| BackgroundFrom1994,the International Association for the Study of Pain(IASP)defined Neuropathic Pain(NP)as an abnormal pain caused by a primary lesion or dysfunction in thenervous system,and is characterized as spontaneous pain,hyperalgesia,and allodynia.This definition was updated by the IASP in 2011.They tended to believe that neuropathic pain is defined as pain caused by a lesion ordisease of the somatosensory system.NeuPSIG suggested that the prevalence of this pain is about 3.3%?8.2%.Although there is no systematic data on the quality of life of patients with neuropathic pain in China,the influence of neuropathic pain on the quality of life of patients is obvious.Long-term pain will not only affect patients' sleep,work and life ability,but also increase the incidence of depression,anxiety and other emotional disorders.Neuropathic pain usually was divided into two types,peripheral pain and central pain,and different types of pain may have similar or common pathogenesis As the pathogenesis of neuropathic pain is so complex,it may include the change of anatomical structure and the damage of tissue function,often caused by a variety of mechanisms.These mechanisms may include peripheral sensitization,central sensitization,deactivation of the descending inhibitory system,activation of spinal glial cells,and changes in ion channels.Several pathological changes may be involved,including nerve injury,neurogenic inflammation,abnormal peripheral nerve excitability,abnormal sympathetic nervous system and changes in neural plasticity.It is worth noting that central sensitization is an important pathogenesis of neuropathic pain,and the maintenance of neuropathic pain mainly depends on central sensitization.Central sensitization refers to the abnormal increase of excitability or synaptic transmission of pain-related neurons in the spinal cord and above,including the increase of spontaneous discharge activity of neurons,the expansion of the sensory field,the reduction of threshold of external stimulation,and the enhancement of response to supraliminal stimulation,etc.,thus amplifying the transmission of pain signals.Spontaneous pain,hyperalgesia,allodynia and so on.At the same time,the abnormal changes of ion channels are also involved in the occurrence of neuropathic pain.Studies have shown that after nerve injury,the ?2-? subunit on the calcium ion channel in the posterior horn of the spinal cord is highly expressed,the calcium ion channel is abnormally open,and the calcium ion influx increases,leading to increased excitatory neurotransmitter release and overexcitation of neurons,resulting in hyperalgesia and allodynia.Neuropathic pain is a continuous process that may recur and require long-term treatment.However,the current treatment status is not satisfactory,about half of patients with neuropathic pain can not fully alleviate the pain.Currently,for many patients,the pharmacotherapy of neuropathic pain is challenging and the effective treatment is lacking.Thus,some new approaches are required to control pain and its consequences in clinical practice.In clinical practice,root neuralgia is a common type of pathologic neuralgia,and pain caused by spinal ganglion or spinal nerve root compression induced by disc herniation and intervertebral foramen stenosis is a common type of root neuralgia.In rats,chronic compression of the dorsal root ganglia(CCD)effectively triggers radicular pain.Mechanical pain sensitivity,thermal pain sensitivity and abnormal pain were found in the behavior test after the establishment of the CCD model in rats.At the same time,abnormal discharge occurred in DRG neurons,and with the increase of spontaneous discharge of DRG neurons,the action potential and current threshold decreased accordingly.These changes are mediated by several types of ion channels and pathways that regulate either cellular excitability or synaptic transmission.Thus,when ion channel function or the expression of protein kinases within the pathway are inhibited,neuron excitability may decrease,relieving pain symptoms and discomfort.Previous