| Postoperative pain is a clinically common form of acute pain,and the acupuncture has been used for many years to treat pain.Accumulating evidence indicated the efficacy of acupuncture for postoperative analgesia on oral operations,orthopedic surgeries,and limbs and gynecological surgeries.But the mechanisms underlying the effect of acupuncture are still unclear.Researchers have done a lot of work in exploring the mechanism of acupuncture in relieving postoperative pain at the spinal cord level,and our previous studes have indicated that electroacupuncture(EA)at Futu(LI18),Hegu(LI4)-Neiguan(PC6)could alleviate the cervical incisional pain,which was related with the decreased spinal algesic substances such as substance P(SP),NK-1R,COX-1,PGE2,5-HT1A,cAMP,CREB expressions,and also with the upregulation of spinal 5-HT2AR,and downregulation of spinal activities of microcytes and astrocytes.Amount of further work still need to do to reveal the mechanisms:.In recent years,dorsal root ganglion(DRG)has been identified by more and more experts as a key therapeutic target for pain neuromodulation,and its roles in sensory regulation and pain modulation have received increasing attention.While whether EA could exert the analgesic effect by regulating the functions of nerve cells in DRG,especially the contribution of the gamma-aminobutyric acid(GABA)ergic modulation on the acupuncture analgesia in DRG is poorly understood.DRG aggregates the greatest proportion of primary sensory neruosns,and the DRG neurons initiates the nociceptive impulse.Satellite glia cells(SGCs)closely envelope the cell body of sensory neurons,providing the basis for information exchange between nerve cells in DRG.We hypothesize that acupuncture can mediate the expressin of GABA of DRG,and enhances the communication between GABAergic and nociceptive neurons,between GABAergic neurons and SGCs by regulating the function of GABARs,and finally weakens the transmission of nociceptive information to the spinal cord and exerts analgesic effect.Based on this hypothesis and previous studies,using behavioral,immunofluorescence staining,molecular biological methods,we conducted experimants to verify the mechanisms from the following aspects in the cervical incional rat model,so as to provide experimental basis for better understanding of EA in relieving postoperative pain and the mechanism of acupuncture anesthesia from the perspective of communication between neuron-neuron and neuron-SGC in DRG.Part 1 Effect of electroacupuncture on thermal pain threshold and expression of calcitonin-gene related peptide,substance P and y-aminobutyric acid in the cervical dorsal root ganglion of rats with incisional neck painObjective:Acupuncture therapy effectively reduces post-surgical pain,but its mechanism of action remains unclear.The aim of this study was to investigate whether expression of GABA and the neuropeptides SP and calcitonin gene related peptide(CGRP)in the primary sensory neurons of cervical DRG are involved in EA-induced analgesia in a rat model of incisional neck pain.Methods:The pain model was established by making a longitudinal midline neck incision in 60 rats.Another 15 rats underwent sham surgery(normal group).Postincision,15 rats remained untreated(model group)and 45 rats underwent EA(frequency 2/100Hz,intensity 1 mA)at bilateral LI18,LI4-PC6 or ST36-GB34(n=15)each for 30min at 4h,24h,and 48h post-surgery,followed by thermal pain threshold(PT)measurement.30min later,the rats were euthanased and cervical(C2-6)DRGs removed for measurement of immunoreactivity and mRNA expression of SP/CGRP and the GABAergic neuronal marker glutamic acid decarboxylase 67(GAD67).Results:Thermal PT was significantly lower in the model group versus the normal group and increased in the LI18 