Investigation of the effects on neural membrane lipid profile of in vitro chronic excitotoxicity
Project no. 541 · Status: Kapanmış · 2015
Summary
Our main objective is to investigate the cell death and change of membrane lipid composition on hippocampal glia, neuron and organotypic slice cultures depending on the chronic glutamic acid application, and meanwhile to determine the potential effects of some anti-epileptics (phenytoin, levatiracetam) and oxytocin. Primer cells and organotypic slice cultures prepared from hippocampus and cortex region of new-born rat brains. Glial and neuronal cells were incubated with media included respectively, 0-1000µM and 0-100 µM glutamic acid for tenand seven days. Followed by determination of the effective dose of glutamate, 3 agents known with their cytoprotective and antiepileptic properties; oxytocin, levatiracetam and phenytoin, were added with glutamate and protective effects of these agents were tested. MTT was utilized to evaluate toxicity. To analyse membrane lipids; cells were homogenized and extracted. Composition of membrane lipids were determined by HPLC. It’s seen that glutamate caused a dose-dependent decrease in cell viability on glial and neuronal cultures, and the glutamate dose, which will be used at following assays, was determined as 100-10 µM, respectively. It’s observed that glutamate-induced cell death is reduced on glial and neuronal cells when glutamate was added to the culture medium together with protective agents. Membrane lipid ratios were determined by HPLC analysis as percentages of following lipid groups; cholestrol esters, cholestrol, phosphatidylethanolamine, phosphatidylinositol/serine, phosphatidylcholine, sphingomyelin. Additionally, the changes of these percentages when protective agents applied were monitored. It’s seen that glutamate application on neuronal cells changed the membrane lipid composition, especially there is a statistically significant variation on cholestrol level. Phenytoin demostrated a significant alteration on membrane lipid ratios. Furthermore, the deterioration of neuronal microenvironment and glial cells in the epileptogenetic process which we mentioned in our hypothesis have been observed in that the change in membrane lipid composition is different in glial cells according to the isolated neural culture in the protected microenvironment.