Fig. 8. SNX25⁺ macrophages in the DRG did not contribute to pain sensitivity.

a, Confocal microscopy of the DRG (L4, naive) immunolabeled for PGP9.5 (green) and MHC-II (red) in WT and Snx25^+/− mice. Right, magnified views of boxed areas in the middle panels. Scale bar, 100 μm. b, Confocal microscopic images of the DRG (L4, naive) immunolabeled for macrophage markers (CD206 or F4/80, red) and SNX25 (red) in WT mice. Scale bar, 50 μm. The graph shows the proportion of SNX25⁺ cells in each specific marker-positive cell population. The numbers in the columns indicate actual cell numbers counted. n = 11 sections from three different mice. c, Confocal microscopy of a DRG (L4) section stained for GFP (green) and fluorescent Nissl (red) in WT mice that received BMT from GFP mice. Arrowheads denote BM-derived GFP⁺ cells. Scale bar, 100 μm. d, Confocal microscopy of the DRG (L4) double labeled for GFP and MHC-II in the mice shown in c. Arrowheads denote double-labeled cells. Scale bar, 100 μm. e, Percentages of MHC-II⁺ cells among GFP⁺ cells and of GFP⁺ cells among MHC-II⁺ cells (5 weeks after transplantation) (n = 5 DRG sections from three different mice). f, Confocal microscopy of the sciatic nerve double labeled for GFP and CGRP in the mice shown in c. Arrowheads denote BM-derived GFP⁺ cells. Scale bar, 100 μm. g, Confocal microscopy of the sciatic nerve double labeled for GFP and MHC-II in the mice shown in c. Arrowhead denote a GFP⁺MHC-II⁺ cell. Scale bar, 100 μm. h, Confocal microscopy of the DRG (L4) labeled for fluorescent Nissl (red) and immunolabeled for YFP (green) after injection of 4-OHT directly into DRGs in Snx25^Cx3cr1-cKO;Ai32^Tg/+ mice (left) and Snx25^fl/fl;Ai32^Tg/+ mice (right). Scale bar, 100 μm. Bottom, confocal microscopy of the DRG (L4) immunolabeled for MHC-II (red) and YFP (green) after injection of 4-OHT directly into DRGs. Arrowheads indicate cells double labeled for MHC-II and YFP and counterstained with DAPI (blue). Scale bar, 100 μm. i, Confocal microscopy of the DRG immunolabeled for YFP, CD206 and SNX25 in Snx25^fl/fl;Ai32^Tg/+ mice and Snx25^Cx3cr1-cKO;Ai32^Tg/+ mice. Arrowheads denote CD206⁺SNX25⁺ macrophages. Scale bar, 50 μm. The graph shows the fluorescence intensities of SNX25 in CD206⁺ macrophages in Snx25^Cx3cr1-cKO;Ai32^Tg/+ mice (n = 7 sections from three different mice) and Snx25^fl/fl;Ai32^Tg/+ mice (n = 6 sections from three different mice). P = 0.001. j, VF thresholds before (n = 6) and after (n = 6) 4-OHT injection in Snx25^Cx3cr1-cKO;Ai32^Tg/+ mice (P = 0.382), Snx25^fl/fl;Ai32^Tg/+ (n = 7) and Snx25^Cx3cr1-cKO;Ai32^Tg/+ (n = 6) mice (P = 0.348) and in contralateral (n = 6) and ipsilateral (n = 6) sides of Snx25^Cx3cr1-cKO;Ai32^Tg/+ mice (P = 0.218). g, gram. k, Confocal microscopy of the hind paw skin, the DRG and the spinal cord immunolabeled for NGF (green) and F4/80 (red) in WT mice. Arrowheads denote double-positive cells and arrows denote NGF⁻F4/80⁺ cells. Scale bar, 50 μm. l, Proportion of NGF⁺ cells (black column) in F4/80⁺ cells. Numbers inside columns are the actual numbers of cells counted. n = 6 sections from three different mice. Results are represented as mean ± s.e.m. Statistical analyses were performed using the two-tailed Student’s t-test. Representative of three independent experiments (a–d,f–i,k).

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