| Objective: According to the International Symposium on Pain Research(IASP),the pain can be divided into two components: sensory-discriminative and emotional-affective.The sensation of pain senses the nature of the pain,the strength and the position of the pain;the emotional reaction of the pain includes anxiety,disgust,fear and other bad mood.A large number of studies have shown that the sensory and emotional responses of pain are produced by different central mechanisms and are mediated by different neurotransmission pathways.Human and animal behavior,electrophysiology,brain imaging and other related treatment have provided evidence that the anterior cingulate cortex,which locates in the frontal lobe of the brain,involves in pain-induced disgust formation,and plays an important role in the behavioral manifestations of adverse emotions,especially in the rostral ACC.Excision of rat bilateral r ACC avoids the conditioned place avoidance(CPA)of animals caused by the emotional aspect of pain.Some clinical and animal studies have shown that electroacupuncture stimulation can alleviate the emotional response of humans and animals;existing information and our past work shows that traditional acupuncture or modern electroacupuncture treatment can activate the body’s opioid receptors to relieve pain.Opioid receptors are divided into three types: μ-receptor,δ-receptor,κ-receptor.Previous studies have shown that electroacupuncture can relieve pain in rats through activation of μ-opioid receptor,but it is not clear whether the pain can be relieved by activating δ-opioid receptor,we will delve into this study.The animal model used in this experiment is the model that complete Freund’s adjuvant(CFA)induced conditional position avoidance(C-CPA).It combined with r ACC injected different concentrations of naltrindole,which is the δ-opioid receptor antagonist.In this way,we can measure the C-CPA behavioral response and heat pain behavior(paw withdrawal latency,PWL).We will discuss whether the antagonism of δ-opioid receptor in r ACC can reverse the effect of electroacupuncture on relieving pain.Methods: 1.To establish an animal model of chronic persistent inflammatory pain induced by CFA Adult male SD(Sprague Dawley)rats weighting 250-270 g were injected with 0.08 ml complete Freund’s adjuvant(CFA)subcutaneously in the left plantar to establish an inflammatory pain model.The control group was injected with 0.08 ml saline(NS)and noninjection of foot in the left.2.To set up CFA-induced conditioned place avoidance model The rats were randomly divided into 3 groups:(1)the uninjected control group;(2)the saline(NS)-injected control group;(3)the CFA-injected(0.08ml)experimental group.(N=6 to 8/group).The C-CPA model was established by using CFA-induced chronic inflammatory pain as a specific condition and coupling with a distinguishable environment.We compared the rats’ time to stay in the pain environment before and after CFA-injection and calculated the avoidance score(CPA Score)to determine whether the rats had CPA responses.3.Electroacupuncture stimulation Adult male SD rats were randomly divided into electroacupuncture EA and sham EA.After the injected with NS / CFA,we inserted the acupuncture needles into the rat’s Huantiao point(GB30),fixed with the tape on the thigh.We used the wire to connect acupuncture needles and electro-acupuncture instrument,starting electroacupuncture stimulation after turnning on the power.4.Detection of Paw Withdrawal Latency(PWL)The PWL of each group was measured on the first day and the third day of CPA respectively.We observed the effects of CFA on the pain response of rats and the change of PWL value after injection of different doses of naltrindole and electroacupuncture stimulation.5.To detect the expression levels of the δ-opioid receptor and phosphorylation of NR1 / NR2 A / NR2 B subunit by western-blot.After the completion of the behavioral test,we detect the expression level of δ-opioid receptor and phosphorylated NR1 / NR2 A / NR2 B subunit in the r ACC region of each group by Western-blot.6.Grouping experiments According to the left plantar subcutaneous injection with CFA / NS,r ACC district administration / NS and stimulation of Huantiao point.The rats were divided into the following eight groups.1)injection of NS in the left plantar,injection of NS in the r ACC,EA;2)injection of NS in the left plantar,microinjection of NS in the r ACC,sham EA;3)injection of CFA in the left plantar,microinjection of NS in the r ACC,EA;4)injection of CFA in the left plantar,microinjection of NS in the r ACC,sham EA;5)injection of NS in the left plantar,microinjection of 1 / 2.5 / 5 / 10 μg/μl naltrindole in the r