Comprehensive Experimental Analysis of Tear Fluid Composition and Structure by Using Novel Physical Methods with Diagnostic Potential for Inflammatory Diseases

Purpose Tear fluid gained attention as a representative biological fluid. Its simple and non-invasive collection methods as well as richness of candidate biomarkers made it a potential diagnostic tool for different diseases such as dry eye. Synchronous fluorescence spectroscopy is a highly sensitive analytical tool that results in narrowing and enhanced peak resolution, and has a potential role in disease diagnosis, biomarker identification, and therapeutic monitoring. We applied synchronous fluorescence spectroscopy to monitor variations of tear fluid composition during the development of dry eye disease and to evaluate the potential effects of phytotherapy. Methods Dry eye model was induced in Chinchilla rabbits by instillation of 1% atropine sulfate ophthalmic solution. Then, the tear fluid was collected at 3, 7, and 14 days and subjected to synchronous fluorescence spectroscopy. Phytotherapy was achieved by topical instillation of 20 µl of water extracts of pomegranate peel or green tea powders. Results The fluorescence results revealed changes in the structure of tear fluid over time and the eye is subjected to toxification due to oxidative stress. In addition, dry eye disease was found to affect the metabolic/energetic state of the eye. On the other hand, phytotherapy led to enhancement of the metabolic/biosynthesis state due to activation of flavin adenine dinucleotide-associated proteins. Conclusion There was change in the electrical conductivity of tear fluid proteins. In the case of dry eyes, they became electrical insulators, while in the case of treatment with extracts, their electrical conductivity properties improved. The effects of phytotherapy can be related to the high content of ellagic acid and anthocyanin of pomegranate extract, while in green tea, they are related to catechins and phenolic compounds.

Background. The global prevalence of dry eye disease (DED) continues to rise. Alterations in the composition of the tear film are a key factor in the development of this condition. In recent years, a growing body of evidence has emerged on the underlying mechanisms of DED pathogenesis, driven by increasing interest in tear fluid as a material for clinical and diagnostic studies. Purpose: to outline the main research directions concerning tear biomarkers of DED, as well as their role in prognosis, development of novel treatment approaches, and monitoring therapeutic effectiveness. Materials and methods. A literature search was conducted in PubMed for articles published from 2000 to 2024. The selection was carried out using a comprehensive sampling method with the following keywords: tear, dry eye disease, Sjögren’s syndrome, biomarkers, cytokines, oxidative stress, enzymes, extracellular vesicles, microRNAs, NETosis. A total of 60 articles were included in the review. Results. Literature data on tear composition changes in DED indicate the presence of both general inflammatory alterations and disease-specific features. The review summarizes findings on tear content changes involving oxidative stress markers, apoptosis-related factors, proteases and their inhibitors, cytokines, lipids, and nucleic acids. Tear hyperosmolarity is a hallmark of DED, contributing to the enhancement of inflammation, recruitment of neutrophils into the tear film, and the formation of neutrophil extracellular traps (NETs). Elevated levels of inflammation markers such as matrix metalloproteinase-9, lactoferrin, myeloperoxidase, and elastase are indicative of disease severity. Differences in tear composition between Sjögren-related and non-Sjögren DED have diagnostic potential. Furthermore, tear microRNAs show promise as severity markers and differential diagnostic tools in DED. Conclusion. Available literature convincingly demonstrates that tear components can serve as biomarkers for diagnostic, prognostic, and therapeutic monitoring purposes in dry eye disease. The investigation of DED biomarkers paves the way for personalized approaches to the treatment of this condition.

The present study aims at determining whether enzymes of urea synthesis are expressed in the human lacrimal gland and in tissues of ocular surface (conjunctiva, cornea), to give evidence for the hypothesis that urea can be locally formed from ocular tissues and is important for the composition of the tear fluid. The presences of enzymes (arginase 1, 2 and agmatinase) that directly contribute to the formation of urea were investigated in the lacrimal gland and tissues of ocular surface by RT-PCR and immunohistochemistry. We collected tear fluid, aqueous humour, and blood samples from a total of 38 subjects, and tear fluid samples from a total of 78 subjects, with and without dry-eye syndrome (DES, keratoconjunctivitis sicca), and determined the urea concentration. The enzymes arginase 1, 2 and agmatinase were expressed in all tissues examined except for arginase 1, which was not expressed in the cornea. There was no correlation of urea concentration in tear fluid with aqueous humour and blood plasma (r = 0.13, p = 0.58 and r = 0.45, p = 0.05 respectively). However, correlation of urea concentration between aqueous humour and blood plasma was highly significant (r = 0.7, p = 0.0001). The concentration of urea in the tear fluid of patients with DES compared to healthy control group was significantly reduced (p < 0.0001). Enzymes that are directly involved in the formation of urea are expressed in ocular tissues. This may imply that in the ocular surface is a well-coordinated system of enzymes that can produce urea which might be independent of external urea supply.

