In-depth analysis of the human tear proteome

The tears, a critical body fluid of the surface of the eye, contain an unknown number of molecules including proteins/peptides, lipids, small molecule metabolites, and electrolytes. There have been continued efforts for exploring the human tear proteome to develop biomarkers of disease. In this study, we used the high speed TripleTOF 5600 system as the platform to analyze the human tear proteome from healthy subjects (3 females and 1 male, average age: 36±14). We have identified 1543 proteins in the tears with less than 1% false discovery rate, which represents the largest number of human tear proteins reported to date. The data set was analyzed for gene ontology (GO) and compared with the human plasma proteome, NEIBank lacrimal gland gene dataset and NEIBank cornea gene dataset. This comprehensive tear protein list may serve as a reference list of human tear proteome for biomarker research of ocular diseases or establishment of MRM (Multiple Reaction Monitoring) assays for targeted analysis. Tear fluid is a useful and an accessible source not only for evaluating ocular surface tissues (cornea and conjunctiva), inflammation, lacrimal gland function and a number of disease conditions, such as dry eye as well as response to treatment.     

The value of tear film breakup and Schirmer's tests in preoperative blepharoplasty evaluation

The authors published their article on the value of tear film breakup and Schirmer's tests in preoperative blepharoplasty evaluation in 1989. The purpose of this update is to expand the original article in light of refinements and experiences in subsequent years. It was concluded in the 1989 article that the abnormal tear film breakup and Schirmer's tests were not good predictors of possible postblepharoplasty dry eye complications. It was concluded in the review that the anatomy and the history including scleral show, lagophtholmus, negative vector, snap test, previous surgery, increased blinking, dryness, grittiness and pain were more important predictors of postoperative dry eye problems than the ocular tests. The authors' opinion remains so, and they do less preoperative tear film testing and place more emphasis on the history and the anatomy as predictors of potential problems. In this follow-up to the 1989 article, the authors include a survey from several surgeons as to how they evaluate their patients for elective aesthetic blepharoplasty, which in large measure confirms the authors' practice.     

Dynamics of tear fluid desiccation on a glass surface: a contribution to tear quality assessment

Background

Fern-like crystalloids form when a microvolume of tear is allowed to dry out at ambient conditions on a glass surface. Presence of crystalloids in tear “microdesiccates” is used to evaluate patients with Dry-Eye disease. This study aims to examine morphologically the desiccation process of normal tear fluid and to identify changes associated with accelerated tear evaporation. Tear microdesiccates from healthy (Non-Dry Eye) and Dry Eye subjects were produced at ambient conditions. Microdesiccate formation was monitored continuously by dark-field video microscopy. Additionally, accelerated desiccation of tear samples from healthy subjects was conducted under controlled experimental conditions. Particular morphological domains of tear microdesiccates and their progressive appearance during desiccation were compared.

Results

In normal tear microdesiccates, four distinctive morphological domains (zones I, II, III and transition band) were recognized. Stepwise formation of those domains is now described. Experimentally accelerated desiccation resulted in marked changes in some of those zones, particularly involving either disappearance or size reduction of fern-like crystalloids of zones II and III. Tear microdesiccates from Dry Eye subjects may also display those differences and be the expression of a more synchronous formation of microdesiccate domains.

Conclusion

Morphological characteristics of tear microdesiccates can provide insights into the relative rate of tear evaporation.     

Mucins and TFF peptides of the tear film and lacrimal apparatus

The three-dimensional organization of the tear film, which is produced and drained by the different structures of the ocular adnexa, is essential for maintainance and protection of the ocular surface. This is facilitated by a class of large, highly glycosylated, hydrophilic glycoproteins, the mucins, which are usually expressed in association with a class of peptides having a well-defined, structurally conserved trefoil domain, the mammalian trefoil factor family (TFF) peptides. In this review, the latest information regarding mucin and TFF peptide function and regulation in the human lacrimal system, the tear film and the ocular surface is summarized with regard to mucous epithelia integrity, rheological and antimicrobial properties of the tear film and tear outflow, age-related changes and certain disease states such as dry eye, dacryostenosis and dacryolith formation.     

