Dataset DOI: 10.5061/dryad.tdz08kq9s

Description of the data

Various behavioral quantifications, histological quantifications, and images to support the associated PNAS paper.

Files and variables

File: qPCR_k14.prism

Description: In tamoxifen-injected KRT14-CreER; Ai162 mice and littermate Cre negative controls, a ventral 2x2 cm patch of skin was harvested. The epidermis was separated from the whole skin by incubating in Trypsin-EDTA 0.15% for 45 minutes. Isolated cells from the epidermis and whole mRNA were extracted using a RNeasy Mini kit from Qiagen after which was reverse transcribed into cDNA using Superscript IV (Thermo Fisher Cat # 18091200). qPCR analyses were carried out with gene specific primers and fluorescently labeled Taqman probes (ThermoFisher Scientific) for Tslp (Mm01157588_m1), Il33 (Mm00505403_m1), Edn1 (Mm00438659_m1) and Postn (Mm01284919_m1). Relative expression level was calculated using the 2−ΔΔCT method. β-actin (Mm02619580_g1) was used as the internal control for each sample.

File: Crowther-Kashem_2025_Data.pzfx

Description: Data for all other behavioral and histological quantifications as graphed in Crowther et al. 2025 PNAS

The data presented in the tables of the prism spreadsheet represent measurements from specific anatomical sites. Figures 1B, 1F, 1G, 1H, 1I, 2B, 2D, 2H, 2I, 3B, 4E, 4H, 4K, S1C, S2A, S3F, and S10 show measurements of scratching behavior or lesion size in the shoulder area. Figure 1E displays measurements collected from skin probing at the shoulder. Figures 1J and S1D show behavioral assessments conducted following injections administered to the cheek area. Figure 6B presents measurements of scratching behavior at any body site, while Figure 6C shows lesion size measurements at the ventral neck. Supplemental Figures 1E-G and 6F-G present data from tests performed on the hind paws, and Supplemental Figure 6H shows results from tail testing. Supplemental Figure 9 provides a quantitative analysis of scratching distribution across various body sites. Figure 3A is a histological quantification of neuronal number. Specifically the measurements were collected as described below.

Measurement of mouse behavior

The experimenter was blinded to the extent possible for all mouse behavior experiments. Mice were acclimatized to the behavior room and habituated to the experimental apparatus for 30 minutes for 3 straight days. Chemical injections were performed with a 30G hypodermic needle.

Documenting scratching

Mice were acclimatized as above, placed in opaque white cylinders over a platform, and recorded via an Imaging Source camera (DMK 37BUX252, lens TCSL 0418 5MP) for 15 or 30 minutes in the dark. In some settings, mice were recorded with a handheld Sony video camera on a mirrored platform for a similar duration. Scratching bouts were scored on video playback and were defined as a single hindpaw motion that contacts either the cheek or the shoulder/back. The end of a bout was defined when the hindpaw was lowered.

Chloroquine-induced scratching

Mice were acclimatized for 30 minutes, and then 200 µg of chloroquine (Sigma 1650000) was injected intradermally into the cheek or intra-lesional area of the shoulder or in a comparable area in a Cre negative mouse without lesions. Scratching was measured for 30 minutes, as described above.

Von Frey measurement of mechanical sensitivity

For all groups, we recorded 3 days of baseline mechanical sensitivity. Animals were habituated on a wire mesh for 2 hours, after which we used von Frey filaments (sizes 1.65, 2.44, 2.84, 3.22, 3.61, 3.84, 4.08, and 4.31) to measure mechanical withdrawal thresholds, using the up-down method. These filaments correspond to the following weights: 0.008, 0.004, 0.07, 0.16, 0.4, 0.6, 1, and 2 g, respectively.

Capsaicin-induced nocifensive behaviors

Mice were acclimatized for 30 minutes, and then 20 µg of capsaicin (Sigma M2028) in 20 µL of 7% Tween-80, 10% ethanol in phosphate-buffered saline (PBS) was injected intradermally into the cheek. Wiping was measured for 30 minutes in a setting comparable to that used to analyze scratching.

Hargreaves measurement of heat sensitivity

We acclimatized the mice for 30 min in Plexiglass cylinders. The mice were then placed on the glass of a Hargreaves apparatus, and the latency to withdraw the hind paw from the heat source was recorded. Each paw was tested five times, and we averaged latencies over the five trials. Hargreaves tests were done 1 hour after the von Frey measurement.

Acetone-induced measurement of cold sensitivity

Mice were habituated for 30 minutes on a mesh in plexiglass cylinders. Next, we used a syringe to squirt 50 µl of acetone onto the plantar surface of the hind paw. The responses of the mice directly after the application of acetone were recorded on video for 30 seconds. Each paw was tested five times, and we measured events spent lifting, licking, or flinching the paw. Results are displayed as the total events across the five trials.

