very sensitive to ischemia and/or hypoxia in liver tissue[18,19]. Therefore, factors related to ischemia and/or hypoxia will definitely influence their metabolism[20].…”