Oral Administration of Royal Jelly Restores Tear Secretion Capacity in Rat Blink-Suppressed Dry Eye Model by Modulating Lacrimal Gland Function

Tears are secreted from the lacrimal gland (LG), a dysfunction in which induces dry eye, resulting in ocular discomfort and visual impairment. Honey bee products are used as a nutritional source in daily life and medicine; however, little is known about their effects on dry eye. The aim of the present study was to investigate the effects of honey bee products on tear secretion capacity in dry eye. We selected raw honey, propolis, royal jelly (RJ), pollen, or larva from commercially available honey bee products. Tear secretion capacity was evaluated following the oral administration of each honey bee product in a rat blink-suppressed dry eye model. Changes in tear secretion, LG ATP content, and LG mitochondrial levels were measured. RJ restored the tear secretion capacity and decrease in LG ATP content and mitochondrial levels to the largest extent. Royal jelly can be used as a preventative intervention for dry eye by managing tear secretion capacity in the LG.     

The effects of PEP-1-FK506BP on dry eye disease in a rat model

As FK506 binding proteins (FK506BPs) are known to play an important role in the regulation of a variety of biological processes related to cell survival, this study was designed to examined the protective effects of FK506 binding protein 12 (FK506BP) on low humidity air flow induced dry eye in a rat model using transduced PEP-1-FK506BP. After the topical application of PEP-1-FK506BP, tear volumes were markedly increased and significant prevention of cornea damage was observed compared with dry eye rats. Further, immunohistochemical analysis demonstrated that PEP-1-FK506BP markedly prevented damage to the cornea, the bulbar conjunctiva, and the palpebral conjunctiva epithelial lining compared with dry eye rats. In addition, caspase-3 and PARP expression levels were found to be decreased. These results demonstrated that topical application of PEP-1-FK506BP significantly ameliorates dry eye injury in an animal model. Thus, we suggest that PEP-1-FK506BP can be developed as a new ophthalmic drop to treat dry eye diseases. [BMB Reports 2015; 48(3): 153-158]     

The value of tear film breakup and Schirmer's tests in preoperative blepharoplasty evaluation

The results of tear film breakup (BUT) and Schirmer's I and II tests were retrospectively analyzed on 146 patients undergoing elective blepharoplasty over a 41-month period. These tests were evaluated in conjunction with ocular history, orbital and periorbital anatomy, and Bell's phenomenon in order to determine their value, if any, in identifying patients at risk of developing a post-blepharoplasty dry eye complication. One-hundred and six patients (73 percent) had test results that were within normal limits, and two of these patients (1.9 percent) complained postoperatively of a transient gritty or burning sensation. Forty patients (27 percent) had abnormal results to one, two, or all three tests, and two of these patients (5 percent) also complained postoperatively of a transient gritty or burning sensation. These four symptomatic patients all had preoperative dry eye histories and abnormal orbital and periorbital anatomy. When analyzed alone, an abnormal tear film breakup (BUT) or Schirmer's test was not a good predictor of possible postblepharoplasty dry eye complications. An abnormal preoperative ocular history or abnormal orbital and periorbital anatomy proved to be the best predictor for the possible development of a postblepharoplasty dry eye complication.     

