Strong Impact of Chronic Cerebral Hypoperfusion on Neurovascular Unit,Cerebrovascular Remodeling,and Neurovascular Trophic Coupling in Alzheimer’s Disease Model Mouse

Abstract

Although chronic cerebral hypoperfusion (CCH) may affect Alzheimer’s disease (AD) pathogenesis, the mechanism remains elusive. In the present study, we investigated the role of CCH on an AD mouse model in neurovascular unit, cerebrovascular remodeling, and neurovascular trophic coupling. Moreover, examined protective effect of galantamine. Alzheimer’s disease transgenic mice (APP23) were subjected to bilateral common carotid arteries stenosis with ameroid constrictors for slowly progressive cerebral hypoperfusion. CCH exacerbated neuronal loss and decrease of α7 subunit of nicotinic acetylcholine receptors (α7-nAChRs) expression in hippocampus and thalamus at 12 months. Meanwhile, CCH greatly induced advanced glycation end products expression, and blood-brain barrier leakage through observing IgG and MMP9 expressions. Furthermore, a significant number of dramatic enlarged cerebral vessels with remodeling, BDNF/TrkB decreased in neurovascular trophic coupling. The present study demonstrated that CCH strongly enhanced primary AD pathology including neurodegeneration, neurovascular unit disruption, cerebrovascular remodeling and neurovascular trophic coupling damage in AD mice, and that galantamine treatment greatly ameliorated such neuropathologic abnormalities.

Keywords

Alzheimer’s disease APP23 mice cerebrovascular remodeling chronic cerebral hypoperfusion galantamine neurovascular unit neurovascular trophic coupling

INTRODUCTION

Alzheimer’s disease (AD) is the most common cause of dementia, which becomes more prevalent with increasing age. In our recent study, AD occupies 69% among all dementia in elderly population more than 75 years old [1]. AD is associated with cholinergic neuronal loss and characterized by deficits in memory and cognition [2, 3].

Although AD results primarily from neurodegenerative changes, an increasing age enhances the pathogenic contribution of cerebrovascular parameters [4]. Between 60 and 90% of AD patients exhibit cerebrovascular pathologies including cerebral amyloid angiopathy, blood-brain barrier (BBB) disruption, and microvascular degeneration [5, 6]. The alterations in cerebrovascular function in AD can be reflected by chronic brain hypoperfusion and altered neurovascular trophic coupling [7].

Two-hit vascular hypothesis of AD speculates that the neurovascular unit (NVU) dysfunction and chronic cerebral hypoperfusion (CCH) potentiate AD pathogenesis, which lead to amyloid-β (Aβ) deposition and aggregation into the brain, and consequently tau protein hyperphosphorylation [8, 9]. The important role of CCH in dementia has already immerged to the front edge of neurology research.

Advanced glycation end products (AGEs) have been implicated in the chronic complications of diabetes mellitus and have been reported to play an important role in the pathogenesis of AD [10]. A previous study detected AGEs, which were identified immunohistochemically, in senile plaques and neurofibrillary tangles from patients withAD [11].

Galantamine is a centrally acting acetylcholinesterase inhibitor, which actually acts as an allosterically potentiating ligand for nicotinic acetylcholine receptors (nAChRs) [12] and exhibits neuroprotective effects in vitro [13] and in vivo [14]. In our previous and others’ clinical studies, galantamine has strong protective effect on cognitive function in patients with either AD or vascular dementia complicated by cerebrovascular pathology [15 –17].

In the present study, therefore, we aimed to investigate the effects of CCH on neurodegeneration, NVU dysfunction, cerebrovascular remodeling, and neurovascular trophic coupling damage in AD mice, moreover, to examine protective effect of galantamine in this AD mice with CCH model.

MATERIALS AND METHODS

Animals

APP23 mice overexpress human APP with the Swedish mutation (KM670/671NL) driven by a Thy1 promoter [18], which can be considered a valid model for AD as they mimic several pathological hallmarks [19], and both cognitive and behavioral alterations typical for AD patients [20, 21].

Male wild type (WT) (C57BL/6J) and the above APP23 mice with an average age of 30±1 days (d) (mean±SD) were used at the start of treatment. Animals were accommodated in standard mouse cages under conventional laboratory conditions with a 12/12 h (h) light–dark cycle and constant room temperature at around 23°C and humidity for 3 months (M). Food and water were present ad libitum. All animal experiments were performed in compliance with a protocol approved by the Animal Committee of the Graduate School of Medicine and Dentistry, Okayama University (OKU#2012325). This is a part of whole project mainly focusing on NVU, cerebrovascular remodeling and neurovascular trophic coupling in this mice model.

