The Effects of Fisetin on Cyclosporine-Treated Dry Eye Disease in Dogs.

Dry eye disease (DED) is a chronic debilitating ophthalmological disease with the current therapeutic options focused on the suppression of the symptoms. Among the possibilities of how to improve DED therapy, polyphenols have shown an enormous capacity to counteract DED functional changes. The study aimed to specifically target pathophysiological mechanisms by the addition of fisetin to the cyclosporine treatment protocol. We examined dog patients with DED on cyclosporine treatment that were administered 0.1% fisetin or fisetin-free eye drops. For the assessment of fisetin effects, tear film production and matrix metalloproteinase 9 (MMP-9) were studied in the tear film. Tear production was not recovered after 7 or 14 days (9.40 mm ± 6.02 mm, p = 0.47; 9.80 mm ± 6.83 mm, p = 0.53, respectively). MMP-9 levels significantly increased after 7 days and then dropped after 14 days (775.44 ng/mL ± 527.52 ng/mL, p = 0.05; 328.49 ng/mL ± 376.29 ng/mL, p = 1.00, respectively). Fisetin addition to cyclosporine DED treatment was not able to restore tear fluid production but influenced molecular pathological events through MMP-9.

Derivative Three-Dimensional Synchronous Fluorescence Analysis of Tear Fluid and Their Processing for the Diagnosis of Glaucoma.

BACKGROUND: Sensitive and rapid diagnosis of the early stages of glaucoma from tear fluid is a great challenge for researchers. METHODS: Tear fluid was analyzed using three-dimensional synchronous fluorescence spectroscopy (3D-SFS). Our previously published results briefly describe the main methods which applied the second derivative to a selected synchronous spectrum Δλ = 110 nm in distinguishing between healthy subjects (CTRL) and patients with glaucoma (POAG). RESULTS: In this paper, a novel strategy was used to evaluate three-dimensional spectra from the tear fluid database of our patients. A series of synchronous excitation spectra were processed as a front view and presented as a single curve showcasing the overall fluorescence profile of the tear fluid. The second derivative spectrum provides two parameters that can enhance the distinction between CTRL and POAG tear fluid. CONCLUSIONS: Combining different types of 3D-SFS data can offer interesting and useful diagnostic tools and it can be used as input for machine learning and process automation.

Effect of tear fluid sampling and processing on total protein quantity and electrophoretic pattern.

Human tears contain more than 1500 proteins that could be diagnostically relevant. To date, numerous candidates on a biomarker of protein origin were identified for ocular and systemic diseases. However, the suitable sampling method is still the subject of discussion. To address the need for a description of sampling methods properties for possible clinical analyses, we studied a total protein concentration and electrophoretic pattern of tear fluid collected by capillary tubes, Schirmer strips, cellulose microsponges, and flushing. The total protein concentration was 4.339 µg/µL ± 1.905 µg/µL, 0.967 µg/µL ± 0.117 µg/µL, 0.022 µg/µL ± 0.016 µg/µL, and 0.008 µg/µL ± 0.006 µg/µ for the capillary tubes, Schirmer strips, flushing, and cellulose microsponges, respectively. Sodium dodecyl sulfate polyacrylamide electrophoresis showed the different patterns of tear proteins obtained by the above-mentioned sampling methods. These differences could originate from the use of a bigger amount of extraction reagent that was not used in the case of capillary tubes, and retention of the proteins by strips and sponges. Taken together, capillary tubes, Schirmer strips, cellulose microsponges, and flushing represent sensitive and convenient sampling methods for tear fluid collection. For the isolation of proteins from strips and sponges, and for the flushing, less than 100 µL of a reagent should be used to ensure the sufficient concentration of the biomarkers in a trace amount.

Tear fluid biomarkers in major depressive disorder: Potential of spectral methods in biomarker discovery.

Spectroscopic methods represent a group of analytical methods that demonstrate high potential in providing clinically relevant diagnostic information, such as biochemical, functional or structural changes of macromolecular complexes that might occur due to pathological processes or therapeutic intervention. Although application of these methods in the field of psychiatric research is still relatively recent, the preliminary results show that they have the capacity to detect subtle neurobiological abnormalities in major depressive disorder (MDD). Methods of mass spectrometry (MALDI-TOF MS), zymography, synchronous fluorescence spectroscopy (SFS), circular dichroism (CD) spectroscopy, Fourier-transform infrared (FTIR) spectroscopy and atomic force microscopy (AFM) were used to analyze the human tear fluid of subjects with MDD. Using MALDI-TOF MS, two diagnostically significant peaks (3747 and 16 411 m/z) were identified with an AUC value of 0.89 and 0.92 in tear fluid of subjects with MDD vs controls, respectively. We also identified various forms of matrix metalloproteinase 9 in subjects with MDD using zymography and synchronous fluorescence spectra (SFS) showed a significant increase in fluorescence intensity at 280 nm. CD spectra were redshifted in tear fluid of subjects with MDD vs healthy controls. FTIR spectroscopy showed changes in the positions of peaks for amide A, I, II in tear fluid of subjects with MDD vs controls. Moreover, atomic force microscopy (AFM) showed different pattern in the crystal structures of tear fluid components in subjects with MDD. SFS, CD, FTIR spectroscopy, AFM and MALDI-TOF MS confirmed, that the human tear fluid proteome could be helpful in discriminating between the group of subjects with MDD and healthy controls. These preliminary findings suggest that spectral methods could represent a useful tool in clinical psychiatry, especially in establishing differential diagnosis, monitoring illness progression and the effect of psychiatric treatment.

