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Purpose: To investigate corneal sub-basal nerve morphology changes in primary Sjogren's syndrome (SS) dry eye (SSDE) patients and determine the association with disease severity at microstructural level., Methods: Twenty-eight eyes of 17 SSDE and 82 eyes of 47 age- and sex-matched non-SS dry eye (NSSDE) patients were included. The Ocular Surface Disease Index questionnaire (OSDI), Schirmer's test (ST), tear meniscus height (TMH), non-invasive breakup time (NIBUT), meibomian gland (MG) morphology, and ocular staining score (OSS) were assessed. In vivo confocal microscopy was performed to observe corneal sub-basal nerve morphology (length, reflectivity, width, and tortuosity). Associations between clinical features and nerve parameters were analysed., Results: SSDE patients more frequently had increased nerve reflectivity (151.12 ± 17.07 vs. 139.37 ± 14.31 grey value), width (4.45 ± 0.87 vs. 3.92 ± 0.81 μm), tortuosity (132.90 ± 8.04 vs. 129.50 ± 7.33 degree), and higher reflectivity, width, and total nerve grades than NSSDE individuals (all P < 0.05). Significant associations were found between nerve reflectivity/width and anti-SSA [OR = 1.139 (1.013-1.281)/1.802 (1.013-4.465)]/labial gland biopsy [OR = 1.046 (1.002-1.161)/1.616 (1.020-3.243)]. Higher nerve width was associated with increased OSDI [β = 0.284 (0.187-0.455)], MG score [β = 0.185 (0.109-0.300)] and OSS [β = 0.163 (0.020-0.345)], but decreased NIBUT [β =  - 0.247 (- 0.548 ~  - 0.154)]. Higher nerve total grade was associated with increased OSDI [β = 0.418 (0.157-0.793)] and OSS [β = 0.287 (0.027-0.547)], but decreased ST [β =  - 0.410 (-0.857 ~  - 0.138)]., Conclusions: Corneal nerve morphology changes associated with clinical features in SS patients. These changes may facilitate severity evaluation and management of the disease., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Purpose: The local application of antiinflammatory and immunosuppressive agents is an effective method for the treatment of ocular graft-versus-host disease (oGVHD); however, we noticed that some patients with oGVHD did not respond to topical therapy as well as many others. This study aimed to determine whether tear cytokines were associated with therapeutic effects in oGVHD., Methods: Forty patients with chronic oGVHD were enrolled and grouped as responders (n = 24) and nonresponders (n = 16) based on the clinical response to 1 month of topical treatment. Tear samples were collected from each participant before and after treatment, and the tear concentrations of 7 cytokines (IL-2, IL-6, IL-8, IL-10, IL-17A, TNF-α, and ICAM-1) were measured using microsphere-based immunoassay analysis. Differences between pretreatment and posttreatment tear samples were analyzed using the Wilcoxon test., Results: No significant differences in ophthalmic symptoms or cytokine levels were observed between responders and nonresponders at baseline. After 1 month of topical treatment, ocular surface parameters (including Ocular Surface Disease Index, National Institutes of Health eye score, best-corrected visual acuity, corneal fluorescein staining score, and fluorescein tear film break-up time) were significantly ameliorated in responders, but not in nonresponders. Moreover, none of the cytokines exhibited significant alteration in nonresponders, whereas the tear levels of IL-6 (P = 0.031) and IL-8 (P = 0.037) exhibited significant decreases in responding patients., Conclusions: Our results revealed that tear IL-6 and IL-8 levels were significantly altered in response to topical oGVHD treatment., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

The glycosylation of proteins is one of the most common post-translational modifications (PTMs) and plays important regulatory functions in diverse biological processes such as protein stability or cell signaling. Accordingly, glycoproteins are also a consistent part of the human tear film proteome, maintaining the proper function of the ocular surface and forming the first defense barrier of the ocular immune system. Irregularities in the glycoproteomic composition of tear film might promote the development of chronic eye diseases, indicating glycoproteins as a valuable source for biomarker discovery or drug target identification. Therefore, the present study aimed to develop a lectin-based affinity method for the enrichment and concentration of tear glycoproteins/glycopeptides and to characterize their specific N-glycosylation sites by high-resolution mass spectrometry (MS). For method development and evaluation, we first accumulated native glycoproteins from human tear sample pools and assessed the enrichment efficiency of different lectin column systems by 1D gel electrophoresis and specific protein stainings (Coomassie and glycoproteins). The best-performing multi-lectin column system (comprising the four lectins ConA, JAC, WGA, and UEA I, termed 4L) was applied to glycopeptide enrichment from human tear sample digests, followed by MS-based detection and localization of their specific N-glycosylation sites. As the main result, our study identified a total of 26 N glycosylation sites of 11 N-glycoproteins in the tear sample pools of healthy individuals ( n = 3 biological sample pools). Amongst others, we identified tear film proteins lactotransferrin (N497 and N642, LTF), Ig heavy chain constant α-1 (N144 and 340, IGHA1), prolactin-inducible protein (N105, PIP), and extracellular lacritin (N105, LACRT) as highly reliable and significant N glycoproteins, already associated with the pathogenesis of various chronic eye diseases such as dry eye syndrome (DES). In conclusion, the results of the present study will serve as an important tear film N-glycoprotein catalog for future studies focusing on human tear film and ocular surface-related inflammatory diseases.

In recent years, anti-inflammatory therapy has become a significant part of the complex approach to treatment of patients with dry eye syndrome (DES), with cyclosporine preparations becoming increasingly important in the structure of the therapy. Taking into account the immunosuppressive effect of cyclosporine A, which is realized through hindering the activation of T-lymphocytes in the tissues of the ocular surface, its topical application in DES has a pronounced pathogenetic focus. Numerous clinical studies have shown that instillations of cyclosporine into the conjunctival cavity contribute to an increase in total tear production, as well as recovery of the density of goblet cells in the conjunctiva of DES patients. The positive effect of cyclosporine A instillations has been convincingly demonstrated in the complex therapy of patients with vernal and atopic corneal conjunctivitis, Thygeson's superficial punctate keratitis, autoimmune keratitis, meibomian gland dysfunction, etc. However, one significant problem associated with cyclosporine A instillations is the irritating effect of the drug. That prompted the development of a drug that is safe and tolerable during instillations into the conjunctival cavity - preservative-free 0.1% cyclosporine A labelled Ikervis (Santen, Japan). The drug carrier is artificial tear Cationorm (Santen), which has an advantage of stabilizing the tear film and protecting the ocular surface from the irritating effect of cyclosporine. According to numerous clinical studies, Ikervis instillations can improve the effectiveness of complex therapy in patients with DES (especially secondary to Sjögren syndrome, Stevens-Johnson syndrome, graft-versus-host disease), with allergic diseases of the cornea and conjunctiva (spring, atopic corneal conjunctivitis), with corneal transplant disease, and other similar conditions. The high efficacy and safety of Ikervis constitute the reason to recommend it for wide clinical use.

Diabetic retinopathy (DR) is a vascular complication of diabetes that can lead to partial or complete loss of vision. Early detection and treatment of DR can prevent blindness. Regular clinical examination is recommended for DR diagnosis; however, it is not always possible or feasible due to limited resources, expertise, time, and infrastructure. Several clinical and molecular biomarkers are proposed for the prediction of DR including microRNAs. MicroRNAs are a class of small non-coding RNAs that are found in biofluids and can be measured using reliable and sensitive methods. The most commonly used biofluid for microRNA profiling is plasma or serum; however, tear fluid (tears) is also demonstrated to contain microRNAs. MicroRNAs isolated from tears present a non-invasive source for DR detection. Different methods of microRNA profiling are available including digital PCR-based methods that can detect up to a single copy of microRNA in the biofluids. Here, we describe microRNA isolation from tears using manual method as well as using a high-throughput automated platform followed by microRNA profiling using digital PCR system., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Tear proteomic analysis has become an important tool in medical and veterinary research. The tear collection method could influence the tear protein profile. This study aims to evaluate the protein profiles of dog tears collected using microcapillary tubes (MT), Schirmer tear strips (ST), and ophthalmic sponges (OS)., Methods: The tear samples were collected using MT, ST, and OS. Tear protein profiles were analyzed using two-dimensional electrophoresis (2-DE) and the different protein spots' expression was compared. Fourteen protein spots were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS)., Results: Tear protein concentrations ranged from 2.80 to 4.03 μg/μL, with no statistically significant differences among collection methods. Protein expression in each collection method differed in terms of both the number and intensity of the spots. There were 249, 327, and 330 protein spots found from tears collected with MT, ST, and OS, respectively. The proteins albumin, haptoglobin, and lactoferrin identified from OS were found to have higher spot intensities than other methods of collection. The use of MT demonstrated the downregulation of nine proteins., Conclusions: The recent study supported that tear protein analysis is affected by different tear collection methods. Although ST is commonly used for tear collection, it provides insufficient information to study particular tear proteins., (© 2022. The Author(s).)

