| Background: Central sensitization is the potential pathogenesis of chronic migraine(CM)and is related to persistent neuronal hyperexcitability.Dysfunction of excitatory amino acid transporter 2(EAAT2)leads to the accumulation of glutamate in the synaptic cleft,which may contribute to central sensitization by overactivating glutamate N-methyl-D-aspartate(NMDA)receptors and enhancing synaptic plasticity.However,the therapeutic potential of CM by targeting glutamate clearance remains largely unexplored.The purpose of this study was to investigate the role of EAAT2 in CM central sensitization and explore the effect of EAAT2 expression enhancer LDN-212320 in CM rats.Method:1.Healthy adult Sprague–Dawley rats(male,250–300 g)were injected with dural inflammatory soup(IS)for 7 consecutive days to construct a rat model of CM.The establishment of the CM rat model was evaluated by measuring the changes of the pain thresholds and the proteinexpression level of calcitonin gene-related peptide(CGRP)in the TNC sites of CM rats.2.The glutamate concentration was measured by high-performance liquid chromatography(HPLC)in the TNC of CM rats.Then,quantitative real-time polymerase chain reaction(q-PCR)and Western blot were performed to examine EAAT2 expression,and the localization of EAAT2 in astrocytes and the immunoreactivity of astrocytes were detected by immunofluorescence staining.3.After the lateral ventricles were given low,high two doses of EAAT2 expression enhancers LDN-212320 and Ceftriaxone(Cef),the changes of mechanical and thermal pain thresholds and the protein expression levels of EAAT2 and CGRP at the TNC sites were detected in CM rats.4.A low-dose of EAAT2 expression enhancer LDN-212320 was chosen for subsequent experiments.After LDN-212320 was administered to the lateral ventricle,the protein expression levels of CGRP were examined by Western blot,and the mean fluorescence intensity of CGRP and substance P(SP)and the immunoreactivity of astrocytes were detected by semi-quantitative immunofluorescence.5.After a low-dose of LDN-212320 was given to the lateral ventricle of CM rats,the glutamate concentration in TNC were detected by HPLC,and the protein expression levels of NR2 B,pNR2B-Y1472,pNR2B-Y1474 and the synapse related proteins postsynaptic density 95(PSD95),synaptotagmin1(syt-1),synaptophysin(Syp)were examined by western blots.In addition,the dendritic spine density of TNC neurons were observed by Golgi-Cox staining and the synaptic ultrastructure was observed by transmission electron microscope(TEM).6.After the CM rats were treated with LDN-212320 in the lateral ventricles,the expression changes of central sensitivity-related proteins c-Fos,CAMP response element binding protein(CREB),pCREB-133,pCREB-129 were detected by western blots.Results:1.After a seven-day repeated dural IS infusions.Compared with the rats in the sham group,the mechanical pain thresholds of the periorbital and hind paws and the thermal pain thresholds of the hind paws in the CM group were significantly decreased,while the protein expression levels of CGRP were increased in TNC.Both results of behavioral and biomarker indicated that a pain model associated with CM was successfully built after continuous IS stimulations.2.Elevated glutamate concentration and decreased EAAT2 expression were found in the TNC of CM rats.Immunofluorescence results showed that EAAT2 was mainly located in astrocytes,and the astrocytes in CM group were activated.3.After treatment with LDN-212320 and Cef in the lateral ventricle,the pain thresholds and the protein expression of EAAT2 in CM rats were significantly increased,while the protein expression of CGRP decreased.In addition,it was found that LDN-212320 had a better effect than Cef in up-regulating EAAT2 protein expression and alleviating allodynia.4.Treatment with a low-dose LDN-212320 in the lateral ventricle reduced the CGRP protein level,attenuated the mean fluorescence intensity of CGRP and SP and the activation of astrocytes in the TNC sites of the CM group.5.In addition,treatment with LDN-212320 reduced the glutamate concentration and the expression of phosphorylated NR2 B,and restored the changes in synaptic structure and the expression of synaptic-associated proteins in the TNC of CM rats.6.Low-dose of LDN-212320 in the lateral ventricle reduced the expression of central sensitization-related proteins in the TNC,and alleviated the central sensitization in CM rats.Conclusion: Taken together,our study demonstrated downregulated expression of EAAT2 in a rat model of CM,the novel compound LDN-212320 could restore the protein expression of EAAT2 through translation activation.The recovery of EAAT2 expression can reduce the transmission of nociceptive signals and regulate synaptic plasticity to alleviate central sensitization.Upregulation of EAAT2 expression has a protective effect in CM rats,and LDN-212320 may have clinicaltherapeutic potential. |
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