studies by my research group(fund number:81071597)showed that expression of transient receptor potential vanilloid receptor 4(TRPV4),a subfamily of transient receptor potential ion channels,was significantly increased in DRG after CCD in rats,and that TRPV4 could be involved in hyperexcitability,hyperalgia and abnormal pain of DRG caused by CCD.Subsequently,we found(fund number:81401862)that intervention on TRPV4 expression and function could affect the degree of neuropathic pain caused by continuous compression of dorsal root ganglion in rats.However,in the process of participating in the sensitivity to pain,what factors regulate the activation of TRPV4 and the intervention need to be further studied.DRG neurons are the first level of sensory afferent neurons,and pain information transmission starts from primary sensory neurons in DRG,which are also known as nociceptors.The central end of the DRG nerve fibers will transmit the nociceptive information to the second-level neurons located in the dorsal horn of the spinal cord.These neurons will send out nerve fibers to form pain pathways such as the lateral spinal-thalamic tract,and further transmit the nociceptive information to the thalamus and brain step by step,and finally produce pain after processing and integration.The dorsal horn of spinal cord is the site where pain signals are integrated and amplified,and is also an important part of the pain transmission pathway.Therefore,as an indispensable and important part of the nociceptive information transmission pathway,the dorsal horn of spinal cord is in urgent need of further study.Therefore,in the process of exploring the pathogenesis of neuropathic pain,we should not only study peripheral sensitization,but also the role of central sensitization in pain.Peripheral nerve injury produces neuropathic pain as well as phosphorylation of mitogen-activated protein kinase(MAPK)family in dorsal root ganglia(DRG)and dorsal horn.The MAPK family consists of three major members:extracellular signal-regulated kinases(ERK),p3 8,and c-JNK,which represent three separate signaling pathways.After nerve injury,ERK,p38,and JNK are differentially activated in neurons,microglia,and astrocytes of the dorsal horn and gracile nucleus,and in injured large DRG neurons,leading to the synthesis of pro-inflammatory or pro-nociceptive mediators,thereby enhancing and prolonging pain.In particular,ERK activation in spinal cord dorsal horn neurons by nociceptive activity plays a critical role in central sensitization by regulating the activity of glutamate receptors and potassium channels and inducing multiple gene transcriptions.In several animal models of neuropathic pain,MEK1 has been identified as a novel target for the treatment of neuropathic pain.Some pharmacological inhibitors of MEK1,such as PD 198306,U0126,and PD98059,have proven effective to alleviate pain by inhibiting the activation of ERK signaling.Thus,targeting ERK can be considered as a promising therapeutic target for the treatment of neuropathic pain.Gene therapy,which aims to replace a defective or a deficient protein at therapeutic or curative levels,has the tremendous potential to completely cure with a single treatment,diseases previously classified as untreatable,or disorders that could be managed but not corrected by drugs.Lentivirus-mediated gene therapy can provide a unique tool to integrate small RNA interference(RNAi)expression constructs with the aim to locally knockdown expression of a specific gene,enabling to assess the function of a gene in a very specific neuronal pathway.Lentivirus-mediated expression of short hairpin RNA(shRNA)was shown to be long-term and mediate stable RNAi in a dose-dependent manner,therefore,providing a convenient way to integrate RNAi expression constructs.For example,delivery of shRNA through lentivirus vector results in persistent knockdown of gene expression in the brain.Therefore,the use of lentiviruses for stable expression of shRNA is a powerful aid to probe gene functions in vivo and for gene therapy of diseases of the nervous system.Both