| Objective:Chronic persistent pain has attracted attention as a disease that has a major impact on the economy and society.Chronic pain not only brings unbearable pain to patients,but also produces more negative negative emotions that affect people’s physical and mental health.Compared with pain perception,the nerve conduction pathways that mediate pain emotions and the central mechanism they produce are very different.A large number of preclinical studies have suggested that the anterior cingulate cortex(ACC)medial to the frontal lobe of the brain is involved in the treatment of pain mood and ACC receives signals from the medial thalamus and cortical areas,among which r ACC regulates the negative emotions of inflammatory pain.Experimental studies of animal behavior,electrophysiology and other related experiments also showed that the anterior cingulate cortex is mainly involved in emotional functions and plays an important role in the treatment of pain emotional components.Experimental studies have observed that chemically damaging the head of ACC can inhibit the conditional position avoidance response(CPA)caused by formalin.There are mainly three types of opioid receptors in brain regions of ACC: μ-opioid receptor,δ-opioid receptor and κ-opioid receptor.Our previous studies have demonstrated that the activation of μ-and δ-opioid receptors in rACC brain region can down-regulate the phosphorylation of NMDA receptor subunits in rACC brain regions,reverse increased neuronal firing frequency of rACC induced by complete Freund’s adjuvant(CFA),inhibite CPA response and play a role in alleviating painful emotional responses,but it is not clear whether the activation of κ-opioid receptors has a similar effect to μ-and δ-opioid receptors,so we will explore it.In this study,CPA-induced Conditioned Position Avoidance(C-CPA)model was used as a measurement standard of pain emotion in rats,and the microinjection of different concentrations of κ-opioid receptor agonist Dynorphin A was administrated in rACC brain regions..Behaviour test,in vivo multichannel electrophysiological recording techniques and Western blot technique will be used to explore whether the activation of the κ-opioid receptor in the rACC brain region can relieve the affective pain in rats.Methods:1.To establish an inflammatory pain modelAdult 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 observe the CFA-induced conditioned place avoidance(C-CPA)behavioral responsesRats were randomly divided into 3 groups:(1)the Naive group;(2)the saline(NS)-injected group;(3)the CFA-injected(0.08ml)experimental group.(n=6 to 8/group).CFA was injected in the left hind paw of rats to produce persistent inflammatory pain,coupled with a recognizable and memoryable environment to produce conditioned place avoidance reaction model.We compared the rats’ residence time in the pain side of the apparatus before and after “pain environment” acclimation and calculated the avoidance score(CPA Score)to determine whether the rats appeared aversion responses.3.Detection of Paw Withdrawal Latency(PWL)The PWL of rats in each group was measured on the first day and the second day after the injection of CFA on the left paw,and the changes of thermal pain in rats after the injection of CFA and rACC with different concentrations of Dynorphin A were observed4.The in-vivo multi-channel technique was used to record,process and analyze the signal changes of rACC neurons.5.To detect the expression levels of the κ-opioid receptor and phosphorylation of GluN1 / GluN2 A / GluN2 B subunit by western-blot.After the completion of the behavioral test,we detect the expression level of κ-opioid receptor and phosphorylated GluN1 / GluN2 A / GluN2 B subunit in the r ACC region of each group by Western-blot.6.Experimental groupingAccording to left hind paw subcutaneous injection with CFA /NS,rACC brain areas are injected different concentrations.The rats were divided into the following twelve groups.12 groups rats(n=6-8/group)=2(CFA/NS)× 6(five different concentrations of κ-opioid receptor agonist /NS)Results:1.Intracerebral injection of kappa-opioid receptor agonist Dynorphin A in rat rACC inhibited CFA-induced conditional avoidance response(1)After subcutaneous injection of CFA into the left paw,rats’ thermal paw reflex latency(PWL)was significantly reduced and the pain model was successfully established.In CFA group,the PWL on the third day was significantly lower than that of the first day(P<0.05);In NS group and no injection group,there was no difference between PWL of the third day and the baseline value of the first day(P>0.05).These showed the injection of CFA into the left hind paw caused inflammatory pain response in rats.