【Abstract】Aim To investigate the effects of Ginsenosides on dentate granule neurogenesis after global ischemia reperfusion in the adult rat brain, and to discuss the potential mechanisms of Ginsenosides in neurogenesis after ischemia. Methods Male mature Sprague Dawley rats were subjected to 4-vessel occlusion (4-VO) model. By using Bromodeoxyuridine ( BrdU ) labelling method , the number of neural precursor cells in the dentate gyrus were compared between intervention group of Ginsenosides and control group at 3 d, 7 d,14 d,21 d, 28 d after ischemia. Results Ginsenosides significantly increased the number of BrdU labeled cells in the dentate gyrus 7, 14, and 21 d after ischemia（P < 0.01）, but did not influence the number at the 3rd and the 28th d . Meanwhile, there were not significantly difference between the ischemia contral group and intervention group of contral（P > 0.05）.Concluaion The Ginsenoside can promote neurogenesis on dentate granule, and the accommodational mechanisms of enhanced neurogenesis are possibly related to many biological activity of Ginsenosides
【Key words】Ginsenoside; Neurogenesis; Neural precusor cell; Bromodeoxyuridine; Cerebral ischemia
For a Long time ,people have thought that the nerve cells in man’s brain will survive all his life.Once encountering injuries，the brain will lose nerve cells for ever.And the lost nerve cells can only be repaced by glial cells,because mature nerve cells can’t divide .Following the rapid development of the biomedicine technology,recently people found that there is the phenomenon of neurogenesis in mature man’s brain tissue,and this phenomenon will last all his life.This discovery break the traditional viewpoint that nervre cells is ultimate cells and can’t regenerate[1-3]. Correlated studies have confirmed that brain injury caused by ischemia can induce the increase of neurogenesis level in mature brain tissue.This discovery brings hope to look for new therapy of ischemic cerebrovascular disease,and the method of promoting neurogenesis of mature brain maybe brings a new therapy for ischemic brain injury.Therefor we investigated the effection of Ginsenoside on granule neurogenesis in the adult rat in vivo,and discussed the potential mechanisms of Ginsenoside’s acceleration on the proliferation and differentiation of neural precusor cells.We hoped to offer the theoretic basis of application of Ginsenoside on ischemic cerebrovascular disease.
Material and Method
1.Groups of experimental animals: We used 45 male mature Sprague Dawley rats that were supplied by Experimental Animals Center of the Forth Military Medical University and which body weight was 200~250g.These SD rats were randomly divided into ischemia control group、intervention group of contral and intervention group of Ginsenosides.And then, each group were divided into five groups by time points of 3d 、7d、14d、21d、28d after 4-VO model was maded.
2.Model of ischemia animals: According to related literatures, the rats were subjected to 4-vessel occlusion (4-VO) model. Cerebral ischemia lasted 15 min.The standard of this model is silent brain waves of cerebral ischemia under EEG monitoring,and those rats that did not fit to this standard were not considered.
3.Administration of BrdU、Ginsenoside and Method of Tissue Picking: Ginsenosides (100 mg/kg, supplied by the Department of Phytochemistry of Bethune Medical University) were injected into abdominal cavities of intervention group of Ginsenosides on 30min before and after ischemia, and then twice each day in the following 2 days ; meanwhile saline of same dosage were injected into intervention group of contral by the same way. BrdU were injected into abdominal cavities of both ischemia groups 24h before specified time points twice with a interval of 2h .The rats were anaesthetized by soluble pentobarbitone (40 mg/kg) at every specified time point, instillated with saline of 100 ml and 4% citromint (pH 7.4) of 500 ml through ascending aorta. Then we broke rats’ heads and took out of brains. The brain tissue were fixed in 4% citromint through a night , sedimentated in 20% sucrose liquid 4℃.We choose hippocampus to get coronal serial section using cryomicrotome, and selected one section each 150μm whose thickness was 30μm, and all selected sections were collected in 0.01 mol/L KPBS.
4.Method of Immunohistochemistry: The selected sections were put into 50% Formamide (Sigma)/2×SSC(65℃ 2h)、2N HCL(37℃ 30min)、mice Monoclonal antibody of BrdU (1:1000，Sigma)4 ℃ by turns and incubated for 36h.Then put in anti-antibody marked by anti-bition of mouse (1:500，Sigma) in room temperature for 2h. After that, put in compounds of ABC(1:500，Sigma) in room temperature for 2h.After developed by augmentation method of DAB, sections were banked up. Sections were washed by 0.01MPBS among above-mentioned steps. Another series of sections were made according to above-mentioned method without antibody as contral.
作者:admin@医学,生命科学 2010-12-12 17:11