| Objective:Epilepsy and migraine are similar in clinical characteristics,neuronal excitability,aura,treatment and so on,are prone to comorbidity.However,due to the lack of animal models,current studies on the comorbidity of epilepsy and migraine are mainly focused on clinical studies.This study intends to use the previously established rat model of epilepsy and migraine to explore the tissue and pathophysiological mechanism of the comorbidity.The genetic mechanism is the recognized mechanism of comorbidity of epilepsy and migraine,but for the larger number of sporadic comorbidity of epilepsy and migraine,the inflammatory mechanism is most likely.Studies have found that toll-like receptor 4(TLR4)induces innate immunity,stimulates the production of inflammatory cytokines,and is involved in the pathological processes of epilepsy and migraine.Our previous monopathy study on epilepsy and migraine found that GABA receptor alpha 1(GABAARα1)expression was decreased after epilepsy and migraine-like seizures in rats.Therefore,in this study,the inflammatory mechanism was used as the entry point for comorbidity of epilepsy and migraine.The temporal cortex and hippocampus were selected as the epilepsy areas of interest,and trigeminal ganglion and medullary dorsal horn were selected as the head pain areas of interest to explore the mechanism of GABAARα1-TLR4 pathway in epilepsy-migraine comorbidity and the relationship between upstream and downstream.Methods:1.Establishment of animal model: Intraperitoneal(I.P)lithium chloride-pilucapine was injected to establish a rat model of epilepsy.The superior sagittal sinus parietal dura was surgically exposed,after catheterization,inflammatory soup(IS)was given daily to stimulate the dura to establish a rat migraine model.2.Grouping: the rats were randomly divided into control group,migraine group,epilepsy group and epilepsy-migraine comorbidity group(comorbidity group).3.Behavioral approach: after intraperitoneal injection of pilucapine induced epilepsy,the latency and maximum seizure level of epileptic seizures in rats were observed.After stimulating the dura mater with IS and inducing migraine-like attack,the number of scratches was observed and the facial mechanical withdrawal threshold was measured with von-frey filaments.4.Western blot: tthe tissues of each group of rats were lysed,the protein solution was extracted after centrifugation,the concentration was determined by BCA method,the amount of the upper sample was calculated,the film was transferred after SDS-PAGE electrophoresis,the skim milk was sealed,the primary and secondary antibodies were incubated according to the molecular weight of the target protein,and the gray value was calculated by membrane scanning to compare the differences between the groups.5.Immunofluorescence: sections of the brain tissues of each group were taken for antigen repair,after the immunohistochemical pen carefully circled along the tissue block,the tissue self-luminescence quench agent was added,sealed in the wet box,and the primary and secondary antibodies were incubated respectively.Finally,the slices were sealed,and the fluorescence intensity values were calculated by reading the slices under the fluorescence microscope to compare the differences in the composition.6.Co-immunoprecipitation: the medulla oblongata dorsal horn of comorbidity rats tissue was lysed,then the protein solution was extracted,objective protein antibody was added with corresponding species IgG(negative control group)and protein solution at a ratio of 1:50.After incubation,protein A/G agarose magnetic beads were added,incubation again,then,PBS was used to wash them thoroughly,supernatant was extracted after centrifugation,and SDS-PAGE electrophoresis was carried out.The film was transferred,and the antibody was incubated,then the film scaned to determine whether GABAARα1 and TLR4 could form receptor heteromeric complexes.7.Drug intervention: the behavioral differences of comorbidity,comorbidity+Muscimol and comorbidity+TAK-242 were observed after the intervention of Muscimol and TAK-242,including migraine behavior(the number of scratches and the facial mechanical withdrawal threshold),epilepsy behavior(the latency of seizure and the Racine scale level).The GABAARα1 and TLR4 expression in temporal cortex,hippocampus,medullary dorsal horn and trigeminal ganglion,and the corresponding changes in different groups of rats were detected by Western Blot.Results:1.Compared with the control group,the number of scratches in the migraine group increased,and the facial mechanical withdrawal threshold decreased,with statistically significant difference(P<0.01),suggesting that the inflammatory soup model could sensitize the rats’ head and face to pain..Compared with the control group,there was no statistically significant difference in the number of scratches and mechanical withdrawal threshold in the epilepsy group(P>0.05),suggesting that epileptic seizures had no effect on the headache behavior of the rats.Compared with the migraine group,the number of scratches in the comorbidity group increased,with a statistically significant difference(P < 0.01),suggesting that the epilepsy-migraine