Proteomic analysis revealed the altered tear protein profile in a rabbit model of Sjögren's syndrome‐associated dry eye

Sjögren's syndrome (SS) is an autoimmune disease that results in pathological dryness of mouth and eye. The diagnosis of SS depends on both clinical evaluation and specific antibodies. The goal of this study was to use quantitative proteomics to investigate changes in tear proteins in a rabbit model of SS‐associated dry eye, induced autoimmune dacryoadenitis (IAD). Proteomic analysis was performed by iTRAQ and nano LC‐MS/MS on tears collected from the ocular surface, and specific proteins were verified by high resolution MRM. It was found that in the tears of IAD rabbits at 2 and 4 weeks after induction, S100 A6, S100 A9, and serum albumin were upregulated, whereas serotransferrin (TF), prolactin‐inducible protein (PIP), polymeric immunoglobulin receptor (pIgR), and Ig gamma chain C region were downregulated. High resolution MRM with mTRAQ labeling verified the changes in S100 A6, TF, PIP, and pIgR. Our results indicated significant changes of tear proteins in IAD rabbits, suggesting these proteins could potentially be used as biomarkers for the diagnosis and prognosis of dry eye. Several of these proteins were also found in the tears of non‐SS dry eye patients indicating a common basis of ocular surface pathology, however, pIgR appears to be unique to SS.     

Selenium Compound Protects Corneal Epithelium against Oxidative Stress

The ocular surface is strongly affected by oxidative stress, and anti-oxidative systems are maintained in corneal epithelial cells and tear fluid. Dry eye is recognized as an oxidative stress-induced disease. Selenium compound eye drops are expected to be a candidate for the treatment of dry eye. We estimated the efficacy of several selenium compounds in the treatment of dry eye using a dry eye rat model. All of the studied selenium compounds were uptaken into corneal epithelial cells in vitro. However, when the selenium compounds were administered as eye drops in the dry eye rat model, most of the selenium compounds did not show effectiveness except for Se-lactoferrin. Se-lactoferrin is a lactoferrin that we prepared that binds selenium instead of iron. Se-lactoferrin eye drops suppressed the up-regulated expression of heme oxygenase-1, cyclooxygenase-2, matrix metallopeptidase-9, and interleukin-6 and also suppressed 8-OHdG production in the cornea induced by surgical removal of the lacrimal glands. Compared with Se-lactoferrin, apolactoferrin eye drops weakly improved dry eye in high dose. The effect of Se-lactoferrin eye drops on dry eye is possibly due to the effect of selenium and also the effect of apolactoferrin. Se-lactoferrin is a candidate for the treatment of dry eye via regulation of oxidative stress in the corneal epithelium.     

Identification of hemopexin in tear film

Human tear fluid is a complex mixture of aqueous lipids, proteins, enzymes, and other biochemical and cellular elements. By conventional comparative proteomic approaches, we investigated the proteome in human tear fluid and compared the tear protein profile of normal control subjects with that of patients suffering from the ocular inflammatory disease vernal keratoconjunctivitis (VKC). Collected tear samples were directed to two-dimensional polyacrylamide gel electrophoresis protein separation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide identification. Six differentially expressed proteins—interleukin 4, phospholipase A2, albumin, lactoferrin, hemopexin, and lipocalin—were displayed. Hemopexin had not been reported previously in tear film. Enzyme-linked immunosorbent assay confirmed that hemopexin concentrations were significantly higher in VKC tear samples and increased with disease stages. The results implied clinical interest of hemopexin in the tear proteome and eye diseases.     

