## 2 Results
### SeNrs Induce the Generation of Ca\({}^{2+}\) Signals in Cortical Astrocytes
(**B**) Cytogram demonstrating the viability of cortical astrocytes after OGD/R (2h and 24-h reoxygenation) and after 24-h pre-incubation with 1 or 5 \(\upmu\)g/mL SeNrs and OGD/R. \(X\)-axis—the intensity of PI fluorescence; \(Y\)-axis—the intensity of Hoechst 33342 fluorescence. Cells were stained with the probes 24-h after the OGD/R. (**C**) Effect of 24-h pre-incubation with 1 or 5 \(\upmu\)g/mL SeNrs on the induction of necrosis and apoptosis. The number of replicates for cell cultures was 3, the number of cover-slips with cells for each sample was 5. Statistical significance was assessed using \(t\)-tests. Comparison of experimental groups vs OGD/R: n/s—data not significant (\(p>0.05\)), * \(p<0.05\), ** \(p<0.001\). Comparison of experimental groups with each other is indicated in red. mRNA expression by more than two times for the genes encoding the _Hif1 alpha_ subunit of the hypoxia-induced transcription factor and the _Nrf-2_ transcription factor, which is involved in a complex regulatory network and plays a pleiotropic role in the regulation of metabolism, inflammation, autophagy, proteostasis, mitochondrial physiology, and immune responses (Figure 4A). Preincubation of astrocytes with SeNrs and subsequent OGD/R led to an increase in the expression of IL-10, which has an anti-inflammatory effect, and simultaneously to a decrease in the level of expression of the tumor necrosis factor _Tnfa_. In addition, after  
Figure 4: The effect of 24-h incubation of mice cortical astrocytes with 1 \(\mu\)g/mL or 5 \(\mu\)g/mL SeNrs on the basal (**A**) and OGD/R-induced (**B**) expression of genes regulating cell death and inflammation pathways. Gene expression in intact cells are 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 \(2^{-\Delta\Delta\text{Ct}}\), where (\(\Delta\Delta\text{Ct}\)) is the difference between the \(\Delta\text{Ct}\) 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 ***\(p<0.001\), n = 3 Comparison of experimental groups with each other is indicated in red. preincubation of cells with SeNrs, an increased level of expression of the _Stat3_, _Nrf2_, and _Hif1 alpha_ genes persisted. (Figure 4B). We have previously shown that spherical selenium nanoparticles activated reactive astrogliosis in neuroglial culture of the cerebral cortex [27]. PCR analysis showed that preincubation of cortical astrocytes with SeNrs led to an increase in the baseline expression of widely used markers of reactive astrocytes _GFAP_ (glial fibrillary acid protein) and _Len2_ (Lipocalin-2), proliferation genes, including late-phase cyclins b1 and b2 (_Ccnb1_ and _Ccnb2_), and _Iff202b_ (interferon-activated gene 202B), which has anti-inflammatory activity (Figure 5A). Preincubation of astrocytes with SeNrs and subsequent OGD/R led to an increase in _GFAP_ expression and suppression of expression of _TNF8F12A_ (TNF receptor superfamily member 12A), which is involved in inflammatory processes in astrocytes (Figure 5B). The results of PCR analysis were confirmed by immunocytochemical staining of astrocytes simultaneously with anti-GFAP and TNF\(\kappa\) antibodies (Figure 6A). The results of image analysis using the ImageJ software ([[https://imagej.net/ij/](https://imagej.net/ij/)]([https://imagej.net/ij/](https://imagej.net/ij/)), accessed on 6 July 2023) package showed that in the control there was practically no fluorescence of secondary antibodies against TNF\(\alpha\) (Figure 6C--Control), with a moderate fluorescence of secondary antibodies against GFAP (Figure 6B--Control), reflecting the level of the studied intracellular proteins.