Analyses of H2O2 production in time during wound healing

Reactive Oxygen Species (ROS) have been traditionally con- sidered as toxic species due to their capacity to damage macromolecules. However, their deleterious effect is lim- ited to high concentrations. At lower levels, further studies have shown that at least hydrogen peroxide (H2O2) acts as a second messenger and results crucial for basic biological functions. Wound healing is one of those: in several animal models, this molecule is emitted in cellular wound margins, leading to neutrophil chemoattraction, while inducing epithe- lial cell migration during skin regeneration. Nevertheless, there are no references to this phenomenon in human ex- perimental set-ups. In this study, human keratinocyte cells (HaCaT), previously modified to express a H2O2-specific ra- tiometric fluorescent sensor (HyPer7) were monitored using live imaging after generating a mechanical-like injury. The time evolution ratio of the probe and the gap area were mea- sured for both cells near the margin and for topologically secondary cells, establishing a relationship between cytosolic H2O2 fluxes and the process of refilling a wound. In addition, comparison of movement rates between HyPer7-expressing and non-expressing HaCaT cells demonstrated a profound dependency of H2O2 availability for the process to occur.