| Acupoint Sensitization is defined as transformation of acupoint from stable state to dynamic state.The underlying mechanism of acupoint sensitization is largely unknown.The phenomenon of "point sensitization" has been recoginized for long time,but the concept had not been proposed until recently.Therefore,there are few mechanism studies.The animal model used for acupoint sensitization research is not yet established.There is a lack of methods for assessing sensitization of experimental animals.Knee osteoarthritis(KOA)is a highly prevalent joint disease.Acupuncture has been widely used in patients with KOA.Studies have shown that stimulation of sensitized acupoint in patients with knee osteoarthritis can achieve better therapeutic effect.In addition,the pathological changes and symptoms of a KOA animal model produced by intra-articular injection of mono-iodoacetate are similar to those of humans.The degree of injury can be managed through different doses and its pathogenesis is progressive over time.It may be a new rat model suitable for acupoint sensitization studies.Previous reports have suggested that the dorsal root ganglion magnifies the afferent information.And HCN2 channels on DRG carried an inward current(Ih)that plays a central role in peripheral sensitization.However,the role of HCN2 conducted Ih in the DRG on acupoint sensitization is not clear.The aim of this study is to establish a rat model of knee osteoarthritis suitable for acupoint sensitization study,screen appropriate indicators to determine acupoint sensitization and explore the role of DRG Ih on acupoint sensitization.Experiment 1: Establish a rat model of knee osteoarthritis suitable for acupoint sensitization study and screen appropriate indicators to determine acupoint sensitizationObjective: To establish a rat model of knee osteoarthritis suitable for acupoint sensitization study and screen appropriate indicators to determine acupoint sensitization.Methods: Rats intra-articularly administered monosodium iodoacetate(MIA,0.3,1 or 3 mg respectively)were evaluated for pain-like behavior and joint pathological changes until 28 days after injury.Then by using the suitable dose(1 mg MIA),the mechanical pain threshold of ST35,ST36,GB37 and non-acupoints was measured by electronic Von Frey stimulation.Mast cells aggregation and degranulation at these acupoints and non-aupocint controls were also tested.Furthermore,local skin tissue protein extracts were tested for histamine,adenosine,2-AG,AEA and CB2 R contents of acupoints were detected with ELISA.All used to screen for appropriate indicators of acupoint sensitization.Results: Both 1 mg and 3 mg MIA induced significant joint damage and persistent pain behavior.But animals accepted 3 mg MIA rapidly developed cartilage and bone damage within 14 days.One mg of MIA produces steady pain behavior and progressive joint damage,which was suitable for acupoint sensitization study.The mechanical pain threshold of bilateral ST35 and ST36 acupoints in the KOA group decreased,while the mechanical pain threshold of GB37 and non acupoint had no significant difference compared with control group.Number of mast cells in the ipsilateral ST35 and ST36 in KOA rats increased compared to normal control,and the rate of degranulation of mast cells on bilateral ST35 acupoints increased,but no such change was observed in GB37 aor non acupoints in KOA animals.No significant differences were found for histamine,adenosine,2-AG,AEA or CB2 R concentration in either side of the acupoint skin in KOA group compared with the control group.Conclusion:The results suggested that 1 mg MIA induced KOA model was suitable for acupoint sensitization research,and acupoint mechanical pain threshold could be used as index for evaluating acupoint sensitization in this animal model.Experiment 2: Effect of electroacupuncture stimulation at sensitized acupoint in rats with knee osteoarthritisObjective: To investigat the effect of electroacupuncture stimulation at sensitized acupoint in rats with knee osteoarthritisMethods: The therapeutic effects of EA treatment on sensitized acupoints(bilateral ST35 and ST36)were evaluated in 3 different senorios: Early-on EA,Mid-term EA and Delayed EA,with treatment started on day 1,day 7 or day 14 after MIA injection.Pathological changes of affected knee and pain related behaviors were tested.Results: Early-on EA treatment provided significant pain relief and reduced joint structure damage in KOA rats.Mid-term EA treatment produced limited pain reduction without significant joint preservation effect,while delayed EA treatment could not provide significant protective effects in either aspects.Conclusion:Electroacupuncture at sensitized acupoints had therapeutic effect against KOA in rats.The effect is related to the time of the beginning of the treatment.Experiment 3: Explore the role of DRG Ih on acupoint sensitizationObjective: To explore the role of DRG Ih on acupoint sensitizationMethods: The KOA model was used to investigate the possible mechanism of acupoint sensitization.Electrophysiological tests were conducted to examine changes in excitability and Ih density for C type and A? fiber current.Furthermore,and the hyperpolarization-activated/cyclic nucleotide-gated 2(HCN2)channel expression of DRG in KOA animals compared to comtrol animals were tested with western blot and immunofluorescent staining.And Ih specific blocker-ZD7288 was used to observe the effect of Ih on the phenomenon of acupoint sensitization with acupoint mechanical pain threshold test in vivo.Results: A substantial increase in Ih density of C-but not A?-nociceptors from bilateral L5 DRG was observed.The expression of HCN2 significantly raised in bilateral L5 DRG,especially in C-nociceptive neurons,after acupoint sensitization.Furthermore,ZD7288 attenuated mechanical threshold reduction in the bilateral sensitizated ST35 of KOA rats without affecting the mechanical threshold of GB37 or non-acupoint.Conclusion:Rusults suggested that increased excitability and upregulation of Ih /HCN2 channels in C-but not A?-nociceptive neurons in the L5 DRG contributes to the phenomenon of acupoint sensitization. |
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