Relationship of Area of Soft Drusen in Retina with Cerebral Amyloid-β Accumulation and Blood Amyloid-β Level in the Elderly

Abstract

Background:

Histopathological studies have confirmed that soft drusen contains amyloid-β (Aβ).

Objective:

To examine the relationship between the area of soft drusen in the macular area and cerebral Aβ accumulation or plasma Aβ level in elderly persons without dementia.

Methods:

Fourteen consecutive patients (18 eyes) aged ≥50 years with macular soft drusen were studied prospectively. From color fundus photographs, the area of soft drusen (pixel) within a 6,000 μm diameter with the macula as center was measured. Standard uptake value ratio (SUVR) was obtained from positron emission tomography using florbetapir, which indicates the ratio of cerebral cortical-to-cerebellar Aβ accumulation. Ratio of plasma Aβ_1-42 to Aβ_1-40 level was calculated.

Results:

Mean age was 73.3±7.6 years. The soft drusen area was 4.32±2.42 mm². The SUVR was 1.08±0.15. Plasma Aβ_1-42/Aβ_1-40 ratio was 0.17±0.08. When SUVR ≥1.10 was defined as positive and <1.10 as negative, the soft drusen area in SUVR-positive patients (6.19±1.14 mm²) was significantly (p = 0.0043) larger than that in SUVR-negative patients (3.13±2.27 mm²). Multivariate regression analysis showed that SUVR positivity correlated with soft drusen area (p = 0.0484) and with Voxel-based Specific Regional Analysis System for Alzheimer’s Disease score (p = 0.0360). However, there was no correlation with gender (p = 0.1921), age (p = 0.2361), Alzheimer’s Disease Assessment Scale score (p = 0.6310), Mini-Mental State Examination score (p = 0.4246), or plasma Aβ_1-42/Aβ_1-40 ratio (p = 0.8398).

Conclusion:

Among elderly persons without dementia, the area of soft drusen was larger in those with more extensive cerebral Aβ accumulation. The area of soft drusen may be a biomarker of cerebral Aβ accumulation.

Keywords

Alzheimer’s disease amyloid dementia florbetapir macular degeneration positron-emission tomography

INTRODUCTION

Age-related macular degeneration (AMD) is broadly divided into two types: neovascular AMD in which choroidal neovascularization occurs in the macula and geographic AMD in which the macula becomes progressively atrophic [1]. For both types, the development of soft drusen (≥63 μm in diameter) in the preclinical stage has been reported to be a risk factor of AMD [2 –5]. Histopathological studies have confirmed that soft drusen contains amyloid-β (Aβ) [6 –8].

In Alzheimer’s disease (AD), Aβ accumulates in the neurons of the hippocampus and cerebral cortex, and destruction of the neurons leads to decrease in brain function [9 –11]. Since AD is a progressive disorder, early diagnosis and treatment are important [12, 13]. Aβ is also found in normal brain and retina, but is increased with aging [14]. Because the eye is an extension of the brain, there is a possibility that abnormal Aβ accumulation may be the common preclinical finding of both AMD and AD [5 –8].

Aβ in the brain can be localized by positron emission tomography (PET) using florbetapir as ligand [15, 16]. However, the issues of this method include radiation exposure, high cost of PET machine and reagents, and high technical skill required for reagent preparation; consequently, the test is available in a small number of facilities.

Measurement of plasma Aβ_1-42 and Aβ_1-40 levels is a less invasive test and has been reported to be a useful biomarker of dementia risk [17 –19]. On the other hand, one study found no association between the presence or absence of soft drusen and AD [20]. However, we hypothesized that quantitative measurement of soft drusen may be a useful and less invasive biomarker. In this study, we measured the area of soft drusen using color fundus photographs and analyzed the relationship with plasma Aβ_1-42/Aβ_1-40 ratio and cerebral Aβ accumulation quantified using PET.

MATERIALS AND METHODS

Design

This prospective observational study was conducted between June 2013 and February 2016 at Nippon Medical School Hospital and Nihon University Hospital. The study was conducted in accord with the ethical standards of the institutional research committee and in accord with the Helsinki declaration of 1975. This study was approved by the ethics committee of Nihon University Hospital (dated May 9, 2013; approval number 130501). Written informed consent was obtained from all patient subjects.

Inclusion and exclusion criteria

Inclusion criteria were patients aged 50 years or above with either unilateral AMD and at least one soft drusen in fellow eye, or at least one soft drusen in both eyes. All subjects were evaluated by one ophthalmologist and one neurologist, and confirmed to have no dementia from history taking and diagnostic interview. In this research, we studied soft drusen (≥63 μm in diameter) in the macular area as described below. Even extremely large drusen (>125 μm) were included as soft drusen. On the other hand, small drusen (<63 μm), which are also called hard drusen, were excluded from the study.