AD mice with CCH model

In vitro study, ameriod constrictors (ACs) with an inner diameter (D) of 0.75 mm and an open gap (G) of 0.5 mm (26 mg weight, Research Instruments NW, Lebanon, OR, USA) were immersed in 0.9% sodium chloride (NaCl) or in 0.9% NaCl + 6 g/dL albumin (Alb) both at 37°C. The pictures of the ACs were captured at 3 h, 1, 7, and 28 d to mimic in vivo study (Fig. 1A). A preliminary in vivo study was simultaneously performed, and the constrictor images of in vivo at 7 and 28 d were also recorded (Fig. 1A).

For in vivo study, four groups were designed in this study: WT mice (WT + sham surgery, n = 10), APP23 group (APP23 + sham surgery, n = 17), CCH group (APP23+CCH, n = 12), and galantamine-treated group (APP23+CCH+Gal, n = 10). APP23 mice develop both meningeal and parenchymal amyloid deposits after 6 M of age [18, 22]. For giving a surgery of CCH, neck incision was made and the ACs were applied to bilateral common carotid arteries (BCCAs) at age 4 M in the APP23+CCH and APP23+CCH+Gal groups (Fig. 1B).

Galantamine treatment

On the 15 d after the ACs surgery, APP23+CCH+Gal group mice began to receive galantamine (5 mg/kg; 1 mg/ml in ultrapure water, 0.15 ml; Takeda Pharmaceutical Company Limited, Osaka, Japan) once daily until sacrifice by oral gavages (Fig. 1C).

Tissue preparation

At ages 6 and 12 M, 4 groups mice were deeply anesthetized by intraperitoneal injection of pentobarbital (40 mg/kg) and transcardially perfused with 20 ml of ice-cold phosphate-buffered saline (PBS), and then 20 ml of ice-cold 4% paraformaldehyde (PFA) in 0.1 mol/L phosphate buffer. The brains were removed and post-fixed in 4% PFA overnight. After postfixation overnight, 50-μm-thick sections were cut with a vibrating blade microtome (VT1000S; Leica, Heidelberg, Germany).

Histochemistry and immunohistochemistry

We focused on the morphological and pathological changes of cells and blood vessels in the cerebral cortex (CTX), hippocampus (HI), and thalamus (TH) in this study. For Nissl staining, brain sections were immersed in 0.1% cresyl violet for 5 min at room temperature, and which were dehydrated in graded alcohol, coverslipped with microcoverglass. For single immunohistochemical analysis, brain sections were immersed in 0.6% periodic acid to block intrinsic peroxidase, and were treated with 5% bovine serum in 50 mM PBS, pH 7.4, containing 0.1% triton to block any non-specific antibody responses, the following primary antibodies were used: Rat anti-α7 nicotinic acetylcholine receptor (α7-nAChR) antibody (1:200; Abcam); mouse anti-N(omega)-(carboxymethyl) lysine (CML) antibody (1:200; COSMO BIO); mouse anti-N(omega)-(Carboxymethyl) arginine (CMA) antibody (1:100; COSMO BIO) and goat anti-MMP9 (1:1000; R&D Systems). To estimate the expressions of α7-nAChRs, CML, CMA, or MMP9, the sections were incubated with the respective first antibody, and then incubated with an appropriate biotin-labeled secondary antibody (1:500). To estimate the mouse IgG, sections were incubated with biotin-labeled goat anti-mouse IgG antibody (1:500), after incubation with an ABC Elite complex (Vector Laboratories, Burlingame, CA, USA), then incubated with DAB. The treated sections were analyzed with a light microscope (Olympus BX-51; Olympus Optical, Tokyo, Japan).

Immunofluorescent analysis

To determine the damage of NVU, Aβ peptide deposition and changes of neurovascular trophic coupling, double immunofluorescence studies were performed for collagen IV plus glial fibrillary acidic protein (GFAP), Aβ₄₀ plus GFAP, brain-derived neurotrophic factor (BDNF) plus tropomyosin receptor kinase B (TrkB), and BDNF plus N-acetylglucosamine oligomers (NAGO). Lycopersicon esculentum lectin (LEL) is a glycoprotein with affinity for NAGO, which mature vascular endothelial cells express [23]. We used following primary antibodies: Rabbit anti-collagen IV antibody (1:200; Abcam); goat anti-GFAP antibody (1:200; R&D Systems); anti-Aβ40 antibody (BA27; Wako); sheep anti-BDNF antibody (1:100; Abcam); rabbit anti-TrkB (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA); and biotinylated LEL (1:200;Vector). Then immunoreactions were visualized using fluorescent secondary antibody or horseradish peroxidase-conjugated antibody with diaminobenzidine reaction. The treated sections were scanned with a confocal microscope equipped with an argon and HeNe1 laser (LSM-510; Zeiss, Jena, Germany).

