The influence of chronic cerebral hypoperfusion on cognitive function and amyloid β metabolism in APP overexpressing mice.
ABSTRACT Cognitive impairment resulting from cerebrovascular insufficiency has been termed vascular cognitive impairment, and is generally accepted to be distinct from Alzheimer's disease resulting from a neurodegenerative process. However, it is clear that this simple dichotomy may need revision in light of the apparent occurrence of several shared features between Alzheimer's disease and vascular cognitive impairment. Nevertheless, it still remains largely unknown whether the burden of vascular- and Alzheimer-type neuropathology are independent or interdependent. Therefore, we investigated whether chronic cerebral hypoperfusion influences cognitive ability or amyloid β deposition in amyloid precursor protein (APP) overexpressing transgenic mice.
Two months old mice overexpressing a mutant form of the human APP bearing both the Swedish and Indiana mutations (APP(Sw/Ind)-Tg mice), or their wild-type littermates, were subjected to chronic cerebral hypoperfusion with bilateral common carotid artery stenosis (BCAS) using microcoils or sham operation. Barnes maze test performance and histopathological findings were analyzed at eight months old by 2 × 2 factorial experimental designs with four groups.
BCAS-operated APP(Sw/Ind)-Tg mice showed significantly impaired learning ability compared to the other three groups of mice. Two-way repeated measures analysis of variance showed a synergistic interaction between the APP genotype and BCAS operation in inducing learning impairment. The cognitive performances were significantly correlated with the neuronal densities. BCAS significantly reduced the density of Nissl-stained neurons and silver-stained cored plaques in the hippocampus of APP(Sw/Ind)-Tg mice but increased the amount of filter-trap amyloid β in the extracellular-enriched soluble brain fraction, compared to those from sham operated mice.
The results suggest interaction between chronic cerebral hypoperfusion and APP(Sw/Ind) overexpression in cognitive decline in mice through enhanced neuronal loss and altered amyloid β metabolism.
- [show abstract] [hide abstract]
ABSTRACT: Alzheimer's disease (AD) and vascular dementia (VaD) are the two most common forms of dementia. In Europe, 800,000 people have a diagnosis of VaD out of 3.7 million people with clinical dementia. These two dementia types share many common pathological, symptomatic and neurochemical features, and cholinergic treatments that have demonstrated robust, broad-ranging and long-term efficacy in AD are now being assessed for the treatment of dementia related to cerebrovascular disease (CVD). There has been recent recognition that dementia in the elderly is a continuum of pathologies, with pure AD and VaD representing the two extremes, and 'mixed' dementia (AD with CVD) in between and perhaps comprising the majority of cases. 'Mixed' dementia is rarely diagnosed in the clinic, however, as the majority of diagnostic procedures are biased toward a diagnosis of AD. Here, the risk factors, pathophysiological mechanisms and clinical symptoms of AD and VaD are described, identifying their overlap as well as some of the differences in both cognitive and noncognitive symptoms. Important findings indicating the high prevalence of 'mixed' dementia in the clinical dementia population are also discussed. In particular, evidence of a causal connection between stroke or CVD and AD is addressed. Regarding effective therapeutic management of dementia patients, further concerted epidemiological study of these related dementia types should aid in clinical decisions on the applicability of cholinergic treatments.Journal of the Neurological Sciences 12/2002; 203-204:29-34. · 2.24 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The main stumbling block in the clinical management and in the search for a cure of Alzheimer disease (AD) is that the cause of this disorder has remained uncertain until now. Evidence that sporadic (nongenetic) AD is primarily a vascular rather than a neurodegenerative disorder is reviewed. This conclusion is based on the following evidence: (1) epidemiological studies showing that practically all risk factors for AD reported thus far have a vascular component that reduces cerebral perfusion; (2) risk factor association between AD and vascular dementia (VaD); (3) improvement of cerebral perfusion obtained from most pharmacotherapy used to reduce the symptoms or progression of AD; (4) detection of regional cerebral hypoperfusion with the use of neuroimaging techniques to preclinically identify AD candidates; (5) presence of regional brain microvascular abnormalities before cognitive and neurodegenerative changes; (6) common overlap of clinical AD and VaD cognitive symptoms; (7) similarity of cerebrovascular lesions present in most AD and VaD patients; (8) presence of cerebral hypoperfusion preceding hypometabolism, cognitive decline, and neurodegeneration in AD; and (9) confirmation of the heterogeneous and multifactorial nature of AD, likely resulting from the diverse presence of vascular risk factors or indicators of vascular disease. Since the value of scientific evidence generally revolves around probability and chance, it is concluded that the data presented here pose a powerful argument in support of the proposal that AD should be classified as a vascular disorder. According to elementary statistics, the probability or chance that all these findings are due to an indirect pathological effect or to coincidental circumstances related to the disease process of AD seems highly unlikely. The collective data presented in this review strongly support the concept that sporadic AD is a vascular disorder. It is recommended that current clinical management of patients, treatment targets, research designs, and disease prevention efforts need to be critically reassessed and placed in perspective in light of these important findings.Stroke 05/2002; 33(4):1152-62. · 6.16 Impact Factor
