| Pain is an unpleasant subjective sensation and emotional experience caused by the sensory nervous system being stimulated by real or potential tissue injury,and is the reflex protection and pathological reaction of the body to the harmful sensation.The harmful stimulation is a kind of energy liberation,which is the process of the receptor energy conversion causing the nerve impulse.The trigeminal neuralgia(trigeminal neuralgia)is one of the most common peripheral cranial neuralgia,which mainly manifests as a kind of transient paroxysmal acute pain in the dominant region of trigeminal nerve.Clinically,TG usually occurs on a unilateral,severe,transient,characteristic pain that is distributed by one or more branches of the fifth cranial nerve.Previous studies have confirmed that pain is an acute attack and lasts for a few seconds to a few minutes.After each episode there is a period of intolerance,during which stimulation of the region does not cause pain.The frequency of pain may occur from 4-5 to hundreds of a day.There will be months or years of pain relief period.The pain is quite severe and is described as intense,sharp,tingling,or shooting,similar to an electric shock.Usually TG can be caused by the non pain physical stimulation of the specific area(trigger point or region)of the trigeminal nerve,and these areas are located on the same side of the pain.Incentives include eating,speaking,washing your face,blowing hair and brushing your teeth.This situation can affect daily life and even lead to depression,while neurological examination is usually normal.According to the Etiology,TN divided into primary and secondary sensory neurons.At present,the pathogenesis of primary trigeminal neuralgia mainly includes microvascular pressure theory,neurodegenerative theory,epilepsy theory,lesion infection,bone oppression theory,but the detailed pathogenesis is still not clear.At present,the treatment of TN is not ideal,although the traditional Chinese and western medicine have corresponding intervention means.The main part of the peripheral pain of trigeminal neuralgia is the ganglion(trigeminal ganglia,TG),which transmits the sensory signal to the first station of the central nerve,and is the afferent primary sensory neuron(mainly mechanical receptor neurons)in the peripheral nociceptive sensation.TG is divided into three branches: eye branch,maxillary branch,mandibular branch.Because of its simple and acute separation operation,TG is commonly used to study the peripheral injury sensation mechanism.In mice,there were about 2.6-43,000 neurons in the trigeminal ganglion,which were divided into small-diameter cells(15-30?m),medium-diameter cells(30-40?m)and large-diameter cells(> 40?m).The small diameter trigeminal ganglion neurons are usually connected with C fiber and A?,suggesting that it is related to the sensation.Urotensin II is an active peptide that was originally isolated from the tail of the bony fish and was subsequently found in mice,rabbits and the human body.Structurally,the tail of urotensin II is a single chain polypeptide consisting of 12 amino acids,and its active center is a ring structure composed of six amino acids in C-F-W-K-Y-C.A large number of biological studies have shown that urotensin II plays a variety of physiological functions within the organism by combining its endogenous receptors: ingestion and energy stability,stress response,vasoconstriction,sleep and arousal,and injury-induced sensory regulation.In mammals,the endogenous receptor of urotensin II was first cloned from the human and belonged to the specific receptor G-protein coupling receptor(GPR14)family.According to the International Pharmacology Federation receptor and Drug classification rules,GPR14 was subsequently identified as UT-R(urotensin II receptor,U-II receptor).Functionally,UT-R mainly distributes in the cardiovascular and nervous system,which plays an important role in regulating cardiac and cerebrovascular activities.In addition,UT-R has a small distribution in the kidneys,lungs and mesentery,and participates in the metabolism and functional regulation of the tissues.In the central nervous system,UT-R mainly exists in cortex,thalamus,spinal cord,trigeminal nucleus and facial nerve nucleus.Activation of UT-R can participate in anxiety regulation,diet,slow-wave sleep and injury reflex of central spinal cord horizontal loop.In the peripheral system,UT-R is mainly expressed in the heart,vascular smooth muscle,kidney,intestine and peripheral sensory ganglia.Activation of UT-R can encode gastrointestinal ions transport and participate in peripheral injury sensation regulation.In the pain system,recent studies have shown that peripheral sensory neurons are closely related to UT-R.Recent reports have shown that