Study On The Role And Mechanism Of REST/NRSF In Chronic Pain

The International Association for the Study of Pain(IASP)defines pain as "unpleasant feelings and emotional experiences related to actual or potential tissue damage".Pain is a complex and unpleasant feeling accompanied by autonomous,psychological and behavioral responses.The manifestation of acute pain usually disappears in a short time after effective treatment.However,if not treated properly,it can generate pathological changes,thus resulting in the development of acute pain into chronic pain.Chronic pain has become one of the main problems in modern human health and social development.Due to the complexity of its pathogenesis and occurrence mechanism,there has been no good solution in clinical practice,which has brought serious harm to the body and psychology of patients.Therefore,it is imperative for us to find out the molecular mechanism of chronic pain attack.REST/NRSF(repressor element-1 silencing transcription factor/neuronrestrictive silencing factor)is a kind of protein with zinc finger domain.By binding with specific acting element NRSE(neuron-restrictive silencer element),histone in the sequence is deacetylated,thus inhibiting the transcription of target gene.REST / NRSF can affect the expression of target genes with different degrees by interacting with a variety of co-factors,and the interference of its function will result in a variety of pathological states.Some related studies have shown that in the neuropathic pain and bone cancer pain models,the expression level of REST / NRSF in peripheral sensory neurons was significantly increased,and the expression of genes(including Kcnd3,Kcnq2,Scn10 a and Oprm1 that encode Kv4.3,kv7.2,Nav1.8 ion channel and opioid receptor)related to pain in the corresponding REST / NRSF target genes was inhibited at the same time.In addition,some transcriptional changes or relief of painful behavior were also detected when the intrathecal injections of REST antisense oligonucleotides or si RNA were carried out.More and more evidences show that REST / NRSF plays an important role in the occurrence and development of chronic pain.However,there is still a lack of systematic and in-depth understanding for the molecular mechanism of REST / NRSF involved in chronic pain.In order to further study the related mechanism,we used transgenic mice with specific REST / NRSF knockout in dorsal root ganglia neuron to explore the molecular mechanism of REST/NRSF in dorsal root ganglia neuron involved in chronic pain disease.In this thesis,we will explore the problem in two parts: in the first part: we over-expressed REST/NRSF in the dorsal root ganglion,and then investigated the effect on pain behavior in mice;in the second part: we knock out REST/NRSF in the dorsal root ganglion,and then investigated the effect on pain behavior in chronic pain.Part One Effects of REST / NRSF-specific overexpression of mouse dorsal root neurons on pain behaviorObjective: To investigate the effect of overexpression of REST / NRSF in dorsal root neurons(DRG)on pain behavior in mice.Methods:1.Stereotactic technology was used to accurately locate the right L4 dorsal root ganglion of the mouse and locally deliver the virus using a microsyringe.The experiment was divided into three groups: injection of saline group,injection of AAV2 / 9-CMV-Null virus group,injection of AAV2 / 9-CMV-Rest –myc virus group.The pain thresholds of mechanical and heat radiant in mices were measured 1day,7days,14 days,21 days,24 days and 28 days after virus administration.2.Immunohistochemical staining was used to detect the expression levels of REST / NRSF in L4 dorsal root ganglion and spinal cord of mice after 28 days of virus injection.Results:1.On the 1st,7st and 14 st day after the operation,there was no significiants difference in the three groups of mice;on the 21 th,24th,and 28 th days after the operation compared with the group of mice injected with saline,the pain threshold of mechanical stabbing and radiation heat pain of the group injected with AAV2 / 9-cmv-rest-myc virus was significantly decreased(n=10,*P < 0.05),there was no significant difference in pain threshold between AAV2 / 9-cmv-null group and saline group.2.About 25 % of the dorsal root neurons in the L4 dorsal root ganglion of mice injucted with AAV2 / 9-CMV-Rest-myc virus group detected the expression of exogenous REST-myc;No expression of exogenous REST-myc was detected in the L4 dorsal root ganglia of mice injected with AAV2 / 9-CMV-Null virus and saline.No expression of exogenous REST-myc was detected in the spinal cord tissue of the three groups of mice.Conclusions: Over-expression of REST / NRSF in L4 dorsal root neurons in mice resulted in significantly reduced pain thresholds for mechanical tingling and radiant heat pain.Part Two Effects of dorsal root neuron-specific REST / NRSF knockout on chronic pain and pain behaviorObjective: To investigate the effect of dorsal root neuron-specific REST / NRSF knockout on neuropathic pain and inflammatory pain.Methods:1.Construction of Tamoxifen-induced dorsal root neuron-specific REST /NRSF knockout mice.2.CFA inflammatory pain model was established to observe and compare the pain threshold of mechanical stabbing and heat pain in control mice and dorsal root neuron-specific REST/NRSF knockout mice.3.SNI neuropathic pain model was established to observe and compare the pain threshold of mechanical stabbing and heat pain in control mice and dorsal root neuron-specific REST/NRSF knockout mice.4.Single cell-PCR was used to detect the expression levels of REST,Kcnd3,Kcnq2,Scn10 a and Oprm1 of small diameter dorsal root neurons in wild-type mice,SNI models of control mice and SNI model of dorsal root neuron-specific REST/NRSF knockout mice.Results:1.In the CFA inflammatory pain model,the pain thresholds of mechanical stabbing and radiant heat pain in dorsal root neuron-specific REST/NRSF knockout mice were significantly higher than than those in the control group(n=6,*P < 0.05)and there was no significant difference in the pain thresholds of contralateral.2.In the SNI neuropathic pain model,the pain thresholds of dorsal root neuron-specific REST / NRSF knockout mice was significantly higher than that of the control group(n=6,*P < 0.05),and there was no significant difference in the pain thresholds of contralateral mechanical stabbing.Moreover,the number of REST-positive neurons in the dorsal root ganglion of SNI neuropathic pain model in wild-type mouse increased significantly.3.Single cell-PCR results showed that the expression levels of REST / NRSF in small diameter dorsal root neurons of SNI neuropathic pain model in wild-type mice was significantly increased,and the expression levels of REST target genes Kcnq2 and Kcnd3 were significantly decreased(n=89,* P < 0.05).The REST expression level in small diameter dorsal root neurons of SNI neuropathic pain model in mice with specific REST/NRSF knockout was significantly reduced,while the expression levels of REST target genes Kcnq2 and Kcnd3 were significantly increased(n=79,* P < 0.05).Conclusions:1.The specific knockout of REST/NRSF in dorsal root neurons of mice can significantly improve the pain threshold of CFA inflammatory pain and SNI neuropathic pain.2.Specific knockout of REST/NRSF in dorsal root neurons can effectively reverse the increased expression in REST/NRSF expression caused by peripheral nerve injury and the down-regulation of its target genes Kcnq2 and Kcnd3.