The Effect of Fluence on Macrophage Kinetics, Oxidative Stress, and Wound Closure Using Real-Time In Vivo Imaging.

Objective: The aim of our study was to quantify the effect of doses delivered by a He:Ne laser on individual macrophage kinetics, tissue oxidative stress, and wound closure using real-time in vivo imaging. Background: Photobiomodulation has been reported to reduce tissue inflammation and accelerate wound closure; however, precise parameters of laser settings to optimize macrophage behavior have not been established. We hypothesized that quantitative and real-time in vivo imaging could identify optimal fluence for macrophage migration, reduction of reactive oxygen species, and acceleration of wound closure. Methods: Larval zebrafish Tg(mpeg-dendra2) were loaded with dihydroethidium for oxidative stress detection. Fish were caudal fin injured, treated with 635 nm continuous 5 mW He:Ne laser irradiation at 3, 9, or 18 J/cm(2) and time-lapsed imaged within the first 120 min postinjury. Images taken 1 and 24-h postinjury were compared for percentage wound closure. Results: A fluence of 3 J/cm(2) demonstrated significant increases in macrophage migration speed, fewer stops along the way, and greatest directed migration toward the wound. These findings were associated with a significant reduction in wound content reactive oxygen species when compared with control wounded fins. Both 3 and 9 J/cm(2) significantly accelerated wound closure when compared with nonirradiated control fish. Conclusions: Wound macrophage activity could be manipulated by applied fluence, leading to reduced levels of wound reactive oxygen species and accelerated wound closure. The zebrafish model provides a means to quantitatively compare wound macrophage behavior in response to a variety of laser treatment parameters in real time.