Case Report: Tear liquid for diagnosis of Alzheimer disease.

Introduction: The common approaches at the diagnosis of Alzheimer Disease(AD) is made with an analysis of the cerebrospinal uid or the last techniques use the study of retinal fundus and the plaques formation, through OCT or more simply with a fundus camera. Tears’ analysis is widely discussed in literature as an essential method to describe molecular and biochemical alterations in different diseases. The aim of our study was the identication with Immunocytochemistry of Amyloid Beta-42 in tears from patients with or without familiarity for Alzheimer disease, in order to make the diagnosis earlier and more accessible compared to other invasive methods. Case presentation: Our study was performed on tears from three phenotypically healthy subjects: two of them were Caucasian with Alzheimer familiarity (48 and 55 years old) and the other one was Asian without Alzheimer familiarity ( 45 years old) and affected by an adenoviral keratoconjunctivitis at the moment of the withdrawal. Tears samples were collected from eye fornix and were examinated by immunocytochemistry (ICC) assay using anti-Amyloid Beta X-42 antibody. Two out of three tears samples showed positive Amyloid Beta-42. Conclusions: Considering that our patients were phenotypically healthy, the identication of Amyloid Beta-42 by ICC could be a candidable method to make the diagnosis of the disease earlier and more accessible and available then other current and invasive methods and it could be candidate to screening method too. Plasma b Amyloid and the Risk of Alzheimer Disease and Elderly Prospective, Population-Based Cohort Study Arch Neurol


Introduction
Alzheimer disease is a neurodegenerative pathology of the central nervous system. The diagnosis is currently performed according to the classical methods through bioptic withdrawal of brain tissue or withdrawal of cerebrospinal liquid, subjecting the patients under a signi cative physical stress and limiting the execution of the practice which cannot be extended to diagnostic screening. Human tears represent an exceptional biomaterial rich in information regarding the health status of eyes and, more generally, of whole body functionality. This is mainly due to the presence in tears of a large variety of organic components including proteins, lipids, metabolites, nucleic acids, and electrolytes, whose concentrations can be altered in pathologies of whole body too [1,2]. An increasing attention is presently given to the analysis of this human body uid. The small amounts of substances considered and the typical low concentration of organic compounds hamper the access to a direct analysis by biochemical methods, so the use of tears in diagnosis is in progress. We focused our attention on tears analysis [3], to detect the presence of Amyloid Beta-42 in order to hypothesize a less invasive method and more rapid diagnosis of Alzheimer disease. Native beta-amyloid is a transmembrane protein with a short cytoplasmic domain that undergoes proteolytic clevage by secretase on its N-terminal intraluminal domain; clevated protein found in the extracellular uid and in tears [4]. Recent studies have demonstrated that Amyloid Beta-42 and Amyloid Beta-40 have different conformation and assembly states; the rst one is associated with the formation of plaques and parenchymal damage in AD respect the classical vascular AD that associated to Amyloid Beta-40 [5]. We focused our study on Amyloid Beta-42. This plaques are formed from an alter composition of the chemical barrier, and the changes in the retinal vasculature and retinal morphology were detected in the eyes of patient with AD so it was Page 3/8 observed a relationship between beta-amyloid deposition in the retina, in brain and AD [6]. In that way the eyes are a good indicator for the study of AD and his progression [8]. In this context, the detection of Amyloid Beta-42 in tears could be a useful method for an early and less invasive diagnosis of AD [7].

