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. 2021 Mar 10:14:665-679.
doi: 10.2147/JPR.S293462. eCollection 2021.

S100A4 in Spinal Substantia Gelatinosa from Dorsal Root Ganglia Modulates Neuropathic Pain in a Rodent Spinal Nerve Injury Model

Xin Jiang #  1 , Wenqi Zhao #  1 , Tiantian Zhao #  2 , Mei Yang  1 , Hongbin Yuan  1 , Jun Qian  2 , Zhenghua Xiang  3
Affiliations

S100A4 in Spinal Substantia Gelatinosa from Dorsal Root Ganglia Modulates Neuropathic Pain in a Rodent Spinal Nerve Injury Model

Xin Jiang et al. J Pain Res. .

Abstract

Purpose: To detect the spatio-temporal expression of S100A4 in a spinal nerve ligation (SNL) rat model. Also to figure out which other molecules directly interact with S100A4 to explore the possible mechanisms which might be involved in neuropathic pain.

Methods: Seven-week-old male SD rats were used for the SNL model construction. Immunofluorescence and Western blotting were used to detect the spatio-temporal expression of S100A4 in the model. S100A4 was co-labeled with a number of related molecules and marker molecules that can distinguish between cell types. After intrathecal injection of S100A4 neutralizing antibody, the behavioral changes of SNL rats were recorded, and molecular changes compared. The direct interaction between S100A4 and other related molecules was verified by co-immunoprecipitation (co-IP) to explore its possible mechanism.

Results: After spinal nerve ligation, the content of S100A4 in the dorsal root ganglion (DRG) and spinal dorsal horn increased significantly. Intrathecal injection of S100A4 neutralizing antibody could effectively relieve the mechanical pain in rats. co-IP revealed a direct interaction between S100A4 and RAGE.

Conclusion: The content of S100A4 in the DRG and spinal dorsal horn of SNL rats increased, compared with that of the control group. Intrathecal injection of S100A4 neutralizing antibody could effectively relieve the mechanical pain in SNL rats. S100A4 may be involved in the production of neuropathic pain through RAGE or other ways, but the specific mechanism needs to be further studied.

Keywords: S100 calcium binding protein A4; neuropathic pain; receptor for advanced glycation end products; spinal nerve ligation model.

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Conflict of interest statement

The authors declare no potential conflicts of interest with respect to this work, the research, authorship, or publication of this article.

Figures

Figure 1
Figure 1
Expression of S100A4 in the DRG of SNL rats. After model building, the results of DRG immunofluorescence, the value of integral optical density, and the number of positive cells were compared (n=3). The integral optical density was of no significant difference on the 3rd, 7th, and 14th day after SNL compared with that of the control group. The number of positive cells in the SNL group was significantly higher than that of the control group on the 7th day after operation. **P<0.01, bar=200 μm.
Figure 2
Figure 2
Expression of S100A4 in the spinal cord of SNL rats. The cross-section of the spinal cord of SNL rats was studied by double-labeling immunofluorescence. The picture shows the merge results of red and green channels. The red fluorescence represented Iba1 (inflammatory marker), which was brighter on the operation side, and the contralateral side is the control side. The green fluorescence represented S100A4. The dotted line area was the substantia gelatinosa of the spinal cord dorsal horn where S100A4 expressed, and S100A4 was expressed more on the operation side. The other marginal green fluorescent areas were the white matter of the spinal cord. Ruler=500 μm.
Figure 3
Figure 3
Expression of S100A4 in the spinal dorsal horn after SNL. (A) Immunofluorescence study of the changes of S100A4 in spinal dorsal horn after SNL. The fluorescence intensity of S100A4 increased significantly on the 3rd, 7th, and 14th day after SNL compared with that of the control group (*P< 0.05, **P<0.01, ***P<0.001). Green: FITC, bar=200 μm. (B) WB analyze of expression of S100A4 in the spinal dorsal horn after SNL. GAPDH was used as internal reference. The expression level of S100A4 increased significantly on the 7th, 14th, 21st, and 28th day after SNL, compared with that of the control group (***P<0.001).
Figure 4
Figure 4
Co-labeling of S100A4 and certain cell markers in DRG of SNL rats. S100A4 co-labeled with IB4, CGRP, and P2X3 in the DRG of SNL rats. Results showed that S100A4 was partially co-labeled with the three markers (the specific co-labeling proportion was: 62.5% of IB4 positive cells, 33.3% of CGRP positive cells, and 50% of P2X3 positive cells were co-labeled with S100A4, respectively, whereas the proportion of S100A4 positive cells co-labeled with IB4, CGRP and P2X3 were 19.2%, 33.3%, and 10%, respectively). White arrows in the pictures refer to the common label area. Bar=200 μm.
Figure 5
Figure 5
Co-labeling of S100A4 and certain cellular markers in spinal dorsal horn of SNL rats. S100A4 co-labeled with Iba1, GFAP, and IB4 in the spinal dorsal horn of SNL rats. Results showed that S100A4 was partially co-labeled with the three markers. White arrows in the picture refers to the common label area. Bar=200 μm.
Figure 6
Figure 6
Changes of paw withdrawal threshold in each group after intrathecal intervention. The changes of PWT in each group were measured before and on the 1st, 3rd, 5th, and 7th day after SNL operation. Compared with the SNL/saline group, intrathecal injection of S100A4 neutralizing antibody could increase the PWT on the 3rd, 5th, and 7th day after operation. ** P<0.01, and *** P<0.001, compared with CTRL/saline; ΔΔ P<0.01 and ΔΔΔ P<0.001, compared with SNL/saline. N=10.
Figure 7
Figure 7
Changes of S100A4 expression in each group after intrathecal intervention. Western blotting results of S100A4 expression changes in each group after intrathecal intervention. GAPDH was used as internal reference. (A) The expression of S100A4 in the DRG of the three groups. The expression level of S100A4 in the DRG of SNL/antibody and SNL/saline groups were significantly higher than that of the CTRL/saline (control) group. Besides, the expression level of S100A4 in the DRG of the SNL/antibody group was significantly lower than that of the SNL/saline group. (B) The expression of S100A4 in the spinal cord of the three groups. The expression level of S100A4 in the spinal cord of the SNL/antibody and SNL/saline groups were significantly higher than that of the control group. Similarly, the expression level of S100A4 in the spinal cord of the SNL/antibody group was significantly lower than that of the SNL/saline group. *P<0.05. **P<0.01. ***P<0.001.
Figure 8
Figure 8
Interaction between S100A4 and RAGE in rats. (A) Results of co-IP showed that the corresponding molecules could be detected in the immune complexes pulled by S100A4 or RAGE antibody, while those pulled down by ineffective IgG could not detect these two molecules. (B) Immunofluorescence of the spinal dorsal horn showed that S100A4 and RAGE co-labeled with each other in the substantia gelatinosa of the spinal dorsal horn. White arrow: hint of co-labeled point, no significant. Red: Cy3. Green: FITC. Scale=200 μm.
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