Graves' ophthalmopathy (GO), the most frequent extrathyroidal manifestation of Graves' disease (GD), can lead to a significant decline in the quality of life in patients. Exosomes, which contain proteins, lipids and DNA, play important roles in the pathological processes of various diseases. However, their roles in Graves' ophthalmopathy are still unclear. We aimed to isolate exosomes and analyze the different exosomal proteins. Tear fluids were collected from twenty-four GO patients, twenty-four GD patients and sixteen control subjects. The numbers of tear exosomes were assayed using nanoparticle tracking analysis. A Luminex 200 kit and ELISA kit were used to confirm the different cytokine concentrations in serum. Extraocular muscle from GO patients and controls was extracted, and western blotting was used to assay the levels of Caspase-3 and complement C4A. Our study demonstrated that the number of tear exosomes differ from GD patients and control. The expression levels of cytokines, including IL-1 and IL-18, were significantly increased in the tear exosomes and serum from GO patients compared with GD patients and controls. The levels of the exosomal proteins Caspase-3, complement C4A and APOA-IV were significantly increased in GO patients compared to GD patients and controls. Orbital fibroblasts from GO patients showed significantly higher levels of Caspase-3 and complement C4A than those from controls. The levels of serum APOA-IV in GO patients were significantly higher than those in GD patients and controls. Specific proteins showed elevated expression in tear exosomes from GO patients, indicating that they may play important roles in GO pathogenesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shi, Zhao, Xin, Hou, Wang, Xie, Li and Yang.)

Background: Many studies indicate a significant role of GDNF in the pathogenesis of the mood disorders, including bipolar disorder (BD) and major depressive disorder (MDD). Potentially, neurotrophic factors in lacrimal fluid (LF) could become biomarkers of various specific disorders. The aim of this study was to assess GDNF levels in LF and blood serum (BS) of patients with a current depressive episode (cDE)., Methods: We studied the glial cell line-derived neurotrophic factor (GDNF) concentration in the LF and BS of 39 healthy controls and 137 patients with a current depressive episode (cDE) (both subgroups members were 20-49 years): BD - 46 patients, MDD - 91 patients., Results: GDNF concentration in BS of women with MDD was significantly lower than in men. In BD patients, univariate linear regression analysis revealed significant correlations between GDNF concentration in the LF and the use of anxiolytics or antidepressants. These correlations were confirmed by the multivariate linear regression analysis. A significant correlation between GDNF concentrations in the LF and BS was found in controls., Limitations: The unequal proportion of men in the BD group did not permit adjusting GDNF concentrations for sex. The collected LF was stimulated, which could influence GDNF levels. It should also be noted that the patients included in the study were not treatment- naïve., Conclusions: Our findings suggest that GDNF concentration in LF could be a biomarker of the cDE (both unipolar and bipolar), though the sensitivity of this potential biomarker may be lower in depressive patients with anxiety symptoms., Competing Interests: Conflict of interest Authors declare they have no conflict of interest with respect to this study., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Diabetic retinopathy (DR), as one of the most important causes of blindness in Western societies, is a common micro-vasculopathy associated with diabetes. There is growing evidence of the role of inflammation in its development. This study was designed to measure cytokines in patients with diabetes with different stages of retinopathy .In this study, tear concentrations of three types of cytokines with different angiogenic properties including IL-1RA, IL-8, and TNF-α were measured in patients with diabetes without retinopathy, with non-proliferative retinopathy, with proliferative retinopathy, and in a healthy control group. The results showed that concentrations of TNF-α and IL-8 were higher in the tear sample of diabetics than in the control group and the concentrations of these cytokines were higher in patients with more advanced stages of diabetes, while the tear level of IL-1RA was significantly lower in diabetics. Based on these findings, it can be concluded that diabetes and its progression of severity affects the tear levels of IL-1RA, IL-8, and TNF-α inflammatory cytokines., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Purpose: The aim of this study was to evaluate in vivo the tear film in infantile nephropathic cystinosis patients with corneal crystals treated with topical viscous cysteamine hydrochloride (Cystadrops ® )., Methods: Ten eyes of five patients with nephropathic cystinosis aged from 10 to 35 years were included in this study. The patients were under treatment with viscous cysteamine hydrochloride formulation containing 3.8 mg/mL cysteamine (vCH 0.55%, equivalent to 0.55% CH; Cystadrops ® ; Recordati rare Diseases, Puteaux, France) to reduce corneal crystal density. Five age and sex matched individuals were randomly selected as control group. Tear osmolarity testing (TearLab TM ) was performed to assess the in vivo osmolarity of patients under treatment and compared to control group values. Tear film break-up time (TBUT) and basic tear secretion (Schirmer test) were also assessed., Results: Mean tear osmolarity was 294.8 mOsms/L (±10.4), with a mean absolute difference of 1.85 mOsms/L (±2.13) between the eyes. There was no statistically significant difference between the osmolarity readings of cystinosis and the control group (294.8 ± 10.4 vs 299.4 ± 6.2mOsm/L, respectively; p = 0.39). The mean TBUT was 10.2 ± 0.83 s in the study group versus 10 ± 0.7 s in controls (p = 0.62). The mean Schirmer test score was 9.2 ± 0.83 mm in the patients versus 10.2 ± 0.83 mm in the controls (p = 0.14)., Conclusions: The TearLab TM osmolarity system test showed good reliability and precision in repeated measurements. This is the first report using the TearLab osmolarity system to assess tear film in patients with cystinosis treated with vCH 0.55%. TearLab TM examination showed that the use of vCH 0.55% drops does not determine alterations of the tear film quality.

Neurotoxic chemotherapy has been shown to be associated with reduced corneal nerves and ocular surface discomfort. Substance P is a neuropeptide expressed by sensory nerves including those in the densely innervated cornea. It is involved in both pain signaling and the regulation of epithelial and neural health. While its levels in tear fluids have been used as a neuropathic biomarker in diabetes, investigations of tear concentrations of substance P in chemotherapy-induced peripheral neuropathy have not been explored. The current cross-sectional study assessed substance P expression in tears of patients following neurotoxic chemotherapy treatment. Patients treated with paclitaxel (n = 35) or oxaliplatin (n = 30) 3-24 months prior to assessment were recruited along with healthy controls (n = 25). Flush tear collection, in-vivo corneal confocal microscopy and neurotoxicity assessments were also conducted. Enzyme-linked immunosorbent assays were used to measure substance P concentrations in collected tears, while total protein content (TPC) was measured with the bicinchoninic acid method (BCA). General linear models were used for statistical analysis. Substance P concentration was reduced in paclitaxel-treated patients [Median (Interquartile range, IQR): 1.11 (0.20-2.24) ng/ml)] compared to the oxaliplatin group [4.28 (1.01-10.73) ng/ml, p = 0.02]. Substance P expressed as a proportion of TPC was also lower in the paclitaxel group [0.00006 (0.00001-0.00010) %] compared to the oxaliplatin group [0.00018 (0.00008-0.00040) %, p = 0.005]. Substance P concentration and its percentage in TPC were also reduced in the paclitaxel group when compared to healthy controls [4.61 (1.35-18.51) ng/ml, p = 0.02; 0.00020 (0.00006-0.00060) %, p = 0.04, respectively]. Higher cumulative dose of paclitaxel was correlated with a reduction in substance P concentrations (r = -0.40, p = 0.037), however no associations were found with corneal nerve parameters or neuropathy severity (p > 0.05). While these findings show evidence for the dysregulation of tear film substance P following paclitaxel treatment, longitudinal studies should be conducted to investigate how substance P levels in tears change during treatment., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Trace elements exposure is proposed to play a role in the pathogenesis of the systemic disease. Emerging studies have suggested that trace metal exposure may contribute to dry eye disease. Our study primarily aimed to investigate the association between trace metal exposure in tear samples and the presence of dry eye metrics in the shipyard industry. Overall, 84 eligible participants from the shipyard industry were included in this cross-sectional study. The parameters for identifying dry eye symptoms included O.S.D.I., SPEED, N.I.B.U.T., and ocular surface conditions, such as tear meniscus height, eye blinking, and meibomian gland area were performed by S.B.M. sistemi ocular surface analyzer. The concentration of tear trace elements was detected by inductively coupled plasma mass spectroscopy (ICP-MS). The association between tear trace elements and dry eye parameters was investigated using regression models. Participants in the exposure group had significantly higher levels of tear Pb than the control group. In the exposure group, tear Pb was significantly associated with increased SPEED and O.S.D.I. score with beta coefficients of 0.144 (95% CI 0.092, 0.197), 0.121 (95% CI 0.049, 0.194), respectively, and decreased lower and upper meibomian gland area with beta coefficients of - 0.158 (- 0.283, - 0.033) and - 0.228 (- 0.396, - 0.061), respectively. Tear trace elements exposure is considered to impact the appearance of dry eye metrics. Improving the occupational environment and monitoring the ocular surface health may benefit workers under exposure to trace elements., (© 2022. The Author(s).)

