Article Figures & Data
Figures
- Figure 1.
Anatomical evidence that the L5 spinal nerve regenerates in the SNL model. a, Schematic of the SNL model showing the site of L5 spinal nerve ligation and transection distal to L5 DRG and proximal to its merger with the L4 spinal nerve to form the sciatic nerve. The dotted line indicates the regenerated nerve segment. In separate experiments, the tracer dextran biotin (injected into the cut end of the spinal nerve) or DiI (injected into the paw) was injected just after the spinal nerve transection. b, c, In situ dissecting microscope images showing the regenerated segment of the L5 spinal nerve (arrows) just distal to the injury sites observed on day 20 (b) and day 70 (c). d, e, Whole-mount images of the L5 DRG showing DiI transported from the hindpaw (red) on day 20 (d; observed in only one of four rats) and day 70 (e; observed in six of six rats). f, g, DRG sections showing colocalization (yellow; arrows) of DiI (red) with labeling for NF200 (f; green; marker for myelinated cells; repeated in five rats) or Substance P (SP; g; green, marker for nociceptive cells; repeated in five rats). Sections were obtained 70 d after spinal nerve ligation and transection. DiI could still be observed at the paw injection site at this time. h, Longitudinal section of sciatic nerve distal to the original transection site, just below the knee, showing dextran biotin-positive fibers 70 d after spinal nerve transection (repeated in four rats). The regenerating, dextran biotin-positive nerve (“reg N.”) appeared somewhat segregated from the dextran biotin-negative original intact nerve (“orig N.) i, Cross-section of paw skin, showing dextran biotin-positive fibers 35 d after spinal nerve transection (repeated in five rats). Scale bars: d, e, 250 µm; f–i, 100 µm.
- Figure 2.
Regeneration of L5 spinal nerve is functional and can be reduced by semaphorin 3A. a, Schematic of the in vivo fiber recording setup. Filaments were teased out of the dorsal root, disconnected centrally. After observing spontaneous activity, receptive fields were examined by applying mechanical stimuli to proximal (above the knee) and distal (lower leg and paw) regions. b, c, Average number of proximal (b) and distal (c) receptive fields per filament detected at the indicated times after SNL, SNL plus perfusion of the injury site with vehicle, or SNL plus perfusion of the injury site with semaphorin 3A (“Sema3A”). In c, values for semaphorin 3A group are zero. d, Average number of spontaneously active fibers per filament (excluding normal muscle spindle and joint receptor activity) detected in the same experiments. N = 3 or 4 rats/group with 72 to 116 filaments per group, except for the resected POD14 group, which contained only two animals. e, Example of spontaneous activity, and activity evoked by mechanical stimulation (Stim) of a receptive field (RF; arrows). f, In situ image of regenerated nerve on day 28 in animal perfused with semaphorin 3A (tubing removed before image was taken); note that the segment is thin and fine (compare Fig. 1b; observation was repeated in eight rats). g, Paw cross-section showing dextran biotin-labeled fibers (red) observed 28 d after SNL in a vehicle-perfused rat (repeated in four rats). h, Dextran biotin-labeled fibers were absent in paw cross-sections obtained from semaphorin 3A-perfused rat (repeated in four rats). Scale bar, 100 µm.
- Figure 3.
Local perfusion of the injury site in vivo with semaphorin 3A reduces pain behaviors in the SNL model. Baseline behaviors (average of two measurements) are plotted on day 0. Immediately after the ligation and transection of the L5 spinal nerve, fine tubing was led from a 14 d osmotic pump to the cut end of the spinal nerve to deliver either vehicle or semaphorin 3A (16 µg/ml), as indicated by the shaded bar. a, Threshold for withdrawal responses to von Frey filaments (F(1,10) = 137.58). b, Dynamic allodynia (withdrawal responses to light stroking with a fine cotton wisp; F(1,10) = 91.08). c, Cold allodynia (withdrawal responses to a drop of acetone; F(1,10) = 13.77). *p < 0.05; **p < 0.01; ***p < 0.001, significant difference between the two groups at the indicated time points, two-way repeated-measures ANOVA, with Bonferroni’s post-test). n = 6 male rats/group. PWT, paw withdrawal threshold.
- Figure 4.
