Contribution of reactive oxygen species to cerebral amyloid angiopathy, vasomotor dysfunction, and microhemorrhage in aged Tg2576 mice

Abstract

Cerebral amyloid angiopathy (CAA) is characterized by deposition of amyloid β peptide (Aβ) within walls of cerebral arteries and is an important cause of intracerebral hemorrhage, ischemic stroke, and cognitive dysfunction in elderly patients with and without Alzheimer's Disease (AD). NADPH oxidase-derived oxidative stress plays a key role in soluble Aβ-induced vessel dysfunction, but the mechanisms by which insoluble Aβ in the form of CAA causes cerebrovascular (CV) dysfunction are not clear. Here, we demonstrate evidence that reactive oxygen species (ROS) and, in particular, NADPH oxidase-derived ROS are a key mediator of CAA-induced CV deficits. First, the NADPH oxidase inhibitor, apocynin, and the nonspecific ROS scavenger, tempol, are shown to reduce oxidative stress and improve CV reactivity in aged Tg2576 mice. Second, the observed improvement in CV function is attributed both to a reduction in CAA formation and a decrease in CAA-induced vasomotor impairment. Third, anti-ROS therapy attenuates CAA-related microhemorrhage. A potential mechanism by which ROS contribute to CAA pathogenesis is also identified because apocynin substantially reduces expression levels of ApoE-a factor known to promote CAA formation. In total, these data indicate that ROS are a key contributor to CAA formation, CAA-induced vessel dysfunction, and CAA-related microhemorrhage. Thus, ROS and, in particular, NADPH oxidase-derived ROS are a promising therapeutic target for patients with CAA and AD.

Keywords: Alzheimer's disease; NADPH oxidase; cerebral amyloid angiopathy; reactive oxygen species; vasomotor dysfunction.

Fig. 1.

Apocynin and tempol attenuate oxidative stress and restores cerebrovascular dysfunction. (A) RNA extracted from leptomeningeal arteries of 12-mo-old Tg2576 and littermate WT mice was subjected to qPCR to compare transcript levels for Nox isoforms and Sod2, and calculated as relative expression to Gapdh gene. *P < 0.05 vs. WT mice. (B–F) Twelve-month-old Tg2576 and littermate WT mice were treated with apocynin (Apo), tempol (Tem), or vehicle (Veh) for 10–12 wk (n = 5–6). (B). Coronally sectioned brain tissues were subjected to immunolabeling with the oxidative stress marker anti–3-nitrotyrosine antibody. Elevated levels of 3-nitrotyrosine immunoreactivity were noted in neurons and cerebral vessel walls in the vehicle-treated Tg2576 mice. This immunoreactivity was attenuated in both apocynin- and tempol-treated Tg2576 mice. (C) Cortical tissue samples (30 µg per lane) were subjected to immunoblotting with a marker for oxidative stress, SOD2. Data indicate mean ± SEM, *P < 0.05 vs. vehicle-treated Tg2576 mice. (D–F) Live pial vessel responses to VSMC-dependent vasodilators SNAP (D) and constrictor PGF2α (E), and EC-dependent vasodilator acetylcholine (F) were assessed via closed cranial window and video microscopy. The percent change in vessel diameter was determined. Data indicate mean ± SEM; *P < 0.05 vs. WT:Veh group; ^#P < 0.05 vs. Tg2576:Veh group.

Fig. 2.

Apocynin preferentially reduces CAA loads, but not parenchymal plaque loads in aged Tg2576 mice. Twelve-month-old Tg2576 and littermate WT mice were treated with apocynin (Apo), tempol (Tem), or vehicle (Veh) for 10–12 wk (n = 5–6). (A) Live imaging of amyloid deposition with methoxy-X04 staining demonstrated that amyloid deposition both in the pial vessels (arrowheads) and in the parenchymal tissues (arrows) were noted in vehicle-treated Tg2576 mice. There was a marked decrease in CAA load but not in plaque load in apocynin-treated mice. (B) Histological assessment in brain sections further confirmed that CAA loads (arrowheads in B) were significantly reduced by apocynin treatment, whereas parenchymal plaque loads (arrows in B) were not affected. (C and D) CAA was quantified by measuring percent coverage of resorufin-positive vessels in the cortex (C) whereas neuritic plaque load was calculated by subtracting CAA load from the total X04–positive amyloid load in the cortex (D). Data indicate mean ± SEM, *P < 0.05 by ANOVA.

