Studies On The Mechanism Of NMDA Receptor Mediating The Affective Pain In The Anterior Cingulate Cortex

Objective:According the latest pain definition by the IASP,pain is a distressing experience associated with actual or potential tissue damage with sensory,emotional,cognitive,and social components.The affective pain is one of four main components of chronic pain,it seriously impact on human health and living quality.However,its mechanism is not clear yet.Many studies and our previous works have showed that rostral anterior cingulate cortex(r ACC)is involved in the formation of the pain-induced aversion.Clinical imaging studies have shown that the r ACC region may be activated by unwanted irritations and unpleasantness caused by pain.Some studies have confirmed that NMDA receptors play an important role in synaptic transmission,long-term potentiation,learning and memory,epilepsy,and pain.ACC brain regions have a large number of ionotropic glutamate receptors;it has also been found that the Glu N2 A and Glu N2 B subunits of the NMDA receptor in the r ACC region contribute to the formation of formalin-induced CPA(F-CPA)behavior.This indicates that NMDA receptors in the ACC brain region play an important role in pain and aversion,but NMDA receptors mainly have several subunits of NR1,NR2 A and NR2 B.It is not clear how different subunits play a role in pain and aversion.In the studies,the effect of NMDA will be further explored in the CFA-induced model.The laboratory has successfully induced CPA response(C-CPA)by subcutaneous injection of CFA in the previous study.This study will use this model to combine in vivo multi-channel electrophysiological recording,western blot protein detection and double-labeling immunofluorescence techniques have further studied the role of different subunits of NMDA receptors in pain and aversion.Methods:1.To build up an inflammatory pain model induced by CFAWe used male Sprague-Dawley rats(weighing 250-260g)to inject CFA subcutaneously into their left hind sole.The control group was subcutaneously injected with the same dose of normal saline(NS).2.To establish the C-CPA model.Divided into three groups:(a)blank control group(b)a saline group;(c)CFA-injected model group,n = 6-12/group.The injected dose of saline or CFA is 0.08 ml/head.We injected CFA as an analgesic substance into the skin of the left hind paw rats,causing chronic inflammatory pain response in rats,and combined with distinguishable environmental conditions.The rats developed an aversive behavioral response to the injured side environment,is successfully modelled.3.The effect of injection of APV or DNQX on pain in r ACC.In this experiment,rats were divided into 22 groups,(1)CFA model group.(2)A saline-injected group.(3)CFA was injected into the left hind paw and 0.5/2/8/16 nmol/ul APV or DNQX was given in r ACC.(4)NS was injected into the left hind paw,and 0.5/2/8/16 nmol/ul APV or DNQX were given in r ACC.4.Surveying the paw withdrawal thermal latency(PWL)Measure the first and third day's pain response.5.The in-vivo multi-channel technique was used to record,process and analyze the signal changes of r ACC neurons.6.To detect the expression levels of the NMDA receptor and phosphorylation of NR1/NR2A/NR2 B submit by western-blot technique.After the completion of the behavioral test,we detect the expression level of NMDA receptor and phosohorylated NR1/NR2A/NR2 B subunit in the r ACC region of each group by western-blot technique.7.Immunofluorescence staining.Results:1.Research results of behavioral observation1.1 Subcutaneous injection of CFA in the left paw significantly reduced the value of PWL.CFA was injected into the left hind paw of the rat,NS was administrated in r ACC region to setup a model of chronic pain,and the NS was injected into the left hind paw in another group of rats as control.The value of PWL in Day 1 and Day 3 were measured,and it was found that after the injection of CFA.On the third day,the value of PWL was significantly shortened(P<0.05),and there was no significant change in the other two groups.1.2 In the pain environment,CFA-injected rats stayed less time than in the non-pain environment.There was no significant difference in the duration of Day3 in the pain matching room between the non-injection control group and the NS control group.There