peripheral activation of UT-R significantly induced pain.Behavioral studies showed that in rat neuropathic pain model(CCI),urotensin II local injection dose-dependently increased thermal and mechanical pain sensitivity.This effect was abolished by UT-R blockade.In CFA-induced inflammatory pain model,the blockade of UT-R significantly inhibited mechanical and heat pain hypersensitivity.At the molecular level,UT-R is mainly expressed in sensory neurons(TG and DRG)and can be combined with the physical properties of G protein Gi/o-protein,which in turn directly or indirectly regulated the release of CGRP.The above results show that UT-R participates in the regulation of peripheral pain signal transduction.The chronic inflammatory stimulation can increase the level of urotensin II(UT-R endogenous ligand)in TG,indicating that UT-R may be involved in TG-mediated pain signal transduction.In human trigeminal vascular system,the study showed that p substance(SP)and CGRP were mainly found in the middle and small diameter TG neurons,suggesting that there might be a common localization of pain-related media(such as CGRP,SP,etc.)in the middle and small TG neurons expressing UT-R.The results indicated that UT-R involved in the occurrence of trigeminal neuralgia and pathophysiological regulation.Part I: Roles and mechanisms of UT-R in trigeminal neuralgia Objective: We study the effect of UT-R on the mechanical threshold of CFA-induced inflammatory pain model as well as the CCI-ION induced neuropathic pain model in mouse trigeminal neuralgia(TN),and clarify the underlying mechanism related to protein kinase regulation.We further elucidate the mechanism of UT-R involved in pain regulation and to develop the academic basis for its clinical application.Methods:(1)The mechanical pain threshold was detected in mice under the condition of 1 days,3 days,7 days,14 days and 28 days after ligation in CCI-ION model.To select the most significant time point of pain threshold,the mechanical pain thresholds of 1 h,2 h and 3 h were detected;(2)The CFA facial inflammatory pain model was prepared,and the threshold of mechanical pain was detected at different points of time(1 d,2 d,3 d,5 d,7 d,and 14 d).We selected the minimum time point(2 d)of the threshold,the expression of UT-R was detected by protein electrophoresis;(3)The CFA facial inflammatory pain model was prepared in mice,the threshold of mechanical pain was detected at 2 d.The trigeminal ganglion was injected with urotensin II(1 nmol),The mechanical pain thresholds at 1 h,2 h,and 3 h were detected respectively;(4)The co-expression of UT-R with NF-200,IB4 and CGRP in trigeminal ganglion neurons was detected by immunofluorescence double staining;(5)The T-type calcium channel blocker Z941 as well as the A-type K-channel blocker 4-AP abolished mechanical hypersensitivity induced by urotensin II.Results:(1)The CCI-ION model significantly reduced the mechanical pain threshold of mice,from ligation 7 days with the mouse head escape threshold significantly reduced.This effect has been able to sustain till 28 days;(2)Intra-TG injection of palosuran significantly reversed the CCI-ION induced mechanical pain threshold decrease.This effect reaches the peak within 1 hour and lasts to 2 hours;(3)Subcutaneous injection of CFA in the region of the trigeminal nerve area significantly induced a decrease in the mechanical pain threshold,which is most evident at 2 days and lasts until the 6th day.The CFA model can significantly increase the expression of UT-R protein in trigeminal ganglion in 2 days;(4)Intra-TG injection of urotensin II can induce the reduction of facial mechanical pain threshold,which can be abolished by the UT-R antagonists as well as the T-type calcium channel blockers TTA-P2 and Z941;(5)In mouse CFA inflammatory pain model,Intra-TG injection of UT-R antagonist reversed the pain threshold and lasted for 2 hours;(6)The Cav3.2 interference si RNA of trigeminal ganglion can significantly reduce the expression of Cav3.2.Knockdown of Cav3.2 reversed the CFA induced mechanical pain hypersensitivity.Application of the UT-R receptor antagonist can not further enhance Cav3.2 effect,suggesting that Cav3.2 may be one of the molecular targets of UT-R;(7)Immunofluorescence staining showed that UT-R and IB4 and CGRP were co-expressed in the small diameter trigeminal ganglion neurons,while the NF200 were less co-stained.In addition,UT-R and Cav3.2 were also expressed in the middle and small diameter TG neurons.Conclusion: Urotensin II significantly reduced the pain hypersensitivity in CCI-ION model of trigeminal neuralgia as well as decreased the mechanical pain threshold of peripheral inflammatory pain through UT-R and Cav3.2 channels.Part II: Regulation of UT-R on the excitability