ERK and TRPV4 are involved in the occurrence mechanism of various pathological pain,such as primary trigeminal neuralgia,masticatory pain caused by temporomandibular joint dysfunction and bone and joint headache,etc.,but they are not pain sensitive and spontaneous pain caused by CCD.Studies have confirmed that,after CCD,the expression of TRPV4 significantly increased,TRPV4 channel opening also increased,and the increase of Ca2+ influx led to the increase of intracellular calcium peak.The increase of intracellular Ca2+ can activate the MAPK family.Subsequently,it was found that the expression of p-erk was significantly increased after CCD compared with the normal group,and ERK was activated and exerted biological effects.However,the application of MEK1 inhibitor U0126 could significantly reduce the expression of p-erk,and along with the decreased excitability of DRG neurons,the current threshold of its action potential was also increased.The increased excitability of DRG neurons induced by TRPV4 channel opening may also be affected by ERK expression and biological function.Previous studies of our research group have confirmed that TRPV4 and p38 are involved in mediating the generation of neuropathic pain after CCD,and there is an interaction between TRPV4 and p38.Therefore,it is urgent to explore the mechanism of ERK-TRPV4 pathway in the sensitivity of CCD induced pain in rats,which will provide an important basis for the targeted inhibition of ERK in the treatment of neuropathic painChapter IEffect of ERK on TRPV4 expression and function in primary dorsal root ganglion neurons of rats with chronic compression Objective1.To construct the shRNA vector of lentivirus interfering with ERK and screen the effective viral fragments interfering with ERK expression,and to study whether interfering with ERK would affect the expression and function of TRPV4 in primary neurons of dorsal root ganglion after CCD in rats.2.To study whether TRPV4 and ERK in rat dorsal root ganglion primary cells interact with each other.Methods and Materials1.CCD Model and Surgical Procedure.Wistar rats:healthy adult male rats weighing about 150-180g.With 10%chloral hydrate(rat body weight,0.3 mL/100 g)abdominal cavity after anesthesia,and then cut back in rats between iliac spine on both sides of the skin,about 2 cm incision,fully exposed deep fascia and muscle,exposed outside the right side of the L4,L5,segmental intervertebral hole,selects the stainless steel shaped like a "U" shape steel bar,the oppression of the corresponding segment of the L4 and L5 vertebral between outside the hole.The incision was washed and sutured layer by layer.Postoperatively,no self-injury,complete loss of sensation or inability to walk occurred in rats.2.DRG Neuron Culture.Rats were anesthetized by 10%chloral hydrate,and the L4 and L5 ganglia were incubated with collagenase and trypsin(Sigma-Aldrich,St.Louis,MO),thoroughly mixed and enzymatically digested in a 37 C incubator for 30 min.The DRGs were then suspended in 50%DMEM/F12 and 50%Neurobasal A medium,collected by centrifugation and mechanically dissociated using glass pipettes with small tip diameters.The neurons in suspension were collected,plated onto poly-L-lysine-coated Petri dishes with glass bottoms,and cultured for 24 hours at 37 ? in 95%humidity with 5%CO2.Then,the culture medium was exchanged with Neurobasal medium containing 15%fetal bovine serum(Gibco Invitrogen),100 U/ml mycillin(Sigma-Aldrich)to avoid bacterial infection and 5 ?mol/L cytosine arabinoside(Sigma-Aldrich)to inhibit nerve fibroblast growth.For subsequent studies,the neurons were cultured for 12-24 hours.3.Cellular Immunofluorescence and Neuron Identification.After 72 hours of extracted and culturing,the slides of DRG neurons were fixed with 4%polyformaldehyde,permeated with 0,5%Triton X-100,and overnight at 4 0C with mouse anti-rat NeuN polyclonal antibody(1:500,Abcam,USA).594 fluorescent labeled donkey anti-mouse IgG(H+L)-red fluorescent second antibody(1:400,Thermo Invitrogen,USA)was stained with DAPI for 10 minutes at 37 0C.The anti-fluorescence