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ABSTRACT: The integrity of the cerebral vasculature is crucial to the maintenance of cognitive functions during ageing. Prevailing evidence suggests that cerebrovascular functions decline during normal ageing, with pronounced effects in Alzheimer's disease (AD). The causes of these changes largely remain unknown. While previous studies recorded ageing-related impairments, such as atherosclerosis and loss of innervation in basal surface arteries of the brain, it only recently has been realized that a number of subtle alterations in both the intracranial resistance vessels and the smaller capillaries is apparent in both ageing animals and humans. The dominant changes include alterations in composition of connective tissues and smooth muscle of large vessel walls, thickening of the vascular basement membrane, thinning of the endothelium in some species, loss of endothelial mitochondria and increased pericytes. Some of these attributes appear more affected in AD. Other abnormalities entail profound irregularities in the course of microvessels, unexplained inclusions in the basement membrane and changes in unique proteins and membrane lipids associated with the blood-brain barrier. Brain imaging and permeability studies show no clear functional evidence to support the structural and biochemical anomalies, but it is plausible that focal and transient breach of the blood-brain barrier in ageing, and more notably in AD, occurs. Thus, circumscribed neuronal populations in certain brain regions could become vulnerable. Furthermore, the characteristic deposition of amyloid in vessels in AD may exacerbate the decline in vascular function and promote chronic hypoperfusion. Although not explicit from current studies, it is likely that the brain vasculature is continually modified by growth and repair mechanisms in attempts to maintain perfusion during ageing and disease.Pharmacology [?] Therapeutics 02/1996; 72(3):193-214. · 7.79 Impact Factor
The Influence of Chronic Cerebral Hypoperfusion on
Cognitive Function and Amyloid b Metabolism in APP
Mahito Yamada1, Masafumi Ihara1*, Yoko Okamoto1, Takakuni Maki1, Kazuo Washida1, Akihiro
Kitamura1, Yoshiki Hase1, Hidefumi Ito1, Keizo Takao2, Tsuyoshi Miyakawa3, Raj N. Kalaria4, Hidekazu
Tomimoto5, Ryosuke Takahashi1
1Department of Neurology, Graduate School of Medicine, Kyoto University, Kyoto, Japan, 2Section of Behavior Analysis, Center for Genetic Analysis of Behavior, National
Institute for Physiological Sciences, Okazaki, Aichi, Japan, 3Division of Systems Medical Science, Institute for Comprehensive Medical Science, Fujita Health University,
Toyoake, Aichi, Japan, 4Institute for Ageing and Health, WRC, Campus for Ageing & Vitality, Newcastle University, Newcastle-upon-Tyne, United Kingdom, 5Department
of Neurology, Graduate School of Medicine, Mie University, Tsu, Mie, Japan
Background and Purpose: Cognitive impairment resulting from cerebrovascular insufficiency has been termed vascular
cognitive impairment, and is generally accepted to be distinct from Alzheimer’s disease resulting from a neurodegenerative
process. However, it is clear that this simple dichotomy may need revision in light of the apparent occurrence of several
shared features between Alzheimer’s disease and vascular cognitive impairment. Nevertheless, it still remains largely
unknown whether the burden of vascular- and Alzheimer-type neuropathology are independent or interdependent.
Therefore, we investigated whether chronic cerebral hypoperfusion influences cognitive ability or amyloid b deposition in
amyloid precursor protein (APP) overexpressing transgenic mice.
Methods: Two months old mice overexpressing a mutant form of the human APP bearing both the Swedish and Indiana
mutations (APPSw/Ind-Tg mice), or their wild-type littermates, were subjected to chronic cerebral hypoperfusion with bilateral
common carotid artery stenosis (BCAS) using microcoils or sham operation. Barnes maze test performance and
histopathological findings were analyzed at eight months old by 262 factorial experimental designs with four groups.
Results: BCAS-operated APPSw/Ind-Tg mice showed significantly impaired learning ability compared to the other three
groups of mice. Two-way repeated measures analysis of variance showed a synergistic interaction between the APP
genotype and BCAS operation in inducing learning impairment. The cognitive performances were significantly correlated
with the neuronal densities. BCAS significantly reduced the density of Nissl-stained neurons and silver-stained cored plaques
in the hippocampus of APPSw/Ind-Tg mice but increased the amount of filter-trap amyloid b in the extracellular-enriched
soluble brain fraction, compared to those from sham operated mice.
