| Objective To investigate the expression and distribution of chemokine CCL2 and its major receptor CCR2 in the dorsal root ganglion(DRG) and spinal cord following lumbar disc herniation-induced neuropathic pain in rats, and to explore their role in the regulation of chronic neuropathic pain.Methods Lumbar disc herniated rat model was made by implantation of the autologous nucleus pulposus(NP), which was harvested from the coccygeal vertebra of each tail, on the left L5 nerve root just proximal to the dorsal root ganglion. Both mechanical allodynia and thermal hyperalgesia were tested at different time points on the ipsilateral hind paw after LDH surgery. The expression of chemokine CCL2, CCR2, and TNF-α mRNA was detected by Real-time PCR in the ipsilateral side DRG and L3-5 spinal segments. The protein level of ATF3, ED-1, CCL2, and CCR2 were examined by immunofluorescence. The cellular distribution of CCL2 and CCR2 was defined by immunofluorescence double-staining in the DRG and spinal segments. The protein expression of glial fibrillary acidic protein(GFAP) was examined by Western blot. The effect of CCR2 antagonist, RS504393 and TNF-α antagonist, Etanercept on pain hypersensitivity was checked by behavioral testing after intrathecal administration.Results(1) NP induced rapid mechanical allodynia and delayed heathyperalgesia. On the ipsilateral paw of the NP animals, the paw withdrawal threshold(PWT) dropped at 1 day, remained at the peak level for more than 14 days and slightly recovered at 28 days after surgery; the paw withdrawal latency(PWL) was decreased at 5 days after surgery, maintained at 21 days and fully recovered at 28 days.(2) NP induced neuronal injury and macrophage infiltration in rat DRG.(3) NP induced the mRNA and protein upregulation of chemokine CCL2 in DRG. The mRNA increase was shown at 3 d and maintained for more than 21 days. The protein has significant increase at 5 days and 10 days after surgery. Immunostaining showed that CCL2 was colocalized with CGRP, IB4 and NF200.(4) In DRG, the mRNA expression of the major receptor of CCL2, CCR2 was increased from 1 day, peaked at 10 days and maintained at relatively high level at 21 days. Immunofluorescence showed that the protein has significant increase at 5 days and 10 days after NP application. Immunofluorescence double staining showed that CCR2 was colocalized with MAP2 and ED-1, but not with GFAP.(5) NP induced the upregulation of CCL2 mRNA and protein in the spinal cord. The upregulation of CCL2 mRNA was shown from 1 d and lasted for 21 days. NP application increased CCL2-IR at 5 days, 10 days and 21 days. Double staining further showed that CCL2 was mainly expressed in GFAP-positive astrocytes, but not in NeuN-positive neurons or OX-42-positive microglia. Western blot show that GFAP in the spinal cord expression was increased from 1 day and maintained for more than 7 days. Immunostaining further showed more intensive GFAP staining in the NP animals than that in the sham animals at 10 days after surgery.(6) NP also increased the mRNA and protein expression of CCR2 in the spinal cord. CCR2 mRNA upregulation was shown at 3 days, 5 days and 10 days, but not at 1 day or 21 days after surgery. The results of immunofluorescence further confirmed the upregulation of CCR2 protein at 5 days and 10 days. Double staining showed CCR2-IR was colocalized with the neuronal marker NeuN.(7) Intrathecal injection of CCR2 antagonist, RS504393 at 3 days or 10 days significantly attenuated NP-induced mechanical allodynia.(8) NP induced rapid TNF-? mRNA upregulation in the DRG and spinal cord. Intrathecal injection of TNF-α inhibitor, etanercept before NP application delayed mechanical allodynia and decreased the mRNA expression of CCL2 and CCR2.Conclusion CCL2/CCR2 signaling in the DRG and spinal cord is involved in LDH-induced pain via mediating interaction between different cells. TNF-? antagonist and CCR2 antagonist attenuated radicular neuropathic pain in the development and maintenance phase, respectively. |
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