Peripheral sensory axons have impaired regeneration after burn injury.

(A) Schematic of larval zebrafish injury. Gray dashed line denotes area used to measured axon density to the right of the notochord. (B) Confocal max-projected images of sensory axons in uninjured, transected, and burned Tg(Ngn1:GFP-Caax) caudal fins 24 hpw. (C) Quantification of axon density for uninjured, transected, and burned larvae in the wound area 24-96 hpw. N>20 larvae per condition from 4 replicates. (D) Quantification of sensory perception for uninjured, transected, and burned larvae 24-96 hpw. N>32 larvae per condition from 4 replicates. (E) Confocal time series images of axonal damage, indicated by calcium-positive punctae (black dots), in Tg(Elavl3:GCaMP5) larvae following either transection or burn injury. Each series follows one representative larva over 6 hpw. (F) Quantification of axon damage area in transected and burned larvae 6 hpw. N>12 larvae per condition from 2 replicates. (G) Images of larvae either transected or burned in the presence of FM 1-43 dye. White dashed box denotes area of uninjured tissue in which axonal damage appears in H. (H) Images show axonal damage following transection or burn injury. Red dashed box corresponds to the tissue region highlighted in G. In all cases, scale bars=20 µm.*p<0.05, **p<0.01, ***p<0.001, ns=not significant.

The burn wound microenvironment contributes to impaired sensory axon regeneration.

(A) Schematic of two-wound experiment design. (B) Confocal max-projected images of FM dye staining following secondary transection in the two-wound experiment at 5 mpw and 6 hpw. (C) Images of sensory axons in larvae subjected to an initial transection or burn injury followed by subsequent transection either early (5 mpw) or late (6 hpw). (D) Quantification of axon density in wounded tissue 24 hpw from larvae wounded as in B. N>28 larvae per condition from 3 replicates. (E) Quantification of sensory perception in wounded tissue 24 hpw from larvae wounded as in B. N=24 larvae each from 3 replicates. In all cases, scale bars=20 µm. *p<0.05, ***p<0.001, ns=not significant.

Burn injury induces coordinated keratinocyte and sensory axon movement.

(A) Confocal max-projected time-series images of Tg(Krt4:UtrCH-GFP) larvae after either transection or burn injury. Yellow pseudocolored cells and colored tracks highlight keratinocyte displacement. Scale bar=20 µm. (B) Quantification of keratinocyte movement distance over 1 hpw. N= 8 larvae each collected from 3 replicates. (C) Confocal max-projected images of superficial and basal keratinocytes in Tg(Krt4:Lifeact-mRuby) labeled larvae. Left, superficial keratinocytes. Middle, basal keratinocytes. Right, Merge. Superficial and basal cell images were taken from the same z-stack and pseudocolored to match the appropriate cell layer. Dashed lines outline one individual keratinocyte. Scale bar=10 µm. (D) Confocal max-projected time-series images of sensory axons and basal keratinocytes in dual-labeled Tg(Krt4:Lifeact-mRuby); Tg(Ngn1:GFP-Caax) larvae unwounded or after burn. Arrows highlight coincident movement between keratinocytes and associated sensory axons. Unless otherwise stated, scale bar=20 µm. ***p<0.001.

The Arp 2/3 inhibitor impairs early keratinocyte movement and alters the spatial distribution of reactive oxygen species signaling.

(A) Confocal max-projected images of control or CK666-treated transiently injected Tg(Krtt1c19e:Lifeact-mRuby) larvae. Arrows point to lamellipodia in the control larva, and lack of lamellipodia in the CK666-treated larva. Scale bar=10 µm. (B) Plot of keratinocyte speed over 1 hpw as treated in A. N=10 larvae each collected from 3 replicates. (C) Plot of keratinocyte distance moved over 1 hpw as treated in A. N=10 larvae each collected form 3 replicates. (D) Confocal sum-projected time-series images of hydrogen peroxide level (Pfbsf intensity) in 1 larva over 1 hpw in the indicated treatment. (E) Quantification of Pfbsf intensity in the wound or fin area of the represented larva after burn injury as treated in D over 1 hpw. N=1 representative larva per condition. (F) Confocal max-projected images of sensory axons 24 hpw in larvae wounded in control medium or CK666. (G) Quantification of axon density 24 hpw in larvae treated as in J. N>22 larvae per condition from 4 replicates. (H) Quantification of sensory perception 24 hpw in larvae treated as in J. N=32 larvae per condition from 4 replicates. Unless otherwise specified, scale bars=20 µm. ns=not significant.

Treatment with isotonic solution inhibits keratinocyte migration and dampens ROS signaling.

