Fig 2: ACG-RGI/KLT promoted angiogenesis and regeneration of the sciatic nerve. (A) NF200 and CD31 immunofluorescence staining of regenerated nerves at 3 weeks after surgery. (B) WB of GAP43, CD31, and VEGF in regenerated nerves at 3 weeks after surgery. (C-E) Statistical analyses of GAP43, CD31, and VEGF, respectively. Data are presented as the mean ± standard error of the mean, n = 3 for each group. * p<0.05, ** p<0.01.

Fig 3: Immunofluorescence view of transient receptor potential mellastatin type 8 (TRPM8) expression after resiniferatoxin (RTX) pretreatment. Perineural administration with vehicle or RTX (0 or 1 µg) was performed before spinal nerve ligation (SNL). To evaluate the protein expression level of TRPM8 located in the DRG, the ipsilateral (left) L5 and L6 DRG were isolated 4 weeks after SNL and confocal immunofluorescence analysis was performed. Mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; blue) counterstain was dispersed over the sections before mounting. a TRPM8 expression (red; arrows) in the DRG of sham-operated control group rats. b TRPM8 expression (red; arrows) in the DRG of RTX 0 µg group rats. TRPM8 expression in the DRG was up-regulated and the co-localization of TRPM8 expressing neurons with NF200-positive neurons (green) was increased (arrow heads). c TRPM8 expression (red; arrows) in the DRG of RTX 1 µg group rats. TRPM8 expression was suppressed compared to that of the RTX 0 µg group

Fig 4: TrkB-positive sensory neurons are putative mechanoreceptors. a–d Double immunofluorescence of DRG sections from TrkBCreERT2::Rosa26RFP mice with a NF200, b Ret, visualized using TrkBCreERT2::Rosa26RFP::RetEGFP triple transgenic mice, c IB4, d quantification of staining on mouse DRG sections; TrkB+ cells account for ~10% of all DRG neurons and all co-express NF200 or NF200 + ReteGFP, while they are negative for IB4, CGRP, and TH. e–h Double immunofluorescence of human DRG sections stained with antibodies against TrkB and e NF200, f Ret, g TrkA. h Size distribution for human DRG neurons expressing TrkB, NF200, and TrkA. i Section through the lumbar spinal cord of TrkBCreERT2::AvilmCherry mice stained with IB4. j TrkB+ lanceolate endings in a section of the back hairy skin of TrkBCreERT2::Rosa26SnapCaaX labeled with Snap Cell TMRstar (red), NF200 (green), and DAPI (blue). k Section from the glabrous skin of TrkBCreERT2::Rosa26ChR2YFP (red) stained with anti-S100 a marker for Meissner’s corpuscles (green) and DAPI (blue) showing TrkB+ innervation. l Section from human glabrous skin stained with antibodies against TrkB (red) and NF200 (green), and DAPI (blue). Scale bars, a–c and i 50 µm, e–g and j–l 40 µm.

Fig 5: BDNFSNAP-IR700 photoablation promotes local retraction of TrkB+ afferents. a–c Substantial loss of TrkBCreERT2 positive afferents (red), but persistence of other fibers (green) upon BDNFSNAP-IR700-mediated photoablation. a Innervation of paw hairy skin prior to ablation, arrows show lanceolate endings. b Loss of TrkBCreERT2 afferents after ablation, arrows show PGP9.5 fibers. c Re-innervation of skin by TrkBCreERT2 afferents at 24 days post ablation. d DRG section from control TrkBCreERT2 mouse labeled for RFP (red) and NF200 (green). e DRG section from photoablated TrkBCreERT2 mouse labeled for RFP (red) and NF200 (green). f Quantification of the proportion of hair follicle innervation and DRG neurons positive for TrkB following photoablation in the paw and the quantification of PGP9.5+ free nerve endings showing the numbers of free nerves remain unaffected. Representative skin sections from control and BDNFSNAP-IR700 photoablated mice labeled with the indicated antibodies. TrkB-positive cells are indicated in red and DAPI-positive nuclei in blue. g, h Keratinocytes labeled with K14 (green). i Quantification of the number of K14+ cells. j, k Dendritic cells and dermal antigen-presenting cells labeled with MHC-II (green) and l the quantification for MHC-II+ cells. m, n Mast cells and epithelial and endothelial progenitor cells labeled with CD34 (green). o Quantification of the number of K14+ cells. Scale bars, 40 µm. Error bars indicate SEM