Aqueous and Plasma Levels of Phosphorylated Tau 181 in Individuals with Normal Cognition

Abstract

Background:

Plasma and cerebrospinal fluid (CSF) levels of p-tau181 have been associated with Alzheimer’s disease (AD). The retina and vitreous have shown measurable quantities of phosphorylated tau 181 (p-tau181). The aqueous humor, which can be collected during cataract surgery, may have measurable concentrations of p-tau181.

Objective:

To determine whether p-tau181 is detectable in the aqueous humor and if so, whether it is associated with other measures that might be consistent with AD such as higher plasma p-tau181 concentration and lower Montreal Cognitive Assessment (MoCA-BLIND version 7.1) score.

Methods:

Aqueous humor samples, blood samples, and MoCA-BLIND scores were collected from patients who did not carry a clinical diagnosis of cognitive impairment at the time of cataract surgery. Aqueous p-tau181 concentrations and plasma p-tau181 concentrations were then measured using ultra-sensitive single-molecule assay ELISA technology. A rank-transformed mixed-effects multivariate regression model was used to determine associations between aqueous concentrations, plasma concentrations, and MoCA-BLIND scores.

Results:

16 eyes of 16 participants were enrolled with an average age of 71.6. Average MoCA-BLIND score was 20.6/22, average aqueous p-tau181 concentration was 6.4 pg/mL, and average plasma p-tau181 concentration was 3.1 pg/mL. Higher plasma p-tau181 was significantly associated with higher aqueous p-tau181 (p = 0.02). Aqueous p-tau181 and plasma p-tau181 were negatively associated with MoCA-BLIND scores (p = 0.005 and p = 0.001 respectively) in these patients.

Conclusions:

Aqueous p-tau181 is positively correlated with plasma p-tau181 and is negatively correlated with MoCA-BLIND scores. Further study in individuals with mild cognitive impairment or AD characterized by cerebrospinal fluid and volumetric MRI metrics may yield further insights.

Keywords

Alzheimer’s disease aqueous humor enzyme-linked immunosorbent assay tau proteins

INTRODUCTION

Alzheimer’s disease (AD) is the most prevalent form of dementia in the Western world. It is defined by the accumulation of amyloid-β (Aβ) plaques and tau neurofilaments in neurons, and this contributes to inflammation, cellular damage, and cognitive decline [1, 2]. Due to the burden of disease, there has been a concerted scientific effort to identify the specific proteins involved in the pathogenesis of AD. These include Aβ₄₀, Aβ₄₂ [3], p-tau181, p-tau217, p-tau231 [4], and neurofilament light chain [5]. The investigation of these biomarkers has primarily focused on their concentrations in cerebrospinal fluid (CSF) and plasma to aid in the diagnosis and monitoring of disease progression along the AD continuum.Particularly, plasma p-tau181 has been identified as a potential biomarker for identifying AD [6] and even serves as a biomarker for brain amyloid burden in cognitively normal adults [7]. However, there has been limited focus on intraocular levels of p-tau181.

Over the past decade, there has been an increasing body of literature highlighting the relationship between retinal imaging metrics and various neurodegenerative diseases associated with cognitive impairment [8 –10]. Particularly, tau neurofibrillary tangles and amyloid plaque deposition in the retina have been identified in the early stages of dementia [11, 12]. In addition to the retina, one study reported that total tau in the vitreous humor is significantly elevated in eyes of patients with AD compared to controls [13, 14]. Additional work has explored the association between these biomarkers in aqueous humor and AD [15]. highlighting the ability to detect amyloid-beta plaques in the aqueous through mass spectroscopy [16], enzyme-linked immunosorbent assay (ELISA) [17], and multiplex immunoassays [18]. However, no study has investigated the burden of AD biomarkers in the aqueous humor in a prospective clinical study. Compared to the vitreous, the aqueous humor is much more readily accessible both in the clinic setting and during certain commonly performed ophthalmic procedures such as cataract surgery. Because of this, the aqueous may serve as a more potentially translatable source of intraocular fluid to measure these evolving biomarkers for the assessment of AD.