of TG neurons Objective: We determine the modulation of urotensin II on the excitability of trigeminal ganglion neurons in mice and elucidate the mechanisms to provide a direct experimental basis for revealing the effect of UT-R on peripheral sensory pain.Methods:(1)Action potentials of small-diameter neurons were recorded by using the whole-cell patch clamp in acute isolated trigeminal ganglion neurons in mice;(2)The current clamp was used to record the action potential of small diameter trigeminal ganglion neurons isolated acutely in 1-5 hours.The statistics of 1 sec were recorded,the discharge frequency,half width,resting film potential and action potential threshold of the cells in the control and urotensin II-treated groups were analyzed;(3)Applying 1 msec stimulation,the action potentials of small diameter trigeminal ganglion neurons were recorded by current clamp mode,and the effects of urotensin II on the AHP parameter were studied.(4)The receptor as well as the downstream signal transduction pathway of urotensin II on the neuronal excitability were determined by using UT-R antagonists and different protein kinase inhibitors;(5)After incubation with the T-type calcium channel blocker Ni Cl2 with trigeminal ganglion neurons,the effect of urotensin II on neuronal excitability was studied;(5)After incubation with the A-type potassium channel blocker 4-AP with trigeminal ganglion neurons,the effect of urotensin II on neuronal excitability was determined.Results:(1)Applying 1 sec stimulation to TG neurons,urotensin II significantly increased the firing frequency of small diameter trigeminal ganglion neurons,while there was no significant change in the peak amplitude of action potentials;(2)Applying 1 msec stimulation to TG neurons,0.1 ?M urotensin II significantly increased the action potential AHP of the small diameter trigeminal ganglion neurons;(3)Addition of T-type calcium channel blocker Ni Cl2 in extracellular fluid,the hyperexcitability induced by 0.1 ?M urotensin II can be abolished by A-type potassium channel blocker 4-AP;(4)Pre-incubation of TG neurons with the PKC inhibitor GF109203 X abolished the 0.1 ?M urotensin II-induced neuronal hyperexcitability;(5)With the A-type transient potassium channel blocker 4-AP added to extracellular fluid,pre-incubation of TG neurons with the T-type calcium channel blocker Z941 completely abolished the 0.1 ?M urotensin II-induced neuronal hyperexcitability;(6)The hyperexcitability of small-diameter trigeminal ganglion neurons induced by 0.1 ?M urotensin II was completely suppressed by pretreatment of TG neurons with the PKA inhibitor KT-5720.Conclusion: Urotensin II increased neuronal excitability of TG neurons through UT-R and the downstream protein kinase pathway,which will activated regulates different ion channels to change the whole cell current in TG neurons respectively.Part III: Regulation of UT-R on A-type potassium channel and T-type calcium channel current Objective: We further determine the regulation of urotensin II on the voltage-gated ion channels of trigeminal ganglion neurons in mice and elucidate the underlying cellular and molecular mechanism.We would like to reveal the roles of UT-R in modulation of both the excitability of small diameter TG neurons and trigeminal neuralgia in mice.Methods: The acute isolated trigeminal ganglion(TG)neurons were recorded with the voltage clamp mode.The effect of urotensin II on the different voltage gated ion channels and the signaling pathway mechanism were studied.The concentrations of urotensin II used in the present study were 1 n M,10 n M,0.1 ?M,1 ?M,and 10 ?M.(1)By using different extracellular fluid and the corresponding stimulation of the square wave,different voltage gated ion(sodium,potassium,calcium)channel currents were recorded.(2)By using electrophysiology(double stimulation square wave)and pharmacological methods to separate A-type potassium channel currents,we study the effects of urotensin II on A-type potassium channel current density and elucidate the underlying mechanisms.(3)By using different protein kinase blockers(HBDDE,U0126,PD98059 etc),we further clarify the molecular mechanism of urotensin II on A-type potassium channel currents.(4)The effect of urotensin II on PKC activity in TG cells was detected by ELISA.(5)The activity of MAPK kinase was determined by western blot analysis.The relevant mechanisms were further determined.(6)Adenovirus packaged sh RNA interference(GFP-tag)was used to knockdown the PKC alpha subtype expression.By using sh RNA interference method to infect cultured TG neurons,the small diameter TG neurons of green fluorescence were selected by inverted microscope,and A-type potassium channel current was recorded in the whole cell voltage clamp to study the effect of urotensin II on current density.