comorbidity model was more obvious than the migraine model in hypersensitivity to pain.2.Compared with the control group,the positive expression of microglia in trigeminal ganglion increased in the migraine group(P<0.01),suggesting that the inflammatory mechanism was involved in the migraine-like attack in the inflammatory soup rat model.Compared with the control group,the positive expression of microglia in trigeminal ganglion increased in the epilepsy group,with a statistically significant difference(P < 0.01),indicating that the seizure of epilepsy also activated the inflammatory process in the area of interest for headache.Compared with the migraine group and the epilepsy group,the positive expression of microglia in trigeminal ganglion increased in the comorbidity group(P < 0.01),suggesting that the inflammatory response in the comorbidity group was more obvious than that in the monotherapy group.3.The expression of TLR4 and GABAARα1 was detected by western blot.Compared with the control group,the TLR4 expression in the temporal cortex and hippocampus,as well as in the medullary dorsal horn and trigeminal ganglia was all increased in the migraine and epilepsy groups,while the expression of GABAARα1 was decreased in the migraine and epilepsy groups,with statistically significant difference(P<0.05).Compared with the migraine and epilepsy groups,the TLR4 expression in the above regions of the comorbidity group was increased,and the expression of GABAARα1was decreased,with statistically significant difference(P < 0.05),suggested that the expression of TLR4 was increased and the expression of GABAARα1 was decreased,which could occur in both single migraine/epilepsy and comorbidity4.The expression and distribution of TLR4 and GABAARα1 was detected by immunofluorescence method.Compared with the control group,the positive expression of TLR4 in the temporal cortex,trigeminal ganglion and medullary dorsal horn of rats in the migraine and epilepsy group was increased,and the positive expression of GABAARα1 was decreased,with statistically significant difference(P<0.05).Compared with the migraine epilepsy groups,the positive expression of TLR4 in the temporal cortex,trigeminal ganglion and medullary dorsal horn of the rats was increased,and the positive expression of GABAARα1 was decreased,with statistically significant difference(P<0.05),indicated that the expression of TLR4 was increased and the expression of GABAARα1 was decreased,which could occur in both single migraine/epilepsy and comorbidity.5.By Co-immunoprecipitation,it was confirmed that there was a direct interaction between GABAARα1 and TLR4.6.After pretreatment with GABAAR agonist Muscimol,the latency period of epileptic seizures was prolonged and the level of seizures decreased in the epileptic phase of comorbidity,with statistically significant difference(P < 0.05),suggested that activation of GABAAR could inhibit epileptic seizures.After pretreatment with TLR4 receptor antagonist TAK-242,there was no significant difference(P>0.05)in the latency and level of epileptic seizures in the epileptic phase of comorbidity,suggested that inhibition of TLR4 had no effect on epileptic seizures.After pretreatment with Muscimol and TAK-242,the number of head scratches decreased and the facial mechanical withdrawal threshold increased during the comorbidity period of epileptic-migraine,with statistically significant difference(P < 0.01),indicated that activation of GABAAR/inhibition of TLR4 could both inhibit migraine-like attacks.7.The expression of GABAARα1 and TLR4 in temporal cortex,hippocampus,medullary dorsal horn and trigeminal ganglion were detected by western blot.After pretreatment with GABAAR agonist Muscimol,the TLR4 expression in rats in the comorbidity+Muscimol group was lower than that in the comorbidity group,,while the expression of GABAARα1 was higher,with statistically significant difference(P <0.05).After pretreatment with TLR4 receptor antagonist TAK-242,the TLR4 expression in the comorbidity+TAK-242 group was lower than that in the comorbidity group,with statistically significant difference(P < 0.05),but the expression of GABAARα1 was not significantly changed(P > 0.05).All of these suggested that activation of GABAAR could reverse the decrease of GABAARα1/increase of TLR4 caused by comorbidity,while inhibition of TLR4 could only reverse the increase of TLR4 caused by comorbidity,without affecting the expression of GABAARα1.Conclusions:1.The comorbidity model was not a simple superposition of the two separate diseases,it could simulate the pathophysiological phenomenon that epileptic seizure would facilitate the onset of headache.2.The inflammatory mechanism is involved in the facilitative effect of epilepsy on headache.Comorbidity of epilepsy and migraine could lead to the decrease of GABAARα1 expression and the increase of TLR4 expression.3.There was a direct interaction between GABAARα1 and TLR4.4.GABAARα1 reducing could cause epileptic seizures,which is the initiating factor of comorbidity of epilepsy and migraine,could lead to increase in TLR4 expression and facilitate migraine-like seizures. |
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