Apart from its potent antioxidant property, recent studies have revealed that melatonin promotes PI3K/Akt phosphorylation following focal cerebral ischemia (FCI) in mice. However, it is not clear (i) whether increased PI3K/Akt phosphorylation is a concomitant event or it directly contributes to melatonin's neuroprotective effect, and (ii) how melatonin regulates PI3K/Akt signaling pathway after FCI. In this study, we showed that Akt was intensively phosphorylated at the Thr308 activation loop as compared with Ser473 by melatonin after FCI. Melatonin treatment reduced infarct volume, which was reversed by PI3K/Akt inhibition. However, PI3K/Akt inhibition did not inhibit melatonin's positive effect on brain swelling and IgG extravasation. Additionally, phosphorylation of mTOR, PTEN, AMPKα, PDK1 and RSK1 were increased, while phosphorylation of 4E-BP1, GSK-3α/β, S6 ribosomal protein were decreased in melatonin treated animals. In addition, melatonin decreased apoptosis through reduced p53 phosphorylation by the PI3K/Akt pathway. In conclusion, we demonstrated the activation profiles of PI3K/Akt signaling pathway components in the pathophysiological aspect of ischemic stroke and melatonin's neuroprotective activity. Our data suggest that Akt phosphorylation, preferably at the Thr308 site of the activation loop via PDK1 and PTEN, mediates melatonin's neuroprotective activity and increased Akt phosphorylation leads to reduced apoptosis.

The circadian rhythm is driven by a master clock within the suprachiasmatic nucleus which regulates the rhythmic secretion of melatonin. Bmal1 coordinates the rhythmic expression of transcriptome and regulates biological activities, involved in cell metabolism and aging. However, the role of Bmal1 in cellular- survival, signaling, its interaction with intracellular proteins, and how melatonin regulates its expression is largely unclear. Here we observed that melatonin increases the expression of Bmal1 and both melatonin and Bmal1 increase cellular survival after oxygen glucose deprivation (OGD) while the inhibition of Bmal1 resulted in the decreased cellular survival without affecting neuroprotective effects of melatonin. By using a planar surface immunoassay for PI3K/AKT signaling pathway components, we revealed that both melatonin and Bmal1 increased phosphorylation of AKT, ERK-1/2, PDK1, mTOR, PTEN, GSK-3αβ, and p70S6K. In contrast, inhibition of Bmal1 resulted in decreased phosphorylation of these proteins, which the effect of melatonin on these signaling molecules was not affected by the absence of Bmal1. Besides, the inhibition of PI3K/AKT decreased Bmal1 expression and the effect of melatonin on Bmal1 after both OGD in vitro and focal cerebral ischemia in vivo. Our data demonstrate that melatonin controls the expression of Bmal1 via PI3K/AKT signaling, and Bmal1 plays critical roles in cellular survival via activation of survival kinases.

Repetitive transcranial magnetic stimulation (rTMS) has gained interest as a non-invasive treatment for stroke based on the data promoting its effects on functional recovery. However, the exact action mechanisms by which the rTMS exert beneficial effects in cellular and molecular aspect are largely unknown. To elucidate the effects of high- and low-frequency rTMS in the acute-ischemic brain, we examined how rTMS influences injury development, cerebral blood flow (CBF), DNA fragmentation, neuronal survival, pro- and anti-apoptotic protein activations after 30 and 90 min of focal cerebral ischemia. In addition, inflammation, angiogenesis, growth factors and axonal outgrowth related gene expressions, were analyzed. Furthermore, we have investigated the effects of rTMS on post-acute ischemic brain, particularly on spontaneous locomotor activity, perilesional tissue remodeling, axonal sprouting of corticobulbar tracts, glial scar formation and cell proliferation, in which rTMS was applied starting 3 days after the stroke onset for 28 days. In the high-frequency rTMS received animals reduced DNA fragmentation, infarct volume and improved CBF were observed, which were associated with increased Bcl-xL activity and reduced Bax, caspase-1, and caspase-3 activations. Moreover, increased angiogenesis, growth factors; and reduced inflammation and axonal sprouting related gene expressions were observed. These results correlated with reduced microglial activation, neuronal degeneration, glial scar formation and improved functional recovery, tissue remodeling, contralesional pyramidal tract plasticity and neurogenesis in the subacute rTMS treated animals. Overall, we propose that high-frequency rTMS in stroke patients can be used to promote functional recovery by inducing the endogenous repair and recovery mechanisms of the brain.

Effects of normobaric oxygen and melatonin on reperfusion injury: role of cerebral microcirculation