Endothelial nitric oxide synthase regulates brain-derived neurotrophic factor expression and neurogenesis after stroke in mice

Abstract

Here, we investigate the effects of endothelial nitric oxide synthase (eNOS) on angiogenesis, neurogenesis, neurotrophic factor expression, and neurological functional outcome after stroke. Wild-type and eNOS knock-out (eNOS-/-) mice were subjected to permanent occlusion of the right middle cerebral artery. eNOS-/- mice exhibited more severe neurological functional deficit after stroke than wild-type mice. Decreased subventricular zone (SVZ) progenitor cell proliferation and migration, measured using bromodeoxyuridine, Ki-67, nestin, and doublecortin immunostaining in the ischemic brain, and decreased angiogenesis, as demonstrated by reduced endothelial cell proliferation, vessel perimeter, and vascular density in the ischemic border, were evident in eNOS-/- mice compared with wild-type mice. eNOS-deficient mice also exhibited a reduced response to vascular endothelial growth factor (VEGF)-induced angiogenesis in a corneal assay. ELISAs showed that eNOS-/- mice have decreased brain-derived neurotrophic factor (BDNF) expression but not VEGF and basic fibroblast growth factor in the ischemic brain compared with wild-type mice. In addition, cultured SVZ neurosphere formation, proliferation, telomerase activity, and neurite outgrowth but not cell viability from eNOS-/- mice were significantly reduced compared with wild-type mice. BDNF treatment of SVZ cells derived from eNOS-/- mice restored the decreased neurosphere formation, proliferation, neurite outgrowth, and telomerase activity in cultured eNOS(-/-) SVZ neurospheres. SVZ explant cell migration also was significantly decreased in eNOS-/- mice compared with wild-type mice. These data indicate that eNOS is not only a downstream mediator for VEGF and angiogenesis but also regulates BDNF expression in the ischemic brain and influences progenitor cell proliferation, neuronal migration, and neurite outgrowth and affects functional recovery after stroke.

Figure 1.

Quantification fields of view in the ischemic border in mice.

Figure 2.

eNOS-deficient attenuated functional recovery after stroke: wild-type (n = 11) and eNOS knock-out (n = 14) mice were subjected to permanent MCAo. Functional tests were performed before MCAo and 1, 3, and 7 d after MCAo. A, mNSS test. B, Foot-fault test. ^*p < 0.05 versus wild-type MCAo control.

Figure 3.

eNOS-deficient mice exhibit reduced angiogenesis after stroke. Angiogenesis was indicated by BrdU-immunoreactive endothelial cells, vascular density, and perimeter in the ischemic border at 7 d after stroke. A, B, BrdU immunoreactivity in the ischemic border in wild-type (WT) control MCAo mice (A) and in eNOS^-/- MCAo mice (B). D, E, vWF immunoreactivity vessels in the ischemic boundary area in wild-type mice (D) and eNOS^-/- mice (E).C, F, G, Quantitative data that eNOS-deficient mice significantly (p < 0.05) decrease the number of BrdU-immunoreactive cells in vessels (C), vascular perimeter (F), and density (G) in the ipsilateral hemisphere compared with wild-type control MCAo. ^*p < 0.05 compared with wild-type MCAo control. H-J, TERT immunostaining in the ischemic border in wild-type mice (H) and eNOS^-/- mice (I). J, Quantitative data that eNOS-deficient mice significantly (p < 0.05) decrease the number of TERT-immunoreactive cells in vessels in the ipsilateral hemisphere compared with wild-type control MCAo. K, Corneal angiogenesis assay in control wild-type mice. L, M, Corneal angiogenesis assay treated with VEGF in wild-type mice (L) and eNOS^-/- mice (M), respectively. N, Quantitative data that eNOS-deficient mice significantly (p < 0.05) reduce VEGF-induced angiogenesis in the cornea compared with wild-type mice treated with VEGF. Scale bars: B, E, 100 μm; I, 50 μm.

Figure 4.

eNOS-deficient mice exhibit decreased neurogenesis after stroke. BrdU, Ki-67, DCX, and nestin are expressed in the ipsilateral SVZ at 7 d after MCAo. A, D, G, M, Ki-67 (A), BrdU (D), DCX (G), and nestin (M) expression in wild-type MCAo mice, respectively. B, E, H, N, Ki-67 (B), BrdU (E), DCX (H), and nestin (N) expression in eNOS^-/- mice, respectively. C, F, I, O, Quantitative data of Ki-67-positive (C), BrdU-positive (F), DCX-positive (I), and nestin-positive (O) cell number or percentage of area in the ischemic brain. J-L, DCX (K) with BrdU (J) double immunoreactivity (red arrow, DCX-positive cell; green arrow, BrdU-positive cell). Scale bars: B, H, N, 50 μm; L, 20 μm.

Figure 5.

SVZ neurosphere formation, proliferation, and survival and neurite outgrowth measurements. A-C, SVZ neurosphere formation in wild-type mouse (A), eNOS^-/- mouse (B), and eNOS^-/- mouse SVZ neurosphere cells treated with BDNF (C). D, E, Quantitative data of primary (D) and secondary (E) SVZ neurosphere formation. F, Quantitative data of a BrdU proliferation ELISA. G, Results of livedead viability measurement. H-J, TUJ1-positive cell neurite outgrowth in cultured SVZ neurosphere cells of wild-type mice (H), eNOS^-/- mice (I), and eNOS^-/- mice treated with BDNF (J). K, quantitative data of SVZ neuronal cell neurite outgrowth. Scale bar, 100 μm.

Figure 6.

SVZ tissue explant cell migration and neurite outgrowth. A, B, Wild-type mouse SVZ explant migration at 3 DIV (A) and 5 DIV (B). C, D, eNOS^-/- mouse SVZ explant migration at 3 DIV (C) and 5 DIV (D). E, F, Quantitative data of SVZ tissue explant migration distance (E) and migration area (F). Scale bar, 500 μm.