ACC,EA;6)injection of NS in the left plantar,microinjection of 1 / 2.5 / 5 / 10 μg/μl naltrindole in the r ACC,sham EA;7)injection of CFA in the left plantar,microinjection of 1 / 2.5 / 5 / 10 μg/μl naltrindole in the r ACC,EA;8)injection of CFA in the left plantar,microinjection of 1 / 2.5 / 5 / 10 μg/μl naltrindole in the r ACC,sham EA.Results: 1.Subcutaneous injection of CFA will change thermal pain behavior in rats.The group of injection of CFA in rat left plantar compared with the group of injection of NS and the control group of uninjection,In the CFA group,PWL of the first day after treatment was significantly shorter than the third day(post 7.23±1.65 s VS pre 12.54±0.72 s,P<0.05,n=6),the group of injection of NS(post 15.62±2.99 s VS pre 14.35±1.05 s,P<0.05,n=6),and the control group of uninjection(post 13.66±2.22 s VS pre 14.27±1.05 s,P>0.05,n=6),there was no significant change in PWL compared with the respective baseline values.This shows that injection of CFA in rat left plantar can cause inflammatory pain.2.Subcutaneous injection of CFA will induce to produce CPA reaction The control group of uninjected plantar(post 275.18±34.35 s VS pre 324.83±34.35 s,P>0.05,n=6)and subcutaneous injection of saline group(post 287.32±41.35 s VS pre 320.42±65.66 s,P>0.05,n=6),there was no significant difference in the duration of stay in the "pain environment" matching room between the third day and the first day,likewise,there is no difference between the two groups’ avoidance scores(P>0.05).Subcutaneous injection of CFA group(post 193.83±69.4s VS pre 315.3±34.6s,P<0.05,n=6),the time of staying in the "painful environment" on the third was significantly less than the first day,so the rats had a disgusting reaction to the environment.This suggests that injection of CFA in the posterior foot of the rat can cause a CPA response that producing emotional pain.3.Electroacupuncture stimulation relieves CFA-induced conditioned place avoidance(CPA)responses in rats Rats were injected with CFA in the left hind paw and then given electrical stimulation(EA)or sham acupuncture stimulation(sham EA)in Huantiao point.After the injection of CFA and pseudo-electroacupuncture in rats,the time of the "painful environment" on the third day was shorter than on the first day,there was significant differences,(post 174.67±31.03 s VS pre 323.03±84.94 s,P<0.05,n=6);after true electroacupuncture stimulation,the time of the "painful environment" on the first and third day was no significant difference,(post 370.4±80.34 s VS pre 400.57±81.03 s,P>0.05,n=6).The CPA score of EA group was significantly smaller than sham EA group.There was significant difference between the two groups.(EA group-77.23±35.38 s VS sham EA group-148.4±31.15 s,P<0.05,n=6).This suggests that electroacupuncture stimulation can relieve CFA-induced CPA responses in rats.After injection of NS on the left hind paw of the rats,the bilateral Huantiao points were given electrical stimulation(EA)or sham acupuncture stimulation(sham EA).There was no significant difference between the two groups in the duration of stay in the pain environment on the third day and the first day,sham electroacupuncture group(post 306.63±30.25 s VS pre 309.8±35.62 s,P>0.05,n=6);true electroacupuncture group(post 278.34±41.07 s VS pre 315.16±51.77 s,P>0.05,n=6).There was no statistically significant difference between the two groups(NS+EA group-30.67±6.36 s VS NS+sham EA group 12.33±1.86 s,P>0.05,n=6).This shows that electroacupuncture stimulation does not cause normal rat disgust or reward response.After the left hind paw rats were injected with CFA,the bilateral Huantiao were given electrical stimulation(EA)or sham acupuncture stimulation(sham EA),on the third day,the rats thermal pain behavioral response were measured,there was no statistical difference between groups,EA group(post 8.97±1.09 s VS pre 7.34±0.52 s,P>0.05,n=6);sham EA group(post 8.53±0.86 s VS pre 7.63±0.26 s,P>0.05,n=6).Electroacupuncture stimulation of bilateral Huantiao point does not affect chronic inflammatory pain by the CFA caused in rats.4.Injection of δ-opioid receptor antagonist naltrindole in r ACC reversed the role of electroacupuncture relieving emotional pain The hind paw was injected with CFA,r ACC was injected with δ-opioid receptor antagonist naltridole or NS,no electroacupuncture stimulation.The time of staying in the "painful environment" on the third day compared to the first day: naltrindole group(post 241.5±26.32 s VS pre 373.68±35.65 s,P<0.05,n=7);NS group(post 183.63±22.60 s VS pre 318.67±37.61 s,P<0.05,n=6);there was no significant difference about the CPA score between the naltrindole group and the NS group(-92.76±12.63 s VS-70.42±11.80 