Coupling Fluid and Solute Dynamics Within the Ocular Surface Tear Film: A Modelling Study of Black Line Osmolarity

We present a mathematical model describing the spatial distribution of tear film osmolarity across the ocular surface of a human eye during one blink cycle, incorporating detailed fluid and solute dynamics. Based on the lubrication approximation, our model comprises three coupled equations tracking the depth of the aqueous layer of the tear film, the concentration of the polar lipid, and the concentration of physiological salts contained in the aqueous layer. Diffusive boundary layers in the salt concentration occur at the thinnest regions of the tear film, the black lines. Thus, despite large Peclet numbers, diffusion ameliorates osmolarity around the black lines, but nonetheless is insufficient to eliminate the build-up of solute in these regions. More generally, a heterogeneous distribution of solute concentration is predicted across the ocular surface, indicating that measurements of lower meniscus osmolarity are not globally representative, especially in the presence of dry eye.Vertical saccadic eyelid motion can reduce osmolarity at the lower black line, raising the prospect that select eyeball motions more generally can assist in alleviating tear film hyperosmolarity. Finally, our results indicate that measured evaporative rates will induce excessive hyperosmolarity at the black lines, even for the healthy eye. This suggests that further evaporative retardation at the black lines, for instance due to the cellular glycocalyx at the ocular surface or increasing concentrations of mucus, will be important for controlling hyperosmolarity as the black line thins.     

Duplex Tear Film Evaporation Analysis

Tear film thinning, hyperosmolarity, and breakup can cause irritation and damage to the human eye, and these form an area of active investigation for dry eye syndrome research. Recent research demonstrates that deficiencies in the lipid layer may cause locally increased evaporation, inducing conditions for breakup. In this paper, we explore the conditions for tear film breakup by considering a model for tear film dynamics with two mobile fluid layers, the aqueous and lipid layers. In addition, we include the effects of osmosis, evaporation as modified by the lipid, and the polar portion of the lipid layer. We solve the system numerically for reasonable parameter values and initial conditions and analyze how shifts in these cause changes to the system’s dynamics.     

The Effect of the Lipid Layer on Tear Film Behaviour

This paper investigates the effect of surfactants during tear film deposition and subsequent thinning. The surfactants occur naturally on the surface of the tear film in the form of a lipid layer. A lubrication model is developed that describes lipid spreading and film height evolution. It is shown that lipids may play an important role in drawing the tear film up the cornea during the opening phase of the blink. Further, nonuniform distributions of lipids may lead to a rapid thinning of the tear film behind the advancing lipid front (shock). Experiments using a fluorescein dye technique and using a tearscope were undertaken in order to visualise the motion of the lipid layer and any associated shocks immediately after a blink. It is found that the lipid layer continues to spread upwards on the cornea after the opening phase of the blink, in agreement with the model. Using the experimental data, lipid particles were tracked in order to determine the surface velocity and these results are compared to the model predictions.     

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.     

Phospholipid transfer protein is present in human tear fluid

The human tear fluid film consists of a superficial lipid layer, an aqueous middle layer, and a hydrated mucin layer located next to the corneal epithelium. The superficial lipid layer protects the eye from drying and is composed of polar and neutral lipids provided by the meibomian glands. Excess accumulation of lipids in the tear film may lead to drying of the corneal epithelium. In the circulation, phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) mediate lipid transfers. To gain insight into the formation of tear film, we investigated whether PLTP and CETP are present in human tear fluid. Tear fluid samples were collected with microcapillaries. The presence of PLTP and CETP was studied in tear fluid by Western blotting, and the PLTP concentration was determined by ELISA. The activities of the enzymes were determined by specific lipid transfer assays. Size-exclusion and heparin-affinity chromatography assessed the molecular form of PLTP. PLTP is present in tear fluid, whereas CETP is not. Quantitative assessment of PLTP by ELISA indicated that the PLTP concentration in tear fluid, 10.9 +/- 2.4 microg/mL, is about 2-fold higher than that in human plasma. PLTP-facilitated phospholipid transfer activity in tears, 15.1 +/- 1.8 micromol mL(-)(1) h(-)(1), was also significantly higher than that measured in plasma. Inactivation of PLTP by heat treatment (+58 degrees C, 60 min) or immunoinhibition abolished the phospholipid transfer activity in tear fluid. Size-exclusion chromatography of tear fluid indicated that PLTP eluted in a position corresponding to a size of 160-170 kDa. Tear fluid PLTP was quantitatively bound to Heparin-Sepharose and could be eluted as a single peak by 0.5 M NaCl. These data indicate that human tear fluid contains catalytically active PLTP protein, which resembles the active form of PLTP present in plasma. The results suggest that PLTP may play a role in the formation of the tear film by supporting phospholipid transfer.     

