An Experiment On The Neuroprotective Effect Of Different Dose Of Topiramate On Hypoxic-ischemic Brain Damage In Newborn Rats

Research background(Hypoxic-ischemic encephalopathy,HIE) was a newborn time common disease,which caused the newborn death and important attributed to the forward nervous system growth barrier.Its pathogenesis was not still clear about,therefore did not have the powerful treatment.To pay more attention to pathogenesis and effective intervention,would improve the prognosis and population quality of HIE.Topiramate was a new kind of effective antiepileptic.A lot of experiments had indicated that topiramate played a vital role in the cerebellum protection after brain damage. More and more research indicated that topiramate played a vital role in protection of hypoxic-ischemic damage brain and would be used to treat HIBD clinically in future.But referring to the mechanism of protection,dosage and side effect,there was few studies.The glial cell line-derived neurotrophic factor(GDNF) was a new subtribe member of TGF-βsuper family,which could have nerve nutrition effect,improve and promote repairing and regeneration of the damage neuron.The brain,which shorted of blood and oxygen,would be a possibly start factors of GDNF,which could increased secretion.Based on the earlier research,we thought that there was few GDNF expression in HIBD sham-operation group in 7day Wistar rat and GDNF was started to rise at 12h,elevated at 2d,reached the peak at 3d,recedes at 5d,but the level wa still high in the simple oxygen deficit lacks blood group.Some research found that,the astrocyte could express Na⁺ and the Ca²⁺ channel,and also GluR and GABAR in its membrane,in view of the fact that holds the topiramate could suppresses the activation of Na⁺ or/and in the Ca²⁺ channel and regulate the balance of GABA/Glu.Therefore,we guessed that topiramate would be effective through the corresponding acceptor,which expressed in the astrocyte.It also could protect astrocyte and promotes the GDNF secretion,which could protect the function of neuron.The earlier research of our team also confirmed this point.Given the topiramate at different times, we observated the different GDNF secretion.This earlier period research had proven that,as topiramate used in HIBD,the treatment time window was short.Lacking the blood after the oxygen deficit in 2h,and also the prevention medication was better than after damage applies;The topiramate the brain protective function was related to the GDNF secretion directly.Many animal experiments study showed topiramate had the obvious treatment function to lack the blood to the brain.The effective dosage was between 50mg/kg-200mg/kg,and had no side effect on edge system and skeleton growth.This research was through established the HIBD animal model,the observation brain damage and topiramate treatment about the brain organization and pathology changes and GDNF dynamic expression.Further discuss the topiramate brain protective function,the mechanism,and the effective dosage, providing the powerful theory and the experimental basis and making a new HIE treatment way.Objective1.To observe the brain organization aspect and so on pathology change of various newborn mat treatment groups in the same time.2.To observe the expression of GDNF,and dynamic changes of newborn rat after HIBD. With changes of the different dosage topiramate,the changes of the GDNF expression.3.To discuss the mechanism of topiramate.Methods1.120 newborn on 7th the age Wistar rat were divided into the sham-operation group,the pure oxygen deficit stochastically lacks the blood group and topiramate treatment group, topiramate treatment group were weighted,and then given medicine 50mg/kg,100mg/kg and 150mg/kg especially.2.Made the HIBD animal model.3.Given the medicine and the methods(1) Topiramate treatment group:Topiramate was dissolve into sterile water and maked into 2%solution.Using the small stomach fills into stomach.Each the different dosage topiramate treatment group separately 30min,then given 1 time.After the HIBD model completed,instantly given 1 time.12h after the surgery each one time,until 5d.(2)The pure oxygen deficit stochastically lacks the blood group and the sham-operation group were given the equal volume physiological saline at identical time spot.(3) Each group executes 1/4 animal separately at 12h,2d,3d,5d after HIBD. 4.Using HE dyeing,Immunochemistry and Western Blotting,we observed.Results1.HE dyeing findingsIn sham-operation group,brain organizational structure level and the cell outline were clear and the nucleus was in the middle,Nepali body was peri-nucliar.In the pure oxygen deficit lacks the blood group,lacking the blood after the oxygen deficit 12h and also lacking the blood,side cerebral cortex and the corpus striatum appeared the slight pathological changes.The 2d pathological changes were most serious,the cerebral cortex, the corpus striatum,the seahorse and the cerebral ganglion displayed a great necrosis area. The 3d pathological changes were reduced,which appeared the spongiocyte proliferation. The 5d almostly did not see the breakage cells and could see big pieces of scar.After 50mg/kg,100mg/kg and 150mg/kg topiramate treatment group,the 12h above spot pathological change not to be obvious,2d appears the slight pathological change,displays increases the width for few intercellular space,the cell nucleus concentration,3d and 5d had not seen the obvious focus of infection.150mg/kg topiramate treatment,the group brain damage was prior to 100mg/kg topiramate treatment group,and 100mg/kg topiramate treatment group was prior to 50mg/kg topiramate treatment.2.Immunochemistry findingssham-operation group only showed the few GDNF masculine cell expression,the coloration was shallow,distribution sparsely in the entire cerebral cortex,the corpus striatum,the seahorse,the two-sided symmetry and did not see the obvious dynamic change in various time light the examination;The pure oxygen deficit lacks the blood group,after 12h of damage,there were to GDNF expression in side cerebral cortex,2d further rised,3d reached the peak,5d was descented to normal and only extremely few GDNF expression; 50mg/kg,100mg/kg and 150mg/kg topiramate treatment group,after lacking the blood side cerebral cortex 12h,it could see many GDNF expression,2d further increased,3d reached the peak,5d reduced,but also obviously many expressions,and 150mg/kg topiramate treatment group,the expression of GDNF was obvious,in each time the expression of GDNF was obviously higher than the pure oxygen deficit to lack the blood group (P＜0.05).The GDNF expression was related to the dosage of topiramate,which was statistics significance(P＜0.05).3.Western blotting findingsThe pure oxygen deficit lacks the blood group and with topiramate treatment groups all had the GDNF protein expression banding.150mg/kg topiramate treatment GDNF protein expression was higher than the pure oxygen deficit to lack the blood group obviously, 100mg/kg topiramate treatment group was slightly weak,50mg/kg topiramate treatment group was worst.The application biology gel electrophoresis image analysis system (Alphaimager 2200) scans the quota,through calculated various histone expression,and the relative integral valve to make the protein the comparative content,discovered after statistics processes:50mg/kg,100mg/kg and 150mg/kg topiramate treatment group at various time to light the protein expression to be higher than the pure oxygen deficit to lack the blood group,had statistics significance(P＜0.05);The GDNF expression increased with holds the topiramate ester to become the dosage related,had statistics significance(P＜0.05).Conclusion1.Topiramate could obviously reduce the brain cell dropsy,the liquefication and necrosis, which was caused by oxygen deficit and short of blood.It was also showed the good brain protective function and the dosage dependent.2.Topiramate would be increased the GDNF expression after HIBD,and its expression degree was related to the dosage of topiramate.3.Topiramate's brain protective mechanisms may be related to GDNF increased,which were secreted by spongiocyte.