Abstract
INTRODUCTION
Alzheimer’s disease (AD) is pathologically characterized by amyloid plaques and neurofibrillary tangles. Amyloid β-protein 1–42 (Aβ42) and, to a lesser extent, amyloid β-protein 1–40 (Aβ40), are the primary components of amyloid plaques [1]. Reduced levels of cerebrospinal fluid (CSF)-Aβ42 are observed in AD patients, reflecting mismetabolism of Aβ or deposition of Aβ42 proteins in amyloid plaques [2]. In contrast, levels of CSF-Aβ40 are normal in AD patients [2].
Cerebral amyloid angiopathy (CAA) is the deposition of Aβ in the cerebral arteries, commonly found in older persons and in AD patients [3, 4]. In CAA, Aβ40 affects vessel walls more than Aβ42 [5, 6]. The reduced CSF levels of both Aβ42 and Aβ40 have been reported in non-demented patients with CAA compared with controls and even compared with patients with AD [7, 8]. Brain microbleeds indicate the presence of CAA when observed in lobar regions of the brain and of hypertensive arteriopathy when observed in deep locations [9].
In patients with AD, lobar microbleeds were reported to be associated with lower concentrations of CSF-Aβ42 [10], on the other hand, the concentrations of CSF-Aβ40 have not been investigated [10]. To reveal associations of CAA-related lobar microbleeds with CSF-Aβ40 and other markers in AD, we have investigated the relationships between the presence and location of microbleeds and CSF levels of Aβ40, Aβ42, total tau protein (tau), and phosphorylated tau protein (ptau) in AD patients.
MATERIALS AND METHODS
Study population
From 1,168 consecutive patients who visited our academic memory clinic between April 2001 and September 2015, we recruited 485 consecutive patients diagnosed as having probable AD according to the National Institute of Neurological and Communicative Disorders Association criteria (NINCDS-ADRDA) [1]. We excluded patients who had no CSF analysis or T2*-weighted MRI imaging. We also excluded patients with a history of acute stroke within 6 months of the CSF collection date, because patients with acute stroke show a transient marked increase of CSF-tau levels, that then returned to normal levels 3–5 months after the stroke [11]. Finally, 88 AD patients were enrolled for analysis (51 males, 37 females; age at the time of CSF collection, 47–83 years; mean±SD is 68.0±8.3 years). Patients with microbleeds only in the cerebral cortex/subcortex or the cerebellar cortex were defined as the group of cortical microbleeds that were considered to be CAA-related microbleeds according to the Boston criteria of CAA-related hemorrhage [12]; patients with microbleeds only in the basal ganglia, thalamus, brainstem, and deep white matter were defined as the group of deep microbleeds; and patients with microbleeds in both cortical and deep microbleeds were defined as the group of mixed microbleeds.
Neuropsychological tests
Cognitive profiles were assessed using the Clinical Dementia Rating (CDR) [13], the Mini-Mental State Examination (MMSE) [14], and the Japanese translation of the Wechsler Memory Scale-Revised (WMS-R) [15]. MMSE was performed for all patients. WMS-R was not performed for 13 AD patients for personal reasons.
ApoE and CSF biomarkers
An apolipoprotein E (ApoE) phenotype was determined for each patient using isoelectric electrophoresis, as described by Kamboh et al. [16]. After CSF was collected via lumbar puncture, it was centrifuged and preserved at –80°C. Sandwich enzyme-linked immunosorbent assays (ELISA) were used to determine CSF-Aβ40 (Human Amyloid β (1–40) Assay Kit; IBL, Gunma, Japan), CSF-Aβ42 (Innotest β-amyloid (1–42); Fujirebio, Belgium), CSF-tau for total tau (Innotest hTAU-Ag; Fujirebio), and CSF-ptau for phosohorylated tau (Innotest Phospho-tau (181p); Fujirebio), according to the manufacturer’s instructions.
