2.3.1. Cellular Stress and Apoptosis Proteins

The expression of heat shock protein 70 kDa (Hsp70) in neural and glial brain cells has constitutional and inductive options. Thus, Hsp70 expression is significantly upregulated in ischemic conditions [29]. In this study, analysis of the intensity of immunofluorescent brain cortex staining with antibodies against Hsp70 revealed a different response of brain cells in the area of ischemic damage in experimental animals when compared with intact animals (Figure 3A,G). The Hsp70 expression level in control (NaCl) (Figure 3B,G) and Ad5-GFP (Figure 3C,G) groups was 27.375 [24.940; 28.649] and 34.361 [31.315; 35.612], respectively, and was significantly higher than that of intact animals (18.422 [17.786; 19.510]). In the therapeutic groups with preventive gene therapy with Ad5-VEGF-GDNF-NCAM (20.419 [18.510; 22.950]) (Figure 3E,G) and UCB-MC+Ad5-VEGF-GDNF-NCAM (18.169 [17.462; 18.964]) (Figure 3F,G), Hsp70 immunoexpression was lower compared to the control groups (*p* < 0.05) and did not differ from intact group (*p* < 0.05). In the UCB-MC+Ad5-GFP group (Figure 3D,G), the expression level of Hsp70 (17.651 [17.648; 20.832]) was lower than the control groups and did not differ from the intact and therapeutic groups.

**Figure 3.** Immunoexpression of Hsp70 in the rat brain cortex, 21 days after the middle cerebral artery occlusion. Immunofluorescent staining with antibody to heat shock protein 70 kDa in intact (**A**), control NaCl (**B**) and Ad5-GFP (**C**), and therapeutic UCB-MC+Ad5-GFP (**D**), Ad5-VEGF-GDNF-NCAM (**E**) and UCB-MC+Ad5-VEGF-GDNF-NCAM (**F**) groups. Arrows indicate cytoplasmic localization of Hsp70 (red glow) in brain cells. Arrowheads point to cell nuclei visualized using DAPI (blue glow). Scale bar in (**A**–**F**) = 50 μm; scale bar in the insert = 20 μm. (**G**)—comparative analysis of fluorescence intensity level of Hsp70 in experimental groups; \*—*p* < 0.05.

Activation of Caspase3 enzyme characterizes the irreversible phase of nuclear DNA degradation and subsequent cell death. In the present study, the activity of the pro-apoptotic protein Caspase3 was studied in the ischemic brain cortex in experimental rats (Figure 4A–F). It was found that the therapeutic groups Ad5-VEGF-GDNF-NCAM (12.00 [9.00; 13.00]) (Figure 4D,F) and UCB-MC+Ad5-VEGF-GDNF-NCAM (9.00 [7.50; 9.00]) (Figure 4E,F) had significantly less Caspase3-positive cells than the control NaCl (25.00 [22.00; 27.00]) group (Figure 4A,F). The number of apoptotic cells in the UCB-MC+Ad5-VEGF-GDNF-NCAM group was also lower than the Ad5-GFP control group (15.50 [11.75; 19.00]) (Figure 4B,F) (*p* < 0.05). In the UCBC+Ad5-GFP group (6.00 [3.25; 9.75]) (Figure 4C,F), the content of Caspase3-positive cells was lower than in the control groups (*p* < 0.05) and did not differ from other therapeutic groups.

**Figure 4.** Count of Caspase3-positive cells in the rat brain cortex, 21 days after the middle cerebral artery occlusion. Immunofluorescent staining with antibody to Caspase3 in control NaCl (**A**) and Ad5-GFP (**B**), and therapeutic UCB-MC+Ad5-GFP (**C**), Ad5-VEGF-GDNF-NCAM (**D**) and UCB-MC+Ad5-VEGF-GDNF-NCAM (**E**) groups. Arrow indicates nuclear localization of Caspase3 (red glow) in brain cells. Arrowheads point to cell nuclei visualized using DAPI (blue glow). Scale bar in (**A**–**E**) = 50 μm; scale bar in the insert = 20 μm. (**F**)—comparative analysis of Caspase3-positive cells number in experimental groups; \*—*p* < 0.05.
