Complement Receptor 3 Pathway and NMDA Receptor 2B Subunit Involve Neuropathic Pain Associated with Spinal Cord Injury

Abstract

Background: Neuropathic pain (NP) after spinal cord injury (SCI-evoked NP) is clinically challenging; the underlying mechanisms are not fully understood, leading to a lack of promising treatment options. NP occurs in only a subset of patients with SCI. The injured spinal cord exhibits a series of histopathological changes, and the complement system has been shown to play an important role in these processes. In addition, NMDA receptor subunit 2B (NR2B) is involved in the development and maintenance of NP. This preliminary study was performed to investigate the correlations of the complement receptor 3/complement component 3 (CR3/C3) pathway and NR2B with SCI-evoked NP.

Methods: A trauma-induced SCI animal model was established and SCI-evoked NP was evaluated by behavioural analysis. Transcriptome analysis was performed to identify genes in the CR3/C3 pathway related to synaptic modification, while the expression and distribution of NR2B in the injured spinal cord, and the relation to NP, were examined by immunohistochemical analysis.

Results: Nine of seventeen SCI rats (52.9%) developed NP. C3 mRNA expression was significantly decreased in SCI-evoked NP rats and significantly increased in the non-NP SCI rats. C1q mRNA and CR3 mRNA expression were significantly increased in all SCI rats, but higher levels of expression were observed in the non-NP SCI rats. NR2B mRNA expression was significantly increased in the SCI-evoked NP rats and significantly decreased in the non-NP SCI rats. In addition, significantly elevated expression of NR2B-positive cells was seen in lamina II of the superficial dorsal horn in SCI-evoked NP rats in comparison with non-NP SCI rats.

Conclusion: NP occurred in only a subset of SCI rats, and the CR3/C3 pathway and NR2B were involved in SCI-evoked NP. Further studies are required to determine the mechanisms underlying the SCI-evoked NP associated with the CR3/C3 pathway and NR2B.

Keywords: NMDA receptor 2B subunit; complement component 3; complement receptor 3; neuropathic pain; spinal cord injury.

Figure 1

Schematic illustration of the time window of the experiment.

Figure 2

Pain-evoked NP developed in a subset of SCI rats.

Figure 3

RNA-Seq analysis of rat spinal cords.

Figure 4

Relative NR2B, CR3, C3 and C1q mRNA expression levels in rat spinal cord. (A) The NR2B mRNA level was significantly increased in SCI-evoked NP rats and significantly decreased in non-NP SCI rats in comparison with sham controls. (B) The C3 mRNA level was significantly increased in non-NP SCI rats and significantly decreased in SCI-evoked NP rats in comparison with sham controls. (C) The CR3 mRNA level was significantly increased in all SCI rats, but was higher in non-NP SCI than SCI-evoked NP rats. (D) The C1q mRNA level was significantly increased in all SCI rats, but was higher in non-NP SCI than SCI-evoked NP rats. (E) The NR2B, CR3, C3 and C1q mRNA expression levels are shown. *Significant difference compared with sham rats, P < 0.05; ^#Significantly higher in non-NP SCI than SCI-evoked NP rats, P < 0.01.

Figure 5

Immunohistochemistry of NR2B in the superficial dorsal horn of rostral, epicentral and caudal sections from SCI-evoked NP rats with significantly decreased expression compared to non-NP SCI rats (10×). NR2B-positive cells were mainly present in lamina II of the superficial dorsal horn of SCI-evoked NP rats. A few NR2B-positive cells were scattered in the other laminae of the superficial dorsal horn of SCI-evoked NP rats, and the entire superficial dorsal horn of non-NP SCI rats. Green fluorescence indicates NR2B-positive cells, blue fluorescence indicates all cells, and merged images show the distribution of NR2B-positive cells and all cells in the superficial dorsal horn. *Indicates the epicentre of SCI. Scale bar, 0.1 mm.

Figure 6

The number and ratio of NR2B-positive cells increased significantly in lamina II of the spinal cord of SCI-evoked NP rats. (A) The merged image illustrates the boundaries of laminae I–IV in the superficial dorsal horn, and the distribution of NR2B-positive cells and all cells. Green fluorescence indicates NR2B-positive cells and blue fluorescence indicates all cells (10×). (B–E) Analyses of NR2B-positive cell expression in laminae I–IV, respectively. (F–I) Analyses of the ratio of NR2B-positive cells to all cells in laminae I–IV, respectively. Significant differences between SCI-evoked NP rats and non-NP SCI rats, *P < 0.05, ^#P < 0.01.