Figure 8.

Functional blood vessel formation and proangiogenic marker induction by N-NAM in the postischemic brain. N-NAM (5 µg) or scN-NAM (5 µg) was administered intranasally three times daily at 4, 5 and 6 days after 60 min of MCAO, and IgG or fluorescein isothiocyanate (FITC)-dextran (60 mg/kg) was injected intravenously (i.v.) 1 h prior to sacrifice 7 days post-MCAO (Fig. 7a). Coronal brain sections were prepared and stained using biotynylated rat anti-IgG antibody for IgG staining (b) or FITC-dextran images acquired with confocal microscopy (c). IgG-positive area or FITC intensity were measured using Scion Image 4.0 (https://scion-image.software.informer.com/) or ImageJ (https://imagej.nih.gov/ij/download.html), respectively. Representative images are shown (upper panels in b and c), and the intraparenchymal leakage area are presented as the means ± SEMs (n = 7 for IgG staining and n = 12 (3 consecutive planes from 4 animals) for FITC-dextran image). (d,e) Tissue lysates were prepared from the asterisked regions in (a) 7 days post-MCAO and immunoblotted for Ang1 and Ang2. (f) Schematic diagram of a proposed mechanism showing how N-NAM induce angiogenesis in HUVECs. Ang1, angiopoietin-1; Ang2, angiopoietin-2; MMP-9, metalloprpoteinase 9; VEGF, vascular endothelial growth factor; eNOS, endothelial nitric oxide synthase. The results are representative of three independent experiments. Scale bars, 200 or 50 μm in C, **p < 0.01, *p < 0.05 versus theN-NAM + PBC control group.