DRG contains two molecularly distinct macrophage populations. (A) scRNA-seq analysis of 2668 CD45⁺ DRG cells. n = 3 mice. (B) Differential gene expression between CD163⁻ and CD163⁺ macrophages using Venice algorithm. (C) UMAP of monocyte/macrophage/DC clusters and their expression of key transcripts. (D) Validation of Fcrls and Ccr2 expression in separate subsets of Cx3cr1⁺ macrophages using RNAscope in DRG sections. Purple arrows indicate Fcrls⁺Ccr2⁻ cells and turquoise arrows indicate Fcrls⁻Ccr2⁺ cells. Images are representative of n = 3 mice. Scale bar, 50 and 5 μm (inset). (E) UMAP of live, CD45⁺ DRG cells analyzed by flow cytometry and expression heatmap of selected markers in all myeloid populations. n = 4 mice pooled. The experiment was performed three times. (F) Subclustering of CD64⁺CX3CR1⁺ macrophages from flow cytometry data. Histograms of key markers in resulting CD163⁻ and CD163⁺ macrophage clusters. (G and H) (G) Representative immunostaining and (H) quantification of Iba1⁺CD163⁻ and Iba1⁺CD163⁺ macrophages in DRG parenchyma. n = 12 mice. Scale bar, 100 μm. (I) Center-of-mass distance to nearest CD31⁺ blood vessel for CD64⁺CD163⁻ and CD64⁺CD163⁺ macrophages. Values are individual macrophages from n = 3 mice. Mann Whitney test. ***P < 0.001. (J) Immunostaining of CCR2 and MRC1 in DRG sections, displaying non-overlapping expression in CD163⁻ and CD163⁺ macrophages. Scale bar, 50 μm.