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The necessity of animal-free performance tests for novel ophthalmic formulation screening is challenging. For this, we developed and validated a new device to simulate the dynamics and physical-chemical barriers of the eye for in vitro performance tests of topic ophthalmic formulations. The OphthalMimic is a 3D-printed device with an artificial lacrimal flow, a cul-de-sac area, a support base, and a simulated cornea comprised of a polymeric membrane containing poly-vinyl alcohol 10 % (w/v), gelatin 2.5 % (w/v), and different proportions of mucin and poloxamer, i.e., 1:1 (M1), 1:2 (M2), and 2:1 (M3) w/v, respectively. The support base is designed to move between 0° and 50° to replicate the movement of an eyelid. We challenged the model by testing the residence performance of poloxamer®407 16 % and poloxamer®407 16 % + chitosan 1 % (PLX16CS10) gels containing fluconazole. The test was conducted with a simulated tear flow of 1.0 mL.min -1 for 5 min. The OphthalMimic successfully distinguished PLX16 and PLX16C10 formulations based on their fluconazole drainage (M1: 65 ± 14 % and 27 ± 10 %; M2: 58 ± 6 % and 38 ± 9 %; M3: 56 ± 5 % and 38 ± 18 %). In conclusion, the OphthalMimic is a promising tool for comparing the animal-free performance of ophthalmic formulations., 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 © 2024 Elsevier Inc. All rights reserved.)

In recent times, tear fluid analysis has garnered considerable attention in the field of biomarker-based diagnostics due to its noninvasive sample collection method. Tears encompass a reservoir of biomarkers that assist in diagnosing not only ocular disorders but also a diverse list of systemic diseases. This highlights the necessity for sensitive and dependable screening methods to employ tear fluid as a potential noninvasive diagnostic specimen in clinical environments. Considerable research has been conducted to investigate the potential of Raman spectroscopy-based investigations for tear analysis in various diagnostic applications. Raman Spectroscopy (RS) is a highly sensitive and label free spectroscopic technique which aids in investigating the molecular structure of samples by evaluating the vibrational frequencies of molecular bonds. Due to the distinct chemical compositions of different samples, it is possible to obtain a sample-specific spectral fingerprint. The distinctive spectral fingerprints obtained from Raman spectroscopy enable researchers to identify specific compounds or functional groups present in a sample, aiding in diverse biomedical applications. Its sensitivity to changes in molecular structure or environment provides invaluable insights into subtle alterations associated with various diseases. Thus, Raman Spectroscopy has the potential to assist in diagnosis and treatment as well as prognostic evaluation. Raman spectroscopy possesses several advantages, such as the non-destructive examination of samples, remarkable sensitivity to structural variations, minimal prerequisites for sample preparation, negligible interference from water, and the aptness for real-time investigation of tear samples. The purpose of this review is to highlight the potential of Raman spectroscopic technique in facilitating the clinical diagnosis of various ophthalmic and systemic disorders through non-invasive tear analysis. Additionally, the review delves into the advancements made in Raman spectroscopy with regards to paper-based sensing substrates and tear analysis methods integrated into contact lenses. Furthermore, the review also addresses the obstacles and future possibilities associated with implementing Raman spectroscopy as a routine diagnostic tool based on tear analysis in clinical settings., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

An extensive exploration of lacrimal fluid molecular biomarkers in understanding and diagnosing a spectrum of ocular and systemic diseases is presented. The chapter provides an overview of lacrimal fluid composition, elucidating the roles of proteins, lipids, metabolites, and nucleic acids within the tear film. Pooled versus single-tear analysis is discussed to underline the benefits and challenges associated with both approaches, offering insights into optimal strategies for tear sample analysis. Subsequently, an in-depth analysis of tear collection methods is presented, with a focus on Schirmer's test strips and microcapillary tubes methods. Alternative tear collection techniques are also explored, shedding light on their applicability and advantages. Variability factors, including age, sex, and diurnal fluctuations, are examined in the context of their impact on tear biomarker analysis. The main body of the chapter is dedicated to discussing specific biomarkers associated with ocular discomfort and a wide array of ocular diseases. From dry eye disease and thyroid-associated ophthalmopathy to keratoconus, age-related macular degeneration, diabetic retinopathy, and glaucoma, the intricate relationship between molecular biomarkers and these conditions is thoroughly dissected. Expanding beyond ocular pathologies, the chapter explores the applicability of tear biomarkers in diagnosing systemic diseases such as multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, and cancer. This broader perspective underscores the potential of lacrimal fluid analysis in offering non-invasive diagnostic tools for conditions with far-reaching implications., (Copyright © 2024. Published by Elsevier Inc.)

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.)

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.)

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.)

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.)

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: The purpose of this investigation was to analyze and compare the composition of meibum between type 2 diabetics with dry eye disease (DED) and control subjects to better reveal the pathologic mechanisms of the meibomian gland degeneration (MGD) and DED in type 2 diabetes mellitus (T2DM)., Methods: 90 subjects were divided into the following 4 groups: DM-DED group: T2DM patients with DED (n = 30); DM control group: DM patients without DED (n = 18); DED group: DED patients without DM (n = 26); naive control group: normal subjects (n = 16). The lipid composition of meibum samples collected from these subjects was analyzed by high-pressure liquid chromatography-mass spectrometry (HPLC-MS) system. The content of lipid features from 12 major lipid classes was compared among the 4 groups., Results: A significantly lower level of triacylglycerols (TG) and wax esters (WE) was found between DM-DED patients and normal controls (P < 0.01), whereas the level of Cholesteryl Ester (CE) in DM-DED patients increased compared with DED patients (P < 0.05). The level of (O-acyl)-omega-hydroxy fatty acids (OAHFA) in DM-DED patients was significantly lower than that in normal controls (P < 0.01). An opposite higher level of phospholipids (PLs) was observed in DM-DED patients than that in normal controls (P < 0.01)., Conclusions: T2DM could influence the expression of meibum lipids to further aggravate DED and MGD. Lower expression of TG,WE and OAHFA, higher expression of CE and PLs were discovered in meibum lipids of T2DM-DED., (Copyright © 2021. Published by Elsevier Ltd.)