Sjögren’s Syndrome-Like Ocular Surface Disease in Thrombospondin-1 Deficient Mice

The rationale of the research is driven by the severity of dry eye syndrome (DES) in the pterygium recurrencies development as well as by the necessity to investigate tear dysfunction and methods for its optimal correction in this patient population.&#x0D; Purpose of the study. To assess the impact of tear dysfunction indices on the development of recurrent pterygium.&#x0D; Materials and methods. We observed 60 patients (67 eyes) with recurrent pterygium. Patients were divided into four observation groups depending on the number of recurrencies. In order to study the dynamics of the DES manifestations during the postoperative period, pathogenetic therapy was used, which included a tear fluid substitute. All patients underwent a comprehensive assessment of subjective and objective DES indices before and after surgery.&#x0D; Results. A positive dynamics of subjective manifestations and objective indices of DES under the action of a tear substitute after surgery was reliably confirmed. A decrease in the number of patients with type III and IV crystallization after surgery was confirmed. Conclusion. The obtained data indicate an increase in the mucin content in the tear fluid composition, which leads to a stabilization of the tear film and to a decrease in the DES intensity.

This paper describes evolutionary divergence in composition of tear fluid among some mammals, and discusses the implications of these differences with regard to the choice of appropriate animals for use as models in ophthalmic research. For the first time a comprehensive investigation of tear fluid in the chimpanzee (Pan troglodytes) is presented in which tear fluid was collected during narcosis of eight chimpanzees. Total protein in chimpanzee tear fluid (8.8±0.3 g/l) is not significantly different from total protein of human tear fluid (10.0±0.6 g/l). The values in tear fluid for lysozyme (6.2±1.5 mg Hen Egg Lysozyme, HEL/ml), peroxidase (115±18 U/ml), and amylase (3.5±0.4 U/ml) in chimpanzees were significantly different from those of human lysozyme (11.8±1.6 mg HEL/ml), peroxidase (70±5 U/ml), and amylase (1.0±0.2 U/ml). Polyacrylamide gelelectrophoresis of tear fluid of the chimpanzees shows in comparison with human tear fluid an additional low-molecular protein (<14 kiloDalton).

BackgroundThe recent development of ultrasensitive immunological methods has allowed for the detection of biomarkers related to neurodegenerative disorders in blood, e.g., phosphorylated tau (p‐tau). This allows for a cost‐effective and scalable assessment of the causes behind cognitive decline. Nevertheless, other biofluids, e.g., tear fluid, have the potential to surpass these metrics. Previously, tear fluid from patients with neurodegenerative conditions has been explored both with mass‐spectrometry and immunoassays. Hereby, we established a method for the detection of p‐tau181, p‐tau217 and p‐tau231 in tear fluid from patients.MethodA total of 40 participants with neurodegenerative diseases (amyotrophic lateral sclerosis [n = 11], dementia [n = 9], Parkinson’s disease [n = 20]) and control subjects without clinical signs of neurodegeneration [n = 20], were recruited at the Department of Neurology at the Klinikum rechts der Isar, München, Germany. Tear fluid from both eyes was collected with Schirmer strips. The strips from both eyes were combined and eluted by centrifugation (16,000g) in sample buffer. The eluate was then analysed for p‐tau181, p‐tau217 and p‐tau231 by Single molecule array (Simoa). Results were normalised to tear fluid running length.ResultAll tear fluid samples were above the lower limit of detection (LLOD) for all p‐tau assays ([mean] p‐tau181, 92.1pg/ml; p‐tau217, 0.398pg/ml; p‐tau231, 52.9pg/ml) improving on previous attempts found in literature. There was a highly significant correlation between all p‐tau biomarkers in tear fluid (r = 0.90–0.98; P&lt;0.001). The normalised concentration of tear fluid p‐tau had a significant positive correlation with age (R p‐tau181 = 0.28, R p‐tau217 = 0.27, R p‐tau231 = 0.39; P&lt;0.05) but there was no association with the corresponding p‐tau in serum. No significant group differences in tear fluid p‐tau were observed but a trend towards increased serum p‐tau181 was found in the neurodegenerative group (6.37pg/ml) compared to controls (4.37pg/ml).ConclusionIn this pilot study, we describe quantifiable levels of p‐tau181, p‐tau217 and p‐tau231 in tear fluid samples. However, the detectable levels of p‐tau in tear fluid do not seem to correlate to p‐tau levels in serum. Further studies will show p‐tau in tear fluid of patients with suspected AD, stratified by CSF biomarkers, and thus establish the relevance of tear fluid as a diagnostic biofluid in AD.