Knockdown of GAP43 in the L5 dorsal root ganglion reduces pain behaviors and GAP43 upregulation in the SNL model. siRNA directed against GAP43 or control nontargeting siRNA was injected into the L5 DRG immediately prior to implementing the SNL model. a–f, Sections were obtained 4 d later. DRG sections from cellular regions (a–c) or axonal regions (d–f) stained for GAP43 (red) and neuronal marker NeuN (green) from normal (a, d), SNL + control siRNA-injected (b, e) and SNL + GAP43-siRNA injected (c, f) DRGs. g, h, Summary data of GAP43 intensity from cellular (g) and axonal (h) regions. ***p < 0.001, significant differences between control siRNA groups and both other groups; ANOVA with Dunnett’s post-test. g, F(2,12) =107.70; h, F(2,12) = 62.77; n = 5 rats/group (3 males, 2 females), with 19–33 soma region sections per rat and 6–12 axonal regions per rat. Effect of siRNA injection on pain behaviors: i, Threshold for withdrawal responses to von Frey filaments (dotted line indicates baseline; F(1,10) = 119.37). j, Cold allodynia (withdrawal responses to a drop of acetone; F(1,10) = 10.77). k, Dynamic allodynia (withdrawal responses to light stroking with a fine cotton wisp; F(1,10) = 124.59). l, Guarding behavior score (maximum value is 3; F(1,10) = 4.94). *p < 0.05; **p < 0.01; ***p < 0.001, significant difference between the two groups at the indicated time points (two-way repeated measures ANOVA with Bonferroni’s post-test). N = 6 rats/group (3 males, 3 female). PWT, paw withdrawal threshold.
- Figure 5.
Semaphorin 3A applied to the injury site reduces regeneration and pain behavior in the SNI model. a1–b, Longitudinal sections of the neuroma region at the spared nerve injury site taken 28 d after nerve injury, stained for regeneration marker GAP43 (red). a1–b, The injury site was locally perfused with vehicle (a1, a2) or semaphorin 3A (16 µg/ml; b) for the first 14 d after the injury. a1, a2, The same nerve, taken at different depths. The suture can be seen as the two dark regions in a1 and the larger dark region in a2 and b. c, d, Grayscale rendering of portion of the images in a2 and b with sharpness enhancement to emphasize fiber structure (and to further increase the signal from b). Scale bar, 500 µm. e–h, Effect of perfusing the injury site in the spared nerve injury model with vehicle or semaphorin 3A for the first 14 d after injury on pain behaviors. e, Threshold for withdrawal responses to von Frey filaments (F(1,10) = 120.19). f, Guarding behavior score (maximum value is 3; F(1,10) = 31.33). g, Dynamic allodynia (withdrawal responses to light stroking with a fine cotton wisp; F(1,10) = 70.30). h, Cold allodynia (withdrawal responses to a drop of acetone; F(1,10) = 47.98). *p < 0.05; **p < 0.01; ***p < 0.001, significant difference between the two groups at the indicated time points (two-way repeated-measures ANOVA with Bonferroni’s post-test). N = 6 rats/group (4 males, 2 female). PWT, paw withdrawal threshold.
- Figure 6.
Applying semaphorin 3A to the injury site reduces established pain in the spared nerve injury model. Averaged baseline behaviors are plotted on day 0. Starting on day 13, an osmotic pump added during a second surgery was used to locally perfuse the injury site with semaphorin 3A or vehicle (ACSF) for 14 d, as indicated by the shaded bar. Behaviors were measured 1 d prior to and 2 d after the pump surgery in order to demonstrate that the behavioral effects were significant within 2 d. a, Static allodynia (threshold for withdrawal responses to von Frey filaments; dotted line indicates baseline; F(1,9) = 104.40). b, Dynamic allodynia (withdrawal responses to light stroking with a fine cotton wisp; F(1,9) = 103.40). c, Guarding score (maximum possible value is 3; F(1,9) = 42.49). d, Cold allodynia (withdrawal responses to a drop of acetone; F(1,9) = 33.03). *p < 0.05; **p < 0.01; ***p < 0.001, significant difference between the two groups at the indicated time points. N = 4 male and 4 female rats (vehicle group); N = 3 male and 4 female rats (semaphorin 3A group). PWT, paw withdrawal threshold.
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