Purpose: The dog is an important animal model for tear dysfunction diseases, however to-date the electrolyte composition of the dog's tears is unknown. The aim of this study was to analyze the electrolyte content of canine tears and compare it to serum and plasma., Methods: Tear samples were collected from 18 eyes of 9 dogs. Blood for serum was collected in tubes with no anticoagulants; plasma was obtained by using two different anticoagulants: Citrate-Phosphate-Dextrose (CPD) and heparin. The electrolytes were measured in all samples, analyzed, and compared., Results: Most of the electrolyte values in tears were statistically different (P < 0.05) from electrolyte values in serum and plasma. Potassium and chloride values were significantly higher in tears compared to serum and plasma, while calcium and phosphate values were significantly lower. Sodium values in tears were higher than in serum and heparinized-plasma, but lower than CPD-plasma. Bicarbonate values were lower in tears compared to serum and heparinized plasma, but was not statistically different than CPD-plasma. While magnesium values were lower in tears compared to serum and heparinized-plasma, the difference was not statistically different., Conclusions: Herein, we report for the first time the electrolyte composition of the canine tears and its comparison to serum and plasma., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Predictive, preventive and personalized medicine (PPPM) is a current concept in healthcare based on the analysis of biomarkers through non-invasive methods. Biomarkers for inflammation and oxidative stress are especially used for screening. Quantification of tear total protein content is important to identify potential, specific biomarkers, such as malondialdehyde concerning oxidative stress. The Schirmer strip test is an accessible and simple method for tear analysis. However, it is limited by the low concentration of biomarkers in the human tear. In this preliminary study, different procedures were compared for the extraction of tear proteins and malondialdehyde. Schirmer strips were used to obtain tears from healthy subjects. Ionic strength and surfactant agents were assessed, as well as different centrifugation parameters. Finally, several volumes of n-butanol on the process of malondialdehyde extraction were evaluated. The results showed that ionic strength strongly influences the extraction process, although most studies have suggested that surfactant agents are the most relevant factor; the most efficient results were obtained using a 2 M solution of NaCl in phosphate buffered saline. Regarding centrifugation, leaving the Schirmer strip tip left hanging outside the tube cap and using 1000 rpm was the best option, which is a lower centrifugation speed than the usually reported on literature. Moreover, 250 μL was the optimal n-butanol volume for malondialdehyde extraction. The importance of this study relies on the increasing relevance of the biomarkers in the field of PPPM and the need of a standardized method to extract biomarkers from the tears, to optimise its use., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Sex steroids impact regulation of the ocular surface tissues and thus influence dry eye. Most tissues in the human body synthesise and metabolise active sex steroids at levels required by the tissue. This is likely to also be the case for humans in ocular surface tissues. This study investigated the presence and quantities of selected sex steroids, in addition to sex steroid precursors and metabolites, in human tears. Detection of sex steroids in tears is challenging due to trace level analyte concentrations and low volumes of available tears. Immunoassays have previously been employed to assess sex steroids in tears, however, this approach only allows a single analyte to be measured and can overestimate concentrations. This study evaluated ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) and tandem mass spectrometry (MS/MS) methods for the concurrent detection and estimation of fourteen sex steroid and metabolite compounds in human tears. Basal tears were collected and pooled from 5 healthy pre-menopausal women (total 100 μL). Following protein precipitation and centrifuging, extract volumes equivalent to 14 μL of pooled tears were analysed. A Thermo Scientific Q Exactive™ Plus MS was used to compare novel high-resolution MS atmospheric pressure chemical ionization (APCI) and electrospray ionisation (ESI) methods for detection of fourteen target sex steroids, including dehydroepiandrosterone-sulphate (DHEAS), testosterone, oestradiol and their metabolites, using standards and pooled tears. The MS was programmed to switch between positive and negative polarity ionization modes at designated times through the UPLC run, in order to detect each analyte at optimal sensitivity. Analytes were analysed using APCI in standard mixtures at concentrations of 0.1, 0.3, 1, 3, 10, 30, 100 and 1000 pg/mL, per component, to determine the limits of detection (LOD) and quantification. Both parallel reaction monitoring (PRM) MS/MS and selected ion monitoring (SIM) MS, were evaluated by plotting narrow range mass-to-charge ratio (m/z) chromatograms for each analyte. An APCI UPLC-MS method to simultaneously measure 14 sex steroids using standards was successfully developed following comparative evaluation of all available LC-MS techniques. Preliminary experiments found APCI to be more sensitive than ESI using standards. Narrow m/z range SIM MS resulted in better sensitivity, in tear samples, than PRM. One sex steroid and two androgen metabolites were detected, with the developed APCI UPLC-MS method, and their concentrations estimated in human tears that had been extracted with a protein crash. Progesterone, androsterone-glucuronide (ADT-G) and 3αDiol-G were successfully detected in the tear extract and their concentrations in the pooled tear sample were estimated (with 95% confidence intervals) to be 0.10 ± 0.03 pg/μL, 30.9 ± 18.3 pg/μL and 9.8 ± 4.3 pg/μL respectively. The concentrations of the remaining 11 sex steroids in the tear sample were below the LODs of the method. This work shows that high mass resolution UPLC-MS can detect certain sex steroids and metabolites in tears, but that sensitivity of the technique and the low available tear volumes limit its application to a broader range of sex steroids. The investigation of sex steroids in tears and ocular surface tissue will aid understanding of the influence of sex steroids on ocular surface tissues facilitating better targeted treatment for dry eye., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The meibomian gland is the major contributor to the tear film lipid layer. It is generally accepted that meibomian gland secretions, i.e, meibum, play a critical role in the homeostasis of the tear film. Lipid components of meibum and their structure, as well as functions were intensively studied. However, the proteins from meibum have not attracted enough attention. This review summarizes current knowledge about protein components of the meibum, particularly their function on tear film and ocular surface, and changes in the proteins during meibomian gland dysfunction (MGD)., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Meibomian glands secrete meibum, which gives rise to the lipid layer of the tear film and thereby prevents excessive evaporation of tear fluid. Meibomian gland dysfunction (MGD) is a major causative condition of evaporative dry eye, which is more common than the aqueous-deficient type of dry eye. Noninvasive meibography relies on infrared light and an infrared-sensitive camera to reveal the morphology of meibomian glands in both the upper and lower eyelids, whereas tear interferometry allows both qualitative and quantitative evaluation of the lipid layer of the tear film. These two techniques not only provide valuable clinical information related to dry eye but also allow clinical evaluation of MGD. Tear interferometry also has the potential to distinguish the condition of the tear film between normal individuals and dry eye patients. Furthermore, combined evaluation of the noninvasive breakup time of the tear film and the interferometric fringe pattern as determined by tear interferometry allows classification of the subtype of dry eye disease., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Meibum is a lipid-rich secretion that is produced by fully differentiated meibocytes in the holocrine Meibomian glands (MG) of humans and most mammals. The secretion is a part of a defense mechanism that protects the ocular surface from hazardous environmental factors, and from desiccation. Meibomian lipids that have been identified in meibum are very diverse and unique in nature. The lipid composition of meibum is different from virtually any other lipid pool found in the human body. In fact, meibum is quite different from sebum, which is the closest secretion that is produced by anatomically, physiologically, and biochemically related sebaceous glands. However, meibum of mice have been shown to closely resemble that of humans, implying similar biosynthetic mechanisms in MG of both species. By analyzing available genomic, immunohistochemical, and lipidomic data, we have envisioned a unifying network of enzymatic reactions that are responsible for biosynthesis of meibum, which we call meibogenesis. Our current theory is based on an assumption that most of the biosynthetic reactions of meibogenesis are catalyzed by known enzymes. However, the main features that make meibum unique - the ratio of identified classes of lipids, the extreme length of its components, extensive ω-hydroxylation of fatty acids and alcohols, iso- and anteiso-branching of meibomian lipids (e.g. waxes), and the presence of rather unique complex lipids with several ester bonds - make it possible that either the activity of known enzymes is altered in MG, or some unknown enzymes contribute to the processes of meibogenesis, or both. Studies are in progress to elucidate meibogenesis on molecular level., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Dysfunction of the meibomian glands alters secreted meibum quantitatively and qualitatively that can lead to damage to the ocular surface epithelium. In response to an unstable tear film cause by meibomian gland dysfunction, ocular surface epithelium is damaged and expresses inflammatory cytokines leading to secondary ocular inflammation. In turn, inflammatory disorders of the palpebral conjunctiva and lid margin may affect the structure and function of meibomian gland. The disorders include allergic conjunctivitis, long-term usage of contact lenses, dermatological diseases that affect conjunctival homeostasis, Stevens-Johnson's syndrome or chemical burning of the ocular surface and lid margin., (Copyright © 2017. Published by Elsevier Ltd.)