The conjunctival lymphoid follicle in mucosal immunology

The conjunctiva forms a continuous mucosal surface from the eyelid margin to the cornea, and makes contact with airborne antigens and those on the adjacent eyelid skin and preocular tear film. Conjunctival lymphoid follicles (CLF) undergo hyperplasia upon conjunctival infection by a specific array of pathogens; infection-associated enlargement of draining preauricular lymph nodes suggests that CLF participate in the afferent limb of acquired immune responses for the ocular surface. In this review, we examine the evidence for classification of CLF as part of the common mucosal immune system, and explore the possible therapeutic implications.     

Biophysical investigations of the structure and function of the tear fluid lipid layers and the effect of ectoine. Part B: Artificial lipid films

The tear fluid lipid layer is present at the outermost part of the tear film which lines the ocular surface and functions to maintain the corneal surface moist by retarding evaporation. Instability in the structure of the tear fluid lipid layer can cause an increased rate of evaporation and thus dry eye syndrome. Ectoine has been previously shown to fluidize lipid monolayers and alter the phase behavior. In the current study we have investigated the effect of ectoine on the artificial tear fluid lipid layer composed of binary and ternary lipid mixtures of dipalmitoyl phosphatidylcholine (DPPC), cholesteryl esters and tri-acyl-glycerols. The focus of our study was mainly the structural and the biophysical aspects of the artificial tear fluid lipid layer using surface activity studies and topology analysis. The presence of ectoine consistently causes an expansion of the pressure–area isotherm indicating increased intermolecular spacing. The topology studies showed the formation of droplet-like structures due to the addition of ectoine only when tri-acyl-glycerol is present in the mixture of DPPC and chol-palmitate, similar to the natural meibomian lipids. Consequently, the hypothesis of an exclusion of tri/di-acyl-glycerol from the meibomian lipid film in the presence of ectoine in the subphase is confirmed. A model describing the effect of ectoine on meibomian lipid films is further presented which may have an application for the use of ectoines in eye drops as a treatment for the dry eye syndrome.     

A biophysical study of tear film lipid layer model membranes

The tear film lipid layer (TFLL), the final layer of the human tear film is responsible for surface tension reduction while blinking, water evaporation retardation and maintaining the stability of the tear film. The study of the composition-structure-function relationship of TFLL is paramount, as a compromised structure of TFLL leads to the emergence of dry eye disease (DED) which is one the most prevalent ophthalmic surface diseases of the modern world, associated with chronic pain and reduced visual capability. In this model membrane study, a systematic approach is used to study the biophysical properties of TFLL model membranes as a function of composition. Three mixed-lipid model membranes are studied along with their individual components comprising cholesteryl oleate (CO), glyceryl trioleate (GT), L-α-phosphatidylcholine (egg PC) and a free fatty acid mixture. The models become progressively more complex from binary to quaternary mixtures, allowing the role of each individual lipid to be derived. Langmuir balance, Brewster Angle Microscopy (BAM) and Profile Analysis Tensiometer (PAT) are used to study the surface activity and compression-expansion cycles, morphology, and rheological behaviour of the model membranes, respectively. Evidence of multilayering is observed with inclusion of CO and a reversible collapse is associated with the GT phase transition. An initially more coherent film is observed due to the addition of polar PC. Notably, these individual behaviours are retained in the mixed films and suggest a possible role for each physiological component of TFLL.     

Longitudinal changes in tear fluid lipidome brought about by eyelid-warming treatment in a cohort of meibomian gland dysfunction

Meibomian gland dysfunction (MGD) is a leading cause of evaporative dry eye and ocular discomfort characterized by an unstable tear film principally attributed to afflicted delivery of lipids to the ocular surface. Herein, we elucidated longitudinal tear lipid alterations associated with disease alleviation and symptom improvement in a cohort of MGD patients undergoing eyelid-warming treatment for 12 weeks. Remarkably, eyelid-warming resulted in stark reductions in lysophospholipids (P < 0.001 for lyso-plasmalogen phosphatidylethanolamine, lysophosphatidylcholine, and lysophosphatidylinositol), as well as numerous PUFA-containing diacylglyceride species in tears, accompanied by significant increases in several PUFA-containing phospholipids. These changes in tear lipidomes suggest that eyelid-warming leads to diminished activity of tear phospholipases that preferentially target PUFA-containing phospholipids. In addition, treatment led to appreciable increases (P < 0.001) in O-acyl-ω-hydroxy-FAs (OAHFAs), which are lipid amphiphiles critical to the maintenance of tear film stability. Longitudinal changes in the tear lipids aforementioned also significantly (P < 0.05) correlated with reduced rate of ocular evaporation and improvement in ocular symptoms. The foregoing data thus indicate that excess ocular surface phospholipase activity detrimental to tear film stability could be alleviated by eyelid warming alone without application of steroids and identify tear OAHFAs as suitable markers to monitor treatment response in MGD.     

