Selective activation of the G protein‐coupled estrogen receptor has been proposed to avoid some of the side effects elicited by the activation of classical estrogen receptors α and β. Although its contribution to neuroprotection triggered by estradiol in brain disorders has been explored, the results regarding ischemic stroke are contradictory, and currently, there is no consensus on the role that this receptor may play. The present study aimed to investigate the role of GPER in the ischemic insult. For that, primary cortical cultures exposed to oxygen and glucose deprivation (OGD) were used as a model. Our results demonstrate that neuronal survival was strongly affected by the ischemic insult and concurrent GPER activation with G1 had no further impact. In contrast, OGD had a smaller impact on astrocytes survival but G1, alone or combined with OGD, promoted their apoptosis. This effect was prevented by the GPER antagonist G15. The results also show that ischemia did not change the expression levels of GPER in neurons and astrocytes. In this study, we also demonstrate that selective activation of GPER induced astrocyte apoptosis via the phospholipase C pathway and subsequent intracellular calcium rise, whereas in neurons, this effect was not observed. Taken together, this evidence supports a direct impact of GPER activity on the viability of astrocytes, which seems to be associated with the regulation of different signaling pathways in astrocytes and neurons.
After decades of effort, there are no effective clinical treatments to induce the recovery of ischemia-injured tissues, and among the several strategies that have been explored, repetitive transcranial magnetic stimulation has proven to be one of the most promising, with beneficial effects in limb motor function, aphasia, hemispatial neglect, or dysphagia. Despite the clinical evidences, little is known about the mechanisms underlying those effects. The present study aimed to explore the cellular and molecular effects of high-frequency repetitive magnetic stimulation (HF-rMS) on an in vitro model of ischemia. Using primary cortical cultures exposed to oxygen and glucose deprivation followed by reperfusion, we observed that HF-rMS treatment prevents the ischemia-induced neuronal death by 21.2%, and the neurite degeneration triggered by ischemia. Our results also demonstrate that with this treatment there is an increase of 89.2% on the number cells expressing ERK1/2, of 20.1% on the number of cells expressing c-Fos, and a synaptogenic effect, through an increase of 62.9% in the number of synaptic puncta as well as of 49.4% in their intensity. Interestingly, our results indicate that astrocytes are crucial to the beneficial effects triggered by HF-rMS after ischemia, thus suggesting a direct effect of HF-rMS on these cells. The modulation of astrocytes with this non-invasive brain stimulation technique is a promising approach to promote the recovery of ischemia-induced injured tissues; however, it is essential to understand how these effects can be modulated in order to optimize the protocols and enhance the beneficial outcomes.
Cell cultures are characterized by their simplicity, controllability, and ability to provide detailed basic information on how a particular cell population responds to specific stimuli or insult. These characteristics led to their extensive application in the study of molecular interactions and represent a valuable tool in the study of different pathologies. However, due to the lack of interactions between the different components that form an in vivo system, the results obtained in pure cell cultures not always translate what occurs in vivo. In this context, the use of co-cultures has the advantage of allowing the study of interactions between different types of cells present in a tissue, which in many situations are determinant for the effects obtained. The present study aimed to characterize cortical neuron-glia and neuron-enriched primary cultures and evaluate their response to an ischemic insult. Cell viability was assessed by the MTT assay and cell number/phenotype was analyzed by immunocytochemistry in control cultures and in cells subjected to 4 h of oxygen and glucose deprivation. The results obtained demonstrate that astrocytes have a substantial impact on the injury induced by an ischemic insult, thus suggesting that the crosstalk between glia and neurons is crucial to the neuronal protection in conditions of ischemia.
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