To examine the relation between changes in the free fatty acid (FFA) composition of human meibum and both objective signs and subjective symptoms of meibomian gland dysfunction (MGD), we analyzed the FFA content of meibum collected from both MGD patients and control subjects. Thirty-eight patients with MGD (13 men and 25 women; mean age ± SD, 66.9 ± 15.0 years) were evaluated. Various objective signs and subjective symptoms of MGD were assessed. Meibum was analyzed by liquid chromatography-Fourier transform mass spectrometry, and the relation between the FFA composition of meibum and each objective sign and subjective symptom was examined by principal component analysis (PCA). No relation was apparent between the FFA composition of meibum and individual subjective symptoms or objective signs of MGD. However, a PCA score plot for meibum samples grouped on the basis of the severity of both telangiectasia and plugging of meibomian gland orifices revealed clear separation of mild and severe groups. This separation of the two groups was largely due to a significantly increased linoleic acid content in meibum of the severe group (3.56%, versus 0.70% of total FFAs in the mild group). The relative amount of linoleic acid in meibum was thus associated with the severity of telangiectasia and plugging of gland orifices in MGD, suggesting that this FFA might contribute to the pathogenesis of these signs., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This review critically evaluates a broad range of literature in order to show the relationship between meibum, tear lipids and the tear film lipid layer (TFLL). The relationship of meibum composition to dry eye syndrome is briefly discussed. The review also explores the interactions between aqueous and the TFLL by examining the correlations between meibomian lipids and lipids extracted from whole tears, and by considering protein adsorption to the TFLL from the aqueous. Although it is clear to the authors that a normal tear film resists evaporation, an emerging idea from the literature is that the main purpose of the TFLL is to allow the spread of the tear film and to prevent its collapse onto the ocular surface, rather than to be an evaporative blanket. Current models on the possible structure of the TFLL are also examined., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The main focus in clinical proteomics is the discovery of new protein or peptide biomarkers which are correlated with a certain disease. Tear proteins have been investigated extensively in the past and distinct relations between the levels of certain tear proteins to different disorders have been demonstrated. In this review we attempt to summarize proteomic technologies for biomarker identification in tears and some disease related biomarkers in tear fluids that were discovered through different proteomic techniques in different conditions like dry eye, Sjogren's syndrome, contact lens wearers, glaucoma, diabetic retinopathy or cancer. Proteomic analysis of tear fluid has proven to be a promising to gain more information about the pathogenesis of diseases and lead to new diagnostic possibilities. Furthermore, biomarkers represent promising targets for drug development and can be used to monitor the disease state or treatment responses, and accordingly improve the standards of patient care., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The tear film coats the cornea and conjunctiva and serves several important functions. It provides lubrication, prevents drying of the ocular surface epithelia, helps provide a smooth surface for refracting light, supplies oxygen and is an important component of the innate defense system of the eye providing protection against a range of potential pathogens. This review describes both classic antimicrobial compounds found in tears such as lysozyme and some more recently identified such as members of the cationic antimicrobial peptide family and surfactant protein-D as well as potential new candidate molecules that may contribute to antimicrobial protection. As is readily evident from the literature review herein, tears, like all mucosal fluids, contain a plethora of molecules with known antimicrobial effects. That all of these are active in vivo is debatable as many are present in low concentrations, may be influenced by other tear components such as the ionic environment, and antimicrobial action may be only one of several activities ascribed to the molecule. However, there are many studies showing synergistic/additive interactions between several of the tear antimicrobials and it is highly likely that cooperativity between molecules is the primary way tears are able to afford significant antimicrobial protection to the ocular surface in vivo. In addition to effects on pathogen growth and survival some tear components prevent epithelial cell invasion and promote the epithelial expression of innate defense molecules. Given the protective role of tears a number of scenarios can be envisaged that may affect the amount and/or activity of tear antimicrobials and hence compromise tear immunity. Two such situations, dry eye disease and contact lens wear, are discussed here., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Biochemical changes brought about by the influence of the contact lens on the tear film are conveniently split into two categories. Firstly, the lens can remove or reduce the levels of specific components in the tear film, and secondly, the lens can augment the tear film, by stimulating the influx of new components or increasing the level of existing components. The most obvious tear film components for study in this context are lipids, proteins, mucins and electrolytes. The interactions are affected by the properties of the lens, the characteristics of the individual wearer and the wear schedule. An additional complicating factor is the fact that the lens is many times thicker than the tear film and any immobilised tear components will be more extensively exposed to oxygen and UV radiation than is the case in the absence of a lens. It is arguably the lipoidal components that are most markedly affected by lens wear, since their immobilisation on the lens surface markedly increases their susceptibility to autoxidative degradation. The limited information that is available highlights the importance of subject specificity and suggests that lipid oxidation phenomena are potentially important in contributing to the 'end of day' discomfort of symptomatic contact lens patients. It is clear that tear lipids, although regarded as relatively inert for many years, are now seen as a reactive and potentially important family of compounds in the search for understanding of contact lens-induced discomfort. The influence of the lens on tear proteins shows the greatest range of complexity. Deposition and denaturation can stimulate immune response, lower molecular weight proteins can be extensively absorbed into the lens matrix and the lens can stimulate cascade or upregulation processes leading either to the generation of additional proteins and peptides or an increase in concentration of existing components. Added to this is the stimulating influence of the lens on vascular leakage leading to the influx of plasma proteins such as albumin. The evidence from studies of mucin expression in tears is not consistent and conclusive. This is in part because sample sources, lens materials and methods of analysis vary considerably, and in some cases the study population numbers are low. Expression levels show mucin and material specificity but clear patterns of behaviour are elusive. The electrolyte composition of tears is significantly different from that of other body fluids. Sodium and potassium dominate but potassium ion concentrations in tears are much higher than in serum levels. Calcium and magnesium concentrations in tears are lower than in serum but closer to interstitial fluids. The contact lens provides the potential for increased osmolarity through enhanced evaporation and differential electrolyte concentrations between the anterior and posterior tear films. Since the changes in ocular biochemistry consequent upon contact lens wear are known to be subject-dependent - as indeed is wearer response to the lens - pre-characterisation of individual participant tear chemistry in clinical studies would enhance understanding of these complex effects., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Tear film stability decreases with age however the cause(s) of the instability are speculative. Perhaps the more saturated meibum from infants may contribute to tear film stability. The meibum lipid phase transition temperature and lipid hydrocarbon chain order at physiological temperature (33 °C) decrease with increasing age. It is reasonable that stronger lipid-lipid interactions could stabilize the tear film since these interactions must be broken for tear break up to occur. In this study, meibum from a pool of adult donors was saturated catalytically. The influence of saturation on meibum hydrocarbon chain order was determined by infrared spectroscopy. Meibum is in an anhydrous state in the meibomian glands and on the surface of the eyelid. The influence of saturation on the surface properties of meibum was determined using Langmuir trough technology. Saturation of native human meibum did not change the minimum or maximum values of hydrocarbon chain order so at temperatures far above or below the phase transition of human meibum, saturation does not play a role in ordering or disordering the lipid hydrocarbon chains. Saturation did increase the phase transition temperature in human meibum by over 20 °C, a relatively high amount. Surface pressure-area studies showing the late take off and higher maximum surface pressure of saturated meibum compared to native meibum suggest that the saturated meibum film is quite molecularly ordered (stiff molecular arrangement) and elastic (molecules are able to rearrange during compression and expansion) compared with native meibum films which are more fluid agreeing with the infrared spectroscopic results of this study. In saturated meibum, the formation of compacted ordered islands of lipids above the surfactant layer would be expected to decrease the rate of evaporation compared to fluid and more loosely packed native meibum. Higher surface pressure observed with films of saturated meibum compared to native meibum suggests greater film stability especially under the high shear stress of a blink., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The factors responsible for the conjunctivitis and iritis associated with acute ocular infection and post enteric inflammatory disease are not fully known. The pro-inflammatory activity of unilateral topical application of muramyl dipeptide (MDP; the smallest bio-active Gram-positive and Gram-negative bacterial cell wall component) was investigated in adult rabbits. The resultant bilateral conjunctivitis/iritis and pyogenic responses were characterized. Bilateral symptoms were graded by slit lamp examinations; tear fluid, Schirmer tests (tear production), blood and aqueous humor (AH) samples were obtained from MDP-treated and untreated rabbits. MDP concentration, gamma-glutamyltranspeptidase activity (GGT; key enzyme in glutathione recapture, xenobiotic detoxification, eicosanoid synthesis and neutrophil function), protein concentration, and tear cell density, cytology, and immunofluorescent antibody reactivity to GGT and calreticulin (CRT; MDP-binding protein) were determined. MDP was cleared from ipsilateral tears and serum by 6 h, but was undetected in mock-treated contralateral tears. Bilateral signs of acute transient pyogenic conjunctivitis, characterized by tearing, lid edema, conjunctival hyperemia, chemosis and leukocytic infiltrate with iritis (erythema and aqueous flare) were detected. Milder symptoms occurred in the mock-treated contralateral eyes. Bilateral symptoms, tear production, tear protein, GGT activity, and mucopurulent discharge (containing up to 2.5-5.0 × 10(6) cells/mL) were elevated 4-8 h post MDP and resolved to near pre-treatment levels by 24 h. Tear GGT activity and protein levels were higher in MDP-treated and mock-treated contralateral eyes than in eyes of untreated adult rabbits (p's < 0.001). Elevated tear GGT activity was associated with histopathology and increased vascular and epithelial permeability to serum protein, GGT-positive epithelia cells, macrophages and heterophils. Repeat MDP applications induced recurrent induction and resolution patterns of bilateral conjunctivitis/iritis and tear GGT activity, but ipsilateral GGT responses were lower. The results suggest unilateral topical MDP application to adult rabbit eyes induces a bilateral acute pyogenic conjunctivitis/iritis (PCI) characterized by increased vascular and epithelial permeability similar to acute bacterial conjunctivitis in man. The detection of CRT/GGT positive heterophils in tears suggests efferocytosis (phagocytosis of dead/dying cells). Tear GGT activity may be a useful means to quantify MDP-induced toxicity and extraocular inflammation., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