Quantitative analysis

For each measurement, we analyzed three separated sections per brains and four randomly selected regions per section (i.e., n = 9–12 measurements per mouse). For the semiquantitative evaluation of Nissl, CML, CMA, MMP9, and IgG staining intensity, CTX, HI, and TH were measured with an image processing software (Scion Image, Scion Corporation, Frederick, MD, USA). For the quantification of α7-nAChRs-positive and BDNF/TrkB double-positive cells, we counted the number of positive cells in three fields (cells/mm²) including CTX,HI, and TH.

To analyze collagen IV/GFAP remodeling in NVU, thickness and inner diameter of cerebrovascular were evaluated in HI and TH, and the intensity of collagen IV or GFAP was measured simultaneously.

Statistical analysis

All data were expressed as mean±SD. Statistical analyses were performed using 1-factor analysis of variance followed by a Tukey–Kramer postcomparison. Differences with a probability value of p < 0.05 were considered statistically significant.

RESULTS

ACs shrink in vitro and in vivo

The gap (G) of AC progressively shrank at 3 h, and disappeared at 1 d in vitro (Fig. 1A), the internal diameter (D) significantly shrank at 7 d to 60.0±2.7% (n = 3) and 28 d to 48.0±5.3% (n = 3), These were similar to that in vivo at 7 d to 61.3±8.0% (n = 3) and 28 d to 50.7±6.7% (n = 3) (Fig. 1A), with which ACs led to a slowly progressive stenosis of BCCAs (Fig. 1B).

Neuropathologic change in AD with CCH model

Nissl staining was examined in the CTX, HI, and TH (Fig. 2A). Analysis of pixel intensity indicated no difference among four groups in the indicated areas at 6 M (Fig. 2B, left). However, compared with WT group, Nissl staining intensity of APP23, APP23+CCH, and APP23+CCH+Gal groups significantly decreased in the CA1, polymorph layer, dentate gyrus (PoDG), and TH at 12 M (Fig. 2B, right). The strongest decrease of Nissl staining intensity was partially recovered by galantamine treatment at 12 M (Fig. 2B, ^*p < 0.05 versus WT, ^**p < 0.01 versus WT; ^# #p < 0.01 versus APP23; §p < 0.05 versus APP23+CCH, §§p < 0.01 versus APP23+CCH).

Nicotinic acetylcholine receptors immunohistochemistry

nAChRs, particularly the α7 subunit, are highly expressed in brain regions relevant to cognitive and memory functions. The three APP23 groups showed a remarkable decrease of α7-nAChRs-positive cells in the CA1, PoDG, and TH at 12 M compared with WT group (Fig. 3A). Such decrease was strongest in APP23+CCH group, which galantamine treatment greatly recovered (Fig. 3B, ^*p < 0.05 versus WT, ^**p < 0.01 versus WT; ^# #p < 0.01 versus APP23; §p < 0.05 versus APP23+CCH).

Advanced glycation end products change

CML is reported as a major antigenic AGEs structure, and as a major product of oxidative degradation of glycated proteins and unsaturated fatty acids, representing an integrative biomarker for oxidative stress. CML is localized in the cytoplasm of neurons, astrocytes, and microglia in both aged and AD brains. CMA is a CML analogue, and a major AGE in collagen. Expression of CML and CMA were examined in the CTX and TH (Fig. 4A). Analysis of pixel intensity indicated the three APP23 groups showed a remarkable increase of CML than WT group at 6 M, but no difference among the three APP23 groups. However, at 12 M, APP23+CCH strongly introduced CML expression, which was greatly recovered by galantamine treatment (Fig. 4B). The expression of CMA showed no difference among four mice groups at 6 M, but APP23+CCH strongly introduced CMA expression in vascular, perivascular, and senile plaques, which was greatly recovered by galantamine treatment at 12 M (Fig. 4C, ^*p < 0.05 versus WT, ^**p < 0.01 versus WT; ^#p < 0.05 versus APP23, ^# #p < 0.01 versus APP23; §p < 0.05 versus APP23+CCH, §§p < 0.01 versus APP23+CCH).

