The Role Of Vesicular Glutamate Transporter2 In Neuropathic Pain And Its Expressional Regulation Mechanism

Background Glutamate plays an important role in sense,transmission and regulation of pain signals.Up-regulation of glutamate release has been proposed to be a critical mechanism of neuropathic pain.Vesicular glutamate transporters(VGLUTs),which transport glutamate into pre-synaptic vesicles,play a crucial role in the regulation of glutamatergic neurotransmitter release.Among the three VGLUT isoforms,VGLUT2 was abundantly expressed in the dorsal root ganglia(DRG),spinal cord,brainstem,and thalamus,which are all key components of the nociceptive pathways.VGLUT2 deficiency in heterozygous knockout mice substantially attenuates neuropathic pain symptoms,and the loss of VGLUT2 in homozygous knockout mice leads to reduction in frequency and amplitude of EPSC in thalamic neurons.In addition,VGLUT2 deficiency in DRG-conditioned knockout mice attenuates thermal allodynia,but not mechanical allodynia.These studies suggest that VGLUT2 is associated with pain,especially neuropathic pain,but its role in thalamus,spinal cord and DRG may be different.Previously,we also demonstrated that VGLUT2 expression is significantly upregulated in spinal cord and some supraspinal regions including thalamus,and Chicago Sky Blue 6B(CSB6B),a VGLUT inhibitor,inhibits spared nerve injury(SNI)induced glutamate release and mechanical allodynia.However,the inhibitory effects of CSB6 B are not selective among VGLUT isoforms and the specific role of VGLUT2 in pain regulation is hard to be dissected.On the other hand,the expressional regulation mechanism of VGLUT2 remains unknown.Some studies reported that Wnt/?-catenin signaling pathway regulates the function of glutamatergic neurotransmission;meanwhile,classical and non classical Wnt signaling pathway plays an important role in neuropathic pain.Therefore,only those questions are identified,1)How does the expression of VGLUT2 change during the development of neuropathic pain? 2)What are the effects of VGLUT2 interference on neuropathic pain? 3)Whether the role of Wnt/?-catenin signaling in neuropathic pain is conduct through VGLUT2? Then it will provide necessary theoretical basis for determining whether VGLUT2 can be a target for the treatment of neuropathic pain.Objective The purpose of this study is to further identify the correlation between VGLUT2 and neuropathic pain,and to investigate the role of Wnt/?-catenin signaling in VGLUT2 expressional regulation and neuropathic pain.Methods To elucidate the association between VGLUT2 and neuropathic pain,we determined the expression and cell distribution characteristics of VGLUT2 under pathological conditions firstly,then developed two lentivirus enveloped VGLUT2 targeting sh RNA and delivered them respectively into spine or VPL of thalamus to study the role of VGLUT2 in neuropathic pain and glutamate release.Finally,we investigated the effect of Wnt/?-catenin signaling on VGLUT2 protein expression and pain behavior by using Wnt agonist and inhibitor in animals with neuropathic pain.1.Changes of VGLUT2 expression in spinal cord after neuropathic pain The mice SNI model was used to simulate the neuropathic pain caused by neurotrauma in clinic.Von Frey filaments were used to test the changes of mechanical threshold in the lateral plantar surface of the injured hind-paw(the sural nerve innervation area).After successful establishment of the model,the expression of VGLUT2 protein in spinal cord was detected by Western blotting and immunohistochemistry.Immunofluorescence double labeling was also used to observe the co-localization of VGLUT2 with neurons(Neu N,MAP2),microglia(IBA1)and astrocyte(GFAP)to study the distribution of VGLUT2 in different types of cells in the spinal dorsal horn of mice after neuropathic pain.2.The effect of lentivirus-sh RNA-VGLUT2 on neuropathic pain and glutamate release Take advantage of the Invitrogen BLOCK-i T? RNAi Designer Website,three VGLUT2 sh RNA interference sequences were designed,and then packaged into lentivirus plasmid.The lentivirus-sh RNA-VGLUT2 was transfected into primary cultured neurons to detect the effect of sh RNA-VGLUT2 on VGLUT2 gene,protein expression and glutamate release.Validated lentiviral-sh RNA-VGLUT2 was injected into the VPL of the thalamus or spinal cord on 7 day before or 4 day after SNI operation,and mechanical threshold was detected.Synaptosomes from spinal cord and VPL were extracted 7 days after SNI,and depolarization induced glutamate release was detected.3.Changes of Wnt/?