Fig. 3.

Apocynin and tempol restore VSMC-dependent cerebrovascular dysfunction in CAA-affected vessels. Twelve-month-old Tg2576 and littermate WT mice were treated with apocynin, tempol or vehicle for 10–12 wk. Live pial vessel responses to VSMC-dependent vasodilator (SNAP) was assessed via closed cranial window and video microscopy. (A) Representative images of pial arteriolar responses to SNAP and acetylcholine in Tg2576 mice treated with vehicle or apocynin. In the vehicle-treated Tg2576 mice, vasodilatory responses were abolished in CAA-affected vessel segments (arrowheads) compared with CAA-free vessel segments (arrows). In the apocynin-treated Tg2576 mice, however, vasodilatory responses were apparent in both CAA-affected and CAA-free vessel segments. (B and D) Relationship between CAA coverage vs. vasodilatory responses to SNAP (B), and acetylcholine (D). Percentage of CAA coverage within 25-µm longitudinal vessels (eight consecutive segments per brain) was assessed as described in the Methods. Data indicate mean ± SEM; *P < 0.05 vs. vessel segments with <20% CAA load; ^#P < 0.05 vs. vehicle-treated group having corresponding % CAA coverage. (C and E) Vascular reactivity to SNAP (C) and acetylcholine (E) was also compared between vessel segments having CAA [CAA (+)] vs. those without CAA [CAA (−)]. Data indicate mean ± SEM; *P < 0.05 vs. vessel segments without CAA in vehicle-treated group; ^#P < 0.05 vs. vessel segments with CAA in vehicle-treated group.

Fig. 4.

Apocynin and tempol do not affect CAA-induced VSMC loss. Twelve-month-old Tg2576 and littermate WT mice were treated with apocynin (Apo), tempol (Tem), or vehicle (Veh) for 10–12 wk (n = 5–6). Amyloid deposition and VSMCs in leptomeningeal vessels were stained with methoxy-X04 and phalloidin-Alexa 488, respectively, and imaged with two-photon microscopy. (A) Number of VSMCs per 100-µm longitudinal vessel segment was counted. (B) Correlation between CAA severity and VSMC loss was plotted. Data indicate mean ± SEM; *P < 0.05 by ANOVA.

Fig. 5.

Apocynin attenuates CAA-associated microhemorrhage in aged Tg2576 mice. Twelve-month-old Tg2576 and littermate WT mice were treated with apocynin (Apo), tempol (Tem), or vehicle (Veh) for 10–12 wk (n = 5–6). (A) Brain sections were subjected to Prussian blue staining to detect microhemorrhage (blue in color) and counterstaining with Nuclear Fast Red. Representative images from brain sections of WT mice (a) and aged Tg2576 mice (b) are seen. (B) Number of microhemorrhagic profiles per section was assessed. *P < 0.05 vs. WT:Veh group; ^#P < 0.05 vs. Tg2576:Veh group.

Fig. 6.

Effects of apocynin on levels of Aβ and ApoE in young mice. Six-month-old Tg2576 and littermate WT mice were treated with apocynin (Apo) or vehicle (Veh) for 12 wk (n = 4–6). Levels of Aβ₄₀ and Aβ₄₂ in the cortex (A) and CSF (B), ratio of Aβ₄₀/Aβ₄₂ (C), and levels of ApoE in the cortex (D) were determined by ELISAs. Data indicate mean ± SEM; *P < 0.05 by ANOVA.

Fig. 7.

Apocynin attenuates activation of astrocytes and microglia in aged Tg2576 mice. Twelve-month-old Tg2576 and littermate WT mice were treated with apocynin (Apo), tempol (Tem), or vehicle (Veh) for 10–12 wk. Activation of both astrocytes and microglia were noted in Tg2576 mice as determined by immunolabeling with cell type-specific markers for activated astrocytes (GFAP) and microglia (CD45) (A). Both GFAP- and CD45-positive immunoreactivity was significantly reduced in apocynin-treated Tg2576 mice (n = 5–6 per group) (B and C). *P < 0.05 as determined by ANOVA.