(O-acyl) ω-hydroxy fatty acids (OAHFAs) are a recently found group of polar lipids in meibum. Since these lipids can potentially serve as a surfactant in the tear film lipid layer, the surface properties of a molecule of this lipid class was investigated and compared with a structurally related wax ester and a fatty acid. (O-oleyl) ω-hydroxy palmitic acid was synthesized and used as the model OAHFA. It was spread either alone or mixed with human meibum on an artificial tear buffer in a Langmuir trough, and pressure-area isocycle profiles were recorded at different temperatures and compared with those of palmityl oleate and oleic acid. These measurements were accompanied by fluorescence microscopy of meibum mixed films during pressure-area isocycles. The pressure area curves indicated that pure films of the model OAHFA are as surface active as oleic acid films, cover a much larger surface area than either palmityl oleate or oleic acid and show a distinct biphasic pressure-area isocycle profile. The OAHFAs appeared to remain on the aqueous surface and show only a minor re-arrangement into multi-layered structures during repetitive pressure area isocycles. All these properties can be explained by OAHFAs binding weakly to the aqueous surface via an ester group and strongly via a carboxyl group. By contrast, the pressure area profiles of palmityl oleate films indicate that they form multi-layers and oleic acid presumably forms micelles and desorbs into the subphase. When mixed with meibum, similar features as for pure films were observed. In addition, meibum-OAHFA films appeared very homogeneous; a feature not seen with other mixtures. In conclusion these data support the notion that the tested OAHFA is a very potent surfactant which is important in spreading and stabilising meibomian lipid films., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Recent NMR studies suggest that unsaturation may contribute to tear film instability in adults and loss of cholesteryl esters and squalene could reduce tear film stability in adults with meibomian gland dysfunction. The proton resonances were tentatively assigned in those studies. In this current investigation, meibum from seven infants and children, one adult and a pool of adult meibum have been analyzed using an NMR spectrometer with greater sensitivity and spectral resolution. The goals of this work are to confirm/correct the previous assignments and to determine possible age-related changes in composition. The initial resonance assignments were confirmed using heteronuclear single quantum correlation spectroscopy. Because there were no significant interferences in the spectral region corresponding to the resonances for cholesteryl and wax esters, the areas of these resonances were used to calculate their molar ratios. We calculated a wax ester:cholesteryl ester molar ratio of 1:0.57 ± 0.05 for all our meibum samples and there were no age-related differences. At lower film thicknesses, the rate of evaporation measured in vitro was lower for wax esters mixed with a long chain cholesteryl ester compared to wax esters alone. However, the film thicknesses tested were non-physiological. Longer chain cholesteryl esters increase the interactions between hydrocarbon chains. Hydrocarbon chains were more saturated in meibum from infants and children compared to adults. Unsaturation may contribute to tear film instability in adults. Loss of cholesteryl ester and squalene could destabilize tear film in adults with meibomian gland dysfunction., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The tear film covers and protects the ocular surface. It contains various molecules including a large variety of proteins. The protein composition of the tear fluid can change with respect to various local and systemic diseases. Prior to the advent of the proteomic era, tear protein analysis was limited to a few analytical techniques, the most common of which was immunoelectrophoresis, an approach dependent on antibody availability. Using proteomics, hundreds of tear proteins could potentially be identified and subsequently studied. Although detection of low-abundance proteins in the complex tear proteome remains a challenge, advances in sample fractionation and mass spectrometry have greatly enhanced our ability to detect these proteins. With increasing proteomic applications, tears show great potential as biomarkers in the development of clinical assays for various human diseases. In this chapter, we discuss the structure and functions of the tear film and methods for its collection. We also summarize potential tear protein biomarkers identified using proteomic techniques for both ocular and systemic diseases. Finally, modern proteomic techniques for tear biomarker research and future challenges are explored.

