The Effect of Fluence on Neutrophil Kinetics in Zebrafish Wounds Using Real-time In Vivo Imaging

Abstract

Background: Neutrophils participate in a cooperative defense strategy with macrophages following tissue injury. Application of low dose electromagnetic radiation via 635 nm wavelength can enhance macrophage recruitment to the wound, decrease tissue levels of ROS and speed healing. The impact of this wavelength on neutrophil activity in vivo is not well-described. Given that wound healing can be enhanced if collateral damage by neutrophils within the wound is decreased, we investigated how neutrophil kinetics changed during these accelerated reparative responses. Methods: Zebrafish, transgenic for the fluorescent neutrophil marker, mpx-dendra2, underwent defined fin tissue injury, and were randomized to one of two laser treatment groups, 3 J/cm2 (n=57), or 18 J/cm2 (n=69) or to the control group, 0 J/cm2 (n=164). Electromagnetic wave exposures were administered by a 635-nm continuous 5mW He:Ne laser with recipients randomized by dose delivered. Fish were three-dimensionally time-lapse imaged 30-120 minutes post injury (mpi) and wound healing documented at 24 hours post-injury. Individual neutrophil movement was tracked according to distance from wound center. Results: The lower treatment fluence, 3 J/cm2, significantly decreased neutrophil migration speed into the wound, increased reverse migration, and promoted stasis outside the adjacent wound area when compared to control and higher energy doses. The 3 J/cm2 treatment groups also exhibited more rapid wound closure when compared to control or higher fluence. Conclusions: Unlike our previous work in macrophages in which low fluence treatment enhanced the speed of forward directed migration into the wound, the response of neutrophils was decreased speed into the wound, increased reverse migration, and stasis outside the areas of the wound edges. These findings advance the notion that low fluence treatments reduce neutrophil inflammatory responses within the wound by their reduced presence within the wound. Reduced neutrophil-mediated collateral damage may work in concert with enhanced macrophage wound activity to promote faster wound healing.