| Objective: Spinal dorsal horn(SDH)is the primary transit station for noxious information transmission,and glutamate is the main neurotransmitter in the primary sensory afferent fibers and excitatory interneurons of the posterior horn of the spinal cord.Maintenance of glutamate homeostasis in SDH synapses is essential for the transmission of noxious information and the formation of hyperalgesia.The homeostasis of glutamate in synapses depends on the balance of glutamate release and clearance.The clearance of glutamate in synaptic clefts mainly depends on the high affinity glutamate transporter system.Glutamate transporter-1(GLT-1),which is mainly expressed on astrocytes,is responsible for about 90% of glutamate uptake and removal.The release of glutamate in synapses clefts is mainly determined by a low affinity glutamate transporter system —— vesicular glutamate transporters(VGLUT).Glutamate taken up by astrocytes is then transmitted to neurons through glutamate-glutamine cycle and is taken up and stored in synaptic vesicles by VGLUT,which express on synaptic vesicles.During synaptic activity,synaptic vesicles release glutamate by exocytosis.At present,three VGLUT subtypes,VGLUT1,VGLUT2 and VGLUT3,have been cloned from the central nervous system.Studies have shown that down-regulation of expression or function of GLT-1 plays a role in the development and maintenance of neuropathic pain.Therefore,modulation of GLT-1 may be a new target for the prevention and treatment of neuropathic pain.In 2005,Rothstein et al reported that beta-lactam antibiotics,such as ceftriaxone(Cef),could specifically regulate GLT-1 expression and glutamate uptake.According to this report,our previous study has found that Cef can enhance the uptake of glutamate by up-regulating the expression of GLT-1 in the dorsal horn of spinal cord in CCI rats and play a role in relieving chronic neuropathic pain caused by CCI.But little is known about the mechanism exists after the effect of GLT-1 up-regulation.Further researches should be done for fully understanding about Cef's effect on neuropathic pain.VGLUT and GLT-1 together maintain the glutamate homeostasis in synapses.VGLUT plays an important role in perceptive and nociceptive transmissions.However,the role of VGLUT subtypes in neuropathic pain is still unclear.Considering that GLT-1 is closely related to VGLUT through glutamate-glutamine cycle.We hypothesize that after Cef up-regulates GLT-1 expression,it affects the quantity of VGLUTs and the transmission of noxious information and the formation of hyperalgesia,thus exerting the effect of relieve chronic neuropathic pain.Based on this hypothesis,this study was designed to observe the expression of VGLUT subtypes in SDH of CCI rats after Cef treatment and their relationship with GLT-1.And to provide further experimental evidence for the mechanism of Cef's anti-pain effect and the role of GLT-1 and VGLUT played in neuropathic pain.Methods: Ninety healthy male Sprague-Dawley rats weighing 290±20 g were randomly divided into the following six groups:(1)Naive group(n=6): Six normal rats were sacrificed.Right L4-L6 segment of SDH was taken out.The expression of GLT-1 and VGLUT subtypes was analyzed by Western blot.(2)Sham group(n=30): Right sciatic nerves of rats were exposed but not ligated.The changes of thermal withdrawal latency and mechanical withdrawal threshold were observed on 1 d before operation,and 1 d,4 d,7 d,10 d,14 d,17 d,21 d after the operation.Right L4-L6 segment of SDH was taken out at 1 d(n=6),4 d(n=6),7 d(n=12)and 21 d(n=6)after operation.The expression of GLT-1 and VGLUT subtypes was analyzed by Western blot.(3)CCI group(n=36): Rats were subjected to sciatic nerve constriction injury(CCI)on the right.The changes of thermal withdrawal latency and mechanical withdrawal threshold were observed on 1 d before operation,and 1 d,4 d,7 d,10 d,14 d,17 d,21 d after the operation.The rats were sacrificed on 1 d(n=6),4 d(n=6),7 d(n=12),14 d(n=6)and 21 d(n=6)postoperative days.Western blot was used to detect the expression of GLT-1 and VGLUT subtypes in L4-L6 segment of SDH.(4)CCI+i.p.NS+i.t.DW(n=6): CCI was performed on the right sciatic nerve of 6 rats.Normal saline(NS),0.72 ml/kg,was injected into the abdominal cavity of rats on the day after operation,once a day for 7 consecutive days.Seventy-two hours after the first intraperitoneal injection,10 ?l double distilled water(DW)was injected into the spinal canal of rats.The thermal withdrawal latency and mechanical withdrawal threshold were observed 1 d before operation,4 d and 7 d after operation.And L4-L6 segment of SDH was taken out 7 d after CCI.The expression of GLT-1 and VGLUT subtypes was detected by Western blot.(5)CCI+i.p.Cef+i.t.DW(n=6): CCI was performed on the right sciatic nerve of rats.Cef was injected into the abdominal cavity of rats on the day after operation,200 mg/kg once a day for 7 consecutive days.Other operations is same as that in CCI+i.p.NS+i.t.DW group.