quenching tablets were sealed and the images were observed and collected under fluorescence microscope.4.Lentivirus-mediated shRNA Preparation.The lentivirus-mediated ERK-silencing shRNA sequences were inserted into a lentivirus-mediated RNAi expression system(GenePharma Co.,Ltd.,Shanghai,China).In the preparation phase,we used a lentivirus-mediated RNAi expression system with GFP,which enabled infection efficiency to be conveniently evaluated.were provided by GenePharma.In addition,a non-silencing ERK-shRNA sequence(5'-TTCTCCGAACGTGTCACGTTT-3')was used as a negative control,which does not target any human genes in mice or rats,as determined by screening with NCBI RefSeq.After DRG neurons from the normal rats had been cultured for two days,the cells were infected with the four ERK-shRNA lentiviruses(Lv-shERK group)or the negative control lentivirus(Lv-shNC group)according to the recommended multiplicity of infection(MOI).After lentiviral infection,the DRG neurons were observed under a fluorescence microscope,and the transfection efficiency of ERK Lentivirus-mediated shRNA was evaluated at 72 hours post infection.Subsequently,at five days post infection,we selected the one most effective sequence,as determined by western blotting and RT-PCR.The lentiviral transfection experiments were performed as reported in a previous study.5.Detection of cellular activity of DRG neurons.The primary DRG neurons were extracted and inoculated in 96-well culture plate with a cell density of 1 × 104/hole.After overnight incubation in 37 C incubator,the adherent cells grew well.The absorbance values(OD values)of 490 nm at 24h,48h,72h,96h and 120h after lentivirus infection were detected by MTT method.The OD values of DRG neurons cultured in normal medium were 100%.The percentage of OD values of each group was obtained by comparing the OD values of DRG neurons cultured in normal medium with those of DRG neurons cultured in normal medium.6.Western Blot Analysis for the expression of protein in DRG primary neurons.The total protein of each group was extracted,Then,the sample of total protein was separated by 5%and 10%SDS-PAGE.Proteins were transferred to polyvinylidene fluoride membranes.The membranes were incubated with the primary antibody at 4? overnight followed by secondary antibodies for 1 h.The signal was detected using the ImmobilonTM Western Chemiluminescent HRP Substrate.To detect and evaluate the transfection effect of lentivirus interfering ERK,and observe the expression changes of TRPV4,ERK and P-ERK protein.7.RT-qPCR.Total mRNA of DRG cells was extracted from each group,and the effect of lentiviral interference on ERK expression at gene level was detected by Real-time quantitative-PCR.The changes of total RNA expression of ERK and TRPV4 in DRG primary cells after lentivirus transfection into CCD were detected.8.Calcium Imaging Technology.Five days after lentivirus transfection,the fluorescence intensity was observed by laser confocal microscopy,and the maximum fluorescence intensity(Fmax)and minimum fluorescence intensity(Fmin)of single cell were recorded respectively to calculate the relative concentration of intracellular Ca2+.The changes of calcium concentration in DRG were observed.The[Ca2+]i was reported as a pseudo-ratio(AF/F),?F/F =(F-Fbase)/(Fbase-B).9.Reagents.For both in vitro and in vivo studies,four days after CCD surgery,the ERK-silencing shRNA sequences(GenePharma Co.,Ltd.,Shanghai,China)were given to the experimental groups.Calcium imaging was performed using the fluorescent calcium indicator dye Fluo-3/acetoxymethyl ester(Fluo-3/AM,Sigma,USA).The TRPV4 inhibitor ruthenium red(RR,Sigma,Germany,recommended concentration = 1 ?mol/L)and the TRPV4 agonist 4a-PDD(CST,USA,recommended concentration = 3 ?mol/L)were dissolved in dimethyl sulfoxide(DMSO),and the final experimental dilutions into normal saline were made on the day of the experiment.Results1.Identification of primary neurons in rat dorsal root ganglion.After 72 hours of culture,the primary DRG neurons were observed under the microscope.Most of the primary DRG neurons