| Objective:Glutamate is the most important excitatory neurotransmitter in mammalian central nervous system.The accumulation of glutamate in synaptic cleft and the overactivation of glutamate receptor are of important mechanisms for various pathological processes of nervous system,including neuropathic pain.Glutamate is generated by glutamine in the cytoplasm of presynaptic neurons under the catalyzation of glutaminase.Via vesicular glutamate transporters(VGLUTs),glutamate is taken up,stored in synaptic vesicles and then released into synaptic cleft in the form of exocytosis.Studies have shown that the number and function of VGLUTs play an important role in regulating glutamate content in the synaptic cleft.The clearance of glutamate in the synaptic cleft is mainly dependent on the uptake of Na~+-dependent high-affinity glutamate transporters(Excitatory amino acid transporters,EAATs)distributed in neurons and glial cell membranes.After uptake into glial cells by EAATs,glutamate is metabolized into glutamine.Glutamine is transported back to neurons and again converted into glutamate,which is assembled into synaptic vesicles by VGLUTs.Therefore,VGLUTs and EAATs are two important targets for the regulation of glutamate levels in synaptic cleft.To this day,five types of EAATs have been discovered,namely,GLAST(EAAT1),GLT-1(EAAT2),EAAC1(EAAT3),EAAT4,EAAT5.And three types of VGLUTs have been identified,namely,vesicular glutamate transporter 1(VGLUT1),vesicular glutamate transporter 2(VGLUT2),and vesicular glutamate transporter 3(VGLUT3).Studies have shown that glial glutamate transporter-1(GLT-1),which is responsible for approximately 90%of glutamate uptake and clearance in the synaptic cleft,and VGLUT2 play important roles in the formation of pain and hyperalgesia.For example,multiple pain models are accompanied by downregulation of GLT-1 expression or function.After using GLT-1 specific agonist ceftriaxone(Cef),we observed with the upregulation of GLT-1expression and function,the neuropathic pain and hyperalgesia were alleviated.The role of VGLUT2 in the formation of pain and hyperalgesia is not consistent,and may be related to the type and/or nature of pain.For example,studies have found that the basic pain threshold and inflammatory pain behavior of VGLUT2half knockout mice are not affected.After selective sciatic nerve injury(SNI),a reduction in mechanical hyperalgesia occurs.However,after chronic constriction injury(CCI)of the sciatic nerve,there was no significant change in mechanical hyperalgesia.Our study found that accompanied with hyperalgesia,the expression of VGLUT2 in spinal dorsal horn in rats was significantly downregulated after CCI.Therefore,the exact role of VGLUT2 in the formation of neuropathic pain remains to be determined.Furthermore,the relationship between VGLUT2 and GLT-1 remains unclear.In our previous study,after the application of GLT-1 specific agonist Cef,we found that accompanied with the upregulation of GLT-1,the expression of VGLUT2 was also upregulated in CCI rats.Moreover,GLT-1 antisense oligonucleotides can inhibit the upregulation of VGLUT2 induced by Cef.These results preliminarily suggest that the downregulation of GLT-1,which in turn leads to the downregulation of VGLUT2,is involved in the development of chronic neuropathic hyperalgesia in CCI rats.However,the impact of VGLUT2 downregulation on GLT-1 upregulation and anti-nociceptive effect induced by Cef after CCI is still unclear.Thus,the present study is to observe the changes in the effect of Cef on chronic neuropathic hyperalgesia and spinal dorsal horn GLT-1 expression in CCI rats,after the downregulation of VGLUT2via VGLUT2 shRNA.The results of the present study will provide experimental support for studying the role of VGLUT2 in the development of neuropathic pain and its relationship with GLT-1.Methods:1.Design and effect evaluation of VGLUT2 gene downregulation in rats’spinal dorsal hornGene Pharma company limited was entrusted to design and construct three VGLUT2 shRNA lentivirus vector sequences and one negative control sequence(i.e.,shRNA-668,shRNA-1267,shRNA-1732,and shRNA-NC),using VGLUT2(Slc17a6)m RNA of rat as a template.The silencing efficacy of these three sequences was evaluated at both the cellular and global levels.1.1 Silencing efficacy evaluation at cellular level(1)Determine the optimal culture duration and multiplicity of infection(MOI)for virus transduction of rats’cortical neuronsThe primary cultured rats’cortical neurons were divided into two groups:3-day transduction group and 7-day transduction group.The 3-day transduction group was transduced with shRNA-NC lentivirus vector on the 3rd day of rat cortical neuron culture,with MOI settings of 10 and 20.The 7-day transduction group was transduced with shRNA-NC lentivirus vector on the 7th day of rat cortical neuron culture,with MOI settings of 5,10 and 20.After 3 days of transduction,all cells were observed for transduction efficiency and neuronal status using an inverted fluorescence microscope.Culture duration with good neuronal survival and MOI with high transduction efficiency were selected for subsequent experiments.