(2)Injection of CFA subcutaneously on the left paw causes CPA responses in ratsThe CFA group showed that the time spent on the pain side was significantly shorter on the third day before and after the pain environment matching(P<0.05),which showed rat produced aversive reaction.In no injection group and the NS group,there was no significant difference between the time of the third day in pain-side and that of the first day before and after the pain environment matching(P>0.05),at the same time,there was no difference between the two avoidance scores(P>0.05).These suggested that injection of CFA in the posterior foot of the rat could cause a CPA response.(3)Injection of κ-opioid receptor agonist Dynorphin A in rACC brain can inhibite C-CPA responseThe left paw subcutaneous injection of NS and r ACC injection of κ-opioid receptor agonist Dynorphin A or NS,which were NS + κ-opioid receptor agonist group and NS + NS group.These two groups of rats were conditioned and adapted in the resident environment(pain environment)after footpad injection.Compared with the first day,the residence time in pain side on second day after environmental adaptation(the third day of the experiment)had no difference(P>0.05),and there was no significant difference between the two groups avoidance scores(P> 0.05).These suggested that injection of Dynorphin A in rACC itself does not affect the CPA response in rats.CFA was injected subcutaneously in the left paw and different concentrations κ-opioid receptor agonist Dynorphin A or NS were injected into the rACC.Each group of rats was injected with CFA on the sole of the foot and matched with the pain environment.In 5 μg/μL and 10 μg /μL Dynorphin A groups,the resident time in the pain side on the third day before and after the pain environment matching compared to the first day had no significant difference(P>0.05),while resident time of the CFA+NS,CFA+0.5 μg/μL,1 μg/μL,2.5 μg/μL groups in the pain side on the third day were less than the first day,and the difference was statistically significant(P<0.05).The avoidance score was significantly different between CFA+NS group and CFA+5 μg/μL Dynorphin group(P<0.05);and CFA+NS group and CFA+10 μg/μL Dynorphin group avoidance fraction was also statistically significant(P<0.05),while avoidance score both CFA+5 μg/μLand CFA+10 μg/μL Dynorphin A groups had no significant difference(P>0.05),These showed that both high concentration of 5 μg/μL and 10 μg/μL Dynorphin A played a role in inhibiting CPA response.Rats on the left side of the foot were injected with CFA,and rACC was injected 0.5 / 1 / 2.5 / 5/10 μg/μL Dynorphin A / NS.PWL value in each group of rats on the third day were less than that of the first day,and all had significant differences(P<0.05).These suggested that Dynorphin A did not affect the thermal pain caused by CFA.2.Injection of κ-opioid receptor agonist Dynorphin A in rACC brain can inhibite the CFA-induced addition in firing rate of rACC neurons(1)After CFA was injected subcutaneously on the left hind paw of rats,the frequency of neuronal firing in the r ACC brain area increased.In the CFA group,the discharge frequency of neurons in rACC brain regions in pain-side and non-pain-side on the third day after conditioning were different,and the frequency of pain side was significantly higher than non-pain side.The difference was statistically significant(P<0.05).The frequency of neuronal firing in the rACC brain area on the third day after matching in the CFA group was significantly higher than that in the NS group and Naive group(P<0.05).These suggests that injection of CFA in the posterior foot of the rat could increase the frequency of neuronal firing in r ACC brain regions.