| The latest definition of the International Association for pain is"pain is a painful experience of the sensory,emotional,cognitive,and social dimensions associated with tissue damage or potential tissue damage."This definition divides pain into four dimensions:sensory,emotional,cognitive,and social.Among these,pain emotion refers to negative emotions such as disgust,anxiety,and fear caused by noxious stimulation,which brings greater injury experience to patients.It has been proved that some drugs can control the pain-related aversion without affecting the judgment of the pain intensity,indicating that there is a certain difference in the brain structure and the conduction mechanism between the pain sensation and the pain emotion.It has also been experimentally demonstrated that the anterior cingulated cortex?ACC?located on the medial frontal lobe of the brain,especially its rostral rACC,is involved in pain-induced aversion.Previous research data and our previous studies have shown that the activation of NMDA receptors within the rACC is involved in the formation of pain-averse emotion,and opioid receptors are involved in the process of electroacupuncture analgesia,with three major subtypes:?-,?-and?-opioid receptors.Previous results in our laboratory have confirmed that activation of?-and?-opioid receptors in rACC can mediate electroacupuncture to relieve the affective pain in rats,but whether?-opioid receptor can mediate electroacupuncture to relieve the affective pain is not clear.In this study,we used behavioral science,pharmacology,molecular biology and multi-channel electrophysiological techniques to study the role of electroacupuncture in activation of?-opioid receptors in r ACC to alleviate the affective pain.Methods:1.Inflammatory pain model250g-270g adult male SD?Sprague Dawley?rats were selected and 0.08ml CFA was injected subcutaneously on the left hind foot for establishing an inflammatory pain model2.Conditioned place avoidance modelThe chronic inflammatory pain induced by CFA and"pain environment"were coupled,compare the stay time of the rats in the"pain environment"before and after the coupling to determine whether the C-CPA model was successfully established.3.Electroacupuncture stimulationEnsuring the location of the Huantiao?GB30?,the acupuncture needles are inserted into the location,giving electric stimulation4.Measurement of paw withdrawal latency?PWL?On the first and third days of CPA training,the PWL of each group was measured.The hot plate temperature increased to 50 degrees.The rats were put into plexiglass,and the time of licking or raising their hind limbs was recorded.Each animal was tested 3 times,and the interval was 15-20 minutes.5.Multi-channel electrophysiology recording in vivoA multi-channel electrode with a drug tube was made to carry out the multi-channel electrode embedding surgery.The electrode was buried in the rat rACC brain region.After a week of recovery,the rats were divided into groups and treated accordingly.We used in-vivo multi-channel technique to record neuronal discharge in rACC brain region.6.Western-blot.Samples of rat rACC brain region were taken and the phosphorylation level of?-opioid receptor and NMDA receptor in the rACC brain region of each experimental group was detected by Western-blot.7.Grouping experimentsGroup method using a two factor,grouped as follows:16 groups?n=6-8/group?=2?CFA,physiological saline?×4??-antagonists:2/10/20nmol/?l nor-BNI,normal saline?×2?shamEA,EA?.Results:Previous experiments in our laboratory have shown that CFA injected into the left hind paws of rats can induce thermal hyperalgesia and C-CPA reaction,and produce pain emotions;CFA can cause increased frequency of discharge of rACC neurons in pain side;CFA can Increase levels of phosphorylation of the NR1/NR2A/NR2B subunit in rACC;electroacupuncture stimulation reverses CFA-induced CPA response,inhibition of the discharge frequency of rACC neurons and the level of phosphorylation of NR1/NR2A/NR2B subunits in rACC[29,46].With these results as a precondition,we obtained the following results.1.Research results of behavioral observation.1.1 Successful establishment of C-CPA modelThe injection of CFA into left hind paw and rACC injected with NS,sham electroacupuncture group?CFA+NS+shamEA?in rats for third days was significantly reduced in the"pain environment"residence time than the first day?P<0.05?;in the NS+NS+EA group,before and after coupling in"pain environment"residence time,no significant difference?P>0.05?,with significant difference compared to?CFA+NS+shamEA?group in avoidance scores?P<0.05?.1.2 electroacupuncture stimulation relieves pain moodThe injection of CFA into left hind paw and rACC injection of NS,really EA group,before and after coupling in the"pain environment"residence time,no significant difference?P>0.05?;in the CFA+NS+shamEA group,rats for third days significantly reduced in the"pain environment"residence time than the first day?P<0.05?,two group with significant difference compared to avoidance scores?P<0.05?.1.3 antagonistic effect of?