Amphibians such as frogs or salamanders have a remarkable ability to regenerate amputated limbs and tails. The regenerative process involves the formation of endogenous pluripotent stem cells, which then expand and differentiate into the tissue types that give rise to the regenerated body part. The complex interplay of the cell types and signals involved in this regenerative response to the injury are not fully known and there is considerable interest in identifying all the necessary steps. The ultimate hope is that by identifying the specific mechanisms of injury response and regeneration, one might be able to activate similar repair processes in humans, who lack the extraordinary regenerative capacity of amphibians.

The recent paper “Amputation-induced reactive oxygen species are required for successful Xenopus tadpole tail regeneration” by Nick Love and colleagues published online in the journal Nature Cell Biology on January 13, 2013 elegantly demonstrates that reactive oxygen species (ROS), also known as oxygen radicals or oxidants, play a critical role in the regeneration of amphibian tails. Using a rather elegant approach, the researchers generated Xenopus tadpoles with a genetically integrated sensor of the oxidant-sensitive protein HyPerYFP that emits fluorescence upon contact with ROS, and is thought to be rather specific for the oxidant H2O2, more commonly known as hydrogen peroxide. This allowed them to study the hydrogen peroxide levels in all cells of the live tadpole, while it was responding to an injury. They found that within 6 hours after the tail amputation, the residual tail tissue was flooded with high levels of the hydrogen peroxide and that as the tail started growing back, the regenerative edge of the growing tail continued to show high levels of this oxidant.

After excluding the possible confounding phenomenon that the increase in ROS was merely a bystander effect of increases in inflammatory cells, the researchers then performed a pivotal set of experiments in which they used anti-oxidants to see if these would impact the tail regeneration. The researchers first utilized pharmacological inhibitors that reduce the production of oxidants as well as the therapeutic antioxidant MCI-186 (its trade-name is Edaravone and is marketed for use in patients in Japan). These pharmacological agents were all very effective in terms of lowering the hydrogen peroxide levels in the regenerating tail, but they also significantly impaired the regeneration itself. In another intriguing set of experiments, the researchers treated the tadpoles with these agents immediately after the injury and then withdrew them after three days, to see if the regeneration would set in after their removal. Interestingly, when the tails were exposed to agents that prevented the generation of the oxidants, the regenerative program remained blocked even when they were removed. On the other hand, the antioxidant scavenger that soaks up oxidants being produced did not permit regeneration while it was present, but regeneration resumed after the antioxidant was removed.

The researchers also performed complementary genetic experiments in which they reduced oxidant revels by suppressing the enzymes that produce oxidants. The results all point to an important conclusion: There is a burst of oxidants that are released after injury and that are necessary to initiate the regenerative program. The exact molecular targets of the oxidant hydrogen peroxide that enable regeneration remain unknown, but some of the data in the paper points to the Wnt protein pathway as a potential oxidant-sensitive regenerative signal in the tadpole tail.

One has to bear in mind that this work was performed in tadpoles and may not be necessarily fully applicable to the human setting, but Wnt is a key regulator of stem cell renewal, differentiation and regeneration in human tissues. This does suggest that there may be some key similarities between the tadpole regeneration pathways and those found in humans. Despite the shared Wnt signals in tadpoles and humans, building a bridge from this work in Xenopus tadpoles to research and therapeutic applications in humans will be quite challenging. After all, the elegance of this study lies in the genetically integrated oxidant sensor that allows live tracking of oxidants as well as the fact that tadpoles can regenerate whole limbs and tails. Current tools do not permit real-time tracking of human oxidant levels in tissues and humans can usually only regenerate very small amounts of tissue, such as superficial skin injury.

Nevertheless, this work is an important milestone in understanding the role of oxidants as promoters of regeneration and it is very likely that at least some similar pro-regenerative role of oxidants may also be present in human tissues. One of the most important take home messages of this work is that we need get rid of the common “oxidants are bad guys and antioxidants are good guys” myth. Oxidants can be harmful in some context, but they can also serve as important regenerative signals. Indiscriminate use of antioxidants can actually impair these important endogenous signals. Instead of consuming large quantities of non-specific antioxidants, we need to use antioxidants in a very targeted, context-specific and perhaps time-limited manner so that they only prevent oxidative damage without affecting beneficial oxidant signaling.

Image credit: Image of a Xenopus hybrid from Figure S1 in Narbonne P, Simpson D, Gurdon J (2011). “Deficient Induction Response in a Xenopus Nucleocytoplasmic Hybrid“. PLOS Biology.

Love, N., Chen, Y., Ishibashi, S., Kritsiligkou, P., Lea, R., Koh, Y., Gallop, J., Dorey, K., & Amaya, E. (2013). Amputation-induced reactive oxygen species are required for successful Xenopus tadpole tail regeneration Nature Cell Biology DOI: 10.1038/ncb2659