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(Stroke. 1999;30:613-618.)
© 1999 American Heart Association, Inc.
Original Contributions |
ß-Amyloid Load Is Not Influenced by the Severity of Cardiovascular Disease in Aged and Demented Patients
From the Department of Neuroscience and Neurology (A.I., S.H.) and A.I. Virtanen Institute (R.P.), Kuopio University; and Department of Pathology (A.I.), Division of Diagnostic Services, Chromosome and DNA Laboratory (H.S., M.A.), and Department of Neurology (S.H.), Kuopio University Hospital, Kuopio, Finland.
Correspondence to Irina Alafuzoff, MD, PhD, Departments of Neuroscience and Neurology and Pathology, Kuopio University, P-O-B 1627, Fin 70 211 Kuopio, Finland. E-mail irina.alafuzoff{at}uku.fi
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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Background and Purpose—This study was conducted to analyze the association between reported risk factors for Alzheimer's disease, apolipoprotein E
4 allele, and
cardiovascular disease and neuropathological changes
essential for the diagnosis of Alzheimer's disease. Methods—Our data are based on clinical and postmortem evaluations of a cohort of nondemented (n=118) and demented (n=107) individuals. A cardiovascular index was calculated at autopsy to estimate the extent of cardiovascular disease. Neuropathological lesions such as senile/neuritic plaques, neurofibrillary tangles, ß-amyloid load, cerebral amyloid angiopathy, and the load of paired helical filaments were determined.
Results—The aforementioned neuropathological lesions did
not show any positive significant correlation with
cardiovascular index. In contrast, the extent of
Alzheimer's lesions was significantly higher in those
nondemented and demented patients carrying the apolipoprotein E 4
allele than in those without this allele.
Conclusions—Our results demonstrate that the apolipoprotein
E 4 allele, but not cardiovascular disease,
indeed influences the extent of Alzheimer's lesions seen in
the brain tissue of demented patients as well as
asymptomatic controls.
Key Words: aged • Alzheimer's disease • apolipoproteins • risk factors
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The most common type of dementia, Alzheimer's disease (AD), is characterized by ß-amyloid (ß-A4) aggregates that are seen as senile/neuritic plaques (SP/NP)1 2 3 4 and by paired helical filament (PHF) constituents of neurofibrillary tangles (NFT).2 4
One known risk factor for AD is the genotype of
apolipoprotein E (ApoE), a protein regulating lipid
metabolism in the central nervous
system.5 6 7 There are 3 different alleles for
ApoE: 2, 3, and 4. The 4 allele is
significantly more common in demented patients than in aged
controls.5 8
In recent years, AD has been reported to be associated with vascular risk factors including hypertension, coronary heart disease, and atrial fibrillation.9 10 An epidemiological study11 revealed an association between AD and atherosclerosis. Furthermore, an interaction between atherosclerosis and ApoE genotype has been suggested to be of importance in the etiology of AD.11
The aforementioned findings prompted us to study whether the severity of cardiovascular disease displays any association with the histopathological changes seen in AD, ie, whether cardiovascular disease has an association with the final diagnosis of AD confirmed at autopsy.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Postmortem samples (Kuopio Brain Bank) obtained since 1991 were evaluated. All nondemented cases (n=118) sampled and those sampled demented cases fulfilling criteria of the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) for possible, probable, or definite AD (n=107)4 were included. The patients had been clinically assessed by neurologists, and the diagnosis of AD was based on the criteria of the National Institute of Neurological Disorders and Stroke–Alzhemier's Disease and Related Disorders Association12 and those of the Diagnostic and Statistical Manual of Mental Disorders, Revised Third Edition (1987) (Table 1
).
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The ApoE genotype was analyzed by
means of polymerase chain reaction, as described
earlier,13 14 with the genomic DNA being extracted
from blood or brain tissue samples (Table 1
).
At necropsy, performed in Kuopio University Hospital, a
cardiovascular index (CVI) was calculated. This CVI was
a score ranging from 0 to 15, based on a semiquantitative estimation of
grossly notable cardiovascular pathology at autopsy
(Tables 2 and 3). The heart weight and
atherosclerosis of the coronary arteries,
aorta, and circle of Willis were graded on a 4-step scale from 0 to 3.