Conclusions: The results suggest interaction between chronic cerebral hypoperfusion and APPSw/Indoverexpression in
cognitive decline in mice through enhanced neuronal loss and altered amyloid b metabolism.
Citation: Yamada M, Ihara M, Okamoto Y, Maki T, Washida K, et al. (2011) The Influence of Chronic Cerebral Hypoperfusion on Cognitive Function and Amyloid b
Metabolism in APP Overexpressing Mice. PLoS ONE 6(1): e16567. doi:10.1371/journal.pone.0016567
Editor: Mark Mattson, National Institute on Aging Intramural Research Program, United States of America
Received October 29, 2010; Accepted January 5, 2011; Published January 27, 2011
Copyright: ? 2011 Yamada et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Grants-in-Aid for Exploratory Research and Integrative Brain Research (IBR-shien) from the Japanese Ministry of Education,
Culture, Sports, Science and Technology (http://www.mext.go.jp), and a grant from the Mitsubishi Pharma Research Foundation (http://di.mt-pharma.co.jp/
zaidan/default.htm) and by Global COE Program "Center for Frontier Medicine" funded by the Japanese Ministry of Education, Culture, Sports, Science and
Technology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Insufficient blood supply to the brain has been shown to lead to
cognitive dysfunction . Cognitive impairment resulting from
such cerebrovascular insufficiency has been termed ‘vascular
cognitive impairment’ (VCI) , and is generally accepted to be
distinct from Alzheimer’s disease (AD) resulting from a neurode-
generative process. However, it is clear that this simple dichotomy
may need revision in light of the apparent occurrence of several
shared features between AD and VCI. For instance, the two
disorders increase in prevalence with age, frequently occur
concomitantly, and overlap considerably in their symptomatology,
pathophysiology, and comorbidity . Indeed, cerebral hypoper-
fusion as a result of vascular risk factors such as hypertension,
diabetes mellitus, hypercholesterolemia, and smoking is a common
vascular component among AD risk factors . Consistent with
this, the microvessels in AD neocortex are frequently narrowed,
degenerate [4,5], and amyloid-laiden , suggesting a pivotal role
of cerebrovascular factors in AD. Furthermore, cerebral hypoper-
fusion is known to potentiate other deleterious modifiers of AD
PLoS ONE | www.plosone.org1 January 2011 | Volume 6 | Issue 1 | e16567
such as oxidative stress, mitochondrial dysfunction, and neuroin-
flammation [7,8]. Thus, neurovascular changes may be key factors
in the upstream stage of pathological cascade of AD.
Accordingly, the Nun study has shown that the risk of dementia
increases by more than 20 times in AD if the patients exhibit
cerebral infarction . The Rush Memory and Aging Project
suggested that mixed brain pathologies, mainly comprising of AD
pathology and cerebral infarctions, account for most of dementia
cases in community-dwelling older persons . Consistent with
this, the MRC Cognitive Function and Ageing Study showed
attributable risk at death for dementia included small vessel disease
(12%), multiple vascular pathologies (9%), and cerebral amyloid
angiopathy (7%) in addition to neocortical neuritic plaques (8%)
and neurofibrillary tangles (11%) . In this respect, the
multifactorial aspects of cognitive impairment may be incorporat-
ed into the dynamic polygon hypothesis, which takes into account
the contribution of strokes of all sizes, as well as white matter
hyperintensities, in parallel to those of plaques and tangles .
Nevertheless, it still remains largely unknown whether the
burden of vascular- and AD-type neuropathology are independent
or interdependent. Elaboration on this point of contention is vital
in clarifying the wider question of whether vascular brain injury
has additive effects on AD pathogenesis. In this study, we therefore
examined whether chronic cerebral hypoperfusion influences
cognitive function and amyloid b (Ab) neuropathology in
APP overexpressing mice [8,13,14,15]. This may determine
whether the burden of vascular- and AD-type neuropathology is
interdependent in the development of dementia syndrome,
and may provide evidence linking chronic hypoperfusion with
Materials and Methods
Animals, treatments, and surgical procedures
We used human APP-Tg mice J20 overexpressing the familial
AD-linked mutation carrying a mutant form of the human APP
bearing the both Swedish (K670N/M671L) and the Indiana (V717F)
mutations (APPSwInd) , imported from the Jackson Laboratory
(USA). This J20 high expressing transgenic line is different from the
J9 low expressing line used in our earlier work [5,17]. Mice were
screened for transgene expression by PCR, and heterozygous mice
were mated with nontransgenic C57BL/6J mice. All male
heterozygous transgenic mice were given access to food and water
ad libitum. At 2 months of age, mice of heterozygosity and their non-
Tg littermates were subjected to either sham operation or bilateral
common carotid artery stenosis (BCAS) using microcoils (four
groups) [8,13,14,15,17]. Under halothane anesthesia (2%), the
common carotid arteries were exposed through a midline cervical
incision, and a microcoil with a diameter of 0.18 mm was appliedto
the bilateral common carotid artery (while maintaining the rectal
temperature between 36.5 and 37.5uC). Those in the control group
were sham-operated, which involved bilateral exposure of the
common carotid arteries only. Body weight, rectaltemperature, and
blood pressure of tail artery was monitored in BCAS- and sham-
operated mice. After the operation, the mice were housed in cages
with a 12-hour light/dark cycle (lights on at 7:00 AM) with access to
food and water ad libitum. At 6 months post-BCAS, the mice were
tested for altered behavior. This study was carried out in strict
accordance with the guidelinesfor animal experimentationfromthe
Animal Research Committee, Kyoto University. The protocol was
approved by the Animal Research Committee, Kyoto University
(Permit Number: MedKyo08526). The raw data of the behavioral
tests have been disclosed in the mouse behavioral phenotype
in 8-month-old male mice (6 months after operation), as previously
described [8,18,19]. Ear twitch, whisker touch, and righting
reflexes were also evaluated.