(7)Cells were clamped at-110 mv and were depolarized to-30 mv,with nifedipine and omega-conotoxin in the extracellular solutions.By using the whole cell voltage clamp technique,we investigate the effects of urotensin II on T-type calcium channel currents.(8)Adenovirus-packaged Gq-protein sh RNA was chosen to knochdown the Gq-protein and was used to study whether or not Gq participates in the regulation of T-type calcium channels.(9)By using different signal pathway blockers,we study the signaling transduction of T-type calcium channels regulated by urotensin II.(10)The effects and mechanisms of urotensin II on Cav3 subtype were studied by using HEK293 transfected system.Results:(1)Bath application of 0.1 ?M U-II decreased the peak amplitude of IA by 34.1 ± 4.3 % in small TG neurons,while IDR was not significantly affected.After washout,the amplitude of IA partially recovered to the level before the U-II treatment within the 5 min.(2)Addition of U-II(0.1 ?M)induced a significant hyperpolarizing shift in the steady-state inactivation potentials of IA by ~8.3 m V,while the voltage-dependent activation potential was unaffected.(3)RT-PCR analysis revealed that the UT-R transcripts were present in mouse TGs.Immunoblot analysis of the mouse TG protein lysates further confirmed the endogenous expression of UT-R.Palosuran(1 ?M),a potent and specific antagonist of UT-R,alone had no significant effect on IA in small TG neurons,while treatment of TG neurons with palosuran(1 ?M)prevented the 0.1 ?M U-II-induced IA decrease.(4)We found that 0.1 ?M U-II significantly enhanced PKC activity(~1.9-fold)in mouse TG cells.This effect was prevented by the specific UT-R antagonist palosuran(1 ?M).Dialysis of small TG neurons with PKC 19-36(10 ?M),a peptide inhibitor of PKC,completely abolished the 0.1 ?M U-II-induced IA decrease,while intracellular application of its inactive analogue(IA-PKC 19-36,10 ?M)did not elicit any effect.Similar attenuation of the U-II-induced IA response was observed for application of the classical PKC isoform antagonist,Go6976.Dialysis of TG cells with the fast Ca2+ chelator BAPTA(20 m M),but not its inactive analogue dn-BAPTA,prevented the U-II-induced IA decrease.(5)Pre-incubation of TG neurons with 200 n M LY333531,a specific inhibitor PKC? isoform,did not affect the IA decrease induced by 0.1 ?M U-II.Contrastingly,pretreating TG neurons with HBDDE(1 ?M),a PKC? and PKC? inhibitor,completely abolished the 0.1 ?M U-II-mediated decrease in IA.Similar attenuation of the U-II-induced IA response was obtained for intracellular application of the specific PKC? peptide inhibitor(PKC?-IP).Dialysis of small TG neurons with 10 ?M PKC?-IP completely abolished the IA decrease induced by U-II.Knockdown of PKC? in TG neurons leaded to the attenuation of the U-II-mediated decrease in IA,indicating the involvement of PKC? signaling in the U-II-induced IA response.(7)The PKC?-dependent ERK activation might be involved in U-II-mediated responses.(8)The UT-R-mediated IA decrease subsequently increased the excitability of small TG neurons.(9)U-II significantly increased T-type calcium channel currents in small TG neurons through activation of UT-R and the downstream PKA signaling pathway.(10)In HEK293 transfected system,U-II significantly increased Cav3.2 channel currents,without any effects on Cav3.1 and 3.3.Conclusion: U-II regulates T-type calcium channels and A-type potassium channels through activating UT-R receptor and the downstream PKA and PKC?-ERK pathway,which then participates in the regulation of action potential firing of trigeminal neurons.In summary,this project identified the UT-R in peripheral sensory regulation of trigeminal neuralgia in mice,suggesting the peripheral TG neuronal UT-R in the pathophysiology and peripheral pain regulation of trigeminal neuralgia.Through the study of the functional regulation and mechanism of the primary afferent neurons of trigeminal neuralgia,we propose that UT-R regulated TG neuron function and participated in the trigeminal neuralgia.Furthermore,the hypothesis of peripheral pain regulation of trigeminal neuralgia is deepened,and the understanding of UT-R function is helpful for the breakthrough of clinical treatment of the trigeminal neuralgia.In addition,by using behavioral,immunohistochemical,molecular biology and electrophysiological studies,we elucidate molecular mechanisms of the UT-R function in TG.Therefore,we can systematically study the peripheral regulation mechanism of UT-R in trigeminal neuralgia,and might provide a new molecular target for exploring the clinical treatment of trigeminal neuralgia and developing the ideal therapeutic drug. |
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