| Objectives:1.We aimed to establish a model of chronic epilepsy-migraine comorbidity and observe the ultrastructural changes of neurons in ventrolateral periaqueductal gray（vlPAG）region.Then local injection of lentivirus carrying brain-derived neurotrophic factor（BDNF）into vlPAG was performed to explore the protective effect of BDNF overexpression on vlPAG neurons and the behavioral manifestations in rats.2.To investigate the alterations of the neural transmission from vlPAG to pedunculopontine nucleus（PPN）in the rat model of chronic epilepsy-migraine comorbidities.3.To explore the neuroimaging mechanism of migraine symptoms in patients with temporal lobe epilepsy（TLE）by comparing the functional connectivity of periaqueductal gray（PAG）between migraine patients with TLE and those without migraine.Methods:Part 1:Epilepsy rat model was induced by intraperitoneal injection of lithium-pilocarpine in adult rats,migraine rat model was induced by repeated intraperitoneal injection of nitroglycerin,and epilepsy-migraine comorbidity model was induced by lithium chloride-pilocarpine-nitroglycerin.After modeling,the expression of c-Fos labeled positive neurons in trigeminal nucleus caudalis（TNC）of spinal cord and CGRP labeled positive neurons in trigeminal ganglion（TG）were detected by immunohistochemistry.Serum CGRP concentrations were detected by enzyme-linked immunosorbent assay（ELISA）,and the behavioral changes of rats in each group were monitored,including the number of scratching head,pain threshold and electroencephalogram（EEG）.Through the above technical means to evaluate the feasibility of each model.Part 2:According to the modeling method in the first part,rat models of epilepsy,migraine and epilepsy-migraine comorbidities were constructed respectively.The pathological ultrastructure and apoptosis of vlPAG neurons were observed by HE,TUNEL staining and transmission electron microscopy.Part 3:Before the establishment of each model,the lentivirus carrying BDNF was injected into the vlPAG region in advance,while the control group was injected with empty virus or normal saline.The expression of BDNF and tyrosine kinase receptor B（Trk B）in vlPAG region was detected by immunofluorescent staining and western blot（WB）.The morphology and apoptosis of neurons in vlPAG region were observed by HE and TUNEL staining.Immunohistochemical staining was used to detect the expression of c-Fos and CGRP positive neurons in the TNC area of the spinal cord and TG.Serum CGRP concentrations were detected by ELISA,and behavioral changes were monitored in each group,including the number of head fleeces,pain threshold,electroencephalogram,etc.The above detection methods were used to investigate the mechanism of BDNF overexpression in vlPAG on chronic epilepsy-migraine comorbidities.Part 4:After modeling,fluoro gold（FG）was injected into the PPN of the brain stem of rats in each group.Brain perfusion and dissection was performed 2 days later.The expression of the c-Fos and vesicular glutamate transporter 1（v GLUT1）in the vlPAG of each group were counted by immunofluorescence technique.The c-Fos and v GLUT1labeled with reverse tracer FG and the average immunofluorescence intensity of FG were counted.In addition,the expression levels of c-Fos and v GLUT1 in vlPAG region were analyzed by detecting the gray value of WB.Part 5:A total of 32 TLE patients were recruited in this study,including 16 cases of TLE with migraine（Ew M）,16 cases of TLE without migraine（Ewo M）,and another 14 healthy subjects were recruited as controls.We collected and collated the basic information and resting functional magnetic resonance imaging（f MRI）data of all subjects.The resting-state functional connectivity（Rs FC）based on the seed of PAG among the three groups was compared using the f MRI data.The frequency and severity of migraine attacks were assessed using the Migraine Disability Assessment Questionnaire（MIDAS）and Visual Analogue Scale/Score（VAS）.The data were processed and analyzed using DPARSF and SPSS17.0 software.Pearson correlation analysis was used to evaluate the correlation between Rs FC values and the frequency and severity of migraine attacks（VAS）in TLE patients.All data were processed and analyzed using DPARSF and SPSS17.0 Software.Results:Part 1:The behavioral monitoring results in each group were as follows:compared with the control group,the number of head scratching in the epilepsy group was slightly increased,and the difference was not statistically