Barnes maze test for reference memory.
maze test was performed in the 8-month-old male mice (6 months
post-operation). The task was conducted on ‘dry land’, a white
circular surface, 1.0 m in diameter, with 12 holes equally spaced
around the perimeter (O’ Hara & Co., Tokyo, Japan) [20,21]. The
circular open field was elevated 75 cm from the floor. A black
Plexiglas escape box (1761367 cm) containing paper cage
bedding on its floor was located under one of the holes. The
hole above the escape box represented the target, analogous to the
hidden platform in the Morris task. The location of the target was
consistent for a given mouse but was randomized across mice. The
maze was rotated daily, with the spatial location of the target
unchanged with respect to the visual room cues, in order to
prevent bias based on olfactory or proximal cues within the maze.
The first training session was started when wild type mice and
BCAS-operated mice were 8-months-old (6 months after the
BCAS operation). One trial per day for 7 successive days and two
trials per day for next 6 successive days were conducted except for
no trial on day 6 and one trial on the last day. A probe trial was
conducted 24 hours after the last training session without the
escape box in order to confirm that this spatial task was performed
based on navigation using distal environment room cues. Time of
latency to reach the target hole, number of errors, distance to
reach the target hole, and time spent around each hole were
recorded by video tracking software (see ‘Image Analysis’). To
assess long-term retention, a second probe trial was applied a week
after probe test 1 and additional one session of retraining.
The applications used for the behavioral
studies were based on the National Institutes of Health’s Image
program (available at http://rsb.info.nih.gov/nih-image/) and
ImageJ program http://rsb.info.nih.gov/ij/, which were modified
for each test by Tsuyoshi Miyakawa (available from O’hara & Co.,
Neurological screening was conducted
were deeply anesthetized with sodium pentobarbital and were
perfused transcardially with 0.01 mol/L phosphate-buffered saline
(PBS) and then with a fixative containing 4% paraformaldehyde
(PFA) and 0.2% picric acid in 0.1 mol/L phosphate buffer (PB,
pH 7.4). The brains were post-fixed in 4% PFA in 0.1 mol/L PB,
andwerestoredin20%sucrose in0.1 mol/LPB(pH 7.4).Posterior
(hippocampal) portion of each brain was dissected in consultation
with a brain map . The coordinates from the bregma measured
from –1.8 to –2.2 mm for histochemical analyses. The posterior
portion was embedded in paraffin and sliced into 6 mm-thick
coronalsectionsand thensubjected tohematoxylinandeosin(H&E)
staining and modified Bielschowsky staining. For immunohis-
tochemistry, serial sections cut on a microtome were incubated
overnight at 4uC with anti-amyloid beta 42 (Ab42; Calbiochem;
diluted1:500)after retrieval by formicacid for 2 minutesand 0.05%
trypsin for 15 minutes. The sections were subsequently treated with
the appropriate biotinylated secondary antibodies (diluted 1:200;
Vector Laboratories, Burlingame, CA) and visualized with 0.01%
diaminobenzidine tetrahydrochloride and 0.005% H2O2 in
50 mmol/L Tris HCl (pH 7.6).
Measurement of neuronal density.
in the cerebral cortex and the hippocampus was counted blindly
by a second investigator (Y. Okamoto). Nissl-stained coronal
The neuronal density
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sections of the brain (–1.8 to –2.2 mm from the bregma) obtained
from the four groups (n=4–7) were used for counting the neurons
by setting regions of interest in the cerebral cortex (the watershed
area of anterior and middle cerebral artery) and the hippocampal
CA1 and CA3 regions.
