| Objective:Pain is defined by International Association for the Study of Pain(IASP)as a painful experience in the sensory,emotional,cognitive and social dimensions associated with tissue damage or potential tissue damage,in which the emotional response to pain is now considered to be an important component of pain differentiated from sensation,especially in persistent and intractable pain.The anterior cingulate cortex(ACC),especially the rostral part of it(rACC),is considered to be a key component of the emotional circuit.Many studies have shown that electro-acupuncture(EA)can alleviate pain.Recent studies have shown that EA can also alleviate pain aversion(pain),but its mechanism is still unclear.It is well known that EA can activate endogenous opioid system to relieve pain,so we propose a hypothesis that EA may alleviate the pain aversion via the μ-opioid receptor activation in rACC region in rats.In this study,the conditioned position avoidance(CPA)test induced through CFA-injected rats acclimating in a specific place was used to investigate the rats’ pain aversion.We will apply CPA test,western blot and in vivo multichannel techniques to explore the mechanism of EA alleviating the pain aversion.Methods:1.To establish the CFA-induced conditioned place avoidance(C-CPA)modelA conditional position avoidance model(C-CPA)was established by matching CFA-induced chronic inflammatory pain stimulation with a specific environment.2.Detection of Paw Withdrawal Latency(PWL)First,the hot plate temperature was raised to 50 degrees.Then the rats were put into the instrument.The time of first licking the left foot or lifting the hind limb was observed and recorded.3.Electroacupuncture(EA)CFA or NS was subcutaneously injected into the left hindpaw to setup a chronic pain model.The acupuncture needle was inserted into the position of the bilateral Huantiao acupoint(GB30)of rat.The acupuncture needle was connected to the electro-acupuncture instrument,and the parameters were set to turn on the power supply for electro-acupuncture stimulation.4.In vivo observation of the discharge activities in rACC region by multi-channel electrophysiological technique.The discharge activities of neurons in rACC brain area was observed and recorded before and after administration.5.Western blotThe phosphorylation levels of three subunits NR1,NR2 A and NR2 B of rACC,the expression of μ-opioid receptor,NR1,NR2 A and NR2 B in rACC region were detected by western blot technique.6.Immunofluorescence stainingThe co-expression of μ-opioid receptor and NR1,NR2 A and NR2 B in rACC neurons were detected by immunofluorescence.Results: 1.Co-expression of μ-opioid receptor and NR1,NR2 A and NR2 B in rat rACC neurons.The subunits NR1,NR2 A and NR2 B of NMDA receptor and μ-opioid receptor in ACC neurons were co-expressed in the same neuron by immunofluorescence.2.To establish CFA-induced conditioned place aversion(C-CPA)model.Compared with NS group,the PWL value of rats in CFA group was significantly shortened on the third day after treatment(P < 0.05).There was no significant difference between NS group and rats in pain side on the third day(P > 0.05).In CFA group,rats in pain side stayed on the third day compared with the first day(P > 0.05).The retention time was significantly shortened(P < 0.05),and the avoidance score of CFA group was higher than that of NS group(P < 0.05).3.EA can alleviate the C-CPA response.In the sham EA group,the time spent on the pain side on the third day was significantly shorter than that on the first day(P < 0.05).There was no significant difference between the time spent on the pain side on the third day and the time spent on the first day in the EA group(P > 0.05).The avoidance score of the sham EA group was higher than that of EA group,and the difference was statistically significant(P < 0.05).4.EA does not change the thermal pain caused by CFA.The shamEA group and the EA group measured the thermal pain behavioral response of the rats on the third day,and there was no significant difference between the PWL values(P > 0.05).5.EA down-regulates the expression of p-NR1,p-NR2 A and p-NR2 B by activating the μ-opioid receptor in rACC.Compared with the sham EA group,the expression of μ-opioid receptor protein increased significantly(P < 0.05),while the expression of p-NR1,p-NR2 A and p-NR2 B protein decreased significantly(P < 0.05).6.Effect of EA on the increase of pain side discharge frequency in rACC brain induced by CFA.The firing frequency of neurons in the sham EA group was significantly higher than that in the non-pain side on the third day after condition matching(P < 0.05).There was no significant difference between the pain side and the non-pain side on the third day after condition matching(P > 0.05).7.Injection of CTOP,a μ-opioid receptor antagonist,into rACC reverses the effect of EA on reducing aversion in rats.Rats were injected with CFA or NS in the sole of their feet,NS or CTOP,a μ-opioid receptor antagonist,in rACC,and then EA.In the saline group,there was no significant difference in the time spent on the pain side on the third day and the time spent on the first day(P > 0.05).In the CFA and saline group,there was no significant difference in the time spent on the pain side on the third day and the time spent on the first day(P > 0.05).In the 2 nmol/μl CTOP group,the time spent on the pain side on the third day and stayed on the first day.There was no significant difference in the duration of stay(P > 0.05).In the 5 / 10 / 20 nmol/μl CTOP group,the duration of stay on the pain side on the third day was significantly shorter than that on the first day(P < 0.05)and the avoidance score was significantly higher than that in the other two groups(P < 0.05).8.Effect of intra-rACC injection of CTOP,a μ-opioid receptor antagonist,on the level of p-NR1,p-NR2 A and p-NR2 B.The level of p-NR1,p-NR2 A and p-NR2 B protein in EA group was significantly lower than that in sham EA group(P < 0.05);the expression of p-NR1,p-NR2 A and p-NR2 B protein in 5 / 10 / 20 nmol/μl CTOP group was significantly higher than that in NS group and 2 nmol/μl CTOP group(P < 0.05).9.CTOP,a μ-opioid receptor antagonist,was injected into rACC to block the effect of EA reversing the increase of CFA-induced discharging.There was no significant difference in neuronal discharge frequency between pain side and non-pain side on the third day after conditional matching(P > 0.05).There was no significant difference in neuronal discharge frequency between pain side and non-pain side on the third day after conditional matching(P > 0.05).There was no significant difference in pain side and non-pain side on the third day after conditional matching(P > 0.05).In low dose 2 nmol/μl CTOP group,the pain side was compared with non-pain side on the third day after conditional matching.There was no significant difference in the discharge frequencies of menstrual elements(P > 0.05).The discharge frequencies of pain side neurons were significantly increased on the third day after conditional matching in the injection of 5 / 10 / 20 nmol/μl CTOP(P < 0.05),and there was a significant difference in the discharge frequencies of pain side neurons compared with those of the other two groups on the third day(P < 0.05).Conclusion:Increased levels of p-NR1,p-NR2 A and p-NR2 B in rACC brain area induced by CFA injection into rat sole are the basis of increased neuronal discharge activity,and the latter is the cause of CPA response,causing pain in rats.The phosphorylation levels of NR1,NR2 A and NR2 B decreased after activation of the μ-opioid receptor in rACC brain area by EA stimulation,which reversed the enhancement of neuronal electrical activity and CPA response in rats,and inhibited the occurrence of pain aversion. |
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