Background & Aims
Fibromyalgia (FM) is one of the most common chronic widespread pain conditions characterized by complex symptomatology, including depression 1. Despite the uncertain aetiology, oxidative stress has taken a leading role in the pathophysiology of FM 2. The proalgesic transient receptor potential ankyrin 1 (TRPA1) channel has been identified as an oxidative stress sensor, including hydrogen peroxide (H2O2) and the by-product of lipid peroxidation, 4-hydroxynonenal (4-HNE) 3,4. Recently, the contribution of TRPA1 expressed in Schwann cells in sustaining painful hypersensitivity has been highlighted in different mouse models of chronic pain. ROS generated by different cellular sources, including macrophages, activate the Schwann cell TRPA1, activating NADPH oxidase (NOX) 1 and amplifying the oxidative stress levels that sustain neuroinflammation and chronic pain 5–9. Thus, we have explored the involvement of this neuroinflammatory pathway in the reserpine-induced FM model.
Methods
Reserpine (1 mg/kg) subcutaneously (s.c.) or vehicle (0.1% acetic acid in saline; 10 ml/kg) was injected once daily/3 consecutive days in C57BL/6J male mice (UNIFI #1194/2015PR). On the 4th day after the first reserpine dose, the paw withdrawal threshold (PWT) using von Frey filaments, acetone drop scores and the immobility time in the forced swimming test (FST) were evaluated. Mice were intraperitoneally (i.p.) administered with the NOX inhibitor (apocynin, 100 mg/kg), selective NOX1 inhibitor (ML171; 60 mg/kg) or vehicle (4% DMSO and 4% Tween 80 in saline; 10 ml/kg) 5,10. Cre-mediated ablation of Trpa1 in PLP-expressing Schwann cells mice treated with tamoxifen (10 mg/ml/once daily/5 consecutive days; i.p.) was used. After the behavioural tests, the sciatic nerves were dissected, and H2O2 levels or immunofluorescence staining for F4/80 (MA516624) or 4-HNE (ab48506) was performed 8. Data were presented as mean±SEM and analyzed by one- or two-way ANOVA-Bonferroni post hoc test.
Results
Reserpine induced oxidative stress (H2O2 levels and 4-HNE staining increase), and neuroinflammation (F4/80+ macrophages increase) in the sciatic nerve tissue, mechanical allodynia (demonstrated by a reduction in PWT), cold allodynia (demonstrated by a cold score increase) and depression-like behaviour (demonstrated by FST immobility time increase) in C57BL/6J mice. The non-selective NOX inhibitor apocynin reversed reserpine-evoked intrasciatic H2O2 increase, mechanical and cold allodynia and depression-like behaviour. Neuroinflammation (F4/80+ macrophages), 4-HNE staining, H2O2 levels, mechanical and cold allodynia and depression-like behaviour elicited by reserpine were abolished by ML171 administration. In mice with selective deletion of TRPA1 in Schwann cells (Plp1-CreERT+/Trpa1fl/fl), mechanical and cold allodynia, depression-like behaviour and increases in intrasciatic F4/80+ cell number and 4-HNE levels were reduced as compared with control mice (Plp1-CreERT-/Trpa1fl/fl).
Conclusions
The reserpine causes monoamine depletion and sensory changes such as mechanical and thermal hypersensitivity and other symptoms, including depression, also reported by FM patients 11,12. Here, we observed that the increase in neuroinflammation induced by reserpine was closely associated with oxidative stress biomarkers and pain. It has been proposed that the TRPA1 in peripheral nerve not only acts as a neuronal sensor for the transmission of painful signals but also acts as a sensor for oxidative stress 4. Specifically, activated Schwann cells TRPA1 promotes an intracellular pathway that terminates with NOX1-dependent ROS release and macrophage recruitment in the sciatic nerve, thus promoting sustained painful hypersensitivity 6–9,13. Our data underlines the contribution of Schwann cell TRPA1 and NOX1 to the neuroinflammation that sustains reserpine-associated pain. Therefore, Schwann cells TRPA1 blockade could represent a new therapeutic target in treating FM-related behaviours.
