| Background and objectiveIschemic cerebral vascular disease (ICVD) seriously endanger human's health, it is one of the most common illnesses with features of high mobility and disability rate. At present, the research on ischemic cerebral vascular disease were focused mainly in local, but the damage about the remote brain tissue and cells with ischemic infarction are less. In recent years, some researchers have shown that cerebral infarction was not only a focal damage, but also caused secondary damage in areas remote from the ischemic territory, which will retard the recovery of neurological function.Glial fibrillary acidic protein (GFAP) is one of the main intermediate filament proteins in astrocytes, as a marker of mature astrocytes activation protein whose increasing expression can partly reflect the damage of brain tissue. Tyrosine hydroxylase (TH) distribute mainly within mesencephalic substantia nigra compacta, which was usually regarded as a marker of dopaminergic neurons in the brain. The decline of TH's activity not only can decrease the formation of DA which promote the DA neurons cell degeneration and necrosis,but also can increase the sensitivity of the cells of exogenous poisons. The Ginkgo biloba extract (EGb761), a standardized extract, is a complex mixture of ingredients including 24% flavonoid and 6% terpenoid, etc. Nowadays so many studies about the effect of EGb761 were focus on the injured neuroglial cells in cerebral ischemia injury, but few of them concentrate on glial cells against ischemia damage. furthermore its protective effect on remote sites of injury and its mechanism were not clear.Consequently, we established the rats'models with cerebral ischemia-reperfusion injury and observed effect of EGb761 on the expressions of GFAP, TH and the apoptosis change, to provide the theoretical basis for clinical treatment.Materials and Methods1 Experimental animals and grouping:54 clean grade healthy male Sprague Dawley rats, weighting 220±30g,from the Experimental Animal Center of Zhengzhou University, were randomly divided into three groups:the sham group, the model group and treated by EGb761 group, each group had 18 rats. Each group was further divided into three different points at time 1d,3d,7d, each time point with 6 rats.2 Model establishment:The model of focal cerebral ischemia-reperfusion injury was made by occluding middle cerebral artery and reperfusing (MCAO/R) referenced from Longa's with improvement. The sham group was operated same as the other two groups except without inserting lines. Horner's signs in the same side and front limbs paralysis of the other side were regarded as criterion of whether models were succeed or not.3 Animal dosage:The Ginkgo biloba extract (EGb761, Ginaton,40mg/tabla,Dr. Willmar Schwabe GmbH & Co., Germany) was made into powder, dissolved in distilled water and blended into concentration of 5mg/ml solution, after that EGb761 were delivered intragastric administration of 24 hours in accordance with 0.5ml/100g, while the sham group and ischemia group were delivered with the same amounts of saline. Each group was given once a day until sacrifice.4 Specimen collections:The rats were separately anesthetized by 10% chloral hydrate (2.5ml/kg) at 1d,3d,7d, perfused with physiological saline and 4% paraformaldehyde through heart. Subsequently, the brain-tissues of rats were took out,fixed, embedded, sectioned and processed for HE staining or GFAP, TH immunohistochemical staining and TUNEL detection.5 Observation indexes:(1) HE staining was performed to observe pathological changes of brain tissue under light microscope; (2) The expression of GFAP, TH in rat substantia parts by immunohistochemical staining;(3)Detected of cell apoptosis by TUNEL.6 Statistic analysis:The experimental data were expressed by mean±standard deviation (x±s) and the data were handled with SPSS 16.0 statistic software. Significance level is judged byα=0.05.Results1 The results of pathological section with HE staining:The most neurons were clearly dense and lined tidily but in sham-operated group cells were in normal shape, the pathological changes in cell morphology of normal was not significant, while, the model group,the neurons were distributed irregularly. A number of neurons died or lost and there often was a blank area around the cell. The structure destruction of neurons and neuronal degeneration and necrosis in EGb761 group were reduced obviously lightly than model group the informal cells.2 The GFAP immunohistochemisty result:expression of GFAP-positive cells in sham group were few, had no significant differences between groups. The expression of GFAP-positive cells in the model group separately at Id,3d,7d were: 12.17±2.04; 19±1.78,25.33±2.12; EGb761 groups:10±1.41,14.33±2.73, 17.16±1.47. Above them we found that the model groups for each time point number of GFAP-positive cells are more than the other group and differences were statistically significant (P<0.01).3 TUNEL:The nuclei of apoptosis cell were coloring, brown. There were few apoptosis in sham group. Numbers of apoptosis were expressed at each time points, the model group:12.23±4.97,39.40±6.94,26.18±6.53; EGb761 group:7.31±1.92,20.26±3.7±4.61,12.23. At 3d there were most apoptosis cells and the model group was more than the EGb761 group, there was statistically significant differences (P<0.01). 4 The immunohistochemisty TH result:Tyrosine of enzyme for cytoplasm and cell membrane coloring, positive cells display a deep brown or dark brown. The positive cells at 1d,3d,7d in each group were:sham group23.39±1.14,24.87±1.98, 23.79±2.23; mod el group11.67±1.36,8.87±2.43,6.15±1.09; EGb761group 18.68±1.29,14.38±0.71,10.17±1.82,Thepositive cells at each time point after ischemia-reperfusion compared with the sham group were more, but fewer than EGb761 group, the differences was statistically significant(P<0.05).Conclusions:1. The areas remote from the ischemic-reperfusion territory took on excessive activation of astrocytes, reducing DA neurons and apoptosis.2. EGb761 can inhibit ultra-reactionary astrogliosis and reduce cell apoptosis after cerebral ischemia-reperfusion injury.3. EGb761 can protect the dopamine neurons and glial cells which located in the areas remote from the ischemic after acute cerebral ischemia-reperfusion. |
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