| Objective: Glial glutamate transporter-1(GLT-1)is the main transporter for maintaining glutamate homeostasis in the synaptic cleft.It is responsible for approximately 90% of glutamate uptake and clearance in the synaptic cleft.Downregulation of GLT-1 expression or function has been found to be involved in the formation and maintenance of a variety of pain and hyperalgesia,including neuropathic pain.Rothstein et al.reported thatβ-lactam antibiotics,such as ceftriaxone(Cef),could specifically upregulate GLT-1 expression and glutamate uptake.On the basis of this report,our previous study has found that Cef could play an anti-nociceptive role in CCI rats via upregulating GLT-1 expression and enhancing its glutamate uptake in spinal dorsal horn.However,the underlying mechanism of this anti-nociceptive effect of Cef remains to be further determined.Glutamate,took in astrocytes by GLT-1,is returned to the presynaptic nerve endings via the glutamate-glutamine cycle,where it is assembled into vesicles by vesicular glutamate transporters(VGLUT)and is released into the synaptic cleft again.Therefore,through glutamate-glutamine cycle,VGLUT and GLT-1 are closely linked together,maintaining the glutamate homeostasis within the synapses.Among VGLUTs,type II vesicular glutamate transporter,namely VGLUT2,is mainly distributed in lamina I,lamina II,and deep lamina IV and V in spinal cord,and plays an important role in the transmission and processing of sensory and nociceptive information.Our previous study has shown that accompanied with the development of thermal and mechanical hyperalgesia,VGLUT2 expression was significantly decreased in the spinal dorsal horn of rats with chronic constriction injury of sciatic nerve(CCI).Cef plays an anti-nociceptive role in CCI rats by upregulating GLT-1 and then VGLUT2 expression.Although there has been some evidence that VGLUT2 is involved in pain,the results are inconsistent.For example,it was found that,the whole body VGLUT2 gene knockout heterozygous mice showed a reduction in mechanical and cold hyperalgesia in the model of spared nerve injury(SNI).However,these heterozygous mice only showed a reduction in cold hyperalgesia in the model of CCI.Therefore,the exact role of VGLUT2 in neuropathic pain and the exact mechanism by which Cef upregulates VGLUT2 to relieve hyperalgesia remain to be further explored.The composition of VGLUT2 immunoreactive nerve endings in spinal dorsal horn is complex,which includes the endings of primary afferent neurons as well as the endings of intrinsic neurons in the spinal cord.Different nerve endings play different roles in the transmission of nociceptive information and in the formation of hyperalgesia.It is generally believed that unmyelinated C fibers are involved in the transmission of pain information.According to the difference of neurotransmitters,C fibers can be divided into isolectin B4(IB4)positive non-peptidergic nerve fibers and calcitonin gene-related peptide(CGRP)positive peptidergic nerve fibers.The changes of VGLUT2 in these primary afferent endings,which are directly involved in the transmission of nociceptive information,are still unknown.Thus,the purpose of this study is to observe the changes of VGLUT2 expression in different types of spinal C fibers in Cef-treated CCI rats,and the modulation relationship between VGLUT2 and GLT-1,so that to provide further experimental evidence for clarifying the mechanism of Cef against chronic neuropathic pain,as well as the role of GLT-1 and VGLUT2 in neuropathic pain.Methods: One hundred and eight healthy male Sprague-Dawley rats,weighing 290±20g were randomly divided into the following five groups:(1)Sham group(n=12): The right sciatic nerve of rats was exposed without ligation.The changes of the thermal withdrawal latency and mechanical withdrawal threshold of the rats were observed 1 d before operation,4 d and 7 d after operation.At 7 d after operation,L4-L6 segments of rats’ spinal dorsal horn were obtained.The expression of GLT-1 and VGLUT2 was detected by western blot.The colocalized expression of IB4 and VGLUT2,CGRP and VGLUT2,was detected by double immunofluorescence assay.(2)CCI group(n=12): Chronic constriction injury was performed on the right sciatic nerve of rats.The changes of the thermal withdrawal latency and mechanical withdrawal threshold of the rats were observed 1 d before operation,4 d and 7 d after operation.At 7 d after operation,L4-L6 segments of rats’ spinal dorsal horn were obtained.The expression of GLT-1 and VGLUT2 was detected by western blot.The colocalized expression of IB4 and VGLUT2,CGRP and VGLUT2,was detected by double immunofluorescence assay.