| BackgroundNeuropathic pain is characterized by severe pain,difficulty in effective control and long duration,and severely damage the patient’s quality of life.The refractory characteristic of neuropathic pain is related to its complex mechanism.At present,the research on the mechanism of neuropathic pain is mainly at spinal cord level,and the peripheral mechanisms of neuropathic pain are mainly at electrophysiological changes such as abnormal discharge of nerve fibers and activation of ion channels.BTX-A is a macromolecular protein neurotoxin produced by Botox,which performed antinociceptive effect through retrograde axonal transport to spinal cord from peripheral nerve.Based on this phenomenon,we supposed that whether there is an axonal transport of pain-causing molecules between nerve fiber and neurons in peripheral nerve injury.The axonal transport mechanism may also be the cause of neuropathic pain.So,we suppose that confirmed pain-causing molecules,including CXCL13,CXCR5 and GAT-1 may be transported from the nerve injury site to the DRG neuron cell body or spinal cord through axon transport after nerve injured.PurposesIn our study,we explored the analgesic effect of BTX-A in CCI rats,and blocked the axonal transport of sciatic nerve using colchicine.Then we observed the influence of colchicine on the analgesic effect of BTX-A,the occurrence of neuropathic pain and the transport of the pain-causing molecules.So as to discover the new mechanisms of neuropathic pain,and provide new ideas and research targets for the treatment of chronic pain.Methods1.We establish the CCI model and examined the expression of CXCL13,CXCR5 and GAT-1.Rats were anesthetized with 10%chloral hydrate(3.5 ml/kg)by intraperitoneal injection.The left hind limb of each rat was shaved and disinfected with iodophor.The sciatic nerves on left side were exposed by making a skin incision and blunt dissecting through the connective tissue.And then,the sciatic nerves were separated by a glass needle.Four ligatures are tied loosely around the sciatic nerve at 1mm intervals with 4-0 surgical wire,to just occlude but not arrest epineural blood flow.The sham surgery was implemented using a similar procedure without ligation.The mechanical paw withdrawal threshold(PWTs)and thermal paw withdrawal latency(PWL)were tested at before operation,1,3,5,7,10,14,17 and 21 days after surgery.And the western blotting was used to exam the expression of CXCL13,CXCR5 and GAT-1 in spinal cord,L4-6DRG,sciatic nerve and plantar subcutaneous tissue at 0(sham),3,7,10,14 and 21 days.2.Explore on the effect and mechanism of BTX-A in neuropathic pain.The male SD rats were divided into five groups randomly:Sham group,CCI+NS group,CCI+4 U/Kg BTX-A group,CCI+7 U/Kg BTX-A group,CCI+10 U/Kg BTXA group.The rats were given injection at 7 days after surgery and the PWTs and PWLs were tested at before operation,1,3,5,7,10,14 and 21 days after surgery.The expression of CXCL13,CXCR5 and GAT-1 was examined at 21 days in spinal cord,L4-6DRG,sciatic nerve and plantar subcutaneous tissue to screen suitable concentration.The male SD rats were divided into four groups randomly:Sham+NS group,Sham+10 U/Kg BTX-A group,CCI+NS group,CCI+10 U/Kg BTX-A group.The injection was given to rats at 7 days after surgery,and we tested the PWTs at before operation,1,3,5,7,10,14 and 21 days after surgery.The spinal cord,L4～6DRG,sciatic nerve and plantar subcutaneous tissue were collected at 10,14 and 21 days after surgery,and the expression of CXCL13,CXCR5 and GAT-1 was detected by western blot to indicate the dynamic changes.3.Explore the role of axonal molecular transport in neuropathic pain.The male SD rats were divided into five groups randomly:Sham group,CCI+NS+NS group,CCI+NS+10 U/Kg BTX-A group,CCI+COL+NS group,CCI+COL+10 U/Kg BTX-A group.The 10 U/Kg BTX-A or NS was injected to rats through intra plantar at 7 days after surgery,as well as the 10 mmol/L colchicine or NS was used to treat the sciatic nerve.And we tested the PWTs of rats at before operation,1,3,5,7,10,14 and 21 days after surgery.We collected the spinal cord,L4～6DRG,sciatic nerve,and plantar subcutaneous tissue at 21 days after surgery.And the expression of CXCL13,CXCR5 and GAT-1 was examined by western blot.To further explore the peripheral axonal transport mechanism of neuropathic pain,we designed the western blotting bands with the loading order of plantar,sciatic nerve,L4～6DRG and spinal cord,and examined the expression of CXCL13,CXCR5 and GAT-1 in Sham group,CCI+NS+NS group,CCI+NS+10 U/Kg BTX-A group,CCI+COL+NS group and CCI+COL+10 U/Kg BTX-A group.Results1.The expression of CXCL13,CXCR5 and GAT-1 proteins were increased in the spinal cord,DRG,sciatic nerve and plantar subcutaneous tissue in CCI rats.The PWTs and PWLs of ipsilateral were significantly decreased from 3 days after surgery in CCI group compared