Purpose: To provide a balanced literature review of the studies that have evaluated the effect of contact lenses on meibomian gland (MG) health., Methods: A PubMed.gov literature search was conducted on or before May 15, 2021. No other time constraints were applied. Search terms included the following: "meibomian gland(s)" plus "contact lens(es)" or "meibography" plus "contact lens(es)". Only full text articles written in English were considered. The reference lists of recovered papers were used to identify articles missed during the primary search. Included articles were required to discuss the impact of contact lenses on MG morphology or function and were graded according to the level of evidence presented., Results: The literature indicates that contact lenses impact MG function; however, the data are equivocal regarding contact lenses inducing MG structural changes. The literature likewise indicates that the mechanism(s) by which contact lenses impact the MGs are likely multifactorial. Recent data suggests that MGs may have some plasticity. Detected differences between studies likely stem from varied populations evaluated, study designs, and the duration of the evaluation periods., Conclusions: With this literature review finding conflicting relationships between MG health and contact lens use, future longitudinal studies with standardized clinical MG assessments are needed to determine the true impact of contact lenses on MG health. Until these data are obtained, contact lens wearers should undergo a full MG evaluation, especially because recent data suggest that MG treatments may restore MG structure and function., (Copyright © 2021 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.)

Purpose: The probability of the coexistence of conjunctivochalasis and pseudoexfoliation syndrome (PES) in the same individual may increase with aging. We investigated the effects of conjunctivochalasis accompanied by PES on the ocular surface (OS) and anterior segment (AS) structures., Methods: Cases with only conjunctivochalasis were determined as Group 1 (n = 62), cases with conjunctivochalasis accompanied by PES as Group 2 (n = 45), and healthy individuals as Group 3 (n = 56). The OS and AS parameters of the groups were compared., Results: There were a higher grade-3 conjunctivochalasis rate (17.7% vs. 46.7%, p = 0.039), a greater "mean grade of conjunctivochalasis" value (1.72 ± 0.24 vs. 2.29 ± 0.32, p = 0.036), and a higher "total conjunctivochalasis score" (4.27 ± 1.13 vs. 6.12 ± 1.35, p = 0.025) in Group 2 than in Group 1. Additionally, Group 2 had a shorter tear film break-up time (TBUT) (9.17 ± 2.53 vs. 5.41 ± 1.32, p = 0.010) and a greater OS disease index (OSDI) score (16.28 ± 3.15 vs. 27.36 ± 4.12, p = 0.037) than Group 1. Moreover, both Group 1 and Group 2 had shorter TBUTs (Group 1-3: p = 0.004; Group 2-3: p < 0.001) and greater OSDI scores (Group 1-3: p = 0.042; Group 2-3: p = 0.019) compared to Group 3. The groups' ocular surface staining scores, Schirmer 1 tests, central corneal thicknesses, keratometries, axial lengths, anterior chamber depths, lens thicknesses, and intraocular pressures were similar (p > 0.05)., Conclusions: To our knowledge, this was the first study comprehensively investigating the effects of conjunctivochalasis accompanied by PES on the OS and AS structures together. We found that conjunctivochalasis might cause the OS disease, while the presence of PES in conjunctivochalasis cases might worsen both the OS disease and conjunctivochalasis findings., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Purpose: To investigate the alteration in lipid composition of meibum, objective clinical signs, and subjective symptoms associated with aging and meibomian gland (MG) dysfunction (MGD)., Methods: In 10 MGD patients [4 males/6 females, mean age: 65.6 ± 7.9 years (range: 50-79 years)] and 24 healthy volunteer subjects [young subjects: 6 males/6 females, mean age: 25.7 ± 3.8 years (range: 20-35 years), elderly subjects: 6 males/6 females, mean age: 58.4 ± 7.5 years (range: 50-79 years)], three objective clinical signs were evaluated: MG orifice obstruction, meibum score, and tear film lipid layer interference pattern. Subjective symptoms were analyzed via a 15-item questionnaire. After careful collection of meibum samples, comprehensive lipid analysis was performed via liquid chromatography-mass spectrometry. Data was analyzed via JMP® ver. 13 (SAS Institute, Inc., Cary, NC) statistical analysis software., Results: In the MGD patients and elderly subjects, there was a significant decrease in non-polar lipids such as cholesterol esters (ChEs), while a significant increase in polar lipids [cholesterol (Ch), (O-acyl)-ω-hydroxy fatty acid (OAHFA), and free fatty acid (FA)] in total lipids (Tukey-Kramer test: p < 0.05). Triglyceride was significantly increased only in MGD patients (p < 0.05). Symptom scores representative of vision quality (i.e., blurred vision/haziness) were significantly negatively-correlated with the ratio of the non-polar lipid ChE, while significantly positively correlated with the polar lipids Ch, OAHFA, and FA (Spearman's rank correlation coefficient: p < 0.05)., Conclusions: Our findings revealed that both MGD and aging affect the composition ratio of major meibum lipids, resulting in the appearance of subjective symptoms., (Copyright © 2021. Published by Elsevier Inc.)

Background: Aqueous deficiency dry eye disease is a chronic and potentially sight-threatening condition, that occurs due to the dysfunction of the lacrimal glands. The aim of this review was to describe the various recent developments in the understanding, diagnosis and treatment of lacrimal gland insufficiency in aqueous deficiency dry eye disease., Methods: A MEDLINE database search using PubMed was performed using the keywords: "dry eye disease/syndrome", "aqueous deficient/deficiency dry eye disease", "lacrimal gland" and "Sjogren's syndrome". After scanning through 750 relevant abstracts, 73 eligible articles published in the English language from 2016 to 2021 were included in the review., Results: Histopathological and ultrastructural studies have revealed new insights into the pathogenesis of cicatrising conjunctivitis-induced aqueous deficiency, where the lacrimal gland acini remain uninvolved and retain their secretory property, while significant ultrastructural changes in the gland have been observed. Recent advances in diagnosis include the techniques of direct clinical assessment of the lacrimal gland morphology and secretion, tear film osmolarity, tear film lysozyme and lactoferrin levels, tear film interferometry and lacrimal gland confocal microscopy. Developments in the treatment of aqueous deficiency dry eye disease, apart from the nanoparticle-based tear substitutes, include secretagogues like diquafosol tetrasodium and rebamipide, anti-inflammatory topical agents like nanomicellar form of cyclosporine and lifitegrast, scleral contact lenses, neurostimulation, and acupuncture for increasing the amount of tear production, minor salivary gland transplantation, faecal microbial transplantation, lacrimal gland regeneration and mesenchymal stem cell therapy., Conclusions: Significant advances in the understanding, diagnosis and management of lacrimal gland insufficiency and its role in aqueous deficiency dry eye disease have taken place within the second half of the last decade. Of which, translational breakthroughs in terms of newer drug formulations and regenerative medicine are most promising.

Purpose: This study investigated structural changes in rat meibomian glands following repeated and sustained application of external pressure on the eyelids using a magnet and then subsequent removal of the external pressure., Methods: Twenty-eight Sprague-Dawley rats were used. The upper eyelid was externally compressed using a pair of magnets. One magnet was placed inside the upper eyelid, another was placed outside the eyelid, and varying periods of pressure were investigated. Untreated eyes were used as controls. Meibography was performed, and the transverse eyelid tissue sections were stained with hematoxylin and eosin and anti-cytokeratin 5 antibody at one hour, two and four weeks after removing the magnets., Results: Meibography showed increased meibomian gland loss (30.0 ± 5.0%), and tissue sections showed decreased area of secretory acini (0.04 ± 0.08 mm 2 ) at one hour after applying external pressure using magnets versus in the control eyes (5.0 ± 5.0% and 0.08 ± 0.08 mm 2 , respectively). On the other hand, there was no meibomian gland loss or reduction of the area of secretory acini at two and four weeks after removing the magnets in comparison with the control eyes., Conclusions: Repeated and sustained application of external pressure on the eyelid could induce meibomian gland loss; however, this meibomian gland loss can be restored when the external pressure is removed. Therefore, the repeated application of external pressure on the eyelid is a safe treatment method for obstructive MGD., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.)