Exclusion criteria for PET examination were as follows: patients with claustrophobia or implanted with devices (such as pacemaker and aneurysm clip) and patients who had been exposed to radiation exceeding 15 mSv in one year through occupational exposure or radiation therapy.

Examinations

AD was evaluated using Voxel-based Specific Regional Analysis System for Alzheimer’s disease (VSRAD) score [21] or Alzheimer’s Disease Assessment Scale (ADAS) [22]. Screening for dementia was performed using the Mini-Mental State Examination (MMSE) [23].

A PET scanner system, Eminence SET-3000GCT/X (Shimadzu Corp., Kyoto, Japan), was used to measure regional brain radioactivity. Florbetapir was used as imaging ligand in PET study. For quantitative analysis of florbetapir-PET images, we used the same method as described previously [24]. Mean cortical, which contained 6 regions (medial orbital frontal, temporal, anterior and posterior cingulate, parietal lobe, and precuneus), and whole cerebellar ROI templates were applied to all PET scans to calculate mean regional cerebral-to-cerebellar standard uptake value ratio (SUVR), which indicates the degree of Aβ accumulation.

Using color fundus photographs, the area of soft drusen was measured within a diameter of 6000 μm diameter with the macula as center [1, 2]. We measured the soft drusen area using ImageJ software (National Institutes of Health, Bethesda, MD). Soft drusen was defined as those with a diameter ≥63 μm, and the total area was measured.

As markers of AD, plasma Aβ_1-42 and Aβ_1-40 levels were measured. A sandwich Aβ enzyme-linked immunosorbent assay kit was used (Wako, Osaka, Japan). The sensitivity of the assay was 0.019 pmol/L (dynamic range, 1.0–100 pmol/L) for Aβ_1-40, and 0.06 pmol/L (dynamic range, 0.1–20 pmol/L) for Aβ_1-42. The plasma Aβ_1-42/Aβ_1-40 ratio was calculated.

Statistics

Statistical analyses were performed using SPSS software version 21 (SPSS, Inc., Chicago, IL). Values are expressed as mean±standard deviation (SD) or percentage. Mann-Whitney U test or chi-square test was used to compare two groups. Multivariate regression analysis was used to identify factors that correlate independently with SUVR positivity. p values less than 0.05 were considered to be statistically significant.

RESULTS

Baseline data

Eighteen eyes of 14 patients (9 males and 5 females) aged 73.3±7.6 (range 59–87) years were studied (Table 1). Bilateral soft drusen was found in 4 patients, while unilateral soft drusen with AMD in the contralateral eye was observed in 10 patients. A VSRAD score high than 2 is considered to indicate a suspicion of AD. The VSRAD scores of all the patients were lower than 2. In the MMSE (maximum score: 30), a score of 23 or lower suggests dementia. One patient had a score of 23, while all other patients had scores of 26 or higher. In the ADAS (maximum score: 70), scores of 15 or higher indicate cognitive impairment. All patients had scores of 8 or lower.

Table 1

SUVR, plasma Aβ_1-40/Aβ_1-42 ratio, and area of soft drusen

No.	Sex	Age (y)	Eye	VSRAD	MMSE	ADAS	SUVR	Plasma Aβ_1-42/Aβ_1-40	Area of soft drusen (mm²)
1	M	71	Right	0.86	28	4	1.49	0.27	5.10
2	M	78	Right	1.28	29	6	1.20	0.29	6.11
3			Left					0.10	5.10
4	M	74	Right	1.05	29	2	1.15	0.10	7.35
5			Left					0.11	6.05
6	M	71	Left	0.51	29	2	1.12	0.11	8.10
7	F	87	Right	1.58	23	8	1.11	0.21	5.52
8	F	64	Right	0.57	26	6	1.07	0.17	2.13
9	F	74	Right	0.39	30	3	1.05	0.19	3.27
10			Left					0.28	4.38
11	M	77	Left	1.04	28	5	1.04	0.11	3.37
12	F	69	Right	0.85	30	–	1.04	0.11	0.29
13	M	78	Right	1.28	29	6	1.03	0.13	3.92
14	M	65	Right	0.5	29	–	1.02	0.10	8.11
15	M	75	Right	0.89	29	2	0.99	0.10	0.58
16			Left					0.13	0.72
17	F	59	Left	0.46	29	5	0.92	0.17	2.83
18	M	84	Right	0.88	30	2	0.88	0.34	4.81

F, female; M, male; VSRAD, Voxel-Based Specific Regional Analysis System for Alzheimer’s Disease score; MMSE, Mini-Mental State Examination score; ADAS, Alzheimer’s Disease Assessment Scale score; SUVR, standard uptake value ratio; Aβ, amyloid-β.