We sought to determine whether aqueous p-tau181 is correlated with plasma p-tau181 and Montreal Cognitive Assessment (MoCA-BLIND version 7.1) scores in individuals with normal cognition and no known eye disease other than age-related cataracts.

MATERIALS AND METHODS

Study approval was obtained from the Institutional Review Board. For each patient, written informed consent was obtained prior to the collection of research-related data. The tenants of the Declaration of Helsinki and Health Insurance Portability and Accountability Act regulations were followed.

This prospective study enrolled patients without neurocognitive disease who were undergoing cataract surgery in one otherwise healthy eye with no known retinal disease. On the day of cataract surgery, patients were asked to participate in the study before entering the preoperative area. If the subject decided to participate, the written informed consent process was completed. Prior to receiving any eye drops, the participants then completed the MoCA-BLIND (version 7.1) assessment administered by a trained member of the iMIND study’s key personnel. Scores were reported on a scale of 22 points with a score > 18 indicative of normal cognition. MoCA-BLIND is a validated measure of cognitive ability that removes items from the MoCA assessment that require visual ability [19]. This was chosen so that cognitive assessment scores would not be confounded by reduced vision due to the participants’ visually significant cataracts. Demographic information including age, sex, and self-identified race was also collected.

One Vacutainer tube of blood (sodium citrate-treated, light blue top) was drawn by preoperative nursing staff by either a direct needle stick or through the patient’s intravenous access line inserted just prior to cataract surgery. Once collected, all blood samples were centrifuged for 10 min at 1000–2000× g at 2–8°C. The resulting supernatant was micropipetted and transferred in 0.5 ml aliquots to microcryovials for storage in a secure –80°C freezer. Patients then proceeded to the operating room for their scheduled cataract surgery. At the start of the cataract surgery procedure, the surgeon created an anterior chamber paracentesis through clear cornea as well as a side port incision, and one sample of aqueous humor was collected from the eye using a 27 g cannula connected to a tuberculin syringe. The surgeon collected as much aqueous as deemed clinically safe. All included patients had at least 100μL collected, which was the volume required for sample analysis. Care was taken to avoid the introduction of any blood into the collected aqueous sample. The sample was then transferred directly from the tuberculin collection syringe into a freezer-safe microcryovial, labelled, and placed on dry ice for study-staff transport to the same secure –80°C freezer a few minutes away from the operating room. When ready for batch analysis, the specimens were removed from the freezer for thawing.

The aqueous samples were then centrifuged for 10 min at 1000–2000× g at 2–8°C in preparation for analysis. The samples were then processed by trained study staff using ultra-sensitive single-molecule assay ELISA (SiMoA) technology on the Quanterix SR-X machine (Quanterix, Middlesex, MA) to determine the concentrations of p-tau181 proteins in each sample. Aqueous and plasma p-tau181 samples were run in duplicate, and samples were run in multiple batches. The SiMoA technology used antibody-coated paramagnetic beads to capturep-tau181 proteins in the respective aqueous and plasma sample wells [20]. Then, a conjugated detection antibody was added to develop fluorescent immunocomplexes of bead, p-tau181 protein, and a detection antibody. Because there are more beads than p-tau181 protein molecules in each sample, each bead will either be bound to one protein or no proteins. The sample is then loaded onto a cartridge containing hundreds of thousands of microwells. Each well holds one bead, and thus, once the fluorescent substrate is added, the number of microwells with fluorescence is directly proportional to the quantitative measure of p-tau181 in each sample [20 –22].

Statistical analysis was performed using Stata (18.0, StataCorp LLC, College Station, TX). Mean concentrations of aqueous and plasma p-tau181 were computed. A rank transformation of aqueous concentration and plasma concentration was performed to test for nonparametric relationships between aqueous concentration and plasma concentration, aqueous concentration and MoCA-BLIND score, and plasma concentration and MoCA-BLIND score using a multivariate mixed-effects regression model. MoCA-BLIND scores were not transformed because they are already discrete variables.