Imaging protocol and MRI measurement
MRI was performed using a 1.5-T system (Sygna Horizon; General Electric Medical Systems, Milwaukee, WI), which consisted of an axial T2*-weighted gradient echo sequence (19 slices; field of view, 200 mm; acquisition matrix, 256×192; slice thickness, 6 mm; interslice gap, 1.5 mm; echo time/repetition time, 20/800 ms; flip angle, 20) and an axial fluid-attenuated inversion recovery (FLAIR) sequence.
Microbleeds were defined as homogeneous and round lesions of less than 5 mm in diameter showing as hypointensity on T2*-weighted images. This definition is consistent with standards for measuring manifestations of cerebral small vessel disease [17]. Microbleeds were counted throughout the brain and were divided into the following categories: the cerebral cortex/subcortex (frontal, parietal, temporal, and occipital), cerebellar cortex, thalamus, basal ganglia, deep white matter, and brain stem. Leukoaraiosis severity was rated using the visual rating scale proposed by Fazekas et al. [18]. For periventricular hyperintensity (PVH), scores correspond to the following characteristics: 0 = no changes; 1 = a cap or a pencil-thin lining; 2 = smooth halo; and 3 = irregular changes extending into the deep white matter. For deep white matter hyperintensity (DWMH), scores correspond to the following characteristics: 0 = no lesion; 1 = punctate foci; 2 = beginning confluent foci; and 3 = confluent changes. The extent of leukoaraiosis was determined on the FLAIR image. All measurements were made by consensus of two independent experienced neurologists blinded to clinical information.
Standard protocol approvals and patient consents
This study was conducted with the approval of the medical ethics review board of Kanazawa University, Kanazawa, Japan (approval number 775). All participants provided written informed consent.
Statistical analyses
Differences in patient characteristics between the four groups of cortical, deep, mixed, and no microbleeds were assessed by Kruskal-Wallis test or χ2 test and residual analysis. Comparisons of CSF amyloid and tau markers, and Aβ ratio between the four groups were performed by Kruskal-Wallis test. Multivariate logistic regression models were used to analyze the independent effects of CSF amyloid and tau markers on the effects of difference between the four groups. The model was adjusted for age, sex, ApoE ɛ4 presence, and PVH and DWMH scores. The correlations between CSF amyloid and tau markers and number of cortical or deep microbleeds were analyzed by Spearman’s rank correlation test. Diagnostic accuracy for CSF markers was assessed by using receiver operating characteristic (ROC) analysis; the optimal cut-off was where the point of both sensitivity and specificity reached maximum vertical distance between the ROC curve and the diagonal line, which is known as Youden index [19]. PVH and DWMH scores among the patient groups were assessed using χ2 test and residual analyses.
Data were reported as mean±SD unless otherwise specified. A p value <0.05 was considered significant. In the residual analysis, an absolute value of standardized residual of more than 1.96 was consideredsignificant. The SPSS software package (version 23; SPSS Inc., Chicago, IL) was used to perform all statistical analyses.
RESULTS
Patient characteristics
Among the 88 patients with probable AD, 34 patients had microbleeds, classified into the groups of cortical (n = 15), deep (n = 16), and mixed microbleeds (n = 3); 54 had no microbleeds. Clinical characteristics of each group are shown in Table 1. There were no significant differences between the groups of cortical, deep, mixed, and no microbleeds for the gender ratio, age, education period, rate of other vascular risk factors, prevalence of ApoE ɛ4 carriers, MMSE scores, CDR scores, or WMS-R scores.
Comparison of CSF-Aβ40, CSF-Aβ42, CSF-tau, and CSF-ptau levels
Due to insufficient volume of the CSF samples, CSF-Aβ40 could not be measured in 15 patients, and CSF-tau could not be measured in 7 patients.