NVU dysfunction in AD mice with CCH

Mouse IgG was detected in Aβ plaques, within neurons, glial cells, and blood vessel wall of the CTX and TH (Fig. 5A). Analysis of pixel intensity indicated the three APP23 groups showed a remarkable increase of IgG than WT group at 6 M, but no difference among the three APP23 groups. However, at 12 M, APP23+CCH greatly increased IgG staining, which was greatly recovered by galantamine treatment (Fig. 5B). The expression of MMP9 was observed within neurons, glial cells, and blood vessel wall of the CTX and TH (Fig. 5C). Compared with WT group, MMP9 expression significantly increased in the three APP23 groups at 6 and 12 M, with the strongest increase in APP23+CCH at 12 M. Galantamine treatment greatly recovered such a strong increase of MMP9 (Fig. 5D, ^*p < 0.05 versus WT, ^**p < 0.01 versus WT; ^#p < 0.05 versus APP23, ^# #p < 0.01 versus APP23; §p < 0.05 versus APP23+CCH, §§p < 0.01 versus APP23+CCH).

NVU remodeling in AD mice with CCH

To observe NVU remodeling, collagen IV/GFAP double immunofluorescent analysis was performed in HI and TH at 6 and 12 M (Fig. 6A, B). NVU remodeling with morphological changes of collagen IV and astrocyte were found in APP23+CCH group at 12 M especially in TH (Fig. 6B). Meanwhile, Aβ accumulations were found in AD with CCH mice brain in the form of parenchymal amyloid plaques and small cerebrovascular deposits, and many GFAP-positive cells were localized around Aβ (Fig. 6C). Significant increases of inner diameter of parenchymal vessels, thickness of collagen IV, and intensity of collagen IV and GFAP were found in APP23+CCH group at 12 M (Fig. 6D–G, ^**p < 0.01 versus WT; ^# #p < 0.01 versus APP23; §§p < 0.01 versus APP23+CCH).

Neurovascular trophic coupling change in AD mice with CCH

BDNF plays a trophic link between cerebral endothelium and neuronal survival. TrkB is BDNF’s specific cognate receptor, a member of the tyrosine kinase family implicated in neuronal development and plasticity. BDNF/TrkB and NAGO/BDNF double staining was performed for evaluating such neurovascular trophic coupling change (Fig. 7A, B). BDNF/TrkB double-positive cells significantly decreased in the CA1, PoDG, and TH at 12 M in the three APP23 groups (Fig. 7A), with the strongest decrease in APP23+CCH. Galantamine treatment greatly recovered such a decrease. NAGO/BDNF double staining showed an increase of inner diameter of parenchymal vessels, morphological change of vascular endothelial cells in APP23+CCH group in the TH, and a decreased BDNG expression in endothelia of neurovascular coupling (Fig. 7B). Quantitative analysis of BDNF/TrkB double-positive cells showed there were no differences among the four mice groups at 6 M, and showed a remarkable decrease in the CA1, PoDG, and TH at 12 M in the three APP23 groups. Such decrease was the strongest in APP23+CCH group, which was greatly recovered by galantamine treatment (Fig. 7C, ^*p < 0.05 versus WT, ^**p < 0.01 versus WT; ^# #p < 0.01 versus APP23; §p < 0.05 versus APP23+CCH).

DISCUSSION

CCH has been used as an animal model for the study of vascular dementia and aged brain with white matter changes [24, 25]. In the present study, we first took an AD mouse plus CCH model to examine the pathological changes of neural cells, NVU, cerebrovascular remodeling, and neurovascular trophic coupling. The present CCH model showed a slowly progressive BCCAs stenosis with ACs (Fig. 1). BCCAs were significantly shrank at 28 d in vivo to 50.7±6.7% after the CCH surgery (Fig. 1), and we found exacerbations of motor and cognitive dysfunctions of AD with additive CCH in this mice model. This mice model is thus ideal to investigate the contribution of CCH to cognitive deficits and AD pathology.

Nissl-stained neuronal densities indicated that APP23+CCH group showed the strongest neuronal loss in the CA1, PoDG, and TH at 12 M than other groups (Fig. 2). The possible reasons are that CCH may promote neurodegeneration through neuronal energy failure, production of reactive oxygen species, and proinflammatory cytokines by activated microglial cells that, in turn, damage the neuronal cells [26 –31].

α7-nAChRs play important roles in regulating neuronal excitability [32] and are linked to Aβ deposition and AD pathogenesis [33]. Galantamine shows multiple actions on various neurons through allosteric potentiating ligand action by nAChR [34, 35]. The present study first showed a remarkable decrease of the main subunit of nAChR (α7) expression in the AD mice with CCH at 12 M (Fig. 3), and galantamine treatment greatly recovered the decrease, which could be related to the long-term benefits of galantamine in this model.

