## 2 Results
### SeNrs Induce the Generation of Ca\({}^{2+}\) Signals in Cortical Astrocytes
Thus, the SeNrs-induced activation of reactive astrogliosis and the cytoprotective effects of SeNrs during OGD and reoxygenation occur due to endocytosis of nanoparticles into astrocytes and activation of the Ca\({}^{2+}\) signaling system of cells. In the absence of these two components, the protective effects of SeNrs were completely abolished. Figure 8: The effect of an inhibitor of actin-dependent pathways of endocytosis (Cytochalasin D, Cyto D, 10 \(\upmu\)M) on astrocyte Ca\({}^{2+}\) signals upon application of SeNrs (5 \(\upmu\)g/mL) (**A**), on the cytoprotective effects of SeNrs (5 \(\upmu\)g/mL) upon application oxygen-glucose deprivation (OGD) (**B–D**), activation of  
reactive astrogliosis (**E**,**F**), and expression of phosphoinositide 3-kinase (PI3K) (**E**,**G**). (**A**) Ca2+ signals of cortical astrocytes upon application of 5 \(\upmu\)g/mL SeNrs (black curve), 10 \(\upmu\)M Cytochalasin D (Cyt D, green curve), and 5 \(\upmu\)g/mL SeNrs after 2-h preincubation of cells with 10 \(\upmu\)M Cytochalasin D (Cyto D + SeNrs, red curve). Averaged Ca2+ signals for several tens of astrocytes are shown. At the end of the experiment, 10 \(\upmu\)M ATP was added to the cells. (**C**) Effects of 24-h preincubation of cortical astrocytes with Cytochalasin D (10 \(\upmu\)M) on OGD-induced Ca2+ signals. At the beginning of the experiment, a short-term application of 35 mM KCl and 10 \(\upmu\)M ATP was added to detect neurons and astrocytes, respectively. F340/F380--The ratio of Fura-2 fluorescence in the 340 nm registration channel to the 380 nm fluorescence registration channel. (**C**,**D**) Effects of 24-h preincubation of cortical astrocytes with Cytochalasin D (10 \(\upmu\)M) on OGD-induced cell death. Astrocyte culture staining after 40 min OGD with propidium iodide (**C**) and necrotic death analysis (**D**). Abbreviations for panels (**B**-**D**) OGD--oxygen-glucose deprivation for 40 min; SeNrs + OGD--astrocytes were preincubated for 24-h with 5 \(\upmu\)g/mL SeNrs and then OGD was created; Cyto D + OGD--cells were preincubated for 24-h with 10 \(\upmu\)M Cytochalasin D and then OGD was created; Cyto D_SeNrs + OGD--astrocytes were preincubated for 2 h with 10 \(\upmu\)M Cytochalasin D and then 5 \(\upmu\)g/mL SeNrs were added for 24-h (Cyto D was maintained in the culture medium for 24-h) and then OGD was created. (**E**-**G**) Effects of 24-h preincubation of cortical astrocytes with Cytochalasin D (10 \(\upmu\)M) on SeNrs and OGD/R-induced astrocyte reactivation (**E**,**F**) and PI3K expression (**E**,**G**). (**E**) Immunostaining of glial fibrillary acidic protein (GFAP, green) and PI3K (blue) in cortical astrocytes after 24-h of incubation with 5 \(\upmu\)g/mL SeNrs (SeNrs group), after 24-h of preincubation with 10 \(\upmu\)M Cytochalasin D and 5 \(\upmu\)g/mL SeNrs (Cyto D + SeNrs group), after 24-h of incubation with 5 \(\upmu\)g/mL SeNrs and OGD/R (SeNrs + OGD/R group), and after 24-h of preincubation with 10 \(\upmu\)M Cytochalasin D with 5 \(\upmu\)g/ mL SeNrs and OGD/R (Cyto D_SeNrs + OGD/R group). Draq5 nuclei staining (red). (**F**,**G**) Intensity levels of GFAP (**F**) and PI3K (**G**) were determined by confocal imaging. We analyzed individual cells that had fluorescence of secondary antibodies. The quantitative data reflecting the level of GFAP or PI3K expression are presented as fluorescence intensity values in summary bar charts (mean +/ - SEM). The values were averaged by 150-200 cells for each column. Each value is the mean \(\pm\) SE (n \(\geq\)\(3,p\) < 0.05). Statistical significance was assessed using paired \(t\)-tests. Comparison with control, *** \(p\)-level < 0.001, n/s--data not significant (\(p\) > 0.05).