| Objective: In order to investigatethe change of expression oftransient receptor potentialmelastatin8(TRPM8), we establish a ratmodel of thenitroglycerin migraine,prescribe some prophylaxis drugs (flunarizine, citicoline,combination of those two drugs). The aim of this experiment is to elucidate the roleofTRPM8channel in the mechanism of migraine, and to provide evidence for theusing of citicoline as a preventive treatment drug of migraine in clinical.Methods:50healthy male Sprague-Dawley (SD) rats (clean grade, two orthree-month-old, weight at the range of180-200g),were randomly divided intoblank control group (n=10) and nitroglycerin model group (n=40) with theproportion of1:4. Blank control group injected saline (10mg/kg) at the necksubcutaneous, model group according to Tassorelli method: cervical subcutaneousinjection of nitroglycerin (10mg/kg)once a week. Then the model groups (40rats)were randomly divided into four groups again, there were model controlgroup (n=10,injected saline10ml/kg), flunarizine group(n=10, lavage injection1.25mg/kg),citicoline group (n=10, sodium0.1g/kg), combined group(n=10, flunarizine1.25mg/kg, citicoline sodium0.1g/kg). From the second week, the every modelgroup were injectedthe nitroglycerin at neck subcutaneous per week (10mg/kg), andat the same time give different drug lavage every day. At5th week, allthe rats wereinjected the nitroglycerin or saline and given lavage injected respectively.4hourslater,10%chloral hydrate was used to anesthetize the rats, as well as thetrigeminalnerve ganglion was quicklyremoved and fixed with4%paraformaldehyde at4℃.We usedHEstainingmethod to observe the change of the trigeminal nerve ganglionstructure.To detect the expressions of glutamate receptor in migraine rats,Immunohistochemical(IHC)was carried. The other tissue was immediately stored inliquid nitrogenuntil the TRPM8protein level was analyzed via western blot method. Results: The behavior gradedisplay that the model control group was significantlyhigher thanthe blank group (P<0.01)， the nitroglycerin migraine modelwasestablished successful. Eachdrug prophylaxis group waslower thanmodel controlgroup (P<0.01),flunarizine group was lower thanciticoline group (P<0.01),combinedgroup waslowest than model control group (P<0.01). HE stainingdisplay that thestructure of trigeminal ganglionhas not special change in each group. Based on theresults of IHC, expressions of glutamate receptor in each model group wasobviouslyhigherthan the blank group (P<0.01), citicoline groupwas lower thanflunarizinegroup (P<0.01),combined group was lower thanciticoline group (P<0.01), combinedgroup was negative expression of glutamate receptor (P<0.01). Therewasstatisticallysignificant difference in each group (P<0.01),citicoline groupwaslower thanflunarizinegroup (P<0.01),combined group was lower thanciticolinegroup(P<0.01), combined group was most lower thanciticoline group andflunarizinegroup(P<0.01), the OD values took the same method showed thatexpression of TRPM8was statistically significant difference in each group (P <0.01). Western blot analysis demonstrated that the level of TRPM8protein inmigraine model control group obviously higher than blank group, flunarizine groupwas a bit lower thanmodelcontrol group, citicoline groupreduced obviously, andcombined group was lowest than model control group. The grey value accord withnormal distribution, the single factor variance analysis showed that expression ofTRPM8was statistically significant difference in each group (P <0.01). There waspositive correlation between the expression of TRPM8andbehavior grade(r=0.822,P<0.01),it illustrated that the moreexpression of TRPM8, the higherwas behavior grade.There was positive correlation between the expression ofTRPM8and glutamate receptor(r=0.794,P<0.01), it implied that TRPM8andglutamate receptor take animportant role in the mechanism of migraine.Conclusion:(1) chronic migraine for trigeminal nerve ganglion has nosignificant damage on the anatomical structure.(2)The finding implied that theup-regulationof TRPM8protein and activation of glutamate receptors may beinvolved in themechanism of migraine attacks.(3) This experiment has proved thatciticoline as neural-protective drug, could prevent the function of nerve, it has asignificant roleto hinder migraine of the neuropathic pain, and combined classicpreventive drug has a more remarkable effect. |
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