A possible link between AD and diabetes mellitus includes AGEs, advancing age, as well as oxidative stress and hypercholesterolemia [36, 37], with oxidative and inflammatory actions of AGEs [38]. In the present study, the biomarkers of AGEs, such as CML and CMA were first examined, and APP23+CCH greatly induced CML (neuronal) and CMA (vascular) expressions at 12 M, and a progressive deposit of CMA in Aβ plaques (Fig. 4).

BBB leakage and altered transport due to hypoperfusion/hypoxia are key cerebrovascular dysfunction pathways associated with AD pathobiology [39], and AD affects multiple cell types within the NVU, including brain vascular cells (endothelial cells, vascular smooth muscle cells and pericytes), glial cells (astrocytes and microglia), and neurons [40]. In the present study, IgG and MMP9 expressions were significantly increased in the three APP23 groups especially in the APP23+CCH group (Fig. 5), suggesting that CCH induced NVU damage in AD mice. Our present data was in accordance with previous reports of Aβ vascular deposition along the perivascular space by cerebral hypoperfusion [41, 42]. Previous studies reported that dysfunction of NVU caused CCH; on the other hand, the present study showed that CCH caused NVU damage in AD mice. Our study further explained the bidirectional relationship between CCH and NVUdamage.

Our present results also newly suggested a NVU remodeling in this combined APP23+CCH model (Fig. 6). In case of simple cerebral amyloid angiopathy (CAA), Aβ depositions were primarily present around the tunica media, then leading to thickening of tunica media and an accumulation of collagen fibers in the media [43], and finally resulting in muscle cell degeneration and dilation of the lumen with Aβ deposits outside the vessels [44]. In our study, a significant number of vessels with dramatic enlargement were found especially in the thalamus, with morphological changes of the collagen IV and astrocyte in APP23+CCH group at 12 M (Fig. 6B). It was probably due to a severer hypoperfusion in the thalamus than cerebral cortex in this mouse model. Furthermore, we newly found that Aβ accumulates in the form of parenchymal amyloid plaques and cerebral vascular deposits, and many GFAP-positive cells were localized around Aβ (Fig. 6C), indicating that the present CCH dramatically accelerated the essential pathology of CAA pathology in AD. Different pathological mechanisms lead to different manifestations of cerebrovascular remodeling. Chronic hypertension induces cerebrovascular hypertrophic remodeling, the media thickens to encroach into the lumen, resulting in increased media cross-sectional area and media/lumen ratio [45]. Chronic hypoperfusion induces cerebrovascular remodeling with a reduced diameter comprised of vascular smooth muscle cell [46]. CAA induces Aβ around the tunica media, muscle cell degeneration, and dilation of the lumen with Aβ deposits outside the vessels [22]. Compared with CAA, CCH+CAA induces vessels with dramatic enlargement, muscle cell losing with enhanced Aβ depositions, morphological changes of the collagen IV, and astrocytes localized around leaked Aβ.

In AD, amyloid may trigger oxidative stress in brain endothelium via RAGE receptors, and perturbations in amyloid transport mechanisms further add to a positive feedback loop that damages both neuronal and vascular compartments [47]. BDNF is one of the many molecules that link the endothelium and the neuroblasts. In the present study, BDNF/TrkB and NAGO/BDNF double staining suggested that endothelium may lose the ability to provide trophic support to neurons, CCH could induce neurovascular trophic coupling dysfunction in this model (Fig. 7). The possible reasons may be as follows (Fig. 8): 1) CCH potentiates Aβ accumulations through reducing cerebral blood flow in AD brain, 2) Aβ triggers oxidative stress in brain endothelium that damages both neuronal and vascular compartments [47], 3) Overexpression of AGEs activated MMP9 that may degrade TrkB integrity in neurons, disrupting neurovascular trophic coupling and leaving the brain vulnerable to injury [48].

In summary, the present study demonstrated that CCH strongly enhanced primary AD pathology including neurodegeneration, NVU remodeling, and neurovascular trophic coupling damage in AD mice, and that such neuropathologic abnormalities were greatly ameliorated by galantamine treatment.

Footnotes

ACKNOWLEDGMENTS

This work was partly supported by Grant-in-Aid for Scientific Research (B) 25293202, (C) 15K09316 and Challenging Research 15K15527 and Young Research 15K21181, and by Grants-in-Aid from the Research Committees (Mizusawa H, Nakashima K, Nishizawa M, Sasaki H, and Aoki M) from the Ministry of Health, Labour and Welfare of Japan.

Authors’ disclosures available online ().

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