Figure 1.
Partial Sciatic Nerve Ligation (PSNL) produces hind paw mechanical hypersensitivity in rats.
Withdrawal thresholds of the ipsilateral hind paw following PSNL were assessed over time with von Frey filaments in sham and PSNL rats for the periods indicated. Data are expressed as mean ± SEM. n = 5/group. ** p<0.01 compared with pre-surgery (one-way ANOVA followed by Tukey-Kramer post hoc test). †† p<0.01 compared with sham group (Student’s t-test).
Figure 2.
Antinociceptive effect of anti-HMGB1 monoclonal antibody on PSNL-induced mechanical hypersensitivity in rats.
Effects of intravenous injection of either anti-HMGB1 monoclonal antibody (anti-HMGB1, 2 mg/kg) or control IgG (2 mg/kg) on nociceptive behaviors over time in rats with either sham or PSNL surgery (A, E: 3 days, B, F: 7 days, C, G: 14 days, D, H: 21 days). A single dose of anti-HMGB1 monoclonal antibody increased withdrawal thresholds in rats with a PSNL, but not sham, surgery. Data are expressed as mean ± SEM. n = 5/group. * p<0.05, ** p<0.01 compared with sham group (Student’s t-test). † p<0.05 compared with pre-operation (one-way ANOVA followed by Tukey-Kramer post hoc test).
Figure 3.
Expression level of HMGB1 was increased in rat lumbar spinal cord dorsal horn after PSNL.
A. The expression of HMGB1 in the ipsilateral spinal dorsal horn of sham (post-Sham 3–21 days) and PSNL (post-PSNL 3–21 days) rats at the indicated periods were measured by Western blotting. The optic densities of HMGB1 were normalized to that of sham rats at the corresponding time point. n = 4/group. B, C. Immunolabeling of HMGB1 within spinal dorsal horn of sham and PSNL rats at 3 (B) and 21 days (C) following operation. Scale bar = 200 µm. D, E. Quantitative analysis of HMGB1 expression at 3 (D) and 21 days (E) following operation. Data indicate the relative mean immunofluorescence intensity of spinal dorsal horn. Data are expressed as mean ± SEM. n = 3/group. *, ** p<0.05, 0.01 compared with sham group (Student’s t-test).
Figure 4.
Distribution of HMGB1 expression in spinal dorsal horn neurons and glial cells.
A-F. Double-labeling immunohistochemistry for HMGB-1 and NeuN (A, B, a marker for neurons), GFAP (C, D, a marker for astrocytes) or Iba1 (E, F, a marker for microglia) in the spinal dorsal horn of sham (A, C, E) and PSNL (B, D, F) rats 21 days following operation. DAPI was used to show nuclear localization. Scale bar = 10 µm. G. Quantitative analysis of HMGB1 expression level in neurons, astrocytes and microglia at 21 days following operation. Data indicate the relative mean immunofluorescence intensity of a single cell. H. Quantitative analysis of the area of HMGB1 expression of neurons, astrocytes and microglia at 21 days following operation. Mean ratios of the area of HMGB1 immunofluorescence to the area of DAPI immunofluorescence are shown. Data are expressed as mean ± SEM. n = 6/group. * p<0.05 compared with sham group (Student’s t-test).
Figure 5.
Effect of anti-HMGB1 monoclonal antibody on activation of spinal dorsal horn glial cells after PSNL.
A, B. Immunofluorescence photomicrographs of spinal dorsal horn astrocytes (A) and microglia (B) from rats treated 2 hrs after with either anti-HMGB1 monoclonal antibody (2 mg/kg) or control IgG (2 mg/kg). Sham and PSNL rats were treated with antibody 21 days after operation. Scale bar = 200 µm. C, D. High-power fields demonstrating morphological change of astrocytes (C) and microglia (D). Scale bar = 20 µm. E, F. Levels of GFAP (E) and Iba1 (F) in the ipsilateral dorsal horn were quantified by Western blotting analysis. The panels indicate representative blots. The graph in lower panels indicates quantitative data for each blot. Protein levels were normalized to levels of β-actin. Data are expressed as mean ± SEM. n = 5/group. *, ** p<0.05, 0.01 compared with sham-control IgG group, † p<0.05 compared with PSNL-control IgG group (one-way ANOVA followed by Tukey-Kramer post hoc test).
Figure 6.
Effect of anti-HMGB1 monoclonal antibody on cFos expression in the spinal dorsal horn.
A. Immunohistochemistry of spinal dorsal horn from rats 21 days following either a sham or PSNL surgery. The expression of cFos was evaluated 2 hrs after treatment with either anti-HMGB1 monoclonal antibody (2 mg/kg) or control IgG (2 mg/kg). Scale bar = 200 µm. B. cFos in the ipsilateral dorsal horn was quantified by Western blotting. The panels above the graph indicate representative blots. The cFos data were quantified as a ratio of cFos to β-actin. Data are expressed as mean ± SEM. n = 5/group. * p<0.05 compared with sham-control IgG group, † p<0.05 compared with PSNL-control IgG group (one-way ANOVA followed by Tukey-Kramer post hoc test).