Measurement of cored plaque density.
cored plaques was counted blindly by a second investigator
(Y. Okamoto) in the cortex and the whole hippocampus. The
density of cored plaques per unit area was calculated by dividing
the number of cored plaques by the number of whole pixels of
each captured image.
Measurement of Ab42-stained area.
were captured by using digital microscope system, BZ-9000
(Keyence, Osaka, Japan). A unified threshold value (155/255) was
used to binarize the images. Captured images were separated into
the cortex, the hippocampus and the other area by the Photoshop
7.0 (Adobe Systems Inc.). Image analysis was performed by ImageJ
1.40 g software (NIH). The number of Ab42-positive pixels was
divided by the entire pixels to yield %Ab42-stained area. The
person performing the analysis was blinded to the animal groups.
The density of
Protein extraction and filter-trap assay
The protein samples for the filter assay were extracted
according to the method proposed by Lesne et al , with some
modification. Briefly, hemi-forebrains were harvested in Tris-
buffered saline (pH 7.6) and a protease inhibitor cocktail (Sigma,
USA). Soluble, extracellular-enriched proteins were collected from
homogenized lysates following centrifugation for 40 min at
100,0006g. The protein concentration of the samples was
measured according to the Bradford method and equal amount
of protein was subjected to filter assay.
The extracellular-enriched proteins from the brains of four pairs
of sham-operated or BCAS-treated littermates were subjected to
vacuum filtration through a 96-well dot blot apparatus (Bio-Rad
Laboratories, USA) containing 20 nm pore-sized nitrocellulose
membranes. The resultant membranes were then incubated with
primary antibody (6E10; diluted 1:1000) at 4uC overnight. The
membranes were then blocked by TBS containing 4% skim milk,
and incubated with HRP-linked anti-mouse IgG secondary
antibody (Mouse TrueBlot ULTRA, eBioscience; diluted 1:1000)
for 1 h. The membranes were developed with the ECL plus
Western Blotting Analysis System (GE Healthcare). The membrane
was digitally captured with LAS-3000 (Fuji, Tokyo, Japan). The
LAS-3000 imaging system has a CCD camera which is equipped
with shading correction without automatic gain control, and has an
ample and linear dynamic range (from 0 to 4.0 OD values). Images
were processed using Multi Gauge v3.1 software (Fujifilm). The
intensity of each spot was determined by measuring the AUC-BG/
mm2(AUC: area under the curve, BG: background).
Statistical analysis was conducted using StatView (SAS
Institute). Data were analyzed by two-way repeated measures
analysis of variance (ANOVA) or one-tailed t-test, unless otherwise
noted. A one-tailed t-test was used for two-group comparison
based on the clear directional prediction. Values in the graphs are
expressed as mean6SEM. p,0.05 was considered statistically
accomplished within 15 minutes on each mouse. By day 10
post-BCAS, less than 10% of the mice had died. The APP
genotype did not affect the survival rate. There were no significant
differences among the four groups of mice in terms of their
physical characteristics such as body weight, temperature and
blood pressure. Mean body weight in non-Tg/sham, non-Tg/
BCAS, APPSw/Ind-Tg/sham, and APPSw/Ind-Tg/BCAS mice was
25.8 g, 26.5 g, 24.5 g, and 23.7 g at the operation (2-month-old),
respectively, with no significant differences among the four groups.
There were also no significant differences in sensory-motor
reflexes (percent with quick response of ear twitch, normal
response of whisker twitch and acoustic startle response) and
physical strength (grip strength and wire hang).
Reference memory test.
performed to investigate reference memory at 6 months post-
BCAS (8-month-old). There were significant differences between
the APPSw/Ind-Tg/BCAS mice and the other three groups of mice;
the APPSw/Ind-Tg/BCAS mice exhibited prolonged latency to
reach the desired target (Figure 1A), increased number of errors
(Figure 1B), and increased distance in reaching the desired target
(Figure 1C) compared to the other groups of mice. These results
suggest that the learning of the APPSw/Ind-Tg/BCAS mice was
significantly impaired. In two-way repeated measures ANOVA,
there was a synergistic interaction between genotype and BCAS.
The mutant APP gene and BCAS synergistically worsened
learning ability (Figure 1A–C).
In the probe trial conducted 24 hours after the last training
session, the time spent around the target hole by the APPSw/Ind-
Tg/BCAS mice was significantly shortened (p=0.0083) compared
to the non-Tg/BCAS mice (p=0.6881). There was no interaction
between the mutant APP genotype and BCAS operation
(p=0.540). To assess the long-term retention of spatial memory,
a second probe test was conducted 7 days after the last training
trial. Both the mutant APP genotype and BCAS operation
shortened the time spent around target hole (APP, p=0.0104;
BCAS, p=0.0202), but there was no interaction between
genotypes and surgery conditions (p=0.2617).