(2)Silencing efficacy evaluation and screenPrimary cultured rats’cortical neurons were divided into five groups:Control group,shRNA-NC group,shRNA-668 group,shRNA-1267 group,and shRNA-1732 group.The control group consisted of cultured normal cortical neurons without any treatment.The remaining groups were transduced with corresponding shRNA or shRNA-NC lentivirus vectors on the 3rd day of cortical neuron culture.Cells in each group were harvested on the 6th day of culture.The expression of VGLUT2 m RNA was detected using RT-q PCR to determine the silencing efficacy of each shRNA sequences.Meanwhile,the expression of VGLUT1 m RNA was detected to determine the specificity of shRNA silencing effect.1.2 Silencing efficacy evaluation at global levelSixty healthy male Sprague-Dawley rats,weighed 270±20 g,were randomly divided into five groups(n=12):Naive group,shRNA-NC group,shRNA-668 group,shRNA-1267 group,and shRNA-1732 group.The naive group consisted of normal rats without any treatment.Other groups were intrathecally injected with corresponding shRNA or shRNA-NC lentivirus vectors.According to the injection amount of lentivirus,these groups were further divided into 5μl and 10μl subgroups.The animals in each group were tested pain behavior 1 day before intrathecal injection and 2,6,and 9 days after injection.On the 9th day after injection,L4-L6 segments of the spinal dorsal horn were taken and the expression of VGLUT2 protein was detected using Western Blot to further determine the effective silencing shRNA sequence of VGLUT2.2.Changes in the effect of Cef on chronic neuropathic hyperalgesia and spinal dorsal horn GLT-1 expression in CCI rats after the downregulation of VGLUT2Thirty-six healthy male Sprague-Dawley rats,weighed 270±20 g,were randomly divided into six groups(n=6):(1)Sham group:The right sciatic nerve of rats was exposed without ligation.(2)CCI group:Chronic constriction injury was performed on the right sciatic nerve of rats.(3)CCI+NS+shRNA-NC group:Chronic constriction injury was performed on the right sciatic nerve of rats.shRNA-NC 5μl were intrathecally injected to rats 2 d before the operation.And the rats were intraperitoneally injected with normal saline(NS)from the day of surgery,0.72 ml/kg,once a day for consecutive 7 days.(4)CCI+Cef+shRNA-NC group:Chronic constriction injury was performed on the right sciatic nerve of rats.And the rats were intraperitoneally injected with Cef from the day of surgery,200 mg/kg,once a day for consecutive 7 days.Other protocols are the same as those in CCI+NS+shRNA-NC group.(5)CCI+Cef+shRNA-668 group:Chronic constriction injury was performed on the right sciatic nerve of rats.shRNA-668 5μl were intrathecally injected to rats 2 d before the operation.Other protocols are the same as those in CCI+Cef+shRNA-NC group.(6)CCI+Cef+shRNA-1732 group:Chronic constriction injury was performed on the right sciatic nerve of rats.shRNA-1732 5μl were intrathecally injected to rats 2 d before the operation.Other protocols are the same as those in CCI+Cef+shRNA-NC group.The animals in each group were tested pain behavior 3 day before operation,4 and 7 days after operation(i.e.,1 day before intrathecal injection,6and 9 days after injection).On the 7th day after operation(i.e.,on the 9th day after intrathecal injection),L4-L6 segments of the spinal dorsal horn were taken and the expression of VGLUT2 and GLT-1 protein was detected using Western Blot.Results:1.Design and effect evaluation of VGLUT2 gene downregulation in rats’spinal dorsal horn1.1 Silencing efficacy evaluation at cellular level(1)Determine the optimal culture duration and MOI for virus transduction of rats’cortical neuronsBoth the 3-day transduction group and the 7-day transduction group showed that the GFP fluorescence expression of lentivirus was the strongest when the MOI was 20.So does the transduction efficiency,which could up to more than 80%.However,the activity of neurons in the 7-day transduction group was significantly affected,which would affect subsequent m RNA detection.So,the protocol,that lentivirus was transduced on the 3rd day of cortical neuron culture with MOI settings of 20,was used for subsequent experiments.