RESULTS

The total number of cognitively normal patients with MoCA-BLIND scores, aqueous p-tau181 values, and plasma p-tau181 values enrolled was 16. Mean age of study participants was 71.6 years, with a standard deviation of 6.7 years. MoCA-BLIND scores were, on average, 20.6 with a standard deviation of 1.1. Mean body mass index (BMI) was 25.2 with a standard deviation of 5.2. Of enrolled participants, 69% were female. Over 80% of study participants were white with no single minority group representing more than 10% of the study population.

Aqueous humor and plasma p-tau181 concentrations were measurable to very low (as low as 0.5 pg/mL) for each using SiMoA ELISA technology. The summary statistics of patient demographics, MoCA-BLIND scores, aqueous p-tau181 concentrations, and plasma p-tau181 concentrations are included in Table 1. In the overall study population, aqueous p-tau181 concentrations were, on average, about twice as high as plasma p-tau181 concentrations (6.4 pg/mL versus 3.2 pg/mL respectively).

Table 1

Patient demographics

Patients Undergoing Cataract Surgery (n = 16)
	Mean	Standard Deviation	Median	Range
Age (y)	71.6	6.7	71	60– 83
MoCA-BLIND	20.6	1.1	20.5	19– 22
Plasma p-tau181 Concentration (pg/mL)	3.2	4.0	1.99	0.66– 17.7
Aqueous p-tau181 Concentration (pg/mL)	6.4	6.1	3.92	0.59– 17.2
Body mass index (kg/m²)	25.2	5.2	24.2	18.5– 40.2
	N	Percentage
Race
Asian	1	6%
Black	1	6%
White	13	81%
Not Reported	1	6%
Sex
Female	11	69%
Male	5	31%

The results of the rank transformed multivariate mixed-effects regression between MoCA-BLIND score, aqueous p-tau181 concentration, and plasma p-tau181 concentration are summarized in Table 2. The multivariate regression model controlled for age, race, sex, and BMI. The BMI was included because it has been shown previously to be associated with AD biomarkers [23]. Figure 1 shows a plot of rank transformed aqueous p-tau181 concentrations versus rank transformed plasma p-tau181 concentrations. As the plasma p-tau181 concentration increased, the aqueous concentration also increased. This association is statistically significant with a correlation coefficient of 0.42 (p = 0.02). Figure 2 shows a plot of rank transformed aqueous p-tau181 concentrations and MoCA-BLIND scores. As the rank of aqueous p-tau181 concentration increased, MoCA-BLIND score decreased, suggesting a statistically significant decline in neurocognitive function as aqueous p-tau181 concentrations rose (regression coefficient of –0.13, p = 0.005). A similar association of increasing plasma p-tau181 rank and decreasing MoCA-BLIND scores is illustrated in Fig. 3 (regression coefficient of –0.12, p = 0.001).

Table 2

Regression results of MoCA-BLIND Score, Serum p-tau181 and Aqueous p-tau181

Regression coefficients from multivariate mixed-effects rank transformed regression model
Dependent Variable	Explanatory variable	Estimate^*	95% CI^*	p ^*
Aqueous p-tau181^**	Plasma p-tau181^**	0.42	(0.76–0.78)	0.02
MoCA-BLIND score	Plasma p-tau181^**	–0.12	(–0.19– –0.05)	0.001
MoCA-BLIND score	Aqueous p-tau181^**	–0.13	(–0.21– –0.04)	0.005

^*Multivariate regression estimates, confidence intervals, and p-values provided are controlling for age, race, sex, and BMI using a mixed-effects regression model.

^**Regression results include rank transformation of concentrations aqueous p-tau181 (pg/mL) and plasma p-tau181 (pg/mL) to model for nonparametric relationships.

