## 2 Results
### SeNrs Induce the Generation of Ca\({}^{2+}\) Signals in Cortical Astrocytes
Preincubation of astrocytes with 5 ug/mL SeNrs resulted in an increase in the fluorescence level of anti-GFAP antibodies (Figure 6A,B) without a significant increase in the level of fluorescence of anti-Tn\(\kappa\) antibodies (Figure 6C, SeNrs). Staining of astrocytes 24-h after OGD/R showed increased levels of GFAP (Figure 6B--OGD/R) and TNF\(\alpha\) (Figure 6C--OGD/R) compared with the control. However, OGD/R after preincubation of astrocytes with 5 ug/mL SeNrs resulted in an increase in the level of GFAP protein (Figure 6B--SeNrs + OGD/R) with a decrease in the level of Tn\(\kappa\) (Figure 6C--SeNrs + OGD/R) compared with the experimental group astrocytes subjected to OGD/R alone. Figure 5: The effect of 24-h incubation of mice cortical astrocytes with 1 ug/mL or 5 ug/mL SeNrs on the basal (**A**) and OGD/R-induced (**B**) expression of genes encoding markers of reactive astrogliosis. Gene expression in intact cells is marked with a dashed line for panel (**A**). Gene expression in OGD/R cells without preincubation with SeNrs compounds is marked with a dashed line for panel (**B**). The results are expressed as \(2^{-\Delta\Delta\text{CI}}\), where (\(\Delta\Delta\text{CI}\)) is the difference between the \(\Delta\text{CI}\) values between the gene under experiment and a housekeeping gene. Statistical significance was assessed using _t_-tests. Comparison of experimental groups with control: n/s—data not significant (\(p>0.05\)), \({}^{*}\)\(p<0.05\), \({}^{**}\)\(p<0.01\), and \({}^{***}\)\(p<0.001\). Comparison of experimental groups with each other is indicated in red. The number of samples is 3. These immunocytochemical staining data correlate well with the results of PCR analysis of OGD/R-induced expression of genes involved in cell death (Figure 4) and the activation of reactive astrogliosis (Figure 5). Phosphoinositide 3-kinase is known to be involved in the activation of the defense mechanisms of brain cells [29; 30; 31]. Immunocytochemical staining of cortical astrocytes with antibodies against GFAP and PI3K showed (Figure 7) that the preincubation of astrocytes for 24-h with 5 ug/mLSeNrs caused an increase in the level of not only GFAP (Figure 7B--SeNrs), but also the level of PI3K (Figure 7--SeNrs). Whereas after OGD/R, on the contrary, there was a decrease in the number of cells stained with antibodies against PI3K, as well as the level of  
Figure 6: Effect of 24-h preincubation of cortical astrocytes with 5 ug/mL SeNrs on baseline and OGD/R induced levels of tumor necrosis factor-alpha (TNFa) expression. (**A**) Immunostaining of glial fibrillary acidic protein (GFAP, green) and TNFa (blue) in cortical astrocytes in the control, after 24-h of incubation with 5 ug/mL SeNrs, after OGD/R and OGD/R after 24-h of incubation with SeNrs. Draq5 nuclei staining (red). (**B,C**) Intensity levels of GFAP (**B**) and TNFa (**C**) were determined by confocal imaging. We analyzed individual cells that had fluorescence of secondary antibodies. The quantitative data reflecting the level of GFAP or TNFa expression are presented as fluorescence intensity values in summary bar charts (mean +/ – SEM). The values were averaged by 200 cells for each column. The results obtained after immunostaining agree well with the data of fluorescence presented in panel (**A**). 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, **\(p\) < 0.01, n/s—data not significant (\(p\) > 0.05). antibody fluorescence in cells (Figure 7C, OGD/R). In contrast, the preincubation of astrocytes with SeNrs contributed not only to an increase in the GFAP level (Figure 7B, SeNrs + OGD/R), but also to an increase in the content of PI3K (Figure 7C, SeNrs + OGD/R) after OGD/R. Figure 7: Effect of 24-h pre-incubations of cortical astrocytes with 5 \(\upmu\)g/mL SeNrs on baseline and OGD/R induced levels of phosphooinositide 3-kinase (PI3K) expression.