Atorvastatin induction of VEGF and BDNF promotes brain plasticity after stroke in mice

Abstract

Molecular mechanisms underlying the role of statins in the induction of brain plasticity and subsequent improvement of neurologic outcome after treatment of stroke have not been adequately investigated. Here, we use both in vivo and in vitro studies to investigate the potential roles of two prominent factors, vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF), in mediating brain plasticity after treatment of stroke with atorvastatin. Treatment of stroke in adult mice with atorvastatin daily for 14 days, starting at 24 hours after MCAO, shows significant improvement in functional recovery compared with control animals. Atorvastatin increases VEGF, VEGFR2 and BDNF expression in the ischemic border. Numbers of migrating neurons, developmental neurons and synaptophysin-positive cells as well as indices of angiogenesis were significantly increased in the atorvastatin treatment group, compared with controls. In addition, atorvastatin significantly increased brain subventricular zone (SVZ) explant cell migration in vitro. Anti-BDNF antibody significantly inhibited atorvastatin-induced SVZ explant cell migration, indicating a prominent role for BDNF in progenitor cell migration. Mouse brain endothelial cell culture expression of BDNF and VEGFR2 was significantly increased in atorvastatin-treated cells compared with control cells. Inhibition of VEGFR2 significantly decreased expression of BDNF in brain endothelial cells. These data indicate that atorvastatin promotes angiogenesis, brain plasticity and enhances functional recovery after stroke. In addition, VEGF, VEGFR2 and BDNF likely contribute to these restorative processes.

Figure 1

Quantification field of view in the ischemic border.

Figure 2

Atorvastatin promotes functional recovery. Mice were treated with or without atorvastatin (10 mg/kg) starting at 24 hours after stroke daily for 14 days (n=9/group). Functional tests were performed before MCAO and 1, 7, and 14 days after MCAO. *P<0.05 versus MCAO control. (A) mNSS test; (B) Foot-fault test; (C) Corner test.

Figure 3

Angiogenesis and BDNF expression. Bromodeoxyuridine-reactive endothelial cells were significantly increased in the atorvastatin-treated group (B) compared with control (A, P<0.05). Arrows (A and B) show BrdU-positive endothelial cells. Cerebral vascular perimeters (vWF) were significantly increased in the atorvastatin-treated group (E) compared with the control group (D, P<0.05). Arrows (D and E) show vWF-positive vessels. (C) and (F) show quantification data of BrdU and vWF, respectively. (G) to (H) show double immunostaining BrdU (G, arrow) reactive cells colocalized with vWF (H, arrow) in the vessel. (J) to (K) show BDNF immunostaining in the ischemic boundary area at 14 days after MCAO treated with saline (J) and atorvastatin (K), respectively. (L) shows quantitative data of BDNF density in the ischemic boundary area. (M) to (O) show double immunostaining BDNF-reactive cell (M; arrow, BDNF-positive cell) colocalized with MAP2 (N; MAP2-positive cell). (O) shows merged from (J) and (K). Brain-derived neurotrophic factor immunoreactive cells were also present in endothelial cells (P; arrow, BDNF-positive endothelial cell). Scale bar (A), (D) and (J)=100 μm. Scale bar (I) and (N)=50 μm and P=20 μm.

Figure 4

DCX, TUJ1 and synaptophysin expression. DCX, TUJ1 and synaptophysin are expressed in the ischemic boundary area at 14 days after MCAO treated with atorvastatin (10 mg/kg). (A), (E) and (H) show DCX (A), TUJ1 (E) and synpatophysin (H) expression, respectively, in control saline-treated animal. (B), (F) and (I) show DCX (B), TUJ1 (F) and synpatophysin (I) expression, respectively, in an atorvastatin-treated animal. (D), (G) and (J) show quantitative data of DCX (D), TUJ1 (G) and synaptophysin (J) density in the ischemic boundary area. (C) shows DCX immunoreactive cells around vessel (arrow: DCX-positive cells; arrow head: vessel). Scale bar (A), (E), (H)=100 μm. Scale bar (C)=20 μm.

Figure 5

SVZ explant cell migration. (A) Control; (B) BDNF treatment; (C) atorvastatin treatment; (D) anti-BDNF antibody treatment; (E) atorvastatin with anti-BDNF antibody treatment; (F) quantitative data of SVZ cell migration. Scale bar (A)=200 μm.

Figure 6

VEGFR2 and BDNF expression in the cultured endothelial cells. VEGFR2 (B) and BDNF (E) immunoreactive cells were increased in the atorvastatin-treated group compared with control group (A: VEGFR2; D: BDNF). (C) and (F) quantitative data of percent of VEGFR2 (C) and BDNF (F) immunoreactive cells in cultured mouse brain endothelial cell. Scale bar (A)=50 μm.