## 2 Results
### SeNrs Induce the Generation of Ca\({}^{2+}\) Signals in Cortical Astrocytes
(**B**) Dependence of the amplitude of Ca\({}^{2+}\) responses of astrocytes on the growth of SeNrs concentration and its approximation by a sigmoid function. To plot dose dependencies, we used the results of Ca\({}^{2+}\)-dynamic measurements on three independent cell cultures. (**C**) Ca\({}^{2+}\) signal generation by cortical astrocytes upon application of 5 \(\upmu\)g/mL SeNrs in a calcium-free medium. (**D**) Suppression of Ca\({}^{2+}\) signals of astrocytes upon application of 5 \(\upmu\)g/mL SeNrs after depletion of Ca\({}^{2+}\) stores in the endoplasmic reticulum with 10 \(\upmu\)M thapsigargin (TG). F340/F380—The ratio of Fura-2 fluorescence in the 340 nm registration channel to the 380 nm fluorescence registration channel. Panels (**C**,**D**) show the Ca\({}^{2+}\) signals of astrocytes in one experiment and their average value (bold red curve). The Anti-Apoptotic and Anti-Inflammatory Effects of SeNrs during Ischemia/Reoxygenation Correlate with Reactive Astrogliosis  
Preincubation of cortical astrocytes with SeNrs at concentrations of 1 \(\upmu\)g/mL or 5 \(\upmu\)g/mL led to the suppression of apoptosis and necrosis in a more toxic model of ischemia-OGD for 2 h followed by 24-h reoxygenation-(OGD/R) (Figure 3). OGD/R caused the induction of late stages of apoptosis (cells are characterized by a high level of Hoechst 33342 fluorescence simultaneously with the appearance of some membrane permeability to  
Figure 2: Effect of pre-incubation of cortical astrocytes with 1 or 5 \(\upmu\)g/mL of SeNrs on Ca\({}^{2+}\) signal generation and survival after OGD. (**A**) Ca\({}^{2+}\) signals astrocytes during OGD (40 min) and OGD after 24-h pre-incubation with 1 or 5 \(\upmu\)g/mL of SeNrs. The absence of Ca\({}^{2+}\) signals on KCl indicates the absence of neurons in the cell culture. Ca\({}^{2+}\) signals from astrocytes averaged over several dozens of cells are presented. The experiments were performed in three replicates on three separate cell cultures. F340/F380—The ratio of Fura-2 fluorescence in the 340 nm registration channel to the 380 nm fluorescence registration channel. (**B**) Images of cortical astrocytes loaded with propidium iodide (PI) after 40 min OGD. OGD conditions induced by omitting O\({}_{2}\) in glucose-free media with argon bubbling. The red dots represent the PI-stained nuclei of necrotic cells. BF—bright-field microscopy. (**C**) Effect of 24-h pre-incubation with SeNrs on the cell viability after 40 min OGD. Black asterisks indicate the differences between the experimental groups compared with the OGD group. Differences between experimental groups are marked by red. *** \(p<0.001\), n/s—insignificant differences. propidium iodide (PI)) in 40% of astrocytes (Figure 3A,B) and the appearance of necrosis (high level of PI fluorescence) in 60% or more astrocytes (Figure 3C). Additional analysis of the effects of SeNrs on the induction of necrosis and apoptosis was performed using the Apoptosis/Necrosis Detection Kit (ab176750, Abcam, Cambridge, UK) (Supplementary Figure S2). It turned out that 24-h after OGD/R, necrosis was registered in 62% of cells, and apoptosis in 27% of astrocytes. Preincubation of astrocytes with SeNrs at 1 \(\upmu\)g/mL or 5 \(\upmu\)g/mL dose-dependently reduced apoptosis to 20% and 14% of cells, and necrosis to 14% and 10% of cells, respectively (Supplementary Figure S2B). An analysis of the expression of genes encoding proteins that regulate cell death showed that the preincubation of astrocytes with 1 \(\upmu\)g/mL SeNrs led to increased expression of 7 of the 15 genes studied (Figure 4A, blue columns), while 5 \(\upmu\)g/mL SeNrs affected the expression of 11 genes (Figure 4A, green columns). SeNrs contributed to an increase in  
Figure 3: The effect of 24-h pre-incubation of cortical astrocytes with 1 or 5 \(\upmu\)g/mL of SeNrs on the OGD/R-induced cell death. (**A**) Double staining of cells with Hoechst 33342 (HO342), propidium iodide (PI), merge HO342 with PI. OGD/R—induction of OGD (2 h) and reoxygenation (24-h) without preincubation with SeNrs.