The Establishment Of The Model Of Chronic Migraine And Expression Of ASICs,P/Q Type Calcium Channels

Objective Migraine is a common neurovascular disease with multiple factors,population prevalence is about 15%,and its clinical manifestation is recurrent headache.Migraine incidence is ranging in age from 25 to 55 years old,which seriously influence the patient's life and work.Although migraine is popular,its pathophysiological mechanism is not clear,so effective drug therapy remains to be explored.Migraine is a kind of subjective feeling,so all of the overall level of the research on mechanism of migraine must be validated.But existing migraine models are still limited.This study intends to repeatedly stimulate mice dura mater with acid solution,similar to the pathogenesis of chronic migraine,to obtain better chronic migraine model.In addition,the research on the preliminary migraine model to explore the possible roles of ASICs,P/Q type channel in migraine.Materials and Methods1.C57BL/6 mice were divided into four groups according to random number method:control group(Sham),negative control group(PH=7.4 synthetic interstitial fluid,PH 7.4 SIF),the experimental group(PH=6.0 synthetic interstitial fluid,PH 6.0 SIF),the positive control group(inflammatory soup,IS group),six in each group.PH=6.0 SIF continuously stimulate the dura mater in the experimental group and mice in the negative control group were given PH=7.4 SIF,while positive control group were given IS.After day intermittent dosing eight times,watch the headache behavior in mice;immunohistochemical staining to detectSp5C c-FOS gene and PAG calcitonin gene related peptide(CGRP)expression.2.To explore the effect of amiloride and AMG-9810 on acid-induced chronic migraine.C57BL/6 mice were divided into three groups according to random number table method:PH 6.0group,PH 6.0 + Amiloride group,PH 6.0 + AMG-9810 group,six in each group.Three reagents on alternate days continuously stimulate mice dura mater,respectively,8 times after the treatment,observation of headache behavior in mice,test the expression of Sp5C c-FOS gene.3.After the last dose for 2 hours,take the negative control group and experimental group mice trigeminal ganglion and trigeminocervical complex,using quantitative PCR,Western blotting,to detect the mRNA and protein expression of ASICs subtypes(ASICla,ASIC3),P/Q type calcium channel.4.Further explore the role of ASICs subtypes(ASICla,ASIC3)and P/Q type calcium channel in acid-induced chronic migraine model.Results1.observation after the last dose,the experimental group of mice PH 6.0 vs PH 7.4 negative control group were more likely to scratchfacial skin,the phenomenon of irritability and no obvious difference was found compared with inflammatory soup positive control group.The comparative difference within 2h duration between experimental group of mice(736 + 66 secs.)and the negative control group(127?18 sees.)was statistically significant(t = 21.824,P = 21.824),and no statistically significant was found between experimental group of mice and the positive control inflammatory group(747 + 73 secs.),(t = 0.277,P= 0.277);The comparative difference in Sp5C c-FOS positive neurons between experimental group of mice(121±18.7)and the negative control group(14±2.8)was statistically significant(t=113.893,P?0.000),and no statistically significant was found between experimental group of mice and the positive control inflammatory group(124±19.8),(t=-0.240,P= 0.816);The comparative difference in the periaqueductal gray CGRP neurons between experimental group of mice(42±2.8)and the negative control group(811.4)was statistically significant(t= 26.336,P=0.000),and no statistically significant was found between experimental group of mice and the positive control inflammatory group(4012.8),(t= 1.225,P= 0.249).2.It was found that PH 6.0 + Amiloride group mice were not prone to boredom?compared with PH 6.0group and PH 6.0 + AMG-9810 group mice were agitated the same as the PH 6.0 group mice.Statistics of duration of wiping and scratching in 2h,PH 6.0 + Amiloride group(343±45secs.)wasdecreased significantly than PH 6.0 group(736±66 secs.)and the difference was statistically(t = 12.05,P = 0.000);there was no statistical differences between PH 6.0 + AMG-9810 group(721 ±79 secs.)and PH 6.0 group(t = 0.34,P = 0.741).As for c-FOS positive cells number in Sp5C,PH 6.0 + Amiloride group(42 ±4.5)significantly reduced compared with thePH6.0 group(121 ± 18.7)by immunohistochemical staining andthe difference was statistically difference(t=10.072,P=10.072).While,there was no statistical differences between PH 6.0 +AMG-9810 group(117± 17.2)and the PH 6.0 group(t = 0.448,P = 0.448).3.The relative mRNA expression levels of ASIC la,ASIC3 andP/Q type calcium channel in the trigeminal ganglion of Acid-induced chronic migraine is PH 7.4 negative control group(1.59±0.07),(1.65-0.08)and(1.6±0.17)times,respectively.Andthe differenceswere statistical significant(t = 19.74,P = 19.74;t = 18.878,P =0.000;t = 7.761,P = 0.0015).The relativeprotein expression levelsof ASICla,ASIC3 andP/Q type calcium channel in trigeminocervical complexof Acid-induced chronic migraineis PH 7.4(1.67±0.23),(1.74±0.24)and(1.30±0.05)times,respectively.And the differenceswere statistical significant(t = 5.01,P = 5.01;t = 5.352,P = 0.033;t =5.774,P = 0.0287).4.Behavioral observation found thatPH 6.0 + KN-93 group mice were not prone to boredom compared with PH 6.0group.Statistics of duration of wiping and scratching in 2h,PH 6.0 + KN-93 group(437±44 sec)was decreased significantly than PH 6.0 group(736 + 66 secs.)and the difference was statistically(t=9.220,P=0.000);and there was statistical differences between PH 7.4 group(127 ± 18secs.)andPH 6.0 +KN-93 group(t=-15.908,P=0.000).c-FOS positive cells number in Sp5C,PH 6.0 +KN-93 group(40±3.4)significantly reduced compared with the PH 6.0 group(121118.7)by immunohistochemical staining and the difference was significant(t=-10.456,P=0.000).And there was statistical differences betweenPH 7.4 group(14±2.8)andPH 6.0 + KN-93 group(t = 0.448,P = 0.448).p-CaMKII/CaMKII ratio in PH 6.0 group higher than PH 7.4 group by Western blotting,and the difference was statistically significant(t= 10.595,p = 10.595).p-CaMKII/CaMKII ratio in PH 6.0+KN-93 groups reduced(0.55 + 0.05)times compared with PH 6.0 group,and the difference was statistically significant(t = 15.731,p = 15.731).Conclusion1.Repeatedly discontinuous acid stimulation on dura mater can be successfully established mice model of chronic migraine;2.ASICs instead of TRPV1 channels are involved in the formation of acid induced chronic migraine model;3.In acid induced migraine model,acid activates ASICs in the dura afferent fibers,further activates the P/Q type calcium channels,through Ca:MKII phosphorylation and activation of CaMKII/cAMP response element binding protein(CREB)pathway,regulate the expression of c-FOSgene.