Signal Mechanisms Mediated The Roles Of Chemokine CCL2 In CCD-induced Neuronal Hyper-excitability And Mechanical Hyperalgesia

Chemokines are central to the innate immune response following tissue damage, injury and some diseases. CC chemokine ligan2/CCL2 (formerly named monocyte chemoattractant protein-1,MCP-1) is little expressed in the normal central nervous system, however, it has been demonstrated that CCL2 could be induced in the brain during ischemia, excitotoxic injury and trauma. The involvement of chemokines in neuropathic pain processing has recently been established in animal models. Evidence of chemokine's contribution to chronic pain includes the up-regulation of CCL2 and its respective receptor, CCR2, in many subpopulations of sensory neurons following peripheral nerve injury such as in a chronic compression of the dorsal root ganlia (CCD) model. Activation of CCR2 by CCL2 elicits membrane depolarization in CCD–injured neurons of the dorsal root ganglia (DRG) and the application of specific chemokine receptor antagonists for CCR2 successfully reverses nociceptive pain behavior. These data suggest that CCL2 might play a major role in the neuropathic pain, but the underlying signal mechanisms of CCL2 have not been elucidated yet.We examined signal mechanisms that mediated the roles of chemokine CCL2 in CCD-induced neuronal hyper-excitability and mechanical hyperalgesia. The main results are as follows:1. As assessed by Western blot analysis, the phosphorylated PLCβ3 expression level was significantly increased in the acutely dissociated CCD neurons after CCL2 treatment for 1 min. Also, the phosphorylated ERK in intact L4/L5 DRGs of rats was induced by CCD injury and pEKR was mainly located in P2X3-IR positive CCD neurons as revealed by immunohistochemistry.2. Behavioral tests showed that intradermal injection of chemokine CCL2 enlarged hyperalgesia in CCD rats, as evidenced by the reduced threshold to mechanical stimuli. The pretreatment by intraplantar administration of CCR2 inhibitor RS 102895, PKC inhibitor chelerythrine, PLC inhibitor U73122 and MEK inhibitor U0126 completely blocked CCL2-induced mechanical hyperalgesia, respectively, while the pretreatment of the PKA inhibitor H89 failed.3. CCL2 reduced the current threshold in CCD neurons. The pretreatment of CCD neurons with CCR2 antibody and CCR2 antagonist RS 102895 impaired CCL2-induced decreases in current threshold. The pretreatment of CCD neurons with U73122, chelerythrine chloride, 2-APB, BABTA and U0126 blocked the effects of CCL2 on cellular excitability, respectively. However, the pretreatment of CCD neurons with PKA inhibitor H89 failed to impair CCL2-induced hyperexcitability. The treatment of U0126 suppressed hyper-excitability in CCD-injured DRG neurons.4. The whole-cell patch-clamp recordings showed that TTX-sensitive sodium channel in CCD neurons was activated when voltage is held at a holding potential of -50mV, and reached maximum when membrane voltage is held at -30mV.Pre-application of CCL2 for 1 min could significantly enhance the TTX-sensitive sodium current amplitude but has no effects on TTX-resistant sodium current. In conclusion, these results suggest that CCL2 play an important roles in CCD-induced pain behaviors by enhancing the excitability of nociceptive DRG neurons, which is dependent on PLC/PKC ,PLC/IP3R/Ca2+ and MAPK signaling pathways.