The Barnes maze test was
reduced in the APPSw/Ind-Tg/BCAS mice with increment in the
following order: non-Tg/sham . non-Tg/BCAS . APPSw/Ind-Tg/
sham . APPSw/Ind-Tg/BCAS mice in the cerebral cortex and
hippocampal CA1 and CA3 (Figure 2). There were significant
differences in neuronal density between non-Tg/sham and APPSw/
Ind-Tg/BCAS or APPSw/Ind-Tg/sham mice in the cerebral cortex,
between non-Tg/sham and APPSw/Ind-Tg/BCAS mice in the
hippocampal CA1 region, and between non-Tg/sham and
APPSw/Ind-Tg/sham mice in the hippocampal CA3 region. There
was no interaction in neuronal density between mutant APP
overexpression and BCAS in the three areas (cortex, p=0.969;
CA1, p=0.873; CA3, p=0.510). Mutant APP overexpression and
BCASseemed to have additive effects on the neuronal density inthe
cerebral cortex and CA1 region.
The neuronal densities in the cerebral cortex and hippocampal
CA1 and CA3 were significantly and inversely correlated with the
cognitive performances on the Barnes maze test, such as time of
latency to reach the target hole, number of errors and distance to
reach the target hole (trials 16–18) (Table 1).
Modified Bielschowsky pathology.
was no amyloid pathology in the brains of the BCAS or sham
operated mice. The analyses subsequently involved comparing
The density of Nissl-stained neurons was
In non-Tg mice, there
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APPSw/Ind-Tg/sham and APPSw/Ind-Tg/BCAS mice. Modified
Bielschowsky staining showed that the number of silver-stained
cored plaques was lower in the APPSw/Ind-Tg/BCAS mice
compared to the APPSw/Ind-Tg/sham mice (compare Figure 3A
and 3B, and Figure 3C and 3D). The difference was significant in
the hippocampus (p=0.018) but not in the cerebral cortex
(p=0.354) (Figure 3I). Diffuse plaques were numerous in the
APPSw/Ind-Tg/sham mice but fewer in the APPSw/Ind-Tg/BCAS
mice (Figure 3E and 3F). The marked difference in diffuse plaques
was clearly evident without further need for quantification. By
morphologically different between the two groups of mice
(Figure 3G and 3H).
The Ab1-42 immunostaining showed
similar findings to modified Bielschowsky staining. The number
of Ab-positive plaques was lower in the APPSw/Ind-Tg/BCAS mice
compared to the APPSw/Ind-Tg/sham mice (compare Figure 4A
and 4B, and Figure 4C and 4D). The %Ab42-stained area showed
a decrement in the APPSw/Ind-Tg/BCAS mice compared to the
APPSw/Ind-Tg/sham mice in both the cortex and hippocampus.
coredplaques(senile plaquecores)were not
Figure 1. Interaction between APP overexpression and chronic cerebral hypoperfusion. Barnes maze test showed that APPSw/Ind-Tg/BCAS
mice exhibited prolonged latency to reach the desired target (A), increased number of errors (B), and increased distance before reaching the desired
target (C) compared to the other groups.
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(Figure 4I). However, in contrast to modified Bielschowsky
staining, Ab immunostaining showed intense positivity for both
diffuse plaque (Figure 4E and 4F) and cored plaque (Figure 4G
and 4H). The Ab-positive diffuse plaques was less frequently
observed in the APPSw/Ind-Tg/BCAS mice compared to the
APPSw/Ind-Tg/sham mice, while cored plaques showed no
morphological differences between the two groups of mice
(Figure 4G and 4H).
differencereached significancein thehippocampus
The filter-trap assay showed that BCAS-treated mice had
significantly higher levels of filter-trap Ab (p=0.012) in the
extracellular-enriched, soluble brain fraction, as shown in the four
littermate pairs (Figure 5).
This behavioral, histological, and biochemical study demon-
strates that chronic cerebral hypoperfusion accelerates reference
memory impairment in conjunction with hippocampal neuronal
loss in the brain of APPSw/Indmice. Chronic cerebral hypoperfu-
sion (6 months) or APPSw/Indoverexpression impaired reference
memory in mice , but a novel finding presented here is that
chronic cerebral hypoperfusion and APPSw/Ind overexpression
interdependently disrupted reference memory (Figure 1). Although
a threshold for behavioral deficits may have been crossed when a
certain number of hippocampal neurons are lost (threshold effect),
the results suggest that burden of vascular- and AD-type lesions
interdependently contribute to the development of components of
the dementia syndrome, and strengthen the notion that vascular
risk factors, if present, should be thoroughly controlled in clinically
probable AD patients . The vascular-type lesions reproduced
in the BCAS model are oligemic e.g. non-infarctional indicating
that chronic hypoperfusion may accelerate AD neuropathology in
a latent manner over an extended period of time via enhanced
neuronal loss and altered Ab metabolism. Although we did not
focus on aging aspects in particular, this effect is likely to be more
pronounced in older animals .
