*2.2. Correlated Astrocytic Response to Ischemic Influence*

The next stage of our study aimed to analyze changes in the adaptive capacity of the astrocytic network in response to modeling ischemia-like conditions. Hypoxia is one of the most common experimental models of brain cell damage [18]. Several studies have shown a significant decrease in the viability and functional activity of brain cells in models of 10-min acute hypoxia in vitro [18–20]. However, astrocytes are considered more resistant to hypoxic damage than neurons. In this regard, to create stress conditions that affect astrocytic network functional activity, we adapted a model of oxygen-glucose deprivation ("ischemia") lasting for 30 min (see the materials and methods). The results showed a significant decrease in the viability of primary astrocyte cultures in the "ischemia" group (see Supplementary Materials 1, Supplementary Materials Table S1), whereas blockage of Cx43 during ischemia modeling maintained the viability of astrocytes at the intact culture level ("control" 98.25 [96.99; 99.197]%, "ischemia" 95.59 [94.55; 97.35]%, "ischemia+Gap19" 99.21 [97.61; 100.0]%).

The number of cells that manifested calcium activity matched the control group and was 56.178 [53.258; 67.692]%. Furthermore, no statistically significant differences in the frequency and duration of Ca2+ oscillations were detected (1.612 [1.25; 2.01] osc./min and 10.967 [10.854;11.708] s, respectively) (Figure 3B,C).

Changes in the pattern of response to ATP stimulation in astrocytic cultures after ischemia-like condition modeling are of particular interest (see Supplementary Materials 1, Supplementary Materials Table S1). Under normal conditions, an increase in the frequency of Ca2+ oscillations after ATP application was observed. In contrast, the frequency of Ca2+ events in response to ATP application was decreased significantly in the "ischemia+ATP" group (1.156 [1.052;1.249] osc./min) (*p* < 0.05, Wilcoxon rank-sum test) (Figure 3C). The percentage of working cells and the duration of Ca2+ oscillations did not change. Therefore, ischemia leads to the loss of the functional response of astrocytes to the stimulating effect of ATP.

Analyzing astrocytic connectivity in the culture after ischemia modeling is of particular interest. While the primary astrocytic cultures in the post-ischemic period exhibited calcium events, their degree of synchrony and correlation was substantially different compared with that of the control cultures. In particular, the modeled ischemia-like conditions manifested as a significant decrease of all the parameters that characterized the connectivity of astrocytes. The number of direct long-distance connections was reduced by a factor of 30 (from 3.95 [3.1; 4.47] to 0.13 [0.07; 0.32]) (Figure 2). In the "ischemia" group, the average value of the maximum correlation of the shift between the average calcium level of neighboring astrocytes over time was 0.110 [0.107; 0.123], which is 2 times lower than that in the "control" group (0.242 [0.226; 0.263]; *p* < 0.05, the KS test). The number of long-distance connections in the "ischemia" group was 0.087 [0.087; 0.091] ("control" 0.096 [0.094; 0.099], *p* < 0.05, the KS test). Importantly, ischemia leads to changes in the astrocytic response to ATP stimulation in the post-ischemic period. Characteristic examples of changes in the dynamics of astrocytic calcium activity after ATP application in normal conditions and in model ischemia-like conditions are presented in Figure 7. Despite the statistically significant increase in distant connections after ATP application, similar to the control group (Figure 2), the average correlation level remained unchanged (Figure 4), which indicates the impairment of the functional astrocytic network.

**Figure 7.** Characteristic examples of the dynamics of Oregon Green calcium sensor fluorescence in primary monoastrocytic cultures in vitro. (**A**,**B**)—normal conditions, PBS application; (**C**,**D**)—normal conditions, ATP applications; (**E**,**F**)—ischemic model, ATP application. (**A**,**C**,**E**)—examples of culture images segmented according to astrocyte positions. (**B**,**D**,**F**)—dynamics of calcium sensor fluorescence in selected astrocytes, note the correspondence between the astrocyte numbering and line colors. Vertical black lines indicate the moments of PBS or ATP application.

The frequency characteristics, particularly the average low-frequency component of calcium intensity, of the astrocytic network were also changed significantly after ischemia-like condition modeling. The average frequency of base-level oscillations was increased by a factor of 3 relative to the control values on day 7 after ischemia influence and was 0.0219 [0.0206; 0.0237] (*p* < 0.05, the KS test). Moreover, the signal delay rate between astrocytes was reduced significantly ("control": 24.81 [21.32; 26.85], "ischemia": 16.98 [16.96; 20.84], *p* < 0.05, the KS test).

Investigation of the role of connexin 43 in the regulation of astrocytic resistivity to ischemic conditions revealed that the Cx43 blockade during ischemia modeling improves the viability of cells in the post-ischemic period. On the seventh day from the start, the number of viable cells in the "ischemia+Gap19" group was not distinguishable from that in the control group (Supplementary Materials 1).

Assessment of the functional calcium activity in the "ischemia+Gap19" group did not reveal significant changes in the main parameters of functional Ca2+ activity compared with the "ischemia" and "control" group values on day 7 after induction of the ischemia model (Figure 3).

In contrast, the application of Gap19 during ischemia modeling maintained all parameters of network connectivity. The average number of long-distance connections of an astrocyte, the average value of the correlation between pairs of astrocytes, average low-frequency component of calcium intensity, and the signal delay rate were preserved at the level of the control values. Furthermore, partial normalization of the response to ATP application was observed, namely, the increasing average correlation of astrocytic activity, although the number of distant connections of a single astrocyte and the frequency of calcium oscillations were not significantly different. Thus, the blockade of hemichannels during ischemic modeling preserves the connectivity of astrocytes in the post-hypoxic period.
