735: Piezo2 Deletion in Sensory Neurons: assessment of pain-relevant behaviors in mice

Topic: Basic Science
Subtopic: Pain category (ICD-11), Nociceptive Pain , , , , , , , , , , Assessment and Measurement of Pain/Nociception, Transgenic models, Behavioral studies

Background and Aims: The mechanosensitive ion channel Piezo2 is essential for light touch perception, as deletion in sensory neurons results in profound deficits in innocuous mechanical sensation while sparing nociceptive responses [3]. The voltage‑gated sodium channel NaV1.8 is predominantly expressed in nociceptive neurons and plays a central role in peripheral pain signaling. In nociceptor‑associated neurons, Piezo2 knockdown has been reported to alleviate pain‑related behaviors in osteoarthritis models [2]; however, it remains unclear whether similar effects extend to other pain contexts or neuronal populations. Importantly, behavioral baseline phenotyping is required to distinguish primary genetic effects from model‑specific pain alterations. Prior to applying Piezo2 conditional knockout models in disease‑specific pain paradigms, it is therefore essential to establish baseline sensory and pain‑related behaviors. Here, we assessed behavioral outcomes following Piezo2 deletion in NaV1.8 and in Advillin‑positive sensory neurons.

Methods: NaV1.8‑Cre+/- Piezo2fl/fl and Advillin‑CreERT2+/- Piezo2fl/fl mice were generated in‑house. Tamoxifen was used to induce recombination in the Advillin‑CreERT2 model and was administered to both Cre‑positive and Cre‑negative mice to control for treatment effects. In the NaV1.8 cohort, pain‑relevant behaviors were assessed using facial grimace scoring automatically analyzed with PainFace [1], periorbital von Frey testing, a vertical climbing assay, a rotarod test assessing motor coordination, and a hot‑plate test. Hindpaw mechanical sensitivity was assessed using von Frey testing, with intraplantar hypertonic saline serving as a positive control. In the Advillin cohort, facial grimace behavior, heat sensitivity, and motor coordination were assessed 28 days after completion of tamoxifen treatment. Group comparisons were performed using t‑tests or two‑way ANOVA, and group sizes ranged from 10 to 21 animals per genotype and cohort.

Results: In the NaV1.8‑Cre cohort, Piezo2 deletion did not affect baseline grimace scores, periorbital withdrawal thresholds, vertical climbing behavior, or hot‑plate responses compared with Cre‑negative control mice. Following intraplantar injection of hypertonic saline, both genotypes displayed a significant reduction in plantar withdrawal thresholds relative to baseline (P ≤ 0.001). In contrast, mice with Piezo2 deletion in sensory neurons (Advillin‑CreERT2 cohort) exhibited a significantly higher baseline grimace score than control animals (P ≤ 0.001).

Conclusions: Establishing intact baseline sensory behavior is critical for the interpretation of pain‑related outcomes in genetic models. In the present study, inducible Piezo2 deletion in Advillin‑positive sensory neurons was associated with altered baseline facial grimace behavior, consistent with a disruption of normal sensory processing. In contrast, Piezo2 deletion confined to NaV1.8‑expressing neurons did not affect baseline sensory or pain‑related behaviors, confirming intact nociceptive sensitization, with no differences observed between genotypes supporting the suitability of this model for subsequent pain‑related behavioral investigations.

References: [1] McCoy ES, Park SK, Patel RP, Ryan DF, Mullen ZJ, Nesbitt JJ, Lopez JE, Taylor-Blake B, Vanden KA, Krantz JL, Hu W, Garris RL, Snyder MG, Lima L V., Sotocinal SG, Austin JS, Kashlan AD, Shah S, Trocinski AK, Pudipeddi SS, Major RM, Bazick HO, Klein MR, Mogil JS, Wu G, Zylka MJ. Development of PainFace software to simplify, standardize, and scale up mouse grimace analyses. Pain 2024;165:1793–1805.

[2] Obeidat AM, Wood MJ, Adamczyk NS, Ishihara S, Li J, Wang L, Ren D, Bennett DA, Miller RJ, Malfait AM, Miller RE. Piezo2 expressing nociceptors mediate mechanical sensitization in experimental osteoarthritis. Nat Commun 2023;14.

[3] Ranade SS, Woo SH, Dubin AE, Moshourab RA, Wetzel C, Petrus M, Mathur J, Bégay V, Coste B, Mainquist J, Wilson AJ, Francisco AG, Reddy K, Qiu Z, Wood JN, Lewin GR, Patapoutian A. Piezo2 is the major transducer of mechanical forces for touch sensation in mice. Nature 2014;516:121–125.

Presenting Author: Karina Obelitz-Ryom
Submitting Author: Karina Obelitz-Ryom

Senior Author: Sarah Louise Tangsgaard Christensen

IASP 2026 Global Year theme of Neuropathic Pain: No

• Rikke Holm Rasmussen: PhD - Danish Headache Center
• Sarah Louise Tangsgaard Christensen: PhD - Danish Headache Center