significant（^P>0.05）.The number of head scratching increased in both the migraine group and the comorbid group（*P<0.01）,while the number of head scratching was significantly higher in the comorbid group than that of the migraine group（△P<0.05,*P<0.01）.The paw withdrawal latency latency（PWL）of each model group was lower than that of the control group（*P<0.05）,while PWL in the comorbid group was significantly lower（^P<0.01）.Compared with the epilepsy group,the frequency and duration of epileptic seizures in the comorbid group were significantly increased（*P<0.01,△P<0.01）.Immunohistochemical results were as follows:compared with the control group,the expression of c-Fos in TNC of spinal cord and CGRP in trigeminal ganglion in epilepsy group increased slightly,and the difference was not statistically significant（P>0.05）.While both migraine and comorbid group showed increases of varying degrees(*P<0.05,**P<0.01),Compared with the migraine group,c-Fos and CGRP positive neurons in the comorbid group were significantly increased（*P<0.05）.The c-Fos and CGRP positive neurons in comorbid group were significantly increased compared with epilepsy group(**P<0.01).ELISA results showed that the serum CGRP concentration in the migraine,epilepsy and comorbid groups increased in different degrees when comparing with the control group(*P<0.05,**P<0.01).Compared with the monopathic groups,serum CGRP concentration were significantly increased in the comorbid group(**P<0.01).Part 2:HE staining showed that the injured nerves in vlPAG area were round or oval in shape,while TUNEL positive cells presented a compact nuclear condensation with a color of dark blue.Compared with the control group,the vlPAG neurons were all presented different degrees of injury in the model groups,and the TUNEL positive cells were increased（△P<0.05,*P<0.01）;compared with the monopathic groups,the number of TUNEL-positive cells in the comorbid group was significantly higher（*P<0.01）,indicating that the degree of neuron injury is more serious.Transmission electron microscopy showed that vlPAG nucleus was round with clear nuclear membrane,complete organelles and normal myelin sheath structure in the control group.In both migraine and epilepsy groups,partial bilayer nuclear membrane fusion,coarse endoplasmic reticulum expansion and swelling,mitochondrial cristal fusion,partial vacuolation,uneven thickness of myelin sheath of myelin nerve,and stratified lamellar structure were observed;while the ultrastructural changes of the neurons were more obvious in the comorbid group,including unclear nuclear membrane,chromatin edge collection,sparse organelles,swelling of rough endoplasmic reticulum,disappearance of inner and outer membranes of mitochondria,obvious vacuolation,appearance of giant mitochondria,thinning and breakage of myelin sheath of myelinated nerves.Part 3:After NS,AAV and AAV-BDNF intracranial injection for 3 weeks,Immunofluorescence staining in vlPAG showed that the AAV-BDNF injection subgroups exhibited significantly higher average optical densities of BDNF-and Trk B-positive neurons than those of the AAV or NS injection group（*P<0.05,^P<0.01）.In addition,the average optical densities of BDNF-and Trk B-positive cells in the comorbidity subgroup was significantly lower than that of the corresponding monomorphic subgroup（*P<0.05,^P<0.01）.Consistent with the results of immunofluorescence staining,the AAV-BDNF injection subgroups exhibited higher expression levels of both proteins compared to the AAV or NS injection group（*P<0.05,^P<0.01）.Additionally,the expression of both proteins in the comorbidity subgroups were significantly lower than that of the corresponding monomorphic subgroups（*P<0.05,^P<0.01）.Behavioral test results showed that PWL was not significantly different among the control subgroups（△P>0.05）.AAV-BDNF injected rats in both the monomorphic and comorbidity groups exhibited significantly lengthen PWL compared to NS or AAV vector injected animals.