| Background:Pain refers to a series of unpleasant feelings and emotional experiences after tissue or nerve injury.The persistent chronic pain is a kind of trouble and torture to human beings.Chronic pain has become a major public health problem that seriously affects the quality of human life.Patients with chronic pain often experience spontaneous ongoing pain,hyperalgesia and allodynia.Although considerable progress has been made in understanding chronic pain in the past decades,treatment options were still largely limited to opioids and nonsteroidal anti-inflammatory drugs.Moreover,intervention with these therapeutics is associated with serious central side effects,e.g.addiction,tolerance,peptic ulcer,which in turn hamper medical adherence.Thus,improved understanding of mechanisms contributing to chronic pain and search for new therapeutic targets is critical for achieving better treatment outcomes with least side effects.Ttyhl is a five time transmembrane volume regulated Cl-channel and is regulated by cell swelling,and its C-terminal can bind calcium ion.Given the mainly restricted localization in neural tissues,the association of Ttyhl with fundamental cellular processes as well as several brain diseases have created a heightened interest.Some studies have shown that Ttyhl plays an important role in the development of embryonic stem cells,and its expressions were significantly up-regulated under several pathological conditions such as epilepsy and childhood brain tumors,suggesting that Ttyhl has important functions in physiological and pathological conditions.In addition,the ectopic expression of Ttyhl in human renal epithelial cells leads to the production of long,branched filaments,while the overexpression in cultured rat hippocampal neurons leads to strong neurofibrillation and complex dendrites.These studies suggest that Ttyhl may be closely related to the structural plasticity of neurons.Of note,among the neural tissues,Ttyhl is predominantly expressed in the dorsal root ganglion(DRG)and spinal cord,where the first-order primary sensory neurons and the first synapse are located in the nociceptive pathway.However,its functional significance in pain information transmission is not clear.There is a great deal of evidence supporting that primary sensory neurons and their synaptic plasticity with spinal dorsal horn(SDH)neurons are considered as the cellular basis of chronic pain.However,it remains elusive whether Ttyhl regulates pain and sensitization,if so,what mechanisms underlie this regulation.Objective:we sought to address the role of Ttyhl in pain circuits at the DRG and spinal levels by combining models of inflammatory pain,behavioral surveys,biochemical analyses,electrophysio logical recordings as well as genetic manipulations.We also try to explore the expression and distribution of Ttyhl in DRG and spinal cord,its role in pain information transmission and pain sensitization,as well as the underlying molecular mechanism.Methods:1.We detected the expression of Ttyhl in DRG and dorsal horn of spinal cord,as well as in CFA induced inflammatory pain by using immunofluorescence in situ hybridization,qRT-PCR and WB technique.2.We determined the role of Ttyhl in the sense of peripheral sensory information under normal physiological conditions and the pain sensitization under pathological conditions by using pain behavior test.3.We determined the effect of Ttyhl on the excitability of DRG small cells under normal physiological conditions and inflammatory pain pathological conditions by using gene knockout(Ttyh1-/-)mice,combined with electrophysiology.4.We determined the effect of Ttyh1 on the activity and excitability of neurons in spinal dorsal horn under normal physiological conditions and inflammatory pain pathological conditions by using Ttyhl-/-mice,combined with immunofluorescence and electrophysiology.5.We investigated the effect of Ttyhl on excitatory synaptic transmission and synaptic plasticity of the projecting neurons in the dorsal horn of the spinal cord by using Ttyhl-/-mice,combined with electrophysiology.6.Using virus specificially knock out Ttyhl in DRG nociceptive neurons,pain behavior test was used to clarify the role of Ttyhl in the sense of peripheral sensory information under normal physiological conditions and the pain sensitivity under pathological conditions.Results:1.The results of immunofluorescence in situ hybridization showed that Ttyhl was widely expressed in almost all DRG neurons,and co-labeled with CGRP,IB4 and NF200.In addition to the expression in the cell body of DRG neurons,Ttyhl is also expressed in the central terminals of DRG neurons.In addition,the expression of Ttyhl was also significant expression in the neurons of spinal dorsal horn.The results of Qrt-PCR and WB experiments showed that the expression of mRNA and protein of Ttyhl in DRG and dorsal horn of spinal cord were significantly upregulated in inflammatory pain induced by CFA injection.2.In the basal state,mechanical pain threshold and thermal pain threshold were lifted for Ttyh1-/-mice as compared with their littermate controls.Under pathological condition,Ttyh1+/+mice often experienced spontaneous ongoing pain,hyperalgesia and allodynia,but which were weakened in Ttyh1-/-mice.3.Small DRG neurons in Ttyh1-/-mice exhibited more negative RMP,lower firing frequency evoked by a depolarizing current step and higher rheobase required to evoke an AP as compared to those from Ttyh1+/+mice in both basal and CFAinflamed state.4.Both the expression of c-Fos and pERK in the dorsal horn of spinal cord in Ttyh1/-mice and the excitability and hyperactivity of spinal dorsal horn neurons projecting to PAG were significantly reduced as compared to those from Ttyh1+/+mice in both basal and CFA-inflamed state.5.Ttyh1-/-mice showed a reduced basal synaptic transmission,as characterized by a mild downward shift of input-output curve(I-O curve)as compared to Ttyh1+/+mice in the basal state.The magnitude of this synaptic potentiation evoked by CFA was largely reduced in those from Ttyh1-/-mice,although they also got potentiated to some degree after inflammation.In striking contrast to Ttyh1+/+mice,the same conditioning stimuli did not evoke LTP in spinal-PAG projection neurons in Ttyh1/-mice and even led to long-term depression(LTD)of C-eEPSCs.The analysis of PPR and mEPSCs showed that the effect of Ttyhl on synaptic transmission and plasticity was mainly mediated by presynaptic mechanism.6.Analysis of acute withdrawal responses to paw pressure and thermal stimuli revealed that mice expressing AAV2/8-shRNA Ttyh1 in nociceptors exhibited blunted responses in comparison with those expressing AAV2/8-shRNA scramble,which is similar to Ttyh1-/-mice.Conclusions:1.Ttyhl is intensively expressed in DRG and spinal dorsal horn,and plays an important role in synergetic perception of peripheral nociceptive stimulation in basal state.The expression of Ttyhl in DRG and spinal dorsal horn was significantly up-regulated,which revealed that Ttyhl may play a significant role in pain sensitization.This result is the first time to confirm that Ttyhl is the key medium of normal pain perception and pain sensitization in the process of chronic inflammatory pain.2.Ttyh1 plays an important role in the transmission of pain information by regulating the excitability of DRG neurons and spinal dorsal horn neurons,as well as the synaptic transmission of primary afferent synapses in basal state.3.Ttyhl is involved in mediating the hyperactivity of peripheral nociceptive neurons.This result is the first to clarify the mechanism of Ttyhl mediated in pain sensitization by enhancing peripheral sensitization in inflammated state.In addition,Ttyhl also mediates the activity and hyperactivity of neurons in the dorsal horn of the spinal cord.4.Ttyhl facilitates pain via enhancement of spinal synaptic transmission and plasticity between primary sensory neurons and spinal dorsal horn neurons by regulating presynaptic mechanism of presynaptic transmitter release probability.5.Specifically knocking out Ttyhl in DRG nociceptive neurons,its pain behavior is similar to global Ttyh1-/-mice,that is to say,it can significantly reduce the pain sensitive behavior under inflammatory pain,suggesting that Ttyhl can be the most important peripheral target of pain transmission and pain sensitization. |
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