When SUVR ≥1.10 was defined as positive, gender distribution (male/female) was not significantly different between SUVR-positive and SUVR-negative patients (4/1 versus 5/4, p = 0.3604) (Table 2). Age was not significantly different between SUVR-positive and SUVR-negative patients (76.2±6.7 versus 71.7±8.0 years, p = 0.3846). The VSRAD score was not significantly different between SUVR-positive and SUVR-negative patients (1.1±0.4 versus 0.8±0.3, p = 0.1420). MMSE score was not significantly different between SUVR-positive and SUVR-negative patients (27.6±2.7 versus 28.8±1.2, p = 0.3267). ADAS score was also not significantly different between SUVR-positive and SUVR-negative patients (4.4±2.6 versus 4.1±1.8, p = 0.9335).

Table 2

Analyses by stratifying patients according to standard uptake value ratio

Parameter	SUVR (≥1.1) (5 cases 7 eyes)	SUVR (<1.1) (9 cases 11 eyes)	p-value
Gender (male/female)	4/1	5/4	0.3604^*
Age (y), mean±SD	76.2±6.7	71.7±8.0	0.3846^**
VSRAD, mean±SD	1.1±0.4	0.8±0.3	0.1420^**
MMSE, mean±SD	27.6±2.7	28.8±1.2	0.3267^**
ADAS, mean±SD	4.4±2.6	4.1±1.8	0.9335^**

^*Chi-square test. ^**Mann Whitney test. SUVR, standard uptake value ratio; VSRAD, Voxel-Based Specific Regional Analysis System for Alzheimer’s Disease score; MMSE, Mini-Mental State Examination score; ADAS, Alzheimer’s Disease Assessment Scale score.

Outcome measure

Data are expressed as mean±SD (range). The area of soft drusen was 4.32±2.42 (0.29–8.11) mm². The SUVR was 1.08±0.15 (0.88–1.49). Plasma Aβ_1-42/Aβ_1-40 ratio was 0.17±0.08 (0.10–0.34).

When SUVR ≥1.10 was defined as positive [25], the area of soft drusen in SUVR-positive patients was significantly larger than that in SUVR-negative patients (6.19±1.14 versus 3.13±2.27 mm², p = 0.0043) (Fig. 1). Plasma Aβ_1-42/Aβ_1-40 ratio was not different between SUVR-positive patients and SUVR-negative patients (0.17±0.07 versus 0.16±0.10, p = 0.6407) (Fig. 2).

Fig.1

Boxplots showing the relationship of area of soft drusen with cerebral amyloid-β accumulation. When standard uptake value ratio (SUVR) ≥1.10 is defined as positive, soft drusen area is significantly larger in SUVR-positive than that in SUVR-negative patients. ^*p = 0.0043.

Fig.2

Boxplots showing the relationship of plasma Aβ_1-42/Aβ_1-40 ratio with cerebral amyloid-β accumulation. Plasma Aβ_1-42/Aβ_1-40 ratio is not different between SUVR-positive and SUVR-negative patients. ^*p = 0.6407.

Multivariate regression analysis showed that SUVR positivity correlated with soft drusen area (p = 0.0484) and with VARAD score (p = 0.0360). However, there was no correlation with gender (p = 0.1921), age (p = 0.2361), ADAS score (p = 0.6310), MMSE score (p = 0.4246), or plasma Aβ_1-42/Aβ_1-40 ratio (p = 0.8398).

Figures 3 and 4 show two representative cases with SUVR higher than 1.10 and lower than 1.10, respectively.

Fig.3

A 74-year-old male without dementia (No. 4). A) Area of soft drusen within a 6000 μm diameter with the macula as center is 7.35 mm². B) Positron emission tomography-computed tomography using ¹⁸F-AV45 (florbetapir) yields standard uptake value ratio (SUVR) of 1.15 (SUVR-positive defined as SUVR ≥1.10). SUVR indicates the ratio of cortical to cerebella Aβ accumulation.