Additionally, cardiovascular thrombus/embolus and
lesions consistent with old or acute myocardial infarct were
noted. A score of 15 was consistent with a patient with major
pathology of the heart muscle and severe generalized cardiac and
cranial atherosclerosis, whereas a low score for the
CVI would represent a patient with minor changes in the heart
muscle and vessel walls.
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The brains were weighed, evaluated for grossly detectable lesions
and vessel abnormalities, fixed in 10% buffered formalin for at least
1 week, and cut in coronal slices. Brain specimens taken from 6 defined
cortical regions (frontal [Brodmann 9], temporal [Brodmann 22]),
parietal [Brodmann 39], precentral, occipital cortices, and gyrus
cinguli); 4 subcortical gray matter regions (striatum, basal forebrain
including amygdala, thalamus, and hippocampus); and 5 infratentorial
regions (midbrain including substantia nigra, pons including locus
ceruleus, medulla, vermis, and cerebellar cortex) were embedded in
paraffin. Seven-micrometer-thick sections were stained with
hematoxylin-eosin and modified Bielschowsky silver impregnation. All
cases were classified into neuropathological diagnostic
groups as recommended by CERAD4 (Table 3).
NFT and SP/NP were also quantified as described previously by
Mölsä et al in 198715 (Table 4). The scoring of lesions (counts
of NFT and SP/NP) from 0 to 10 was performed under light microscopy
with a x100 magnification (area, 0.92 mm2)
on 5 randomly selected fields in each cortical region. The score was
the sum of scores in frontal, temporal, and parietal cortices.
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The ß-A4 aggregates and PHF-
expression in the gray matter
were visualized with the use of immunohistochemical methodology. The
sections were deparaffinized and rehydrated according to routine
procedure. For ß-A4 staining, the sections were pretreated with 80%
formic acid at room temperature for 6 hours. For immunohistochemical
staining, monoclonal antibody to human ß-A4, at a dilution of 1:100
(DAKO, M872), monoclonal antibody to human PHF- at a dilution of
1:100 (Innogenetics, BR-03), and the streptavidin–alkaline phosphatase
system (Histomark Kit, 71–00-39) were used. The reaction product,
the streptavidin-biotin complex, was visualized with Vector-Red (Vector
Labs, SK-5100).
The quantification of ß-A4 expression was performed under light
microscopy at x40 magnification, with the use of the NIH Image system
for PC. ß-A4 expression was estimated in temporal and parietal
cortices within the total thickness of gray matter on 3 randomly
selected fields, with the ß-A4 load being reported as stained area
fraction (Table 4). ß-A4 expression in vessel walls, ie,
cerebral amyloid angiopathy (CAA) and PHF- expression, was
quantified under light microscopy at x40 magnification and was scored
on a 4-step scale from 0 to 3 (none=0, some=1, moderate=2, or
extensive=3). In a case scored 1, occasional positively stained fibrils
were seen; in a case scored 2, several stained fibrils were noted with
additional threads; and in a case scored 3, numerous fibrils and
threads were noted. Accordingly, for CAA, in a case scored 1, few
occasionally affected vessels were found; in a case scored 2, 
25% of
the vessels seen in the field were affected; and in a case scored 3,
>50% of the vessels in the microscopic field were affected. The
extent of CAA was evaluated in the leptomeninges and
the parenchyma, and the amount of PHF- expression was
estimated in temporal and parietal cortices (Table 4).
The SPSS program for Windows was used for statistical analysis. The differences were analyzed by Student's t test. The correlation between individual variables was estimated with the use of Pearson's correlation tests.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Alzheimer's lesions were noted in 40% of the nondemented individuals, and in 18% the extent of these Alzheimer's lesions was sufficient for a histopathological diagnosis of possible AD (Table 3
).
The calculated CVI (Table 2) showed significant
correlation (r=0.3, P<0.05) with the premortem
estimated Hachinski score. Both the Hachinski score and the CVI were
significantly lower in demented than in nondemented individuals (Tables 1 and 3). Neither the Hachinski score nor the CVI was
significantly influenced by the ApoE 4 allele.
SP/NP and NFT scores, ß-A4 and PHF- loads, and the extent of
CAA were significantly higher in demented subjects than in controls
(Table 4).
