Background & Aims

Neuropathic pain is a devastating condition that, according to a recent study of UK Biobank data(1), affects more than 9% of the population. Mechanistically, a primary driver of neuropathic pain is abnormal activity in sensory nerves. However, the timing and ultimate causes of this activity are still not fully elucidated(2). Local immune cells have been implicated by many groups as a potential source of pro-algesic mediators, which then go on to drive peripheral neuron sensitization(3). Meanwhile, mesenchymal cell populations are less well-studied(4), but are also starting to be implicated(5). Here we present novel data that point towards a potential role for pericytes, mural cells which wrap around capillaries to help control vascular and immune cell function.

Methods

To investigate mesenchymal cell function in neuropathic pain, the partial sciatic nerve ligation (PSNL) model was used. Mice underwent unilateral PSNL or sham surgery; ipsilateral and contralateral sciatic nerve was collected 5 days or 2 months post-injury. Tissue was enzymatically digested, and mesenchymal cells isolated for single-cell RNA sequencing. To examine changes in pericyte numbers at the protein level, PSNL was performed in mice, and sciatic nerves collected and enzymatically digested on day 10 for analysis using flow cytometry. Additionally, PSNL was performed on PdgfrbCre x TdTomato mice, which were then perfused with FITC-albumin gelatin 5 days post-injury. Sciatic nerves were cleared and stained with DAPI for imaging on a confocal microscope. Finally, human sciatic nerve pericytes were treated with inflammatory cytokines in-vitro for 4 hours. Media was collected and analysed using a Luminex assay.

Results

Single-cell RNAseq showed an increase in the number of Notch3 positive cells in sciatic nerve following acute and chronic nerve injury. These Notch3 positive cells, likely pericytes or vascular smooth muscle cells, were found to express known pro-algesic mediators, such as Il6 and Ngf. The increase in the number of pericytes following nerve injury was confirmed at protein level using flow cytometry. We have also performed tissue clearing to allow further confirmation of these changes in morphologically intact sciatic nerve. Finally, Luminex assay of conditioned media from cultured human sciatic nerve pericytes showed that stimulation with TNF? induces IL6 and CCL2 release from these cells, which is further enhanced in the presence of TNF? and IL17 combined.

Conclusions

Our data demonstrate an increase in the number of sciatic nerve pericytes in a mouse model of neuropathic pain, both at acute and chronic timepoints. These cells were also shown to be a significant source of key pro-algesic mediators, suggesting that pericytes may be involved in the development of chronic pain following nerve injury. Preliminary studies in human sciatic nerve pericytes grown in culture suggest that our pre-clinical findings may translate to human cells.

References

1Baskozos, G. et al. Epidemiology of neuropathic pain: an analysis of prevalence and associated factors in UK Biobank. Pain Rep 8, e1066, doi:10.1097/pr9.0000000000001066 (2023).
2Choi, D. et al. Spontaneous activity in peripheral sensory nerves: a systematic review. Pain, doi:10.1097/j.pain.0000000000003115 (2023).
3Fiore, N. T., Debs, S. R., Hayes, J. P., Duffy, S. S. & Moalem-Taylor, G. Pain-resolving immune mechanisms in neuropathic pain. Nature Reviews Neurology 19, 199-220, doi:10.1038/s41582-023-00777-3 (2023).
4Shinotsuka, N. & Denk, F. Fibroblasts: the neglected cell type in peripheral sensitisation and chronic pain? A review based on a systematic search of the literature. BMJ Open Sci 6, e100235, doi:10.1136/bmjos-2021-100235 (2022).
5Singhmar, P. et al. The fibroblast-derived protein PI16 controls neuropathic pain. Proc Natl Acad Sci U S A 117, 5463-5471, doi:10.1073/pnas.1913444117 (2020).
6Hofer, D. M. et al. Rethinking the definition of chronic postsurgical pain: composites of patient-reported pain-related outcomes vs pain intensities alone. PAIN 163 (2022).

Presenting Author

Julia Vlachaki Walker

Poster Authors

Julia Vlachaki Walker

PhD

Kings College London

Lead Author

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Sara Villa

Kings College London

Lead Author

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Zoe Hore

Kings College London

Lead Author

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Laura Fedele

Kings College London

Lead Author

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Irene Zebochin

Kings College London

Lead Author

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Yuening Li

Kings College London

Lead Author

Franziska Denk

Kings College London

Lead Author

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Topics

  • Models: Chronic Pain - Neuropathic