Fig. 1

Rank of Aqueous p-tau181 Concentration versus Rank of Plasma p-tau181 Concentration. A scatter plot of the rank transformation of aqueous p-tau181 concentration plotted against the rank transformation of plasma p-tau181 concentration. A linear line of best fit was added to highlight a positive relationship with a 95% confidence interval in gray.

Fig. 2

MoCA-BLIND Score versus Rank of Aqueous p-tau181 Concentration. A scatter plot of the rank transformation of aqueous p-tau181 concentration plotted against MoCA-BLIND score. A linear line of best fit was added to highlight a negative relationship with a 95% confidence interval in gray.

Fig. 3

MoCA-BLIND Score versus Rank of Plasma p-tau181 Concentration. A scatter plot of the rank transformation of plasma p-tau181 concentration plotted against MoCA-BLIND score. A linear line of best fit was added to highlight a negative relationship with a 95% confidence interval in gray.

DISCUSSION

The eye is a lens through which we can observe and image neurosensory tissue that reflects changes that are also occurring in the brain in individuals with neurodegenerative disease. Previous research has demonstrated links between the retina and established measures of neurodegenerative disease [8 , 12].

Bai et al. (2022) correlated Mini-Mental State Examination (MMSE) scores with these aqueous biomarker concentrations and only found a significant association between neurofilament light chain and MMSE scores [24]. However, this study enrolled primarily patients with neovascular age-related macular degeneration and because there was already a link between retinal pathologies such as neovascular age-related macular degeneration and AD biomarkers in the aqueous [18 , 25], it is difficult to determine whether the cause of elevated AD biomarkers was due to neurocognitive disease or neovascular age-related macular degeneration, especially when the study did not find an association of aqueous AD biomarkers and MMSE. Our study is the first investigation to measure levels p-tau181 in the aqueous humor of neurocognitively normal patients’ eyes that, other than age-related cataracts, were normal.

The significant association between aqueous p-tau181 and plasma p-tau181 concentrations supports the suggestion that aqueous p-tau181 may be another potential bodily fluid from which this AD biomarker can be quantitated, since plasma p-tau181 has been established in prior studies as a potential biomarker for identifying AD [6]. This is further supported by a similar association between MoCA-BLIND scores and plasma p-tau181 in our study. This association between plasma p-tau181 and MoCA-BLIND scores is consistent with previous findings that increasing plasma p-tau181 is associated with lower MoCA scores (Fig. 3) [26]. There was also a significant association between aqueous p-tau181 concentrations and MoCA-BLIND scores, and as aqueous p-tau181 concentrations increased, MoCA-BLIND scores decreased (Fig. 2). Because this association is significant and is consistent with the proposed pathogenesis of AD in which p-tau181 aggregation contributes to neurotoxicity and symptoms of AD [27, 28], it is reasonable to conclude that aqueous p-tau181 levels may be a useful biomarker for the detection of AD, although not as easily attainable as blood at this time.

An important strength of this study is that all included eyes were otherwise normal with no known eye disease, other than age-related cataracts. Because ophthalmic conditions such as age-related macular degeneration and glaucoma have been associated with AD biomarkers in the aqueous humor [18], narrowing the study population to those individuals who only have age-related cataracts provides a robust study cohort that allows us to more neatly consider the significance of aqueous p-tau181 as a potential biomarker of AD. Moreover, the inclusion of only cognitively normal participants as measured by MoCA-BLIND scores and medical history is helpful in the early investigation of aqueous p-tau181 as a potential biomarker for AD as it removes the effect of other, potentially confounding, neurologic and ophthalmic conditions that may affect neurologic assessment and aqueous and plasma p-tau181 concentrations.

