Effect Of Electroacupuncture Intervention On Synaptic Plasticity And Related Protein Expression In Amygdala In Pain-Negative Affection Rats

BACKGROUNDPain is an unpleasant subjective feeling and emotional experience associated with peripheral tissue damage or potential damage,which leads to emotional changes like anxiety,fear and averseness.Pain has multiple dimensions including sensation,emotion,cognition,etc.As an important part of pain,pain-related memory has been paid more and more attention in recent years.Studies have revealed that the limbic system is mainly involved in emotional expression of pain,and the amygdale,known as the"emotional brain",is an important nucleus of the limbic system in generating emotional reactions,detecting salient stimuli and adjusting mood,anxiety,fear related behaviors,autonomic activity and hormone levels.Results of fMRI researches showed a signal enhancement of the amygdala in response to a strong emotional response caused by pain stimulation,which means the amygdala is responsible for processing information related to the emotional components of pain.Chronic pain is associated with changes in pain sensitivity,cognition,and mental activities,in addition to changes in the structure and function of the brain.Chronic pain leads to reorganization of brain anatomical structure and changes of gray matter density.During chronic pain process,amygdala function changes in synaptic plasticity may be associated with changes in synaptic proteins.Glutamate is a major excitatory neurotransmitter in the nervous system,and participates in synaptic plasticity,being regarded as one of the mechanisms underlying processing of learning and memory.GABA is a major inhibitory neurotransmitter in the central nervous system,and is rich in the amygdale,allowing the control of amygdala output through direct inhibition,feedforward inhibition and disinhibition.It was demonstrated that GABAA receptor-mediated synaptic inhibition is lost or impaired in a model of arthritic pain that contributes to the pain-related increase of excitatory transmission,and GABAB-receptors reduce synaptic transmission between parabrachial axon terminals and central nucleus of amygdala-Locus coeruleu(CeA-LC)neurons by inhibiting N-type calcium channels,modulating both fear-memory formation and pain perception.PSD-95 is an important structural protein in postsynaptic density,which participates in central sensitization of neuropathic pain by connecting the glutamate NMDA receptor located on the cell membrane and the signal transduction system in the cell.The Piccolo protein is located in the presynaptic cytoplasmic matrix,which plays an important role in the neurotransmitter transmission.Acupuncture is an effective therapy for chronic pain,and is widely used in clinical practice.Abundant clinical evidence showed that acupuncture is also effective in improving depression,anxiety,fear and other emotional disorders.It has a positive effect on pain induced mood,and can relieve the negative emotion of chronic neuropathic pain.Our previous studies observed in chronic neuropathic pain rats that repeated electroacupuncture(EA)alleviates pain sensory component and emotional component which may be related to the up regulation of expression of corticotropin-releasing factor receptor 1(CRF-1R),CRF-2R,N-methyl-D-aspartate(NMDA)receptor subtype NR2B,Gamma-amino butyric acid(GABA)receptor subtype A and C genes and muopioid receptor(MOR)protein,as well as the down-regulation of Ionotropic AMPA receptor GluAl protein.However,its mechanism is still not clear.In this research,in chronic neuropathic pain-induced negative affection(CNPNA)model rats,we observed the effect of EA stimulation of "Zusanli"-"Yanglingquan" on changes of emotional and sensory components of pain behavioral reactions,and expression levels of GABAA receptor subtype ?2,GABAB1,Glu-NMDA receptor subtype NR1,PSD95 and Piccolo genes and proteins with quantitative RT-PCR and western blot,and validated the involvement of GABAA and GABAB receptor proteins in EA-induced amelioration of sensory or/and emotional components by intra-amygdaloid microinjection of their antagonists.In addition,we also observed ultrastructural changes of synapses of neuronal cells in the amygdaloid nucleus by using a transmission electron microscopy,so as to reveal the mechanisms of EA analgesia from integrated functional and structural plasticity.Materials and MethodsPart OneA total of 56 Male Wistar rats(230g-250g)were randomized into normal control,model,EA,and anesthesia+EA(A+EA)groups(n=14 in each group,8 for quantitative RT-PCR and 6 for Western Blot).The CNPNA model was established by ligation of the left sciatic nerve and repeated electrical stimulation of the soles in a pain-paired compartment.For acquisition of conditioned place aversion(CPA),the conditioning procedure comprised a pretest session on day 1,followed by 3 consecutive training days in a CPA apparatus.EA(1mA,2/15Hz)was applied to bilateral“Zusanli"(ST 36)and“Yanglingquan"(GB 34)for 30min,once daily for 7 days.After the EA intervention,the rats were put into the conditioned place aversion(CPA)box for 20 minutes,and two more hours later,removed to the unconditioned control box for 20 minutes.The thermal pain threshold(paw withdrawal latency,PWL)of the bilateral paws was measured by using a Tail-Flick Unit.The time spent in the conditioned control box was determined by using the aforementioned CPA-paired