The proteomic profile of tear fluid is of fundamental interest in eye research. In this study we optimized the tear sample preparation method for two-dimensional (2D) analysis and determined the protein profile of tear fluid from healthy males and females. To find the most efficient method for tear sample preparation, four widely applied precipitation methods and ultrafiltration were compared. Of these, TCA precipitation & ultrafiltration resulted in efficient sample concentration and desalting. Use of a nonionic wetting agent, Tergitol NP7, in rehydration solution during isoelectric focusing improves protein separation in 2D gel electrophoresis considerably. Using this optimized method, tear protein profile was analyzed from healthy males and females. Of the thirty six tear proteins identified by LC-MS/MS, seven tear proteins were found to be significantly up regulated in the healthy female tear samples when compared to the male tear samples. These results indicate that the tear protein profile differs with respect to the sex. Mostly, the up regulated proteins are involved in the local immune defense; implying that there may be a sex difference in the ability to defend against infection at the anterior segment of the eyes of normal individuals., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Proteins are very important components in tears. Their phosphorylation is an important posttranslational modification affecting biological activity. Using proteomic techniques, this study was designed to analyze phosphoproteins found in open eye basal tears from normal human subjects. Proteins in tear samples were separated in 1-dimensional (1D) and 2-dimensional (2D) gels and phosphoproteins were selectively stained with Pro-Q diamond dye before visualization of all proteins using Sypro Ruby. Potential phosphoproteins in 2D gels were identified by liquid chromatography-mass spectrometry (LC-MS/MS) after trypsin digestion and phosphopeptide enrichment using titanium dioxide (TiO(2)) columns. The tryptic digests of the tear samples were also analyzed to identify phosphoproteins directly by LC-MS/MS after phosphopeptide enrichment. The major phosphoprotein stained by Pro-Q diamond in the gels and identified by LC-MS/MS from the spots was tear lipocalin. Tear lipocalin was separated into 3 different isoforms and one phosphorylation site (serine at position 24) was identified in one of the isoforms. Prolactin-induced protein, nucleobindin-2 and lipophilin C were also stained with Pro-Q diamond although no phosphorylated peptides from these proteins could be found using LC-MS/MS. Direct analysis of the tear tryptic digests by LC-MS/MS identified a further 12 potential phosphoproteins with tear lipocalin predominant. Four phosphorylation sites (position 24 (serine), 32 (serine), 34 (threonine) and 36 (tyrosine)) were identified for tear lipocalin using this method. These results indicate that tear lipocalin is the predominant phosphoprotein in normal human basal tears. Nucleobindin-2, prolactin-induced protein and lipophilin C also appear to be phosphorylated in basal tear samples., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Lactoferrin plays an important role in the defense against infections, including herpes simplex virus (HSV) keratitis. We studied the impact of three single nucleotide polymorphisms in the human lactoferrin gene on the susceptibility to HSV infections of the eye and the severity of such infections. Lactoferrin gene polymorphisms were determined by PCR combined with restriction fragment length analysis in 105 HSV keratitis patients and 145 control subjects. Bilateral tear samples were harvested from 50 patients and 40 healthy controls and tear lactoferrin concentrations were determined by ELISA. Patients' records were used to acquire information about the severity of the HSV keratitis. The frequencies of the Glu561Asp polymorphism, but not those of the Ala11Thr and Lys29Arg polymorphisms, differed significantly between patients and control subjects with an under-representation of the Asp561 allele in the patient group. Furthermore, the values for best corrected visual acuity, frequency of recurrences since onset, and average duration of clinical episodes did not differ among patients with various lactoferrin genotypes. In addition, tear lactoferrin concentrations were the same in patients with HSV keratitis and healthy controls and also did not differ among patients with various lactoferrin genotypes. Lactoferrin Glu561Asp polymorphism is associated with the susceptibility to HSV keratitis with a protective role for lactoferrin variants comprising Asp561. However, no beneficial effects of this lactoferrin variant on the clinical outcome of ocular HSV keratitis were noted.

To evaluate the use of stationary phase protein array technology for tear analysis and to characterize the distribution of inflammatory mediators in normal and allergic tears in the open and closed eye states. Microcapillary tube collected Open (OTF) and closed eye tear fluid (CTF) samples from normals (N), from individuals with various active chronic ocular and other allergies (CA), as well as from an individual subsequent to unilateral induction of an acute allergic conjunctivitis were assayed using membrane arrays that were optimized to allow the detection of GM-CSF, ILs-1 alpha, 1 beta, 2-10, 12-13, INF gamma, MCP-1 and TNFalpha in clinical size samples. The protocol of a micro-well plate array specific for ILs-2, 4, 5, 8, 10, 12, 13, TNFalpha and INF gamma was modified to minimize the impact of tear matrix effects. This was used to carry out parallel analysis on selected samples. By optimizing the protocol as well as the composition of a membrane array it proved possible to significantly increase the signal-to-noise ratio and sensitivity of assay allowing for the detection of some inflammatory mediators into the sub-picogram range. This provided sufficient sensitivity to allow the assay of clinically obtainable size samples. Analysis revealed that OTF from most Ns contained a high level of IL-8 and faint signals if any for the other probed proteins. In contrast, OTF samples from most CA individuals with and without ocular symptoms exhibited to varying degrees detectable levels of most of the other probed entities. The difference between normal and pathological tears and the levels of signals became far more pronounced in the CTF compared to the OTF samples. Use of the micro-well plate assay kit without modification revealed two tear matrix effects that profoundly impact the ability to obtain meaningful ELISA data. Modifying the assay protocol reduces but does not eliminate these artifacts making it possible to approximate the concentration of many of the probed entities. The obtained data is consistent using both methodologies revealing elevated levels of IL-8 and other cytokines in approximately 60% of the OTF samples from the CA population. Other than a modest increase in IL-8, no change could be observed in the profile of OTF after induction of an acute allergic reaction.

