Autofluorescence in BrdU-positive cells and augmentation of regeneration kinetics by riboflavin

The earthworm, Eudrilus eugeniae, has a prodigious ability to regenerate lost segments. The skin of the worm has an outermost epidermal layer followed by a thick circular muscle layer and an innermost thin longitudinal cell layer. During the process of regeneration, the circular muscle layer decreased in thickness, and longitudinal cell layer increased. The histological analysis of the regenerated worm shows that the longitudinal cell layer forms the regeneration blastema. BrdU-labeling retention assay confirmed that the circular muscle and longitudinal cell layers have BrdU-positive cells, which migrate from the adjacent segments to the regeneration blastema. In addition, it was noted that the cells of the earthworm, E. eugeniae, have the property of autofluorescence. Autofluorescence was found in the cytoplasm, but not in the nucleus. It has been also found that the major source for autofluorescence is riboflavin. Further, it was also demonstrated that supplementation with riboflavin increases the rate of regeneration, while regeneration was hampered by reduced levels of riboflavin. The importance of riboflavin in regeneration was also confirmed by rescue assay. In addition, it was also identified that BrdU-positive cells are highly fluorescent compared to the surrounding cells.