Several studies have reported that chronic ischemia/hypoxia
mechanistically contribute to AD pathogenesis via alteration of Ab
metabolism. In Swedish mutant APP transgenic mice (APP23),
long-term hypoxia has been shown to markedly increase Ab
deposition and neuritic plaque formation and potentiate the
memory deficit by increasing b-site APP cleaving enzyme 1
(BACE1) gene transcription and expression, primarily mediated by
the binding of hypoxia-inducible factor-1a to the BACE1
promoter [25,26]. BACE1 activation and resultant Ab40 over-
production has also been reported in Tg2576 mice following
energy insufficiency by pharmacological agents (insulin, 2-
deoxyglucose, 3-nitropropionic acid, or kainic acid) . Such
findings collectively suggest that the energy/oxygen deficiency
facilitates AD pathogenesis by BACE1 elevation and Ab
In this study, the degree of Ab deposition and cored plaque
formation was suppressed following chronic cerebral hypoperfu-
sion and APPSw/Ind overexpression, although this interaction
resulted in the augmentation of reference memory impairment
and hippocampal neuronal loss. This group has previously
reported that chronic cerebral hypoperfusion increased the level
of aggregated Ab of no less than 200 nm in diameter in the soluble
extracellular-enriched brain fraction of a relatively low-expressor
line (J9) of APPSw/Indmouse . The current study, which used a
high-expressor line (J20) of APPSw/Indmouse, also demonstrated
that the amount of filter-trap Ab in the extracellular-enriched,
soluble fraction significantly increased following 6 months of
cerebral hypoperfusion. By contrast, insoluble Ab species that
cause senile plaques were reduced in this J20 line after cerebral
hypoperfusion but had not been analysed in the J9 line due to their
scarcity in the brain . Therefore, soluble, but not insoluble, Ab
species may play a direct role in neurotoxicity or neuronal loss and
resultant behavioral abnormalities in the hypoperfused APPSw/Ind
mice. Insoluble plaque cores are known to be largely inactive but
sequester synaptotoxic Ab dimers . Insoluble Ab deposition
may be disassembled by the inflammation caused by cerebral
hypoperfusion as ischemia is known to activate macrophages and
reduce senile plaques . Alternatively, chronic cerebral
hypoperfusion may interrupt Ab aggregation process, causing a
shift in Ab solubility. Thus, this study suggests that insoluble Ab1–
42 may not be the causative factor, but rather a resultant
epiphenomenon, in cognitive deterioration in the APPSw/Ind
mice. These findings are consistent with a recent report that found
Ab oligomer-induced pathology in the absence of amyloid plaques
in APP-Tg mice expressing the E693Delta mutation . They
also concur with the findings of several earlier studies that found
environmental enrichment consistently prevented cognitive de-
cline in AD model mice, but changed the amount of senile plaques
variably, according to the experimental conditions (the senile
plaques increased , decreased , or remained the same 
despite the consistent cognitive improvement).
Ischemia increases the vulnerability of neurons to Ab peptide by
impairing calcium regulation . Cellular calcium homeostasis is
also disrupted by AD-causing mutations in presenilin-1 and APP
in cultured neurons and transgenic mice . Therefore, the
Table 1. Significant inverse correlations of the cognitive performances with the neuronal densities.
Latency r=20.458 (p,0.0001)r=20.291 (p=0.0149) r=20.290 (p=0.0152)
Number of errors r=20.379 (p=0.0012)r=20.301 (p=0.0116) r=20.319 (p=0.0072)
Distancer=20.417 (p=0.0003)r=20.314 (p=0.0083) r=20.272 (p=0.0237)
Figure 2. Neuronal density in the cerebral cortex and the hippocampal CA1 and CA3 areas. (A) The schematic illustration of the regions
of interest depicted in the cerebral cortex (size, 0.0560.5 mm) and the hippocampal CA1 and CA3 areas (0.0560.25 mm each) of the coronal section
of the brain. (B) The density of Nissl-stained neurons was lowest in APPSw/Ind-Tg/BCAS mice.
Linkage between Ischemia and Neurodegeneration
PLoS ONE | www.plosone.org6 January 2011 | Volume 6 | Issue 1 | e16567
synergistic interaction between ischemia and neurodegeneration
may be mediated by calcium dysregulation. In accordance with
this hypothesis, presenilin-1 mutant knock-in mice exhibit greater
severity of brain injury and poorer behavioural outcome after focal
cerebral ischemia/reperfusion ; moreover, neuronal elevation
of intracellular calcium levels is enhanced after glucose deprivation
and chemical hypoxia in cortical cell cultures from the same
animals . Thus, disturbance in calcium homeostasis may
provide a mechanistic link between ischemia and neurodegener-
ation, and may underlie the observed synergistic interaction
between APP overexpression and hypoperfusion in cognitive
Figure 3. Chronic cerebral hypoperfusion affected the number
of silver-stained cored plaques in the APPSw/Ind-Tg mice.
