A Study Of The Activation Of Neural Progenitor/Stem Cells In Rat's Hippocampus After Prolonged Seizure And The Effect Of BDNF And Glutamate On Neural Progenitor/Stem Cells

PARTⅠPROLIFERATION AND APOPTOSIS OF NEURAL PROGENITOR/STEM CELLS AFTER STATUS CONVULSION IN THE ADULT RAT HIPPOCAMPUSObjective: To explore the proliferation and apoptosis of neural progenitor/stem cells (NPCs) after status convulsion(SC)in the adult rat hippocampus.Methods: Seizures were induced in adult Wistar rats injected with lithium and pilocarpine intraperitoneally and controlled 30 minutes later. Rats were sacrificed at 6 time points (1, 3, 7, 14, 28, 56 days) after SC. Each rat was injected with bromodeoxyuridine (BrdU) intraperitoneally 1 day before killed. The expression of BrdU and neuroepthelial stem cell protein( nestin ) were determined by immunohistochemistry to mark the proliferation of NPCs. Double-label immunofluorescence of nestin and in situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) were used to assess the survival of newly generated NPCs.Results:In the normal hippocampus formation, only a small amount of BrdU positive and nestin positive cells were found in dentate gyrus, not CA1 and CA3 region. One day after SC , the amount of BrdU positive cells began to increase in the CA1 region and dentate gyrus, the former peaked at 7 days , began to decrease at 14 days after , and reached normal at 28 days after, while the latter increased up to 20-fold at 14 days after and reached normal at 56 days after SC . A large amount of BrdU positive cells were observed in CA3 region at 7 days following SC. The number of BrdU and nestin positive cells of the same region at the same time point had no statistical significance. TUNEL positive nuclei were observed in almost all of the nestin positive cells in CA1 region within the first 3 days after SC, but didn't exist in dentate gyrus during the the whole experiment process.Conclusion:SC stimulates the proliferation of inherent NPCs in hippocampus within a certain time window, and parts of the newly generated cells appear to migrate from proliferation area into the injuried area.PARTⅡCHARACTERIZATION OF EXCITABILITY AND VOLTAGE-GATED ION CHANNELS OF NEURAL PROGENITOR/STEM CELLS IN RAT HIPPOCAMPUS Objective: To segregate and culture the neural progenitor/stem cells (NPCs) in rat hippocampus in vitro and clarify the characterization of excitability and voltage-gated ion channels of NPCs.Methods: NPCs were obtained from Wistar rats at embryonic day 15-16 and cultured in serum-free medium, then the neurospheres were harvested, fixed and immunostained for nestin (the marker of NPCs); NPCs were induced to differentiate with 1% fetal calf serum contained medium; Cells were cultured on coverslips coated with poly-L-lysine with DMEM/F12 medium containing EGF and bFGF before the experiment. All the following studies were then performed 6-24 hours after transferring the cells to the coverslips coated with poly-L-lysine. The membrane potential changes of NPCs were detected with fluorescent dye DiBAC4(3) (bis-(1,3-dibutylbarbituric acid) trimethine oxonol) by confocal laser scanning microscope. Under the current-clamp, spontaneous discharge was detected and the action potential was to be elicited. Under the voltage-clamp, the NPCs'ion channel currents were detected.Results: NPCs obtained from embryonic rats could be passaged for 3 times and were nestin positive which indicated the ability of self-proliferation and they could be induced to differentiate into neurons (Tuj-1 immunostaining positive) and astrocytes (GFAP immunostaining positive). NPCs cultured on coverslips coated with poly-L-lysine within 6-24 hours were still in undifferentiated status. The changes of fluorescent intensity of DiBAC4(3) stain after KCl stimulation were not obvious which indicated that the NPCs was inexcitable. Spontaneous discharge was not detected and the action potential was failed to be elicited. These findings were consistent with the result from DiBAC4(3) staining. Under the voltage-clamp, the NPCs expressed two types of outward K+ currents with no evidence for Na+ currents. An outward delayed rectifier type K+ current and outward transient K+ current were elicited.Conclusion: NPCs in hippocampus of rats could be cultured in vitro. The findings demonstrate NPCs'electrophysiological properties: the electrical inexcitability indicated by the presence of two types of K+ currents and the absence of Na+ currents.PARTⅢTHE EFFECT OF BDNF ON THE ENAHANCEMENT OF SURVIVAL, PROLIFERATION