| BackgroundParkinson’s disease(PD)is the second most common neurodegenerative disease and has become one of the leading causes of neurological disability.Currently,the prevalence exceeds 6 million people worldwide and is expected to increase to 12 million by 2050.L-dopa induced dyskinesia(LID)may occur in approximately 50-80% of PD patients treated with its clinical first-line agent levodopa(L-dopa)for more than 5-10 years.LID is characterised by involuntary or choreiform hyperactivity or dystonia of the trunk and limbs,which seriously affects the quality of life of patients and may even lead to disability.In recent years,with the increase in the number of people with LID and related research,some researchers have suggested that there is abnormal synaptic plasticity in the LID brain.This may be associated with the severe loss of dopaminergic(DA)neurons in the substantia nigra of PD models,followed by fluctuating DA stimulation with L-dopa,causing an imbalance between the direct and indirect pathways,and over-activation of the direct medium spiny neurons(d MSN),resulting in altered synaptic plasticity.Glutamate(Glu)plays a key role in the regulation of neurogenesis,synaptic growth,and neuronal survival in the brain.Dysfunctional Glureceptors lead to cognitive impairment,loss of synaptic plasticity,and neuronal damage.Glutamate metabotropic receptor 5(mGluR5)is inv olved in the regulation of synaptic plasticity and has been shown to be most closely associated with LID.Acupuncture has been shown to produce clinically significant improvements in abnormal behavioral profiles in patients with LID,but the exact mechanism is not yet clear.In contrast,electroacupuncture has been shown in studies of other diseases to have a modulatory effect on mGluR5 and to have a modulatory effect on synaptic plasticity.Therefore,this study proposes the hypothesis that electroacupuncture may interfere with abnormal behavior in LID by modulating synaptic plasticity.ObjectiveSD male rats were used to make a PD model using 6-hydroxydopamine(6-OHDA),and then the LID model was replicated on this basis by intraperitoneal injection of L-dopa.The study was carried out to observe the effects of electroacupuncture on the behavior,striatal synaptic structure,synaptic-related protein levels and Glureceptor-related signaling pathways in LID rats,to verify the mechanism of abnormal synapti c plasticity in LID,and to reveal the possible mechanisms of electroacupuncture intervention in LID,to provide a theoretical basis for the clinical treatment of LID.Methods Part Ⅰ.In this experimental part,SD rats were used,and it was proposed to construct a primary model of PD using 6-OHDA first,and then replicate the LID model by intraperitoneal injection of L-dopa on top of the primary model.The rats were divided into five groups: blank control group(Ctrl),sham-operated group(Sham),Parkinson’s disease model group(PD),L-dopa induced dyskinesia model group(LID),and L-dopa induced dyskinesia + electroacupuncture intervention group(LE).The "DU16"," LR3" and " ST36" acupoints were selected for electroacupuncture stimulation in the LE group.The rats in each group were compared by conducting the open field test,suspension test,pole climbing test,forelimb function test,abnormal in voluntary movement(AIM)score,and time to the first black stool to observe the behavioral effects of electroacupuncture on each group.Part Ⅱ.In this part of the experiment,SD rats were used to construct a primary model of PD using 6-OHDA and based on the primary model,the LID model was replicated by intraperitoneal injection of L-dopa,divided into four groups: Ctrl,Sham,LID,and LE.The " GV16"," LR3" and " ST36" acupoints were selected for electroacupuncture stimulation in the LE group.Changes in the tyrosine hydroxylase(TH)content in the substantia nigra and striatum of each group were observed by immunohistochemistry,followed by transmission electron microscopy to observe the changes in dendritic spine density,synaptic gap,synaptic interface curvature,post-synaptic dense material thickness and number of perforated synapses in the synapses of striatal neurons of each group.The morphological changes of the synaptic spines of striatal neurons in each group were then observed by Golgi staining,and the effects of electrodes on the synaptic structure of LID rats were comprehensively evaluated.In addition to the synaptic structure,protein blotting was used to detect the changes in the content of PSD-95,△ FOSB,and p-ERK in the striatum of rats to evaluate the effect of electricity on the synaptic function of LID rats.Due to the importance of Glureceptors in synaptic plasticity,real-time fluorescence quantification was used to detect ionotropic and metabotropic glutamate receptors to screen the key targets of electroacupuncture affecting abnormal synaptic plasticity in LID rats.Part III.The second part of the experiment screened that mGluR5 may be an