Endothelial Nitric Oxide Synthase-Deficient Mice: A Model of Spontaneous Cerebral Small-Vessel Disease

Abstract

Age-related cerebral small-vessel disease (CSVD) is a major cause of stroke and dementia. Despite a widespread acceptance of small-vessel arteriopathy, lacunar infarction, diffuse white matter injury, and cognitive impairment as four cardinal features of CSVD, a unifying pathologic mechanism of CSVD remains elusive. Herein, we introduce partial endothelial nitric oxide synthase (eNOS)-deficient mice as a model of age-dependent, spontaneous CSVD. These mice developed cerebral hypoperfusion and blood-brain barrier leakage at a young age, which progressively worsened with advanced age. Their brains exhibited elevated oxidative stress, astrogliosis, cerebral amyloid angiopathy, microbleeds, microinfarction, and white matter pathology. Partial eNOS-deficient mice developed gait disturbances at middle age, and hippocampus-dependent memory deficits at older ages. These mice also showed enhanced expression of bone morphogenetic protein 4 (BMP4) in brain pericytes before myelin loss and white matter pathology. Because BMP4 signaling not only promotes astrogliogenesis but also blocks oligodendrocyte differentiation, we posit that paracrine actions of BMP4, localized within the neurovascular unit, promote white matter disorganization and neurodegeneration. These observations point to BMP4 signaling pathway in the aging brain vasculature as a potential therapeutic target. Finally, because studies in partial eNOS-deficient mice corroborated recent clinical evidence that blood-brain barrier disruption is a primary cause of white matter pathology, the mechanism of impaired nitric oxide signaling-mediated CSVD warrants further investigation.

Figure 1

Evidence of cerebral hypoperfusion, microinfarction, and amyloid pathology in endothelial nitric oxide synthase (eNOS) model. A and B: Representative section of 18-month–old eNOS^+/− mice doubly immunostained with either anti– β-amyloid (Aβ; 4G8; A) and glucose transporter-1 (GLUT1; B) plus DAPI or 4G8 and CD31 antibodies. C: Quantification of Aβ-positive arterioles in hippocampal fissure. D and E: Representative images of fluorescein isothiocyanate–dextran (2000 kDa) angiography performed in eNOS^+/− mice at young (6 months old; D) and old (18 months old; bilateral section; E) ages, as described previously.White double arrows indicate bilateral nonperfused lesions located in parietal association cortex (asterisks), retrosplenial granular cortex (daggers), and temporal association cortex (arrowheads). F: Quantification of the number of cerebral nonperfusion lesions per mouse. G–I: Representative images of hematoxylin and eosin staining (6 μm thick coronal sections). Blue arrow line indicates magnified fresh infarct lesion, and blue arrows indicate old lesions reminiscent of infarct scars. Notes: Figure is modified from the published figures used in Tan et al, used with permission from Springer Nature and BMC. Data represent means ± SEM (F). n = 5 to 6 mice for each genotype (C); n = 4 to 6 mice for each genotype (F). ∗P < 0.05, ∗∗P < 0.01. Scale bars: 300 μm (A); 50 μm (B); 100 μm (D and H, right panel); 500 μm (E, G, H, left panel, and I).

