THE EFFECT OF OUABAIN AND BUFALIN ON THE NEURONS OF PRIMARY CULTURES OF RAT CEREBRAL CORTEX UNDER GLUCOSE-OXYGEN DEPRIVATION

Objectives: The objective of this project was the comparison of neuroprotective effects of the cardiotonic steroids (CTS) ouabain and bufalin in a primary culture of rat cortical neurons in conditions of glucose-oxygen deprivation. In addition, investigation of the activation of intracellular singnaling cascades associated with the viability of rat cortical neurons in a primary culture was conducted. Material and methods: Modeling of 4-hour glucose-oxygen deprivation, followed by a 20-hour re-oxygenation, was performed on a primary culture of rat cortical neurons (10-12 days in vitro). Ouabain and bufalin in concentrations of 10 nM and 100 nM were administrated 30 min before glucose-oxygen deprivation. Oxidative stress was induced through a 24-hour long incubation of the cul ture with 20 mkM rotenone. Viability of the culture after the experimental procedures was evaluated using the MTT-test. The level of Akt kinase activation was evaluated by comparing the ratio of phosphorylated Akt protein to total protein of the Akt kinase using Western Blot. Results: 30-minute preincubation with 10 nM and 100 nM ouabain, unlike bufalin, protected neurons from death. At the same time, ouabain did not prevent neuronal death when oxidative stress was induced using 20 mkM rotenone. Unlike bufalin, ouabain caused an increase in Akt kinase activation. Conclusion: Ouabain, but not bufalin, has a neuroprotective effect in conditions of glucose-oxygen deprivation. Simultaneously, in our experiments ouabain does not have a neuroprotective effect under direct induction of oxidative stress. The neuroprotective effect of ouabain is likely due to its ability to activate the Akt kinase, which bufalin does not do.

Akt, ouabain, bufalin, ischemia, oxidative stress, Akt