The Role And Related Mechanism Of γ-aminobutyric Acid B2 Receptor In Central Sensitization Of Chronic Migraine

Background: Chronic migraine(CM)has been listed as one of the four most serious chronic dysfunction diseases by WHO and is also one of the most common chronic daily headache(CDH)diseases,characterized by frequent headache attacks and at least 15 headache days per month.Migraine sufferers account for 1-2% of the global population.The result of the long course of illness and intense headache attacks is the huge reduction in the quality of their life and work.However,the pathogenesis of CM is very complex and unclear.Currently,one of the widely accepted views is that neurogenic inflammation leads to overexcitation of neurons,also known as central sensitization,which is the main mechanism of CM.Previous studies have shown that GABABR plays a crucial role in regulating the sensation of pain.Various pain models have shown that GABABR expression is decreased and its activation can significantly improve pain,reflecting that GABABR is closely associated with the central sensitization process of pain.However,the mechanism of central sensitization inCM involving GABABR has not been reported.Thus,the purpose of this research is to probe whether GABABR is able to facilitate the pathophysiological process of CM via central sensitization.Methods:1.Healthy adult Wistar rats(male,250-300 g)were injected with dural inflammatory soup(IS)for 7 consecutive days to establish a rat model of CM.The reliability of the model of chronic migraine was evaluated by measuring the thermal and mechanical pain thresholds of CM rats.2.The m RNA levels of GABAARα2,GABABR1 and GABABR2 in the Periaqueductal gray(PAG)were analyzed by quantitative real-time polymerase chain reaction(q-PCR).Western blot and immunofluorescence staining both were performed to verify the variation of GABABR2 expression.Immunofluorescence staining was conducted to investigate the distribution of GABABR2 in neurons,inhibitory interneurons and microglia cells.3.Immunofluorescence staining was performed to label the inhibitory interneurons at the PAG and determine the number of inhibitory interneurons inCM rats.High-performance liquid chromatography(HPLC)and Western blot were used to determine the concentration of inhibitory neurotransmitter GABA in PAG and the expression of GABA synthetase glutamate decarboxylase 65/67(GAD65/67)inCM rats.4.The variation of mechanical pain thresholds and thermal pain thresholds inCM rats were detected after the low and high doses of GABABR and PKA agonists and inhibitors(Baclofen,CGP35348,H89,8-Br-cAMP)were administered by intraventricular injection.Then,the effective doses of four drugs were applied in rats of Sham group to observe the variation of the pain thresholds of the hindpaw.5.After four drugs were administered to the lateral ventricle of rats inCM group,immunofluorescence staining and Western blot were performed to test the expression levels of calcitonin gene-related peptide(CGRP),c-Fos protein and vesicle glutamate transporter(VGLUT2)in the PAG.HPLC was applied to detect glutamate content in the PAG.6.After the four drugs were treated by intraventricular injection in rats of CM group,the expression levels of protein kinase A(PKA),the total cAMP response binding protein(CREB),the phosphorylated CREB phosphorylated(p CREB)and synaptic cell adhesion molecule 1(SynCAM1)in the PAG were observed by Western blot analysis.7.After CM rats were treated with Baclofen and H89 drugs,Western blot was conducted to determine the levels of postsynaptic density 95(PSD95),synaptophysin(Syp),synaptotagmin1(Syt-1),and brain-derived neurotrophic factor(BDNF)proteins in the PAG.At the same time,electron transmission electron microscopy(TEM)was performed to analyze the synaptic ultrastructural differences in the PAG.Results:1.After a seven-day repeated dural IS infusions,the thermal and mechanical pain thresholds in the CM group robustly declined.The results of behavioral indicated that continuous dural IS infusion successfully established a pain model associated with CM.2.There was no substantial variation in the GABAARα2 m RNA expression in the PAG between the Sham and CM groups.Compared to the Sham group,GABABR1 was sharply elevated,while GABABR2 was significantly down-regulated.Western blot and immunofluorescence staining also confirmed the reduction in GABABR2 protein levels.Additionally,the results from immunofluorescence stain showed that GABABR2 was mainly localized in neurons,partially expressed in inhibitory interneurons,but no obvious expression was observed in microglia cells.3.Compared to the Sham group,inhibitory interneurons,inhibitory transmitter GABA and its synthetase GAD65/67 in the PAG of rats inCM group were significantly reduced,illustrating that the inhibitory function of interneurons is impaired in the PAG of CM rats.4.After low and high doses of GABABR and PKA agonies and inhibitors(Baclofen,CGP35348,H89,8-Br-cAMP)were injected intraventricularly inCM rats,the thermal and mechanical pain thresholds of hindpaw in the rats with high-dose Baclofen and H89 application markedly elevated,while the thermal and mechanical pain thresholds of hindpaw further declined in high-dose CGP35348 and 8-Br-cAMP conditions.In addition,when the effective dose(high dose)of the four drugs was administered in the Sham group,no substantial change in the mechanical and thermal pain thresholds of hindpaw was found.The high dose of four drugs was selected in the subsequent studies.5.The expression levels of glutamate,VGLUT2,CGRP and c-Fos in the PAG of the CM rats were robustly promoted compared to the Sham group.The administration of Baclofen and H89 to the lateral ventricle significantly reduced expression of glutamate,VGLUT2,CGRP and c-Fos in the CM rats,while the treatment of CGP35348 and 8-Br-cAMP induced the opposite result.H89 significantly relieved the elevation of CGRP and VGLUT2 levels evoked by CGP35348 compared to the result of CGP35348 treatment alone inCM rats.6.The levels of PKA,p CREB and SynCAM1 in the PAG of the CM rats were robustly raised compared to the Sham group,and there was no substantial variation in the total CREB protein.After CM rats were administered with four drugs,the protein expression levels of PKA,p CREB and SynCAM1 in the PAG in the Baclofen and H89 treatment groups decreased significantly,while the treatment of CGP35348 and8-Br-cAMP triggered the opposite results.The lateral ventricle injection of the four drugs did not influence the protein expression level of total CREB.7.The expression levels of PSD95,Syp,Syt-1 and BDNF in the PAG of CM rats substantially improved compared to the Sham group.Application of Baclofen and H89 drugs inCM rats markedly decreased PSD95,Syt-1,Syp and BDNF protein expression levels.In addition,TEM observations showed that the synaptic plasticity was enhanced in the PAG of CM rats,and the treatment of Baclofen and H89 ameliorated the deterioration of synaptic structure.Conclusion: This study found the deficit of GABABR2-related inhibitory system function in the PAG of CM rats,and GABABR2 activation can ameliorate glutamate-dependent central sensitization via modulating synaptic plasticity.Besides,this research also provides a novel GABABR2-SynCAM1 signaling-mediated mechanism underlying the glutamate-dependent central sensitization in the PAG of CM rat model,which provides an innovative perspective on the potential therapy targets against CM.