Modified Bielschowsky staining showed cored plaques in the hippo-
campus (A, B, G, H) and the cerebral cortex (C, D), and diffuse plaques in
the hippocampus (E, F) of the APPSw/Ind-Tg/sham mice (n=6; A, C, E, G)
and APPSw/Ind-Tg/BCAS mice (n=4; B, D, F, H). Number of cored plaques
was counted in the cortex and the hippocampus (I). Scale bars: 400 mm
(A, B), 300 mm (C-F), 80 mm (G, H).
Figure 4. Chronic cerebral hypoperfusion reduced the Ab1-42
stained area in the APPSw/Ind-Tg mice. Immunohistochemistry for
Ab1-42 showed intense staining in the hippocampus (A, B, E, F) and in
the cerebral cortex (C, D) of the APPSw/Ind-Tg/sham mice (n=4; A, C, E,
G) and APPSw/Ind-Tg/BCAS mice (n=6; B, D, F, H). (I) The area positive for
Ab1-42 was analysed in the cortex and the hippocampus. Scale bars:
400 mm (A, B), 300 mm (C–F), 80 mm (G, H).
Linkage between Ischemia and Neurodegeneration
PLoS ONE | www.plosone.org7 January 2011 | Volume 6 | Issue 1 | e16567
The cognitive decline arising from human AD or VCI is known to
progress over a period of decades. A report using 3xTg-AD mice (,3
months of age) and APPSw/Indmice (16–17 months) indicated that
blood flow insufficiency should be prolonged enough to accelerate
AD pathology; here, the use of captopril (a commonly used anti-
hypertensive drug) and resultant modest inhibition (,30%) of brain
angiotensin-converting enzyme (Ab-degrading) activity for 28 days
did not affect Ab catabolism . This suggests that cerebral
autoregulatory capacity and redundant Ab-degrading pathways
counteract Ab overproduction triggered by blood flow insufficiency
at least for a short period of time. Consistent with this, the authors
have previously shown anatomical and metabolic abnormalities in
the hippocampus after 6 months of cerebral hypoperfusion . It is
therefore apparent that cerebral hypoperfusion over a prolonged
period oftime(probablyencompassing asignificantproportionoflife)
is necessary to replicate the condition found in humans.
A limitation of this study is the lack of information on the
temporal profile of cerebral blood flow (CBF) in our cohort of the
APPSw/Indmice. This group has previously monitored the CBF of
wild-type C57BL/6J mice immediately after the BCAS operation
up to 3 months post-BCAS, indicating that the CBF temporarily
decreased to 60 to 70% of the control value but gradually
recovered to a level of .80% at 1 to 3 months . Given the
cerebrovascular dysfunction in the J20 line of the APPSw/Indmice
, the CBF may be further jeopardized in the APPSw/Indmice
compared to C57BL/6J mice after BCAS operation. However,
CBF measurement procedures requiring anesthesia after the
behavioral study were avoided in order to minimize the influence
of anesthesia on the correlation analysis between cognitive
performance and histopathological findings. Therefore, future
study should investigate whether APP overexpression accelerates
CBF reduction in the BCAS brain.
Another limitation is that Ab was not measured in a quantitative
fashion and plaque load or neuronal cell counts were determined
withoutusing stereological approaches.However,two different histolo-
gical methods (modified Bielschowsky staining and Ab1-42 immuno-
staining) showed the similar trend of decreasing plaque load after
cerebral hypoperfusion in both the cortex and hippocampus, making a
chance finding less likely. Sampling of each mouse using stereological
methods will further delineate our findings in a future study.
accelerated reference memory impairment and hippocampal neuronal
loss together with reduced Ab deposition and cored plaque formation
but an increased amount of filter-trap Ab in the extracellular-enriched
soluble brain fraction in APPSw/Indmice. This suggests interaction
between chronic cerebral hypoperfusion and APP overexpression for
cognitive decline through altered Ab metabolism.
We would like to thank Dr. Khundakar for his editorial assistance and
comments. We are indebted to Ms. Nakabayashi and Ms. Asada-Utsugi for
their excellent technical assistance.
Conceived and designed the experiments: MY MI KT HT. Performed the
experiments: MY YO. Analyzed the data: MY MI YO TM KW AK YH
HI KT TM RNK HT RT. Contributed reagents/materials/analysis tools:
MY. Wrote the paper: MY MI.
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