Assessment of skin lesion size

Skin lesions were defined as erosive (loss of epidermis) regions of unshaved mice. The maximum width and length of the lesions were measured with a digital caliper (Rexbeti).

Chemical agents and delivery

For acute drug experiments, a baseline measure of scratching was initially recorded over 30 minutes. 24 hours later, scratching over 30 minutes was recorded with the drug on board, generating paired data points for each individual.

Gabapentin (Sigma G154) was injected intraperitoneally at 30 mg/kg after 30 minutes of acclimatization in the test chamber. Mouse behavior was measured 30 minutes after injection for another 30 minutes.

Nalfurafine was injected intraperitoneally at 20 μg/kg after 30 minutes of acclimatization. Mouse behavior was measured 30 minutes after injection for another 30 minutes.

Morphine (Sigma M8777) was injected intraperitoneally at 10 mg/kg after 30 minutes of acclimatization. Mouse behavior was measured 30 minutes after injection for another 30 minutes.

 Cetrizine (Sigma BP837) was injected intraperitoneally at 30 mg/kg after 30 minutes of acclimatization. Mouse behavior was measured 30 minutes after injection for another 30 minutes.

Resiniferatoxin (Adipogen AG-CN2-0534-MC01) was diluted at 1.0 mg/ml in 100% ethanol and then subcutaneously injected with 3 escalating consecutive daily doses of 30, 60, and 100 mg/kg in 300 µL of normal saline. The efficacy of RTX-mediated TRPV1+ sensory nerve ablation was assessed by monitoring tail-flick latency, which increased to a 15-second cut-off.

Clozapine-N-oxide (Cayman Chemical) was added to a solution of 5.0 mg CNO in 40 mL of PBS. This solution was topically painted on the skin every day for 10 days, unilaterally over the skin lesion; the contralateral skin lesion received a similar volume of PBS.

Tamoxifen administration in KRT14-CreER mice

For topical application: 4-hydroxy-Tamoxifen *(Sigma-Aldrich ) was dissolved in ethanol (1.0 mg / 50 ml) at 55 degrees for 15 minutes with vortexing, and aliquots were stored at -20 degrees. First, the mouse was anesthetized using ketamine (80 mg/kg ketamine + 5.0 mg/kg xylazine). Next, both sides of the mouse, spanning from the ear to the lower abdomen, were depilated with Nair, creating two lateral stripes down the back, preserving fur at the dorsal midline. A total of 100 µL of the 4-OHT solution was applied on the right side of the depilated skin using a micropipette. Small droplets of the solution were dispensed and rubbed equally across the depilated area. On the left side, 100 µL of ethanol (vehicle) was similarly applied. Mice were monitored in a holding cage until they were ambulatory and then returned to their home cage; their general health was monitored daily. The liquid application, but not the Nair, was repeated for three days.

For systemic administration: Tamoxifen (Sigma Cat #5648) 100 mg/kg in corn oil (Sigma Cat #8267) was injected intraperitoneally. This dose was repeated every other day for a total of three injections. Mice were returned to their home cage after injection, and their general health was monitored daily.

6-OHDA induced sympathectomy

Mice were intraperitoneally injected with 100 mg/kg 6-OHDA in 0.01% ascorbic acid in phosphate-buffered saline (PBS) once (vehicle is 0.01% ascorbic acid in PBS). Mice were returned to their home cage after injection, and their general health was monitored daily. A measurement of scratching frequency was taken at 1 month post-injection.

Averaged data was compiled and graphed in Prism.

File: Crowther-Kashem_2025_CervicalSpinalCordImages.zip

Description: Primary dataset for the cervical neuronal cell counting (Figure3A)

Histological counts of cervical spinal neurons:

50-micron transverse cryostat sections of the entire cervical enlargement were collected. A regularly spaced subset of sections (one every 150 microns) was selected for DAPI staining and mounted. All selected sections were imaged for endogenous tdTomato and DAPI at 1.6 x 1.6 x 8-micron resolution with eight z-slices using a 10X objective on an FV3000 confocal. Cell counting was performed on at least 3 sections per mouse and averaged.

The HDF5 files contain a hierarchical structure used here for storing multi-resolution image data from microscopy. Modality_LSM is the top group which is the main container for image data and nested groups contain up to resolution level 6 and each level represents the same image at decreasing resolutions (likely for efficient viewing or processing at different scales). On the whole each dataset is a 3D image array (Z, Y, X)

File structure:

(Root folder) Crowther-Kashem_2025_CervicalSpinalCordImages/..

(Individual Sample folders) 2023_05_31_m1129/..

(4X or 10X imaging selection) 2023_05_31_m1129_4x_wholeSlideMap_CycleCode/..

(Individual spinal cord section images) StitchA01G001.oir

Phox2a-Cre; Ai9 with/without Ai162

Phox2b-Cre; Ai9 with/without Ai162

Code/software

Prism Graphpad - Prism Viewer Mode

Images are compatible with FIJI - https://imagej.net/software/fiji/downloads