| 1. Establishment of neuron oxygen glucose deprivation/reoxygenation (OGD/R) model in vitroObjective To obtain pure mouse cerebral cortex neurons by primary culture, and establish a reliable and easy-conducted OGD/R neuron model in vitro to provide necessary theory for the researches of mechanisms of neuron ischemia and treatment.Methods Primary cortex neurons were obtained from embryo (14-15 d) Balb/C mice by the enzyme digestion method. Neurons were firstly cultured in Dulbecco's Modified Eagle's Medium (DMEM) with 20% fetal bovine serum at 37℃in 5% CO2 atmosphere. After 24 h the medium was replaced by DMEM with 2% B27 supplement to facilitate differentiation of neurons and minimize glial growth. Morphology of neurons was observed under microscope. After 10 days of culturing in vitro, immunofluorescence staining ofβ-tubulin was used to identify the purity of neurons. Experiments of OGD/R were performed on cultures at 10 d in vitro. The time of OGD was 4 h followed by 20 h reoxygenation. Then typan Blue (TB) staining was used to detect the cell viability, and lactate dehydrogenase (LDH) leakage ratio in the culture medium was used to evaluate the changes of permeability of cell membrane.Results After 10 days of culture in vitro, the primary cortex neurons showed typical morphologic characters of mature neurons. The neuronal nucleus was large and clear with round or elliptical outline. The nucleolus and the membrane of nucleus were easily found. There were pleomorphic neurons in the medium, and the cytoplasm was bright with many dendrites and axons striking out from the cellular body, which connected each other to form a complicated reticular formation. The percent ofβ-tubulin positive neurons was more than 70%. The results of TB staining showed that 4 h OGD could induce neuron death significantly. The results of LDH leakage ratio were consistent with the results of TB staining.Conclusion Primary cortex neurons could be successfully obtained from the embryo (14-15 d) Balb/C mice by the enzyme digestion method. B27 Supplement not only facilitated differentiation of neurons, but also minimized glial growth. Neuron ischemia reperfusion model induced by OGD 4 h/R 20 h showed moderate and stable damage to cell viability, and was suitable for control research of cerebral ischemia reperfusion injury in vitro.2. Protective effects of Resveratrol (Res) against neuronal injury induced by OGD/R in vitro and effect on matrix metalloproteinase (MMP)-9Objective To confirm the protective effects of Res against neuronal injury induced by OGD/R in vitro, and further research the mechanisms of neuroprotection of Res in cell level, namely the relationship between Res and MMP-9 induced by OGD/R in neuron. Methods Primary mouse cerebral cortex neuronal ischemia reperfusion model was created by OGD 4 h /R 20 h. Stock solution (0.1M) of Res was prepared in dimethylsulfoxide (DMSO) and stored at -20℃. For treatment, the Res was diluted in PBS and added to cultures to give the desired final concentrations (2.5, 5 and 10μM). Untreated cultures received the same amount of the carrier solvent (0.1 % DMSO). The duration of treatment is from OGD/R to the end of the experiment. Cell viability was evaluated by TB staining and LDH leakage ratio. Total protein extraction of cells was used for detection of expression of Peroxisome proliferators-activated receptor (PPAR)α,γand MMP-9 protein by Western blot. Total RNA isolated from cells was used for evaluation of MMP-9 mRNA levels by reverse transcription polymerase chain reaction (RT-PCR).Results The results of TB staining showed that OGD 4 h /R 20 h could induced 50% neuronal death, and 0.1% DMSO did not aggravate neuronal damage. Res treatment could reduce cell death under these conditions, and the neuroprotection of Res for neurons was dose-dependent. These results indicated that treatment with 5μM and 10μM of Res showed better therapeutic outcome, and no harmful effects were found. The results of Western blot and RT-PCR showed that normal neurons only expressed very low level of MMP-9 protein. There were low level of PPAR-αand PPAR-γin normal neurons. OGD 4 h/R 20 h could increase transcription and translation of MMP-9 in neurons. At the same time, expressions of PPAR-αand PPAR-γwere also up-regulated by OGD/R injury. Res could inhibit transcription and translation of MMP-9. Expressions of PPAR-αand PPAR-γalso increased further by Res. Bioactivity of Res on MMP-9, PPAR-αandγwas in concentration-dependent manner.Conclusions Res could inhibit transcription and translation of MMP-9 in OGD/R neuron model and activate expressions of PPAR-αand PPAR-γin concentration- dependent manner.3. Mechanisms of protective effects of Res against neuronal injury induced by OGD/R in vitroObjective To confirm mechanisms of protective effects of Res against neuronal injury induced by OGD/R in vitroMethods Primary mouse cerebral cortex neuronal ischemia reperfusion model was created by OGD 4 h /R 20 h. Neurons were grouped according to different reagents. Stock solution of reagents was prepared in DMSO and stored at -20℃. Reagents were added into cultural medium in different dosage and combination, including Res (10μM), activator of PPAR-γtroglitazone (5μM), activator of PPAR-αwy14643 (5μM), antagonist of PPAR-γGW9662 (10μM), and antagonist of PPAR-αMK886 (10μM). Cell viability was evaluated by TB staining and LDH leakage ratio. Total protein extraction of cells was used for detection of expression of PPAR-α, PPAR-γand MMP-9 protein by Western blot. Total RNA isolated from cells was used for evaluation of MMP-9 mRNA levels by RT-PCRResults The results of TB staining showed that OGD 4 h/R 20 h could induced 50% neuronal death, and 0.1% DMSO did not aggravate neuronal damage. Res, troglitazone and wy14643 could decrease neuronal injury caused by OGD/R. Western blot and RT-PCR showed that Res, troglitazone and wy14643 could inhibit up-regulated expression of MMP-9 in OGD/R conditioin. When neurons were co-cultured in GW9662 or MK886 with agonist of PPARs, inhibition effects of troglitazone and wy14643 on MMP-9 and protection effect on neurons were antagonized. Inhibition of MMP-9 and protection of neurons by Res was partially antagonized by MK886, but which was not affected by GW9662. Conclusions Inhibition effects on MMP-9 and protection effect on neurons by Res has relationship with selective activation of PPAR-α, blockage of PPAR-αactivation can partially bring negative impact to physiological function of Res to MMP-9 and neurons. Though both Res and troglitazone can activate expression of PPAR-γ, they have different effect on down-stream target. It may have relation with complex structure of PPARs.
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