Study On The Mechanism Of MGluR5 In Chronic Neuropathic Pain And Associated Emotional Dosorders

Chronic pain affects many aspects of a patient’s quality of life,including mood,sleep and cognitive processes.Patients are often accompanied by mood disorders such as anxiety and depression.In addition to chronic stress and psychosocial trauma,chronic pain is considered to be the leading cause of emotional disorders.However,as of now,the mechanism of chronic pain associated with anxiety and depression is not fully understood.Metabotropic glutamate receptors(m Glu Rs)are members of the C protein family of G protein-coupled receptors,which regulate the transmission of glutamate and participate in many physiological processes and diseases,including chronic pain and mood disorders.In recent years,more and more studies have focused on the mechanism of excitatory post-synaptic protein mGluR5 in various psychiatric diseases.The study found that mGluR5 changes in the brain in different pain models,and that mGluR5 changes in different brain regions in the same pain model.The use of mGluR5 allosteric modulators can effectively alleviate some chronic pain and anxiety-like and depression-like behaviors.The core brain regions that may be affected are the medial prefrontal cortex(mPFC)and amygdala.However,the mechanism of mGluR5’s involvement in chronic pain and associated emotional disorders has not been fully elucidated.In this study,a selective sciatic nerve injury(SNI)mouse was used as a model,and a variety of research methods were used to explore the mechanism of mGluR5 in chronic neuropathic pain and associated anxiety-like and depression-like behaviors.The specific experiment is divided into the following four parts:Part One: Effect of mGluR5 regulation on chronic neuropathic pain and anxiety-like and depression-like behaviorsObjective: To investigate the effects of m GluR5 regulation on chronic neuropathic pain and related negative emotion.Methods: Neuropathic pain mouse model of SNI was constructed.The mechanical pain sensitivity of the mice was detected by von Frey silk.The anxiety-like behavior of the mice was measured by the open field(OF)test and the elevated plus maze(EPM).The tail suspension test(TST)and the sugar preference test(SPT)were used to detect the depression-like Behavior in Mice.The tracer 18F-FPEP of mGluR5 was used to observe the mGluR5 level in the whole brain of mice by positron emission tomography(PET).By applying mGluR5 allosteric modulators and constructing the cytomegalovirus(CMV)-mGluR5 knockout(KO)mouse and Glutamate Decarboxylase 2(Gad2)-mGluR5 KO mice to observe the behavioral changes after different levels of mGluR5 regulation.Results:1)On the 7th day after surgery,the threshold of mechanical foot contraction in the left lower limb of the mouse was significantly lower than that in the control group(P <0.001),and this reduction trend continued until the 60 th day.On the 60 th day after SNI,the time spent in the central area of OF and the open arm of EPM was significantly reduced compared with the control group(P <0.001),and the immobility time in the SNI group was significantly longer in the TST(P <0.05),the sugar preference rate of the SNI group in SPT was significantly reduced(P <0.05).These results suggest that SNI mice have significant mechanical hyperalgesia and associated anxiety-like and depression-like behaviors.2)The PET results showed that the standard uptake value(SUV)of 18F-FPEP in the whole brain of SNI model mice was significantly increased.3)Systemic administration of mGluR5 negative allosteric modulator basimglurant can effectively alleviate anxiety-like and depression-like behaviors induced by SNI,but it cannot relieve mechanical hyperalgesia.Treatment with systemic administration of ketamine can effectively alleviate SNI-induced anxiety-like and depression-like behaviors,as well as relieve mechanical hyperalgesia.4)CMV-mGluR5 KO mice showed no obvious behavioral abnormalities,but Gad2-mGluR5 KO mice had obvious anxiolytic-like and antidepressant-like behaviors.Regardless of CMV-mGluR5 KO mice or Gad2-mGluR5 KO mice,the mechanical pain threshold did not change significantly.Conclusion: The SNI model has obvious mechanical hyperalgesia associated with anxiety-like and depression-like behaviors at 60 th day.PET showed an increase of mGluR5 in the whole brain of SNI mice.The use of negative allosteric modulators of mGluR5 can effectively relieve anxiety-like and depression-like behaviors.Gad2-mGluR5 KO mice have obvious anxiolytic-like and antidepressant-like behaviors,suggesting that the negative allosteric modulator of mGluR5 may act on GABAergic neurons.The possible mechanism that mGluR5 whole brain reduction has no effect on mechanical pain sensitivity in mice needs more research.Part Two: The mechanism of mGluR5 in mPFC participating in the regulation of chronic neuropathic pain and associated anxiety-like and depression-like behaviorsObjective: To investigate the molecular mechanism of m GluR5 in mPFC on chronic neuropathic pain and related negative emotions.Methods: The expression of mGluR5 was detected by immunofluorescence,immune electron microscopy,Western blot,and RT-PCR.Micro RNA chip screening and database prediction were used to detect micro RNAs related to mGluR5,and dual luciferase reporting experiments and experiments with primary neuron culture in vitro were used to verify the regulation