Optimization of Laser-Based Method to Conduct Skin Ablation in Zebrafish and Development of Deep Learning-Based Method for Skin Wound-Size Measurement
Authors: Siregar Petrus , Liu Yi-Shan , Casuga Franelyne P. , Huang Ching-Yu , Chen Kelvin H.-C. , Huang Jong-Chin , Hung Chih-Hsin , Lin Yih-Kai , Hsiao Chung-Der , Lin Hung-Yu 
Published: February 2024

Skin plays an important role as a defense mechanism against environmental pathogens in organisms such as humans or animals. Once the skin integrity is disturbed by a wound, pathogens can penetrate easily into a deeper part of the body to induce disease. By this means, it is important for the skin to regenerate quickly upon injury to regain its protective barrier function. Traditionally, scientists use rodents or mammals as experimental animals to study skin wound healing. However, due to concerns about animal welfare and increasing costs of laboratory animals, such as rodents, scientists have considered alternative methods of implementing replace, reduce, and refine (3Rs) in experimentation. Moreover, several previous studies on skin wound healing in fish used relatively expensive medical-grade lasers with a low calculation efficiency of the wound area, which led to human judgment errors. Thus, this study aimed to develop a new alternative model for skin wound healing by utilizing zebrafish together with a new rapid and efficient method as an alternative in investigating skin wound healing. First, in order to fulfill the 3Rs concept, the pain in the tested zebrafish was evaluated by using a 3D locomotion assay. Afterward, the obtained behavior data were analyzed using the Kruskal–Wallis test, followed by Dunn’s multiple comparisons tests; later, 3 watts was chosen as the power for the laser, since the wound caused by the laser at this power did not significantly alter zebrafish swimming behaviors. Furthermore, we also optimized the experimental conditions of zebrafish skin wound healing using a laser engraving machine, which can create skin wounds with a high reproducibility in size and depth. The wound closure of the tested zebrafish was then analyzed by using a two-way ANOVA, and presented in 25%, 50%, and 75% of wound-closure percentages. After imparting wounds to the skin of the zebrafish, wound images were collected and used for deep-learning training by convolutional neural networks (CNNs), either the Mask-RCNN or U-Net, so that the computer could calculate the area of the skin wounds in an automatic manner. Using ImageJ manual counting as a gold standard, we found that the U-Net performance was better than the Mask RCNN for zebrafish skin wound judgment. For proof-of-concept validation, a U-Net trained model was applied to study and determine the effect of different temperatures and the administration of antioxidants on the skin wound-healing kinetics. Results showed a significant positive correlation between the speed of wound closure and the exposure to different temperatures and administration of antioxidants. Taken together, the laser-based skin ablation and deep learning-based wound-size measurement methods reported in this study provide a faster, reliable, and reduced suffering protocol to conduct skin wound healing in zebrafish for the first time.