-catenin signaling pathway and its role in the regulation of VGLUT2 expression in neuropathic pain The effect of ?-catenin over-expression,Wnt3 a or XAV939 treatment(Wnt/?-catenin signaling pathway inhibitor)on the expression of VGLUT2 protein was observed in PC12 cells.The effects of intrathecal injection of Wnt agonists or XAV939 on the expression of VGLUT2 protein were observed in mice.To study the regulatory mechanism of VGLUT2 expression in pathological condition,we first detected the expression of Wnt factor and ?-catenin protein in spinal cord at different time points(1,4,7,14,21 days)after SNI.In order to investigate whether Wnt/?-catenin pathway regulates the expression of VGLUT2 and its role in neuropahtic pain behavior,1)XAV939 was intrathecal administered 7 days after SNI surgery,when VGLUT2 protein was significantly up-regulated.The change of mechanical threshold and the expression of VGLUT2 protein were determined;2)the effects of Wnt agonist or Wnt1 on mechanical thresholds were observed 14 days after sh RNA-VGLUT2 administration.Results 1.Changes of VGLUT2 protein expression and cell type location in spinal cord at different time points after SNI The western blot results showed that the SNI surgery induced significant changes in the expression of VGLUT2 protein(F [6,36] = 6.249,p < 0.0001).The expression of VGLUT2 protein increased significantly on postoperative day 7,day 14 and day 21 compared with the naive animals,which increased for 31% ± 9%(p < 0.05),33% ± 11%(p < 0.05),27% ± 7%(p < 0.05)respectively.The results of immunohistochemistry indicated that VGLUT2 in the spinal cord increased significantly 14 days after SNI(p < 0.05).Double immunofluorescence labeling of VGLUT2 with four kinds of cell markers(Neu N,MAP2,IBA1 and GFAP)showed that VGLUT2 was mainly expressed in neurons in the dorsal horn of the spinal cord after SNI(Neu N,MAP2).2.The effect of lentivitiral sh RNA-VGLUT2 on neuropathic pain and the release of glutamate 2.1 Validation of sh RNA sequences against VGLUT2 and sh RNA-VGLUT2 affects glutamate release in primary cultured neurons The lentiviral-enveloped sh RNA-VGLUT2 was transfected into primary cultured neurons.Ten days after the infection,the VGLUT2 m RNA levels were significantly downregulated in all sh RNA-treated cells while protein levels were efficiently affected in cells treated with sh RNA-1 and sh RNA-2.The sh RNA-1 decreased the VGLUT2 m RNA and protein to 16% ± 1.5% and 43% ± 10.3% respectively and sh RNA-2 decreased the VGLUT2 m RNA and protein to 13% ± 1.5% and 20% ± 5.1% respectively.The results of extracellular glutamate release from lentiviral sh RNA-VGLUT2 pre-treated primary cultured neurons show that sh RNA-1(33.82 ± 0.66 ?M vs.20.39 ± 1.17 ?M,p < 0.05)and sh RNA-2(33.82 ± 0.66 ?M vs.18.97 ± 1.09 ?M,p < 0.05)significantly decreased the extracellular glutamate release from primary cultured neuron compared with sh RNA-Sc.2.2 The effect of lentivirus-sh RNA-VGLUT2 on mechanical threshold of mice before or after SNI and the glutamate release from synaptosomes Intrathecal administration of sh RNA-VGLUT2 does not affect the basal mechanical threshold of mice.Intrathecal administration of sh RNA-VGLUT2 7 days before surgery significantly decreased SNI induced allodynia(F [5,168] = 261.78,p < 0.0001),and there was significant difference on 4,7,10,14 day after operation.In another experiment,intrathecal administration of sh RNA-VGLUT2 4 days after operation significantly decreased SNI induced allodynia(F [5,168] = 367.28,p < 0.0001),and there was significant difference on 7,10,14 day after operation.Microinjection of sh RNA-VGLUT2 into VPL 7 days before surgery significantly decreased SNI induced allodynia(F [5,164] = 316.4,p < 0.0001),and there was significant difference on 4,7,10,14 day after operation.In another experiment,Microinjection of sh RNA-VGLUT2 into VPL 4 days after operation significantly decreased SNI induced allodynia(F [5,164] = 284.93,p < 0.0001),and there was significant difference on 7,10,14 day after operation.Glutamate release from spinal synaptosomes is significantly increased by SNI surgery compared with normal animals(7.34 ± 0.42 nmol/mg vs.11.94 ± 0.57 nmol/mg,p < 0.05).Intrathecal injection of sh RNA-VGLUT2 significantly reduced the amount of glutamate release in the spinal synaptosomes of SNI animals(p < 0.05).Microinjection of sh RNA-VGLUT2 in the VPL of the thalamus significantly reduced the amount of glutamate released from VPL synaptosomes(8.12 ± 0.24 nmol/mg vs.7.31 ± 0.21 nmol/mg,p < 0.05).3.The effect of neuropathic pain on the expression of Wnt/?-catenin signaling pathway and its role in the regulation of the expression of VGLUT2 3.1 Changes in expression of Wnt/?-catenin signaling pathway after SNI After SNI,Wnt1 m RNA(F [2,12] = 11.45,p < 0.0001),Wnt1 protein(F [2,10] = 9.279,p < 0.001)and ?-catenin protein(F [3,15] = 6.312,p < 0.001)change significantly.Wnt1 m RNA increased significantly on the 7,14,21 day after operation(p < 0.05),Wnt1 protein increased significantly on 1,4,7,14 and 21 days after operation(p < 0.05),and ?-catenin protein increased significantly at 1,4,7,14 and 21 days after operation.On the 14 day after SNI,the expression of ?-catenin protein increased significantly in the frontal cortex,while the ?-catenin in the nucleus accumbens and amygdala decreased significantly,while the ?-catenin in hippocampus and thalamus did not change.3.2 The effect of Wnt/?-catenin,BDNF signaling pathway on the expression of VGLUT1 and VGLUT2 protein In PC12 cells,?-catenin overexpression or Wnt3 a treatment increased the protein level of VGLUT1 and VGLUT2,while XAV939 treatment reduced VGLUT1 and VGLUT2 protein expression.In animals,intrathecal administration of Wnt agonist or Wnt1 significantly increased the expression level of VGLUT2 protein in the spinal cord.Intrathecal administration of XAV939 significantly decreased the expression level of VGLUT1 and VGLUT2 protein in spinal cord.Intrathecal injection of BDNF significantly increased the expression level of VGLUT1,VGLUT2,and ?-catenin protein in spinal cord.3.3 Regulation of the expression of VGLUT2 protein by Wnt/?-catenin signaling pathway and its effect on pain behavior Intrathecal administration of XAV939 7 days after SNI significantly affected mechanical allodynia in mice(F [2,108] = 401.05,p < 0.0001).After 2 h,the animal mechanical threshold began to rise,and the maximum value was reached after 6 h(0.522 ± 0.071 g vs.0.044 ± 0.015 g,p < 0.01).After 12 h,the threshold restored to the level before administration.Detection of protein expression reveled that the expression of VGLUT2 and ?-catenin was significantly lower than that in the control group after 6 h of treatment,alought the expression level of ?-catenin was still at a decreasing level,VGLUT2 protein returned to the level of before administration after 6 h of treatment.In normal animals,intrathecal injection of Wnt agonist decreased the mechanical threshold at 3,6,9,and 12 h after administration,while sh RNA-VGLUT2 significantly counteracted Wnt agonists induced mechanical allodynia(F [3,84] = 10.56,p < 0.001).Intrathecal administrition of Wnt1 decreased the mechanical threshold at 3,6,9,and 12 h after injection in mormal mice,while sh RNA-VGLUT2 significantly counteracted Wnt1 induced mechanical allodynia(F [3,96] = 38.21,p < 0.0001).Conclusion The VGLUT2 expression increased after SNI surgery in the mice spinal cord,and sh RNA-VGLUT2 attenuated the SNI-induced mechanical allodynia.This suggested that VGLUT2 is an important molecule involved in the development of neuropathic pain.The role of VGLUT2 on pain is due to its inhibition of glutamate release.Under the condition of neuropathic pain,the Wnt/?-catenin signaling pathway regulated the expression of VGLUT2 protein,and this regulation was closely related to neuropathic pain behavior.