Materials And Methods
Our study was performed on three phenotypically healthy subjects: two of them were Caucasian with Alzheimer familiarity (48 and 55 years old) and the other one was Asian without Alzheimer familiarity (45 years old) and affected by an adenoviral keratoconjunctivitis at the moment of the withdrawal. Patients were examinated with fundus camera to highlight retinal plaques as described in literature; only the Caucasian patient presented a strong alteration in the posterior chamber with numerous plaques on retina ( g. a 1 and 2). In this present work, human tear samples were examinated with ICC using anti-Amyloid Beta X-42 antibody; samples were stained with DAB if positive, no staining if negative. (Fig. B 1,2 and 3). NaCl solution was employed as negative control and protein Amyloid Beta-42 as positive control (Fig. C 1 and 2). Our test was not in uenced by irritative and infective phenomena that can occur in tissue during the execution of withdrawal.
Tear's sample were dispensed on Thermo Scienti c adhesion slides, dried at 76°C for 4 hours and hydrated with 100% ethanol for 3 minutes, 95% ethanol for 3 minutes, 70% ethanol for 3 minutes and distilled water for 3 minutes. The area of interest was marked using a PAP pen, which draws an hydrophobic barrier to prevent the waste of reagents by keeping liquid pooled in a small droplet. The endogenous peroxidases were neutralized using peroxidase block (3-4% v/v hydrogen peroxide) for 10 minutes, followed by protein block for 10 minutes (0.4% casein in phosphate-buffered saline, with stabilizers, surfactant and 0.2% bronidox L as preservative) to reduce non-speci c binding of primary antibody and polymer. Samples were incubated with the primary antibody (anti-Amyloid Beta X-42, clone 12F4, a puri ed mouse monoclonal IgG1k in buffer containing 0.1 M Tris-Glycine pH 7.4, 150 mM NaCl with 0.05% sodium azide, Millipore) diluted 1:200 for 60 minutes. Post primary (rabbit anti-mouse IgG < 10µg/mL in 10% v/v animal serum in tris-buffered saline/0.09% Proclin 950) was incubated for 20 minutes, followed by Novolink Polymer (anti-rabbit Poly-HRP-IgG < 25µg/mL containing 10% v/v animal serum in tris-buffered saline/0.09% Proclin 950) for 20 minutes. To avoid the presence of residual reagent from the previous step, starting from peroxidase block, each step was interspersed with a washing with wash buffer (diluted 1:10, < 1%-2-Methyl-2H-Isothiazol-3-One) for 5 minutes. Peroxidase activity was developed with DAB working solution (DAB chromogen 1.74% v/v 3,3'-diaminobenzidine, in a stabilizer solution) diluted 1:20 in DAB substrate buffer and after 5 minutes the excess of reagent was washed with distilled water for 5 minutes. Samples were counterstained with hematoxylin for 30 seconds, washed again with running water for 5 minutes and distilled water for 3 minutes, and dehydrated with 70% ethanol for 3 minutes, 95% ethanol for 3 minutes and 100% ethanol for 3 minutes. Finally, they were covers lipped with synthetic mounting medium and results were interpreted using a light microscope. Immunocytochemistry (ICC) allows the identi cation by light microscopy of an antigen and its location in cells through speci c antigen-antibody reaction. In our study, we employed ICC indirect method: the speci c antigen was recognized by an unlabeled primary antibody which binds the secondary antibody (or post primary), conjugated to the horseradish peroxidase (HRP or polymer) which reacts with the substrate yielding a chromogenic development at the antigen site. Samples were counterstained with hematoxylin, covers lipped and results were interpreted at light microscopy.

Results
We observed that patient 1 and 2, with familiarity for Alzheimer, have Retinal plaques ( gure A 1 and 2) and presence Amyloid Beta-42 residues on ICC samples ( gure B 1 and 2), patient 3 not showed retinal plaques ( gure A1) and not has Amyloid Beta-42 residues in ICC ( gure B3). We have compared the samples 1, 2 and 3 with a negative control and positive control (1mg/5ml of Amyloid Beta-42) and we observe that the expression of Amyloid Beta-42 in sample 1 and 2 is comparable to positive control and the sample 3 is comparable with negative control. We observed that the appearance of retinal plaques were directly linked with the presence of residues of Amyloid Beta-42 in tears [9]. We can also note that the residues of beta 42 is not linked to expression of symptoms in the patients but at the appearance of the retinal plaques. The Amyloid Beta-42 residues in tears could have a predictive value in the diagnosis of AD.

Discussion And Conclusion
Recent studies have investigated the concentration of beta amyloid 42 and other potential biomarkers in tear uid and blood of patients with Alzheimer's disease and other forms of dementia with controversial results [11]. In our study, for the rst time, a high concentration of amyloid beta 42 was found in the tear uid of the two healthy subjects tested with familiarity for Alzheimer's disease. This preliminary data suggests the possibility of being able to identify subjects with a genetic predisposition to the development of the disease early with non-invasive alternatives to cerebrospinal uid that could serve as front-line diagnostics for Alzheimer's disease risk, before the development of any sign of the pathology. The negative test for beta amyloid 42 in the patient with adenoviral keratoconjunctivitis observed also suggests that in ammatory conditions do not cause the production of this substance and therefore are not responsible for false negative ndings. Our results suggest that tear analysis may have a predictive role in the diagnosis of AD until 20 years before [1,7]; we detected the presence of the Beta-42 amyloid protein with DAB staining in the tears of patients with familiarity for AD. To demonstrate that the in ammatory process does not generate a false positivity, we tested a subject with viral conjunctivitis, who gave a negative response to the test. ( Figure 3B) Our ndings suggest that beta-42 amyloid expression is exclusively [7] linked to Alzheimer's disease. The closed relationship between the expression of retinal plaques and the expression of beta-42 amyloid residues in tears sets the stage for a larger study in order to verify the real predictive response of the test we propose.

Declarations
Funding: No funding was received for this study.
Con ict of Interest declaration: The patient consented to participate and have their clinical data published as a case report.
Compliance with Ethical Standards: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Figure 1 retinal plaques 1 patient  CN(1) patient 3 sane and CP (2) Beta Amiloide 5mg/ml