This study evaluated the cytokine levels in unilateral tear samples from both sides in patients with pediatric lacrimal duct obstruction. Fifteen cases of unilateral lacrimal duct obstruction (mean, 26.9 ± 28.7 months old) were enrolled in this study. Tear samples were collected separately from the obstructed side and the intact side in each case before surgery, which was performed under general anesthesia or sedation. The levels of IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ, and IL-17A then were measured in each tear sample. A receiver operating characteristic (ROC) curve was constructed for the IL-6 levels in the tears. We also measured the postoperative tear fluid levels of IL-6 in those cases from which tear fluid samples could be collected after the surgery. Only the IL-6 concentration was significantly higher on the lacrimal duct-obstructed sides, compared to the control sides (P < 0.001). An ROC curve analysis for the IL-6 levels in tears showed a high value for discriminating the lacrimal duct-obstructed side from the control side (area under the ROC curve [AUC], 0.99; 95% confidence interval [CI], 0.968-1). Significant decrease of the tear fluid IL-6 levels was observed in the seven cases from which tear fluid samples also could be collected after the surgery (P = 0.016). The IL-6 level in tear fluid was significantly higher on the sides with lacrimal duct obstruction, compared to the control sides, and could be a biomarker for pediatric lacrimal duct obstruction.

Thirty-two eyes of 16 patients with verified Sjögren's syndrome were examined for clinical signs of dry eye. Tear fluid samples were collected for plasmin assay. Ophthalmologic examinations included estimation of conjunctival or corneal discharge, filament formation and presence of conjunctival or corneal epithelial defects, assessment of tear meniscus height and measurement of tear fluid break-up time, Schirmer test, and fluorescein and Rose-Bengal staining graded by the van Bijsterveld score. Tear fluid plasmin activity (IU/l) was determined by a fluorometric assay and tear fluid flow (microl/min) was measured for calculation of tear fluid plasmin activity release (microIU/min). All patients had relatively dry eyes; the mean Schirmer test value was 5.7 +/- 0.5 mm/5 min. The mean tear fluid break-up time was also low, 7.7 +/- 0.5 s. The mean Bijsterveld score value was 2.5 +/- 0.5. Because collection of tear fluid by microcapillaries for the plasmin assay was difficult due to the low tear fluid flow rate, it was necessary to drop 20 microl of balanced salt solution topically on the cornea to aspirate a tear fluid sample. Despite this, the mean tear fluid plasmin activity was higher than in control individuals (7.75 +/- 1.51 IU/l vs. 0.73, range 0.64-0.80 IU/l). On the basis of these findings we conclude that elevated tear fluid proteolytic activity may play a role in the pathology of dry eye/ocular surface disease.

To understand the pathophysiology of dry eye disease (DED), it is necessary to characterize proteins in the ocular surface fluids, including tear fluid (TF) and lacrimal fluid (LF). There have been several reports of TF proteomes, but few proteomic studies have examined LF secreted from the lacrimal gland (LG). Therefore, we characterized the proteins constituting TF and LF by liquid chromatography mass spectrometry. TF and LF were collected from patients with non-Sjögren syndrome DED and from healthy subjects. Through protein profiling and label-free quantification, 1165 proteins from TF and 1448 from LF were identified. In total, 849 proteins were present in both TF and LF. Next, candidate biomarkers were verified using the multiple reaction monitoring assay in both TF and LF of 17 DED patients and 17 healthy controls. As a result, 16 marker proteins were identified (fold-change > 1.5, p-value < 0.05), of which 3 were upregulated in TF and 8 up- and 5 down-regulated in LF. In conclusion, this study revealed novel DED markers originating from the LG and tears by in-depth proteomic analysis and comparison of TF and LF proteins.

