Figure 2.

MCP1 positive macrophages are reduced in aging, and MCP1 is a potent promoter of axonal outgrowth. (A) Selection of ligands expressed in nerves before and after the injury as per microarray dataset (12). Note the decrease in aged nerves of some (Il6, Ccl2) but not other (Igf-1, Lif) cytokines and growth factors known to be expressed by macrophages (19). *p < 0.03 for the Il6 graph (vs. PBS), *p < 0.05; ***p < 0.0001 for the MCP1 graph (vs. PBS). (B,C) Representative IHC images (B) and supporting quantification (C) demonstrating that neurite outgrowth was increased in dorsal root ganglion (DRG) neurons cultured in vitro with MCP1 vs. PBS, IL1β, and IL6. IL6 showed significant improvement in the mean outgrowth per neuron compared to PBS (C). The percent of neurons with outgrowth, mean outgrowth per neuron, and branches per neuron were all shown to be enhanced with MCP1. **p < 0.007, **p < 0.002 and ***p < 0.0001 (vs. PBS) for graphs 1–3, respectively. Scale bar, 200 μm. (D,E) Representative IHC images (D) and quantification (E) demonstrated that MCP1 is primarily expressed by macrophages (Ccr2, green) in the injured nerve, and not Schwann cells (Sox2-GFP, green), axons (NF, green) and other nucleated cells. Scale bar, 5 μm. (F) Experimental design for macrophage RNAseq analysis. Macrophages were FACS collected from young and aged Cx3cr1^GFP/Ccr2 ^RFP mice 3 days after injury. Cells were lysed and sequenced as per the methods section. (G) Macrophage RNAseq analysis revealed no significant difference between the Mcp1 FPKM values in young vs. aged groups. FPKM, Fragments Per Kilobase of transcript per Million mapped reads. ns = no significance. (H,I) Representative IHC images (H) and supporting quantification (I) demonstrated less MCP1+ macrophages per field of view (FOV) between aged and young injured nerves. **p < 0.005. Scale bar, 10 μm. All error bars indicate ± SEM.