Remote Ischemic Preconditioning Plasma Prevents Neutrophil Infiltration by Increased Anti-oxidative Capacity in the Zebrafish
Chung Ho Leung, University of Toronto; Stephen Lam, University of Toronto; Xiao-Yan Wen, University of Toronto; Christopher Caldarone, University of Toronto; Ori Rotstein, University of Toronto
Oxidative stress and neutrophil infiltration following hemorrhagic shock contribute to organ dysfunction in trauma patients. We have previously reported that transient ischemia/reperfusion in the limb known as remote ischemic preconditioning (RIPC) attenuates lung and liver injury in a murine model of hemorrhagic shock. Further, RIPC liberates humoral factors in the plasma that reduces neutrophil infiltration in a zebrafish model of tailfin injury. We hypothesized that plasma from RIPC animals might exert an anti-oxidant effect as a mechanism leading to reduced neutrophil migration in the zebrafish.Hypothesis:
RIPC plasma prevents neutrophil infiltration through up regulation of anti-oxidative capacity.Methods:
C57BL/6 mice were subjected to RIPC by occlusion of the left hindlimb with a tourniquet for four cycles of 5-min ischemia and 5-min reperfusion. Control or RIPC plasma were collected and microinjected into 3 days-post-fertilization Tg(mpx:EGFP) zebrafish. In these fish, neutrophils are labelled with GFP. After 18h, complete tailfin transection was performed. Neutrophil infiltration at the transected area was assessed at peak migration and reactive oxygen species (ROS) production at the site of injury was assessed over a time course using the CellROX DeepRed fluorescent dye. Fluorescence imaging was used to study neutrophil migration and oxidative stress. Data are shown as mean ± SEM of n determinations.Results:
Tailfin transection in zebrafish microinjected with control plasma resulted in a significant rise in ROS production at the site of injury which peaked at 55 to 65 min post-transection (1024711 ± 153172 MFI, n = 3) and neutrophil infiltration (12 ± 2, n = 6). By contrast, zebrafish microinjected with RIPC plasma had significantly lower ROS production compared to zebrafish microinjected with control plasma (714,777 ± 103254 MFI, n = 3, p < 0.05) and significantly lower neutrophil count (5 ± 1, n = 6, p < 0.01). In support of the conclusion that reduction of neutrophil migration was related to the anti-oxidant effect of RIPC plasma, administration of anti-oxidant N-acetylcysteine prior to tailfin transection prevented the ROS production and subsequent neutrophil infiltration.Conclusions:
ROS is a chemokine that mediates neutrophil migration in the zebrafish. RIPC liberates humoral factor(s) that may prevent neutrophil infiltration through attenuation of local ROS production. Induction of antioxidant capacity represents a novel mechanism underlying the anti-inflammatory effect of RIPC.