Meniscal Tear Film Fluid Dynamics Near Marx’s Line

Extensive studies have explored the dynamics of the ocular surface fluid, though theoretical investigations are typically limited to the use of the lubrication approximation, which is not guaranteed to be uniformly valid a-priori throughout the tear meniscus. However, resolving tear film behaviour within the meniscus and especially its apices is required to characterise the flow dynamics where the tear film is especially thin, and thus most susceptible to evaporatively induced hyperosmolarity and subsequent epithelial damage. Hence, we have explored the accuracy of the standard lubrication approximation for the tear film by explicit comparisons with the 2D Navier–Stokes model, considering both stationary and moving eyelids. Our results demonstrate that the lubrication model is qualitatively accurate except in the vicinity of the eyelids. In particular, and in contrast to lubrication theory, the solution of the full Navier–Stokes equations predict a distinct absence of fluid flow, and thus convective mixing in the region adjacent to the tear film contact line. These observations not only support emergent hypotheses concerning the formation of Marx’s line, a region of epithelial cell staining adjacent to the contact line on the eyelid, but also enhance our understanding of the pathophysiological consequences of the flow profile near the tear film contact line.     

The Lipid Layer: The Outer Surface of the Ocular Surface Tear Film

The outer layer of the tear film—the lipid layer—has numerous functions. It is a composite monolayer composed of a polar phase with surfactant properties and a nonpolar phase. In order to achieve an effective lipid layer, the nonpolar phase, which retards water vapor transmission, is dependent on a properly structured polar phase. Additionally, this composite lipid layer must maintain its integrity during a blink. The phases of the lipid layer depend on both lipid type as well as fatty acid and alcohol composition for functionality. Surprisingly, the importance of the composition of the aqueous layer of the tear film in proper structuring of the lipid layer has not been recognized. Finally, lipid layer abnormalities and their relationship to ocular disease are beginning to be clarified.     

Structure and orientation of pancreatic colipase in a lipid environment: PM-IRRAS and Brewster angle microscopy studies

Colipase is a key element in lipase-catalyzed dietary lipids hydrolysis. Although devoid of enzymatic activity, colipase promotes pancreatic lipase activity in the physiological intestinal conditions by anchoring the enzyme on the surface of lipid droplets. Polarization modulation infrared reflection absorption spectroscopy combined with Brewster angle microscopy studies was performed on colipase alone and in various lipid environments to obtain a global view of both conformation and orientation and to assess lipid perturbations. We clearly show that colipase fully inserts into a dilaurin monolayer and promotes the formation of lipid/protein domains, whereas in a phospholipid environment its insertion is only partial, limited to the polar head group. In a mixed 70% phosphatidylcholine/30% dilaurin environment, colipase adsorbs to but does not penetrate deeply into the film. It triggers the formation of diglyceride domains under which it would form a rather uniform layer. We also clearly demonstrate that colipase adopts a preferred orientation when dilaurin is present at the interface. In contrast, at a neutral phospholipid interface, the infrared spectra suggest an isotropic orientation of colipase which could explain its incapacity to reverse the inhibitory effects of these lipids on the lipase activity.     

Extensive characterization of human tear fluid collected using different techniques unravels the presence of novel lipid amphiphiles

The tear film covers the anterior eye and the precise balance of its various constituting components is critical for maintaining ocular health. The composition of the tear film amphiphilic lipid sublayer, in particular, has largely remained a matter of contention due to the limiting concentrations of these lipid amphiphiles in tears that render their detection and accurate quantitation tedious. Using systematic and sensitive lipidomic approaches, we validated different tear collection techniques and report the most comprehensive human tear lipidome to date; comprising more than 600 lipid species from 17 major lipid classes. Our study confers novel insights to the compositional details of the existent tear film model, in particular the disputable amphiphilic lipid sublayer constituents, by demonstrating the presence of cholesteryl sulfate, O-acyl-ω-hydroxyfatty acids, and various sphingolipids and phospholipids in tears. The discovery and quantitation of the relative abundance of various tear lipid amphiphiles reported herein are expected to have a profound impact on the current understanding of the existent human tear film model.     

