1.EFFECTS OF HYPOXIC-PRECONDITIONING ON Fos AND Jun EXPRESSION AND APOPTOSIS IN CULTURED RAT HIPPOCAMPAL NEURONS AFTER ANOXIA-REOXYGENATION in vitro
Aishi DING ; Fuzhuang WANG ; Ming FAN
Acta Anatomica Sinica 2002;0(06):-
Objective To study the effects of hypoxic preconditioning on the Fos and Jun expression and apoptosis in cultured rat hippocampal neurons after anoxia\|reoxygenation. Methods The hippocampal neurons cultured for 12d from control and hypoxic\|preconditioning groups were exposed to anoxia environment (0\^90?l/L N\-2+0\^10?l/L CO\-2) for 4h and than reoxygenated for 24h and 72h.The expression of Fos and Jun were revealed immunocytochemically using the antiserum against Fos and Jun.The apoptosis was examied using the terminal deoxynucleotidyl transferese\|mediated biotinylated deoxyuridine triphosphate nickel and labeling(TUNEL) method and flow cytometric analysis. Results The increased of percentage of Fos and Jun positive neurons and apoptosis neurons in cultured hippocampal neurons after anoxia\|reoxygenation than those in befor anoxia.The precentage of Fos and Jun positive neurons and apoptosis neurons were less in the hypoxic\|preconditioning group than that in control aftre anoxia\|reoxygenation.Conclusion\ Hypoxic\|preconditioning increases tolerance of hippocampal neurons to anoxia and decreases the precentage in Fos and Jun expressing neurons and apoptosis neurons in hippocampus aftre anoxia\|reoxygenation in vitro. \;[
2.Protective effects of adenosine on cultured rat hippocampal neurons after oxygen-glucose deprivation
Aishi DING ; Yan WU ; Liying WU ; Zimin MA ; Ming FAN
Chinese Journal of Pathophysiology 2000;0(08):-
AIM:To investigate the protective effects of adenosine on cultured rat hippocampal neurons after oxygen-glucose deprivation. METHODS:The control and adenosine-treated hippocampal neurons cultured for 12 d were exposed to oxygen-glucose deprivation environment for 0.5-4 h and then cultured with original medium in normoxia for 24 h . The soma area,survival rate, effluxes of lactate dehydrogenase (LDH)and apoptosis of neurons were observed. RESULTS:The soma area, effluxes of lactate dehydrogenase from neurons and apoptosis were increased while survival rate of neurons was decreased after oxygen-glucose deprivation compared with those pre-oxygen-glucose deprivation. Compared with the control, after oxygen-glucose deprivation the soma area, effluxes of lactate dehydrogenase from neurons and apoptosis were decreased, however, the survival rate of neurons was increased in the adenosine group.CONCLUSION: Oxygen-glucose deprivation can lead to the severe damage of cultured hippocampal neurons, and adenosine can reduce neuronal injury induced by oxygen-glucose deprivation.
3.Anti-NR_1 monoclonal antibody inhibits the glutamate-induced intracellular free calcium increase in rat hippocampal neurons
Lanxiang WU ; Changkai SUN ; Ming FAN ; Aishi DING
Basic & Clinical Medicine 2006;0(10):-
Objective To study the effect of mAbN1,a monoclonal antibody against N-methyl-D-aspartate receptor subunit 1(NR1)on Ca2+ influx after glutamate stimulation in cultured rat hippocampal neurons.Methods Excitotoxicity was induced by glutamate in cultured hippocampal neurons.Confocal laser scanning microscopy was used to observe the changes in intracellular free calcium concentration([Ca2+]i)at the level of cultured hippocampal neurons following pretreatment with mAbN1 and MK-801.Intracellular free calcium was imaged after loading cells with the fluorescent dye indicator fluo-3/AM.Results Our findings indicate that as compared with MK-801,mAbN1 can more significantly attenuate the glutamate-induced [Ca2+]i increase,and it has no effect on [Ca2+]i in physiological condition.Conclusion mAbN1 may alter the secondary structure of NR,consequently influence Ca2+ influx in excitotoxicity process.
4.Effect of heat treatment on striatum neurons
Yongqi ZHAO ; Hong LIU ; Yan WU ; Shuhong LIU ; Xueming GE ; Aishi DING ; Ming FAN
Chinese Journal of Pathophysiology 1989;0(05):-
AIM: To determine the effect of heat treatment on rat primary cultured neurons, and give fundamental research for candidate molecule to protect the neurons from heat injury. METHODS: Neurons from rat striatum were primary cultivated in D-MEM with 15% horse serum, and when got mature, cells were identified by immuno-cytochemical staining with neurofilament protein (NF), tyrosine hydroxylase (TH) and neuron specific enolase (NSE) antibodies. Cells in heat treatment groups were put in an 43 ℃ CO 2 incubator for 1 h, and the control groups at 37 ℃ as normal. Striatum neurons were stained with trypan blue and dual fluorscence dye (PI/H33258) immediately followed heat treatment, and necrosis rate of neurons was estimated. At the same time, activated caspase-3 immuno-cytochemical and TdT TUNEL methods were applied to determine apoptosis rate, and cell volume was also identified with micro-photography. RESULTS: During day 7 to day 9, the cultured striatum neurons got mature, and many neuronal fibers starched out and formed neuron network, NF, TH, and NSE staining positive. Treatment at 43 ℃ for 1 h, cell number decreased greatly, while NF+ percentage kept unchanged, and the heat treatment survived neurons were processing cell necrosis and apoptosis, but necrosis percentage was much greater than that of apoptosis. While cell volume kept unchanged after heat treatment. CONCLUSION: Heat treatment greatly affects the growth and survival of the cultured striatum neurons, and the injury effect is most due to cell necrosis process.
5.Neuroprotective effects of nGBE against glutamate excitotoxicity
Jing SUN ; Changkai SUN ; Ming FAN ; Aishi DING ; Lin YIN ; Wei WU ; Xiaotong WANG
Chinese Journal of Clinical Pharmacology and Therapeutics 2000;0(01):-
AIM:To investigate protective effects of novel extract of Ginkgo biloba(nGBE) in different administration modes on glutamate-induced neuronal damage.METHODS: The values of Essential nGBE were obtained by supercritical CO_2 fluid extraction.Based on glutamate excitotoxicity of primary cultures from neonatal Wistar rats hippocampal,by use of Trypan blue dye staining,testing the lactate dehydrogenase leakage from cultured neurons and terminal deoxynucleotidyl transferase-mediated nick end labeling(TUNEL) method,we examined glutamate-induced necrosis and apoptosis.The protective effects of nGBE in different administration modes(pre-treatment and post-treatment) were adopted and compared with the NMDA receptor uncompetitive antagonist MK-801 in acute-treatment.RESULTS:Treatment with nGBE in two administration modes all could increase ratio of surviving neuron,decrease LDH efflux and reduce ratio of neuron apoptosis in different degree,and the benefit of pre-treatment was superior to post-treatment,but inferior to MK-801.CONCLUSION:The extracts of Ginkgo biloba used nowadays in cerebrovascular disease as post-treatment have no prominent effect as far as their mechanism of antiexcitotoxicity.However they may have more value if they were used for precautionary intervention in high-risk population.