| Objective:The First,used the topiramate (TPM) and the flunarizine (FNZ) to prevent and intervent the migraine model of nitroglycerin rats,then stripping the trigeminal ganglion tissue of these rats. The second,we would applicat the immunohistochemical method to detect the changes of adenosine 2A receptor protein expression and the content of calcitonin gene related peptide in these rats trigeminal ganglion.In order to confirm the topiramate may be through inhibiting the expression of A2AR to reduce the release of CGRP to prevent the attack of migraine and also provide the new experimental and theoretical basis for the prevention mechanism about topiramate treat the migraine.Methods:1. Dividing the group:choose 3-4 months and weighing 180-200g of clean grade healthy male Sprague-Dawley (SD) 40 rats,and divide them into 4 groups and 10 rats in each group according to the random number table.They are called control group, model group, topiramate group (hereinafter referred to as TPM group) and flunarizine group (hereinafter referred to as FNZ group).2. Making the model:inject the normal saline in the subcutaneous of the blank group rats about10ml/kg.However,the remaining 3 groups according to the method of Tassorelli inject the nitroglycerin (nitroglycerin, NTG) 10 mg (2ml)/kg in the subcutaneous of rat, by the above methods 1 times a week, in total 5 weeks,and the rats were scored after inhection.3. Drug intervention:from the beginning of the second week, rats were by intragastric administration one time of day:blank group using NS 10ml/kg/d, model group using NS 10ml/kg/d, FNZ group using flunarizine 1.25mg/Kg/d, TPM group using topiramate 9mg/kg/d.4. Seeking the material:In the fifth week,after 4 hours when rats were injected drugs and bolled gastric, inject the chloral hydrate anesthesia 10% 3ml/kg in the intraperitoneal of rats.when rats were completely anesthesia, use the NS to perfusion the heart of rat, open the cranial cavity, separate the brain and dura mater, and expose the trigeminal ganglion of skull base.Last,intercept the middle part of the uptake of intact (about 2-3cm) with 4%paraformaldehyde fixed.These tissue partially preserved in the refrigerator (4 DEG C for HE staining and immunohistochemistry), the other preserved in liquid nitrogen (for Western-blot).5. Target detection:â‘ taking part of trigeminal ganglion tissue were stained with HE, and observed the change of organizational structure under the microscope; â‘¡ immunohistochemistry (SP method)to detect CGRP content variations in the trigeminal ganglion of migraine rats in each group; â‘¢ immunofluorescence was used to detect the expression of A2AR protein in the trigeminal ganglion neurons; â‘£ detection the amount of change of A2AR protein expression of Western-by blot.6. Analysis on the application of SPSS on the detection results of 17 software for statistics.Results:1. Injection of nitroglycerin after the set of behavioral scores in rats:the model group compared with the control group, the difference was statistically significant (P<0.01); two kinds of drug intervention group (FNZ group and TPM group) were compared with the model group, the difference was statistically significant (P<0.01); the two drug intervention groups compared with each other, there was statistical significant differences (P<0.01).2. HE staining showed that the trigeminal ganglion were observed under microscope:the structure is basically the same, no significant difference.3. Immunohistochemical method was used to detect CGRP content:the OD value showed differential expression of CGRP in trigeminal nerve tissue in each group was statistically significant (P< 0.01), the model group was significantly higher than the control group, the FNZ group was higher than that of blank group and TPM group, but lower than that in the model group, the TPM group was higher than that of control group and lower than model group and the FNZ group.4.The expression of A2AR was detected immunofluorescence:in the trigeminal ganglion tissue A2AR protein mainly expressed in neurons, there was significance of the expression of A2AR positive neurons in the trigeminal ganglion were few differences (P< 0.01); the number of fluorescence intensity and positive cell in TPM group were significantly lower than that of model group and FNZ group. Similar with the blank group.5.Western-blot detection of A2AR protein:the gray value showed differential expression of A2AR in each group was statistically significant (P< 0.01), which expressed in the blank group and the TPM group of A2AR protein were significantly lower than that of model group and FNZ group.6.The OD value of CGRP and A2AR gray values for correlation analysis showed that the difference was statistically significant (P<0.01).Conclusion:1.No anatomical structure changes of trigeminal nerve tissue of rats, suggesting that behavior changes in rats may be abnormal functional.2 Activation or up regulation of the A2AR protein and CGRP release may be involved in the pathogenesis of migraine, and their variation trends are consistent.3. Flunarizine which is the prevention and treatment of classic migraine drug may inhibit the trigeminal nerve sensory nerve endings release of CGRP to achieve the effect of prevention and treatment of migraine, but had no influence on the expression of A2AR protein.4. Topiramate may inhibit the activation of A2AR or down regulated the protein expression and inhibition of CGRP release to prevent migraine migraine symptoms and reduce the Curative effect.5. The clinical efficacy of topiramate in migraine may be better than flunarizine. |
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