Quantification of Methylene Blue Exclusion for Tracking of Regenerative Re-Epithelialization

A major difference between the non-regenerative (scar-forming) wound closure found in mammals and the regenerative wound closure (scarless healing) found in many fish and salamanders is that reepithelialization of the injury is much faster in animals that can regenerate appendages [1]. This reepithelialization, in turn, is essential for appendage regeneration. Methylene blue dye has long been used to guide surgical debridement of burn injuries [2] because it is absorbed by damaged tissue but not undamaged or regenerated epithelium. Here, we use microscopy and image analysis to develop a quantitative methylene blue exclusion assay to assess the time course for tr-epithelialization in the regenerating zebrafish fin and axolotl tail. We expand and adapt methods used for the assessment of reepithelialization associated with wound healing in zebrafish [3] to the epithelialization associated with tail regeneration both in zebrafish and axolotls, classic model systems for appendage regeneration.