This study is not without some limitations, however. One potential limitation is that the development of cataracts may possibly be associated with AD [29]; however, this finding is quite unlikely given the widespread development of cataracts around the world as individuals age, many of whom do not develop cognitive-related disorders. Although the study of eyes without other ocular disease is advantageous in this exploratory study, it may also be viewed as a limitation in the broader generalizability of study findings and thus, the inclusion of patients with different ocular diseases will be important in future studies to facilitate the understanding of how these biomarkers may be affected by other ophthalmic disease processes. Another potential limitation is that aqueous volume in the anterior chamber is limited, and careful aqueous collection will be necessary to obtain the required sample volume of 100μL. The use of MoCA-BLIND scores is a commonly used assessment of cognitive function, but it does not provide insight into a patient’s specific risk of developing AD. Therefore, future studies that include AD patients with confirmed CSF biomarkers for AD and corresponding volumetric MRI findings may facilitate the correlation of their aqueous p-tau181 levels with more than MoCA scores and age to further understand our findings and explore the viability of aqueous p-tau181 as a potential biomarker.

In conclusion, increasing aqueous p-tau181 concentrations are associated with decreased MoCA-BLIND scores. These observations are consistent with previous findings that increasing CSF and serum p-tau181 concentrations are associated with cognitive decline. This study serves as a solid base for future investigations of a larger number of more diverse participants, including those with biomarker-confirmed AD. Our findings demonstrate that aqueous specimens obtained at the time of cataract surgery may be used to measure aqueous p-tau181 levels, and that aqueous p-tau181 has the potential to serve as another biomarker for the identification of AD.

AUTHOR CONTRIBUTIONS

Hemal Patel (Conceptualization; Formal analysis; Investigation; Methodology; Project administration; Resources; Software; Validation; Visualization; Writing – original draft; Writing – review & editing); Clayton Ellis Wisely (Conceptualization; Data curation; Funding acquisition; Investigation; Methodology; Project administration; Resources; Supervision; Writing – review & editing); Cason Robbins (Conceptualization; Data curation; Funding acquisition; Methodology; Resources; Supervision; Visualization; Writing – review & editing); Daniel Parker (Conceptualization; Resources; Supervision); Pratap Challa (Data curation; Supervision; Writing – review & editing); Dilraj Grewal (Conceptualization; Funding acquisition; Project administration; Supervision; Writing – review & editing); Sharon Fekrat (Conceptualization; Funding acquisition; Investigation; Methodology; Project administration; Resources; Supervision; Visualization; Writing – review & editing).

Footnotes

ACKNOWLEDGMENTS

We would like to express our gratitude to Sandra S. Stinnett for her contribution to this research. Her expertise in statistical analysis was instrumental in the completion of our project.

FUNDING

Supported by a grant from the VitreoRetinal Surgery Foundation and in part by the Research to Prevent Blindness Unrestricted Grant (Duke University).

Presented in part at the 2021 Association for Research in Vision and Ophthalmology, 2022 American Society of Cataract and Refractive Surgery.

CONFLICT OF INTEREST

C. Ellis Wisely serves as a Glaukos consultant.

Sharon Fekrat has Alcon patent royalties, serves on/received Apellis, Alimera, and Bausch Surgical advisory board/honoraria, is a Glaukos consultant, and has Optos and Genentech research support.

All other authors have no conflict of interest to report.

DATA AVAILABILITY

The data supporting the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

References

Scheltens

, Blennow

, Breteler

, de Strooper

, Frisoni

, Salloway

, Van der Flier

(2016) Alzheimer’s disease. Lancet 388, 505–517.

Bloom

(2014) Amyloid-beta and tau: The trigger and bullet in Alzheimer disease pathogenesis. JAMA Neurol 71, 505–508.

Nakamura

, Kaneko

, Villemagne

, Kato

, Doecke

, Dore

, Fowler

, Li

, Martins

, Rowe

, Tomita

, Matsuzaki

, Ishii

, Arahata

, Iwamoto

, Ito

, Tanaka

, Masters

, Yanagisawa

(2018) High performance plasma amyloid-beta biomarkers for Alzheimer’s disease. Nature 554, 249–254.