Significance: The dewetting process of contact lenses (CLs) is a result of material and solution properties as well as environmental factors. This article describes an investigational approach to observe and describe dewetting characteristics of different CL material and solution combinations., Purpose: This study aimed to determine the in vitro dewetting characteristics of various daily disposable CLs that were assessed using a noninvasive keratograph dewetting procedure (noninvasive keratograph dry-up time). In vitro dewetting data of the same CL materials soaked in saline solution and artificial tear solution (ATS) were measured to determine additional dewetting characteristics., Methods: Noninvasive keratograph dry-up time was measured for six different soft CL materials and three different test conditions, in their specific blister solution, after exposure to saline and an ATS. Twenty CLs of each solution/material combination were assessed after an 8-hour soaking, during a 180-second dewetting observation, and the results were expressed by area under the curve values., Results: Fastest dewetting occurred for all materials when measured out of saline, indicated by the highest averaged area under the curve value of 9243.3 ± 38.3 over all lens materials. Slower dewetting was detected for all materials when measured out of their specific blister solution (7755.9 ± 37.1) and out of ATS (7988.8 ± 40.0). Intragroup results were statistically significantly different for all solutions showing the smallest differences within the ATS group ( P < .001, Kruskal-Wallis test)., Conclusions: A pure saline thin film is not an ideal representation of a complex tear film layer of a healthy human because it lacks any evaporative protection by a lipid layer. The use of an ATS, which more likely mimics the natural tear film, allowed in this experimental in vitro project to decrease the gap to the in vivo field. In vitro dewetting information in connection with the blister solution allows only a theoretical conclusion about the initial lens wear after lens insertion., Competing Interests: Conflict of Interest Disclosure: None of the authors have reported a financial conflict of interest., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Optometry.)

Purpose: To investigate and quantify the effect of recombinant human lubricin (rh-lubricin) on model tear film stability., Methods: A custom-built, interferometry-based instrument called the Interfacial Dewetting and Drainage Optical Platform was used to create and record the spatiotemporal evolution of model acellular tear films. Image segmentation and analysis was performed in MATLAB to extract the most essential features from the wet area fraction versus time curve, namely the evaporative break-up time and the final wet area fraction (A10). These two parameters indicate the tear film stability in the presence of rh-lubricin in its unstressed and stressed forms., Results: Our parameters successfully captured the trend of increasing tear film stability with increasing rh-lubricin concentration, and captured differences in rh-lubricin efficacy after various industrially relevant stresses. Specifically, aggregation and fragmentation caused by a 4-week, high temperature stress condition negatively impacted rh-lubricin's ability to maintain model tear film stability. Adsorbed rh-lubricin alone was not sufficient to resist break-up and maintain full area coverage of the model tear film surface., Conclusions: Our results demonstrate that fragmentation and aggregation can negatively impact rh-lubricin's ability to maintain a stable tear film. In addition, the ability of rh-lubricin to maintain wetted area coverage is due to both freely dispersed and adsorbed rh-lubricin., Translational Relevance: Our platform and analysis method provide a facile, intuitive, and clinically relevant means to quantify the effect of ophthalmic drugs and formulations intended for improving tear film stability, as well as capture differences between variants related to drug stability and efficacy.

The lacrimal film has attracted increasing interest in the last decades as a potential source of biomarkers of physiopathological states, due to its accessibility, moderate complexity, and responsiveness to ocular and systemic diseases. High-performance liquid chromatography-mass spectrometry (LC-MS) has led to effective approaches to tear proteomics, despite the intrinsic limitations in sample amounts. This review focuses on the recent progress in strategy and technology, with an emphasis on the potential for personalized medicine. After an introduction on lacrimal-film composition, examples of applications to biomarker discovery are discussed, comparing approaches based on pooled-sample and single-tear analysis. Then, the most critical steps of the experimental pipeline, that is, tear collection, sample fractionation, and LC-MS implementation, are discussed with reference to proteome-coverage optimization. Advantages and challenges of the alternative procedures are highlighted. Despite the still limited number of studies, tear quantitative proteomics, including single-tear investigation, could offer unique contributions to the identification of low-invasiveness, sustained-accessibility biomarkers, and to the development of personalized approaches to therapy and diagnosis., (© 2021 The Authors. Mass Spectrometry Reviews published by John Wiley & Sons Ltd.)

As the cornea is densely innervated, its nerves are integral not only to its structure but also to its pathophysiology. Corneal integrity depends on a protective tear film that is maintained by corneal sensation and the reflex arcs that control tearing and blinking. Furthermore, corneal nerves promote epithelial growth and local immunoregulation. Thus, corneal nerves constitute pillars of ocular surface homeostasis. Conversely, the abnormal tear film in dry eye favors corneal epithelial and nerve damage. The ensuing corneal nerve dysfunction contributes to dry eye progression, ocular pain and discomfort, and other neuropathic symptoms. Recent evidence from clinical studies and animal models highlight the significant but often overlooked neural dimension of dry eye pathophysiology. Herein, we review the anatomy and physiology of corneal nerves before exploring their role in the mechanisms of dry eye disease., Competing Interests: Declaration of competing interest No competing interests to declare for any of the authors., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Cytokine profiles in tears have become a noninvasive biomarker for various ocular surface diseases. Therefore, the preoperative profile of cytokines in tear samples of 89 primary pterygium patients were obtained from Zhongshan Ophthalmic Center during 2015-2017. Compared to the tear cytokines in primary groups, the concentrations of IL-8, MMP-1, MMP-9, bFGF and VEGF were generally higher in recurrent pterygium group. The five cytokines were used to build diagnostic models by multiple machine learning algorithms, which can accurately distinguish non-recurrent and recurrent samples of primary pterygium patients. Besides, these cytokines were significantly associated with Recurrent-free survival (RFS) time in pterygium patients and further applied to develop a prognostic model which can estimate the prognosis of pterygium after resection. Afterward, a novel nomogram combined risk score of cytokines related biomarker and clinical characteristics was constructed, which manifested ideal accuracies to predict the 1 and 2 years' probability of pterygium recurrent events. Thus, our finding provides a more simple and accurate prediction for early pterygium recurrence after resection. It also affords a useful tool for ophthalmologists to choose the optimal treatment strategies for pterygium patients., Competing Interests: Declaration of competing interest The authors declare that they had no conflicts of interests., (Copyright © 2022. Published by Elsevier Ltd.)

Background and objective : Dry eye disease (DED) is a relatively common disorder associated with abnormal tear film and the ocular surface that causes ocular irritation, dryness, visual impairment, and damage to the cornea. DED is not a life-threatening disease but causes discomfort and multifactorial disorders in vision that affect daily life. It has been reported that all traditional medicinal plants exhibit anti-inflammatory effects on several diseases. We hypothesized that the decoction ameliorated ocular irritation and decreased cytokine expression in the cornea. This study aimed to investigate the molecular mechanisms of DED and discover a therapeutic strategy to reduce corneal inflammation. Material and Methods : We used a DED mouse model with extraorbital lacrimal gland (ELG) excision and treated the mice with a decoction of five traditional medicines: Lycium chinense , Cuscuta chinensis , Senna tora , Ophiopogon japonicus , and Dendrobium nobile for 3 months. The tear osmolarity and the ocular surface staining were evaluated as indicators of DED. Immunohistochemistry was used to detect the level of inflammation on the cornea. Results : After treatment with the decoction for three months, epithelial erosions and desquamation were reduced, the intact of corneal endothelium was maintained, and tear osmolarity was restored in the eyes. The IL-1β-associated inflammatory response was reduced in the cornea in the DED model. Conclusions: These data suggested that a mixture of traditional medicines might be a novel therapy to treat DED.

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular surface gland network. Meibomian glands (MG) secrete lipids that create a covering layer that prevents the evaporation of the aqueous part of the tear film. Neutrophils and extracellular DNA traps populate MG and the ocular surface in a mouse model of allergic eye disease. Aggregated neutrophil extracellular traps (aggNETs) formulate a mesh-like matrix composed of extracellular chromatin that occludes MG outlets and conditions MG dysfunction. Here, a method for inducing ocular surface inflammation and MG dysfunction is presented. The procedures for collecting organs related to the ocular surface, such as the cornea, conjunctiva, and eyelids, are described in detail. Using established techniques for processing each organ, the major morphological and histopathological features of MG dysfunction are also shown. Ocular exudates offer the opportunity to assess the inflammatory state of the ocular surface. These procedures enable the investigation of topical and systemic anti-inflammatory interventions at the preclinical level.

