Facilitation Of Peripheral Nociceptive Signal Transmission In Spinal Cord By Monocyte Chemoattractant Protein-l(CCL2) And Its Underlying Mechanisms

The mechanism of neuropathic pain (chronic) has been a challenge in neuroscience.Clinically, the drugs used to treat chronic pains by simply inhibiting the neurons thattransfer pain signals, have poor efficacy and serious side effects. This suggests that thesensation function of the pain receptors and the transduction function of the pain signals dohave abnormal changes.But the factors and mechanisms are unclear. In recent years, animportant progress in chronic pain research clearly shows that the responses of thenerveinflammation are playing an important role in the emergence and persistence ofchronic pain.Lesions such as peripheral nerve injury, inflammation and demyelination cause chronicpain, followed by the production of various proinflammatory cytokines.The expression ofchemokine and chemokine receptors plays an important role in the pathophysiologicmechanism of chronic pain. Among these chemokines, monocyte chemoattractantprotein-1(MCP-1or CCL2) up-regulated in dorsal root ganglia (DRG) neurons canenhance the receptive ability of the peripheral pain receptors. Thus,it is considered to be anew factor which induces pain. However, the results in literatures and our recent researchessuggest that the function of CCL2in regulating pain signals may act at the spinal cord level.This may be the just reason for the long-lasting and recurrent of inflammatory pain.Therefore, on the basis of our previous studies, the subject aims to identify whether CCL2up-regulated on dorsal root ganglia (DRG) neurons would act as neuromodulator toamplify the peripheral afferent pain signals at the spinal level, which is probably one of thecauses for central sensitization mechanisms of chronic pain. So we must deeply investigatethe role and the mechanism of CCL2directly regulating the excitatory/inhibitory synaptictransmission between the central end of the peripheral nociceptive afferent fibers and thesubstantia gelatinos（aSG）neurons, and its subsequent effects on the maintaining of chronicpain.Our works offer a new and scientific theory, as well as a background to find newtherapies for chronic pain by interfering CCL2/CCR2signal system at the spinal level.The following are the main findings:1.CCL2facilitates pain behavior of the CCD rats on spinal cord level.(1). Behavioral study showed that CCL2could facilitate mechanical pain behavior in CCD rats after intrathecal injecting CCL2，and intrathecal injection of the antagonist ofCCR2(INCB3344,AZ889) could significantly reverse the mechanical pain behavioralmean threshold of CCD rats.2.The expression of CCL2and CCR2are increased in the DRG andspinal cord of CCD rats(1). Immunohistochemistry studys showed that CCL2was increasingly expressed in DRGneurons，and coexpressed with IB4which is the marker of nociceptive neurons；both CCL2and CCR2had high expression in dorsal horn of the spinal cord.(2). Western blot results showed that the CCR2proteins were up-regulated in dorsal hornof the spinal cord of CCD rats.3. Facilitation of peripheral nociceptive signal transmission in spinal cordby monocyte chemoattractant protein-1(CCL2) and its underlyingmechanisms(1). Whole cell patch clamp recordings in spinal cord slice showed that the frequencyof spontaneous excitatory postsynaptic currents (sEPSCs) of the SG neurons in CCD ratswas higher than normal rats.(2). Whole cell patch clamp recordings in spinal cord slice showed that intrathecalinjection of CCR2specific inhibitor INCB3344could decrease the frequency of excitatorysynaptic transmission in dorsal horn of the spinal cord，and had no effect on the amplitudeof sEPSCs.(3). Whole cell patch clamp recordings in spinal cord slice showed that CCR2specificinhibitor INCB3344could decrease excitatory synaptic transmission in dorsal horn of thespinal cord.(4). Whole cell patch clamp recordings in spinal cord slice showed that CCL2couldenhance the frequency and amplitude of spontaneous excitatory postsynaptic currents(sEPSCs), and decrease the frequency and amplitude of spontaneous inhibitorypostsynaptic currents (sIPSCs) of the SG neurons. Further research indicated that CCL2increased AMPA、NMDA currents and decreased GABA currents.(5). Whole cell patch clamp recordings in spinal cord slice showed that CCL2couldenhance the excitability of SG neurons in CCD rats.In conclusion, we investigated the roles and mechanisms of CCL2/CCR2signal system in regulating the transmission of pain signals in spinal cord in CCD rats. The results indicatedthat CCL2/CCR2signaling played an important role in facilitating the excitatory synaptictransmission and decreasing the inhibitory synaptic transmission of the SG neurons,enhancing the excitability of SG neurons,and inducing and maintaining chronic pain. Thus,CCL2/CCR2signal system could amplify the peripheral afferent pain signals at spinal cordlevel, becoming one of the factors to cause central sensitization of chronic pain.Thissystem also played a role in maintaining chronic pain at spinal cord level.