| Background and ObjectiveStroke is the third leading cause of death and the No. 1 cause of adult disability. Complex pathophysiologic process happened in brain tissue after cerebral ischemia occurred, which included interruption of blood, cerebral hypoperfusion, energy depletion, reperfusion injury after blood flow recanalization, free radical reactions, excitatory amino acid toxicity, intracellular calcium overload and so on. All of these will lead to degeneration and necrosis of neurons, delayed neuron death and/or apoptosis. The application of neuroprotectant, which can reduce a series of pathological injury caused by ischemia, is a recent focus of cerebral ischemia perfusion injury.Recent studies demonstrated that progressive decline of the rate between y-aminobutyric acid (GABA) and glutamate (Glu), which result in the imbalance of excitatory and inhibitory neurotransmitter, is one of the important reasons for causing ischemic neuronal injury. GABA is the major inhibitory neurotransmitter in the brain, which fight against Glu, the main excitatory neurotransmitter, in neuronal injury. There are at least three kinds of GABA receptors mediated GABA inhibitory effect, in which GABAa receptor distribute most abroadly and take a leading role in neuroprotection during ischemic neuronal injury. N-methyl-D-aspartate (NMDA) receptor plays an important role in the excitotoxicity of Glu. Influx of calcium caused by excessive excitement of NMDA receptor can induce delayed nerve cell injury, which play the leading status in the pathology of excitotoxicity. NMDAR1 is the necessary function sub-unit of receptor complex and play the important role in the central effect of NMDA.At present, the neuroprotectitive effect of antiepilepsy drugs has been noticed, which probably due to reduce the transmission of excitement or enhance neuronal inhibition and offset abnormal excitability of brain. Topiramate is a novel wildly clinical used antiepileptic drug with multiple pharmacological mechanisms, which include enhancing GABA-mediated inhibitory currents, blocking Ca2+ and Na+ channels, antagonize glutamatergic excitotoxicity through non-NMDA receptors, etc. The application of Topiramate in antiepilepsy has gotten satisfied effects; however, the research about its role in neuroprotection after cerebral ischemic are not fully undertaken. Studying neuroprotective effect of Topiramate and its mechanism in the ischemic neuronal injury have a great a significance, which can provide the theory basis for its clinical application as a neuroprotectant.By administrating Topiramate in a rat model with focal cerebral ischemia, determining the content of GABA and Glu in the cerebral ischemic region through HPLC analysis, detecting receptor-positive cells of infarction hemisphere NMDAR1.GABAa receptor by immunohistochemical staining, observing cell necrosis changes by light microscopy, this study try to explore topiramate's nerve protection and delve into its mechanism, this study try to explore neuroprotection of Toprovide and its mechanism, which will be useful for finding new measure and applying topiramate in the treatment of cerebral infarction in the future.Materials and methodsAnimal groups: 92 healthy male SD rats, weighing 280-320 grams, from Zhengzhou University Laboratory Animal House. The rats were divided into four groups in random.The number of each group is eighteen. A: Sham-operated ; B: Ischemia-reperfusion ; C: Ischemia-reperfusion plus therapeutic topiramate ; D Ischemia-reperfusion plus preventive topiramate .Right middle cerebral artery occlusion (MCAO) animal model was made by ligation according to the way in Kuge's report with a little modification. The sham operated group is the same operation except that it will not plug the nylon thread into the internal carotid artery. Group B , C, and D the rats makes up MCAO model.Groug A: sham treatment, gavage saline(10ml/kg) after 24 hoursGroug B: garage saline(10ml/kg) when reperfusion Groug C: garaged fresh TPM suspension(10mg/ml,100mg/kg) when reperfusionGroug D: garage 3 days before cerebral ischemia-reperfusion(dose: 10mg,once a day); the last time, after garaging 30minutes, do ischemia-reperfusion brain surgery. And the others were anesthetized and perfused with 200ml normal saline and subsequently fixed with 4% paraform to build the paraffin sections,which provided for HE stainning and immmunohistochemistry observation.Production of Specimens: Reperfuse the rats 24 hours later,select 6 rats from each group in random beheading brain, put them on ice plates rapidly and separate right parietal cortex volume, weighting, adding HCLO4 (0.4mol/L)according