were round,elliptical and polygonal,and most of the cells were elliptical and polygonal.The cell bodies were large.Trypan blue staining showed that more than 90%of the cells were active.Immunocytofluorescence staining with Neuronal Marker anti-NeuN and DAPI nucleus staining showed that DRG neurons were labeled red fluorescent cells.2.Effect of Lentivirus on Cell Activity of DRG Neurons.According to the plurality of infections recommended in the Gima Lentivirus Use Manual,MOI=5 was selected to infect DRG neurons with lentiviruses.With MOI=5,lentivirus negative control infected DRG neurons.MTT assay of DRG neurons at 24h,48h,72h,96h and 120h showed that the activity of DRG neurons was not affected.3.Selection of Lentivirus-mediated shRNA ERK-silencing shRNA Sequence in DRG Neurons.To evaluate the lentivirus-mediated shRNA against ERK,rat DRG neurons were used for an in vitro study.Limited study was performed via lentivirus-mediated shRNA knockdown of ERK in DRG neurons.A lentiviral vector system expressing GFP,as a reporter gene,and shRNA against ERK was established.To determine the infection efficiency of the lentivirus in DRG neurons,cells were infected with the Lv-shERK#1,Lv-shERK#2,Lv-shERK#3,Lv-shERK#4 or Lv-shNC,and three days after infection,the cells were observed under a fluorescence microscope.>85%of the DRG neurons expressed GFP,which indicated a high lentiviral infection efficiency.To verify the knockdown efficiency of the four Lv-shERK vectors,ERK protein and mRNA expression levels were detected by western blotting and RT-qPCR analysis in DRG neurons five days post infection.The protein expression levels of ERK were significantly decreased in the Lv-shERK#1-,Lv-shERK#3-and Lv-shERK#4-infected groups compared with the Lv-shNC-infected group(n=6 per group,P<0.01).The mRNA expression levels of ERK1 and ERK2 were also significantly down-regulated in the Lv-shERK#1-,Lv-shERK#3-and Lv-shERK#4-infected groups compared with the Lv-shNC-infected group(n=6 per group,P<0.01).On the basis of the above results,the lentivirus vector and GFP had minimal influence on cell survival;thus,the Lv-shERK#1 sequence(Rat-550,5'-GCTCTTGAAGACACAGCACCT-3')was selected as the most effective ERK-silencing shRNA sequence(Lv-shERK group)and was used for the following in vitro experiments.4.Changes of ERK and TRPV4 Protein in DRG neurons.Four days after CCD surgery,DRG neurons were obtained and cultured in vitro.To investigate whether the expression levels of TRPV4 and ERK were altered,lentivirus-mediated ERK-silencing shRNA was used with the cells,and ERK protein expression in DRG neurons was detected at five days post infection.As demonstrated,ERK,P-ERK and TRPV4 protein expression levels were significantly increased in the DRG neurons of CCD rats compared with those of the control rats(n=6 per group,P<0.01),and these levels were significantly decreased in the Lv-shERK group compared with the Lv-shNC group(n=6 per group,P<0.01).5.Changes in the expression of cell-related genes in DRG neurons.RT-qPCR analysis revealed that ERK1,ERK2 and TRPV4 mRNA expression was significantly up-regulated in the CCD group compared with the control group(n=6 per group,P<0.01),and the expression of ERK1 and ERK2 decreased after lentivirus interference,while the expression of TRPV4 decreasethese levels were significantly down-regulated in the Lv-shERK group compared with the Lv-shNC group(n=6 per group,P<0.01).6.Calcium Changes after Lentivirus-mediated shRNA Inj ection in DRG neurons.Calcium imaging was performed at five days post infection.The challenges with 30%hypotonic solution and 3 ?mol/L 4a-PDD increased the[Ca2+]i in small and medium diameter DRG neurons,and the calcium response was abolished when extracellular Ca2+ was eliminated and following treatment with 1 ?mol/L RR.In response to the 30%hypotonic solution and 3 ?mol/L 4a-PDD,the calcium fluorescence ratio was significantly different between the control and CCD groups.The percentage of DRG neurons that responded to the hypotonic solution was significantly up-regulated,and the hypotonicity-induced