Cerebral Amyloid Angiopathy-Related Microbleeds and Cerebrospinal Fluid Biomarkers in Alzheimer’s Disease

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.

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).