Exploring Cellular Mechanisms of Inflammatory Temporomandibular Joint Pain by Single-Cell Multiomic Sequencing

Temporomandibular Disorders (TMDs) stand as a primary cause of non-dental orofacial pain. However, the etiology and pathophysiology of TMD-like pain remain poorly understood. Orofacial pain including TMD-like pain is intricately linked to the spinal trigeminal nucleus, specifically the caudalis subnucleus of the spinal trigeminal nucleus (Sp5C). In this study, we aimed to identify cell-specific gene targets in the Sp5C for TMD-like pain using single-cell multiomic sequencing. Complete Freund's Adjuvant (CFA) was injected into mouse temporomandibular joint (TMJ) to induce inflammatory TMD-like pain. By conducting single-nucleus RNA sequencing (snRNA-seq) and single-nucleus Assay for Transposase-Accessible Chromatin sequencing (snATAC-seq) of Sp5C tissues, we examined altered cell-specific gene expression and gene activity after CFA treatment, respectively. Totally 18 cell types in the Sp5C tissues were indicated via UMAP clustering, in which microglia and serotonergic neurons exhibited the most significant alterations by CFA-induced TMJ pain. The snRNA-seq showed 202 up-regulated genes and 117 down-regulated genes in microglia (mt-Co1, Cd52 and Apoe: top 3 genes), and 238 up-regulated genes and 90 down-regulated genes in serotonergic neurons (Sgk1, Adipor2 and Rcan2: top 3 genes). The snATAC-seq showed 22 genes with increased activity and 34 genes with decreased activity in microglia (Xylt1, Grk5 and Syne3: top 3 genes), and 32 genes with decreased activity in serotonergic neurons (Zbtb16, Gm28035 and Phyhd1: top 3 genes). Together, this study identified potential gene targets that can be further investigated to determine cell-specific mechanisms for TMD-like pain. Funding: R01DE031255 (F.T.) and R01DE032061 (F.T.).