Fig.4

A 69-year-old female without dementia (No. 12). A) Area of soft drusen within a 6000 μm diameter with the macula as center is 0.29 mm². B) Positron emission tomography-computed tomography using ¹⁸F-AV45 (florbetapir) yields standard uptake value ratio (SUVR) of 1.04 (SUVR-negative defined as SUVR <1.10). SUVR indicates the ratio of cortical to cerebella Aβ accumulation.

DISCUSSION

In a clinical study, PET study using florbetapir showed positive cerebral Aβ accumulation in 76% of patients with AD, 38% in individuals with mild cognitive impairment, and 14% in healthy control subjects [26]. To evaluate Aβ accumulation in the brain or retina, quantitative analysis is required. In the present study, SUVR that indicates the ratio of cortical to cerebella Aβ accumulation was 1.08±0.15 (0.88–1.49) and the area of soft drusen was 4.32±2.42 (0.29–8.11) mm², showing large variations among individual patients. The results of analysis by stratifying patients into SUVR-positive and -negative groups demonstrated that patients with more cerebral Aβ accumulation had significantly larger area of soft drusen. Furthermore, multivariate regression analysis identified soft drusen area as an independent variable associated with SUVR positivity (p = 0.0484).

In AD patients, Aβ plaques were detected earlier in the retina than in the brain, and they accumulated with disease progression [27]. The results of the present study also suggest that the retina may be a sensitive tissue for the detection of AD deposition in the central nervous system. Exposure of cultured human retinal pigment epithelial cells to Aβ induced a significant increase in the expression of VEGF [28], suggesting a possible role of Aβ also in the development of AMD.

The Blue Mountains Eye Study (3,509 participants aged 49–97 years) found an association between cognitive impairment (defined as a MMSE score <24) and late AMD (odds ratio, 3.7; 95% confidence interval, 1.3–10.6) [29]. On the other hand, no significant association was found between cognitive impairment and early AMD. Early AMD was observed in 16.3% of individuals with AD and in 15.6% of those without AD [20]. Whether AMD is significantly more common in AD patients remains undetermined.

Evaluation of the eyes of AD patients by optical coherence tomography and electroretinogram found a significant reduction in thickness of the nerve fiber layer, which was associated with retinal dysfunction [30]. While AMD, ganglion cell degeneration, and decreased thickness of the retinal nerve fiber layer may be the major ophthalmologic findings in patients with AD, these abnormalities may not be observed in patients in the preclinical stage of AD. From the view point of early detection, recognizing cerebral Aβ accumulation by measuring the area of soft drusen is important. Aβ denotes peptides formed by 36–43 amino acids, and is cleaved from the amyloid-beta protein precursor (AβPP) by β- and γ-secretase. Cognitively normal individuals with higher plasma Aβ_1-42 and Aβ_1-40 levels are considered to be at a biomarker of progression to AD [17, 18]. Measuring soluble Aβ levels in plasma would provide an easy method to study Aβ because the procedure is minimally invasive and relatively inexpensive. However, another study found that plasma Aβ_1-42 and Aβ_1-40 levels did not consistently distinguish between probable AD and nondemented control [31]. No consensus has been established. In the present study, plasma Aβ_1-42/Aβ_1-40 ratio was 0.17±0.07 in SUVR-positive patients and 0.16±0.10 in SUVR-negative patients, with no significant difference (p = 0.6407). Furthermore, multivariate regression analysis detected no significant correlation between SUVR positivity and plasma Aβ_1-42/Aβ_1-40 ratio (p = 0.8398). It is possible that plasma Aβ concentration is less sensitive than area of soft drusen in detecting early accumulation of Aβ in the brain.

Measurement using the Image J software is time-consuming and labor intensive. Using the software installed in the optical coherence tomography device for automated detection of drusen may be useful as a screening for the area of soft drusen [32].

The present study had some limitations. The absence of proper controls was one limitation, and this issue has to be addressed in future study. All subjects were evaluated by one ophthalmologist and one neurologist, but grading was not performed in a blinded manner, which may have been a source of bias. Furthermore, the sample size was small, and the study may be underpowered. Despite an apparent male dominance and older age in SUVR-positive patients, there were no significant differences in gender distribution and age between SUVR-positive and -negative patients. This could be caused by the small number of patients studied. An adequately powered study designed to address the above issues is required to validate the present findings.

The present study demonstrated that among elderly subjects without dementia, those with greater cerebral Aβ accumulation had larger area of soft drusen. A large-scale study including patients with dementia is warranted to verify whether area of soft drusen is a biomarker of cerebral Aβ accumulation.

Footnotes

ACKNOWLEDGMENTS

This research received no grant from any funding agency in the public, commercial or not-for-profit sectors.

Authors’ disclosures available online ().

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