important target for electroacupuncture intervention in LID,so this part added CHPG,an agonist of mGluR5,and MPEP,an inhibitor of mGluR5,for validation.The rats were divided into seven groups: Ctrl,CHPG alone(CC group),LID,LE,LID +MPEP(LM),LID + CHPG(LC group),and LID + CHPG +electroacupuncture(LCE group).The intervention consisted of electroacupuncture stimulation at the " DU16"," LR3" and "ST36" acupoints in the LE group.In the LM group,MPEP was injected intraperitoneally.In the LC group,CHPG was injected stereotactically into the brain,and in the LCE group,CHPG was injected stereotactically into the brain and electroacupuncture stimulation was added.Since the literature did not reveal whether CHPG affected the replication of the LID model,this experim ent first chose to construct a CC model by stereotaxically injecting mGluR5 agonist CHPG into the brain of normal SD rats.The rats’ behavioral changes were observed using the open field experiment,water maze experiment,pole climbing experiment,suspension experiment,and AIM score to find the appropriate agonist dose that did not affect the construction of the LID model.MPEP,a mGluR5 inhibitor,has been shown to significantly improve the behavior of LID animal models.Therefore,MPEP was used as a positive drug in part of this experiment to compare the abnormal involuntary activity scores,synaptic structure,and synaptic-related protein changes in the LE and LM groups,and to observe whether the improvement effect of electroacupuncture and mGluR5 inhibitor on LID was similar.Following this,it was observed whether the mGluR5 agonist blocked the therapeutic effect of electroacupuncture on LID.The LE and LCE groups were compared,Golgi staining was used to observe the striatal synaptic structure,and changes in the level of the synapse-associated protein PSD-95 m RNA were quantified by real-time fluorescence to verify whether mGluR5 was a key target of electroacupuncture in improving synaptic plasticity in LID.After verifying that mGluR5 may be a key target of electroacupuncture to improve synaptic plasticity in LID,the m RNA levels of mGluR5,CAMK II,CREB,and BDNF were quantified by real-time fluorescence,and the protein levels of mGluR5 and BDNF and the phosphorylation changes of p-CAMK II and p-CREB were measured by protein blotting to investigate the specific mechanism of electroacupuncture to improve synaptic plasticity in LID.The mechanism of action was investigated.Results Part I.1 Electroacupuncture did not attenuate the ameliorative effect of L-dopa on motor dysfunction in PD ratsCompared with the Ctrl group,there were no significant differences in the results of the Sham group in the open-field experiment,the pole-climbing experiment,the forelimb function and the hanging experiment(all P>0.05);compared with the Ctrl group,the total distance of active movement,the time of active movement,the mean movement speed,the score of the hanging experiment and the forelimb function in the response box of the PD group rats in the open-field experiment were significantly lower(all P<0.01),and the time of the poleclimbing experiment Compared with the PD group,the LID group showed a significant increase in total distance travelled,active movement time,mean movement speed,hanging test score and forelimb function(all P<0.01)and a significant decrease in climbing test time(P<0.01);compared with the LID group,the LE group showed a significant increase in total distance travelled,active movement time,mean movement speed,hanging test score and forelimb function(all P<0.01).Compared with the LID group,there was no significant difference in the total distance of active movement,active movement time,mean movement speed,hanging test score,and pole climbing test time in the LE group(all P>0.05),and there was a trend of gradual increase in the efficacy of forelimb function to stability.This indicates that the effect of electroacupuncture on the motor dysfunction of PD rats was not reduced by the acupuncture points " DU16"," LR3" and " ST36".2 Electroacupuncture improved the AIM score of LID ratsThe Ctrl,Sham,and PD groups did not show AIM throughout the experiment;compared with the Ctrl group,the LID group showed a significant increase in axial,limb,oromandibular,motor,and total AIM scores on days 6,11,16 and 21(all P<0.01);compared with the LID group,the LE group showed a significant decrease in total AIM and motor scores on days 6,11,16 and 21(all P<0.01),limb and axial scores decreased significantly on days 11,16 and 21(all P<0.01),and oromandibular scores decreased significantly on day 21(P<0.01).This indicates that electro-acupuncture points " DU16"," LR3" and " ST36" improved the abnormal involuntary movements of LID rats.3 Electroacupuncture improved the non-motor function of LID ratsIn the open field experiment,there was no significant difference between the Ctrl and Sham groups(P>0.05);the activity time and the number of vertical