The Effections Of Ginkgolide B On Apoptosis And Neurogenesis In Brain Of Hypoxic-ischemic Neonatal Rats

ObjectiveNeonatal hypoxic-ischemic brain damage (HIBD) is what all sorts of perinatal asphyxia caused varying degrees of hypoxia, blood flow reduced even stopped in brain, which led to foetus or new borns brain damage. HIBD is always the cause of perinatal death of fetal and newborn an, the childhood epilepsy, low intelligence as well as cerebral palsy. Neurogenesis is a process by which under certain conditions neural stem cells can proliferate,migrate and differentiate into mature neurons with specific phenotype, and be involved in the restoration process of nerve injury. After studies have demonstrated that there are neural stem cells(NSCs)in the adult mammalian brain, and they have the capability of neurogenesis, research of NSCs is progressing rapidly in recent years. Normally Neurogenesis is a small amount and increases in such conditions as ischemia and seizure,but is not enough to repair damaged brain tissue for some reason such as lower multiplied quantity, apoptosis and a smaller proportion of divided into neurons.So,it will be a new hope for treating many diseases of the central nervous system to study the process of neurogenesis after brain damage and to search methods toinduce NSCs proliferation and differentiation. Ginkgolides is a terpene lactones compound extracted from ginkgo biloba, which has wide pharmacological functions and neuroprotective and repair functions by lowing the apoptosis of nerve cells and promoting neovascularization after injuries in central nervous system. It has recently reported that GB can accelerate the proliferation,migration and differentiation directionally of the NSCs cultured in vitro. The present study is proposed to observe apoptosis and changes of neurogenesis following injur by the HIBD model of neonatal rats, to study the the effect of GB on these,and it is hoped to provide a basis for future studies.MethodsA total of200cleaning grade healthy neonatal aged7-day-old SD rats were randomly divided into sham operated group with48rats, HIBD group with52rats, low dose GB group with50rats and high dose GB group with50rats and duplicated a model of HIBD in the neonatal rat by by the classical Rice. The behaviour of rats were observed in and after modeling process, Low dose GB group and high dose GB group were intraperitoneally injected with GB in dose of5mg/kg、10mg/kg respectively while the other groups was given equal normal saline, once daily for5days.6rats were randomly selected each group and sacrificed on3d,7d,14d,28d respectively. Rats were killed on scheduled time and expression of Caspase-3and VEGF were detected at various points by real-time PCR(RT-PCR).1.Relative expression were calculated with the method of2-△△CT then the difference of each group was compared.2. Rats marked with5bromodeoxyuridine (BrdU) before execution in dose of50mg/kg, once12h for5times and the rats were put to death after4hours of last injection. Nestin, neuron special enolase(NSE) glial fibrillary acidic protein(GFAP) were seen as markers of NSCs, neurons and astrocytes. Then the number of BrdU positive cell were measured by immunohistochemistry and the number of BrdU-Nestin positive cells of double staining in SGZ and BrdU-NSE、 BrdU-NSE double positive cells cortex were investigated by immunofluorescent double staining. The difference of positive cells among each group were compared. Quantitative data presented in a form of x±s were analyzed by SPSS17.0.The measurement data of multiple group were compared with single factor analysis of variance, and any two of several groups was performed with LSD-t West. Pearson’s correlation was used in analyzing the relation of two parameters. To consider P<0.05as statistically significant.Results1. The expression of Caspase-3:The expression of Caspase-3reached the peak at3d, then declined gradually, approaches the contrast values at28d. Compared with the model group, the level of the expression was decreased in the two trial groups, but it reduced dramatically in high-dose group.2. The expression of VEGF:The expression of VEGF significantly increases at3d after HIBD, then declined gradually.The level of the expression were higher in the two trial groups compared with the model group but it was added dramatically in high-dose group.3. The expression of Foxgl:expression were observed at3d after HIBD, peaked at7d, then declined gradually; The level in the two treatment groups are higher than that of the HIBD group; The expression of Foxgl in high-dose group is higher than that in low-dose group, with statistical significance.4. The BrdU positive cells in hippocampus:A few histamine positive cells were found in hippocampus; The number of BrdU+cells increased after HIBD, reached the peak at7d then declined; The number of positive cells were higher in the two trial groups compared with it in the model group; It was higher in high-dose group than in low-dose group.5. BrdU+/nestin+cells in cortex:BrdU+/nestin+cells appeared at3d peaked on7d, then declined gradually;The number of positive cells were higher in the two trial groups compared with it in the model group; The numbers of BrdU+/nestin+cell in high-dose group were increased than those in low-dose group at7d,14d,28d.6. BrdU+/NSE+cell in cortex:There is no significant difference among the four groups at3d; The number of positive cells appeared at3d,peaked on14d, then declined gradually; Those were higher in the two trial groups compared with those in the model group at7d; The numbers of BrdU+/NSE+cell in high-dose group were increased than those in low-dose group at28d, with statistical significance.7. BrdU+/GFAP+cell in cortex:Among all the groups, there was no significant difference of positive cells3d; the positive cells increased at7d,peaked on14d, declined at28d after HIBD; The number of positive cells were higher in the two trial groups compared with it in the model group at7d,14d,28d; The numbers of BrdU+/NSE+cell in high-dose group were increased than those in low-dose group at14d and28d, with statistical significance.Conclusions1. HIBD can induce the apoptosis of cells, promote neovascularization, induce upregulation of Foxgl expression and reduce neurogenesis in brain.2. GB can lower the expression of Caspase-3increase VEGF, facilitate the expression of Foxgl, the proliferation of neural stem cells, the differentiate from stem cell into neuron and finally improve the functional recovery of nervous system.3. There are more significant efficacy in high-dose group than in low-dose group.