A Study On The Neurogenesis In Dorsal Root Ganglions Under Chronic Pain

Chronic pain(such as inflammatory pain,neuropathic pain,and cancer pain)is highly prevalent worldwide and highly comorbid with anxiety and depression.It can ilicit substantial financial and psychological burden to patients.The changes of neural plasticity has been well studied as the mechanism of chronic pain.The most widely studied neuronal mechanisms are hyperexcitability and sensitization of primary sensory neurons(peripheral sensitization)and enhancement of excitatory synaptic transmission in spinal cord,brainstem,and cortical neurons(central sensitization),caused by transcriptional,translational,and post-translational regulation.Other neuronal mechanisms include disinhibition(reduced inhibitory synaptic transmission),descending pathway facilitation(eg,from the brainstem to the spinal cord),and long-term potentiation(LTP)in the cortex and spinal cord.These neuronal mechanisms have been strongly implicated in the development and maintenance ofpersistent pain.Recently,more and more evidence is accumulated that glial cells in DRGs(satellite cells)are involved in the development of chronic pain.In the central nerve system,glial cells usually undergo certain degree of dedifferentiation upon injury.Glial cells in the carotid body and the intestinal ganglions could generate new neurons to adapt to the functional requirement in conditions of chronic hypoxia or inflammation.Considering the facts that satellite cells in DRGs share the same neural crest origin with glial cells in carotid body and intestinal ganglions,and that DRGs receive persistent periphery stimulation under chronic pain,we speculate that satellite cells in DRGs may be neurogenic under chronic pain.If that is the case,satellite cells will be involved in the pathological changes of chronic pain in a way we have not expected.Therefore,this study focuses on the DRG neurogenesis and the potential role of satellite cells in this process under chronic pain.The research consists of four parts:Part one: the activation of satellite cells in the dorsal root ganglion under chronic pain.Objective: To analyze the activation of different satellite cell populations in DRGs mice suffered from chronic pain.Methods: Using immunofluorescent staining and BrdU incorporation experiments,the proliferation and dedifferentiation of different satellite cell populations were observed at 7 and 14 days after chronic pain induction.Results: Both SNI and CFA treatment could induce the proliferation of Sox2-,GFAP-,and PDGFR?-positive satellite cells.In addition,chronic pain could induce the up-regulation of Nestin in Sox2-and GFAP-positive cells,and the up-regulation of Sox10 and Nestin in PDGFR?-positive satellite cells.Conclusion: Chronic pain can induce the activation of satellite cells in DRGs.Part two: neurogenesis in dorsal root ganglions under chronic painObjective: To explore the generation and the types of new neurons in DRGs under the condition of chronic pain.Methods: immunohistochemistry of BrdU and DNA damage repair marker(?-H2 A.X)were adopted.Results: The neurogenesis was observed in the ipsilateral DRGs by double-immunostaining of neuronal markers DCX and NeuNwith BrdU respectively.In neuronal type analysis,BrdU-positive neurons were mainly IB4-or CGRP-positive,but not NF200-positive.The DNA damage marker ?-H2 A.X was mainly expressed by NF200-positive neurons,but not by IB4-or CGRP-positive cells.Conclusion: chronic pain induces neurogenesis in the dorsal root ganglions.New born neurons are mainly IB4-or CGRP-positive nociceptive neurons.Part three: The cellular origin of neurogenesis in DRGs under chronic painObjective: The origin of neurogenesis was traced under the pathological conditions of chronic pain.Methods: By using GFAP-CreER:ROSA/YFP,Sox2-CreER:ROSA-YFP and PDGFR?-CreER:ROSA-YFP transgenic mice,cell lineage tracing was performed to track the origin of new neurons under chronic pain.Results: No neurons were traced in PDGFR?-CreER: Rosa/YFP mice uner chronic pain.Then fate trace was performed in GFAP-CreER:ROSA/YFP mice under the same chronic model.The neuron-like YFP-positive cells are surrounded by satellite cells and can be labeled by NeuN.All the YFP-labeled neurons are IB4-positive.Further,we also traced new neurons using Sox2-CreER:ROSA/YFP mice.Neuron-like YFP-positive cells were IB4 positive and CGRP positive.In addition,blocking sodium channel activity in the periphery on chronic pain can reduce the neurogenesis of DRG neurons from Sox2 positive satellite cells.Conclusion: Under chronic pain,PDGFR?-positive satellite cells are mainly gliogenic.GFAP-positive satellite cells could only generate IB4-positive neurons,while Sox2-positive satellite glial cells could produce both IB4-positive and CGRP-positive neurons.It is suggested that the neurogenesis of DRG neurons induced by chronic pain depends on peripheral afferent.Part four: Functional analysis of glial-derived neuronsObjective: To identify the electrophysiological characteristics of glia-derived new neurons,their response to pain stimulator(capsicin)and expression of TRPV1 were examined.Methods: Whole cell patch clamp recording,intracellular labeling of biocytin and immunohistochemistry were adopted;Results: The YFP-labeled neurons in both Sox2-CreER:ROSA/YFP and GFAP-CreER:ROSA/YFP mice can fire action potentials,although the number of action potentials is obviously less,threshold potential is lower andthe rheobase is higher than that of the pre-exsiting YFP-negative neurons.The resting membrane potential and the amplitude of action potential of new-born neurons showed no significant difference as compared to pre-existing YFP-negative neurons.Approximately44% of YFP-positive neurons respond to pain stimulator capsicin.At the same time,TRPV1 can be found expressed in approximately 18% YFP-positive neurons.Conclusion:These glial-derived neurons have the function of neuronal electrical activity.