(3)CCI+NS+R-ODNs(n=28): chronic constriction injury was performed on the right sciatic nerve of rats.Rats were intraperitoneally injected with normal saline(NS)on the day after surgery,0.72 ml/kg,once a day for consecutive 7 days.At the same time,random oligodeoxynucleotides(R-ODNs)of GLT-1 were intrathecally injected to rats,10 μl(18nmol)per time.According to the times of intrathecal injection,the rats were divided into three subgroups: once,twice and three times injection subgroups.As to once injection subgroup(n=8),the rats were intrathecally injected GLT-1 R-ODNs once on post-operative day 3.In twice injection subgroup(n=12),the rats were intrathecally injected GLT-1 R-ODNs twice on post-operative day 2 and day 5,respectively.In three times injection subgroup(n=8),the rats were intrathecally injected GLT-1 R-ODNs three times on post-operative day 1,day3.5 and day 6,respectively.The changes of the thermal withdrawal latency and mechanical withdrawal threshold of the rats were observed 1 d before operation,4 d and 7 d after operation.At 7 d after operation,L4-L6 segments of rats’ spinal dorsal horn in twice injection subgroup were obtained.The expression of GLT-1 and VGLUT2 was detected by western blot.The colocalized expression of IB4 and VGLUT2,CGRP and VGLUT2,was detected by double immunofluorescence assay.(4)CCI+Cef+R-ODNs(n=28): chronic constriction injury was performed on the right sciatic nerve of rats.Rats were intraperitoneally injected with Cef on the day after surgery,200 mg/kg,once daily for consecutive 7 days.At the same time,GLT-1 R-ODNs were intrathecally injected to rats,10 μl(18nmol)per time.Other protocols are the same as those in CCI+ NS+R-ODNs group.(5)CCI+Cef+AS-ODNs(n=28): chronic constriction injury was performed on the right sciatic nerve of rats.Rats were intraperitoneally injected with Cef on the day after surgery,200 mg/kg,once daily for consecutive 7 days.At the same time,antisense oligodeoxynucleotides(AS-ODNs)of GLT-1 were intrathecally injected to rats,10 μl(18nmol)per time.According to the times of intrathecal injection,they were divided into three subgroups: once,twice,and three times injection subgroups.As to once injection subgroup(n=8),the rats were intrathecally injected GLT-1AS-ODNs once on post-operative day 3.In twice injection subgroup(n=12),the rats were intrathecally injected GLT-1 AS-ODNs twice on post-operative day 2 and day 5,respectively.In three times injection subgroup(n=8),the rats were intrathecally injected GLT-1 AS-ODNs three times on post-operative day 1,day 3.5 and day 6,respectively.The changes of the thermal withdrawal latency and mechanical withdrawal threshold of the rats were observed 1 d before operation,4 d and 7 d after operation.At 7 d after operation,L4-L6 segments of rats’ spinal dorsal horn in twice injection subgroup were obtained.The expression of GLT-1 and VGLUT2 was detected by western blot.The colocalized expression of IB4 and VGLUT2,CGRP and VGLUT2,was detected by double immunofluorescence assay.Results:1.Determination of the regimen of GLT-1 AS-ODNsTo explore the modulation relationship between GLT-1 and VGLUT2 in the anti-nociceptive process of Cef,GLT-1 AS-ODNs were intrathecally used to observe the changes of VGLUT2 expression in spinal dorsal horn,which would suppress the expression of GLT-1 and blocking the anti-nociceptive effect of Cef.Therefore,it is necessary to determine the regimen of GLT-1AS-ODNs.Pain behavioral results showed,compared with baseline,there were no significant changes in thermal withdrawal latency and mechanical withdrawal threshold of sham rats after surgery.Compared with sham group,thermal withdrawal latency and mechanical withdrawal threshold of CCI rats were significantly reduced on post-operative day 4 and day 7(P<0.05),which indicated that thermal and mechanical hyperalgesia had been developed after CCI operation.Compared with CCI group,rats in CCI+NS+R-ODNs group had no significant changes in thermal withdrawal latency and mechanical withdrawal threshold.Compared with each subgroup in CCI+NS+R-ODNs group,the thermal withdrawal latency of CCI+Cef+R-ODNs group was significantly increased on post-operative day 7(P<0.05).The mechanical withdrawal threshold also showed an increased tendency,but it was no statistical significance.These results indicated that intraperitoneal injection of Cef could play an anti-nociceptive role in CCI rats.Compared with each subgroup in CCI+Cef+R-ODNs group,the thermal withdrawal latency in once injection subgroup of CCI+Cef+AS-ODNs group showed a decreased tendency,but it was no statistical significance.The thermal withdrawal latency in twice and three times injection subgroups of CCI+Cef+AS-ODNs group were significantly decreased(P<0.05).The changes of mechanical withdrawal threshold in CCI+Cef+AS-ODNs group were the same as those of thermal withdrawal latency.The above results indicated that twice and three times injection of GLT-1 AS-ODNs can effectively block the anti-nociceptive effect of Cef on CCI rats.Based on the principle of effectiveness and minimal injury,we chose the regimen of twice injection of GLT-1 AS-ODNs in the following experiments.The