## 2 Results
### SeNrs Induce the Generation of Ca\({}^{2+}\) Signals in Cortical Astrocytes
(**A**) Immunostaining of glial fibrillary acidic protein (GFAP, green) and PI3K (blue) in cortical astrocytes in the control, after 24-h of incubation with 5 \(\upmu\)g/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 PI3K (**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 PI3K 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_-level < 0.05, n/s—data not significant (_p_ > 0.05). Thus, preincubation of cortical astrocytes with SeNrs suppresses the processes of necrosis and apoptosis caused by ischemia/reoxygenation conditions. Such a protective effect of selenium nanorods occurs due to an increase in the expression level of key genes encoding anti-apoptotic, anti-inflammatory proteins, and transcription factors. This activates the process of reactive astrogliosis, which correlates with an increase in the level of the protein phosphoinositol-3-kinase and a decrease in the level of TNF\(\alpha\). The Induction of Reactive Astrogliosis and the Cytoprotective Effects of SeNrs Are Abolished upon Inhibition of Endocytosis  
It is known that Cytochalasin D blocks or largely suppresses all actin-dependent pathways of endocytosis, depending on the duration of incubation (from 2 to 24-h) [32; 33]. Preincubation of astrocytes with 10 uM Cytochalasin D (Cyto D) for 2-h leads to complete suppression of Ca\({}^{2+}\) responses to SeNrs application (Figure 8A, red curve). Astrocytes did not respond with \"standard\" Ca\({}^{2+}\) signals to SeNrs application in the form of a gradual increase in [Ca\({}^{2+}\)]i (Figure 8A, black curve), but single Ca\({}^{2+}\) pulses were recorded. Application of 10 uM Cyto D in an acute experiment did not cause the generation of Ca\({}^{2+}\) signals by astrocytes (Figure 8A, green curve). At the same time, the addition of ATP (10 uM) at the end of the experiments indicates the functionality of the Ca\({}^{2+}\) signaling system of astrocytes. After a 24-h preincubation of astrocytes with 5 ug/mL SeNrs, the OGD-induced increase in [Ca\({}^{2+}\)]i was suppressed (Figure 8B, blue curve) and cell death was reduced to 17% (Figure 8C,D, SeNrs + OGD). After a 2-h preincubation of astrocytes with Cyto D SeNrs was added to culture medium at a concentration of 5 ug/mL for 24-h. After this preincubation, OGD induced a biphasic increase in [Ca\({}^{2+}\)]i (Figure 8B, orange curve) which was comparable with OGD experimental group (Figure 8B, red curve). Cell death after OGD was 83% of cells (Figure 8C,D--OGD group), and after preincubation with SeNrs together with Cyto D, necrosis was recorded in 81% of astrocytes (Figure 8C,D--Cyto D_SeNrs + OGD). Preincubation of astrocytes with 10 uM Cyto D for 24-h did not suppress the OGD-induced increase in [Ca\({}^{2+}\)]i (Figure 8B--green curve) and did not reduce astrocyte death (82%) (Figure 8C,D--Cyto D + OGD). Immunocytochemical staining of astrocytes with antibodies against GFAP and PI3K showed that the effect of 24-h preincubation with SeNrs on the increase in the expression of these proteins was abolished after preincubation with Cyto D (Figure 8E-G--Cyto + SeNrs). Similarly, after preincubation of astrocytes with SeNrs and subsequent OGD/R, activation of reactive astrogliosis occurred, recorded by an increase in the level of GFAP and PI3K (Figure 8E-G--SeNrs + OGD/R). However, preincubation of astrocytes with Cyto D abolished this SeNrs-induced increase in GFAP and PI3K expression (Figure 8E-G--Cyto D_SeNrs + OGD/R).