玻璃酸钠联合重组牛碱性成纤维细胞生长因子对糖尿病性白内障超声乳化术后泪膜稳定性的影响

目的:探讨玻璃酸钠联合重组牛碱性成纤维细胞生长因子(b FGF)对糖尿病性白内障超声乳化术后泪膜稳定性的影响。方法:选取2015年1月到2017年1月期间在我院接受治疗的150例糖尿病性白内障患者,根据随机数字表法分为对照组和研究组,各75例。两组均进行常规治疗,在常规治疗的基础上对照组采用玻璃酸钠滴眼液进行治疗,研究组在对照组的基础上加用bFGF眼用凝胶进行治疗,两组均治疗1个月。比较两组术前、术后1周、术后1个月的泪膜破裂时间(BUT)、基础泪液分泌试验(SIt)值、角膜荧光素染色(FL)评分、干眼症状评分和眼表疾病指数(OSDI)评分,并比较两组术前、术后1个月的最佳矫正视力。结果:术后1周,两组患者的BUT明显低于术前和术后1个月,且研究组的BUT高于对照组(P0.05);随时间推移两组患者的OSDI评分持续降低(P0.05),术后1周,两组患者干眼症状评分、SIt值、FL评分明显高于术前和术后1个月,且研究组的OSDI评分、干眼症状评分、SIt值、FL评分均低于对照组(P0.05)。术后1个月两组患者的最佳矫正视力均明显上升,且研究组明显高于对照组(P0.05)。结论:玻璃酸钠联合bFGF能有效提升糖尿病性白内障患者在超声乳化术后泪膜稳定性,利于患者术后视力的恢复,值得临床推广应用。     

Lacrimal Proline Rich 4 (LPRR4) Protein in the Tear Fluid Is a Potential Biomarker of Dry Eye Syndrome

Dry eye syndrome (DES) is a complex, multifactorial, immune-associated disorder of the tear and ocular surface. DES with a high prevalence world over needs identification of potential biomarkers so as to understand not only the disease mechanism but also to identify drug targets. In this study we looked for differentially expressed proteins in tear samples of DES to arrive at characteristic biomarkers. As part of a prospective case-control study, tear specimen were collected using Schirmer strips from 129 dry eye cases and 73 age matched controls. 2D electrophoresis (2DE) and Differential gel electrophoresis (DIGE) was done to identify differentially expressed proteins. One of the differentially expressed protein in DES is lacrimal proline rich 4 protein (LPRR4). LPRR4 protein expression was quantified by enzyme immune sorbent assay (ELISA). LPRR4 was down regulated significantly in all types of dry eye cases, correlating with the disease severity as measured by clinical investigations. Further characterization of the protein is required to assess its therapeutic potential in DES.     

Spectroscopic evaluation of human tear lipids

Infrared and fluorescence spectroscopies were applied to characterize the molecular conformational/structure and dynamics of human meibum (ML) and tear lipids (SSL). ML lipids contained more CC and CH3 moieties than SSL. SSL contained OH groups that were not apparent in the spectra of ML. The CO stretching band observed in the infrared spectra of SSL and ML revealed that the CO groups are not involved in hydrogen bonds. Bands due to the polar moieties CO and PO2- did not change significantly with increasing temperature, suggesting that they may not play an appreciable thermodynamic role in the lipid hydrocarbon chain phase transition. Components in tears bind to SSL and exclude water at the water-lipid boundary where the polar headgroups of phospholipids are located. If similar interactions occur in vivo at the tear film lipid-aqueous interface, they would reduce the rate of evaporation. The results provide a foundation for future studies to assess possible differences with age and sex in tears from normal and dry eye subjects.     

Assessment of thermal dehydration using the human eye: What is the potential?

Human hydration assessment is a key component for the prevention and proper treatment of heat-related fluid and electrolyte imbalances within military, sports and clinical medicine communities. Despite the availability of many different methods for assessing hydration status, the need for a valid method or technology that is simple, rapid, non-invasive, universal (detects both hypertonic and isotonic hypovolaemia) and is applicable for static (single point in time) and dynamic (change across time) hydration assessment is widely acknowledged. The eye is one candidate body region that might afford such a measure given the intricate balance between ocular dynamics (tear and aqueous humor formation) and blood (plasma osmolality and volume), which is considered the criterion measure for hydration assessment. The aim of this review is to introduce and discuss the potential for using ocular measurements for non-invasive hydration assessment, including tear fluid osmolarity (Tosm), non-invasive tear break-up time (NITBUT) and intraocular pressure (IOP). There is a relevant physiological basis for testing the merit of ocular measures for human hydration assessment and recent data indicate that Tosm and IOP may have utility. Further investigations are warranted to determine the degree to which ocular measures can act as accurate and reliable non-invasive hydration status markers.     

