Fig. 9

Clodronate liposomes injected into the CL site of Ccr2^gfp KO mice significantly reduces macrophages but does not prevent the peripheral CL response. A. Diagram depicting a CL paradigm performed on Ccr2^gfp KO animals in which CL macrophages were targeted for ablation by injecting clodronate liposomes into the proximal CL site on the day of the CL (day 0) the next day (day 1) and every other day thereafter. Control animals received vehicle liposome injections instead. B-E. Representative images of regenerating nerves immunostained for regenerating axons with SCG10. Unconditioned regeneration (B, D) was the same for both treatment groups. Conditioned regeneration (C, E) was also the same between treatments and significantly increased compared to contralateral uninjected nerves. The dotted line indicates the center of the crush site which was considered to be 500 µm wide, and the solid line is 3000 µm from the crush. Scale bar is 500 µm. F. Axon regeneration quantified at 100 µm intervals as the fraction of regenerating axons relative to the crush site. G. Mean regeneration distance calculated by integrating SCG10 immunofluorescent staining of regenerating axons. H. Macrophages quantified by percent CD68 positive area in a circle placed on the CL with a diameter equal to the largest width of the uninjured portion of the nerve. Forty-micron sections were used in B-H. I-J. Representative images of a PBS (vehicle) liposome-treated (I) and clodronate liposome-treated (J) Ccr2^gfp KO CL site immunostained for macrophages with F4/80, proregenerative macrophages with Arg1, recruited CCR2⁺ macrophages with GFP, and DAPI. F4/80 is a membrane marker, shown in green, while Arg1, shown in red, and GFP, shown in cyan, are both intracellular. These colors cause proregenerative F4/80⁺, Arg1⁺ macrophages to appear yellow or red ringed by yellow, recruited proregenerative GFP⁺, Arg1⁺ monocyte/macrophages to appear cyan and white, and triple positive cells to appear white surrounded by yellow-green. I’-J’’’’. Individual channel images from I and J. Percent area stained was quantified by outlining the tissue of the entire CL site. K. GFP staining was significantly reduced within the CL site of clodronate liposome treated Ccr2^gfp KO mice compared to vehicle treated mice. L. F4/80 macrophage staining was also significantly reduced in clodronate liposome versus PBS liposome treated mice. M. Despite the significant reduction in macrophages in the CL site of clodronate liposome treated mice, DAPI staining was not altered in the CL site. Scale bar is 500 μm. N = 18–20 per group for B-H and N = 5 per group for I-M. ** p < 0.01; *** p < 0.001. # p < 0.05 between injury conditions within the same treatment group