and LI4-PC6 groups but not the ST36-GB34 group compared with the model group.Additionally,EA at LI18 and LI4-PC6 markedly suppressed neck incision-induced upregulation of mRNA/protein expression of SP/CGRP(P<0.05),and upregulated mRNA/protein expression of GAD67 in the DRGs of C2-6 segments(P<0.05).Conclusion:EA at LI18/LI4-PC6 increases PT in rats with incisional neck pain,which is likely related to downregulation of pronociceptive mediators SP/CGRP and upregulation of the inhibitory transmitter GABA in the primary sensory neurons of cervical DRGs.Part 2 Effects of electro-acupuncture on activities of satellite glial cells of DRG in rats with neck incision painObj ective:To observe the effect of EA intervention on the activities of SGCs in the DRG in rats with incisional neck pain and explore the mechanism of acupuncture anesthesia in thyroid surgery.Methods:SD male rats were randomly divided into control,model,EA-"Futu"(LI18),EA-"Hgu"(LI4)-"Neiguan"(PC6),and EA-"Zusanli"(ST36)="Yanglingquan"(GB34)groups,20 rats each group.Except the normal group,a longitudinal incision,about 1.5cm in length was done along the neck midline in the rats to prepare the model of incisional pain.In the EA-LI18,EA-LI4-PC6 and EA-ST36-GB34 groups,at 4h,24h and 48h after modeling,EA(1mA,2/100Hz)was applied,once a day,continuously for 3 times.In the normal group and the model group,no any intervention was applied.The thermal radiant apparatus was used to detect the thermal PT.The fluorescent immunohistochemistry,quantitative RT-PCR,ELISA and the western blotting(WB)were used to determine the immunoactivity of SGC,the mRNA expressions and protein content of IL-10,TNF-a and IL-6,mRN A expressions of glial fibrillary acidic protein(GFAP,SGC specific protein marker),and the connexin43 protein expression in C2-6 DRG.Results:After EA stimulation at LI18 and LI4-PC6,the thermal PTs were obviously prolonged compared with model group(P<0.05),modeling-induced dramatic increase of the GFAP-immunoactivity of SGC,GFAP mRNA expression,mRNA expression and protein content of IL-1β,1L-6 and TNF-a-in C3-6 DRGs were significantly suppressed(P<0.05),and IL-1β,TNF-a protein in EA-LI18 group and IL-6 protein in EA-LI4-PC6 group were also decreased remarkably(P<0.05),while except the IL-1β and TNF-amRNA expression,no significant changes were found in the EA-ST36-GB34 group compared with the model groups.Conclusion:EA intervention of LI18 and LI4-PC6 can significantly suppress pain reaction of neck incision in the rat,which is closely associated with its effects in down-regulating the activity of SGCs,decreasing the release of pro-inflammatory cytokines in the cervical DRGs.Part 3 GABARs possibly contribute the crosstalk between GABAergic and peptidergic neurons,GABAergic and satellite glial cells in electroacupuncture analgesia on neck incisional pain ratsObj ective:Acupuncture therapy is effective for relieving post-operative pain.Our previous study showed that EA at Futu(LI18)and Hegu(LI4)-Neiguan(PC6)could alleviate incisional neck pain,which was related with its effect in upregulating GABA expression in cervical(C2-6)DRGs,but whether its receptor subsets GABAAα2R and GABABR1 in C2-6 DRGs involve in EA analgesia or not remains unknown.Methods:Ninety male SD rats were randomized into normal control,model,LI18,LI4-PC6 and Zusanli(ST36)-Yanglingquan(GB34)groups.The incisional neck pain model was established by making a longitudinal incision along the midline of the rats’neck.EA was applied to bilateral LI18,LI4-PC6 or ST36-GB34 for 30 min at 4,24,and 48h after operation.The thermal PT of the neck was detected by a tail-flick unit,and the C2-6 DRGs were removed for assaying the immunoactivity of SP,GABAAα2R,GFAP,connexin43 and GABABR1 and the expression of GABAAα2R and GABABR1 