increase in the calcium fluorescence ratio was also significantly enhanced(n = 6,20 times per group,P<0.01)in CCD group compared with controls.In the Lv-shERK group treated with Lv-shERK after CCD surgery,the percentage of responsive cells and the fluorescence ratio(n = 6,20 times per group,P<0.01)were decreased compared with these parameters in the Lv-shNC group.Similar to the hypotonic solution-induced Ca2+ response,the percentage of DRG neurons responsive to 4a-PDD treatment was significantly up-regulated in the CCD group compared with the control group.The calcium fluorescence ratio was significantly enhanced in the CCD group compared with the CON group(n = 6,20 times per group,P<0.01).This percentage and the fluorescence ratio(n = 6,20 times per group,P<0.01)were decreased in the Lv-shERK group compared with the Lv-shNC group.Conclusion1.The effective shERK fragment of ERK interfered by lentiviruses specific to DRG neurons in rats is ERK-shRNA as 550-571,5'-GCTCTTGAAGACACAGCACCT-3'.2.The expression and function of TRPV4 in primary DRG neurons of rats were changed by interfering with ERK by lentivirus-mediated.Chapter ?ERK-TRPV4 pathway is involved in peripheral sensitization and central sensitization of allodynia with chronic compression of the dorsal root ganglion in ratsObjective1.To investigate whether ERK-TRPV4 pathway is involved in the occurrence and development of allodynia after chronic compression of dorsal root ganglion in rats.2.To investigate whether ERK-TRPV4 pathway is involved in the mechanism of peripheral and central sensitization of pain sensitivity after sustained compression of dorsal root ganglion in rats.Methods and Materials1.Animals.Adult male Wistar rats weighing 150-180 g were provided by the Experimental Animal Center of Shandong University.Rats were anesthetized by 10%chloral hydrate(300mg/100g i.p.),then two stainless steel rods were implanted unilaterally into the intervertebral foramen at L4 and L5.Rats in sham-operation group were given the same operation but no steel bar inserting.The rats with autophagy phenomenon,feeling deficiency and disability were eliminated.2.Intrathecal Injection.The rats were anesthetized by inhalation of isoflurane and oxygen in a small animal anesthesia machine.After skin disinfection,L4/L5 intervertebral space was located at the intersection of bilateral iliac spine and spine.The next intervertebral space,L5/L6 intervertebral space,was inserted into the intervertebral space vertically along the front edge of index fingertip,until there was a sense of breakthrough.Lateral rapid tail flick occurred in rats,indicating that the rats entered the subarachnoid space,carefully pumped back,no blood reflux,and 2 uL/s was injected slowly.The volume of each injection was 10 ul.After sheath injection,the microinjector was quickly drawn out and the injection site was pressed for about 10 seconds to prevent cerebrospinal fluid leakage.After the operation and inhalation anesthesia,the righting reflex could be restored within 2 minutes.3.Behavioral testing.(1)Walk gait pattern:1 point:normal gait,no foot deformity on the operative side;2 point:normal gait but obvious foot deformity on the operative side;3 point:slight gait abnormality and/or foot drop on the operative side;4 point:severe gait abnormality in rats,and muscle strength decline on the operative side.(2)Paw withdrawal mechanical threshold(PWMT):Using the ipsilateral hind paw of the animals,behavioral testing was performed prior to the operation and on post-operative days.The paw withdrawal mechanical threshold(PWMT)was evaluated with a BME-404 Mechanical Analgesia Tester(Chinese Academy of Medical Sciences,CAMS,Beijing,China).The probe was pressed with sufficient force against the lateral plantar surface of the hind paw.A positive response was noted when the paw was immediately withdrawn.After at least 5 min,the rats were tested again;the tests were repeated five times,and the average was calculated and used in the statistical analyses.