Supplementary Digital content for Xie et al., Localized sympathectomy reduces peripheral nerve regeneration and pain behaviors in two rat neuropathic pain models
Supplementary Figure 1:
Supplemental Figure 1. Validation of the Iba‐1 antibody. Sections of DRG obtained 28 days after spared nerve injury were obtained without (A) and with (B) pre‐incubation of the antibody with the immunizing peptide overnight at 4°C prior to applying it to the tissue samples. Examples of images obtained under the same settings in a side‐by‐side experiment. Scale bar 200 µm. C, D: examples of DRG sections co‐
stained with the Iba‐1 antibody (red) and an antibody for glutamine synthetase (“GS”; green), a marker for satellite glia cells, showing lack of significant co‐localization. Anti‐glutamine synthetase antibody was from Abcam (catalog ab64613, RRID:AB_1140869) at 1:100 dilution. C, normal L5 DRG; D, L5 DRG 4 days after spinal nerve ligation. Scale bar, 50 µm. E, Iba‐1 signal in normal nerve showing relative lack of signal (compare to neuroma staining in Figure 8). Scale bar, 500 µm.
Supplementary Figure 2
Supplemental Figure 2. Effect of microsympathectomy (mSYMPX) on regeneration as measured with neuronal tracers. A‐C, dextran labelling of regenerating nerve. Dextran was injected into the L5 spinal nerve at the time of ligation. Dextran signal was observed in sciatic nerve sections 3 – 5 cm distal to the injury site 28 days later. A, example of dextran labeling in nerve after spinal nerve ligation (SNL) and sham mSYMPX. B, example of labeling after SNL plus mSYMPX.
Scale bars 100 µm. C, Summary data show the average intensity/area of dextran labeling. ***, p<0.001, t‐test, significantly different from sham mSYMPX. N = 4 rats per group, 9 ‐13 sections per rat. D‐F, fast di‐I labeling of the L5 DRG. Di‐I was injected intradermally into the ipsilateral hindpaw 42 days after spinal nerve ligation and either sham mSYMPX or mSYMPX. DRGs were collected 14 days later and the di‐I signal was observed in whole mount DRG preparation. C, example of DRG after SNL with sham mSYMPX. D, example of DRG after SNL with mSYMPX.
Scale bars, 200 µm. E., summary data show di‐I signal intensity was significantly lower in the
mSYMPX group. *, p<0.05, t‐test. N = 5 rats per group (mSYMPX group) and 4 rats per group (sham mSYMPX group).
Supplemental Figure 3
Supplemental Figure 3: Effect of SNI on tyrosine hydroxylase (TH; red) and Iba‐1 (green)
immunoreactivity in DRG and neuroma. Above: sample images of normal DRG, DRG 14 days after SNI, normal nerve, and the SNI neuroma 14 days after SNI. Scale bars: 200 µm. Summary data: ***, p<0.01, significant difference between Normal and SNI groups, t‐test. For normal DRG TH intensity, signal from TH‐positive small neurons (that disappears after peripheral nerve ligation) was manually excluded from the total intensity and area measurements; this process did not change the overall conclusions.
