Damage-induced basal epithelial cell migration modulates the spatial organization of redox signaling and sensory neuron regeneration

Reviewer #1 (Public Review):

Summary:
In this manuscript, Fister et. al. investigate how amputational and burn wounds affect sensory axonal damage and regeneration in a zebrafish model system. The authors discovered that burn injury results in increased peripheral axon damage and impaired regeneration. Convincing experiments show altered axonal morphology and increased Ca2+ fluxes as a result of burn damage. Further experimental proof supports that early removal of the burnt tissue by amputation rescues axonal damage. Burn damage was also shown to markedly increase keratinocyte migration and increase localized ROS production as measured by the dye Pfbsf. These responses could be inhibited by Arp 2/3 inhibition and isotonic treatment.

Strengths:
The authors use state-of-the-art methods to study and compare transection and burn-induced tissue damage. Multiple experimental approaches (morphology, Ca2+ fluxing, cell membrane labeling) confirm axonal damage and impaired regeneration time. Furthermore, the results are also accompanied by functional response tests of touch sensitivity. This is the first study to extend the role of tissue-damage-related osmotic exposure beyond wound closure and leukocyte migration to a novel layer of pathology: axonal damage and regeneration.

Weaknesses:
The conclusions of the paper claiming a link between burn-induced epithelial cell migration, spatial redox signaling, and sensory axon regeneration are mainly based on correlative observations. Arp 2/3 inhibition impairs cell migration but has no significant effect on axon regeneration and restoration of touch sensitivity.

Pharmacological or genetic approaches should be used to prove the role of ROS production by directly targeting the known H2O2 source in the system: DUOX.

While the authors provide clear and compelling proof that osmotic responses lie at the heart of the burn-induced axonal damage responses, they did not consider the option of further exploring any biology related to osmotic cell swelling. Could osmotic ATP release maybe play a role through excitotoxicity? Could cPLA2 activation-dependent eicosanoid production relate to the process? Pharmacological tests using purinergic receptor inhibition or blockage of eicosanoid production could answer these questions.

The authors provide elegant experiments showing that early removal of the burnt tissue can rescue damage-induced axonal damage, which could also be interpreted in an osmotic manner: tail fin transections could close faster than burn wounds, allowing for lower hypotonic exposure time. Axonal damage and slow regeneration in tail fin burn wounds could be a direct consequence of extended exposure time to hypotonic water.

Reviewer #2 (Public Review):

This is an interesting study in which the authors show that a thermal injury leads to extensive sensory axon damage and impaired regrowth compared to a mechanical transection injury. This correlates with increased keratinocyte migration. That migration is inhibited by CK666 drug treatment and isotonic medium. Both restrict ROS signalling to the wound edge. In addition, the isotonic medium also rescues the regrowth of sensory axons and recovery of sensory function. The findings may have implications for understanding non-optimal re-innervation of burn wounds in mammals.

The interpretation of results is generally cautious and controls are robust.

Here are some suggestions for additional discussion:
The study compares burn injury which produces a diffuse injury to a mechanical cut injury which produces focal damage. It would help the reader to give a definition of wound edge in the burn situation. Is the thermally injured tissue completely dead and is resorbed or do axons have to grow into damaged tissue? The two-cut model suggests the latter. Also giving timescales would help, e.g. when do axons grow in relation to keratinocyte movement? An introductory cartoon might help.

Could treatment with CK666 or isotonic solution influence sensory axons directly, or through other non-keratinocyte cell types, such as immune cells?

Reviewer #3 (Public Review):

Fister and colleagues use regeneration of the larval zebrafish caudal fin to compare the effects of two modes of tissue damage-transection and burn-on cutaneous sensory axon regeneration. The authors found that restoration of sensory axon density and function is delayed following burn injury compared to transection.

The authors hypothesized that thermal injury triggers signals within the wound microenvironment that impair sensory neuron regeneration. The authors identify differences in the responses of epithelial keratinocytes to the two modes of injury: keratinocytes migrate in response to burn but not transection. Inhibiting keratinocyte migration with the small-molecule inhibitor of Arp2/3 (CK666) resulted in decreased production of reactive oxygen species (ROS) at early, but not late, time points. Preventing keratinocyte migration by wounding in isotonic media resulted in increased sensory function 24 hours after burn.

Strengths of the study include the beautiful imaging and rigorous statistical approaches used by the authors. The ability to assess both axon density and axon function during regeneration is quite powerful. The touch assay adds a unique component to the paper and strengthens the argument that burns are more damaging to sensory structures and that different treatments help to ameliorate this.

A weakness of the study is the lack of genetic and cell-autonomous manipulations. Additional comparisons between transection and burns, in particular with manipulations that specifically modulate ROS generation or cell migration without potentially confounding effects on other cell types or processes would help to strengthen the manuscript. In terms of framing their results, the authors refer to "sensory neurons" and "sensory axons" throughout the text - it should be made clear what type of neuron(s)/axon(s) are being visualized/assayed. Along these lines, a broader discussion of how burn injuries affect sensory function in other systems - and how the authors' results might inform our understanding of these injury responses - would be beneficial to the reader.

In summary, the authors have established a tractable vertebrate system to investigate different sensory axon wound healing outcomes in vivo that may ultimately allow for the identification of improved treatment strategies for human burn patients. Although the study implicates differences in keratinocyte migration and associated ROS production in sensory axon wound healing outcomes, the links between these processes could be more rigorously established.