(2)κ-opioid receptor agonist Dynorphin A itself does not affect the discharge activity of rACC brain regionsThe left paw subcutaneous injection of NS and r ACC injection of κ-opioid receptor agonist Dynorphin A or NS,which were NS + κ-opioid receptor agonist group and NS + NS group.The discharge frequency of neurons in rACC brain regions of pain side on the third day after matching of the two group was compared with that of non-pain side,and there was no difference in the discharge frequency of neurons between painful and non-painful side in both groups(P>0.05).These suggested that injection of Dynorphin A in rACC itself does not affect the frequency of neuronal firing in rACC brain regions.(3)κ-opioid receptor agonist Dynorphin A inhibite CFA-Induced increase in neuronal discharge frequencyCFA was injected subcutaneously in the left paw and different concentrations κ-opioid receptor agonist Dynorphin A or NS were injected into the rACC.The discharge frequency of neurons in the pain side on the third day of CFA+5 μg/μL Dynorphin and CFA+10 μg/μL Dynorphin A groups were compared with the discharge frequency of neurons in the non-pain side rACC brain after the pain environment matching,and there was no difference in the discharge frequency of neurons between painful and non-painful side in both groups(P>0.05),while on the third day of CFA+NS,CFA+0.5 μg/μL,1 μg/μL,and 2.5 μg/μL Dynorphin A group,the frequency of pain side was significantly higher than that of non-pain side of the neurons and the difference was statistically significant(P<0.05).Compared with CFA+NS group,the discharge frequency of neurons in rACC brain regions in pain-side of CFA+5 μg/μL Dynorphin and CFA+10 μg/μL Dynorphin A groups were significantly decreased(P<0.05).Electrophysiological results showed that both high concentration of 5 μg/μL and 10 μg/μL Dynorphin A could inhibite the CFA-induced addition in firing rate of rACC neurons.3.Intracerbral injection of κ-opioid receptor agonist Dynorphin A in the rat rACC could up-regulate the expression of κ-opioid receptor and down-regulate the increased phosphorylation levels of NMDA receptor subunits GluN1,GluN2 A,and GluN2 B in rACC brain regions by CFA.r(1)Subcutaneous injection of CFA into the left hind paw of rats increased the phosphorylation levels of NMDA receptor subunits GluN1,GluN2 A,and GluN2 B in rACC brain regions.Compared with CFA+NS group and Na?ve group,the expression levels of GluN1,GluN2 A,GluN2B phosphorylationin in the rACC of of CFA+NS group significantly increased(P<0.05).It showed that CFA increased phosphorylation levels of GluN1,GluN2 A,GluN2B subunits in rACC.(2)The κ-opioid receptor agonist Dynorphin A upregulates the expression level of κ-opioid receptor and down-regulated the increased phosphorylation levels of NMDA receptor subunit GluN1,GluN2 A,GluN2B induced by CFA in rACC.Rats on the left side of the foot were injected with CFA,and rACC was injected different concentrations Dynorphin A.Compared with CFA+NS group,the expression levels of of κ-opioid receptor of CFA+5 μg/μL Dynorphin A group and CFA+10 μg/μL Dynorphin A group were significantly increased(P<0.05),and the phosphorylation of NMDA receptor subunit GluN1,GluN2 A,GluN2B was decreased(P<0.05),while there was no difference between the two groups(P>0.05).Compared with CFA + NS group,the expression of κ-opioid receptor and phosphorylation of NMDA receptor subunit GluN1,GluN2 A,GluN2B in other concentration groups were not significantly different(P>0.05).The results showed that high concentration of κ-opioid receptor agonist Dynorphin A upregulated the expression of κ-opioid receptor and down-regulated the increased phosphorylation levels of NMDA receptor subunit GluN1,GluN2 A,GluN2B induced by CFA in rACC.Conclusion:After injection of CFA in rats,the phosphorylation level of NMDA receptors subunits GluN1,GluN2 A and GluN2 B in brain regions of rACC were up-regulated,and the frequency of neuronal firing was increased,which caused the CPA response in rats and resulted pain emotional reactions.Injection of a certain concentration of κ-opioid receptor agonist DynorphinA in rACC activated κ-opioid receptor,down-regulated the phosphorylation levels of GluN1,GluN2 A,and GluN2 B subunits,and reversed the increase in the frequency of neuronal firing in r ACC brain regions induced by CFA.,which relieved the pain emotional response. |
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