movements in the central grid area of the PD group decreased significantly compared with the Ctrl group(P<0.01),and the number of bowel movements increased significantly(P<0.01);there was no significant improvement in the LID group compared with the PD group(P>0.05);compared with the LID group,the LE Compared with the LID group,rats in the LE group spent more time moving around the central grid area in the response box(P<0.01),increased the number of vertical movements(P<0.05)and significantly decreased the number of bowel movements(P<0.01).This indicates that electro-acupuncture points " DU16"," LR3" and " ST36" can improve the anxiety of LID rats.The time to the first black stool in the Sham group was not significantly different from that in the Ctrl group(P>0.05);the time to the first black stool in the PD group was significantly longer than that in the Ctrl group(P<0.01);the time to first black stool in the LID group was significantly longer than that in the PD group(P<0.01);the time to first black stool in the LE group was significantly longer than that in the LID group(P<0.01).In the LE group,the time to first black stool was significantly reduced compared with the LID group(P<0.01).This indicates that electro-acupuncture points " DU16"," LR3" and " ST36" can promote the intestinal motility of LID rats.Part II.1 Electroacupuncture increased the expression of positive TH signals in the substantia nigra and striatum of the brain in LIDThe positive signals in the substantia nigra and striatum of the rats in the Ctrl and Sham groups were more expresse d,and the neuronal outline was intact;the positive TH signals in the substantia nigra and striatum of the rats in the LID group were significantly reduced,and the positive TH signals in the substantia nigra and striatum of the rats in the LE group were significantly increased after the electroacupuncture intervention.This indicates that electroacupuncture points "DU16"," LR3" and " ST36" can increase the DA energy content in the substantia nigra and striatum.2 Electroacupuncture improves the synaptic structure of the LID striatumThe results of transmission electron microscopy showed that:compared with the Ctrl group,there was no significant difference in the striatal synaptic structure in the Sham group(P>0.05);compared with the Ctrl group,the number of synapses per field of view in the striatal region of the LID group increased significantly(P<0.01),the thickness of the postsynaptic dense material increased significantly(P<0.01),the synaptic gap became narrower(P<0.01),and the curvature of the synaptic interface increased(P<0.01).The striatal synapses were significantly reduced(P<0.01),the post-synaptic dense material thickness was significantly reduced(P<0.01),the synaptic gap was significantly restored(P<0.01),the synaptic interface curvature was reduced(P<0.01),and the number of perforated synapses was decreased(P<0.01)compared to the LID group rats(P<0.01).The results of Golgi staining showed that there was no significant difference in synaptic morphology in the Sham grou p compared with the Ctrl group(P > 0.05);the density of neuronal dendritic spines was significantly increased in the LID group compared with the Ctrl group(P < 0.01),and the density of mushroom-shaped and long and thin spines increased(P < 0.01),while there was no significant change in short and thick dendritic spines(P > 0.05);compared with the LID group,the density of neuronal dendritic spines in the LE group decreased significantly(P<0.01),the density of mushroomshaped and long and thin spines decreased(P<0.01),and the density of short and thick dendritic spines did not change significantly(P>0.05).This suggests that electroacupuncture at " DU16"," LR3" and " ST36" points can improve the morphological structure of striatal synapses in LID rats.3 Electroacupuncture improves the synaptic function of the striatum in LID ratsThe results of real-time fluorescence quantification showed that there was no significant difference between the experimental results of PSD-95 in the Ctrl and Sham groups(P>0.05);compared with the Ctrl group,the expression of PSD-95 gene in the brain striatum of LID rats was significantly increased(P<0.01);compared with the LID group,the expression of PSD-95 gene in the brain striatum of LE group was significantly reduced(P<0.01).The results of Western blot showed that there was no significant difference between the Ctrl and Sham groups(P>0.05),but compared with the Ctrl group,the LID group showed an increase in the amount of LID signature protein △FOSB protein(P<0.01),enhanced phosphorylation of P-ERK(P<0.01),and a significant increase in the postsynaptic-related protein PSD-95(P<0.01)in the striatum of the LID group.Compared with the LID group,△ FOSB,and PSD-95 were significantly lower in the striatum of the LE group(both P<0.01),and P-ERK phosphorylation was weakened(P<0.01).This indicates that electroacupuncture