to 1: 10, using tissue homogenates to ice homogenate 1.5min. precipitating and ice-bathing for 30 minutes, centrifugating (12,000per min, 20 min, 40°C), then dip supernatant into several tube, and put them in a refrigerator to preserve on -70°C, waiting for testing. And the others were anesthetized and perfused with 200ml normal saline and subsequently fixed with 4% paraform to build the paraffin sections,which provided for HE stainning and immmunohistochemistry observation.Observation (1) Ischemia-reperfusion 24 hours later, measure the nerve function deficiency score. About general behavioral observations, carry out the neurological function score by Zausinger six points(2) HE staining: observe brain pathology in light microscopy.(3) Using immunohistochemical technique to observe the expression of NMDAR1 and GABAA (4) Using HPLC analysis to measure the content of GABA and Glu in brain tissue.Statistical Methods : SPSS 10.0 software for data processing, All data is used bymean±standard deviation (x|-±s) to indicate. Several groups are compared by using single-factor analysis of variance, selectα= 0.05 as a significant test.Results(1) sham-operated group doesn't have neurological deficits ; Score of ischemia-reperfusion group the nerve function deficiency is 1.32±0.76; Score of the topiramate prevention of neurological behavioral is 2.97±0.94. Score of topiramate in the treatment of neurological behavioral is 2.11±0.93. Each group had significant difference (P<0.05).(2) In light microscopy topiramate group prevention group infarct area surrounding nerve cells gap widens, cells swelling and shrinkage, and scatter neutrophils infiltration. Ischemia-reperfusion group infarct area surrounding nerve cells gap widened, cell swelling, residual neurons appears karyopyknosis. Organization beacomes loose, short cytoplasm with cell necrosis performance infarction peripheral nerve cell swelling, degeneration and cytoplasm with desalination. Necrotic foci of inflammatory cell infiltration around significantly Topiramate heavy intervention group, cell shape change than the intervention group. And the sham-operated group in light microscope structure clearly nucleolus exist, pathological changes little.(3) In rat brain cortex,Glu. GABA content of the sham-operated group 86.36±7.01uM/L. 5.57±0.36)uM/L; ischemia-reperfusion group 105.87±20.70uM/L. 8.61±1.98 uM/L; Topiramate in the treatment group 75.31±9.87)uM/L. 11.87±2.41uM/L, topiramate group 63.29±7.08 .13.91±2.29 ischemia and reperfusion in rat cerebral ischemic tissue excitatory amino acid glutamate, and the inhibitory amino acid GABA are increased (P<0.01). Topiramate Glu in the intervention group decreases, compared with that in chemia-reperfusion group(P<0.01), while GABA content increases(P<0.01). Among them, Topiramate prevention group is more effective than Topiramate in the treatment group (P<0.01) .(4)the number of GABAa receptor-positive cells in ischemic territory or rats sham-operated group: 58.16±1.72; the ischemia-reperfusion group: 19.67±2.80. the TPM treated group.: 31.50±1.38 ; the TPM prevented group:38.67±1.63 .GABAa receptor-positive cells in the ischemia-reperfusion group descend clearly (p<0.01)compared with that in sham-operated group. GABAa receptor-positive cells in the TPM treated group increased compared with that .in the ischemia-reperfusion group (p<0.05).then the numbers in TPM prevented group increased significantly compared with that .in the ischemia-reperfusion group.The number of NMDAR1 receptor-positive cells in ischemic territory or rats sham-operated group: 35.50±1.38; the ischemia-reperfusion group: 25.33±2.25. the TPM treated group.: 16.00±2.09 ; the TPM prevented group:10.07±1.03. NMDAR1 receptor-positive cells in the ischemia-reperfusion group descend clearly (p<0.01)compared with that.in sham-operated group. NMDAR1 receptor-positive cells in the TPM treated group increased compared with that .in the ischemia-reperfusion group (p<0.05).then the numbers in TPM prevented group increased significantly compared with that .in the ischemia-reperfusion group.Conclusion(1).Topirarnate's protection and treatment on brain ischemia-reperfusion injury (2).The cerebral protection of topiramate is related with the application of time, cerebral protection of therapeutic application of topiramate is weak, cerebral protection of prevetion of topiramate application is strong.(3).One of the important mechanisms of topiramate in the rat ischemia is inhibit excitotoxicity amino acid release, enhance inhibitory amino acid release and change its receptor expression.
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