Neuroprotective Effect Of Astrocytes After Preconditioning With Ischemia In Vitro

Glia cell line-derived neurotrophic factor (GDNF), a recently cloned member of the transforming growth factor-β(TGF-β) superfamily, has been implicated in the survival, morphological and functional differentiation of neurons in vitro and in vivo. The factor may thus have utility in the treatment of human ischemic cerebral vascular disease. The application of exogenous GDNF is limited because GDNF can not traverse the BBB, how to utilize and induce endogenous GDNF became research object. Mechanisms regulating expression of GDNF in normal and diseased brain as a possible means to increase the local availability of GDNF are only beginning to be explored.Part 1: Primary culture of rat cortical neurons and astrocytes and establishment of oxygen glucose deprivation modelObjective: To culture and identify the rats'cerebral cortical astrocytes and neurons, and to set up the OGD model of cortical neurocytes. Methods: Primary cultures of neurons and astrocytes were originated from cerebral cortex of neonatal (12h and 1-2d) rats, and plated at 1.0×106 cells/ml and 2.0×105 cells/ml in DMEM medium with 10% of FBS. Cell presented in the culture were shown to be astrocytes and neurons after characterization by immunocytochemistry, using a primary specific anti-GFAP, NSE antibody. To find out the preconditioning and ischemia time, cell culrures were subjected to OGD injury using hypoxic D-Hank's, and were incubated in an anaerobic chamber which was refitted from a vacuum dryer. Cortial cultures were deprived of oxygen and glucose for 15, 30, 45, 60, 90, 120 and 180min. To terminate the OGD, cells were washed with DMEM and removed from the anaerobic chamber. The rate of cell death and LDH release were detected at 24h after OGD termination. Results: Neurons exposed to 45-60min OGD showed some cells blue-stained. Many neurons body were swollen after 90min OGD. By exposed to 180min OGD, 100% neurons were stained by trypan blue. The astrocytes were not stained during the period of exposure to OGD from 15 to 180min. The LDH release was dependent on the duration of OGD exposure. 30min OGD caused the release of LDH from cultures of neurons and astrocytes, and 60min OGD caused a significant increase on the levels of LDH release (p<0.05). Conclusions: The culture of rats'cerebral cortical astrocytes and neurons were accomplished by combined enzymatic digestion and mechanical dissociation. Neurons and astrocytes could be explored to OGD at the 6th and 9th day, respectively. The model of OGD ischemic preconditioning and ischemia in neurocytes were established by hypoxic D-Hank's and an anaerobic chamber. 30min OGD could be used as preconditioning time, and 90min as ischemia time.Part 2: Expression of GDNF in astrocytes and its receptors in neurons after oxygen glucose deprivationObjective: To observe the effect of ischemic preconditioning on the expression of GDNF and its receptor GFRα1, RET in astrocytes and neurons. Methods: Different treatments were supplied on neurocytes. The neurocytes in preconditioning group were exposed to 30min OGD, and in preconditioning and ischemia group, the neurocytes were exposed to 30min OGD firstly, another 90min OGD were given after 24h reperfusion. The neurocytes that only explored to 90min OGD were ascribed as ischemia group. Different groups were separated according to the various reperfusion time (6h,12h,18h,24h,36h and 48h). The level of GDNF in astrocytes were detected by rt-PCR , Western blot and ELISA, in addition, the level of GDNF receptor in neurons were detected by rt-PCR. Results: In the ischemia group, the expression of GDNF mRNA and protein started to increase at 6h and 12h after reperfusion, respectively. The level of GDNF mRNA was peaked at 24h and 48h, however, that of GDNF protein was peaked at 18h. In the preconditioning and ischemia group, the expression of GDNF mRNA and protein increased markedly at 6h after reperfusion, and the expression tendency of mRNA was the same with that in the ischemia group (p<0.05). The level of GDNF protein was peaked at 24h in the preconditioning and ischemia group. In the ischemia group, the expression of GDNF in the astrocyte medium increased from12h to 48h, and