The roles of phosphoinositide-3 kinases/akt phosphorylation in the neuroprotective effects of melatonin on neuronal survival In-vitro and In-vivo

Melatonin is a well-known circadian rhythm regulatory molecule found to be conserved from lower to higher organisms. Melatonin possesses several functions mainly in the biological mechanisms of aging, antioxidant scavenging and cellular survival. Depending on the background of this study, specifically, the attention was drawn into the neuro-survival abilities of melatonin. In our previous studies, we have observed that melatonin increases the phosphorylation of Akt. However, the significance of increased Akt phosphorylation was not known. In the present study, the role of Phosphoinositide-3 kinase (PI-3K) /Akt signalling pathway in the neuro-protective effects of melatonin was tested on primary cortical neuron culture, as in vitro model, and mice cerebral ischemia in vivo model through the use of Wortmannin as a PI-3K/Akt pathway inhibitor. Primary cortical neuron culture was arrived from new born (DIV 1-3) Balb/c mice and the hypoxia-OGD experiment was applied on day 7 of culturing. Melatonin and Wortmannin treatments were introduced during hypoxia-OGD and reperfusion. 30 mins of Middle Cerebral Artery Occlusion (MCAO) was applied on adult male C57BL mice for the in vivo experiment model. Wortmannin was administered before MCAO and melatonin was injected after 30 mins of MCAO. The cell survival counting analysis was performed from the pictures taken with confocal microscopy (20X, DAPI). Neuronal survival analysis and DNA Fragmentation analyses were found by applying Cresyl violet staining and TUNEL assay on cross sectioned in vivo experimental group mice brains, respectively. The results of this study indicate that specifically in the melatonin group of in vitro experiments the axonal extensions was protected after cortical cells were exposed to the hypoxic and OGD conditions. Similarly, melatonin significantly increased neuronal survival with the percentage of 94.1 % in vivo. In this study, we provide evidence that melatonin increases neuronal survival from hypoxic cell death both in vivo and in vitro. We further reveal that the inhibition of Akt phosphorylation reverses the neuro-protective effect of melatonin.