In nondemented individuals, the ApoE genotype
significantly influenced the scores of SP/NP and NFT and the extent of
ß-A4, CAA, and PHF- load. The degree of these lesions was higher
in patients with the ApoE 4 allele than in those not carrying
this allele (Table 4, Figures 1 and 2). In the demented patients, we
observed similar significant influence of the ApoE
genotype on AD lesions but only for the scores of SP/NP and NFT
and the extent of CAA. The CVI did not have any significant influence
on AD lesions (Table 4, Figures 1 and 2) in
nondemented individuals. In demented patients, the extent of AD lesions
tended to decrease as the CVI increased. With respect to neuronal
pathology, this decrease was significant; PHF- load and NFT counts
were significantly lower in patients with higher CVI values. The
correlations between CVI and the extent of AD lesions without regard to
clinical symptoms or clinicopathological groups are listed in Table 5. All lesions with neuronal degeneration
and scores of SP/NP, NFT, and PHF- showed significant negative
correlation with CVI. A slight dose-dependent influence of ApoE 4
allele on the correlation between CVI and ß-A4 load and PHF-
was noted (Figure 3).
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Significant difference at least
P<0.05, Student's t test and Pearson's
correlation test. |
Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Recent reports have proposed that AD may be associated with vascular risk factors including hypertension, coronary heart disease, and atrial fibrillation. In the Rotterdam Study,11 the extent of atherosclerosis was assessed by ultrasonography of carotid arteries and by the ratio of ankle to brachial systolic blood pressure. According to these indicators, the participants were scored from 0 (no atherosclerosis) to 3 (severe atherosclerosis). Results indicated that atherosclerosis was associated with dementia; the odds ratio for AD in those with atherosclerosis was significantly higher than in those without atherosclerosis.
In the present study, the calculated CVI was based on autopsy
findings in which both presence of atherosclerosis and
the state of the myocardium were estimated. Although the
epidemiological study found a relationship between dementia and
atherosclerosis, this was not confirmed in our autopsy
study, which assessed the situation in the final stage of the disease.
According to our results, the essential neuropathological lesions, ie,
those fundamental for a definite diagnosis of AD, such as SP/NP, NFT,
ß-A4, and PHF- load, did not increase significantly with elevation
of the CVI estimated at autopsy. In 1996, Skoog et al16
published a longitudinal study revealing an association between
elevated blood pressure at age 70 years and the development of dementia
10 to 15 years later. They hypothesized that hypertension causes
hyalinization of the vessel walls in the brain, and subsequent episodes
of hypoperfusion may cause lesions in vulnerable areas, such as the
deep white matter. However, we could not detect an increase in
Alzheimer's lesions associated with increased severity in
extracranial signs of cardiovascular disease in the
final postmortem stage. Our results and the results by Skoog et
al16 indicate that the recently reported association
between dementia and atherosclerosis is not related to
AD but rather to a more complex and heterogeneous group of
dementias, namely, the dementias of vascular origin. When the AD
lesions were related to a well-known risk factor, such as the ApoE 4
allele, its influence was notable and significant. Both nondemented
and demented individuals with the ApoE 4 allele had
significantly more lesions than those without this detrimental
allele. The influence of the ApoE 4 allele was most
significant on neuronal degeneration estimated by NFT counts or PHF-
load. Its influence on the ß-A4 load was noted only as an increased
extent of CAA in demented patients with the ApoE 4 allele
compared with those without this allele and as an increase in
ß-A4 load in the parenchyma of nondemented individuals with the ApoE
4 allele.
In contrast to the influence of the ApoE 4
genotype on the extent of AD lesions, the increase in CVI was
associated with a decrease in the extent of AD lesions. A significant
negative correlation was noted between CVI and AD lesions (SP/NP, NFT,
and PHF-).
We conclude that aggregation of ß-A4 in the brain tissue,
development of PHF-, and the formation of SP/NP and NFT are not
directly dependent on the patient's cardiovascular
status, whereas the ApoE 4 allele is linked with the development
of Alzheimer's lesions. Recently, dementia has been linked to
atherosclerosis in epidemiological studies, but our
study could not find an increase in the extent of AD lesions parallel
with the increased severity of cardiovascular disease.
These findings emphasize the need to identify specific and reproducible
histopathological lesions detected in the brain tissue of demented
individuals resulting from cardiovascular
dysfunction.
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Acknowledgments |
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This study was supported by the Health Research Council of the Academy of Finland and EVO grants 5018 and 5105. We thank Hannu Tianen, Heikki Luukkonen, Tarja Kauppinen, and Tarja Tuunanen for their skillful technical help.
Received September 15, 1998; revision received December 10, 1998; accepted December 10, 1998.
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