(A) Confocal time-series images of basal keratinocyte movement in Tg(Krtt1c19e:acGFP) larvae over 1 hpw after burn injury in the indicated treatment. (B) Plot of basal keratinocyte average speed over 1 hpw treated as in A. N=10 larvae per condition collected from 3 replicates. (C) Distance of keratinocyte movement over 1 hpw treated as in A. N=10 larvae per condition collected from 3 replicates. (D) Confocal sum-projected, heat mapped time-series images of hydrogen peroxide level (Pfbsf intensity) over 1 hpw as treated in A. (E) Quantification of Pfbsf intensity in the wound or fin area of the represented larva after burn injury as treated in D over 1 hpw. N=1 representative larva per condition. (F) Confocal sum-projected images of Pfbsf intensity in the fin and wound zone either 0 or 6 hours following burn injury. Dashed red line denotes the boundary between the wound area and distal fin tissue. Scale bar=50 µm. (G) Quantification of mean Pfbsf fluorescence intensity (MFI) immediately (0 hpw) after burn injury normalized to the control condition. N>27 larvae per condition from 3 replicates. (H) Quantification of mean Pfbsf fluorescence intensity 6 hpw in the indicated region of the fin normalized to the control condition. N>26 larvae per condition from 3 replicates. Unless otherwise indicated, scale bars=20 µM. *p<0.05, ***p<0.001, ns=not significant.

Isotonic treatment improves axon regeneration.

(A) Confocal max-projected images of axon damage in control or isotonic treated Tg(Elavl3:GCaMP5) larvae 0 or 6 hpw. (B) Quantification of axon damage in control and isotonic treated burned fins at 6 hpw as treated in A. N=16 larvae per condition from 3 replicates. (C) Confocal max-projected images of sensory axons in larvae 24 hpw as treated in A. (D) Quantification of axon density 24 hpw in larvae treated as depicted in C. N>30 larvae per condition from 3 replicates. (E) Quantification of sensory perception 24 hpw in larvae treated as in C. N=24 larvae each from 3 replicates. (F) Schematic illustrating the different isotonic treatment paradigms that are being compared. (G) Confocal sum-projected images of pfbsf intensity in control and isotonic +1 hpw treated burned larvae. Dashed red line denotes the boundary between the wound area and distal fin tissue. White dashed line denotes the fin. (H) Quantification of mean Pfbsf fluorescence intensity (MFI) 6 hpw in the indicated region of the fin normalized to the control condition. N=31 larvae per condition from 3 replicates. (I) Confocal max-projected images of sensory axons 24 hpw in burned control or isotonic treated larvae starting 1 hpw. (J) Quantification of axon density 24 hpw in larvae treated as in D. N=29 larvae per condition from 3 replicates. (K) Quantification of sensory perception 24 hpw in larvae treated as in D. N=24 larvae per condition from 3 replicates. Unless otherwise indicated, scale bars=20 µm. *p<0.05, **p<0.01, ns=not significant.

Elavl3-GCaMP5 transgenic fish show sensory axon damage.

(A) Confocal max-projected images of axon damage in Tg(Elavl3:GCaMP5) larval zebrafish caudal fins either untreated or 30 minutes post-treatment with the neurotoxin sodium azide (NaN3, 1.5% final concentration). Sensory neuron damage is indicated by calcium-positive axon fragments (black dots). Dashed black lines denote the fin edge. Black boxes highlight area of inset, shown below. (B) Confocal max-projected images of Tg(Elavl3:GCaMP5) larvae injected with Ngn1 morpholino both before and 5 minutes after the indicated injury. (C) Confocal max-projected images of Tg(Elavl3:GCaMP5) larvae taken from a time series. Red dashed box denotes inset area shown on right of a sensory axon fragmenting over a period of 30 minutes. (D) Confocal max-projected images of Tg(Ngn1:GFP-Caax) larvae taken from a time series. Red dashed box denotes inset area shown on right of a sensory axon fragmenting over a period of 30 minutes. (E) Schematic of Rohon-Beard (green) and Dorsal Root Ganglia (blue) soma localization in 3 dpf zebrafish. Red box denotes area in which the image shown in F was acquired. (F) Representative confocal max-projected image of intact RB and DRG somas 24 hpw in a Tg(Ngn1:GFP-Caax); Tg(Krtt1c19e:Lifeact-mRuby) dual-labeled larva. Arrows denote RB somas, while arrowheads indicate DRG somas. Soma position was unchanged compared to pre-wounding. In all cases, scale bar=20 µm.

Inhibition of early ROS signaling does not significantly improve sensory neuron function.

(A) Confocal time series of basal keratinocyte, Tg(Krtt1c19e:acGFP), movement after the indicated injury. Yellow pseudocolored cells highlight keratinocyte displacement. (B) Confocal max-projected images of sensory axons treated with DPI. (C) Quantification of axon density 24 hpw in larvae treated as stated in F. N>19 larvae per condition from 3 replicates. (D) Quantification of sensory perception 24 hpw in larvae treated as in F. N=24 larvae each from 3 replicates. Unless otherwise indicated, scale bars=20 µM. ns=not significant.

Keratinocyte movement after injury and effect of D-Sorbitol on sensory axon regeneration.

(A) Representative max-projected confocal images of sensory axons in control and isotonic D-Sorbitol treated larvae 24 hpw. (B) Quantification of axon density in wounded tissue 24 hpw. N=28 larvae each from 4 replicates. (C) Quantification of sensory perception 24 hpw. N=24 larvae each collected from 4 replicates. (D) Representative max-projected confocal images of sensory axons in unwounded, drug-treated 4 dpf larvae. (E) Quantification of axon density in larvae treated as stated in E. N>15 larvae each from 3 replicates. In all cases, scale bar=20 µm. **p<0.01, ***p<0.001, ns=not significant.