CCL2 Mediated Hyperexcitability Of Peripheral Sensory Neurons And The Characteristics And Mechanisms Of STDP In Spinal Dorsal Horn Neurons

Objective:Chronic pain is highly prevalent and diverse,and it seriously affects people’s health.Regarding the pathogenesis of chronic pain,the most studied mechanisms are peripheral sensitization of the dorsal root ganglion(DRG)and central sensitization of the dorsal horn of the spinal cord.Peripheral and central sensitization due to plastic changes in the nervous system is an important cause of chronic pain.The common cause of peripheral sensitization is the occurrence of inflammation-related changes,and chemokine C-C motif ligand 2(CCL2)plays an important role in the development of peripheral inflammatory pain,but the exact mechanism by which CCL2mediates peripheral sensory neuron hyperexcitability causing peripheral sensitization is not known.As an important site for the development of chronic pain,the dorsal horn of the spinal cord has a very complex mechanism for synaptic plasticity changes,and the phenomenon of spike timing-dependent plasticity(STDP)provides us with the possibility to regulate the synaptic plasticity changes of spinal cord dorsal horn neurons.Therefore,revealing the mechanisms of peripheral sensitization of DRG and central sensitization of the spinal dorsal horn will hopefully reveal the mechanisms of chronic pain onset and development.Methods:In this study,we first investigated the mechanism of peripheral sensitization of DRG,and observed the effect of I_hcurrent blocker ZD7288 on the pain sensitization response induced by plantar injection of CFA and DRG injection of CCL2in mice by behavioral assay.The excitability and I_hcurrent changes of DRG neurons after CFA and DRG incubation with CCL2 were observed by electrophysiological recordings.The expression distribution of CCR2,the receptor for CCL2,and Hyperpolarization-activated cyclic nucleotide-gated 2(HCN2)channels in DRG neurons after plantar injection of CFA and DRG injection of CCL2 was investigated using immunohistochemical staining techniques.Next,the central sensitization mechanism of the spinal dorsal horn was investigated,and examined the STDP characteristics of the spinal dorsal horn neurons by means of a patch-clamp technique.We investigated the role of pre and postsynaptic NMDA receptors in the regulation of STDP by adding blockers of NMDA receptors using a neuropharmacological approach and establishing transgenic mice with conditional knockout of presynaptic NMDA receptors.We further used the spinal cord stimulation(SCS)technique to regulate the STDP changes in the dorsal horn neurons of the spinal cord.Results:In the first part of this topic,the results of our study are as follows.1.Behavioral assays showed that DRG injection of ZD7288 was effective in relieving mechanical and thermal pain induced by plantar injection of CFA for 24 h.Mice exhibited significant mechanical and thermal pain responses after DRG injection of CCL2,and DRG injection of ZD7288 dose-dependently attenuated this pain-sensitive response.2.After incubation of the DRG with CFA or CCL2 by plantar injection,membrane clamp recordings showed a significant increase in excitability of small diameter neurons in the DRG and a significant increase in I_hcurrent.3.Immunohistochemical staining showed that the number of CCR2~+and HCN2~+co-labeled neurons was significantly upregulated in the DRG after plantar injection of CFA or DRG injection of CCL2,and further analysis revealed that the expression ratio of CCR2~+/HCN2~+co-labeled neurons was upregulated in small diameter neurons.In the second part of this study,the results of our results are as follows.1.Membrane clamp recordings revealed the presence of Hebbian STDP phenomenon in the synaptic connections formed by specific injury sensory neurons and primary injury sensory afferent fibers in the dorsal horn layer I of the spinal cord,and Anti-Hebbian STDP phenomenon in some neurons in the dorsal horn layer II of the spinal cord.2.Pre and postsynaptic NMDA receptors were blocked by perfusion of AP5 during electrophysiological recordings and t-LTP was found to be significantly inhibited.Intrapolar fluid plus MK801 blocked postsynaptic NMDA receptors and t-LTP was also significantly inhibited.In transgenic mice conditionally knocked out of presynaptic NMDA receptors,t-LTP in the dorsal horn of the spinal cord was similarly significantly inhibited.3.SCS stimulation modulates the e EPSC amplitude of spinal cord dorsal horn neurons in both directions.At low intensity stimulation,e EPSC amplitude is significantly suppressed,while at medium to high intensity stimulation,enhancement of e EPSC amplitude is induced.SCS co-stimulation was performed during the induction of t-LTP,and t-LTP was significantly inhibited.When SCS secondary stimulation was performed after t-LTP formation,t-LTP tended to decrease.Conclusion:CCL2/CCR2 upregulates HCN channels in small-diameter neurons in the DRG during peripheral inflammatory pain states,causing an increase in I_hcurrents,which in turn leads to neuronal hyperexcitability.Synaptic plasticity changes in spinal dorsal horn neurons have a bidirectional character of STDP,and such synaptic plasticity changes can be precisely modulated by changing the temporal sequence of pre-and postsynaptic stimuli to achieve interconversion.Pre-and postsynaptic NMDA receptors play an important role in the induction and maintenance of STDP in spinal dorsal horn.SCS stimulation can modulate STDP in spinal dorsal horn neurons,and its possible mechanism is the modulation of complex neural circuit in the dorsal horn of the spinal cord.