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. 2023 Apr 18;120(17):e2211631120. doi: 10.1073/pnas.2211631120

Fig. 4.

Fig. 4.

Chronic widespread pain in mice increases neutrophil trafficking into sensory ganglia. (A) Immunohistochemical staining for S100A9 in L4 DRG sections from control and primed mice (i) demonstrating increased numbers of neutrophils in DRG of primed mice (sections from n = 3 to 4 mice per group; ***P < 0.001 one-way ANOVA). Yellow line in representative L4 DRG images (ii) indicates 100 µm. (B) FACS sorted number of neutrophils (Ly6G/Ly6C double-positive cells) derived from DRG in control (n = 5) vs. primed (n = 6) mice (*P < 0.05 unpaired t test). (C) tSNE analysis of Ly6C/Ly6G double-positive neutrophils obtained from blood of control (n = 6) mice and primed (n = 8) mice. Neutrophil subpopulations labeled for CXCR2, CD62L, CD11b, Ly6G, and Ly6c are illustrated with colors in the expression level heatmap representing median intensity values for relative clustering based on cell density plots. (D) Differential expression of CXCR2, CD62L, CD11b, Ly6G, and Ly6C based on cluster 3 and cluster 5, with the most decreased (−55%) and increased (+101%) numbers of neutrophil subpopulations in primed cells, respectively. (E) tSNE analysis of Ly6C/Ly6G double-positive neutrophils obtained from DRG of control mice (n = 5) and primed (n = 6) mice, with CXCR2, CD62L, CD11b, Ly6G, and Ly6c markers. (F) Differential expression of CXCR2, CD62L, CD11b, Ly6G, and Ly6C based on clusters 3 and 5, with the most decreased (−60%) and increased (+178%) numbers of neutrophil subpopulations in primed cells, respectively (G and H) FACS quantification of CXCR2-positive and CD62L-positive neutrophils (double positive for Ly6G and Ly6C) in (G) blood vs. (H) DRG (*P < 0.05, **P < 0.01 unpaired t test).