Background & Aims
Osteoarthritis is the most common form of arthritis and one of the leading causes of chronic pain and disability worldwide1. We have previously reported that male nociceptor-conditional Piezo2 knock-out mice (NaV1.8cre-Piezo2fl/fl; Piezo2cko), were protected from NGF-induced inflammatory joint pain as well as from mechanical pain in the DMM model of osteoarthritis2. Other reports also demonstrate that inflammatory signals can enhance mechanical signaling through Piezo2, and inhibiting Piezo2 through knockdown was effective in reducing inflammatory pain3,4,5,6. However, whether Piezo2 inhibition protects from mechanical sensitization in both sexes and exactly how Piezo2 inhibition produces this effect is not yet clear. The aim of this study was two-fold: one to assess pain behavior in models of joint pain in both sexes and two to identify whether Piezo2 deletion on nociceptors protects from molecular and cellular changes in the dorsal root ganglia induced by these models of joint pain.
Methods
Male and female WT and Piezo2cko mice were used for this study. Mice received sham, DMM or PMX surgery as previously described2,7. Another model utilized nerve growth factor, NGF, (500ng/5ul) or vehicle injected intra-articularly twice a week for 8 weeks. Prior to surgery or injection and every 4 weeks after, mice were tested for knee hyperalgesia and weight-bearing deficits as described2. Briefly, mice were manually restrained for PAM testing and the experimenter was guided to increase pressure until a pain response was elicited. Weight bearing was assessed using a custom voluntary access static incapacitance meter where animals were assessed while balancing on their hind legs. Immunofluorescent staining of the L3-L5 DRGs following standard protocols was done for F4/80+ macrophages and CGRP+ neurons. Flow cytometric analysis was done on the blood and DRG. Ipsilateral L3-L5 DRGs were pooled from 2 mice/group and underwent staining and flow cytometry for immune cell markers.
Results
Both sexes of WT PMX mice developed weight bearing deficits by 12-weeks, while neither sex of Piezo2cko PMX mice developed asymmetry. At 4, 7, and 12 weeks, WT PMX mice of both sexes displayed knee hyperalgesia, while Piezo2cko PMX mice had significantly less knee hyperalgesia at all time points. Like the PMX model, Piezo2cko mice intra-articularly injected with NGF were protected from knee hyperalgesia and weight-bearing asymmetry at the 4 and 8 week time points compared to WT controls. Immunofluorescent staining of the DRGs demonstrated that WT DMM mice had increased F4/80+ macrophages compared to Piezo2cko DMM mice. Preliminary flow cytometry data of DRGs collected from our PMX study supports these findings in both sexes. Finally, immunofluorescent staining of the DRGs of mice injected with NGF demonstrated that WT mice had increased numbers of CGRP positive neurons compared to Piezo2cko mice.
Conclusions
The current study provides evidence that inhibiting mechanosensation through genetic deletion of Piezo2 on nociceptors is effective in reducing pain behavior in both sexes. Further the study provides preliminary evidence that inhibition of mechanical sensitization of nociceptors through deletion of Piezo2 reduces the production of chemokines and CGRP by nociceptors in the DRG.
References
1.Neogi et al, Arthritis Rheumatol. 2016;68(3):654-61; 2. Obeidat et al, Nat Commun. 2023;14:2479; 3. Eijkelkamp et al, Nat Commun. 2013;4:1682; 4. Romero et al, Nat Commun. 2020;11:2997; 5. Dubin et al, Cell Rep;2(3):511-7; 6. Ranade et al, Nature. 2014;516(7529):121-5. 7. Knights et al, Pain. 2012; 153(2):281-292
Presenting Author
Natalie Adamczyk
Poster Authors
Topics
- Models: Musculoskeletal