AND DIFFERENTIATION INTO NEURONS OF NEURAL PROGENITOR CELLS ISOLATED FROM RATS'HIPPOCAMPUSObjective: To explore the effect of brain derived neurotrophic factor (BDNF) on the neural progenitor/stem cells (NPCs) isolated from rats'hippocampus.Methods: NPCs were obtained from Wistar rats hippocampus at embryonic day 15-16 and cultured in serum-free medium; The single NPC was obtained by a mechanical dissociation and grown for 4 days in medium containing BDNF at the different concentration of 10-200 ng/mL then the number of neurosphere was counted and the diameter of neurosphere was measured; The TUNEL staining positive cells and the level of lactic acid dehydrogenase (LDH) in culture medium were used to evaluate the effect of BDNF on the NPCs'survival;βtubulinⅢ(Tuj-1) immunostaining was used to label the neurons differentiated from NPCs, and the percentage of Tuj-1 positive cells among DAPI positive cells in the BDNF and control group were compared. Additionally, the length of neurite in Tuj-1 immunostaining positive cells was measured.Results:NPCs used in this experiment exhibited the properties of proliferation and differentiation. BDNF at the concentration of 10-200 ng/mL enhanced the proliferation of NPCs at the cell density of 5×105/ mL, while 40 ng/mL BDNF exhibited the strongest proliferation enhancement; The diameter of newly formed neurosphere with 40 ng/mL BDNF was increased obviously while the TUNEL staining positive cells and the level of LDH in the culture medium of 40 ng/mL BDNF group was significantly decreased compared with the control group. In 40 ng/mL BDNF group, more neurons (Tuj-1 positive cells) were observed and average neuritic length in Tuj-1 positve cells were significantly longer compared with the control group. Conclusion: BDNF can promote neural progenitor/stem cells proliferation and induce them differentiation into neurons.PARTⅣSTUDY OF THE DYNAMIC CHANGES OF GLUTAMATE IN RATS'HIPPOCAMPUS AFTER STATUS CONVULSION AND THE EFFECT OF GLUTAMATE ON NEURAL PROGENITOR/STEM CELLSObjective: To explore the dynamic changes of gluatamates in rat hippocampus after status convulsion (SC), and observe the effect of glutamate on the neural progenitor/stem cells in vitro.Methods: Seizures were induced in adult Wistar rats injected with lithium and pilocarpine intraperitoneally and controlled 30 minutes later. Rats were sacrificed at 3 different time points (1, 3, 7 days) after SC, and the level of glutamate in hippocampus at each time point was measured by high performance liquid chromatography (HPLC). NPCs were segregated from Wistar rats'hippocampus at embryonic day 15-16 and cultured in vitro. Immunofluorescence and Western blot were used to detect the expression of NR1 (an essential subunit of NMDA receptor) on NPCs; Calcium imaging by Fluo-3/AM and single channel recording were used to investigate the function of NMDA receptor; The single NPC was obtained by mechanical dissociation and grown for 4 days in medium containing 30μM MK-801 and Glu at the different concentration of 50-1600μM, then TUNEL staining positive cells and the level of lactic acid dehydrogenase (LDH) in culture medium were measured to evaluate the effect of Glu on NPCs'survival; The number of neurosphere was counted and the diameter of neurosphere was measured to evaluate the effect of Glu on NPCs'proliferation.Results: The glutamate content in rats'hippocampus increased at 1 day after status convulsion, peaked at 3 days, and backed to normal level at 7 days after SC; NPCs used in this experiment exhibited the properties of proliferation and differentiation, and expressed NR1. Calcium imaging by Fluo-3/AM showed that the fluorescence intensity increased significantly after NMDA stimulation. Three types of currents were detected with the cell-attached recording: unipolar rectangular pulse discharge, flicking, burst and cluster. The level of LDH in the culture medium of 1600μM Glu group increased significantly compared with other groups. Further study of the newly formed neurosphere showed that there was no great difference in diammetre or apoptosis rate of neurosphere among each groups. Howevere, the number of neurosphere in 800μM Glu increased significantly than that in other groups.Conclusion: The content of glutamate increase in rats'hippocampus after status convulsion within a certain time window; NPCs express functional NMDA receptor; Calcium influx conducted by NMDA receptor activation may participate in the process that Glu promotes the proliferation of neural progenitor/stem cells.