of micro RNAs on mGluR5.Using behavioral pharmacology experiments to observe the effects of local regulation of mGluR5 in mPFC on chronic pain and associated anxiety-like and depression-like behaviors in SNI models.Results: The results of immunofluorescence,immunoelectron microscopy and Western blot showed that compared with the control group,the total protein expression of mGluR5 in mPFC was not significantly different,and the expression of mGluR5 protein on the cell membrane was increased.The SNI model had no effect on the expression of m GluR5 a and m GluR5 b variants in mPFC.Local injection of MPEP in mPFC in SNI mice can effectively alleviate mechanical pain and anxiety-like and depression-like behaviors in mice.Micro RNA chip screening and database prediction combined with dual luciferase reporting experiments and in vitro neuronal primary cell culture experiments suggest that mir RNA-17-3p can directly regulate mGluR5 / GRM5 expression.Local injection of mi RNA-17-3p mimic in mPFC of SNI mice can effectively reduce the expression of GRM5 and relieve mechanical pain sensitivity and anxiety-like and depression-like behaviors in mice.Conclusion: The expression of m GluR5 in the cell membrane of the mPFC brain region of SNI mice is increased.Local injection of negative allosteric modulators of mGluR5 in mPFC can effectively relieve chronic pain and associated anxiety-like and depression-like behaviors.mi R-17-3p can directly regulate mGluR5,and local injection of mi R-17-3p mimic in mPFC can effectively alleviate chronic pain and associated anxiety-like and depression-like behaviors.Part Three: The mechanism of mGluR5 in amygdala participating in the regulation of chronic neuropathic pain and associated anxiety-like and depression-like behaviorsObjective: To explore the effect of m GluR5 in the left and right amygdala on chronic neuropathic pain and related negative emotions.Methods: Combined techniques such as Western blot,immunofluorescence staining,in situ hybridization,RT-PCR,and micro-optical sectioning tomography(MOST)were used to observe and compare the left and right amygdala of normal male mice mGluR5 expression,number of neurons,and blood vessel length.PET technology was used to detect the signal of mGluR5 in the amygdala of the right and left sides of normal male mice and the SNI model.Using behavioral pharmacology experiments to observe the effects of local regulation of mGluR5 levels in the left and right amygdala on mechanical hyperalgesia and associated anxiety-like and depression-like behaviors.Results: Western blot,immunofluorescence staining,in situ hybridization,RT-PCR,and MOST showed that there was no significant difference in the expression of mGluR5,the number of neurons,and the length of blood vessels in the amygdala of the right and left sides of normal male mice.The mGluR5 signal detected by PET in the left and right amygdala of SNI model was significantly increased.For the left injury SNI model,localized injection of MPEP at the central nucleus of the amygdala(Ce A)on the right can effectively relieve mechanical pain and anxiety-like and depression-like behaviors,but Ce A injection on the left cannot.For the right injury SNI model,local injection of MPEP at Ce A on the left can effectively alleviate mechanical pain and anxiety-like and depression-like behaviors,but CeA injection on the right cannot.Conclusion: We did not find the leading role of right Ce A in regulating pain in the SNI model,and Ce A on the left can also participate in the regulation of mechanical pain sensitivity.Our results further demonstrate the role of mGluR5 in relieving chronic neuropathic pain and associated anxiety-like and depression-like behaviors.Part Four: Changes of intestinal flora in mice of mGluR5 knockout,SNI model and ketamine intervention in SNI modelObjective: To investigate the changes of intestinal flora of model mice such as mGluR5 knockout and SNI.Methods: Using the method of 16 Sr DNA sequencing,the changes of intestinal flora of mice under the conditions of CMV-mGluR5 KO mice,SNI 60 d model mice and ketamine intervention were detected.Results: Compared with control mice,the distribution and diversity of intestinal flora of CMV-mGluR5 KO mice did not change significantly.Compared with control mice,the SNI model has significantly increased flora including Betaproteobacteria(class),Burkholderiales(order),Alcaligenaceae(family)and Prevotella(family and genus),the significantly reduced bacteria were Firmicutes(phylum).After ketamine treatment,the significantly increased flora included Firmicutes(phylum),Clostridia(class,order),and the significantly reduced flora included Bacteroides(phylum,class,order),Prevotella(family and genus).After ketamine treatment,the proportion of Firmicutes(phylum),Prevotella(family and genus)changed by the SNI model was effectively reversed.Compared with control mice,the SNI model has reduced flora diversity,and ketamine treatment can reverse the decrease in flora diversity.Conclusion: The intestinal flora of m GluR5 systemic knockout mice did not change significantly.While the SNI model(whole brain mGluR5 PET detection signal is enhanced),the distribution and diversity of rat intestinal flora have changed significantly.Ketamine treatment(whole brain mGluR5 PET detection signal is reduced)exerts antidepressant-like effects,which may be related to regulation.Some intestinal flora are abnormal.