| Background and ObjectiveCerebral vascular disease has become one of the three main fatal diseases because of it's high disability, high mortality and high morbidity. It jeopardizes humans's health since 20 century. The ischemic cerebrovascular disease accounting for70-80% which have complicated causes. Recently, there is an increasing tendency in the morbility of cerebrovascular disease. Owing to this ,more and more scholars have begun studied the neuroprotective agents .The neuroprotective agents can be used with more people. It can complementing the deficiency of only thrombolysis, prolonging the survival period of ischemic cells. It can decrease the infarct volume and ameliorate the prognosis to those people who can not be thrombolysized. So far, there are few efficient and cheap medicines in clinical practice. Therefore, finding eligible neuroprotectives has become one of the urgent problems to be solved .Most experiments of animal have indicated: The neuron death in ischemia/ reperfusion includes necrosis and apoptosis. The conception of apoptosis has come to cause scholar's attention and become one of the hot research points in diverse fields since kerr et al introduced it firstly in 1972. Studies have proved, the happening of the cell apoptosis is related to the expressions of genes, the present studying concentrate on the Bcl-2 gene family, P53 gene, caspases family. Bcl-2 is a suppressive apoptosis gene, and bax is a promoting apoptosis gene. The protein ratio of bal-2/bax determines the degree of cell apoptosis. Topiramate is a new type anti-epileptic,whose chemical structure is completely different from the present anti-epileptic. It belongs to natural monosaccharideic dextrorotary sulphide, easily permeates blood brain barrier . As a wide anti-epileptic spectrum, topiramate have various mechanisms : (1) blocking the voltage-dependent Na~+ channel :blocked action potential induced by neuron's continual depolarization. (2) enhancingγ-aminobutyric acid (GABA) activities:Topiramate can enhance GABAA receptor frequency which are activated by GAB,accelerate the Cl~- entereing cell,which can enhance the suppressive function of neurotransmitter;(3)blocking up AMPA (α-amino-3-hydroxy- 5-methylisoxazole-4-propionic acid )/KA (kainate)type glutamate receptor:Topiramate can decrease the Cl~-entereing cell that through AMPA/KA. The recently study have indicated the neuron protective actions of topiramate in experimental cerebral ischemia attack. But the mechanisms of neuroprotective of topiramate is not clear known.In this experiment, an animal of MACO in rats was established through an occlusion of thread method. Did intragastric administration with topiramate. Then observed the cell injury in hippocampal CA1 under optical microscope, detected the cell apoptosis in hippocampus areas with TUNEL way and the Bcl-2, Bax protein expressions in immunohistochemistry to approaching the topriamate neural protective functions, which can offer theoritic base for the topiramate's clinica application as neural protective agents.Material and methods(1) Group of experimental animalsHealthy adult male SD rats 72, weighting 280-350g, were randomly divided into sham group (A group) , operation group(B group), tropiramate intervention group(C group), Then A B C groups were subdivided reperfusion 6h 12h 24h 48h groups, every group keeps six rats. Before the model made, C group were intragastric administration with topiramate( 100mg/kg/d) which solved the 2ml normal saline. A B groups were intragastric administration with the same volume normal saline;(2) Produce of the modelThe ischemia/reperfusion models were made accoding to the way of reports made by Cao Yongjun and Cheng Yanbin after the third intragastric administration half an hour later. The sham group are made the same operation except the thread should not reach the internal cervical artery;(3) Produce of specimensExecuting rats in different time, fixing with 4% paraformaldehyde, extracting the brains, embedding them with paraffin;(4) Indexes surveyed1) Assessment of the behaviors: All the rats were marked on 6h 12h 24h 48h after being reperfusion;2) Using the HE and TB(toluidine blue) dying to observe the changes of brain neurons; Taking immunohistochemistry(SP) to survey the expressions of bcl-2 and bax and observing the hippocampus cell apoptosis in TUNEL way.