Mucin genes, both secreted (MUC2, MUC5AC, MUC5B, MUC7) and membrane associated (MUC1, MUC4, MUC16), have been reported to be expressed by ocular surface epithelia. The purpose of this study was to comprehensively assay the mucin content of human tear fluid using multiple antibodies for each mucin and to develop a sensitive, semi-quantitative method for the assay of mucins in tears. Tear washes were obtained by instillation of saline onto the ocular surface, followed by collection from the inferior fornix. Tear proteins were separated in 1% agarose gels, transferred to nitrocellulose membrane by vacuum blotting and probed with multiple antibodies recognizing MUC1, MUC2, MUC4, MUC5AC, MUC5B, MUC7 and MUC16. Binding was detected using chemiluminescence, and quantity was determined by densitometry. Serial dilutions of pooled tears from normal individuals were assayed to determine the linear range of detectability. MUC1, MUC4, MUC16, MUC5AC and low levels of MUC2 were consistently detected in human tear fluid, while MUC5B and MUC7 were not. Use of several antibodies recognizing different epitopes on the same mucin confirmed these findings. The antibodies to mucins bound to serial dilutions of tears in a linear fashion (r2 > 0.9), indicating the feasibility of semi-quantitation. MUC5AC in tear fluid had an increased electrophoretic mobility compared to MUC5AC isolated from conjunctival tissue. This study provides clear evidence that the mucin component of tears is a mixture of secreted and shed membrane-associated mucins, and for the first time demonstrates MUC16 in tear fluid. Immunoblots of tears using agarose gel electrophoresis and chemiluminescence detection provide a semi-quantitative assay for mucin protein that will be useful for comparisons with tears from diseased eyes or after pharmacological intervention.

Pseudomonas aeruginosa is a pathogen gaining prevalence in contact lens-related corneal ulcers. Tear outflow protects the ocular surface, where high molecular weight tear glycoproteins bind bacteria for removal from the eye. The purpose of the present study was to identify glycoproteins in human tears involved in the adhesion of ocular P. aeruginosa isolates. Basal human tears were applied to a bacterial adhesion assay involving electrophoretic separation of tear components, transfer to nitrocellulose and incubation with biotin-labelled bacteria. Glycoproteins were further characterised using lectin profiling. The results showed large-dimension agarose gels were imperative for the detection of at least four glycoproteins with a migration >200 kDa, including species not previously identified. P. aeruginosa 6294 preferentially bound to a well-defined glycoprotein near the origin of the gel that, unlike other glycoproteins >200 kDa, reacted with Sambucus nigra lectin (sialic acid alpha2-6) but not WGA lectin (N-acetylglucosamine, sialic acid alpha2-3). Adhesion did not involve free biotin label or hydrophobic interactions. Also, the pre-incubation of separated tear glycoproteins with S. nigra lectin increased subsequent adhesion of 6294 to this tear glycoprotein. The less virulent Paer1 strain showed diffuse adhesion in the S. nigra-reactive region at the gel origin. In conclusion, an overlay adhesion assay was developed that identified slow-migrating sialylated glycoprotein species in human tears preferentially bound by P. aeruginosa ocular strains, and S. nigra lectin seemed to enhance the interaction. The study provides a basis for direct investigation of bacterial adhesion to glycoproteins with an apparent migration >200 kDa in tear fluid.

Mucins are highly glycosylated proteins that are vital to the maintenance of healthy epithelial surfaces including the ocular surface. Mucins act as lubricants, protectants, and mediators of signal transduction. The majority of the O-glycosylation sites on the transmembrane mucin MUC1 are found in a highly polymorphic core region containing a variable number of tandem repeats (VNTR). MUC1 alleles can be divided into size classes that contain small (30-45) or large (60-90) numbers of repeats. Although at least 12 splice variants of MUC1 have been found in other tissues, no splice variants have been reported in human ocular surface tissues. We have used RT-PCR to identify MUC1 splice variants that were then confirmed by sequencing. We here report the presence in some samples of human cornea, conjunctiva, and lacrimal gland of MUC1/B which features canonical splicing between exons 1 and 2 and MUC1/A, a transcript that retains 27bp from the 3' end of intron 1 and is predicted to add 9 amino acids to the MUC1 sequence upstream of the tandem repeat region. Cornea and conjunctiva both contain the MUC1/SEC splice variant that lacks the transmembrane domain and, therefore, results in a soluble, secreted form of MUC1. Cornea and conjunctiva also contain MUC1/Y and MUC1/Z(X) variants that lack the tandem repeat region. Cornea, conjunctiva, and lacrimal gland also contain a previously undescribed MUC1 variant transcript, termed MUC1/YI, that retains 99bp from the 5' end and 27bp from the 3' end of the first intron, resulting in a frame shift and premature stop codon. This transcript is predicted to produce a novel 27 amino acid peptide after signal peptidase cleavage. Analysis of brush cytology samples revealed that the percentage of dry eye patients expressing the MUC1/A variant in the conjunctival epithelium is lower than in normal control donors. Western blotting confirmed that MUC1/A is associated with alleles containing the large size class of tandem repeats. Therefore, we propose that one factor in susceptibility to dry eye disease may be the lengths of the MUC1 VNTR in conjunctival epithelium based on the rationale that longer VNTR provide better lubrication and greater protection of the ocular surface against inflammation.

Nerve growth factor in the tear film and corneal epithelium is hypothesized to play an important role in ocular surface maintenance and corneal wound healing. The purpose of this study was to determine the expression of nerve growth factor and its high affinity (trkA) receptor in tears, cornea, and lacrimal glands of normal dogs, the modulation of nerve growth factor and its trkA receptor during corneal wound healing, and the effect of topical nerve growth factor application on canine corneal epithelial wound healing. In the first of three experiments, the nerve growth factor content of tears, corneal epithelium, lacrimal gland, and 3rd eyelid gland was determined in normal dogs by enzyme-linked immunosorbent assay and the expression of nerve growth factor and its trkA receptor were evaluated in the cornea and lacrimal glands by immunohistochemistry. In a second experiment, unilateral corneal epithelial defects were created, and tissues were evaluated for changes in nerve growth factor or trkA expression for 1 week. In a third experiment, bilateral corneal epithelial defects were created and the right eyes in each animal were treated 4 times daily with either recombinant human nerve growth factor, murine nerve growth factor, or nerve growth factor-blocking antibody. The results of this study showed that nerve growth factor levels in normal dog tears, corneal epithelium, third eyelid gland and lacrimal gland were 15.4+/-4.6 ng ml(-1), 33.5+/-12.3, 52.4+/-17.4 and 48.8+/-9.4 ng g(-1), respectively. NGF and trkA receptors were identified by immunohistochemistry in all tissues examined. After unilateral corneal wounding, nerve growth factor concentration increased in the tears bilaterally for 3 days, especially in the wounded eye, and then returned to pre-wounding values. Nerve growth factor content, and immunohistochemical staining for nerve growth factor and trkA, increased significantly in the ipsilateral cornea epithelium following unilateral wounding. Nerve growth factor concentrations in lacrimal and third eyelid glands also increased bilaterally (p<0.01) after unilateral wounding. Time to wound closure and rate of epithelial migration did not differ significantly between nerve growth factor-treated, nerve growth factor antibody-treated, and control eyes. In conclusion, nerve growth factor is present under resting physiologic conditions in normal canine tears, and nerve growth factor and its trkA receptor are present under resting conditions in normal canine corneal epithelium, lacrimal gland and third eyelid gland. Nerve growth factor is elevated in the tears, cornea, and lacrimal glands after corneal epithelial wounding; however, topical application of nerve growth factor, or its blocking antibody does not modulate corneal wound healing in the normal dog eye.