to peak at 24h (p<0.05). Preconditioning induced GDNF expression in the preconditioning and ischemia group increased gradually at 6h after reperfusion. Expression of GDNF in preconditioning and ischemia group was more significant than that in ischemia group. The GFRα1 mRNA increased at 6h after reperfusion both in preconditioning and ischemia group and in ischemia group, with peak expression at 18h. RET mRNA increased at 12h and 18h after reperfusion in preconditioning and ischemia group and in ischemia group, respectively. The expression of GFRα1 mRNA was higher in preconditioning and ischemia group at 6h than that in the ischemia group, and that of RET mRNA was higher in preconditioning and ischemia group at 12h than that in the ischemia group. Conclusions: OGD preconditioning could induce the expression of GDNF in astrocytes, and could induce the expression of GDNF receptors GFRα1, RET in the neurons. Objective: To observe the efficiency of plasmid vector transfection, and the gene silencing effect of RNAi on GDNF in astrocytes cultured in vitro.Methods: Astrocytes of the second generation were used in the experiment. Short hairpin plasmid vector RNAi pGenesil-1A and B that could specially suppress GDNF expression were constructed. The astrocytes were transfected using Lipofectamine 2000, and the expressions of EGFP were observed after 48h. We chose the plasmid vector which had higher transfection efficiency to transfect astrocytes, OGD preconditioning and ischemia were supplied to astrocytes after 12h. The expressions of GDNF mRAN were detected with rt-PCR at 6h, 12h, 24h and 48h after reperfusion. The control plasmid vector RNAi pGenesil-1C group and normal group were the control groups. Results: The passage culture of astrocytes grew rapidly, and its appearance was the same as that of the primary cultures. The transfection efficiency of pGenesil-1A and pGenesil-1B were 46.49%±5.64% and 13.26%±1.33%, respectively. After GDNF gene silencing, the expression of GDNF mRNA presented a significant reduction, and in the 24h group, the gene silencing effect was most obvious (p<0.05). Conclusions: GDNF RNAi pGenesil-1 could be transfected in passage cultured astrocytes successfully using Lipofectamine2000. RNAi could inhibit the expression of GDNF in astrocytes.Part 4: Oxyen glucose deprivation ischemic preconditioning prevents neuron apoptosis through GDNF secreted by astrocytes Objective: To explore the influence of GDNF secreted by preconditioning astrocytes on the apoptosis of primary cortical neurons and to identify the effect of GDNF on different stage of apoptosis. Methods: ACM1 was collected from normal astrocyte medium. Astrocytes were exposed to preconditioning and ischemia, after 24h reperfusion, astrocyte medium was collected, and named as ACM2. The astrocytes, transfected GDNF RNAi pGenesil-1, werePart 3: RNAi inhibits the expression of GDNF secreted by astrocytes exposed to oxygen glucose deprivation exposed to OGD preconditioning and ischemia, and the astrocyte medium was collected at 24h reperfusion named as ACM3. Neurons were divided into 4 groups: the normal control group, the preconditioning group, the preconditioning and ischemia group and the ischemia group. After the last ischemia, using ACM1, ACM 2 and ACM 3 to culture the neurons, so an interaction system of astrocyte/neuron was established. 24h later, the apoptosis of neurons were detected by staining method of GIEMSA, AO/EB and AnnexinV-FITC/PI FCM technique. Results: The GIEMSA staining method showed clearly that ACM2 could decrease the neuronal apoptosis. The results of AO/EB double staining are similar to GIEMSA staining, there were more necrotic neurons in ischemia group, and the rate of apoptosis in preconditioning and ischemia group was lower than that in the ischemia group(p<0.05). The rate of apoptosis in ACM2 group was lower than in ACM1 and ACM3 groups. The early apoptotic neurons were less in the ACM2 group than in the ACM1 and ACM3 groups by FCM (p<0.05). Conclusions: OGD preconditioning could induce astrocytes to secrete GDNF that could decrease the rate of ischemic neuronal apoptosis. The results of FCM showed that GDNF could play an endogenous protection role through decreasing the rate of neuronal early apoptosis.