Evidence of Polyphenols Efficacy against Dry Eye Disease.

Dry eye disease is a multifactorial pathology compromising the quality of life of patients, resulting in significant damage of the ocular surface and discomfort. The current therapeutical strategies are not able to definitively resolve the underlying causes and stop the symptoms. Polyphenols are promising natural molecules that are receiving increasing attention for their activity/effects in counteracting the main pathologic mechanisms of dry eye disease and reducing its symptoms. In the present review, a deep literature search focusing on the main polyphenols tested against dry eye disease was conducted, analyzing related in vitro, in vivo, and clinical studies to provide a comprehensive and current review on the state of the art. Polyphenols present multiple effects against dry eye diseases-related ocular surface injury. In particular, the observed beneficial effects of polyphenols on corneal cells are the reduction of the pathological processes of inflammation, oxidative stress, and apoptosis and modulation of the tear film. Due to numerous studies reporting that polyphenols are effective and safe for treating the pathological mechanisms of this ocular surface disease, we believe that future studies should confirm and extend the evidence of polyphenols efficacy in clinical practice against dry eye disease and help to develop new ophthalmic drug(s).

Native fluorescence of tear fluid as a tool for diagnostics of glaucoma.

Glaucoma is one of the leading causes of irreversible vision loss worldwide. There is an enormous need for the detection of its early stages and also speeding up and simplifying regular examinations. Among the new diagnostic approaches, the use of tear fluid has been intensively investigated in recent years. For this purpose, we analyzed the tear fluid of patients with glaucoma and related diseases. To sensitively capture the subtle ocular abnormalities related to glaucoma and manifested in tear fluid, we used synchronous fluorescence spectroscopy. In this observational case-control study, we detected significant differences in the intensity of tear fluid fluorescence located at λ ex/Δλ = 280/70 nm between the groups of primary open-angle glaucoma (p < 0.01), suspected glaucoma (p < 0.0001), and ocular hypertension (p < 0.05), when compared to the healthy control group. The signal was not significantly higher in women than in men (p = 0.05), and no correlation was found with age (r = -0.05, p > 0.05), nor treatment (p > 0.05). Taken together, tear fluid fluorescence could serve as a discriminative parameter between patients with glaucoma, related diseases, and healthy control subjects and might contribute to the improvement of diagnostics of these diseases.

Noninvasive diagnostic methods for diabetes mellitus from tear fluid.

Diabetes mellitus and prolonged hyperglycemia can cause diabetic retinopathy. Diabetic retinopathy arises from damage to retinal vessels and, in its final stages, causes blindness. The early stages are often asymptomatic and although regular screening of diabetic patients is recommended, the beginning of diabetic retinopathy is insufficiently detected. The diagnostic potential of fluorescence spectroscopy, infrared spectroscopy and atomic force microscopy as the untraditional methods for diabetes mellitus was investigated using tear fluid. In our pilot study the structural changes of tear fluid of patients with diabetes mellitus after insulin and oral antidiabetic drug treatment was compared with healthy subjects. The results of analysis, infrared spectroscopy and atomic force microscopy confirmed structural changes in tear fluid of patients in comparison with the tear fluid of healthy subjects. Using new experimental laboratory methods in future could contribute to an improvement in diagnosis of diabetes mellitus and other selected ocular diseases using tear fluid.

Diagnostic potential of tears in ophthalmology.

In research circles, there is an increasing need to seek and identify new methods in the diagnosis of pathologies, monitoring the progression of the disease and response to treatment. The sensitivity of detection technologies has improved markedly, and enables the quantification of analyses in very small quantities. Tears represent a biological material with ever increasingly developing possibilities in the diagnosis of various pathologies. Our objective was to compile a basic overview of the diagnostic potential of tears via a summary of the potential lachrymal biomarkers of various pathologies. The article contains descriptions of protein biomarkers studied particularly in recent years, which correlate with a certain ocular pathology (dry eye, allergy, glaucoma etc.). It also summarises the results published to date in the field of systemic pathologies in patients with scleroderma, cystic fibrosis, diabetes mellitus, multiple sclerosis, cancers and Parkinson's disease. It concentrates on proteomic analyses, with the aim of improving the effectiveness of markers which could be used in future also in the timely diagnosis of ocular pathologies in clinical practice.Key words: Tears, proteins, biomarker, ocular pathologies, systemic pathologies.