（*P<0.05,^P<0.05）.In the comorbidity groups,three subgroups exhibited significantly shorten PWL than that of the corresponding monomorphic and control subgroups（^P<0.05）.The subgroup rats in the control group showed less head scratching frequency with no significant difference（△P>0.05）.AAV-BDNF injected rats both in the monomorphic and comorbidity groups exhibited significantly decreased number of head scratching compared to NS or AAV vector injected animals(*P<0.05,**P<0.01).In the comorbidity group,the number of head scratching frequency of the three subgroups was higher than that of the corresponding subgroups in both monomorphic and control groups（^P<0.05）.EEG monitoring data showed significantly increased the spontaneous seizures and seizure duration in the AAV-BDNF injection subgroup than those in the AAV or NS injection subgroups(*P<0.05,**P<0.01).In addition,under the same intervention conditions,the spontaneous seizures and seizure duration in the comorbidity group were higher than those in the epilepsy group（^P<0.05）.Few TUNEL-positive cells were observed in the control group with no difference among the three subgroups（△P>0.05）.The average number of TUNEL positive cells in AAV and NS injection subgroups was significantly higher than that in AAV-BDNF injection subgroups(*P<0.05,**P<0.01).In addition,in the comorbidity subgroup,the average number of TUNEL positive cells was significantly higher than that of the corresponding monomorphic subgroup（^P<0.01）.The expression of c-Fos and CGRP positive neurons were less among control subgroups with no significant difference（△P>0.05）.In all model groups,the average c-Fos and CGRP immunoreactive cells in AAV-BDNF injection subgroups were significantly lower than those in the corresponding AAV or NS injection subgroups(*P<0.05,**P<0.01).In addition,the average c-Fos and CGRP immunoreactive cells in the comorbidity subgroups were significantly higher than that of the corresponding monomorphic subgroups（^P<0.01）.The serum CGRP concentrations was not significantly difference among the control subgroups（△P>0.05）.In all model groups,the serum CGRP concentrations in AAV-BDNF injection subgroups were significantly lower than those in the corresponding AAV or NS injection subgroups(*P<0.05,**P<0.01).In addition,the serum CGRP concentrations in the comorbidity subgroups were significantly higher than that of the corresponding monomorphic subgroups（^P<0.01）.Part 4:Two days after local microinjection of FG in PPN,FG could be found in vlPAG in each group.However,the fluorescence intensity of FG was the highest in the control group,followed by the epilepsy group and migraine group,and the lowest in comorbid group（P<0.01）.Fluorescent staining results of vlPAG showed that the number of FG-traced neurons labeled with c-Fos or with v GLUT1 in each model group were lower than those in the control group,while the decrease was more obvious in the comorbid group（P<0.01）.The variation trend of c-Fos and v GLUT1 detected by Western Blot were basically consistent with the corresponding immunofluorescence results,which showed that decreased c-Fos and v GLUT1 in both the epilepsy and migraine group,while the lowest expression in the comorbid group（P<0.01）.Part 5:Compared to HCs group,the Rs FC between PAG and the left PPN,between PAG and the corpus callosum（CC）was decreased both in Ewo M and Ew M group,while the Rs FC between PAG and the right PPN was increased only in Ewo M group but not in Ew M group.Compared to Ewo M group,the Rs FC between PAG and the right PPN was decreased in Ew M group.Rs FC values between PAG and corresponding abnormal brain regions were extracted for correlation analysis with headache frequency and headache degree of TLE patients.The results showed that the Rs FC value between PAG and left PPN was negatively correlated with migraine attack frequency（r=-0.502,P=0.048）and VAS score（r=-0.526,P=0.037）,suggesting that the lower the functional connection between PAG and left PPN,the higher the frequency and severity of headache attack in TLE patients.However,Rs FC between PAG and corpus callosum had no significant correlation with headache frequency（r=-0.048,P=0.861）and VAS score（r=0.339,P=0.198）.Conclusions:1.The establishment of the epilepsy-migraine comorbidity model induced by the combined application of lithium chloride,pilocarpine and nitroglycerin is helpful to further explore the pathophysiological mechanism of the comorbidities.2.Structural abnormalities and dysfunction in vlPAG are important mechanism of chronic epilepsy-migraine comorbidity,and the local BDNF overexpression in vlPAG region is helpful to inhibit epilepsy and migraine attacks.3.Decreased transmission of the vlPAG-PPN excitatory pathway is involved in the pathogenesis of chronic epilepsy-migraine comorbidity,and the degree of decreased functional connectivity of the PAG-left PPN pathway is correlated with the frequency and severity of headache attacks in TLE patients. |
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