Article Figures & Data
Figures
- Figure 1.
Functional analysis of Sema3A/Ad. U373 cells were transfected with 30 pfu/cell GFP control virus or virus encoding Sema3A. GFP/Ad-transfected U373 cell aggregates shows a contact-mediated inhibition on axonal growth from E10 chicken DRG in medium containing NGF (A), whereas Sema3A/Ad-transfected aggregates display a much stronger chemorepulsive effect on axonal growth (B). Schematic representation of the method used to quantify the total circumferential degrees of DRG axonal growth in relationship to transfected cells (C). D, Axon growth angle of DRG cultured with aggregates secreting Sema3A is significantly smaller (92.5 ± 38; n = 16) compared with GFP control aggregates (222.5 ± 17; n = 6). Values represent mean ± SD of at least three independent experiments. ***p < 0.001, analyzed by unpaired t test. Scale bar, 100 μm.
- Figure 2.
NGF regulates Sema3A chemorepulsion. U373 cells were cotransfected with Sema3A/Ad (30 pfu/cell) alone or together with NGF/Ad at 1:0.04 (1.2 pfu/cell), 1:0.1 (3 pfu/cell), 1:0.2 (6 pfu/cell), and 1:1 (30 pfu/cell) ratios. Western blots of conditioned medium show consistent expression of Sema3A and a dose-dependent expression of NGF (A). C, Example showing slightly stronger than average chemorepulsion mediated by explant transduced with Sema3A/Ad alone. D–G, NGF secreted from aggregates reduced Sema3A repulsion on DRG outgrowth. Cotransfection with equal concentration of NGF/Ad and Sema3A/Ad completely abolished the repulsive effect of Sema3A (G). H, Very little to no chemorepulsion when DRG are grown in the presence of NGF-expressing explants. B, Equal titers of NGF/Ad significantly reverse Sema3A-induced decrease in growth angle (chemorepulsion) compared with the other treatment groups [Sema3A (92.5 ± 38; n = 16); Sema3A/NGF: 1:0.04 (90 ± 21; n = 13), 1:01 (80±10; n = 4); 1:0.2 (122±31; n = 13), and 1:1 (225±115; n = 8); NGF (344±42; n = 7)]. ***p < 0.001, compared with explants transduced with GFP/Ad, NGF/Ad, or Sema3A/NGF (1:1), as determined by a one-way ANOVA, followed by a Tukey post hoc test. Scale bar, 200 μm.
- Figure 3.
Overexpression of NGF induces robust sprouting of CGRP-positive axons without altering other sensory axons. Fourteen days after NGF/Ad treatment, the ipsilateral CTB staining pattern (A–C, brown axons) for myelinated axons is similar to the contralateral (control side) staining pattern (A). The density of CGRP-positive axons (blue) increased dramatically on the NGF/Ad-treated side and extended throughout that spinal cord hemisection. Higher magnifications (B, C) reveal very little overlap in staining between the CTB- and CGRP-labeled axons. D, Immunofluorescent staining of CTB (green) also shows that very few, if any, of these axons colabel for CGRP (red). Overexpression of NGF induced extensive sprouting of CGRP-positive axons into aberrant spinal cord regions, throughout laminas IV–VI (arrows) and around blood vessels (E, F, arrowheads). Nociceptive axons that stain positive for IB4 (green) show no observables prouting after injection of NGF/Ad into the dorsal spinal cord (F). Scale bars: A, 300 μm; B, D, F, 100 μm; C, E, 50 μm.
- Figure 4.
Sema3A inhibits NGF-mediated sprouting of CGRP-positive sensory afferents in adult rat spinal cord. Western blot using 9E10 α-Myc monoclonal antibody and NGF ELISA verified Sema3A expression and dose-dependent NGF expression 10 d after injection of Sema3A/Ad (1.25 × 106 pfu/μl), together with NGF/Ad at different ratios (A, B). Coinjection of GFP/Ad (1.25 × 106 pfu/μl) and NGF/Ad at either a 1:0.2 or 1:0.04 ratio induced robust sprouting within the ipsilateral, but not contralateral, spinal cord (D, F). Coinjection using Sema3A/Ad instead of GFP/Ad shows inhibition of sprouting, which is much greater when combined with lower titers of NGF/Ad (G) than with higher titers of NGF/Ad (E). Insets, Higher magnification shows aberrant axon sprouting within this region and along blood vessels (arrows). C, Are a of CGRP sprouting in the right dorsal quadrant of spinal cords is significantly reduced by Sema3A in the presence of lower concentrations of NGF. The means ± SD for the control and experimental groups at the ratio of 1:0.2 for GFP and NGF and Sema3A and NGF are 52.1 ± 12.6 and 32.8 ± 17.9, respectively, with p = 0.087. Those at the ratio of 1:0.04 for GFP and NGF and Sema3A and NGF are 58.9 ± 25.7 and 21.2 ± 15.5, respectively, with p = 0.002 as determined by a one-way ANOVA, followed by a Tukey post hoc test. Values were calculated from six rats for each treatment. ** p < 0.01. Scale bar, 100 μm.
- Figure 5.
Sema3A inhibits sprouting of SP-containing nociceptive afferents. Two weeks after coinjection of GFP/Ad with NGF/Ad (1:0.04 ratio), robust sprouting of SP-positive fibers within the dorsal spinal cord was apparent (A, B). Coinjection of Sema3A instead of GFP/Ad eliminated aberrant sprouting of SP-positive fibers(C, D). Quantification of SP-containing fiber in the right dorsal horn revealed a significant reduction of fiber growth by Sema3A expression (E). Values represent mean ± SD of six rats for each group. **p < 0.01, analyzed by Student's t test. Scale bar, 100 μm.
- Figure 6.
Changes in nociceptive response after treatment. Nociceptive responses were evaluated by measuring the PWL to thermal stimuli (A,B) or the 50% paw withdrawal threshold to mechanical stimuli (C, D). Measurements were taken from the left (contralateral control) hindpaw (A, C) and the right (experimental) paw (B, D) at –3, 7, and 14 d after coinjection of GFP/Ad or Sema3A/Ad (1.25 × 106 pfu/μl) with NGF/Ad at 1:0.04 ratio. The contralateral hind paw demonstrated no thermal hyperalgesia (A) or mechanical allodynia (C) in either treatment group. Sema3A/Ad treatment significantly reversed NGF-mediated reduction of the 50% withdrawal threshold 14 d after treatment (D) but not the latency of paw withdrawal. Values represent mean ± SD of six rats. **p < 0.01, analyzed by a nonparametric Mann–Whitney U test.
