NOVEL HYDROXYPYRIDINE DERIVATIVE PROTECTS NEURONS AGAINST ISCHEIC DAMAGE

Abstract

In this work, the cytoprotective effect of the original substance 3-hydroxypyridine ascorbate (3-EA) was studied. Cells for mixed cortical neuroglial culture were isolated from the brain (GM) of neonatal NMRI mice. The cell density in culture was adjusted to 15000 cells per 1 cm2. For experiments, 10-day-old cultures were used. To register the intracellular concentration of calcium ions ([Ca2+]i), cells were loaded with an intracellular fluorescent probe Fura-2AM dissolved in Hanks solution at a concentration of 4 μM, followed by 40-minute incubation at a temperature of 370C. Cells were washed three times before the experiment. Potassium chloride was applied to identify neurons, and short-term addition of ATP to the medium was used to detect glial cells (astrocytes). Cell cultures of the mouse cerebral cortex were preincubated for 24 hours with various concentrations (10, 50 and 100 μM) of 3-EA. The amplitude and shape of calcium responses were determined under conditions of oxygen-glucose deprivation (oxygen-glucose deprivation, OGD) and glutamatergic excitotoxicity (GluTox). Using fluorescence microscopy, cell viability tests, and PCR analysis, 3-EA has been shown to dose-dependently inhibit cortical cell death under glutamate excitotoxicity and ischemia/reoxygenation. Preincubation of cerebral cortex cells with 3-EA in the concentration range of 10–100 μM leads to a significant inhibition of the increase in Ca2+ ions in the cytosol ([Ca2+]i) of neurons and astrocytes in modeling glutamate excitotoxicity (GluTox) and oxygen-glucose deprivation (OGD). Decreasing [Ca2+]i and establishing a lower baseline [Ca2+]i inhibits necrotic cell death in an acute experiment.