Ossenkoppele

, van der Kant

, Hansson

(2022) Tau biomarkers in Alzheimer’s disease: Towards implementation in clinical practice and trials. Lancet Neurol 21, 726–734.

Teunissen

, Verberk

IMW

, Thijssen

, Vermunt

, Hansson

, Zetterberg

, van der Flier

, Mielke

, Del Campo

(2022) Blood-based biomarkers for Alzheimer’s disease: Towards clinical implementation. Lancet Neurol 21, 66–77.

Karikari

, Pascoal

, Ashton

, Janelidze

, Benedet

, Rodriguez

, Chamoun

, Savard

, Kang

, Therriault

, Scholl

, Massarweh

, Soucy

, Hoglund

, Brinkmalm

, Mattsson

, Palmqvist

, Gauthier

, Stomrud

, Zetterberg

, Hansson

, Rosa-Neto

, Blennow

(2020) Blood phosphorylated tau 181 as a biomarker for Alzheimer’s disease: A diagnostic performance and prediction modelling study using data from four prospective cohorts. Lancet Neurol 19, 422–433.

McGrath

, Beiser

, O’Donnell

, Yang

, Ghosh

, Gonzales

, Himali

, Satizabal

, Johnson

, Tracy

, Seshadri

(2022) Blood phosphorylated tau 181 as a biomarker for amyloid burden on brain PET in cognitively healthy adults. J Alzheimers Dis 87, 1517–1526.

Yoon

, Thompson

, Polascik

, Calixte

, Burke

, Petrella

, Grewal

, Fekrat

(2019) Correlation of OCTA and volumetric MRI in mild cognitive impairment and Alzheimer’s disease. Ophthalmic Surg Lasers Imaging Retina 50, 709–718.

Robbins

, Grewal

, Stinnett

, Soundararajan

, Yoon

, Polascik

, Liu

, Burke

, Fekrat

(2021) Assessing the retinal microvasculature in individuals with early and late-onset Alzheimer’s disease. Ophthalmic Surg Lasers Imaging Retina 52, 336–344.

10.

Grewal

, Polascik

, Hoffmeyer

, Fekrat

(2018) Assessment of differences in retinal microvasculature using OCT angiography in Alzheimer’s disease: A twin discordance report. Ophthalmic Surg Lasers Imaging Retina 49, 440–444.

11.

Gupta

, Chitranshi

, den Haan

, Mirzaei

, You

, Lim

, Basavarajappa

, Godinez

, Di Angelantonio

, Sachdev

, Salekdeh

, Bouwman

, Graham

, Gupta

(2021) Retinal changes in Alzheimer’s disease- integrated prospects of imaging, functional and molecular advances. Prog Retin Eye Res 82, 100899.

12.

Hart

, Koronyo

, Black

, Koronyo-Hamaoui

(2016) Ocular indicators of Alzheimer’s: Exploring disease in the retina. Acta Neuropathol 132, 767–787.

13.

Vig

, Garg

, Tuz-Zahra

, Xu

, Tripodis

, Nicks

, Xia

, Alvarez

, Alosco

, Stein

, Subramanian

(2023) Vitreous humor biomarkers reflect pathological changes in the brain for Alzheimer’s disease and chronic traumatic encephalopathy. J Alzheimers Dis 93, 1181–1193.

14.

Wright

, Stein

, Jun

, Chung

, McConnell

, Fiorello

, Siegel

, Ness

, Xia

, Turner

, Subramanian

(2019) Association of cognitive function with amyloid-beta and tau proteins in the vitreous humor. J Alzheimers Dis 68, 1429–1438.

15.

Dehghani

, Frost

, Jayasena

, Masters

, Kanagasingam

(2018) Ocular biomarkers of Alzheimer’s disease: The role of anterior eye and potential future directions. Invest Ophthalmol Vis Sci 59, 3554–3563.

16.