Figure 2

Evidence of age-dependent blood-brain barrier leakage and thrombosis in endothelial nitric oxide synthase (eNOS) model. A: Evidence of thrombosis. Representative images of fibrinogen/fibrin (red) and/or CD31 (green) immunohistochemistry in the brain sections of 18-month–old eNOS^+/− mice. Boxed areas: White arrows indicate individual fibrinogen clots inside the arterioles. B: Western blot of eNOS forebrain lysate probed with anti-fibrinogen antibody. Quantification was done on the basis of three mice per genotype, run three times. P value obtained by unpaired t-test. C: Representative images of anti-mouse IgG immunohistochemistry with DAPI counterstaining show parietotemporal cortical vascular extravasations of mouse serum IgG (green) in old eNOS^+/− mice. Mice were perfused transcardially, vibratome sectioned (60 μm thick), followed by immunostaining with Alexa 488–goat anti-mouse IgG antibody. White arrows in the right panel indicate leaky vascular areas. D: Quantification of extravasated mouse IgG based on anti-mouse IgG immunoblot analysis from mice after perfusion. Note less expression of β-actin in hippocampus (Hipp) than in cortex (Cort). Values are normalized to β-actin of 20 μg total proteins. E: Representative image of FITC–dextran angiography taken from 18-month–old eNOS^+/− mice. Angiography taken 60 minutes after injection of FITC-dextran (150 kDa), showing diffusive fluorescent signal as an indication of leaky vessels surrounding the occluded area located in parietal association cortex. F–I: Representative images of Evans blue angiography taken in young eNOS^+/− mice taken at 1.85 to 2.0 mm bregma. Evans blue was injected in 200-μL volume through mouse tail vein. Mice were euthanized 5 minutes later without perfusion, and whole brains were removed and vibratome processed to 100-μm serial sections for fluorescent imaging using rhodamine filter. Notes: Figures in A and C through E are modified from the published figures used in Tan et al, used with permission from Springer Nature and BMC. Data are expressed as means ± SEM (D). n = 4 animals for each genotype (D). ∗P < 0.05. Scale bars: 500 μm (A, left panel); 50 μm (A, right panels); 100 μm (C, left panel); 20 μm (C, right panel); 200 μm (E); 300 μm (F–I).

Figure 3

Myelin loss in endothelial nitric oxide synthase (eNOS) model at 12 months of age. A–F: Representative images of Luxol fast blue (LFB) and Nissl double-stained sections (coronal; 16 μm thick) of brains of eNOS^+/− and control littermates in whole brain (A and D) and frontal cortical regions (B, C, E, and F). B: Red double arrows and the corresponding Roman numerals indicate cortical layers I through V. G and H: Double-staining immunohistochemistry on myelin-binding protein (MBP; red) and NFP-200 (green). I: Quantification of MBP immunofluorescence signals in the cortical region based on three pairs of eNOS^+/− and control mice. J and K: Representative images of MBP immunohistochemistry. The dashed white lines (upper left) delineate the cortical surface. L: Quantification of MBP immunofluorescence signals in the corpus callosum region based on three pairs of eNOS^+/− and control mice. ∗P < 0.05, ∗∗P < 0.01. Scale bars: 1 mm (A and D); 150 μm (B and E); 40 μm (C and F); 50 μm (G and H); 500 μm (J and K).

Figure 4

Mechanistic hypothesis and supporting evidence. A–C: Up-regulated bone morphogenetic protein 4 (BMP4) distribution on blood-brain barrier (BBB) predominantly on descending arterioles in frontal-parietal cortex of endothelial nitric oxide synthase (eNOS)–deficient mice at young age. A: Microscopic image of BMP4-CFP reporter mice on eNOS^+/+ and eNOS^+/− background (5 to 6 months of age) using wide DAPI filter. B and C: Quantification graphs are based on the BMP4-CFP–positive cells (B) and on the fluorescent intensity of the positive cells (C). D: Approximate locations of the occluded lesions and cerebral amyloid angiopathy (CAA; white clouds) and BBB leakage (yellow staggers) in young eNOS^+/− brain; fluorescent image of hemispheric cerebrovasculature shown was taken from young mouse brain with Pdgfrb-positive cells labeled in tdTomato; PDGFRβ-tdTomato mice were derived from crossing between Pdgfrb-Cre with Ai14 (JAX stock number 007914). Pdgfrb: Platelet-derived growth factor receptor beta. E: Schematic diagram depicts the multiple pathologic events occurring along the life span of eNOS^+/− mice. P0: postnatal day 0. We hypothesize that the up-regulated BMP4 is an initial critical pathologic factor derived from BBB, leading to BBB leakage/pericyte degeneration, elevated generation of reactive oxygen species (ROS), astrogliosis, and oligodendrocyte lineage changes that underpin the white matter pathology; these pathologic sequelae are followed by subcortical and ultimately hippocampal neurodegeneration in aging eNOS^+/− mice. n = 3 each for genotype (A). ∗∗P < 0.01, ∗∗∗P < 0.001. Scale bar = 100 μm (A). Amyg, amygdala; Hippo, hippocampus.