References
1.Sarzi-Puttini, P., Giorgi, V., Marotto, D. & Atzeni, F. Fibromyalgia: an update on clinical characteristics, aetiopathogenesis and treatment. Nature Reviews Rheumatology vol. 16 645–660 at https://doi.org/10.1038/s41584-020-00506-w (2020).
2.Meeus, M., Nijs, J., Hermans, L., Goubert, D. & Calders, P. The role of mitochondrial dysfunctions due to oxidative and nitrosative stress in the chronic pain or chronic fatigue syndromes and fibromyalgia patients: peripheral and central mechanisms as therapeutic targets? Expert Opin. Ther. Targets 17, 1081–1089 (2013).
3.Trevisani, M. et al. 4-Hydroxynonenal, an endogenous aldehyde, causes pain and neurogenic inflammation through activation of the irritant receptor TRPA1. Proc. Natl. Acad. Sci. 104, 13519–13524 (2007).
4.Andersson, D. A., Gentry, C., Moss, S. & Bevan, S. Transient Receptor Potential A1 Is a Sensory Receptor for Multiple Products of Oxidative Stress. J. Neurosci. 28, 2485–2494 (2008).
5.De Logu, F. et al. Schwann cell TRPA1 mediates neuroinflammation that sustains macrophage-dependent neuropathic pain in mice. Nat. Commun. 8, 1–16 (2017).
6.De Logu, F. et al. Schwann cells expressing nociceptive channel TRPA1 orchestrate ethanol-evoked neuropathic pain in mice. J. Clin. Invest. 129, 5424–5441 (2019).
7.De Logu, F. et al. Schwann cell endosome CGRP signals elicit periorbital mechanical allodynia in mice. Nat. Commun. 13, 646 (2022).
8.De Logu, F. et al. Macrophages and Schwann cell TRPA1 mediate chronic allodynia in a mouse model of complex regional pain syndrome type I. Brain. Behav. Immun. 88, 535–546 (2020).
9.De Logu, F. et al. Peripheral Nerve Resident Macrophages and Schwann Cells Mediate Cancer-Induced Pain. Cancer Res. 81, 3387–3401 (2021).
10.Marone, I. M. et al. TRPA1/NOX in the soma of trigeminal ganglion neurons mediates migraine-related pain of glyceryl trinitrate in mice. BRAIN 141, 2312–2328 (2018).
11.Nagakura, Y., Oe, T., Aoki, T. & Matsuoka, N. Biogenic amine depletion causes chronic muscular pain and tactile allodynia accompanied by depression: A putative animal model of fibromyalgia. Pain 146, 26–33 (2009).
12.Brum, E. S., Becker, G., Fialho, M. F. P. & Oliveira, S. M. Animal models of fibromyalgia: What is the best choice? Pharmacol. Ther. 230, 107959 (2022).
13.Landini, L. et al. Acetaldehyde via CGRP receptor and TRPA1 in Schwann cells mediates ethanol-evoked periorbital mechanical allodynia in mice: relevance for migraine. J. Biomed. Sci. 30, 28 (2023).
Presenting Author
Evelyne da Silva Brum
Poster Authors
Evelyne Silva Brum, PhD
PhD
Federal University of Santa Maria
Lead Author
Maria Fialho
Federal University of Santa Maria, Santa Maria, RS, Brazil
Lead Author
Daniel Doctor Araújo
PhD
University of Florence
Lead Author
Lorenzo Doctor Landini
PhD
University of Florence
Lead Author
Matilde Doctor Marini
PhD
University of Florence
Lead Author
Mustafa Doctor Titiz
PhD
University of Florence
Lead Author
Pierangelo Doctor Geppetti
PhD
University of Florence
Lead Author
Romina Nassini
University of Florence
Lead Author
Francesco De Logu
Lead Author
Sara Marchesan Oliveira
PhD
Federal University of Santa Maria
Lead Author
Topics
- Models: Musculoskeletal