Goldstein

, Muffat

, Cherny

, Moir

, Ericsson

, Huang

, Mavros

, Coccia

, Faget

, Fitch

, Masters

, Tanzi

, Chylack

Jr. , Bush

(2003) Cytosolic beta-amyloid deposition and supranuclear cataracts in lenses from people with Alzheimer’s disease. Lancet 361, 1258–1265.

17.

Prakasam

, Muthuswamy

, Ablonczy

, Greig

, Fauq

, Rao

, Pappolla

, Sambamurti

(2010) Differential accumulation of secreted AbetaPP metabolites in ocular fluids. J Alzheimers Dis 20, 1243–1253.

18.

Janciauskiene

, Westin

, Grip

, Krakau

(2011) Detection of Alzheimer peptides and chemokines in the aqueous humor. Eur J Ophthalmol 21, 104–111.

19.

Wittich

, Phillips

, Nasreddine

, Chertkow

(2010) Sensitivity and specificity of the Montreal Cognitive Assessment modified for individuals who are visually impaired. J Vis Impair Blind 104, 360–368.

20.

Quanterix Corporation, Quanterix SR-X Brochure, https://www.quanterix.com/wp-content/uploads/2023/03/2023_SR-X-Brochure-final.pdf.

21.

Wilson

, Rissin

, Kan

, Fournier

, Piech

, Campbell

, Meyer

, Fishburn

, Cabrera

, Patel

, Frew

, Chen

, Chang

, Ferrell

, von Einem

, McGuigan

, Reinhardt

, Sayer

, Vielsack

, Duffy

(2016) The Simoa HD-1 analyzer: A novel fully automated digital immunoassay analyzer with single-molecule sensitivity and multiplexing. J Lab Autom 21, 533–547.

22.

Quanterix Corporation, Simoa^® pTau-181 Advantage V2.1 HD-1/HD-X Data Sheet, https://www.quanterix.com/simoa-assay-kits/p-tau181-v2-new/.

23.

Manouchehrinia

, Piehl

, Hillert

, Kuhle

, Alfredsson

, Olsson

, Kockum

(2020) Confounding effect of blood volume and body mass index on blood neurofilament light chain levels. Ann Clin Transl Neurol 7, 139–143.

24.

Bai

, Wan

, Wang

, Wu

, Wang

, Zhang

, Xue

, Xu

, Peng

(2022) Association of cognitive function with neurofilament light chain in the aqueous humor of human eye. Front Aging Neurosci 14, 1027705.

25.

Zhu

, Su

, Wang

, Li

, Liu

, Wang

(2021) Highly expressed amyloid beta-42 of aqueous humor in patients with neovascular macular degeneration. Semin Ophthalmol 36, 9–13.

26.

Shekhar

, Kumar

, Rai

, Kumar

, Singh

, Upadhyay

, Tripathi

, Dwivedi

, Dey

(2016) Estimation of tau and phosphorylated tau181 in serum of Alzheimer’s disease and mild cognitive impairment patients. PLoS One 11, e0159099.

27.

Jack

Jr. , Bennett

, Blennow

, Carrillo

, Feldman

, Frisoni

, Hampel

, Jagust

, Johnson

, Knopman

, Petersen

, Scheltens

, Sperling

, Dubois

(2016) A/T/N: An unbiased descriptive classification scheme for Alzheimer disease biomarkers. Neurology 87, 539–547.

28.

Mielke

, Hagen

, Xu

, Chai

, Vemuri

, Lowe

, Airey

, Knopman

, Roberts

, Machulda

, Jack

Jr. , Petersen

, Dage

(2018) Plasma phospho-tau181 increases with Alzheimer’s disease clinical severity and is associated with tau- and amyloid-positron emission tomography. Alzheimers Dement 14, 989–997.

29.

Hwang

, Longstreth

Jr. , Thielke

, Francis

, Carone

, Kuller

, Fitzpatrick

(2021) Ophthalmic conditions associated with dementia risk: The Cardiovascular Health Study. Alzheimers Dement 17, 1442–1451.