Peripheral neuropathic pain remains challenging to treat, partly due to our limited understanding of the molecular mechanisms at play in humans. In this multicentre cohort study, we describe the local molecular signature of neuropathic pain at the lesion site, using peripheral nerves of patients with Mortons neuroma as a human model system of neuropathic pain. Plantar tibial nerves were collected from 22 patients with Mortons neuroma (18 female, median age 60.0 [IQR 16.0] years) and control nerves from 11 participants (4 females, 58.0 [21.0] years) without a nerve injury. Pre-surgery, we collected data on pain severity, duration and nature (e.g., neuropathic pain inventory, NPSI). RNA bulk sequencing of peripheral nerves identified 3349 genes to be differentially expressed between Mortons neuroma and controls. Gene ontology enrichment analysis and weighted gene co-expression network analyses (WGCNA) revealed modules specific for host defence and neurogenesis. Deconvolution analysis confirmed that the densities of macrophages as well as B-cells were higher in Mortons neuroma than control samples. The findings for T-cells were inconclusive. Modules associated with defence response, neurogenesis and muscle system development correlated with paroxysmal and evoked pain in people with Mortons neuroma. Macrophage cell populations identified by deconvolution analysis as well as single differentially expressed genes (MARCO, CD163, STAB1; indicating the presence of a specific M(GC) subset of macrophages) correlated with paroxysmal pain. Immunofluorescent analyses confirmed the presence of demyelination, higher densities of intraneural T-cells and CD163+MARCO+ macrophage subsets in Mortons neuroma compared to control nerves. Histological CD68+ macrophage density correlated with burning pain. Our findings provide detailed insight into the local molecular signature in the context of human focal nerve injury. There is clear evidence for an ongoing role of the immune system in chronic peripheral neuropathic pain in humans, with macrophages and specifically the M(GC) MARCO+ subset implicated.

The authors have declared no competing interest.

ABS is supported by a Wellcome Trust Clinical Career Development Fellowship (222101/Z/20/Z). IDS and PSS were funded by the Clinical Research Priority Program (CRPP) Pain from the University of Zurich, Switzerland. PSS is supported by postdoctoral fellowships from the International Foundation for Research in Paraplegia (P198F), the Swiss National Science Foundation (P500PB_214416), and Michael Smith Health Research BC (RT-2023-3173). This research was funded in whole, or in part, by the Wellcome Trust [222101/Z/20/Z]. For the purpose of Open Access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. We also received funding from from ONO pharamceuticals Ltd.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

Ethics Committee South Central - Oxford C Research Ethics Committee gave ethical approval for this research (REF 18/SC/0410). Ethics Committee London - Camden & Kings Cross Research Ethics Committee gave ethical approval for this research (REC16/LO/1920) Ethics committee of Canton of Zurich gave ethical approval for this work (2018-02198).

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The RNA sequencing data that support the findings of this study are openly available in GEO following publication in a peer reviewed journal (series number GSE250152). Data on clinical phenotypes are available from the corresponding author upon reasonable request.