2.3.2. Neuroglia Cells

Analysis of GFAP-positive cells (astrocytes), Olig2-positive cells (oligodendrocytes), and Iba1-positive cells (microglia) revealed different reactions of neuroglia cells in the ischemic area of the brain in experimental animals.

Microglial cells are the dominant cell type involved in post-stroke neuroinflammation and the organization of the glial scar. In the intact group, the immunopositive area for microglia cells detected with Iba1 marker was lower (5.42 [4.96; 5.98]) (Figure 5A,G) than in the control NaCl (12.57 [10.31; 16.82]) (Figure 5B,G) and Ad5-GFP (14.43 [12.93; 16.01]) (Figure 5C,G) groups and in the therapeutic UCB-MC+Ad5-GFP (10.07 [7.67; 10.52]]) group (Figure 5D,G) (*p* < 0.05). The Iba1-positive area in the therapeutic Ad5-VEGF-GDNF-NCAM (5.60 [4.89; 6.69]) (Figure 5E,G) and UCBC+Ad5-VEGF-GDNF-NCAM (8.80 [6.37; 9.99]) (Figure 5F,G) groups was lower when compared with the control groups and did not differ from the intact group (*p* < 0.05).

**Figure 5.** Immunoexpression of Iba1 in the rat brain cortex, 21 days after the middle cerebral artery occlusion. Immunofluorescent staining with antibody to microglia specific calcium-binding protein Iba1 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 Iba1 (green glow) in microglial 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 Iba1-positive areas in experimental groups; \*—*p* < 0.05.

Astrocytes in the post-ischemic brain, in association with microglial cells, are involved in the formation of the glial scar, with physical and chemical properties inhibitory for brain recovery. Decreased astrogliosis often correlates with reduced volume of ischemic infarct [30]. The GFAP-positive areas in the brains of the intact group was lower (4.12 [3.36; 4.85]) (Figure 6A,G) than in the control NaCl (9.34 [8.08; 10.15]) (Figure 6B,G) and Ad5-GFP (8.92 [6.25; 12.42]]) (Figure 6C,G) groups (*p* < 0.05). In the therapeutic groups Ad5-VEGF-GDNF-NCAM (4.22 [3.36; 5.19]) (Figure 5E,G) and UCBC+Ad5-VEGF-GDNF-NCAM (3.43 [2.81; 4.19]) (Figure 5F,G), the GFAP-positive areas were lower than in the control (NaCl and Ad5-GFP) groups and did not differ from intact group (*p* < 0.05). In the UCBC+Ad5-GFP (Figure 5D,G) group, the GFAP-positive area did not differ in comparison with all experimental groups (*p* < 0.05).

**Figure 6.** Immunoexpression of GFAP in the rat brain cortex, 21 days after the middle cerebral artery occlusion. Immunofluorescent staining with antibody to astrocyte specific cytoskeletal glial fibrillary acidic protein GFAP 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 GFAP (red glow) in astrocytes. 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 GFAP-positive areas in experimental groups; \*—*p* < 0.05.

Postischemic brain damage is accompanied by the destruction of oligodendrocytes and the subsequent demyelination of neural processes [31]. The number of Olig2-positive cells was significantly reduced in the control NaCl (10.00 [9.00; 11.00]) (Figure 7B,G) and Ad5-GFP (10.00 [9.00; 11.00]) (Figure 7C,G) groups and in the UCBC+Ad5-GFP group (9.00 [9.00; 11.00]) (Figure 7D,G) relative to the intact group (14 [13; 19]) (Figure 7A,G) (*p* < 0.05). The number of Olig2-positive cells in the intact group did not differ from the therapeutic Ad5-VEGF-GDNF-NCAM (12.00 [9.00; 18.00]) (Figure 7E,G) and UCBC+Ad5-VEGF-GDNF-NCAM (14.00 [12.00; 16.00]) (Figure 7F,G) groups. It is important to note that there were more oligodendrocytes in the UCBC+Ad5-VEGF-GDNF-NCAM group than in the control groups and in the UCBC+Ad5-GFP group (*p* < 0.05), but the number did not differ from the Ad5-VEGF-GDNF-NCAM group.

**Figure 7.** Count of Olig2-positive oligodendrocytes in the rat brain cortex, 21 days after the middle cerebral artery occlusion. Immunofluorescent staining with antibody to oligodendrocyte transcription factor Olig2 (red glow) 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 nuclear localization of Olig2 (red glow) in oligodendrocytes. 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 Olig2-positive cells number in experimental groups; \*—*p* < 0.05.