(3)Thermal paw withdrawal latency(TPWL):Thermal allodynia tests were assessed in response to radiant heat by using a BME-410C Thermal Analgesia Tester(CAMS)as described previously.After at least 5 min,the rats were tested again;the tests were repeated five times,and TPWL was calculated for both hind paws and averaged.4.Lentivirus-mediated shRNA Injection.To avoid a GFP-mediated effect in the following investigations,a lentivirus-mediated RNAi expression system without GFP was also provided by GenePharma and was used during the infection phase of in vivo studies.To investigate the optimal intrathecal lentivirus injection dose for rats,different lentivirus titrations were given to rats.The following titers of ERK-shRNA lentivirus were used:10^4 toxic units(TU),10^4 TU,10^5 TU,10^6 TU and 10^7 TU.In addition,10 ?l of reagent was given to each rat to assure.Furthermore,to select the optimal dose for live rats,different concentrations of the lentivirus-mediated ERK-targeting shRNA were provided to the normal rats via intrathecal injection for three consecutive days,and at five days post-injection,western blotting and RT-PCR were used to evaluate shRNA effectiveness.The groups without lentivirus infection were given an equal volume of normal saline.The intrathecal injection studies were performed in accordance with a previous study.5.Western Blot for the expression of protein in DRG and spinal dorsal horn.The L4 and L5 dorsal root ganglia and corresponding segments of the dorsal horn of the spinal cord on the operative side were extracted rapidly from the ice and the total protein was extracted.The signal was detected using the ImmobilonTM Western Chemiluminescent HRP Substrate.To detect and evaluate the transfection effect of lentivirus interfering ERK,and observe the expression changes of TRPV4,ERK and P-ERK protein.6.RT-qPCR.The L4 and L5 dorsal root ganglia and the corresponding spinal dorsal horn of the operative side were sampled quickly,total RNA was extracted and the concentration was determined.Real-time quantitative RT-PCR was used to detect the expression of TRPV4,ERK1 and ERK2 in DRG and spinal dorsal horn tissues.The primer sequence is the same as the first part.7.Immunocoprecipitation.After extracting the total proteins of spinal dorsal horn and DRG,prorein A-argarose method was used to react with anti-TRPV4 antibody and anti-ERK antibody,respectively,for immunoprecipitation.The precipitated complex was analyzed by protein imprinting method to observe whether the target protein existed.8.Immunohistochemistry with NeuN,TRPV4,ERK and P-ERK in spinal cord and DRG.The corresponding spinal cord segments of L4 and L5 were obtained,were removed rapidly after perfusion,post-fixed in the same fixative overnight at 4? and then dehydrated and paraffin infused.A series of 4-?m paraffin sections were cut using a rotary microtome.The sections were heated at 65? for at least 2 h and then deparaffinated.Antigen retrieval was accomplished with citrate buffer in a microwave oven at 92-98? for 15-20 min.The sections were washed in PBS then incubated separately in mouse anti-NeuN polyclonal antibody(1:300,Abcam,USA),rabbit anti-TRPV4 polyclonal antibody(1:200,Abcam,USA),rabbit anti-ERK polyclonal antibody(1:500,CST,USA)and rabbit anti-P-ERK polyclonal antibody(1:200,CST,USA)at 4? overnight.The sections were incubated using secondary antibody,DAB substrate solution and hematoxylin were used to develop the color.Labeled sections were examined under a Leica Quantimet 550 DMRXA automated research microscope(GER).Observe the proteins in the dorsal horn of spinal cord.9.Immunohistofluorescence with ERK-TRPV4 in spinal cord and DRG.The primary antibodies at 4 0C overnight:mouse anti-ERK polyclonal antibody(1:500,CST,USA)and rabbit anti-TRPV4 polyclonal antibody(1:200,Abcam,USA).Then the sections were incubated in Alexa Fluor 488-conjugated Affinipure Donkey Anti-Mouse IgG(H+L)and Alexa Fluor 594-conjugated Donkey Anti-Rabbit IgG(H+L)for 2 h at room temperature.Labeled sections were examined under an Olympus automatic fluorescence microscope using the image analysis system of the microscope(JA)and analyzed using the IPP.6 by