Many ocular surface tests have been developed for the clinical diagnosis of keratoconjunctivitis sicca, and several have been used to 'validate' animal models of Sjogren's and non-Sjogren's dry eye. However, many of these tests themselves have not been systematically studied or standardized, and yet their use in animal models of dry eye is common. This review provides a rational approach and systematic review of the tear film and ocular surface tests described in the literature that may be applicable to the current animal models of dry eye, with particular emphasis on their limitations, along with some suggestions regarding their standardization and applications in eye research.

The interaction between the tear film and the ocular surface epithelium is crucial for the maintenance of ocular surface health; interference with this relationship may cause dry eye. Several diagnostic techniques have been developed to assess the tear film and diagnose dry eye but many of these tests are invasive and modify the parameter which they are designed to measure. Non-invasive or minimally invasive tests may overcome this problem and provide more reproducible and objective data. One test of this kind is meniscometry, which is particularly useful in assessing tear volume indirectly by measuring tear meniscus radius. The newly developed video-meniscometer, which enables calculation of the meniscus radius digitally, is useful for the diagnosis of tear-deficient dry eye. Video-meniscometry also has other applications, to the study of tear and eye drop turnover, determining the indication for punctal plugs and in demonstrating dysfunction of the tear meniscus. Interferometry of the tear film lipid layer is useful in screening and evaluating dry eye severity and in selecting dry eye candidates for punctal occlusion. It is also useful for analysing tear lipid layer pathophysiology more clearly, especially in combination with meniscometry. Meibometry is a minimally invasive technique to quantify the amount of meibomian lipid on the lid margin. Lipid is blotted onto a plastic tape and the change in optical density is used to calculate lipid uptake. Laser meibometry has increased the scope of this technique for the assessment of meibomian gland dysfunction; also, the delivery of lipids from the lid reservoir to the preocular tear film can be analysed using interferometry and laser meibometry. The present report reviews the application of these techniques to the study of tear film physiology and dry eye.

The lipid layer is an essential component of the tear film, providing a smooth optical surface for the cornea and retarding evaporation from the eye. The meibomian lipids which compose it are well adapted for this purpose. They form a thin, smooth film whose thickness, and probably composition, influences the rate of evaporation. Their melting range ensures sufficient fluidity for delivery to the tear film from the lid margin reservoirs, while the film itself may exhibit a higher viscosity at the cooler temperature of the ocular surface. The factors governing lipid film formation during the blink are not fully understood, but one view is that the polar lipids, interacting with the aqueous sub-phase of the tear film, spread in advance of the non-polar components, which form the bulk of the film. The meibomian lipids stabilise the tear film by lowering its free energy; they carry water into the film during its formation and interact with lipid-binding proteins in the aqueous phase, such as tear lipocalin. The lipocalins, complexed with other tear components, may also contribute to the high, non-Newtonian viscosity of the tear film and its low surface tension, features which are essential for tear film stability. Formation of the lipid film is a complex process. Lipid is delivered to the tear film in the up-phase of the blink, more from the lower than the upper reservoir. The lipid layer comes to a stop well after completion of the blink and remains relatively immobile until it is compressed in the down-phase of the blink that follows. Then, it either retains its structure in a series of subsequent blinks, or is completely re-constituted after mixing with the reservoir lipids. Delivery of meibomian lipid to the marginal reservoirs is mainly the result of continuous secretion, under neural and hormonal control, supplemented by lid action. The reservoirs provide a hydrophobic barrier to tear overspill and to contamination by skin lipids which might destabilise the tear film. They probably also provide the chief route for meibomian lipid excretion.

Both for tear films and along the airways within the lung, one has an extremely thin fluid layer overlying a biological substrate; in both cases surfactants either of natural origin, or artificially introduced, are important in driving fluid flows. There is evidence that slip can occur when hydrophilic liquids, similar to mucus or aqueous tear films, overlie hydrophobic epithelium. Utilizing results from recent experimental findings we examine the possible influence of slip upon tear film rupture, important in so-called dry eye, and upon surfactant-induced flows within the lung, used in surfactant replacement therapy.

We investigate the rupture mechanism of a precorneal thin mucus coating sandwiched between the aqueous tear film and the corneal epithelial surface with a monolayer of surfactant overlying the aqueous layer. The Ostwald constitutive relation is employed to model mucus and a linear equation of state describing the relationship between surface tension and surfactant concentration is adopted. Three nonlinear coupled evolution equations governing the transport of surfactant, mucus, and total liquid layer thicknesses, based on lubrication theory and a perturbation expansion technique, have been derived. The resulting equations are solved numerically in order to explore the influence of the rheological properties of mucus, aqueous-mucus thickness ratio, aqueous-mucus interfacial tension, Marangoni number, and surfactant concentration on both the onset of instability and tear film evolution in the presence of van der Waals interactions, which could rupture the tear film. Our results reveal that the influence of rheological properties, aqueous-mucus thickness ratio, and interfacial tension on the time required for film rupture can be significant and varies considerably, depending on the magnitude of the Hamaker constants governing the strength of the van der Waals forces.

The lacrimal urea content was found to be proportional to that of blood, which suggested its possible utilization in the monitoring of hemodialysis as a less invasive method. On the other hand, however, arginase activity was detected in tears, which may influence the urea content independently of blood urea concentration. The feasibility of using lacrimal urea measurement to replace blood urea measurement in the monitoring hemodialysis was also investigated. Blood and tear samples of 35 healthy persons and 43 renal patients undergoing hemodialysis were tested. Tear samples were collected on Schirmer paper strips. After elution the lacrimal urea content was measured by a colorimetric method. The determination of arginase activity was based on the release of urea and ornithine. The correlation between blood and lacrimal urea and arginase was studied by multivariate regression analysis. The lacrimal arginase isoenzyme pattern was investigated by native polyacrylamide gel electrophoresis and Western blotting. The effect of partially isoform-specific inhibitors was also studied. Blood urea levels in blood were significantly higher in the renal patients before dialysis than in the control patients (12.86 +/- 0.59 vs. 6.45 +/- 0.41 mM, p < 0.0001). Blood sera arginase activity was very low. Lacrimal arginase activity was significantly higher in tears than in sera (p < 0.0001 for each group). The tear/serum ratio of urea content was significantly different between controls and renal patients, particularly in postdialytic samples (1.89 +/- 0.07 vs. 3.49 +/- 0.31, p < 0.0001). The correlation between lacrimal and blood sera urea was best in controls (r = 0.89) and was better in predialytic (r = 0.75) than in postdialytic (r = 0.52) samples, depending on the level of arginase activity. In postdialytic samples a stronger correlation (r = 0.77) between tear urea and arginase was observed. Both arginase isoforms were detected in tears, but the extrahepatic (arginase II) isoenzyme was present in higher concentration. In conclusion, the determination of lacrimal urea level as a possible less invasive replacement for blood urea determination could only be utilized in the monitoring of hemodialysis if lacrimal arginase is also measured. Blood urea levels can be correctly determined by using equations, which take into account arginase activity. The accuracy of these equations was checked on a new patient population. Both arginase isoenzymes were observed in lacrimal samples., (Copyright 2003 Elsevier Science Ltd.)