(6)CCI+i.p.Cef+i.t.AS-ODNs(n=6): CCI was performed on the right sciatic nerve of rats.Cef,200 mg/kg,was injected into the abdominal cavity of rats on the day after operation,once a day for 7 consecutive days.Seventy-two hours after the first intraperitoneal injection,the rats were intrathecally injected with GLT-1 antisense oligodeoxynucleotides(AS-ODNs)18 nmol once.The thermal withdrawal latency and mechanical withdrawal threshold were observed 1 d before operation,4 d and 7 d after operation.And L4-L6 segment of SDH was taken out 7 d after CCI.The expression of GLT-1 and VGLUT subtypes was detected by Western blot.Results:1 Changes of pain threshold and expression of GLT-1 and VGLUT subtypes in SDH after CCI1.1 Changes of pain threshold after CCIIn Sham group,the thermal withdrawal latency and mechanical withdrawal threshold of the hind paw of the operated side did not significantly change at 1,4,7,10,14,17 and 21 days after operation.Compared with sham group,after CCI,obvious thermal and mechanical hyperalgesia were observed in rats.It was shown that thermal withdrawal latency and mechanical withdrawal threshold began to decrease from post-operative day one(P<0.05),reached the lowest level on post-operative day 4,and lasted to post-operative day 21 observed.1.2 Changes in the expression of GLT-1 and VGLUT subtypes in rat SDH after CCIWe first observed the effect of sham surgery on the basic expression of GLT-1 and VGLUT subtypes.Western blot results showed that compared with naive group,the expression of GLT-1 and VGLUT subtypes in SDH of rats on 1,4,7 and 21 days after sham operation had no significant changes.Therefore,we chose day 7 after sham operation as the control group of GLT-1 and VGLUT expression changes at different time points.Western blot results showed that compared with sham group,the expression of GLT-1 protein in SDH significantly decreased from postoperative day 4 and lasted to day 21 observed(P<0.05);the expression of VGLUT1 protein in SDH significantly decreased from postoperative day 7 and lasted to day 21 observed(P<0.05);the expression of VGLUT2 protein in SDH significantly decreased from postoperative day 4 and lasted to day 21 observed(P<0.05);and the expression of VGLUT3 protein in SDH significantly decreased from postoperative day 1 and lasted to day 21 observed(P<0.05).These four glutamate transporter proteins decreased in the same time window as the development of hyperalgesia after CCI,suggesting that they might participate in the development and maintenance of chronic neuropathic hyperalgesia induced by CCI.2 Effects of intraperitoneal injection of Cef on chronic neuropathic hyperalgesia and expression of GLT-1 and VGLUT subtypes in SDH of CCI rats2.1 Effect of intraperitoneal injection of Cef on chronic neuropathic hyperalgesia in CCI ratsCompared with CCI group,CCI+ i.p.NS+i.t.DW group had no significant effect on thermal withdrawal latency and mechanical withdrawal threshold in the hind paw of rats 4 and 7 days after operation.Compared with the CCI+i.p.NS+i.t.DW group,the thermal withdrawal latency and mechanical withdrawal threshold of the hind paw of rats on the 4th and 7th day after CCI were significantly increased after intraperitoneal injection of Cef(i.e.CCI+i.p.Cef+i.t.DW group)(P<0.05).After intrathecal injection of GLT-1 AS-ODNs 18 nmol(i.e.CCI+i.p.Cef+i.t.AS-ODNs group),the latency of thermal withdrawal latency and mechanical withdrawal threshold of the hind paw on the 4th and 7th day after CCI decreased again(P<0.05),suggesting that GLT-1 AS-ODNs inhibited the therapeutic effect of Cef on neuropathic pain in CCI rats.2.2 Effect of intraperitoneal injection of Cef on the expression of GLT-1 and VGLUT subtypes in SDH of CCI ratsWestern blot results show that compared with sham group,the expression of GLT-1,VGLUT1,VGLUT2 and VGLUT3 in CCI group significantly decreased on post-operative day 7(P<0.05).Compared with CCI group,the expression of GLT-1,VGLUT1,VGLUT2 and VGLUT3 in CCI+i.p.NS+i.t.DW group do not significantly change after operation.Compared with CCI+i.p.NS+i.t.DW group,the expression of GLT-1,VGLUT1,VGLUT2 and VGLUT3 significantly increased after intraperitoneal injection of Cef(i.e.CCI+i.p.Cef+i.t.DW group)(P<0.05).After intrathecal injection of GLT-1 AS-ODNs 18 nmol(i.e.CCI+i.p.Cef+i.t.AS-ODNs group),the expression of GLT-1,VGLUT1,VGLUT2 and VGLUT3 significantly decreased again on post-operative day 7(P<0.05),which indicated that GLT-1 AS-ODNs inhibited the Cef-induced up-regulation of GLT-1,VGLUT1,VGLUT2 and VGLUT3 in SDH of CCI rats.Conclusions:The down-regulation of VGLUT1,VGLUT2 and VGLUT3 might be involved in the development and maintenance of CCI-induced neuropathic pain in rats.Cef might play a role of anti-neuropathic pain by its up-regulation of VGLUT1,VGLUT2 and VGLUT3 mediated by GLT-1 in CCI rats. |
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