Effects of Variable Energy Delivery on Leukocyte Kinetics and Wound Healing

Wound related complications affect millions per year and cost the healthcare system billions. New therapeutics with better outcomes are needed. Photobiomodulation therapy, an energy-based modality, possesses a paradigm-shifting cost and safety advantage. Varied clinical responses to this therapeutic, however, underscore the need to identify standard energy parameters optimized for enhanced wound healing responses. One target for therapeutic intervention is the macrophage, a critical player in directing the fate of wound healing. The precise energy parameters required to optimize macrophage wound behavior remain ill-defined and present a major gap in the knowledge of the field. The zebrafish in vivo transgenic macrophage model enables tracking of fluorescently marked macrophages in responses to biochemical cues, such as, reactive oxygen species, however real time detection of macrophage behavior using commercially available software is limited. An image analysis software platform, termed Zirmi, was developed to provide real time quantitative measures of cellular kinetics and reactive oxygen species in defined multi-dimensional parameters of space and time. Zirmi, differs from current software solutions that may only provide qualitative, single image analysis, or cell tracking solutions. This approach demonstrated standardized space- and time-based quantitative measures of (1) a soluble oxidative stress fluorescent probe and (2) macrophage recruitment kinetic based changes after tissue injury. The implementation of Zirmi image analysis pipeline performed efficient automated image segmentation methods at execution speeds as high as 10-fold faster than manual image-based approaches. The effect of fluences delivered by a He:Ne laser on individual macrophage kinetics, tissue oxidative stress, and wound closure could then be quantitatively measured. Macrophage velocity and directionality was increased by lowest dose of applied energy and led to reduced levels of wound reactive oxygen species and accelerated wound closure. The software platform in concert with a modified zebrafish wound model provides a strong in vivo system for a pipeline of investigations examining each parameter of photobiomodulation on wound inflammatory cell behavior, wound reactive oxygen species production, and the ultimate outcome, degree and speed of wound closure. This reproducible test system can support necessary quantitative in vivo studies for the optimization of laser therapeutics.