Purpose: To evaluate the presence of SARS-CoV-2 virus in tears of patients with COVID-19 in the early symptomatic stages and to compare two different sampling methods., Materials and Method: In this cross-sectional study, tears sampling was performed in COVID-19 patients admitted within the first 7 days of symptom onset. The samples were collected with both conjunctival swabs and Schirmer strips. Each specimen was analyzed via RT-PCR. The viral load was evaluated in terms of the cycle threshold value. Ocular and systemic symptoms and comorbidities of the patients were also recorded., Results: Forty patients were included. The average time from the initiation of symptoms was 3.15 days. Unilateral conjunctivitis has been observed in 5% of patients and foreign body sensation in 7.5% of patients. No viral RNA was detected in the tear samples of the patients with ocular findings. The positivity rate for SARS-CoV-2 in tears was 2.5% (n = 1). None of the samples collected by Schirmer test strips yielded positive polymerase chain reaction result for SARS-COV-2. The Ct value of the positive conjunctival swab was 36.03 and the nasopharyngeal Ct value of the same patient was 25.68., Conclusion: The SARS-CoV-2 viral shedding rate has been determined as 2.5% in the tears of early symptomatic stage COVID-19 patients. The viral load of the tears was lower than the naso-oropharynx. The conjunctival swab method is recommended in tear collection to evaluate the presence of SARS-CoV-2 by RT-PCR analysis in low viral load tears., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Optical Coherence Tomography (OCT) is a noninvasive, high-speed, high-resolution imaging technology based in the Michaelson interferometry. A near-infrared light beam is used to register the intensity variations for the light backscattered on each sample layer. Due to the high repeatability on corneal measurements, spectral domain OCT (SD-OCT) is the gold standard when talking about in vivo, non-invasive anterior segment imaging. Changes in the morphology of various ocular surfaces such as the cornea, conjunctiva, limbus or tear film with soft (SCL), rigid, corneal or scleral lens (SL) wear can be described by OCT measurements. For instance, evaluation of the corneoscleral region is essential on SL fitting. For orthokeratology lenses central epithelial thinning and peripheral thickening and their regression could be quantified with OCT after Ortho-K lens wear. Blood vessel compression on the landing zone as well as vault thickness and fluid reservoir (FR) turbidity could be imaged with OCT. Tear film evaluation on contact lens wearers is essential because its use could lead to variations on the biochemical components in tears. Changes in tear meniscus dynamics and several parameters such as volume (TMV), tear meniscus height (HMT) and turbidity could be determined with OCT and positively correlated with the instillation of different ophthalmic solutions with Non-Invasive Break Up Time (NIBUT) and Schirmer test values. This manuscript shows the increasing applicability of OCT technology for the in vivo characterization of contact lens fitting and interaction with the ocular surface in a faster, safer and non-invasive way. Future research will still allow exploring OCT imaging to its full potential in contact lens practice, as there is still a significant amount of information contained in the images that are not yet easy to extract, analyze and give clinical value., (Copyright © 2021. Published by Elsevier Ltd.)

Objective: Urea is a component of tear fluid showing a significantly decreased concentration in dry eye disease. The urea content of tear fluid may depend on urea transporters. The purpose of this study was to examine the expression of urea transporter B (UT-B) at the ocular surface and in the lacrimal glands., Methods: UT-B protein and mRNA expression was investigated in human, porcine, and murine samples. Immunohistochemical staining for UT-B was performed on paraffin sections of human, porcine, and murine corneas, eyelids, and lacrimal glands (n = 5 each). Reverse transcriptase polymerase chain reaction was conducted to detect UT-B mRNA in human and murine cornea, conjunctiva, Meibomian gland, and lacrimal gland (n = 5 each)., Results: UT-B protein expression was comparable in all three species. It was found in the corneal epithelium and endothelium, in the conjunctival epithelium, in the end pieces and excretory ducts of the lacrimal gland, Meibomian gland, and in the glands of Moll and Zeis. The glands of Zeis and the Meibomian glands showed intense UT-B signals in the basal layers of the alveolar epithelia and in the cells of the ductal epithelia. UT-B mRNA was detected in all samples analyzed., Conclusion: UT-B is expressed by the cells and tissues of the ocular surface and in the lacrimal glands. Potential changes in urea transporter expression might have implications for the pathogenesis of dry eye disease. Since comparable results were obtained for all species investigated, the presented findings may open the door for DED-relevant experimentation on porcine and murine model systems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Purpose: To determine the relationship between tear film interferometric patterns and properties of lipid, including rheological properties., Design: Prospective, cross-sectional laboratory investigation., Method: This study included 105 subjects (94 dry eye patients and 11 normal participants). The subjects were divided into 3 categories (group 1, normal; group 2, thin; and group 3, irregular) according to interferometric patterns. According to tear interferometric patterns, ultra-performance liquid chromatography (LC) quadrupole-linear ion trap/mass spectrometry (MS)-based analysis was used to investigate lipid profiling of meibum. Rheological properties were examined by using a Langmuir-Blodgett trough with saline solution., Results: Normal subjects showed Pearl-like patterns, and dry eye patients showed either irregular or thin patterns. Group 2 tended to be the evaporative type, and group 3 tended to be the aqueous-deficient type. Lipid profiling using LC-MS identified 280 lipid species of 25 lipid classes. In the meibum of the patient groups, the content of cholesteryl esters and nonpolar lipids was lower than that in the normal group. However, the content of polar lipids such as sphingolipids and phospholipids in the patient groups was higher than that in the normal group. Rheological properties showed that the lift-off areas were comparable among the 3 groups and the surface tension was the highest in group 1, followed by group 3 and group 2., Conclusions: The findings of this study suggest that tear interferometric patterns are associated with lipid profiling of meibum and its rheological properties. These results may contribute toward the development of new treatment modalities., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Obstructive sleep apnoea (OSA) and laryngopharyngeal reflux disease (LPR) are two common diseases that lower patients' quality of life. OSA is defined by cyclic events of airflow obstruction that occur during sleep, while LPR is characterized by upper airway inflammatory signs and symptoms due to the return of gastroduodenal gaseous and liquid elements. pH-metry is the gold standard in LPR diagnosis, but considering its invasiveness among other negative traits, questionnaires that catalog symptoms and signs of the disease such as Reflux Symptoms Index (RSI) and Reflux Finding Score (RFS) are preferred. Moreover, LPR can be evaluated by testing the presence of pepsin in tears, and Narrow Band Imaging (NBI) has been introduced for the early diagnosis of larynx oncological disease. This paper aims to test whether LPR is more frequent in OSA patients than in control ones, performing a non-invasive protocol composed of RSI, RFS test (with light vs. NBI techniques), followed by pepsin detection in tears. 68 LPR patients were enrolled in the study (45 with OSA and 23 without OSA). A strong linear relationship between Apnea-Hypopnea Index (AHI) and Oxygen Desaturation Index (ODI) was found, and patients who presented pepsin in tears had higher values of AHI and ODI in comparison to patients without it. Pathological RFS and NBI showed higher values of AHI and ODI in comparison to the control group. Furthermore, pathological RSI showed higher values of AHI and ODI in comparison to the control group. In conclusion, this diagnostic combined non-invasive protocol may be a good method to perform an early diagnosis of LPR.

Background: Thyroid-associated ophthalmopathy (TAO) is a common orbital inflammatory disease, but the abnormal expression of proteins in tears of TAO patients has not been systematically studied. The purpose of this study is to compare and analyze the total tear protein profile of TAO patients and to provide protein cues for TAO pathogenesis., Methods: Tear samples were isolated from 30 TAO patients with obvious ocular surface damage and 30 healthy control subjects. Tear samples from 30 individuals were mixed and divided into three sample pools. Easy nano-scale LC-MS/MS based on labeling-free quantitative technology was utilized to profile tear proteome., Results: Here, electrospray ionization mass spectra and SDS-PAGE results confirmed the good parallelisms among samples. A total of 313 proteins were obtained from six tear pools, among them, 103 differential abundance proteins (DAPs) were identified, including 99 up-regulated DAPs (including APOA1, HV103, IGH, and Transferrin variant) and four down-regulated DAPs (including FABA, VCC1, NUCB2, and E-cadherin) in the TAO group compared with the control group. GO analysis showed that up-regulated DAPs were mainly enriched in lipid metabolism and platelet molecular function, and down-regulated DAPs were involved in binding, cell junction, and cellular process. KEGG results indicated that DAPs were involved in 117 kinds of signal transduction pathways, among which the immune-related pathway of complement and coagulation cascades had the greatest relevance., Conclusion: In conclusion, label-free LC-MS/MS is an effective strategy for profiling tear proteins component. Our study provides proteins and pathways altered in TAO and provides protein cues for further study on the precise mechanism of TAO pathogenesis., Competing Interests: The authors declare that they have no competing interests., (© 2022 Zhou et al.)