The concentrations of CSF-Aβ40, CSF-Aβ42, and CSF-ptau were significantly lower in the group of cortical microbleeds than in that of no microbleeds (p = 0.001, p = 0.027, p = 0.009, respectively) (Fig. 1, Table 2). The concentrations of CSF-tau tended to be lower in the group of cortical microbleeds than in that of no microbleeds (p = 0.056) (Fig. 1, Table 2).
However, the concentrations of CSF-Aβ40, CSF-Aβ42, CSF-tau, and CSF-ptau did not differ significantly between the groups of cortical, deep, and mixed microbleeds. After adjustment for age, sex, ApoE ɛ4 presence, and PVH and DWMH scores, the results remained unchanged. Spearman’s rank correlation tests showed that the CSF-Aβ40, CSF-Aβ42, CSF-tau, and CSF-ptau were significantly correlated with the number of cortical microbleeds (r = –0.450, p = 0.001; r = –0.287, p = 0.007; r = –0.298, p = 0.007; r = –0.341, p = 0.001, respectively) (Fig. 2). In addition, the CSF-Aβ40 levels were significantly correlated with number of deep microbleeds (r = –0.263, p = 0.024) (Fig. 3).
The ROC analyses were performed for CSF-Aβ40, CSF-Aβ42, CSF-tau, and CSF-ptau (Table 3). The CSF-Aβ40 showed the best separation of patients with cortical microbleeds from those without microbleeds.
Leukoaraiosis on MRI
PVH scores were significantly higher in the groups of cortical microbleeds and mixed microbleeds than in the group of no microbleeds (p = 0.029, Supplementary Table 1). DWMH scores were significantly higher in the groups of deep microbleeds and mixed microbleeds than in the group of no microbleeds (p = 0.007, Supplementary Table 1).
DISCUSSION
The major findings of this study were that: (i) CSF-Aβ40 and CSF-Aβ42 were significantly decreased in AD patients with cortical microbleeds than in those without microbleeds, (ii) AD patients with cortical microbleeds had lower levels of CSF-ptau compared to those without microbleeds; and (iii) CSF-Aβ40 levels well separate patients with cortical microbleeds from those without microbleeds in AD.
This is the first study to demonstrate that AD patients with cortical microbleeds have significantly lower levels of CSF-Aβ40 than those without microbleeds. There has been no study to investigate the association between the location of microbleeds and CSF-Aβ40 in AD patients as this study. Microbleeds are considered indicative of small-vessel blood leakage; lipofibrohyalinosis is the primary vascular change associated with microbleeds situated in deep brain regions, whereas CAA is associated with cortical microbleeds [9]. CSF-Aβ40 and CSF-Aβ42 concentrations were reported to be decreased in non-demented patients with CAA [7, 8], probably due to the deposition of both Aβ40 and Aβ42 proteins in the cerebral vasculature in these individuals. In the present study with AD patients, we revealed that CSF-Aβ40 and CSF-Aβ42 concentrations were decreased with an increase of cortical microbleeds, which would reflect CAA pathology, that is, deposition of both Aβ40 and Aβ42 in the cerebrovasculature. There was a considerable variation in the levels of CSF-Aβ40 and CSF-Aβ42 in patients without microbleeds. We speculate the possibility that the group of no microbleeds would have included patients with various severities of CAA in the absence of CAA-related microbleeds, showing the variously reduced levels of CSF-Aβ40 and CSF-Aβ42. In a previous study [20], the concentrations of CSF-Aβ40 did not differ between AD patients with and without microbleeds, which may be due to the fact that they did not divide the AD patients with microbleeds into subcategories according to their locations as we did in the present study. Regarding the patients with deep microbleeds, interestingly, CSF-Aβ40 concentrations were decreased with an increase the number of deep microbleeds. The mechanism for the reduction of CSF-Aβ40 in AD with deep microbleeds remains unknown. Decreased CSF-Aβ40 levels and increased plasma-Aβ40 levels were reported in patients with vascular dementia and deep microbleeds [20], implying possible some leakage across the blood brain barrier of these proteins related to blood brain barrier dysfunction.
