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- Role of astrocytes in an in vitro model of ischemic strokePublication . Roque, Cláudio André Martins; Baltazar, Graça Maria FernandesIschemic stroke (IS) is the leading cause of complex and serious long-term disability in developed countries, and after decades of effort there are no effective clinical treatments for IS, especially in the subacute and chronic phases. Currently, in these stages of the IS there is no alternative to promote the recovery of brain tissues affected by the ischemic injury. Most of the treatments (e.g., physical therapy, speech therapy, occupational therapy) are applied with the aim of reducing the sequelae left, or to controlling modifiable risk factors (e.g., hypertension, diabetes, coagulopathies). This leads to a need to develop new approaches to recover those areas, reduce the neurological deficits and, if possible, enhance the functions regulated by the affected brain regions. In this context, this work intends to explore two approaches that hypothetically could induce the recovery of the areas affected by ischemia. The first is related to the potent physiological effects of estrogens on central nervous system (CNS) and its participation in several processes such as, neurogenesis, the expression of neuroprotective factors and antioxidant mechanisms, through the evaluation of the potential beneficial effects induced by the selective activation of G protein–coupled estrogen receptor 1 (GPER or GPR30). The second, by evaluating the potential protective effects induced by high frequency repetitive magnetic stimulation (HF-rMS), an approach that has been described as having the ability to correct maladaptive brain plasticity and to enhance neuronal communication during rehabilitation. In both cases the ability to induce neuroprotection in neurodegenerative disorders, such as, Alzheimer´s disease, Parkinson’s disease, and mood disorders, was already demonstrated. To standardize the ischemic damage and evaluate the potential beneficial effects induced by these two approaches several in vitro models of ischemia were developed and characterized. Neuron-enriched, neuron-glia, and astrocyte-enriched primary cortical cultures subjected to oxygen and glucose deprivation (OGD) followed by a reperfusion period, were used as models. The evaluation of the effects induced by GPER activation and by HF-rMS was performed through the assessment of several parameters related cell survival and proliferation, GPER expression, calcium imaging, as well as neurite morphometric and synaptic modifications. Concerning the role of GPER on the ischemic injury, we observe that ischemia did not change the levels of GPER in neurons and astrocytes. Moreover, GPER selective activation had no impact in neuronal survival, whereas it induced the apoptosis of astrocytes, being this effect meditated by the activation of phospholipase C pathway, and the subsequent intracellular calcium rise. These data indicate a direct impact of GPER on the viability of astrocytes, and the coupling of GPER to different signaling pathways in astrocytes and neurons. Our data also shows that HF-rMS reduces the neuronal loss, the initial neurite degeneration and the loss of synaptic markers triggered by ischemia. Interestingly the protective effect triggered by HF-rMS required the presence of astrocytes. Taken together the data obtained suggests that HF-rTMS has the potential to be used as a therapeutic approach to reduce neuronal death and neuronal damage, by limiting neurite degeneration and enhance functional connectivity and synaptic plasticity in the areas affected by the ischemia. Furthermore, our results also suggest that astrocytes play a crucial role on ischemic injury. Astrocytes were more resistant to ischemic periods than neurons in all experiments performed and when they were present the injury was smaller, which indicate an active role in the neuronal protection against ischemia-induced injury. Taking into account their preponderant role in neuronal physiology and the fact that their presence is crucial for the observed beneficial effects induced by HF-rMS it seems evident that astrocytes could have a substantial impact on the protection and recovery of ischemia-induced lesion. Thereby, we hypothesize that astrocytes could be a potential therapeutic target for the treatment of cerebral ischemia and any methodology/approach that potentiate their beneficial effects may be a promising therapeutic approach.