software.To calculate the numbers of eath-positive cell and co-positive cell.10.Immunohistofluorescence with NeuN-ERK and NeuN-TRPV4 in spinal cord.The primary antibodies were combined with mouse NeuN polyclonal antibody(1:300,Abcam,USA).The sections were incubated separately in mixtures of the following primary antibodies at 4 ? overnight:rabbit anti-ERK polyclonal antibody(1:500,CST,USA)and rabbit anti-TRPV4 polyclonal antibody(1:200,Abcam,USA).Then the sections were incubated in Alexa Fluor 488-conjugated Affinipure Donkey Anti-Mouse IgG(H+L)and Alexa Fluor 594-conjugated Donkey Anti-Rabbit IgG(H+L)for 2 h at room temperature.DAPI was used to stain the cell nuclei.Labeled sections were examined under an Olympus automatic fluorescence microscope using the image analysis system of the microscope(JA)and analyzed using the IPP.6 by software.To calculate the quantitative analyses of the numbers of positive neurons.Results1.Selection of Optimal Lentivirus Dose in Rats.To investigate the optimal intrathecal lentivirus injection dose for rats,different lentivirus titrations were given to rats.In the animal experiment,the death rate was more than 50%in the 107 group.Since this high dose disturbed the normal survival of rats,we did not use this group.To select the optimal Lv-shERK dose,ERK mRNA and protein expression levels in rat DRGs were detected by western blotting and RT-qPCR analysis at five days post infection.ERK protein expression was significantly reduced in the 106 TU group(n=6 per group,P<0.05).ERK1 and ERK2 were also significantly lower in the 106 infection group than in the control group,in mRNA expression levels(n=6 per group,P<0.01).On the basis of the above experimental results,an Lv-shERK dose of 106 TU per rat was confirmed as the optimal dose for the following in vivo experiments.2.Changes of Paw withdrawal mechanical threshold(PWMT)and Changes of Thermal paw withdrawal latency(TPWL).Comparing with the CON group,the mechanical stimulation claw reduction threshold of the CCD group decreased significantly from day 4 to day 10(n=6,P<0.05).On the second day after intrathecal injection of lentivirus ERK shRNA interference,the threshold of mechanical stimulation claw contraction reaction was significantly higher than that of lentivirus negative control group(n = 6,P<0.01).There was no significant difference between lentivirus negative control group and CCD group.The latency of thermal stimulation claw contraction began to decrease on the second day after CCD.Compared with the CON group,the latency of mechanical thermal stimulation claw contraction in the CCD group decreased significantly from the fourth day to the twelfth day(n=6,P<0.01),and the sensitivity of rats to heat pain increased.On the 3rd day after lentivirus ERK shRNA interference was given intrathecal injection,the latency of claw contraction response of heat stimulation was significantly higher than that of lentivirus negative control group(n = 6,P<0.01).There was no significant difference between lentivirus negative control group and CCD group.3.Changes of TRPV4 and ERK protein expression after lentivirus ERKshRNA interference in CCD rats.There was no significant difference between the CCD group and the CCD+Lv-shNC group in the dorsal horn of spinal cord in rats.Compared with the CON group,the expression of TRPV4,ERK and P-ERK in the CCD group increased(n=6,P<0.01),while the expression of TRPV4,ERK and P-ERK in the Lv-shERK group decreased(n=6,P<0.01).4.Changes of TRPV4 and ERK1/2 mRNA Genes.There was no significant difference in CCD and CCD+Lv-shNC groups.Compared with CON group,the expression of TRPV4,ERK1 and ERK2 in CCD group was up-regulated(n=6,P<0.01).After lentivirus interference,ERK1 and ERK2 were down-regulated significantly,while TRPV4 also changed with the change of ERK,and the expression of TRPV4 was down-regulated significantly(n=6,P<0.01).1.Compared with negative control.5.Immunocoprecipitation in spinal dorsal horn and DRG.After anti-TRPV4 antibody immune complexes and Western Blot... |
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