Spatially resolved single-cell analysis of transcriptomic changes linked with neuropathic pain in human neuromas
Transcriptomics of rare human neuromas reveals a novel role for endothelial cell dysfunction and HLA-A in neuropathic pain.
Approximately 300,000 peripheral nerve injuries arise each year in Europe.15 In addition to loss of motor and sensory function, a proportion of patients have persistent pain for which there is no reliable treatment.15 This is associated with sleep disturbances, depression, and various debilitating psychological problems.81 Injuries to branches of the trigeminal nerve, most commonly the lingual and inferior alveolar nerves, can occur as a result of routine dental procedures.49 Although peripheral nerves regenerate spontaneously, in some patients, the presence of a gap between the proximal and distal ends, bone fragments, scar tissue, and inflammation may prevent functional reinnervation of the target areas, resulting in the formation of a swollen mass termed neuroma.107 The frequency of lingual nerve injuries during oral and maxillofacial procedures varies between 0.6% and 2%, resulting in anesthesia, paresthesia, or hyperesthesia of the floor of the mouth, the lingual gingiva, and the anterior part of the tongue, affecting everyday activities such as speaking, eating, and drinking and potentially leading to altered taste.5,75,78 In addition, some patients report the presence of neuropathic pain, often described as a burning sensation.5 To treat these symptoms, patients can undergo nerve repair surgery where the neuroma is resected and the nerve ends are surgically reconnected, promoting functional recovery of sensation.5
Human lingual neuromas represent a unique resource to investigate mechanisms linked with neuropathic pain. The comparison between samples from patients who have incurred the same type of injury, where some, but not all, report symptoms of pain, enables the identification of factors specifically linked with neuropathic pain, which are independent from the pathophysiological changes associated with nerve injury and regeneration. Previous work on human neuromas highlighted changes in the molecular expression of selected targets including ion channels, MAP kinases, and inflammatory mediators.9,11 High-throughput bulk transcriptomics has been used to identify overall differences in nontrigeminal neuromas compared to healthy uninjured controls.83 However, detailed characterization of the cellular composition and the transcriptional changes linked with the severity of pain at single cell level in human neuromas has never been performed.
In this work, we sought to characterize the cellular and transcriptional composition of healthy and injured trigeminal nerves and identify molecular changes in human trigeminal neuromas linked with the presence of pain. Single nuclei RNA sequencing (snRNA-seq) was used on samples of healthy and injured human trigeminal nerves to characterize the cellular composition at single-cell resolution. Spatial transcriptomics was performed on a larger pool of human lingual neuromas, including both painful and nonpainful samples, to characterize the transcriptional landscape within the morphological context and identify changes in gene expression localized within the nerve fascicles. Cell–cell interactome analysis was performed to identify changes in signaling networks linked with the presence of pain. RNAscope and immunohistochemistry were used to validate the findings.