mRNA and proteins using immunofluorescence,real-time PCR and WB,respectively.Results:The cervical thermal PT was significantly lower in the model group than the normal group(P<0.001),indicating a hyperalgesia after neck incision,and was considerably increased in both EA-LI18 and LI4-PC6 groups(P<0.001),but not in ST36-GB34 group compared with model group(P>0.05).Immunofluorescence staining showed that GABAAα2R expressed on SP+ neurons,and GABAbR1 on SGCs.EA of LI18 and LI4-PC6 markedly suppressed the modeling induced upregulation of the immunoactivity of SP(P<0.001,P<0.01,respectively),GFAP(P<0.01,P<0.001,respectively)and the protein expression of connexin43 in C3-6 DRGs(P<0.05),and significantly revised neck-incision induced down-regulation of expression of GABAAa2R and GABABR1(P<0.05)mRNAs and proteins.Conclusion:EA of LI18 and LI4-PC6 have an analgesic effect in incisional neck pain rats,which is related to its effects in up-regulating GABAergic inhibitory modulation on nociceptive peptidergic neurons and SGCs and weakening the communication between SGCs in the cervical DRGs in cervical DRGs.Part 4 Contribution of GABAergic modulation in DRGs to acupuncture analgesia in incisional neck pain ratsObjective:To verify the role of GABA receptors in C3-C6 DRG in regulating the communication of nerve cells in DRG during the analgesic effect of electroacupunctureMethods:120 male SD rats were randomized into DMSO+ LI18,DMSO+LI4-PC6,Bicuculline(Bic)+LI18,Bic+LI4-PC6,normal saline(NS)+LI18,NS+LI4-PC6,Phaclofen(Pha)+LI18,Pha+LI4-PC6 groups.The application of Bic and Pha to the C3-C6 DRGs of the rats was made after the basic pain threshold was measured,3 days later,the incisional neck pain model was established by making a longitudinal incision along the midline of the rats’neck.EA was applied to bilateral LI18 or LI4-PC6 for 30 min at 4,24,and 48h after neck operation.The thermal pain threshold(PT)of the neck was detected by a tail-flick unit,and the C2-6 DRGs were removed for assaying the immunoactivity of substance P(SP),NK-1R,GFAP,SP、NK-1R using immunofluoresce,the mRNA expression of GFAP、IL-1β、IL-6、TNF-α、Kir4.1 were analyzed with real-time PCR and the protein expression of Kir4.1 was detected by western blotting,and the protein content of IL-1β、IL-6、TNF-α were measured by Elisa.Results:The cervical thermal PT was significantly lower in the Bic/Pha+LI18 和Bic/Pha+LI4-PC6 groups compared with NS/DMSO+LI18 and NS/DMSO+LI4-PC6 groups(P<0.05).Compared with DMSO+LI18 and DMSO+LI4-PC6 groups,percentage of SP-positive neurons and the immunoactivity of NK-1R and GFAP and the mRNA expression of SP,NK-1R and GFAP in C 3-C 6 DRGs of Bic+LI18 和 Bic+LI4-PC6 groups were markedly increased(P<0.05).Compared with NS+LI18 and NS+LI4-PC6 groups,the immunoactivity and the mRNA expression of GFAP in C3-6 DRGs were significantly increased in Pha+LI18(P<0.05)and Pha+LI4-PC6(P<0.05)groups.Compared with NS+LI18 and NS+LI4-PC6 groups,the protein content and the mRNA expressions of IL-1β、IL-6 and TNF-αwere considerably increased in both Pha+LI18(P<0.05)and Pha+LI4-PC6(P<0.05)groups,and the protein expression of Kir4.1 was also downregulated in the Pha+LI18(P<0.05)and Pha+LI4-PC6(P<0.05)groups.Conclusion:After the GABAAR was blocked,its inhibitory effect on peptidergic SP neurons and NK-1R was weakened.when GABABR was blocked,its inhibition on SGCs was abated,and it is verified that the inhibition of GABABR on SGCs was exerted through the activation of Kir4.1 protein,and the alleviating effects of electroacupuncture at LI18 and LI4-PC6 on cervical incisional pain was mediated by GABAAR and GABABR which regulated the crosstalk between GABAergic and peptidergic neurons,and between GABAergic neurons and SGCs. |
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