with Sham group(P<0.05),and maintained a steady state from 7 to 21 days.There was no significant difference on PWTs and PWLs between CCI group and Sham group in contralateral(P>0.05).These results showed that our CCI surgery was successful.The western blot showed that the expression of CXCL13,CXCR5 and GAT-1 was significantly increased at 7,10,14 and 21 days in spinal cord,DRG,sciatic nerve and plantar subcutaneous tissue(P<0.05).2.BTX-A relieved mechanical and thermal allodynia,decreased the over expression of CXCL13,CXCR5 and GAT-1 in CCI rats.The deferent concentration BTX-A was injected to the rats through intra plantar at 7 days after surgery.The results showed that all of the 4 U/Kg BTX-A,7 U/Kg BTXA and 10 U/Kg BTX-A can relieve the mechanical and thermal hypersensitivity significantly,but the analgesic effect of the 10 U/Kg BTX-A was better than that of 4 U/Kg BTX-A and 7 U/Kg BTX-A.We collected the spinal cord,DRG,sciatic nerve and plantar subcutaneous tissue at 21 days after surgery,and the expression of CXCL13,CXCR5 and GAT-1 was detected by western blot.The results showed that the upregulation of the CXCL13,CXCR5 and GAT-1 in spinal cord,DRG,sciatic nerve and plantar was decreased significantly after medication with BTX-A {P<0.05).The inhibitory effect of 10 U/Kg BTX-A was better than 4 U/Kg BTX-A(P<0.05).3.The dose of 10 U/kg BTX-A has no effect on the mechanical hypersensitivity for sham rats.After the CCI rats were injected with 10 U/Kg BTX-A,the expression of CXCL13,CXCR5 and GAT-1 showed a downward trend among 10d,14d and 21d.The 10 U/Kg BTX-A or normal saline(NS)was injected to the rats through intra plantar at 7 days after surgery.There was no significant difference on PWTs between Sham+NS group and Sham+10 U/Kg BTX-A group(P>0.05).The results showed that the dose of 10 U/Kg BTX-A will not cause leg spasm in rats leading to an increase in the PWTs.The expression of CXCL13,CXCR5 and GAT-1 was examined by western blot at 10,14 and 21 days after surgery to indicate the dynamic changes.The results showed that the expression of CXCL13,CXCR5 and GAT-1 indicated no significant difference between Sham+NS group and Sham+10 U/Kg BTX-A group(P>0.05).What’s more,there was a downward trend among day 10,14,21 about the expression of CXCL13,CXCR5 and GAT-1 in spinal cord,DRG,sciatic nerve and plantar tissue in CCI+10 U/Kg BTX-A group.4.The mechanical hyperalgesia and the expression of CXCL13,CXCR5 and GAT-1 were significantly decreased after the sciatic nerve was treated with colchicine.The sciatic nerve was treated with colchicine(COL)at 7 days after CCI surgery to block the axonal transport of sciatic nerve.The PWTs of CCI+COL+NS group and CCI+COL+BTX-A group were significantly increased at 10 days and maintained at 15 g above(P<0.05).The PWTs of CCI+NS+BTX-A group increased significantly compared with the CCI+NS+NS group(P<0.05).The expression of CXCL13,CXCR5 and GAT-1 in plantar,sciatic nerve,DRG,spinal cord was significantly decreased in CCI+COL+NS group compared with CCI+NS+NS group(P<0.05),the results indicated that colchicine can regulate the expression of inflammatory factors;The expression of CXCL13,CXCR5 and GAT-1 showed no significant difference between CCI+COL+NS group and CCI+COL+BTX-A group(P<0.05).The results showed that the inhibitory effect of BTX-A on the expression of CXCL13,CXCR5 and GAT-1 disappeared after the axonal transport of sciatic nerve was blocked by colchicine.The expression of CXCL13,CXCR5 and GAT-1 in CCI+NS+10U/Kg BTX-A group showed a significant decreased compared with the CCI+NS+NS group(P<0.05).There was no significant difference between the CCI+NS+10U/Kg BTX-A group and the CCI+COL+10U/Kg BTX-A group about the expression of CXCL13,CXCR5 and GAT-1(P>0.05).To further explore the axonal molecular transport mechanism in neuropathic pain,we designed the western blotting bands with the loading order of plantar,sciatic nerve,DRG and spinal cord to exam the expression of CXCL13,CXCR5 and GAT-1.The results showed that the expression of CXCL13,CXCR5 and GAT-1 peaked at the sciatic nerve in CCI+NS+NS group.However,the expression of CXCL13,CXCR5 and GAT-1 peaked at DRG or spinal cord in CCI+COL+NS group.According to these results,we hypothesized that there may be a mechanism that colchicine prevents the transport of CXCL13,CXCR5 and GAT-1 from the injury site to DRG and spinal cord via nerve fibers through blocking the axonal transport of sciatic nerve.ConclusionThe expression of CXCL13,CXCR5 and GAT-1 were increased in CCI rats after CCI surgery.The BTX-A can relieve the hypersensitivity by inhibiting the expression of CXCL13,CXCR5 and GAT-1 in CCI rats through axonal transport.After the sciatic nerve was treated with colchicine,the expression of CXCL13,CXCR5 and GAT-1 was significantly decreased in CCI rats. |
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