| Objective:Neuropathic pain is caused by the peripheral or central nervous system structure damage or dysfunction. Preliminary studies had observed that the immunoreactivity of vascular endothelial growth factor (VEGF) in dorsal root ganglia (DRG) of chronic constriction injury (CCI) rats was enhanced. Treated with Anti-rVEGF antibody (injected to intrathecally the CCI rats), thermal withdrawal latency (TWL) and the mechanical withdrawal threshold (MWT) of the CCI rats were reduced, and the expression of VEGF receptor (VEGFR-2), P2X3 and P2X2/3 receptor of DRG in the CCI rats were reduced. This study wants to observe the effects of VEGF on P2X3 agonist-activated currents and P2X2/3 agonist-activated currents in freshly isolated DRG neurons of rat by whole-cell patch-clamp technique. The relationship between VEGFR-2 and P2X2, P2X3 receptors in DRG and spinal dorsal horn were studied by immunohistochemistry, double-label immunofluorescence and western blotting. The objective is to broaden and deepen the understanding of pathogenesis of neuropathic pain, so as to explore new targets for preventing and treating neuropathic pam.Methods:This study took chronic constriction injury (CCI) rats as neuropathic pain model. Sprague-Dawley male rats were randomly divided into Sham group (A), CCI+PBS group (B) and CCI+Anti-rVEGF antibody group (C). Anti-rVEGF antibody was injected to the rats intrathecally in group C every two days, while PBS was injected into group A and B at the same time. After 14-day consecutive treatment, we isolated the L4-6 DRG and L4/5 spinal dorsal horn immediately from three groups. The expressions of P2X2, P2X3 and VEGFR-2 protein in DRG were detected by double-label immunofluorescence and in spinal dorsal horn were detected by immunohistochemistry, double-label immunofluorescence and western blotting. The the modulation effect of VEGF on P2X3 receptor agonist-activated currents and P2X2/3 receptor agonist-activated currents were determined in freshly isolated DRG neurons of rats by whole-cell patch-clamp technique.Results:(1) Immune fluorescence results showed that VEGFR-2 and P2X2 or P2X3 receptors were co-expressed in the cytoplasm and surface membranes of DRG. The co-expression of VEGFR-2 and P2X2 or P2X3 receptor in the group B exhibited more intense staining than those in group A and C respectively. (2) The majority of the DRG neurons were sensitive to ATP in the concentration range from 1 to 1000μmol·L with a concentration-dependent current. The currents were simulated by a,p-meATP (a selective agonist of P2X2/3 receptor) and blocked by A-317491(a selective antagonist of P2X2/3 receptor). Current traces demonstrated that VEGF(1 nmol·L-1) enhancesα,β-meATP(10μmol·L-1)-activated current and ATP(100μmol·L-1)-activated current markedly. Both of the currents were blocked by vatalanib (a antagonist of VEGF receptors). (3) Immunohistochemistry results showed that the optical density of P2X2, P2X3, VEGFR-2 in L4/5 spinal dorsal horn in group B were enhanced significantly compared with those in group A and C (p<0.05) respectively. (4) Immune fluorescence results showed that VEGFR-2 and P2X2 or P2X3 receptors were co-expressed in the cytoplasm and surface membranes of spinal dorsal horn. The co-expression of VEGFR-2 and P2X2 or P2X3 receptors in the group B exhibited more intense staining than those in groupA and C respectively. (5) By western blotting, the protein levels of P2X2, P2X3 and VEGFR-2 in L4/5 spinal dorsal horn of group B were higher than those in group A and C (p<0.01) respectively.Conclusions:The co-expression of VEGFR-2 and P2X3 or P2X2/3 receptors were increased in DRG neurons and spinal dorsal horn of CCI rats. VEGF enhanced markedly ATP-and a,(3-meATP-activated current. Therefore, there is interaction between VEGFR-2 and P2X3 or P2X2/3 receptors. VEGF can enhance the activation of P2X3 or P2X2/3 receptors in the role of neuropathic pain. |
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