The aim was to determine the association of tear fluid cytokine levels and post-PRK corneal haze evaluated by in vivo confocal microscopy. In addition, the possible association between subbasal neural regeneration and haze formation, or epithelial regeneration were investigated. Twenty eyes of 20 patients (16 women and four men, age 30.7 +/- 7.5 years, range 21-48 years) underwent a myopic PRK. The spherical equivalent (SE) of the intended correction was -4.7 +/- 1.5 D (range -2.75 to -9.00 D). ELISA-methods were used to assess tear fluid concentrations of TGF-beta1, PDGF-BB and TNF-alpha pre-operatively, and post-operatively on day 2 and at 3 months. Tear fluid flow in the collection capillary was recorded, and rates of cytokine release (= tear fluid flow-corrected concentrations) were calculated. In vivo confocal microscopy was performed at 3 months to evaluate the corneal morphology and to determine numerical haze estimate. There was wide interindividual variation between pre-operative and post-operative concentrations and rates of release of TGF-beta1, PDGF-BB and TNF-alpha. Subepithelial haze was observed in all corneas and the mean haze estimate was 506 +/- 401 U (100-1410 U). However, no association was found between tear fluid cytokine levels and post-PRK haze. Regenerating subbasal nerve plexus was found in 18 out of 20 corneas; in two corneas it was absent or could not be visualized due to subepithelial haze. The density of the subbasal nerve fiber bundles had a positive correlation with the epithelial thickness (Pearson correlation, r = 0.56, P = 0.011), but not with the haze estimate or the thickness of the haze area. At 3 months post-PRK, haze could be observed in all patients. The results suggest that tear fluid cytokine analysis, as measured, may not be suitable for screening the potential candidates for haze formation. We did not find any correlation between haze and regeneration of subbasal nerve plexus, but we demonstrated that the regeneration of subbasal nerve plexus might have significant influence on regulation of epithelial healing., (Copyright 2001 Academic Press.)

The expression of three aquaporin (AQP)-type water channels has been reported in the lacrimal gland: AQP5 in the apical membranes of acinar and duct cells, AQP4 in the basolateral membranes of acinar cells, and AQP1 in microvascular endothelia. Recent experiments indicate that water movement through AQP5 in the salivary gland is important in saliva secretion. To investigate the role of aquaporins in lacrimal gland function, basal and pilocarpine-stimulated tear secretion was compared in wildtype mice and knockout mice lacking AQP1, AQP4 and AQP5, as well as AQP3, which was found here to be expressed in the basolateral membrane of acinar cells. Tear fluid was collected in anesthetized mice using microcapillary tubes before and at 4 min intervals after pilocarpine administration. Tear fluid volumes were (in microliter per 4 min, S.E.): 0.69 +/- 0.06 (wildtype mice), 0.70 +/- 0.07 (AQP1 -/-), 0.81 +/- 0.13 (AQP3 -/-), 0.62 +/- 0.14 (AQP4 -/-), and 0.78 +/- 0.09 (AQP5 -/-) (differences not significant). Chloride concentrations (average 155 +/- 13 mM) measured by a fluorescence assay were also not different in tear fluid collected from wildtype vs aquaporin null mice. These findings provide direct evidence against an essential role for aquaporins in lacrimal gland fluid secretion. The requirement for aquaporins in salivary but not lacrimal gland secretion, may involve the substantially slower fluid secretion rate across lacrimal gland acinar cells.

To quantify the presence of inflammatory/fibrogenic cytokines and procollagens type I (PICP) and III (PIIIP) in active and non-active tarsal and limbal forms of vernal keratoconjunctivitis (VKC), tear and blood samples were collected from 27 VKC patients (20 active and 7 non-active) and 15 normal subjects. Upper tarsal conjunctival biopses were obtained from 8 controls and 8 tarsal VKC patients. From biopses of 4 tarsal VKC, fibroblasts were cultured in F12 medium with 10% FCS. TGF-beta 1, IL-1 alpha, IL-1 beta, IL-6 and TNF-alpha, PICP and PIIIP were measured in: (1) tears, (2) homogenized conjunctival tissues, (3) serum, (4) supernatants of tissue cultures at 24 hr, and fibroblast primary passage cultures. Results showed: (1) in tears, TGF-beta 1 and TNF were identified in several active VKC patients without significant differences between the tarsal and the limbal forms. IL-1 beta (27 +/- 51 pg ml-1, P = 0.03) and IL-6 (28 +/- 43 pg ml-1, P = 0.006) were significantly increased in tarsal VKC compared to controls. Both control and non-active VKC tear samples had undetectable levels of all of the above cytokines. PICP and PIIIP were significantly increased in tarsal VKC compared to both limbal VKC and controls. Non-active VKC levels were similar to controls. (2) In homogenized VKC tissues, TGF-beta 1 and IL-6 were both significantly increased compared to controls (P < 0.01) while no increases were observed in IL-1 and TNF-alpha. (3) In serum, IL-1 alpha, IL-1 beta and TNF-alpha were higher in VKC patients compared to controls. (4) In vitro fibroblasts from VKC patients showed an increased production of TGF-beta 1, IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, PICP, and PIIIP over time. Increased levels of TGF-beta 1, IL-1 and IL-6 in VKC tissues and tears indicate a local production of these cytokines in active VKC. Collagen hyperproduction occurs only in active tarsal VKC and may be related to high levels of TGF-beta 1, IL-1 and IL-6. Increased serum levels of IL-1 and TNF-alpha suggests that systemic immunological changes occur in VKC. Cell culture can be used as a model to further study the pathogenesis of VKC and its characteristic local fibroblast activation.

Inflammatory processes are characterized by the dynamic influx of leukocytes. This leukocyte recruitment and activation is thought to be initiated by chemical signals including chemotactic factors. This study was designed to investigate the chemotactic activity in different tear types and bacteria isolated during adverse responses to contact lens wear. Chemotactic activity was determined by quantitating in vitro neutrophil migration using a microchemotaxis chamber. Results demonstrated that tears collected immediately after 8 hours sleep (P<0.001) and tears collected during adverse responses (P<0.001) showed significantly higher chemotactic activity compared to reflex tears. Specific neutralizing antibodies to IL-8, LTB4 and C5a were added to closed eye and adverse response tears. Pre-incubation of closed eye tears with antibodies to IL-8 showed a significant reduction in chemotactic activity (P<0.0001), whereas a significant reduction of PMN migration in adverse response tears was observed after pre-treatment with antibodies to LTB4 (P<0.0001). However no difference in chemotactic activity was observed after incubation with antibody to C5a or irrelevant antibody. Dot blots demonstrated that closed eye tears contained approximately 150 ng ml-1 IL-8 and adverse response tears contained 2 ng ml-1 IL-8. Most Gram negative bacteria isolated from contact lenses caused directed migration of PMNs. Addition of neutralizing antibody to LPS significantly abrogated the chemotactic activity of bacterial cells (P<0.001). Our findings provide evidence that IL-8 during eye closure, and bacterial chemotactic substances and LTB4 during contact lens induced adverse responses, are responsible for the recruitment of PMNs., (Copyright 1998 Academic Press Limited.)