DOI: 10.5061/dryad.tdz08kq9s
title: Data from: Keratinocyte-TRPV1 sensory neuron interactions in a genetically controllable mouse model of chronic neuropathic itch

description: Our understanding of neural circuits that respond to skin dysfunction,
 triggering itch, and pathophysiological scratching remains incomplete.
 Here, we describe a profound chronic itch phenotype in transgenic mice
 expressing the tetracycline transactivator (tTA) gene within the Phox2a
 lineage. Phox2a; tTA mice exhibit intense, localized scratching and
 regional skin lesions, controllable by the tTA inhibitor, doxycycline. As
 gabapentin and the kappa opioid receptor agonist, nalfurafine, but not
 morphine, significantly reduce scratching, this phenotype has a
 pharmacological profile of neuropathic pruritus. Importantly, the Phox2a;
 tTA expression occurs in a spatially restricted population of skin
 keratinocytes that overlaps precisely with the skin area that is
 scratched. Localized Gi-DREADD-mediated inactivation of these
 Phox2a-keratinocytes completely reverses the skin lesions, while inducible
 tTA activation of the keratinocytes initiates the condition. Notably,
 ablation of TRPV1-expressing primary afferent neurons also reduces
 scratching and skin lesions, but this occurs slowly, over a course of two
 months. In contrast, denervation-induced loss of all cutaneous input
 rapidly blocks scratching. These findings identify the cellular,
 molecular, and topographic basis of a robust and chronic sensory
 neuron-dependent and gabapentin-responsive neuropathic itch that is
 initiated by genetic factors within keratinocytes.
description: # Data from: Keratinocyte-TRPV1 sensory neuron interactions in a
 genetically controllable mouse model of chronic neuropathic itch Dataset
 DOI: [10.5061/dryad.tdz08kq9s](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 |
 ID | Genotype | Sex | | ----- | -----------------------------------------
 | ------ | | m919 | Ai9 fl(fl/+), 2a-Phox-Cre(+) | Female | | m920 |
 Ai162(fl/+), Ai9 fl(fl/+), 2a-Phox-Cre(+) | Female | | m922 | Ai162(fl/+),
 Ai9 fl(fl/+), 2a-Phox-Cre(+) | Female | | m1057 | Ai9 fl(fl/+),
 2a-Phox-Cre(+) | Male | | m1058 | Ai162(fl/+), Ai9 fl(fl/+),
 2a-Phox-Cre(+) | Male | | m1059 | Ai9 fl(fl/+), 2a-Phox-Cre(+) | Male | |
 m1060 | Ai162(fl/+), Ai9 fl(fl/+), 2a-Phox-Cre(+) | Male | | m1061 | Ai9
 fl(fl/+), 2a-Phox-Cre(+) | Male | | m1062 | Ai162(fl/+), Ai9 fl(fl/+),
 2a-Phox-Cre(+) | Male | | m1127 | Ai162(fl/+), Ai9 fl(fl/+),
 2a-Phox-Cre(+) | Female | | m1129 | Ai9 fl(fl/+), 2a-Phox-Cre(+) | Female
 | | m1151 | Ai162(fl/+), Ai9 fl(fl/+), 2a-Phox-Cre(+) | Male | | m1153 |
 Ai9 fl(fl/+), 2a-Phox-Cre(+) | Male | | m1311 | Ai9 fl(fl/+),
 2a-Phox-Cre(+) | Female | ### Phox2b-Cre; Ai9 with/without Ai162 | ID |
 Genotype | Sex | | ----- | ----------------------------------------- |
 ------ | | m1429 | Ai162(fl/+), Ai9 fl(fl/+), 2b-Phox-Cre(+) | Female | |
 m1430 | Ai162(fl/+), Ai9 fl(fl/+), 2b-Phox-Cre(+) | Male | | m1431 |
 Ai162(fl/+), Ai9 fl(fl/+), 2b-Phox-Cre(+) | Male | | m1432 | Ai162(fl/+),
 Ai9 fl(fl/+), 2b-Phox-Cre(+) | Female | | m1169 | Ai9 fl(fl/+),
 2b-Phox-Cre(+) | Male | | m1183 | Ai9 fl(fl/+), 2b-Phox-Cre(+) | Male | |
 m1186 | Ai9 fl(fl/+), 2b-Phox-Cre(+) | Male | ## Code/software Prism
 Graphpad - Prism Viewer Mode Images are compatible with FIJI
 - [https://imagej.net/software/fiji/downloads](https://imagej.net/software/fiji/downloads)