A Complex Neuroprotective Effect of Alpha-2 Adrenergic Receptor Agonists in a Model of Cerebral Ischemia-Reoxygenation In Vitro

The mechanism of the neuroprotective action of alpha(2)-adrenergic receptor agonists in a model of oxygen-glucose deprivation (OGD) in vitro was investigated. Using fluorescent microscopy, immunostaining, inhibitor analysis, and real-time PCR analysis, we have demonstrated that OGD evokes a biphasic [Ca2+](i) increase in all cells in the culture. Initial Ca2+ impulse and neuronal Ca2+ oscillations are followed by a slower global increase of [Ca2+](i) in both cell populations. The accumulation of pro-inflammatory factors, such as IL1b and TNF alpha, was observed during 40-min OGD. It was established that the reoxygenation is followed by the hyperexcitation of neurons, caspase-3 activation, and subsequent cell death. We showed that a 24-h pretreatment of cell cultures with selective alpha(2)-adrenergic receptor agonists guanfacine and UK-14,304 abolished a global [Ca2+](i) increase in astrocytes and neurons but did not suppress the first phase of the OGD-induced Ca2+-impulse in astrocytes. In addition, the number of dead cells after OGD was decreased in cell cultures pretreated with the alpha(2)-agonists. Guanfacine inhibited caspase-3 activation and suppressed apoptosis in our experiments. In particular, the expression of antiapoptotic genes Stat3 and Bcl-2 was enhanced after the pretreatment with guanfacine. On the contrary, the expression of proapoptotic genes (Socs-3, p53, fas, and lick) was decreased. Moreover, application of guanfacine evokes Ca2+-response in astrocytes and leads to Ca2+-mediated ATP release and this way suppresses hyperexcitation of the neurons. Thus, the activation of astrocytes and Ca2+-mediated ATP release possibly contribute to the complex neuroprotective effects of the alpha(2)-adrenergic receptor agonists.