Human tear fluid lipidome: from composition to function

We have explored human aqueous tear fluid lipidome with an emphasis to identify the major lipids. We also address the physiological significance of the lipidome. The tears were analysed using thin layer chromatographic, enzymatic and mass spectrometric techniques. To emphasize the physiological aspect of the lipidome, we modelled the spreading of the non-polar tear fluid lipids at air-water interface in macroscopic scale with olive oil and egg yolk phosphatidylcholine. Based on enzymatic analysis the respective concentrations of choline-containing lipids, triglycerides, and cholesteryl esters were 48±14, 10±0, and 21±18 μM. Ultra performance liquid chromatography quadrupole time of flight mass spectrometry analysis showed that phosphatidylcholine and phosphatidylethanolamine were the two most common polar lipids comprising 88±6% of all identified lipids. Triglycerides were the only non-polar lipids detected in mass spectrometric analysis i.e. no cholesteryl or wax esters were identified. The spreading experiments show that the presence of polar lipids is an absolute necessity for a proper spreading of non-polar tear fluid lipids. We provide evidence that polar lipids are the most common lipid species. Furthermore, we provide a physiological rationale for the observed lipid composition. The results open insights into the functional role of lipids in the tear fluid and also aids in providing new means to understand and treat diseases of the ocular surface.     

Biophysical properties of tear film lipid layer I. Surface tension and surface rheology

Tear film lipid layer (TFLL) is the outmost layer of the tear film. It plays a crucial role in stabilizing the tear film by reducing surface tension and retarding evaporation of the aqueous layer. Dysfunction of the TFLL leads to dysfunctional tear syndrome, with dry eye disease (DED) being the most prevalent eye disease, affecting 10%–30% of the world population. To date, except for treatments alleviating dry eye symptoms, effective therapeutic interventions in treating DED are still lacking. Therefore, there is an urgent need to understand the biophysical properties of the TFLL with the long-term goal to develop translational solutions in effectively managing DED. Here, we studied the composition-function correlations of an artificial TFLL, under physiologically relevant conditions, using a novel experimental methodology called constrained drop surfactometry. This artificial TFLL was composed of 40% behenyl oleate and 40% cholesteryl oleate, representing the most abundant wax ester and cholesteryl ester in the natural TFLL, respectively, and 15% phosphatidylcholine and 5% palmitic-acid-9-hydroxy-stearic-acid (PAHSA), which represent the two predominant polar lipid classes in the natural TFLL. Our study suggests that the major biophysical function of phospholipids in the TFLL is to reduce the surface tension, whereas the primary function of PAHSA is to optimize the rheological properties of the TFLL. These findings have novel implications in better understanding the physiological and biophysical functions of the TFLL and may offer new translational insight to the treatment of DED.     

Biophysical investigations of the structure and function of the tear fluid lipid layer and the effect of ectoine. Part A: Natural meibomian lipid films

The tear fluid lipid layer is the outermost part of the tear film on the ocular surface which protects the eye from inflammations and injuries. We investigated the influence of ectoine on the structural organization of natural meibomian lipid films using surface activity analysis and topographical studies. These films exhibit a continuous pressure–area isotherm without any phase transition. With the addition of ectoine, the isotherm is expanded towards higher area per molecule values suggesting an increased area occupied by the interfacial lipid molecules. The AFM topology scans of natural meibomian lipid films reveal a presence of fiber-like structures. The addition of ectoine causes an appearance of droplet-like structures which are hypothesized to be tri-acyl-glycerols and other hydrophobic components excluded from the lipid film. Further the material properties of the droplet-like structure with respect to the surrounding were determined by using the quantitative imaging mode of the AFM technique. The droplet-like structures were found to be comparatively softer than the surrounding. Based on the observations a preliminary hypothesis is proposed explaining the mechanism of action of ectoine leading to the fluidization of meibomian lipid films. This suggests the possibility of ectoine as a treatment for the dry eye syndrome.     

Tear cytokine and ocular surface alterations following brief passive cigarette smoke exposure