s,P>0.05,n=6).Naltrindole itself does not affect rat CPA response.The hind paw was injected with CFA,r ACC was injected with δ-opioid receptor antagonist naltrindole or NS,electroacupuncture stimulation.The time of staying in the "painful environment" on the third day compared to the first day: naltridole group(post 287.50±67.03 s VS pre 388.37±63.10 s,P<0.05,n=11);NS group(post 372.75±71.35 s VS pre 392.76±66.7s,P>0.05,n=11);the CPA score of naltrindole group was significantly higher than the NS group’s,there are significant differences(-100.90±32.45 s VS-20.01±26.53 s,P<0.05,n=6).Naltrindole injection reverses theeffect of electro-acupuncture stimulation on relieving pain.The left plantar was injected with CFA,r ACC was injection with different concentrations(1 / 2.5 / 5 / 10 μg/μl)of δ-opioid receptor antagonist naltrindole,the effect of reversing that electro-acupuncture stimulation relieving emotional pain was different.The time of staying in "Pain environment" on the third day compared with the first day : 1 μg/μl naltrindole group was not statistically different(post 304.6±40.3s VS pre 293.0±25.28 s,P>0.05,n=11);2.5 μg/μl naltrindole group was not statistically different(post 284.3±62.62 s VS pre 375.3±28.0s,P>0.05,n=8);5 μg/μl naltrindole group was statistically different(post 287.5±35.28 s VS pre 388.4±31.08 s,P>0.05,n=11);10 μg/μl naltrindole group was statistically different(post 252.0±29.46 s VS pre 319.1±12.24 s,P>0.05,n=11).Injection of 1 / 2.5 μg/μl naltrindole in r ACC did not have the effect of reversing of electro-acupuncture to relieve pain,but injection 5 / 10 μg/μl naltrindole in r ACC had an important role.The reversal effect of 5 μg/μl naltrindole was more strongly,but compared 10 μg/μl naltrindole group,their effect is not statistically significant.When the rats’ hind paw was injected with CFA,there was no significant difference in PWL between 1 / 2.5 / 5 / 10 μg/μl naltrindole injected in r ACC group and NS group(P>0.05).The PWL value of each group: CFA+NS group 8.94±1.26 s,n=7;CFA+1 μg/μl naltrindole group 9.22±1.15 s,n=6;CFA+2.5 μg/μl naltrindole group 9.0±0.60 s,n=8;CFA+5 μg/μl naltrindole group 10.36±1.44 s,n=6;CFA+10 μg/μl naltrindole group 9.96±1.40 s,n=6;the injection of naltridole within r ACC does not affect CFA-induced thermal hyperalgesia.5.Detection of expression levels of δ-opioid receptor and phosphorylated NR1 / NR2 A / NR2 B subunit in r ACC by western-blot The group of injection with CFA in hind paw,injection with NS in r ACC and real electroacupuncture stimulation in rats(CFA+NS+EA),which compared with the group of injection with CFA in hind paw,injection with NS in r ACC and sham electroacupuncture stimulation(CFA+NS+sham EA),the expression of δ-opioid receptor in r ACC was increasing,which was statistically significant(P < 0.05).Electroacupuncture stimulation can activate δ-opioid receptors in rats’ r ACC.The group of CFA-injection in foot,NS-injected in r ACC and pseudo-electroacupuncture stimulation(CFA+NS+sham EA),was compared with the group of NS-injection in foot,NS-injection in r ACC and given pseudo-electroacupuncture stimulation(NS+NS+sham EA),and the phosphorylation levels of NR1 / NR2 A / NR2 B subunit in r ACC were significantly increasing,with statistical significance(P<0.05).CFA maybe lead to an increase in phosphorylation expression of NR1 / NR2 A / NR2 B subunits in rat r ACC.The group of CFA-injection in foot、NS-injection in r ACC and electroacupuncture stimulation(CFA+NS+EA),was compared with the group of CFA-injection in foot、NS-injection in r ACC and pseudo-electroacupuncture stimulation(CFA+NS+sham EA),and the phosphorylation levels of NR1 / NR2 A / NR2 B subunit in r ACC were obviously reducing,with statistical significance(P<0.05).Electroacupuncture can down-regulate the phosphorylation expression of NR1 / NR2 A / NR2 B subunits in r ACC.Conclusion: 1.Electroacupuncture stimulation activates δ-opioid receptor in r ACC to relieve the CFA-induced emotional reactions.2.The antagonist of the δ-opioid receptor within r ACC can reverse the effect of electroacupuncture stimulation that relieving the CFA-induced pain response,5 / 10 μg/μl of the dose of the naltrindole reversed the effect of electroacupuncture that relieving pain and 1 / 2.5 μg/μl of the dose of naltrindole did not play a reversal effect.3.The phosphorylation of NR1 / NR2 A / NR2 B subunit in r ACC was significantly up-regulated in CFA-induced inflammatory pain model.4.Electroacupuncture can activate the δ-opioid receptor and down-regulate expression of the phosphorylation of the NR1 / NR2 A / NR2 B subunit in r ACC,thereby relieving pain. |
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