Our results also revealed that AD patients with cortical microbleeds had lower levels of CSF-ptau compared to those without microbleeds; furthermore, AD patients with cortical microbleeds tended to have lower levels of CSF-tau compared to those without microbleeds. In a previous study, among ApoE ɛ4-non-carrying patients, AD patients with microbleeds had lower levels of both CSF-tau and CSF-ptau compared with those without microbleeds [21]; the authors hypothesized that, owing to a relatively higher degree of vascular damage, a relatively lower degree of tau tangle pathology is sufficient for a diagnosis of dementia. In contrast, no relationships were found between microbleeds and CSF-ptau or CSF-tau level in other studies [20, 23]. Furthermore, it was reported that CSF-tau and CSF-ptau levels were higher in the AD patients with multiple microbleeds compared with those without microbleeds [24]. Also in the US-ADNI study [25], lobar microbleeds were independently associated with a higher likelihood of having an abnormally high CSF-ptau level. Thus, studies that have examined the relationship between microbleeds and CSF-ptau or CSF-tau levels among AD patients are limited in number and their results have been inconsistent. In our study, there was no significant difference in cognitive functions between the groups with and without microbleeds, which may suggest less severe AD type parenchymal pathology in the groups with microbleeds. Further studies involving tau imaging and neuropathology are required to determine the relationship between microbleeds, CSF-tau or CSF-ptau levels, and cognitive functions.
Furthermore, we have demonstrated that the CSF-Aβ40 levels well separate AD patients with cortical microbleeds from those without microbleeds. Cortical microbleeds were noted in 16.7% –32% of AD patients when examined by T2*-weighted MRI [26–30], and patients with cortical microbleeds are at considerable risk of future symptomatic stroke and stroke-related mortality, indicating that these patients should be treated with the utmost care [23]. Our results indicate that the CSF-Aβ40 would be a useful marker to detect CAA-related cortical microbleeds in AD patients.
In our study, the leukoaraiosis of AD patients with microbleeds showed more severe levels of both PVH and DWMH compared with those without microbleeds, which is in agreement with previous studies [22–24]. However, the possibility that abnormal CSF marker levels in AD patients with cortical microbleeds might have been caused indirectly by leukoaraiosis associated with vascular risk factors is unlikely, because the results of the CSF markers remained unchanged after adjustment for PVH and DWMH scores.
The present study has some limitations. First, the sample size was relatively small, especially for the group of mixed microbleeds. This study was done in a single hospital; additional large-scale multicenter studies are needed. Second, we did not evaluate neuropathology. Further longitudinal studies using MRI, amyloid and tau positron emission tomography, CSF markers, and pathological investigations are required to elucidate the influences of CAA-related microbleeds on AD.
In conclusion, cortical microbleeds in AD are associated with more reduced levels of CSF-Aβ40 and CSF-Aβ42, indicating CAA pathology.
Footnotes
ACKNOWLEDGMENTS
The authors would like to thank the staff in the Department of Neurology of Kanazawa University Hospital and the Medical and Pharmacological Research Center Foundation for their clinical and technical support. The authors would like to thank Enago (
) for the English language review.
This study was supported in part by a Grant for Development of Advanced Technology for Measurement and Evaluation of Brain Functions, Ishikawa Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence (to M.Y.), from Japan Science and Technology Corporation, Japan, by a Grant for the Knowledge Cluster Initiative [High-Tech Sensing and Knowledge Handling Technology (Brain Technology)] (to M.Y.) and Grants-in-Aid for Scientific Research (B) (20390242) (to M.Y.) and for challenging Exploratory Research (15K15336) (to M.Y.) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, by a Grant for Amyloidosis Research Committee from the Ministry of Health, Labour and Welfare, Japan (to M.Y.), and by a Grant for SENSHIN Medical Research Foundation (to M.Y.). The sponsors had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; or preparation, review, or approval of the manuscript.