Image-Based Measurement of H2O2 Reaction-Diffusion in Wounded Zebrafish Larvae

Abstract

Epithelial injury induces rapid recruitment of antimicrobial leukocytes to the wound site. In zebrafish larvae, activation of the epithelial NADPH oxidase Duox at the wound margin is required early during this response. Before injury, leukocytes are near the vascular region, that is, ∼100-300 μm away from the injury site. How Duox establishes long-range signaling to leukocytes is unclear. We conceived that extracellular hydrogen peroxide (H₂O₂) generated by Duox diffuses through the tissue to directly regulate chemotactic signaling in these cells. But before it can oxidize cellular proteins, H₂O₂ must get past the antioxidant barriers that protect the cellular proteome. To test whether, or on which length scales this occurs during physiological wound signaling, we developed a computational method based on reaction-diffusion principles that infers H₂O₂ degradation rates from intravital H₂O₂-biosensor imaging data. Our results indicate that at high tissue H₂O₂ levels the peroxiredoxin-thioredoxin antioxidant chain becomes overwhelmed, and H₂O₂ degradation stalls or ceases. Although the wound H₂O₂ gradient reaches deep into the tissue, it likely overcomes antioxidant barriers only within ∼30 μm of the wound margin. Thus, Duox-mediated long-range signaling may require other spatial relay mechanisms besides extracellular H₂O₂ diffusion.