(2)Silencing efficacy evaluation and screenThe results of RT-q PCR showed that there was no significant difference in VGLUT2 m RNA expression between shRNA-NC group and control group.Compared with shRNA-NC group,the expression of VGLUT2 m RNA in shRNA-668,shRNA-1267 and shRNA-1732 groups was all significantly decreased(P<0.05).The decrease was especially obvious in shRNA-668 and shRNA-1732 groups.Compared with control group,there was no significant change in the expression of VGLUT1 m RNA in shRNA-NC,shRNA-668,shRNA-1267 and shRNA-1732 groups.The above results indicate that the three VGLUT2 shRNA sequences can all effectively and specifically downregulate the expression of VGLUT2 m RNA.The silencing efficacy of shRNA-668 and shRNA-1732 is especially obvious.1.2 Silencing efficacy evaluation at global levelPain behavioral test showed that in intrathecal injection of 10μl lentivirus subgroup,compared with naive group,there were no significant changes in thermal withdrawal latency and mechanical withdrawal threshold on 2 d,6 d,and 9 d after injection in shRNA-NC group.Compared with the shRNA-NC group,the mechanical withdrawal threshold in shRNA-668,shRNA-1267 and shRNA-1732 groups was significantly increased on 6 d and 9 d after injection(P<0.05).However,the thermal withdrawal latency in these three groups did not significantly change at the time points observed after injection.In intrathecal injection of 5μl lentivirus subgroup,compared with naive group,there were no significant changes in thermal withdrawal latency and mechanical withdrawal threshold on 2 d,6 d,and 9 d after injection in shRNA-NC group.Compared with shRNA-NC group,there were also no significant changes in the thermal withdrawal latency and mechanical withdrawal threshold on 2 d,6 d,and 9 d after injection in shRNA-668,shRNA-1267 and shRNA-1732 groups.The above results indicate that intrathecal injection of 10μl VGLUT2 shRNA lentivirus vector will affect the basic mechanical pain threshold of rats,while intrathecal injection 5μl shRNA lentivirus vector will not.So,the scheme of intrathecal injection 5μl shRNA lentivirus vector may be more applicable for subsequent experiments.Western Blot results showed that in intrathecal injection of 10μl shRNA lentivirus subgroups,compared with naive group,there was no significant change in the expression of VGLUT2 in spinal dorsal horn in shRNA-NC group.Compared with shRNA-NC group,the expression of VGLUT2 in spinal dorsal horn was significantly decreased in shRNA-668,shRNA-1267 and shRNA-1732 groups(P<0.05).The decrease was especially obvious in shRNA-668 and shRNA-1732 groups.The change trend of VGLUT2 expression in spinal dorsal horn of intrathecal injection 5μl shRNA lentivirus subgroups was the same as that in 10μl shRNA lentivirus subgroups,i.e.,compared with shRNA-NC group,the expression of VGLUT2 in spinal dorsal horn was significantly decreased in shRNA-668,shRNA-1267,and shRNA-1732 groups(P<0.05).And the decrease was especially obvious in shRNA-668 and shRNA-1732 groups.So VGLUT2 shRNA-668 and shRNA-1732 sequence were chosen for the subsequent VGLUT2 downregulation experiments.2.Changes in the effect of Cef on chronic neuropathic hyperalgesia and spinal dorsal horn GLT-1 expression in CCI rats after the downregulation of VGLUT2Western Blot results of VGLUT2 showed that,compared with sham group,the expression of VGLUT2 in spinal dorsal horn was significantly decreased in CCI group(P<0.05).Intraperitoneal injection of Cef could significantly up-regulate the expression of VGLUT2 in spinal dorsal horn in CCI rats(P<0.05).Intrathecal injection of VGLUT2 shRNA-668 or shRNA-1732 could significantly inhibit the upregulation of VGLUT2 in spinal dorsal horn in CCI rats induced by Cef(P<0.05).Pain behavioral test showed that,compared with sham group,mechanical withdrawal threshold and thermal withdrawal latency were significantly reduced in CCI group(P<0.05).Compared with CCI group,there was no significant change in mechanical withdrawal threshold and thermal withdrawal latency in CCI+NS+shRNA-NC group.After intraperitoneal injection of Cef,mechanical withdrawal threshold and thermal withdrawal latency were significantly increased in CCI rats(P<0.05).However,after intrathecal injection of VGLUT2 shRNA-668 or shRNA-1732,mechanical withdrawal threshold and thermal withdrawal latency were significantly decreased again in Cef-treated CCI rats(P<0.05).The above results indicated that VGLUT2downregulation could inhibit the anti-nociceptive effect of Cef on CCI rats.Western Blot results of GLT-1 showed that,compared with sham group,the expression of GLT-1 in spinal dorsal horn was significantly decreased after CCI operation(P<0.05).Intraperitoneal injection of Cef could significantly upregulate the expression of GLT-1 in spinal dorsal horn in CCI rats(P<0.05).There was no significant influence on the GLT-1 upregulation induced by Cef in CCI rats after intrathecal injection of VGLUT2 shRNA-668 or shRNA-1732.Conclusions:The downregulation of VGLUT2 in rats’spinal dorsal horn could inhibit the anti-nociceptive effect of Cef on CCI rats,which had no significant influence on the GLT-1 upregulation induced by Cef in CCI rats.It can be concluded that the downregulation of VGLUT2 is involved in the development of chronic neuropathic hyperalgesia in CCI rats;the downregulation of VGLUT2 expression may be the downstream mechanism of downregulation of GLT-1 expression in the development of chronic neuropathic hyperalgesia in CCI rats,i.e.,the downregulation of GLT-1,which in turn leads to the downregulation of VGLUT2,is involved in the development of chronic neuropathic hyperalgesia in CCI rats. |
PDF Full Text Request