(5) Statistics analysisThe data was handled with SPSS11.5 statistic software. The experimental data was described by means±standard deviation. The difference of two groups was compared with t-test. The difference of many groups was compared with one-way analysis of variance, significant level is a=0.05.Results(1) the common situationAfter MCAO model made, all the rats behaved low spirit in various degree, eat little, low ability of self-clearance, slow in reaction, sluggish and hemiparasis. The rats of Group B and C decreased the right appendicular muscle power, not stretch right upper extremity while raising tail, revolved, chased tail or dextrad lateroversion after fatal middle cerebral artery occlusion;(2) Behavioral marks: Group A are zero. The behavioral marks of group C are lower than that of group B(P<0.05). Expecially the behavioral marks on reperfusion 48h, group B:1.50±0.22; group C:0.50±0.55(P<0.01);(3) The result of pathological section with HE staining in every group:(1)The hippocampal neuronal cells arranged closely ,have clear layers and more cell amount in group A .(2)In hippocampus neuronal cell of group B, there are unclear cell layers, loose arrangement, fewer cells, and the cell bodies became triangle caused by crenation and degeneration, karyopyknosis, broken to pieces or solvation, vacuolar degeneration in some neurons. The gaps between neuronas and brain tissue was broaden.(3) In group C, the cell morphous changes are less than that of group B, but there are still to some extent cell deletion;(4) TB(toluidine blue) dying: (1)Group A: Hippocampus neural cells have clear layers, closely arrangement, round and big nucleus and rich in Nissl bodies. The cell plasma and nuclui can be spotted easily.(2)Group B : The neuron layers of hippocampus s decreased arranged barely, nissl bodies disappeared,plasma and nuclui became unclear.(3) In group C, the cells and nissl bodies is more than group B. Comparing to group B, the cell morphous changes of group C are better than group B. But group A have more nissl bodies than group B;(5) Bcl-2 with immunohistochemistry: There was no apparent expression in group A. At the same time, there were significant in the difference among group A with group B and C (P<0.05). At the same time, the mean of Bcl-2 positive cells of group B is lower than group C (P<0.05). The mean of Bcl-2 positive cells of every group at 6h: group A, 4.67±1.03; group B, 27.67±1.63; group C, 30.67±1.21. 12h: group A, 5.17±0.98; group B, 43.33±2.16; group C, 50.17±2.32. 24h: group A, 5.17±0.98; group B, 43.33±2.16; group C, 50.17±2.32; 48h: group A, 5.33±1.03; group B, 12.67±1.86; group C, 30.50±1.87;(6) Bax with immunohistochemistry:There was no apparent expression in group A. At the same time, there were significantin the difference among group A with group B and C (P<0.05). At the same time, the mean of Bax positive cells of group B is lower than group C (P<0.05). The mean of Bax positive cells of every group of 6h: group A, 5.00±1.26; group B, 21.50±1.87; group C, 14.83±2.71. 12 h: group A, 4.83±1.17; group B, 33.17±1.94; group C, 25.33±3.00. 24 h: group A, 4.83±1.17; group B, 32.67±1.86; group C, 20.50±2.17; 48h: group A, 4.00±1.10; group B, 18.17±0.75; group C, 12.50±2.43;(7) Results of TUNEL marking :There were few apoptosis cells in group A; At the same time ,the apoptosic cells in group C were lower in contrast to group B(P <0.05). The mean of TUNEL positive cells of every group at 6h: group B, 9.50±1.87; group C, 7.00±1.79. 12h: group B, 13.67±2.16; group C, 9.17±1.47. 24 h: group B, 17.17±2.23; group C, 11.17±1.67. 48h: group B, 20.00±1.41; group C, 13.33±1.68;Conclusions(1)Tropiramate can degrade the behavioral marks in ischemia/reperfusion rats and reduce the symptom of the deletion of neuron.(2)Tropiramate can protect the rat brain via enhancing the expression of bcl-2 ,degrade the expression of bax and decrease the cell apoptosis in hippocampus areas.
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