Temperature-induced conformational changes in human tearlipids hydrocarbon chains

As a first step to characterize human meibum and tear lipids, infrared spectroscopy was applied to characterize the molecular structure/conformation and packing of hydrocarbon chains. Temperature-induced phase transitions were fit to a sigmoid equation and were experimentally reproducible and were similar for multiple samples collected from the same person. No hysteresis was observed. Hydration of polar tear lipids increased their phase transition cooperativity, enthalpy and entropy. Hydrophobic interactions in meibum lipid (ML) were stronger than in tear-fluid lipids (TL), as reflected by the higher entropy and enthalpy of the gel to liquid crystalline phase transition of ML. The results of this study provide further evidence of the differences in the composition and structure of ML and TL. The conformational changes observed in the hydrocarbon chains of ML with temperature suggest that the observed therapeutic increased delivery of ML with eye lid heating could be related to the increased disorder in the packing of the hydrocarbon tails. This work also highlights the power of infrared spectroscopy to characterize molecular structure/conformation, and packing of human tear lipids and provides a basis for future studies of tear film lipid composition-structure-function relationships and lipid-protein interactions in relation to age, sex, and dry eye symptoms.     

Restasis® for the treatment of 'dry eye' in Aotus nancymaae

Background  This case report describes the treatment of three male owl monkeys ( Aotus nancymaae ) diagnosed with chronic dry eye with a topical cyclosporine product, Restasis^®, approved for use in humans. These owl monkeys had ocular disease resulting from procedures performed at a biotechnology company. They were moved to the Center for Neotropical Primate Research and Resources at University of South Alabama to be incorporated into the breeding colony.
Materials and methods  Schirmer tear testing was performed initially and during the course of treatment to monitor efficacy of twice daily administered Restasis^®. The goals of treatment were to reduce pain and/or distress and if possible to quantitatively increase tear production.
Results and discussion  All animals had improvements in conjunctival inflammation and had an increase in tear production.     

Calorie restriction: A new therapeutic intervention for age-related dry eye disease in rats

A decrease in lacrimal gland secretory function is closely related to aging and leads to an increased prevalence of dry eye syndrome. Since calorie restriction (CR) is considered to prevent functional decline of various organs due to aging, we hypothesized that CR could prevent age-related lacrimal dysfunction. Six-month-old male Fischer 344 rats were randomly divided into ad libitum (AL) and CR (−35%) groups. After 6 months of CR, tear function was examined under conscious state. After euthanasia, lacrimal glands were subjected to histological examination, tear protein secretion stimulation test with Carbachol, and assessment of oxidative stress with 8-hydroxy-2 deoxyguanosine (8-OHdG) and 4-hydroxynonenal (HNE) antibodies. CR significantly improved tear volume and tended to increase tear protein secretion volume after stimulation with Carbachol compared to AL. The acinar unit density was significantly higher in the CR rats compared to AL rats. Lacrimal glands in the CR rats showed a lesser degree of interstitial fibrosis. CR reduced the concentration of 8-OHdG and the extent of staining with HNE in the lacrimal gland, compared to AL. Furthermore, our electron microscopic observations showed that mitochondrial structure of the lacrimal gland obtained from the middle-aged CR rats was preserved in comparison to the AL rats. Collectively, these results demonstrate for the first time that CR may attenuate oxidative stress related damage in the lacrimal gland with preservation of lacrimal gland functions. Although molecular mechanism(s) by which CR maintains lacrimal gland function remains to be resolved, CR might provide a novel therapeutic strategy for treating dry eye syndrome.     

