Single-cell RNA sequencing of the post–spinal cord injury dorsal root ganglia in cynomolgus monkeys: elucidation of the cellular immune microenvironment of the central nervous system

Abstract Few studies have investigated alterations in the immune cell microenvironment of the dorsal root ganglia following spinal cord injury and whether these modifications facilitate axonal regeneration. In this study, we used a single-cell RNA sequencing dataset to create a comprehensive profile of the diverse cell types in the dorsal root ganglia and spinal cord of a mid-thoracic contusion injury model in cynomolgus monkeys. Cell communication analysis indicated that specific signaling events among various dorsal root ganglia cell types occur in response to spinal cord injury. Single-cell analysis using dimensionality reduction clustering identified distinct molecular signatures for nine cell types, including macrophage subpopulations, and differential gene expression profiles between dorsal root ganglia cells and spinal cord cells following spinal cord injury. The macrophage subpopulations were categorized into 11 clusters (MC0–MC10) based on differentially expressed genes, with the top 10 genes being ABCA6, RBMS3, EBF1, LAMA4, ANTXR2, LAMA2, SOX5, FOXP2, GHR , and APOD . MC0, MC1, and MC2 constituted the predominant macrophage populations. MC4, MC6, and MC9 were nearly absent in the spinal cord, but exhibited significant increases in the dorsal root ganglia post–spinal cord injury. Notably, these subpopulations possess a strong capacity for regulating axonal regeneration. The developmental progression of dorsal root ganglia macrophages after spinal cord injury was elucidated using cell trajectory and pseudo-time analyses. Genes such as EBF1 (MC6 and MC9 marker), RBMS3 (MC6 and MC9 marker), and ABCA6 (MC6 marker) showed high expression levels in the critical pathways of macrophage function. Through ligand–receptor pair analysis, we determined that the effects of macrophages on microglia are predominantly mediated through interaction pairs (e.g., SPP1-CD44, LAMC1-CD44, and FN1-CD44), potentially facilitating specific cellular communications within the immune microenvironment. The single-cell RNA sequencing dataset used in this study represents the first comprehensive transcriptional analysis of the dorsal root ganglia after spinal cord injury in cynomolgus monkeys, encompassing nearly all cell types within the dorsal root ganglia region. Using this dataset, we evaluated diverse subtypes of macrophages in the post- spinal cord injury dorsal root ganglia area and examined the signaling pathways that facilitate interactions among immune responserelated macrophages in the dorsal root ganglia. Findings from this study provide a theoretical basis for understanding how the immune microenvironment influences the regenerative capacity of dorsal root ganglia neurons after spinal cord injury and offer novel insights into the complex processes underlying the pathobiology of spinal cord injury.