| Purpose:The incidence of chronic pain is estimated to be20-25%worldwide. Few patients withchronic pain obtain complete relief from the drugs that are currently available, and morethan half report inadequate relief. Research on the new strategies for the treatment ofchronic pain is pertinent to improve patients' quality of life. Trigeminal neuralgia is anuncommon disorder characterized by recurrent attacks of lancinating pain in the trigeminalnerve distribution. Typically, brief attacks are triggered by talking, chewing, teeth brushing,shaving, a light touch, or even a cool breeze. The pain is nearly always unilateral, and itmay occur repeatedly throughout the day. It has become an area of intense researchactivity–largely because it represents a disorder with high unmet medical need. It is not asingle entity but rather includes a range of heterogenous conditions that differ in aetiology,location and initiating cause. Neurons at all levels in trigeminal nerves (trigeminalganglion neurons and trigeminal nerve main nuclear, ridge beam nuclear, hypothalamus,the cerebral cortex neurons) improve excitability, enhance the facilitation, reduce painthreshold when TN onset. The high excitability results in neurons paroxysmal greatly firewith light or no stimuli, then it afferent to central neurons casing pain. But these neuronalextreme excitability and the reasons for the firing are not known. P2receptor is a keyprotein involved in pathological pain. Peripheral noxious stimuli activate P2receptor andcause membrane depolarization in sensory dorsal root ganglion (DRG) cells or TG neurons.Lawson et al. Several types of Kv channels have been proposed as potential targetcandidates for pain therapy. It was recently demonstrated that a selective KCNQ/Kv7channel opener (retigabine), selectively reduces the activity of axotomized Aδ/C-fibers, butnot uninjured axons, and human C-fiber axons. Takeda et al showed experiments in theirtrigeminal neuropathic/inflammatory pain model indicated that the common changes in theKv channels, such as IA, IKin rats with neuropathic/inflammatory pain, were significantlysuppressed compared to those in na ve rats. Since previous reports suggested that IAand IKare important for regulating the firing frequency and duration of action potentials in theTRG neuron, respectively.Thus this study observed the mechanism of P2receptors regulateKv channels using the pain behavior, electrophysiology, and molecular biology methodsand so on. in order to find the target treatment for trigeminal neuropathic pain and providesthe basic theory for clinical treatment, that exploring what kind of P2receptors are the regulatiors of IAchannels and which signaling pathway should be through, also involved inthis research.Methods:1. Animals:(1) in normal rats: SD male rats (200-250g) were divided randomly intodifferent groups to do the double-immunostaining of P2Y2and Flurogold or Kv1.4orKv3.4or Kv4.2or Kv4.3. SD male rats (80-120g) were divided randomly into differentgroups to be treated with UTP/ATP, Suramin and U0126.(2) in ION-CCI rats: SD malerats (200-250g) were divided randomly into two groups: one was Sham group, the otherwas ION-CCI group.2. Methods:(1) ION-CCI surgery,(2) target injection through infraorbital nerve,(3) ratbehavior test,(4) TG neurons culture,(5) immunofluorescence and cellularimmunofluorescence,(6) P2Y2antisense,(7) the reverse transcription–polymerase chainreaction analysis,(8) western blotting,(9) electrophysiologyResults:1. normal rats:(1) Fast effect on IAby ATP in the trigeminal neurons: through electrophysiology (a) IAwassignificantly reduced on T type TG neurons, IApeakwas (4.18±0.63) nA(n=14) reducedto (1.34±0.22) nA (n=13),(P<0.05,paired t-test).(b) IAwas not change on S type TGneurons.(c) IAwas inhibited on part of ATP-insensitve TG neurons.(2) P2Y2receptors and Kv1.4, Kv3.4, Kv4.2and Kv4.3were co-expression in high rate,respectively on TG neurons.(3) Long-term effect by ATP on the TG neurons: using electrophysiology we can see:(a)active P2Y2receptors reduced the threshold of action potential on TG neurons:147.1±32.9pA to58.3±10.3pA (control: n=7; UTP: n=9,**P＜0.01). Meanwhile itincreased the number of action potential:(control:1.3±0.9spikes400ms, n=3; UTP:10.8±3.5spikes400ms, n=4,*P <0.05).(b) active P2Y2receptors significantlyreduced IAand it can be blocked by suramin, a selective P2Y2antagonist (Control:0.14±0.01nA vs UTP:0.09±0.01pA, n=20, P <0.05; Suramin:0.13±0.01pA, n=9,P>0.05vs control).(c) the inhibition of active P2Y2receptors on IAcan be blocked byU0126, an ERK antagonist (Control:0.14±0.01nA vs UTP:0.09±0.01pA, n=20, P<0.05; U0126:0.15±0.03pA, n=11, P>0.05vs control).(d) The mRNA expressionof Kv1.4, Kv3.4, Kv4.2and Kv4.3were significantly inhibited by active P2Y2 receptors through RT-PCR.2. ION-CCI rats:(1) The number of neurons which is co-expression of P2Y2and Kv1.4or Kv3.4orKv4.2or Kv4.3significantly reduced.(2) The threshold of whisker pad in ION-CCI rats was dose-dependently recovery bysuramin and maintained45minutes using target injection(saline:0.96±0.4g n=8,Suramin0.3:11.45±2.4g n=7, Suramin3:29.96±4.1g n=6,**P<0.01;##P<0.01vs Suramin0.3).(3) When P2Y2receptors were down regulated by P2Y2antisense, the threshold ofwhisker pad in ION-CCI rats was significantly recovery which was prolonged for120hours(saline:0.49±0.1g n=7; antisense:49.54±8.0g n=5, P<0.001).(4) When P2Y2receptors were down regulated by P2Y2antisense, the mRNAexpression of Kv1.4, Kv3.4, Kv4.2and Kv4.3were increase.(5) When P2Y2receptors were down-regulated by P2Y2antisense, the proteinexpression of pERK was significantly decreased.Conclusion:On TG neurons, active P2Y2receptors could increase the excitability of TG neuronsbecause it can down-regulate the expression of Kv1.4, Kv3.4, Kv4.2and Kv4.3through ERK signaling pathway. Then these results suggest that inhibition of P2Y2receptors could be one of key treatment for the neuropathic pain. And it can supply aTheoretical basis for new target drug treating the neuropathic pain from ourexploration.
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