To compare ocular surface cytokine expression in healthy controls and subjects with moderate dry eye and to study the ability of interleukin (IL)-1beta to modulate cytokine expression in cultured human conjunctival epithelial cells (CECs). Subjective (symptom questionnaire) and objective (tear osmolality, fluorescein tear break-up time [TBUT]) measures of dry eye were determined in five healthy controls and five subjects with moderate dry eye. Tear clearance rates were measured with a fluorophotometer. Enzyme immunoassay and a cytokine bead assay were used to quantify IL-1beta in tear fluid. RT-PCR was performed to detect expression of IL-1beta, IL-6, IL-8, growth-related oncogene (GRO)-beta, intercellular adhesion molecule (ICAM)-1, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and ephrin A5 in conjunctival impression cytology (CIC) samples and in CECs (IOBA-NHC cell line, n=3; primary cultured CEC, n=3) exposed to 10 ng/mL IL-1beta for 6 hours. Subjects with moderate dry eye had significantly higher symptom scores, higher tear osmolality, and shorter TBUT than healthy controls. Subjects with dry eye demonstrated slightly slower tear clearance (13.1% per minute) than healthy controls (15.4% per minute). Very low levels of IL-1beta protein were detected in the tear fluid of both groups. TRAIL was constitutively expressed in CIC samples, whereas IL-1beta, IL-6, and GRO-beta were absent. Weak expression of IL-8 (two healthy, four dry eye), ICAM-1 (four healthy, four dry eye), and ephrin A5 (one healthy, two dry eye) was observed. IL-1beta upregulated its own expression and that of IL-6, IL-8, GRO-beta, and ICAM-1 in cultured CECs but not that of ephrin A5 or TRAIL. The lack of major differences in ocular surface cytokine expression between the two groups of subjects implies other inflammatory pathways or etiologies are involved in moderate dry eye. Although IL-1beta modulated the expression of various cytokines in cultured CECs, its absence in tear fluid and CIC samples suggests that IL-1beta does not play a modulatory role in moderate dry eye.

Sialic Acid in Human Tear Fluid Decreases in Dry Eye

Background:Changes in the biochemical and protein composition of ocular fluid may reflect inflammation due to disruption of the blood-retinal barrier (BRB), as in uveitis. Tear fluid is studied as a...

ObjectiveThis study aimed to investigate changes in the tear metabolome and the therapeutic impact of 0.05% cyclosporine A (CsA) eye drops in patients with dry eye disease (DED) linked to primary Sjögren’s syndrome (pSS).MethodsFifteen patients with pSS-related DED were treated with topical 0.05% CsA for 3 months. Ocular examinations were performed, and tear samples were collected at baseline (T0) and 3 months post-treatment (T1). Differentially expressed metabolites were detected and correlated with clinical parameters.ResultsTopical 0.05% CsA treatment significantly improved the Ocular Surface Disease Index score, lid margin vascularity, conjunctival staining, tear breakup time (TBUT), and lid wiper epitheliopathy (LWE) in DED patients (all p < 0.05). A total of 402 metabolites were identified in pSS patients’ tear fluid, with 64 showing differential expression. Pathway analysis identified significant enrichment in the biosynthesis pathways of phenylalanine, tyrosine, and tryptophan. Additionally, certain metabolites (e.g., lipids and anti-inflammatory molecules) correlated positively or negatively with clinical parameters such as TBUT, LWE, and conjunctival staining.ConclusionThis study underscores significant alterations in tear metabolites at the ocular surface in pSS patients receiving 0.05% topical CsA, offering important insights for managing pSS-related DED clinically.

The diagnosis and monitoring of Sjögren syndrome (SS) is often difficult, requiring a multidisciplinary approach with invasive procedures. Our aim is to elucidate the tear protein alterations of dry eye disease (DED) with primary SS (pSS) and secondary SS (sSS) with the long-term instillation of eyedrops. We collected clinical demographics and tear fluid (TF) samples from DED patients with no autoimmune diseases (non-SS-DED), pSS-DED, and sSS-DED patients, followed by TF screening with tandem mass tagging-labeling gel-free proteomics assay. Bioinformatic analysis via Ingenuity Pathway Analysis was used to identify functional pathways and interacting networks. Validation of candidate proteins with enzyme-linked immunosorbent assay on the tear samples was done. The top functional pathways of the two comparisons (sSS-DED vs. pSS-DED and sSS-DED vs. non-SS-DED) were both associated with inflammation and stress-related signaling. After constructing an interaction network model with the selected candidate proteins, five proteins were identified. A Disintegrin and Metalloproteinase domain-containing protein 10 (ADAM10) was found to be an important candidate biomarker in all groups, followed by epidermal growth factor (EGF) in TF. This study revealed novel DED markers, ADAM10 and EGF, in differentiating between primary and secondary SS patients from tears by in-depth proteomic analysis.