Tear analysis has become an invaluable asset in clinical research in order to identify and quantify novel biomarkers for a wide array of conditions. The present work is intended to take this area of study one step further by implementing an innovative sensing platform through which exploration of low-molecular-weight compounds is conducted outperforming traditional analytical technologies. With this aim, carefully engineered plasmonic nanoassemblies have been synergistically combined with molecular-sieving materials giving rise to size-selective samplers with SERS detection capabilities. These architectures have been then integrated onto hydrogel-based contact lenses and tested in simulated tear fluids in order to evidence their operational features. Through this approach, a prolonged analyte accumulation can be realized, thus providing a competitive advantage in those scenarios where concentration of biomarkers is typically low or minimum sample volumes are not met. Additionally, quenching of metabolic flux and analyte extraction protocols can be circumvented, hence preventing the intrinsic physical and chemical interferences stemming from these procedures. The obtained results render these sensing platforms as promising medical devices, and constitute a great opportunity in order to expand the clinical toolkit in tear analysis.

Background: Coronary artery disease (CAD) detection in asymptomatic patients still remains controversial. The aim of our study was to evaluate the usefulness of ophthalmologic findings as predictors of the presence of CAD when added to cardiovascular classic risk factors (CRF) in patients with acute coronary cardiopathy suspicion., Methods: After clinical stabilization, 96 patients with acute coronary cardiopathy suspicion were selected and divided in two groups: 69 patients with coronary lesions and 27 patients without coronary lesions. Their 192 eyes were subjected to a complete routine ophthalmologic examination. Samples of tear fluid were also collected to be used in the detection of cytokines and inflammatory mediators. Logistic regression models, receiver operating characteristic curves and their area under the curve (AUC) were analysed., Results: Suggestive predictors were choroidal thickness (CT) (OR: 1.02, 95% CI 1.01-1.03) and tear granulocyte colony-stimulating factor (G-CSF) (OR: 0.97, 95% CI 0.95-0.99). We obtained an AUC of 0.9646 (95% CI 0.928-0.999) when CT and tear G-CSF were added as independent variables to the logistic regression model with cardiovascular CRF: sex, age, diabetes, high blood pressure, hypercholesterolemia, smoking habit and obesity. This AUC was significantly higher (p = 0.003) than the prediction derived from the same logistic regression model without CT and tear G-CSF (AUC = 0.828, 95% CI 0.729-0.927)., Conclusions: CT and tear G-CSF improved the predictive model for CAD when added to cardiovascular CRF in our sample of symptomatic patients. Subsequent studies are needed for validation of these findings in asymptomatic patients., (© 2022. The Author(s).)

The Tear Film Lipid Layer (TFLL) acts primarily as an interface between the aqueous layer and air. Tear film lipid is composed of a thin layer of polar lipids that interact with the secretory layer of the underlying mucosa and a thicker layer of non-polar lipids at the air interface. The tear film has a complex structure and composition that protects the cornea, promotes wound healing, and maintains high-quality vision. Plasma Rich in Growth Factor (PRGF) eye drops emerged as an exciting new treatment for corneal epitheliopathies, including aqueous deficient dry eye. The purpose of this study was to compare the lipidomic profile of eye drops obtained from PRGF with tear lipidome to determine whether PRGF drops could be an adequate complement to tears in patients with impaired TFLL. To address this study, tears and blood was collected and processed from healthy donors to obtain PRGF eye drops. Samples were aliquoted and stored at -80 °C until use. The lipid profiles of these samples were analysed by Ultrahigh Performance Liquid Chromatography (UHPLC) using a Vanquish UHPLC system to obtain untargeted lipidome profiles on a Q-Exactive HF-X hybrid quadrupole-Orbitrap mass spectrometer. In PRGF eye drops, 408 lipids were identified in ESI+ mode and 183 in ESI- mode, and they were grouped into 15 different lipid classes from four distinct categories. By contrast, 112 lipid species were identified from tear samples in ESI+ mode and 36 in ESI- mode, belonging to 12 lipid classes from six different categories. The relative abundance of most lipid species was much greater in the PRGF eye drops than in the tear, although there were some lipids present in tears that were not found in the PRGF, such as wax esters and (O-acyl)-ω-hydroxy fatty acids. In summary, these results suggest that the lipids present in PRGF eye drops could serve as a tear supplement in individuals in whom tear lipid composition is altered, although there are differences in the lipid profile of these two fluids., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

When considering eye blinking from a tribological perspective, tear viscosity is expected to play a fundamental role. The application of rheological techniques to describe the tear film dates back to the late 1980s, but there has been a continuous need of reappraisal due to new findings in tear film biochemistry and to the development of new methods of physico-chemical characterization. This review provides an overview on tear rheological behavior by analyzing the peer-reviewed literature on this topic. Specifically, examples of in-vitro and in-vivo viscosity measurements are detailed, highlighting experimental criticalities and the need of a standard convention for rheological techniques to compare data across different studies, of analyses on tears of single individuals even within the limits of the low volume available, and of a further development of in-vivo techniques. Then, the controversial role of specific tear components on viscosity is discussed, together with the alterations associated to dry eye disease and contact lens wear. Finally, an updated focus is reported on the viscosity of artificial tears formulations., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The tear film lipid layer (TFLL) is important to the maintenance of ocular surface health. Surprisingly, information on the individual roles of the myriad of unique lipids found therein is limited. The most abundant lipid species are the wax esters (WE) and cholesteryl esters (CE), and, especially their branched analogs. The isolation of these lipid species from the TFLL has proved to be tedious, and as a result, insights on their biophysical profiles and role in the TFLL is currently lacking. Herein, we circumvent these issues by a total synthesis of the most abundant iso-methyl branched WEs and CEs found in the TFLL. Through a detailed characterization of the biophysical properties, by the use of Langmuir monolayer and wide-angle X-ray scattering techniques, we demonstrate that chain branching alters the behavior of these lipid species on multiple levels. Taken together, our results fill an important knowledge gap concerning the structure and function of the TFLL on the whole., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Most current invasive analytic devices for disease diagnosis and monitoring require the collection of blood, which causes great discomfort for patients and may potentially cause infection. This explains the great need for noninvasive devices that utilize other bodily fluids like sweat, saliva, tears, or urine. Among them, eye tears are easily accessible, less complex in composition, and less susceptible to dilution. Tears also contain valuable clinical information for the diagnosis of ocular and systemic diseases as the tear analyte level shows great correlation with the blood analyte level. These unique advantages make tears a promising platform for use in clinical settings. As the volume of tear film and the rate of tear flow are only microliters in size, the use of microfluidic technology in analytic devices allows minimal sample consumption. Hence, more and more microfluidic tear analytic devices have been proposed, and their working mechanisms can be broadly categorized into four main types: (a) electrochemical, (b) photonic crystals, (c) fluorescence, and (d) colorimetry. These devices are being developed toward the application of point-of-care tests with rapid yet accurate results. This review aims to provide a general overview of the recent developmental trend of microfluidic devices for tear analysis. Moreover, the fundamental principle behind each type of device along with their strengths and weaknesses will be discussed, especially in terms of their abilities and potential in being used in point-of-care settings.