-opioid receptor reversing electroacupuncture on pain reliefThe left hind paw of the rat was injected with NS,and the?-opioid receptor antagonist nor-BNI or NS was given in the brain area of rACC,and the stimulation was carried out by pseudo electroacupuncture.The rats in the condition before and after"pain environment"time,there was no significant difference?P>0.05?;nor-BNI group and NS group compared avoidance scores had no significant difference?P>0.05?,It showed that nor-BNI itself does not affect CPA response in rats.The left hind paw was injected with CFA,the rACC with giving different concentrations of 2/10/20 nmol/?l?-opioid receptor antagonist nor-BNI or NS,electric stimulation.In each group,third days in the"pain environment"in time compared with the basic value:no statistically significant differences in the 2nmol/?l nor-BNI group?P>0.05?;no statistically significant differences in the 10nmol/?l nor-BNI group?P>0.05?;20nmol/?l nor-BNI group had statistically significant difference?P<0.05?.20nmol/?l nor-BNI group and NS group compared,avoid fraction increased significantly?P<0.05?.In the rACC injection of 2/10nmol/?l?-opioid receptor antagonist group,did not observe the reversal role to alleviate the pain emotion of EA,in rACC treated with 20 nmol/?l?-opioid receptor antagonist nor-BNI can reverse the utility of electroacupuncture on emotional pain.2 Neuron discharge characteristics in rACC brain region of rats recorded in vivo multichannel electrophysiological techniqueCFA was injected into the left hind paw of the rat,and different concentrations of2/10/20 nmol/?l?-opioid receptor antagonist nor-BNI were administered within the rACC,and the electroacupuncture-stimulating group?CFA+2/10/20 nmol/?l+EA?.In the 2nmol/?l nor-BNI group,there was no significant difference in the firing frequency of rACC neurons in"painful environment"and"non-painful environment"after conditional coupling?P>0.05?.In the 10 nmol/?l nor-BNI group,the firing frequency of rACC neurons in"painful environment"and"non-painful environment"after conditional coupling was not statistically significant?P>0.05?.In the 20nmol/?l nor-BNI group,the firing frequency of rACC neurons after conditional coupling was significantly higher in the"pain environment"than in the"non-pain environment"?P<0.0001?.The group of injection with CFA in left hind paw,injection with 2/10/20nmol/?l nor-BNI or NS in the r ACC region and real electroacupuncture stimulation in rats?CFA+/NS/2/10/20+EA?,One way ANOVA test indicated the frequency of discharge in the"pain environment"was significantly increased?P<0.0001?.Multiple comparisons showed that:20nmol/L nor-BNI group VS NS group,P<0.0001,with statistical difference.3 Detecting levels of?-opioid receptor and phosphorylated NR1/NR2A/NR2B subunit in rACC by western-blot3.1 Electroacupuncture stimulation activates?-opioid receptor expressionCompared with CFA+NS+sham EA group,the expression of?-opioid receptor in r ACC brain region increased in CFA+NS+EA group?P<0.05?3.2 Antagonistic effects of electroacupuncture stimulation on the expression of?-opioid receptorsCFA+2/10/20nmol/?l+EA group compared with CFA+NS+EA group,?-opioidreceptor the expression level of 2nmol/?l nor-BNI group was not statistically significant?P>0.05?,NR1/NR2A/NR2B subunit phosphorylation level was not statisticallysignificant?P>0.05?;the expression level of?-opioid receptor was significantlydecreased in 10nmol/nor-BNI group rACC?P<0.05?,phosphorylation of NR1/NR2A/NR2B subunit without statistical difference?P>0.05?.20nmol/?l nor-BNI compared with2/10nmol/?l group and NS group,the expression level of?-opioid receptor wassignificantly decreased in the rACC?P<0.05?,phosphorylation level of NR1/NR2A/NR2B subunit was significantly increased?P<0.05?.It was suggested that nor-BNI into rACC could antagonize the?-opioid receptor in r ACC and up regulate the phosphorylation level of NR1/NR2A/NR2B subunits.Conclusion:The results showed that 20 nmol/?l nor-BNI reversed EA in alleviating pain emotion,while the 2/10 nmol/?l nor-BNI did not play a role on reversal.It indicates that?-opioid receptor antagonist can up-regulate level of phosphorylated NR1/NR2A/NR2B subunit in rACC,reverse the role of electroacupuncture inhibit rACC neuronal firing rate and the role of electroacupuncture alleviate CFA induced painful affect. |
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