Purpose: To prospectively investigate the effects of acute passive cigarette smoke exposure on the ocular surface and tear film in healthy non-smokers. Methods: Twelve right eyes of 12 subjects without any ocular diseases were examined before, 5 min, and 24 h after 5 min of passive cigarette smoke exposure in a controlled smoke chamber. Tear samples were obtained before, 5 min and 24 h after smoke exposure to detect tear hexanoyl-lysine (HEL), acrolein and inflammatory cytokine concentrations. Tear evaporation rate, DR-1 tear film lipid layer interferometry, tear film break-up time (TBUT), ocular surface fluorescein staining (FS) and Rose Bengal staining (RB), Schirmer I test were performed before, 5 min, and 24 h after smoke exposure. Conjunctival impression cytology (IC) and brush cytology (BC) were carried out before and 24 h after smoke exposure. Results: Tear evaporation rate, tear lipid spread time, tear film break-up time, and vital staining scores showed significant worsening with passive smoke exposure. Tear HEL and IL-6 concentrations increased significantly 24 h after smoke exposure. Tear acrolein level showed an insignificant increase at 5 min. IC and RT-PCR revealed a significant reduction in goblet cell density, a shift toward higher squamous metaplasia grades and a significant downregulation of MUC5AC mRNA expression at 24 h. Conclusion: Even brief passive exposure to cigarette smoke in healthy non-smoker subjects was associated with adverse effects on the ocular surface health as evidenced by an increase of tear inflammatory cytokines, tear lipid peroxidation products and decrease of mucosal defense resulting in tear instability and damage to the ocular surface epithelia.     

A minisensor for the rapid screening of sucralose based on surface-stabilized bilayer lipid membranes.

This work describes an electrochemical technique that is suitable for the rapid and sensitive screening of the sweetener sucralose based on surface-stabilized bilayer lipid membranes (s-BLMs) composed of egg phosphatidylcholine. The interactions of sucralose with s-BLMs produced electrochemical ion current increases, which appeared reproducible within a few seconds after exposure of the membranes to the sweetener. The mechanism of signal generation was investigated by differential scanning calorimetric studies. The mechanism was found to be associated with alteration of the electrostatic fields of the lipid film. These studies revealed that an increase of the molecular area of the lipids at the membranes and a stabilization of a gel phase structure occurred due to adsorption of the sweetener. Water molecules are adsorbed at the polar headgroups of the lipids, which changes the electrostatic field at the surface of the membranes. The current signal increases were related to the concentration of sucralose in bulk solution in the micromolar range. The present lipid film based sensor provided a fast response (i.e. in the order of a few seconds) to alterations of sucralose concentration (5-50 microm) in electrolyte solution. The electrochemical transduction of the interactions of this artificial sweetener with s-BLMs was applied in the determination of this compound in granulated sugar substitute products using the present minisensor.     

Interaction of phospholipid transfer protein with human tear fluid mucins

In addition to circulation, where it transfers phospholipids between lipoprotein particles, phospholipid transfer protein (PLTP) was also identified as a component of normal tear fluid. The purpose of this study was to clarify the secretion route of tear fluid PLTP and elucidate possible interactions between PLTP and other tear fluid proteins. Human lacrimal gland samples were stained with monoclonal antibodies against PLTP. Heparin-Sepharose (H-S) affinity chromatography was used for specific PLTP binding, and coeluted proteins were identified with MALDI-TOF mass spectrometry or Western blot analysis. Immunoprecipitation assay and blotting with specific antibodies helped to identify and characterize PLTP-mucin interaction in tear fluid. Human tear fluid PLTP is secreted from the lacrimal gland. MALDI-TOF analysis of H-S fractions identified several candidate proteins, but protein-protein interaction assays revealed only ocular mucins as PLTP interaction partners. We suggest a dual role for PLTP in human tear fluid: (1) to scavenge lipophilic substances from ocular mucins and (2) to maintain the stability of the anterior tear lipid film. PLTP may also play a role in the development of ocular surface disease.     

Molecular organization of the tear fluid lipid layer

The tear fluid protects the corneal epithelium from drying out as well as from invasion by pathogens. It also provides cell nutrients. Similarly to lung surfactant, it is composed of an aqueous phase covered by a lipid layer. Here we describe the molecular organization of the anterior lipid layer of the tear film. Artificial tear fluid lipid layers (ATFLLs) composed of egg yolk phosphatidylcholine (60 mol %), free fatty acids (20 mol %), cholesteryl oleate (10 mol %), and triglycerides (10 mol %) were deposited on the air-water interface and their physico-chemical behavior was compared to egg-yolk phosphatidylcholine monolayers by using Langmuir-film balance techniques, x-ray diffraction, and imaging techniques as well as in silico molecular level simulations. At low surface pressures, ATFLLs were organized at the air-water interface as heterogeneous monomolecular films. Upon compression the ATFLLs collapsed toward the air phase and formed hemispherelike lipid aggregates. This transition was reversible upon relaxation. These results were confirmed by molecular-level simulations of ATFLL, which further provided molecular-scale insight into the molecular distributions inside and dynamics of the tear film. Similar type of behavior is observed in lung surfactant but the folding takes place toward the aqueous phase. The results provide novel information of the function of lipids in the tear fluid.