Hypothesis: Although wetting agents have been developed to limit tear film dewetting over contact lenses, systematic analyses correlating wetting agent properties to mechanisms of the tear film destabilization are not readily available. Clarifying destabilization characteristics across key physio-chemical variables will provide a rational basis for identifying optimal wetting agents., Experiments: We employ an in-house, in vitro platform to comprehensively evaluate drainage and dewetting dynamics of five wetting agents across seventeen different formulations and two model tear film solutions. We consider the film thickness evolution, film thickness at breakup, dewetted front propagation, and develop correlations to contact angle to compare the samples., Findings: Zwitterionic wetting agents effectively stabilize the tear film by reducing the film thickness at the onset of dewetting, and delaying the propagation of dewetted regions across the lens. Furthermore, tuning wetting agent surface concentrations and utilizing binary mixtures of wetting agents can enhance wetting characteristics. Finally, despite disparities in wetting agent molecular properties, the time to dewet 50% of the lens scales linearly with the product of the receding contact angle and contact angle hysteresis. Hence, we fundamentally establish the importance of minimizing the absolute contact angle and contact angle hysteresis for effective wetting performance., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Purpose: To detect SARS-CoV-2 RNA in post-mortem human eyes. Ocular symptoms are common in patients with COVID-19. In some cases, they can occur before the onset of respiratory and other symptoms. Accordingly, SARS-CoV-2 RNA has been detected in conjunctival samples and tear film of patients suffering from COVID-19. However, the detection and clinical relevance of intravitreal SARS-CoV-2 RNA still remain unclear due to so far contradictory reports in the literature., Methods: In our study 20 patients with confirmed diagnosis of COVID-19 were evaluated post-mortem to assess the conjunctival and intraocular presence of SARS-CoV-2 RNA using sterile pulmonary and conjunctival swabs as well as intravitreal biopsies (IVB) via needle puncture. SARS-CoV-2 PCR and whole genome sequencing from the samples of the deceased patients were performed. Medical history and comorbidities of all subjects were recorded and analyzed for correlations with viral data., Results: SARS-CoV-2 RNA was detected in 10 conjunctival (50%) and 6 vitreal (30%) samples. SARS-CoV-2 whole genome sequencing showed the distribution of cases largely reflecting the frequency of circulating lineages in the Munich area at the time of examination with no preponderance of specific variants. Especially there was no association between the presence of SARS-CoV-2 RNA in IVBs and infection with the variant of concern (VOC) alpha. Viral load in bronchial samples correlated positively with load in conjunctiva but not the vitreous., Conclusion: SARS-CoV-2 RNA can be detected post mortem in conjunctival tissues and IVBs. This is relevant to the planning of ophthalmologic surgical procedures in COVID-19 patients, such as pars plana vitrectomy or corneal transplantation. Furthermore, not only during surgery but also in an outpatient setting it is important to emphasize the need for personal protection in order to avoid infection and spreading of SARS-CoV-2. Prospective studies are needed, especially to determine the clinical relevance of conjunctival and intravitreal SARS-CoV-2 detection concerning intraocular affection in active COVID-19 state and in post-COVID syndrome., (© 2021. The Author(s).)

Purpose: To evaluate the efficacy of oral pilocarpine (20 mg daily) for the treatment of dry eye in patients with Sjogren's Syndrome. The frequency of side effects reported during the treatment was also investigated., Methods: In this placebo-controlled crossover study, 32 patients with Sjögren's syndrome were enrolled to receive either oral pilocarpine or placebo for 10 weeks. Following a 2-week washout period, the treatment was inverted for each patient for the same duration. Assessments included the quality of life National Eye Institute Visual Function Questionnaire-25 (NEI-VFQ-25), dry eye specific questionnaire Ocular Surface Disease Index, non-invasive breakup time, invasive breakup time with fluorescein, corneal and conjunctival staining patterns with the use of fluorescein and rose bengal staining, Schirmer's test, and tear ferning test., Results: According to the NEI-VFQ-25, there was statistically significant improvement in the quality of life following oral pilocarpine. Similar results were observed for ocular discomfort, as determined by the Ocular Surface Disease Index. All clinical tests showed favorable and statistically significant results following the use of oral pilocarpine. Regarding the analysis of tear samples, there was an improvement in the quality of tear film. This was evidenced by the modification of the patterns observed in the tear ferning test. Side effects were reported by 96.8% and 56.2% of the patients who received pilocarpine and placebo, respectively. Sweating was the most frequently reported side effect (67.74% versus 11.11%, respectively)., Conclusions: Although the treatment was associated with a high frequency of side effects, oral pilocarpine (20 mg daily) was able to relieve discomfort related to dry eyes in patients with Sjögren's syndrome and induce favorable structural changes in the tear film.

Ocular drug delivery remains a grand challenge due to the complex structure of the eye. Here, we introduce a unique platform of ocular drug delivery through the integration of silicon nanoneedles with a tear-soluble contact lens. The silicon nanoneedles can penetrate into the cornea in a minimally invasive manner and then undergo gradual degradation over the course of months, enabling painless and long-term sustained delivery of ocular drugs. The tear-soluble contact lens can fit a variety of corneal sizes and then quickly dissolve in tear fluid within a minute, enabling an initial burst release of anti-inflammatory drugs. We demonstrated the utility of this platform in effectively treating a chronic ocular disease, such as corneal neovascularization, in a rabbit model without showing a notable side effect over current standard therapies. This platform could also be useful in treating other chronic ocular diseases.

Purpose: Upon the outbreak of 2019, novel coronavirus (COVID-19) pandemic confirmed the cases surpassed 20 million. Despite a few reports identified the association of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with ocular manifestations, it may assess the ocular symptoms of patients with the COVID-19 by ophthalmologists facilitate the diagnosis and prevent transmission., Methods: A total of 60 patients with the COVID-19 admitted to Baghiatallah hospital from March 2020 to May 2020 were retrospectively reviewed and analyzed for the ocular manifestations, blood tests, and reverse transcriptase-polymerase chain reaction (RT-PCR) for SARS-CoV-2 using nasopharyngeal and conjunctival swabs., Results: Among 60 included patients with clinically confirmed COVID-19, the median age 58.36 years (IQR: 30-88 years), 27 (45%) were male. Furthermore, 29 (48%) and 5 (8%) patients yielded positive for SARS-CoV-2 on RT-PCR from nasopharyngeal swabs and conjunctival specimens, respectively. Among 60 patients, 10 (16%) and 3 (5%), respectively, had the ocular manifestations and positive results for SARS-CoV-2 on RT-PCR from conjunctival and nasopharyngeal swabs., Conclusion: Although the positive rate of tear RT-PCR rate is not noticeable as nasopharyngeal swabs yet, COVID-19 transmission through the eyes is biologically plausible., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Extracellular vesicles (EVs) in easily accessible body fluids have emerged as a promising source for liquid biopsy. Although tear collection is fast, safe, and noninvasive, EVs of tear fluid are less studied and their involvement in physiological and pathological processes is largely unknown. The aim of present study was to analyze and characterize EVs in tear fluid at the single-particle level to reveal the population heterogeneity. A laboratory-built nano-flow cytometer (nFCM) was used to analyze the purity, size distribution, and particle concentration of EVs isolated from unstimulated tears (basal tears) upon double ultracentrifugation (17 min at 100,000×g, 4 °C) via side scattering detection. The expression of CD9, CD63, CD81, CD47, CD45, CD24, and EpCAM was assessed via immunofluorescent detection. The EV concentration in tear fluid was measured to be 1.1 ± 0.7 × 10 11 particles/mL, which is approximately 100-fold higher than that of plasma EVs. In particular, it was identified for the first time that tears have strong coagulant activity owing to the abundant presence of tissue factor (TF) on tear EVs. The concentration of TF-exposing EVs (4.4 ± 3.1 × 10 10 particles/mL) was found to be approximately 100-fold higher than their counterparts in saliva (4.5 ± 2.1 × 10 8 particles/mL). We postulate that TF-exposing vesicles in tears might play a role in host defense by promoting clot formation and thus reducing the risk of pathogen invasion. The coagulant activity of tears triggered by TF-exposing EVs could provide a new research perspective for ophthalmic research., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The study was to investigate the diagnostic value of ultrasound based on the random forest segmentation algorithm for dry eye disease and the relationship between dry eye degree and tear osmotic pressure. Specifically, 100 patients with dry eye syndrome were selected as the research subjects, and they were divided into group A (conventional ultrasonic detection) and group B (ultrasonic detection based on the random forest segmentation algorithm), with 50 patients in each group. An ultrasonic measurement was used as the gold standard to evaluate the effect of ultrasonic diagnosis. The degree of dry eye was determined by Ocular Surface Disease Index (OSDI) Questionnaire and DR-1 tear film lipid layer (TFLL) test. The tear osmotic pressure was measured, and the relationship between the degree of dry eye disease and the tear osmotic pressure was analyzed. The results showed that the ultrasonic imaging effect and each index based on random forest algorithm were better than the traditional graph cut algorithm. The average central corneal thickness (CCT) values of group A and group B were (27.8 ± 30.6) μ m and (29.1 ± 30.9) μ m, respectively. 95% confidence interval was 22.7-34.2  μ m. In patients with moderate dry eye, the average CCT measured in group A was (-6.31 ± 2.82) μ m, and that in group B was (-6.45 ± 3.06) μ m. The 95% confidence interval of the difference between the two is -7.66~-5.43  μ m. In patients with severe dry eye, the average CCT was (-3.78 ± 1.13) μ m in group A and (-7.09 ± 2.05) μ m in group B ( P < 0.05). The 95% confidence interval of the difference between the two is -7.05~ -5.11  μ m. In spearman correlation analysis, tear osmotic pressure increased with dry eye severity. There was a statistically significant difference between the moderate and the severe ( P < 0.05). Tear osmotic pressure can be a rapid diagnostic index of dry eye severity. Ultrasound based on the random forest segmentation algorithm has high clinical application value in the diagnosis of dry eye syndrome., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2022 Lei Jiang et al.)

