Background & Aims
Osteoarthritis (OA) is a painful disease of synovial joints, characterised by cartilage destruction, tissue damage and synovial inflammation (synovitis) [1]. Synovitis is associated with pain, but poor understanding of the local mechanisms driving OA pain limits novel treatment options.
Within the knee joint, the synovial joint lining is innervated by sensory nerve endings, and locally released inflammatory mediators can activate and sensitise nociceptors and promote axonal sprouting, which can lead to peripheral sensitization associated with OA [2].
Based on the above context, the hypothesis to be tested is that the pathotype of pain associated synovial fibroblasts may promote sensory neuronal excitability, which may drive joint pain.
The aim of our current study is to map the relationship and cellular crosstalk mechanisms between synovial fibroblasts and neurons responsible for nociceptor excitability which may contribute to the sensation of pain.
Methods
To investigate the influence of sensory nerve terminal environment in OA synovium, this study was conducted using primary mouse DRG culture (E16.5) in compartmentalized microfluidic chambers. At div6, sensory neuron axon terminals were exposed for 48h to conditioned media from cultured synovial fibroblasts isolated from the synovium tissue of OA patient knees (reported painful or non-painful anatomical sites). Following the 48h incubation, 25 mM KCl was applied to the axon compartment and a subsequent somal side stimulation was performed. Ca2+ transients were recorded in the cell body, as a proxy marker of neuron excitability. DRG neurons were fixed and immunolabelled with an axonal marker (acetylated ?-tubulin), nerve growth factor and the receptor TrkA.
For siRNA studies, siRNA was applied to the axonal terminals (48h) before stimulation to evaluate their capacity to locally mitigate sensory DRG neuron excitability.
Results
Exposure of the axonal side of DRG neurones to conditioned media from painful OA derived fibroblasts sensitized responses to KCl stimulation (41% increase), compared to exposure to non-painful OA derived conditioned media (13% increase) and control media. Whereas stimulation on the somal side didn’t produce any change in excitability.
Transcriptomic analysis of synovial fibroblasts from painful vs non-painful sites identified differentially expressed genes (including NRN1, CXCL14, INHBA, DNAJB1 and HTRA) which were tested in our functional studies [3]. In order to test if some of these previously identified deregulated markers in fibroblast profiling experiments can contribute to the observed phenotype, we first applied siRNA for NRN1 to the axonal side of DRG neurones. This reduced KCl induced neuronal hyperexcitability (13%) compared to control. Current work will use siRNA to block NRN1 in pain fibroblasts, to assess whether it can prevent the sensitising properties.
Conclusions
Our data demonstrates that the use of the compartmentalised microfluidic chamber can advance our knowledge of the peripheral pain mechanisms and allow the investigation of potential therapeutic targets with peripheral accessibility during OA pain.
Future work includes the study of other identified genes in OA synovial fibroblasts subsets to validate their efficacy in disrupting fibroblasts neuronal crosstalk.
References
1.Yao Q, Wu X, Tao C, Gong W, Chen M, Qu M, Zhong Y, He T, Chen S, Xiao S. Osteoarthritis: pathogenic signalling pathways and therapeutic targets. Signal Transduction and Targeted Therapy 2023; 8: 56.
2.Chakrabarti S, Jadon DR, Bulmer DC, Smith ESJ. Human osteoarthritic synovial fluid increases excitability of mouse dorsal root ganglion sensory neurons: an in-vitro translational model to study arthritic pain. Rheumatology (Oxford) 2020; 59: 662-667.
3.Nanus DE, Badoume A, Wijesinghe SN, Halsey AM, Hurley P, Ahmed Z, et al. Synovial tissue from sites of joint pain in knee osteoarthritis patients exhibits a differential phenotype with distinct fibroblast subsets. EBioMedicine 2021; 72: 103618.
Presenting Author
Jyoti Agrawal
Poster Authors
Jyoti Agrawal
PhD
University of Nottingham
Lead Author
Susanne Wijesinghe
PhD
The University of Birmingham
Lead Author
Caitlin Ditchfield
MSc
The University of Birmingham
Lead Author
Simon Jones
PhD BSc
The University of Birmingham
Lead Author
Victoria Chapman
School of Life Sciences, University of Nottingham
Lead Author
Federico Dajas-Bailador
PhD BSc
University of Nottingham
Lead Author
Topics
- Models: Chronic Pain - Neuropathic