results of western blot showed that compared with sham group,the expression of GLT-1 in spinal dorsal horn after CCI was significantly decreased(P<0.05).Compared with CCI group,there was no significant changes of GLT-1 expression in CCI+NS+R-ODNs group.Compared with CCI+NS+R-ODNs group,the expression of GLT-1 in CCI+Cef+R-ODNs group was significantly increased(P<0.05).Compared with CCI+Cef+R-ODNs group,the GLT-1 expression in CCI+Cef+AS-ODNs group was significantly decreased(P<0.05).The above results indicated that Cef could significantly upregulate the GLT-1 expression in spinal dorsal horn of CCI rats.Intrathecal twice injection of GLT-1 AS-ODNs could block the above effect of Cef,which further confirmed that the intrathecal twice injection regimen could be used in the following experiments.2.The effect of Cef on VGLUT2 expression of spinal dorsal horn and the role GLT-1 played in the processThe results of western blot showed that compared with sham group,VGLUT2 expression of spinal dorsal horn was significantly decreased after CCI operation(P<0.05).Cef could significantly upregulate VGLUT2 expression of spinal dorsal horn after CCI operation(P<0.05).Intrathecal twice injection of GLT-1 AS-ODNs could significantly inhibit the up-regulation of VGLUT2 in Cef-treated CCI rats(P<0.05).The above results indicated that Cef upregulated the expression of VGLUT2 in CCI rats’ spinal dorsal horn via its up-regulation of GLT-1.3.The effect of Cef on the number of IB4 positive C-fiber nerve endings and the expression of VGLUT2 in these endingsThe results of immunofluorescence histochemistry showed that a large number of IB4 immunopositive C fiber nerve endings were distributed in the lamina I and II of the spinal dorsal horn in sham rats.Compared with sham group,the number of IB4 positive C fiber nerve endings were significantly increased in CCI group,which was represented by the increased integrated gray value of IB4 positive signal(P<0.05).Compared with CCI group,the number of IB4 positive nerve endings in CCI+NS+R-ODNs group did not significantly change.Compared with CCI+NS+R-ODNs group,the number of IB4 positive C fiber nerve endings in CCI+Cef+R-ODNs group was significantly decreased,which was represented by the decreased integrated gray value of IB4 positive signal(P<0.05).Compared with CCI+Cef+R-ODNs group,the number of IB4 positive C fiber endings in CCI+Cef+AS-ODNs group was significantly increased again(P<0.05).The results of immunofluorescence colocalization showed that the colocalization fluorescence intensity of IB4 and VGLUT2 in spinal dorsal horn of CCI rats was significantly increased,which was represented by the significantly increased Pearson’s correlation coefficient and Manders’ overlap coefficient(P<0.05).Compared with CCI group,the colocalization fluorescence intensity of CCI+NS+R-ODNs group had no significant difference.Compared with CCI+NS+R-ODNs group,the colocalization fluorescence intensity of CCI+Cef+R-ODNs group was significantly decreased,which was represented by the significantly decreased Pearson’s correlation coefficient and Manders’ overlap coefficient(P<0.05).Compared with CCI+Cef+R-ODNs group,the colocalization fluorescence intensity of CCI+Cef+AS-ODNs group was significantly increased again(P<0.05).The above results indicated that the number of IB4 positive C-fiber nerve endings was significantly increased after CCI operation.And the expression of VGLUT2 in these endings was also upregulated.Cef could inhibit CCI induced increase of IB4 positive fibers and CCI induced up-regulation of VGLUT2 in these fiber endings.The effects of Cef could be blocked by the application of GLT-1 AS-ODNs.4.The effect of Cef on the number of CGRP positive C-fiber nerve endings and the expression of VGLUT2 in these endingsThe results of immunofluorescence histochemistry and immunofluorescence colocalization showed that the changes of CGRP positive C fiber ending numbers and VGLUT2 expression in these endings in spinal dorsal horn of each group were consistent with those of IB4 positive fiber endings.That is,the number of CGRP positive C-fiber nerve endings was significantly increased after CCI operation(P<0.05).And the expression of VGLUT2 in these endings was also upregulated(P<0.05).Cef could inhibit CCI induced increase of CGRP positive fibers and CCI induced up-regulation of VGLUT2 in these fiber endings.The effects of Cef could be blocked by the application of GLT-1 AS-ODNs.Conclusions:The increased expression of VGLUT2 in spinal IB4 positive C fiber endings and CGRP positive C fiber endings might be involved in the development and maintenance of CCI induced chronic neuropathic pain in rats.Cef plays an anti-nociceptive role via upregulating GLT-1 expression,and sequentially down-regulating the expression of VGLUT2 in IB4 positive C fiber endings and CGRP positive C fiber endings. |
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