| Neuropathic pain is a severe disabling disease mainly resulted from peripheral or central nerve injury,which is characterized by a presence of spontaneous pain,allodynia and hyperalgesia.Given that the pathogenesis is extremely complex,the drug therapies clinically used to cure neuropathic pain are always ineffective and even have unpredictable side-effects.Recently,the transcranial direct current stimulation(t DCS)has been suggested as a safe and noninvasive neuro-modulatory technique.This technique modulates neural activity and cortical plasticity via applying small direct currents to specific brain areas,and has been proved to be effective in relieving the refractory pain caused by surgery or spinal cord injury.However,the analgesic mechanisms underlying remain largely elusive,and the analgesic effect of t DCS can be influenced by many factors such as electrode size,currrnt strength and duration,electrodes' positions and polarity,and the course of patients,etc.Besides,it has been shown that many neurotransmitters such as glutamate and GABA mediated the modulation of cortical plasticity by t DCS.Furthermore,the effect of t DCS can be blocked via the inhibition of cortical NMDA receptors.P2X receptors are a group of ionotropic purinergic receptor,which play an important role in pain processing.When the organisms obtain noxious stimulation,massive ATP are released from the injured neural terminals and combined with P2 X receptors,leading to cation influx and cell depolarization.Additionally,P2 X receptors can further activate the pain pathways through the interaction with glutamate receptors.Mounting evidences have shown that the activation of NMDA receptor takes part in the onset and maintenance of central sensitization in spinal cord during neuropathic pain.However,at cortical level,the activation of NMDA receptor and P2X3 receptor has analgesic effect,suggesting an opposite mechanism.Periaqueductal gray(PAG)is a pivotal region that is considered to be vital for pain processing and modulating.Studying the modulation of pain sensation in PAG will behelpful in better understanding the analgesic mechanism of t DCS.Our hypothesis is that t DCS facilitated the changes in cortical neuronal plasticity,further influenced the PAG and activated the descending inhibition pathway,exerting the analgesic effect.In this process,the NMDA receptor and P2X3 receptor may be involved in processing the pain sensation in neocortex and PAG.In this study,we will use the chronic constriction injury of sciatic nerve(CCI)as an animal model to conduct the t DCS manipulation.By screening the best stimulation factors,combining with morphology,molecular biology,electrophysiology and ethology,we will explore the exact role of P2X/NMDA receptors in neuropathic pain modulation and discuss their specific mechanisms in mediating the t DCS's analgesic effect.This study will lead to a better understanding of the mechanisms of neuropathic pain and will be helpful in improving the efficiency of t DCS's analgesic effect.Methods:1.CCI to SD rats was established as a neuropathic pain model,and the pain threshold of paw withdraw mechanical threshold(PWMT)and paw withdraw thermal latency(PWTL)was evaluated by von Frey and Hargreaves tests.2.Four modes of t DCS model was established with different positions and polarity,and the PWTL and PWMT were evaluated to choose the best mode of t DCS under single or repetitive application,and different currrnt strength,duration and intervening time.3.Western blot and immunohistochemistry were used to observe the protein level and distribution changes of P2X3 and NR2 B receptors in primary motor cortex(M1)and PAG after t DCS.4.Whole cell patch-clamp recording was used to observe the excitability changes of PAG neurons after applying the ?,?-me ATP(P2X3 activator)or A-317491(P2X3 inhibitor)or MK-801(NMDA inhibitor)in normal rats,and to observe the excitability changes of PAG neurons after applying the A-317491 or MK-801 in CCI rats.5.By employing the intra-vlPAG micro-cannula planted CCI rats,the PWTL and PWMT were evaluated after administrating the ?,?-meATP,in the condition of pre-applying A-317491 or MK-801.6.A-317491 or MK-801 was intra-vlPAG microinjected in CCI rats after t DCS treatment,and the PWTL and PWMT were evaluated to assess the P2X3/NMDA receptor'seffect on tDCS treatment.Results:1.CCI rat model was successfully established,and the PWMT and PWTL were significantly downregulated compared with normal rats.The most severe reduction was appeared on day 14 after surgery and the pain threshold began to recover after this time point.2.Anode and cathode t DCS rat models were successfully established.Both single Anode and cathode t DCS could increase the PWMT and PWTL of CCI rats,and the analgesic effect of ipsilateral of cathode t DCS was better than other modes,and this effect were vanished within 24 hours.The prolonged analgesic effect was observed under repetitive Anode and cathode t DCS,which was depended on the intensity and duration of current.The contralateral of anode t DCS gained the most stable analgesic effect among the four modes of t DCS.The efficacy of t DCS was also influenced by the intervening time.3.The protein level and distribution range of P2X3 and NR2 B in M1 and vlPAG were all significantly upregulated on day 14 after CCI compared with normal group.Both of the Anode and Cathode tDCS further elevated the expression level of P2X3 and NR2 B in M1 and vlPAG in CCI group.On day 14 after CCI,the expression of Glu R1 was upregulated in M1 and vlPAG,however,this upregulation was alleviated after positive/negative t DCS treatment.4.The whole-cell recording showed that the administration of?,?-me ATPprominently excited the PAG neurons in normal rats,while the A-317491 and MK-801 inhibited these neurons.After CCI,the PAG neurons exhibited a hyper-excited state compared with normal group and the A-317491 and MK-801 also downregulated the excitability of CCI neurons.5.After intra-vlPAG microinjection of A-317491 in CCI rats,the PWMT and PWTL of the ipsilateral hindpaw were unaltered.But the PWTL and PWMT of CCI rats were significantly upregulated after microinjection of ?,?-meATP.Furthermore,the analgesic effect of ?,?-me ATP was positively dose-dependent,and appeared rapidly and decayed slowly.With the pre-administration of A-317491 at same site,the analgesic effect of?,?-me ATP was obviously attenuated,reflected in the significantly smaller upregulation of pain threshold.6.After intra-vl PAG microinjection of MK-801 in CCI rats,the PWMT and PWTL of the ipsilateral hindpaw were significantly upregulated.This analgesic effect is dose-dependent and time-dependent.However,with the pre-administration of MK-801 in vlPAG,the analgesic effect of ?,?-me ATP was also significantly attenuated.7.The intra-vl PAG microinjection of A-317491 or MK-801 significantly blocked the analgesic effect of t DCS in the diffusion time course of the drug from the site of action.Conclusion:1.t DCS is a effective treatment for neuropathic pain,and the reasonable chosen of stimulation factors will improve the analgesic effect.It will exert the best analgesic effect when the contralateral M1 was chosen as the target for repeated anode stimulation.2.In PAG,the functional P2X3 is helpful for analgesia,and the inhibition of P2X3 will downregulate the pain threshold of CCI rats.The analgesic effect of P2X3 is mediated by promoting the presynaptic glutamate release.3.The NMDA receptor is either analgesic or pain-producing in PAG.The inhibition of NMDA receptor can upregulate the pain threshold of CCI rats,suggesting that NMDA receptor is involved in the central pain processing.However,the inhibition of NMDA receptor attenuated the analgesic effect of P2X3 receptor,indicating that this analgesic effect is mediated by NMDA receptor.4.The analgesic effect of t DCS is achieved by upregulating the expression of P2X3/NMDA receptor in M1 and PAG,and by activating the central descending inhibition pathway. |
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