The tear film is a multi-layer fluid that covers the corneal and conjunctival epithelia of the eye and provides lubrication, nutrients, and protection from the outside environment. Tear fluid contains a high concentration of proteins and has thus been recognized as a potential source of biomarkers for ocular disorders due to its proximity to disease sites on the ocular surface and the non-invasive nature of its collection. This is particularly true in the case of dry eye disease, which directly impacts the tear film and its components. Proteomic analysis of tear fluid is challenging mainly due to the wide dynamic range of proteins and the small sample volumes. However, recent advancements in mass spectrometry have revolutionized the field of proteomics enabling unprecedented depth, speed, and accuracy, even with small sample volumes. In this study using the Orbitrap Fusion Tribrid mass spectrometer, we compared four different mass spectrometry workflows for the proteomic analysis of tear fluid collected via Schirmer strips. We were able to establish a method of in-strip protein digestion that identified >3000 proteins in human tear samples from 11 healthy subjects. Our method offers a significant improvement in the number of proteins identified compared to previously reported methods without pooling samples.

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a newly discovered class of endogenous lipids that consist of two acyl chains connected through a single ester bond. Being a unique species of FAHFAs, (O-acyl)-ω-hydroxy fatty acids (OAHFAs) differ from other FAHFAs in that their hydroxy fatty acid backbones are ultralong and their hydroxy esterification is believed to be solely at the terminal (ω-) position. Only in recent years with technological advances in lipidomics have OAHFAs been identified as an important component of the tear film lipid layer (TFLL). It was found that OAHFAs account for approximately 4 mol% of the total lipids and 20 mol% of the polar lipids in the TFLL. However, their biophysical function and contribution to the TFLL is still poorly understood. Here we studied the molecular biophysical mechanisms of OAHFAs using palmitic-acid-9-hydroxy-stearic-acid (PAHSA) as a model. PAHSA and OAHFAs share key structural similarities that could result in comparable biophysical properties and molecular mechanisms. With combined biophysical experiments, atomic force microscopy observations, and all-atom molecular dynamics simulations, we found that the biophysical properties of a dynamic PAHSA monolayer under physiologically relevant conditions depend on a balance between kinetics and thermal relaxation. PAHSA molecules at the air-water surface demonstrate unique polymorphic behaviors, which can be explained by configurational transitions of the molecules under various lateral pressures. These findings could have novel implications in understanding biophysical functions that FAHFAs, in general, or OAHFAs, specifically, play in the TFLL., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Purpose: The unifying characteristic of dry eye is the loss of tear film homeostasis, and the tear lipid layer is a key component for maintaining film stability. The detection of tear lipid is of great significance for the diagnosis of dry eye. In this study, we explored a new test strip for the detection of tear lipid., Methods: The tear lipid test strip was prepared by coating the strip material with hydrophobic nano-silica. We tested its physical properties with iodine vapor chromogenic and cobalt chloride test methods. Its biosafety was evaluated by an ocular irritation test in rabbits. Finally, we established a rabbit meibomian gland dysfunction model and measured both eyes with the tear lipid test strip at the first, third, seventh, 14th, 16th, and 21st day after surgery., Results: The tear lipid test strip had fine lipophilicity and hydrophobicity. It can extract lipid from tear, and the tear lipid can be quantified by measuring the length of lipid infiltration. In the ocular irritation test, the test strip had no obvious eye irritation. The length of lipid infiltration between experimental and control rabbit eyes began to show statistical difference since the third day after surgery., Conclusions: The novel tear lipid test strip has great lipophilicity, hydrophobicity, and biological safety. It might be effectively applied in diagnosis of dry eye., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

A possible approach for the treatment of meibomian gland disease (MGD) can be the supplementation of meibomian gland secretion (MGS) with nonpolar lipids (NPL) rich plant oils. Sesame oil (SO), approximately equal in monounsaturated fat (oleic acid, 40% of total) and polyunsaturated fat (linoleic acid, 42% of total), has shown multiple health benefits due to its anti-inflammatory and antioxidant effects. Thus, the interactions between SO and MGS in surface layers deserve further study. Therefore, pseudobinary films were formed with controlled MGS/SO molar ratios (0%, 10%, 30%, 50%, and 100% SO) at the air/water surface of the Langmuir trough over phosphate buffered saline (pH 7.4) subphase. Surface pressure (π)-area (A) isotherms and Brewster angle microscopy observations showed nonideal interactions where SO aggregates with MGS and complements the NPL stratum of the meibomian layers. The analysis of stress relaxation transients with Kohlrausch-Williams-Watts equation revealed that the supplementation of fixed amount of MGS with excess lipids via SO altered the dilatational elasticity of the films as reflected by the increase of the exponent β. Thus, SO with its unique combination of high oxidative stability and abundance of long polyunsaturated acyl chains might be a useful supplement to MGS layers.

Purpose: Sphingolipids (SPL) play a role in cell signaling, inflammation, and apoptosis. The purpose of this study was to examine meibum and tear SPL composition in individuals with poor versus good meibum quality., Methods: Individuals were grouped by meibum quality (n = 25 with poor quality, case group and n = 25 with good quality, control group). Meibum and tears were analyzed with liquid chromatography-mass spectrometry (LC-MS) to quantify SPL classes. Semiquantitative and relative composition (mole percent) of SPL and major classes, Ceramide (Cer), Hexosyl-Ceramide (Hex-Cer), Sphingomyelin (SM), Sphingosine (Sph), and sphingosine 1-phosphate (S1P) were compared between groups., Results: Demographic characteristics were similar between the two groups. Overall, individuals with poor meibum quality had more SPL pmole in meibum and tears than controls. Relative composition analysis revealed that individuals with poor meibum quality had SPL composed of less Cer, Hex-Cer, and Sph and more SM compared to individuals with good quality meibum. This pattern was not reproduced in tears as individuals with poor meibum quality had SPL composed of a similar amount of Cer, but more Hex-Cer, Sph and SM compared to controls. In meibum, SPL pmole and relative composition most strongly correlated with MG metrics while in tears, SPL pmole and relative composition most strongly correlated with tear production. SPL in both compartments, specifically Cer pmole in meibum and S1P% in tears, correlated with DE symptoms., Conclusion: SPL composition differs in meibum and tears in patients with poor vs good meibum quality. These findings may be translated into therapeutic targets for disease., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Purpose: This study evaluated the presence and roles of cholesteryl esters (CEs) and wax esters (WEs) from human tear film and meibum in meibomian gland dysfunction (MGD)., Methods: Out of 195 enrolled subjects, 164 and 179 subjects provided tear and meibum samples, respectively. Subjects were classified into normal, asymptomatic MGD, MGD, and mixed (MGD & aqueous deficient). The precorneal tear film (PCTF) thinning rate (evaporation) was measured using optical coherence tomography. Lipids extracted from tear and meibum samples were infused into a SCIEX 5600 TripleTOF mass spectrometer. CE and WE intensities quantified with Analyst 1.7 TF and LipidView 1.3 were compared across disease groups in MetaboAnalyst 5.0 and correlated with PCTF thinning rates., Results: The numbers of unique CEs and WEs identified in the samples were 125 and 86, respectively. Unsupervised Principal Component (PC) analysis and supervised Partial Least Square Discriminant analysis exhibited little separation among groups for both CEs and WEs in tears and meibum. Spearman's correlation analyses showed no association between either the first or second PC scores with PCTF thinning rates., Conclusion: The abundances of human PCTF and meibum-derived CEs and WEs were independent of MGD disease status and PCTF thinning (evaporation). CEs and WEs alterations do not contribute to alterations in tear film dynamics in MGD, such as has been demonstrated by the (O-acyl) ω-hydroxy fatty acids (OAHFAs)., (Copyright © 2021 Elsevier Inc. All rights reserved.)