Attenuation of brain response to vascular endothelial growth factor-mediated angiogenesis and neurogenesis in aged mice

Abstract

Background and purpose: Alterations of neuroangiogenic response play important roles in the development of aging-related neurodisorders and affect gene-based therapies. We tested brain response to vascular endothelial growth factor (VEGF) in aged mice.

Methods: Adeno-associated viral vector (AAV)-VEGF, an adeno-associated viral vector expressing VEGF, was injected into the brain of 3-, 12-, and 24-month-old mice. AAV-LacZ-injected mice were used as controls (n=6). Before euthanasia at 6 weeks after vector injection, the mice were intraperitoneally injected with 5-bromodeoxyuridine for 3 consecutive days. The vascular density and the number of neuroprogenitors were analyzed.

Results: Injection of AAV-VEGF increased the vascular density in the brain of 3-, 12-, and 24-month-old mice by 22%+/-7% (AAV-VEGF: 320+/-15 per 10x field versus AAV-LacZ: 263+/-8, P<0.05), 20%+/-8 (AAV-VEGF: 300+/-9 versus AAV-LacZ: 250+/-11, P<0.05), and 7%+/-16% (AAV-VEGF: 257+/-27 versus AAV-LacZ: 236+/-13, P=0.283), respectively. There were more VEGF receptor-positive neuroprogenitors in the subventricular zone of AAV-VEGF-injected 3- (22+/-2) and 12-month-old mice (21+/-5) than that of 24-month-old mice (7+/-1). More 5-bromodeoxyuridine-positive endothelial cells and neuroprogenitors were detected around the injection site and subventricular zone of 3- (13+/-4) and 12-month-old mice (14+/-5) than that of 24-month-old mice (1+/-1). VEGF receptor 2 was upregulated in AAV-VEGF-injected brains of 3- and 12-month-old mice, but not in 24-month-old mice.

Conclusions: The angiogenic and neurogenic response to VEGF stimulation is attenuated in the aged mouse brain, which may be due to reduced VEGF receptor activity.

Figure 1. VEGF and LacZ expression detected at the injection sites

A. Drawings illustrate the position of the vector injection site, sagittal section (left) and coronal section (right). Cc= Corpus callosum; V= ventricle; and BG= basal ganglia. B. Photomicrographs show VEGF and LacZ positive cells (brown, indicated by arrows) in AAV-VEGF (VEGF) or AAV-LacZ (LacZ)-injected brains. The nuclei were counterstained with hematoxylin. Scale bars=100μm. 3M, 12M, and 24M represent 3-, 12-, and 24-month old mice, respectively.

Figure 2. Vascular density in the AAV-VEGF-injected brain

A. Photomicrographs show lectin-stained microvessels in the brain of 3-, 12- and 24-month old mice 6 weeks after AAV vector injection. B. Bar graph shows the fold increase of capillaries in the AAV-VEGF-injected brain vs. AAV-LacZ-injected brain. There were more capillaries in the brain of 3- and 12-month old mice than 24-month old mice (Asterisk, p<0.05). VEGF: number of capillaries in the AAV-VEGF-injected brain; LacZ: number of capillaries in the AAV-LacZ-injected brain. Scale bar = 100μm.

Figure 3. BrdU positive endothelial cells in the vector-injected brain

A. More BrdU positive cells (red) were detected around injection sites of 3- and 12-month old mice than in 24-month old mice after AAV-VEGF injection. Few BrdU-positive cells were detected in AAV-LacZ-injected brains. B. Bar graph shows quantification of BrdU positive cells (per section; asterisk, p<0.05). C. Double labeling shows that BrdU positive cells (red) were colocalized with Lectin-positive vessels (green). Scale bars for A = 50 μm, for C = 25 μm.

Figure 4. VEGFR-2 positive NPCs in SVZ

A. Schematic drawing shows the SVZ region of the mouse brain. B. Photomicrographs show VEGFR-2 positive cells in the SVZ regions. The AAV-VEGF-injected brain had more VEGFR-2 positive cells in the SVZ than in the AAV-LacZ-injected brain. There were more VEGFR-2-positive cells (arrow) in the SVZ of 3- and 12-month old mice than in 24-month old mice after receiving AAV-VEGF injection (per section; asterisk, p<0.05). C. Bar graph shows the numbers of VEGFR-2 positive cells per 40X objective field in the different groups. D. Photomicrographs show BrdU positive cells (red) in the SVZ. E. Bar graph shows the quantification of BrdU positive cells in the SVZ (per section, asterisk, p<0.05). VEGF: AAV-VEGF injected brains; LacZ: AAV-LacZ injected brain. F. Double-label with BrdU (red) and DCX (green) shows most of the BrdU positive cells in SVZ were DCX positive NPCs. Scale bars = 50μm.

Figure 5. VEGFR-2 expression in the vector-injected brain

Representative Western blot image shows VEGFR-2 expression was not increased in the brain of 24-month old mice treated with AAV-VEGF. LacZ: AAV-LacZ-injected brain: VEGF: AAV-VEGF-injected brain. Expression of β-actin was used as internal control.