compartments.The expression levels of GABAAB2,GABAB1,NMDA receptor subunit NR1,and PSD-95 genes and proteins,and piccolo gene in the right amygdala area were determined using quantitative RT-PCR and Western Blot.Part twoFifty Male Wistar rats(230g-250g)were randomized into five groups:normal control,CCI+NA+saline,CCI+NA+EA+GABAA antagonist(Flumazenil),CCI+NA+EA+GABAB antagonist(Phaclofen)groups(n=6).The CNPPNA model was established by ligation of the left sciatic nerve.Five days later,except for the normal group,the rats of the other 4 groups received bilateral cannula implantation in the amygdala(P2.5mm,L4.5mm,H7.7mm)on a stereotaxic instrument according to the coordinate system described by Paxinos and Watson.After 10 days' recovery from operation,the rats were received repeated electrical stimulation of the sole in pain-paired compartments of conditioned control box,once daily for 3 days.Bilateral intra-amygdala microinjection of saline(0.5 ?L/side),Flumazenil(2?g/?L,0.5?L/side),phaclofen(2?g/?L,0.5?L/side),DMSO in saline(5?g/?L,0.5?L/side)were respectively conducted by using a mini-pump at a rate of 0.1?L/min,once daily for 7 days.After the microinjection,EA(1mA,2/15Hz)was applied to bilateral ST 36 and GB34 for 30min,once daily for 7 days.The thermal pain threshold(PWL)of the bilateral paws and the time spent in the aversion-paired compartment in every group was detected 3 and 7 days after EA interventions.Part threeSixteen male Wistar rats were randomized into normal,model,EA and AEA groups(n=4 in each group).The methods of modeling,EA intervention,thermal pain threshold detection and PLA test procedures are the same with those described in part one.At the end of the experiments,the rats were perfused transcardially with 4%paraformaldehyde plus 2%glutaraldehyde.After section of the amygdala tissue,a transmission electron microscopy was used to observe the changes of the structure of the synapses of neuronal cells in the amygdale,followed by measuring the length of the active zones,thickness of the post-synaptic density(PSD),synaptic cleft width and curvature of the synaptic interface.Results(1)After modeling,PWL difference(PWLD)values of the model group were significantly increased(P<0.001),and the time spent in the CPA-paired compartment was considerably decreased compared with the control group(P<0.001).After EA intervention for 3 and 7 days,the PWLD levels of both EA and AEA groups were apparently decreased(P<0.05),and the time spent in the CPA-paired compartment was apparently increased in the EA group(P<0.05),suggesting a pain relief and an improvement of the negative affection after EA intervention.(2)Compared to the normal group,the expression level of GABAA?2,GABAB1,PSD95 genes and proteins were apparently lower(P<0.05),and that of Piccolo gene was down-regulated(P<0.05),while the expression levels of NMDA-NR1 protein was remarkably upregulated(P<0.05).After EA intervention for 7 days,the expression levels of NMDA-NR1,GABAa?2,GABARB1,PSD95 genes and protein were remarkably increased in the EA group(P<0.05).The expression level of Piccolo gene was increased(P<0.05).The expression level of Piccolo gene was apparently lower in the AEA group than in the EA group(P<0.001).(3)Results of intra-Amygdala microinjection of antagonists of GABAA and GABAB receptors showed that after modeling,PWL difference(PWLD)values of the model group were significantly increased(P<0.001)and the time spent in the CPA-paired compartment was considerably decreased(P<0.001).After EA intervention for 7 days,compared with the model group,the PWLD levels and the time spent in the CPA-paired compartment in both CCI+NA+EA+GABAA inhibitor(Flumazenil)and CCI+NA+EA+GABAB inhibitor(phaclofen)groups had no apparent changes(P>0.05),while the pain threshold and time spent in the conditioned control box of the DMSO+EA group were significantly increased(P<0.01),suggesting an elimination of both EA-induced increase of pain threshold and EA-induced prolongation of time spent in the PLA-compartment after blocking GABAA and GABAB receptors in the amygdale region.(4)In comparison with the normal group,the length of the active zones,thickness of PSD and the curvature of the synaptic interface were considerably decreased(P<0.001),and the synaptic cleft width was notably increased(P<0.001)in the model group.Compared with the model group,the length of the active zones and PSD thickness were significantly increased(P<0.001),and the synaptic cleft width was markedly decreased(P<0.01)in both the EA and AEA groups,and the curvature of the synaptic interface was markedly increased in the EA group(P<0.05),indicating an improvement of the synaptic plasticity changes of neuronal cells in the amygdale following EA intervention.Conclusions(1)EA stimulation of ST 36-GB 34 can improve both sensory and affection dimensions of pain in the neuralgia negative affection rats,which is closely related to its effect in activating GABAA and GABAB receptors,and is also probably associated with its effects in up-regulating the expression levels of NMDA-NR1,GABA?2,GABAB1 PSD95 genes and proteins as well as Piccolo gene in the Amygdala region.(2)EA can remodel the synaptic plasticity changes of neuronal cells in the Amygdala by reversing neuropathic pain plus negative affection induced decrease of the length of the active zones,PSD thickness and the curvature of the synaptic interface,and widening of the synaptic cleft in neuropathic pain rats.