points " DU16","LR3" and " ST36" can improve the synaptic function of the striatum in LID.4 Electrical targeting of glutamate receptors in LIDReal-time fluorescence quantification was used to screen glutamate receptor-related genes,and the results showed that mGluR2,mGluR3,mGluR4,and mGluR6 were significantly down-regulated in the LID group compared to the Ctrl group(all P<0.01),NMDAR2 C was down-regulated in the LID group(P<0.05),mGluR5 and NMDAR2 D gene expression was NMDAR1 was upregulated in the LID group(P<0.05);mGluR5 and NMDAR2 D gene expression was significantly reduced in the brain striatum of the LE group compared with the LID group(both P<0.01),and mGluR6 gene expression was significantly upregulated(P<0.01).This suggests that mGluR5,NMDAR2 D,and mGluR6 genes may be important targets for electroacupuncture intervention in LID.However,as mGluR5 has been shown to be closely related to LID,mGluR5 was chosen to be studied in this study,presumably as an important target for electroacupuncture intervention in synaptic plasticity in LID.Part Ⅲ.1 CHPG dose selectionSince it was not clear from the previous literature w hether CHPG affects the replication of the LID model,it was important to find a dose that could increase mGluR5 expression in the striatum in a sustained manner without significant effects on rat behavior.In this study,mGluR5 agonist intervention was performed in the striatal region using stereotactic brain injection of 75 nm CHPG,and the results showed that there were no significant differences in the CC group compared to the Ctrl group(P>0.05)in the open field experiment,suspension experiment,pole climbing experiment,water maze experiment,and AIM score.(P<0.01).Therefore,this dose is the appropriate dose for this study.2 Effects of electroacupuncture and MPEP on synaptic plasticity in LIDThe results of the AIM score showed that electroacupuncture significantly improved the abnormal behavior of LID,similar to the effect of the mGluR5 inhibitor MPEP.In the Golgi staining results,it was found that compared with the LID group,the neuronal dendritic spine density in the LE group was significantly reduced(P<0.01),mushroom-shaped and long and thin spine density was reduced(P<0.01),and short and thick dendritic spine density was not significantly changed(P>0.05);compared with the LE group,the dendritic spine density,mushroom-shaped and long and thin spine density,and short and thick dendritic spine density in the LM group were not significantly different(all P>0.05).In the real-time fluorescence quantification and protein blotting assay,we found that both electroacupuncture and MPEP downregulated the expression of PSD-95,mGluR5,CAMK II,and CREB in the striatum of LID rats(P<0.01),and downregulated the protein content of mGluR5 and BDNF and decreased the phosphorylation of p-CAMK II and p-CREB in the striatum of LID rats(P<0.01).It is suggested that electroacupuncture points " DU16","LR3" and " ST36" have similar effects to MPEP on improving abnormal synaptic plasticity in LID,and mGluR5 may be a key target of electroacupuncture in the treatment of LID.3 CAMK II-CREB-BDNF as a mechanism of action of electroacupuncture in regulating synaptic remodeling in LID ratsThe results showed that when electroacupuncture was combined with CHPG(LCE group),the AIM scores of the LCE group were not significantly different from those of the L ID group(P > 0.05).It was confirmed that mGluR5 was the target of electro-acupuncture at s " DU16"," LR3" and " ST36" points for the treatment of LID.The results of Golgi staining showed that there were no significant changes in the density of dendritic spines,mushroom-shaped and long-thin spines,and short thick dendritic spines in the LC and LCE groups compared with those in the LID group(all P > 0.05);the protein content of mGluR5 and BDNF and the phosphorylation levels of p-CAMK II and p-CREB were not significantly different in the protein blotting assay(all P >0.05).This suggests that CAMK Ⅱ-CREB-BDNF may be the specific mechanism of action of electroacupuncture points " DU16"," LR3" and " ST36" in regulating synaptic remodeling in LID rats.Conclusion1 Electro-acupuncture points " DU16"," LR3" and " ST36" did not diminish the effect of L-dopa on the motor function of PD and improved the abnormal involuntary movements of LID.It also reduces anxiety and improves slow bowel movement in the LID model.2 Electro-acupuncture points " DU16"," LR3" and " ST36" can improve the synaptic structure and function of striatal neurons in LID,and adjust their synaptic plasticity.3 mGluR5 may be a key target of electroacupuncture at the "DU16"," LR3" and " ST36" points to improve synaptic plasticity in the striatum of LID.4 mGluR5-CAMKII-CREB-BDNF may be the specific mechanism of action of electroacupuncture at " DU16"," LR3" and " ST36" points in regulating synaptic remodeling in LID rats. |
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