杞菊地黄汤联合玻璃酸钠滴眼液对肝肾阴虚型干眼症患者眼表功能和泪液MMP-2、MMP-9水平的影响

摘要 目的：观察杞菊地黄汤联合玻璃酸钠滴眼液治疗肝肾阴虚型干眼症的疗效及对眼表功能和泪液基质金属蛋白酶-2（MMP-2）、基质金属蛋白酶-9（MMP-9）的影响。方法：病例来源于2018年6月~2021年3月期间就诊于湖南省直中医医院眼科门诊的干眼症患者,共98例。采用随机数字表法分为对照组和实验组,两组均49例。对照组给予玻璃酸钠滴眼液治疗,实验组在对照组基础上接受杞菊地黄汤治疗,两组均治疗4周。对比两组疗效、中医证候积分、眼表功能和泪液MMP-2、MMP-9水平变化,观察两组治疗期间不良反应发生率。结果：实验组的临床总有效率高于对照组（P<0.05）。实验组治疗4周后眼部干涩不爽、畏光、双目频眨、口干少津、白眼泛红、舌红、苔薄、脉细证候积分均低于对照组（P<0.05）。实验组治疗4周后角膜荧光素染色（FL）评分低于对照组,泪膜破裂时间（BUT）、泪液分泌试验（SIT）水平高于对照组（P<0.05）。实验组治疗4周后泪液MMP-2、MMP-9水平低于对照组（P<0.05）。两组间不良反应发生率无统计学差异（P＞0.05）。结论：杞菊地黄汤联合玻璃酸钠滴眼液治疗肝肾阴虚型干眼症,可促进患者症状缓解,改善眼表功能,降低泪液MMP-2、MMP-9水平,安全可靠。     

Elastohydrodynamics of the Eyelid Wiper

This paper presents an elastohydrodynamic model of the human eyelid wiper. Standard lubrication theory is applied to the fluid layer between the eyelid wiper and ocular surface. The role of the lubrication film is to reduce the shear stresses by preventing solid to solid contact between the eyelid wiper and ocular surface. For the lubrication film to be effective, it is required that the orientation of the eyelid wiper changes between the opening and closing phases of a blink. In order to model this, the hydrodynamic model is coupled with an elastic mattress model for the soft tissue of the eyelid wiper and ocular surface. This leads to a one-dimensional non-linear partial differential equation governing the fluid pressure in the lubrication film. In order to solve the differential equation, a loading condition or constraint equation must be specified. The resulting system is then solved numerically. The model allows predictions of the tear film flux from under the upper eyelid, as well as normal and shear stresses acting on the ocular surface. These factors are important in relation to dry eye syndrome, deformation of the cornea and contact lens design. It is found that the pressure and shear stress under the eyelid act across a length of approximately 0.1 mm which is consistent with clinical observations. It order to achieve a flow of tears from under the upper eyelid during a blink, the model requires that the normal force the eyelid applies to the ocular surface during the closing phase of the blink is significantly higher than during the opening phase of the blink. Electronic Supplementary Material The online version of this article () contains supplementary material, which is available to authorized users.     

Mucin Deficiency Causes Functional and Structural Changes of the Ocular Surface

MUC5AC is the most abundant gel-forming mucin in the ocular system. However, the specific function is unknown. In the present study, a Muc5ac knockout (KO) mouse model was subject to various physiological measurements as compared to its wide-type (WT) control. Interestingly, when KO mice were compared to WT mice, the mean tear break up time (TBUT) values were significantly lower and corneal fluorescein staining scores were significantly higher. But the tear volume was not changed. Despite the lack of Muc5ac expression in the conjunctiva of KO mice, Muc5b expression was significantly increased in these mice. Corneal opacification, varying in location and severity, was found in a few KO mice but not in WT mice. The present results suggest a significant difference in the quality, but not the quantity, of tear fluid in the KO mice compared to WT mice. Dry eye disease is multifactorial and therefore further evaluation of the varying components of the tear film, lacrimal unit and corneal structure of these KO mice may help elucidate the role of mucins in dry eye disease. Because Muc5ac knockout mice have clinical features of dry eye, this mouse model will be extremely useful for further studies regarding the pathophysiology of the ocular surface in dry eye in humans.