β-Amyloid Load Is Not Influenced by the Severity of Cardiovascular Disease in Aged and Demented Patients
Background and Purpose—This study was conducted to analyze the association between reported risk factors for Alzheimer’s disease, apolipoprotein E ε4 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, β-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 ε4 allele than in those without this allele.
Conclusions—Our results demonstrate that the apolipoprotein E ε4 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.
The most common type of dementia, Alzheimer’s disease (AD), is characterized by β-amyloid (β-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: ε2, ε3, and ε4. The ε4 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.
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⇓).
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.
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 ×100 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.
The β-A4 aggregates and PHF-τ expression in the gray matter were visualized with the use of immunohistochemical methodology. The sections were deparaffinized and rehydrated according to routine procedure. For β-A4 staining, the sections were pretreated with 80% formic acid at room temperature for 6 hours. For immunohistochemical staining, monoclonal antibody to human β-A4, at a dilution of 1:100 (DAKO, M872), monoclonal antibody to human PHF-τ 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 β-A4 expression was performed under light microscopy at ×40 magnification, with the use of the NIH Image system for PC. β-A4 expression was estimated in temporal and parietal cortices within the total thickness of gray matter on 3 randomly selected fields, with the β-A4 load being reported as stained area fraction (Table 4⇑). β-A4 expression in vessel walls, ie, cerebral amyloid angiopathy (CAA) and PHF-τ expression, was quantified under light microscopy at ×40 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-τ 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.
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 ε4 allele.
SP/NP and NFT scores, β-A4 and PHF-τ 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 β-A4, CAA, and PHF-τ load. The degree of these lesions was higher in patients with the ApoE ε4 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-τ 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-τ showed significant negative correlation with CVI. A slight dose-dependent influence of ApoE ε4 allele on the correlation between CVI and β-A4 load and PHF-τ was noted (Figure 3⇓).
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, β-A4, and PHF-τ 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 ε4 allele, its influence was notable and significant. Both nondemented and demented individuals with the ApoE ε4 allele had significantly more lesions than those without this detrimental allele. The influence of the ApoE ε4 allele was most significant on neuronal degeneration estimated by NFT counts or PHF-τ load. Its influence on the β-A4 load was noted only as an increased extent of CAA in demented patients with the ApoE ε4 allele compared with those without this allele and as an increase in β-A4 load in the parenchyma of nondemented individuals with the ApoE ε4 allele.
In contrast to the influence of the ApoE ε4 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-τ).
We conclude that aggregation of β-A4 in the brain tissue, development of PHF-τ, and the formation of SP/NP and NFT are not directly dependent on the patient’s cardiovascular status, whereas the ApoE ε4 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.
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.
- Copyright © 1999 by American Heart Association
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