Neuroprotective Effect And Mechanisms Of Fasudil Hydrochloride On Cultured Cortical Astrocytes Subjected To Oxygen-Glucose Deprivation

Aims: To establish rat astrocyte culture in vitro and the model of oxygen-glucose deprivation (OGD), and to determine the neuroprotective effect and the related mechanisms of fasudil hydrochloride (FH) (5μM, 10μM) on cultured astrocytes exposed to OGD. Methods: 1~3 days SD rats were adopted for the primary cultures of cortical astrocyte. Cells were passaged when the primary astrocytes cover the culture flask for about 85%~90%. And then experiments were performed with those 5th day of passaged astrocytes. We used oxygen deprived by Na2S2O4 combined with the incubation-solution without glucose to induce OGD model. Cell injuries were detected by thiazoyl blue tetrazolium bromide (MTT) staining method and measurement of lactate dehydrogenase (LDH) release. Concentrations of extracellular amino acids (Glu, Gly, Tau and GABA) were detected by high performance liquid chromatography (HPLC). DHCF-DA staining was utilized for quantification of intracellular ROS in astrocytes. Results: 1. MTT assay: the percentage of survival rate of control group was 100%, the percentage in OGD decreased to 24% with significant statistics difference (p<0.01). The percentages of survival rate of FH treated OGD groups were increased to 41.2% and 41.5% of control group separately, the statistics difference were significant compared with the OGD group (p<0.01); but there was no significant statistics difference within the two FH treated groups (p>0.05). 2. The release of LDH: there was only a little amount LDH released in the control group. The LDH leakage of OGD group was 210% of control group, and the change was statistics different (p<0.01); compared with OGD group, the groups of FH were both decreased significantly (p<0.01), and the difference was exhibited in a dose dependent manner between the two groups with FH (p<0.05). 3. The concentrations of extracellular amino acids (Glu, Gly, Tau and GABA): compared with control group, all four kinds of amino acids concentration had increased significantly (p<0.01), with Glu improving most obviously, while there was a significant decrease in the groups of FH compared with OGD group (p<0.01). There was no significant statistics difference within the two FH treated group (p>0.05). 4. The production of ROS: contrast to control group, there was a significant increase in the group of OGD; the ROS productions in FH pretreated groups were decreased significantly (p<0.01). Conclusion: FH (5μM, 10μM) exhibited obvious neuroprotection against OGD-induced injury in cultured astrocytes, and the mechanism underlying may be probably by the way of repressing the excitotoxicity induced by excitatory amino acids and reducing the production of ROS.