was no significant difference in avoidance scores between the two groups.CFA(P < 0.05)was injected into the left hind paw.The time spent in Day3 pain environment Matching Room was significantly shorter than that in Day1.There was significant difference between the avoidance scores of the non-injected control group and the NS injection control group(P < 0.05).Therefore,the rats had an aversion reaction to the "pain environment" and stayed less than the "non-pain environment".1.3.AP? could inhibit C-CPA responses,but DNQX could not.In the treatment group of injecting CFA into the left hind foot paw and giving NS in r ACC,the baseline value measured by Day3 CPA was significantly less than that measured by Day1,and the avoidance score was higher,which resulted in aversion to "pain environment".The rats in the hind paw were injected with CFA and injected into the treatment group with different doses of 0.5/2/8/16(nmol/?l)AP? Glu N1?Glu N2 A and Glu N2 B in r ACC.The AP?injection dose was(2/8/16 nmol/?l),and the avoidance score was gradually reduced.There was no significant difference among the three groups,but there was statistical difference compared with NS group(P < 0.05).It showed that the high concentration group inhibited the pain response caused by CFA.That is to say,it alleviates the CPA reaction.There was no change in the avoidance score when the CFA was injected into the hind paws of the rats and the doses of 0.5/2/8/16(nmol/?l)DNQX were different in the r ACC.There was no significant difference compared with the NS group,which indicated that different concentrations of DNQX could not alleviate the pain response caused by CFA.It was shown that AP? could inhibit C-CPA reaction,but DNQX could not.1.4 AP?and DNQX could not change the value of PWL.The PWL of each group of rats injected with CFA and AP? or DNQX by r ACC was significantly shorter than that before treatment(P < 0.05).It shows that APV and DNQX cannot change the value of PWL.2.Electrophysiological resultsOn the first day,the base discharge frequency of the rats was measured.On the second day,the left hind Paw was injected with CFA,and the r ACC brain region was injected with different concentrations(0.5/2/8/16 nmol/?l)of the NMDA receptor antagonist AP?.The frequency of discharge in the r ACC brain regions of different concentrations of mice was observed on the third day.The frequency of action potentials in the “non-pain environment of the CFA+NS group was statistically significant compared with the action potential frequency in the "pain environment"(P<0.05),there was no significant difference in the action potential frequency between the CFA+2/8/16 nmol/?l group in the "non-pain environment" and the action potential frequency in the "pain environment".The frequency of discharge activity of neurons caused by CFA is down-regulated.Among them,the pain side parts were specifically compared,and it was found that the high concentration of the dose significantly inhibited the enhancement of neuronal discharge activity induced by CFA,and the difference was statistically significant(P<0.05).Among them,the CFA + 0.5/2/8/16 nmol/?l DNQX group had a higher discharge frequency in the "pain environment" than the "non-pain environment",and the difference was statistically significant(P<0.05),indicating that it could not be regulated by CFA.The frequency of discharge activity of the induced neurons is increased.3.Molecular biological resultsCFA was injected into the left hind paw of the rat,NS was injected into the r ACC,CFA was injected into the left hind paw,and 0.5/2/8/16 nmol/?l AP? was injected into the r ACC,Compared with CFA+0.5/2/8/16 nmol/?l AP?,the phosphorylation level of each subunit in CFA+NS group was not significantly different from CFA+0.5 nmol/?l AP?;compared with CFA+2/8/16 nmol/?l AP?,the phosphorylation level of each subunit in CFA+NS group decreased significantly(P<0.05,n=3).Conclusion:The antagonism of NMDA receptor by AP? can inhibit the pain response induced by CFA,decrease the firing frequency of increased r ACC neurons induced by CFA,and decrease the phosphorylation level of Glu N1?Glu N2 A and Glu N2 B subunit in r ACC.A smaller dose of AP? could not cause changes,but when the concentration was 2/8/16 nmol/?l,it could inhibit the pain response caused by CFA,but DNQX had no smilar effect.