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.     

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.     

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.     

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.     

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.     

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 Polar Lipids on Tear Film Dynamics

In this paper, we present a mathematical model describing the effect of polar lipids, excreted by glands in the eyelid and present on the surface of the tear film, on the evolution of a pre-corneal tear film. We aim to explain the interesting experimentally observed phenomenon that the tear film continues to move upward even after the upper eyelid has become stationary. The polar lipid is an insoluble surface species that locally alters the surface tension of the tear film. In the lubrication limit, the model reduces to two coupled non-linear partial differential equations for the film thickness and the concentration of lipid. We solve the system numerically and observe that increasing the concentration of the lipid increases the flow of liquid up the eye. We further exploit the size of the parameters in the problem to explain the initial evolution of the system.     

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.     

Study of water vapor and surfactant absorption by lipid model systems using the quartz crystal microbalance

Skin is constantly exposed to surfactants which compromise the essential barrier function of normal healthy skin. To model the interactions of surfactants with the barrier lipids of the stratum corneum (SC), it is essential to develop in vitro and in vivo quantitative measurement methods to predict, evaluate, and demonstrate the effect of the different surfactant chemistries and technologies on skin. In the current work, in vitro water vapor uptake and surfactant absorption onto skin lipid model films were quantitatively studied using a technique based on the piezoelectric effect, the quartz crystal microbalance (QCM). This approach is straightforward and reliable in providing subtle surface/interface related mass change information with high resolution and sensitivity. The results show that barrier properties of the lipid model system may be damaged by surfactant absorption, as well as by long-term water exposure due to alterations to the lipid film structure. Surfactant absorption is found to be concentration dependent even beyond its critical micelle concentration (CMC). QCM results for different surfactant systems are consistent with reported clinical data in showing that clinically milder surfactants (SLES) do not perturb the film as much as clinically harsh surfactants (SDS).     

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.     

Temperature-induced transitions in the structure and interfacial rheology of human meibum

Meibomian lipids are the primary component of the lipid layer of the tear film. Composed primarily of a mixture of lipids, meibum exhibits a range of melt temperatures. Compositional changes that occur with disease may alter the temperature at which meibum melts. Here we explore how the mechanical properties and structure of meibum from healthy subjects depend on temperature. Interfacial films of meibum were highly viscoelastic at 17°C, but as the films were heated to 30°C the surface moduli decreased by more than two orders of magnitude. Brewster angle microscopy revealed the presence of micron-scale inhomogeneities in meibum films at higher temperatures. Crystalline structure was probed by small angle x-ray scattering of bulk meibum, which showed evidence of a majority crystalline structure in all samples with lamellar spacing of 49 Å that melted at 34°C. A minority structure was observed in some samples with d-spacing at 110 Å that persisted up to 40°C. The melting of crystalline phases accompanied by a reduction in interfacial viscosity and elasticity has implications in meibum behavior in the tear film. If the melt temperature of meibum was altered significantly from disease-induced compositional changes, the resultant change in viscosity could alter secretion of lipids from meibomian glands, or tear-film stabilization properties of the lipid layer.     

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.     

Corneal development associated with eyelid opening

The development of the cornea as a tissue initiates as early as five weeks in the human embryo. This development continues gradually until the time of eyelid opening, which is associated with major developmental changes. These events, most easily observed in rodents, which are born with closed eyelids, include alterations in the rate of cell proliferation in the epithelium, stroma and endothelium; differentiation of the epithelium; appearance of a tear film and tear-film-associated proteins; and swelling and thinning of the stroma. Eyelid opening is also associated with numerous alterations in gene expression. These events are the subject of this review. Readers are directed to the article by Wolosin et al., also in this volume, for an in-depth discussion of early corneal development.     

The corneal surface of aquatic vertebrates: microstructures with optical and nutritional function?

The anterior surface of the mammalian cornea plays an important role in maintaining a smooth optical interface and consequently a sharp retinal image. The smooth surface is produced by a tear film, which adheres to a variety of microprojections, which increase the cell surface area, improve the absorbance of oxygen and nutrients and aid in the movement of metabolic products across the outer cell membrane. However, little is known of the structural adaptations and tear film support provided in other vertebrates from different environments. Using field emission scanning electron microscopy; this study examines the density and surface structure of corneal epithelial cells in representative species of the classes Cephalaspidomorphi, Chondrichthyes, Osteichthyes, Amphibia, Reptilia, Aves and Mammalia, including some Marsupialia. Variations in cell density and the structure and occurrence of microholes, microridges, microplicae and microvilli are described with respect to the demands placed upon the cornea in different aquatic environments such as marine and freshwater. A progressive decrease in epithelial cell density occurs from marine (e.g. 29348 cells mm(-2) in the Dover sole Microstomius pacficus) to estuarine or freshwater (e.g. 5999 cells mm(-2) in the black bream Acanthopagrus butcheri) to terrestrial (e.g. 2126 cells mm(-2) in the Australian koala Phascolarctos cinereus) vertebrates, indicating the reduction in osmotic stress across the corneal surface. The microholes found in the Southern Hemisphere lampreys, namely the pouched lamprey (Geotria australis) and the shorthead lamprey (Mordacia mordax) represent openings for the release of mucus, which may protect the cornea from abrasion during their burrowing phase. Characteristic of marine teleosts, fingerprint-like patterns of corneal microridges are a ubiquitous feature, covering many types of sensory epithelia (including the olfactory epithelium and the oral mucosa). Like microplicae and microvilli, microridges stabilize the tear film to maintain a smooth optical surface and increase the surface area of the epithelium, assisting in diffusion and active transport. The clear interspecific differences in corneal surface structure suggest an adaptive plasticity in the composition and stabilization of the corneal tear film in various aquatic environments.     

Formation of 2-D paracrystals of F-actin on phospholipid layers mixed with quaternary ammonium surfactants.

Two-dimensional paracrystalline arrays of F-actin have been formed on positively charged lipid layers composed of phosphatidylcholine (PC) and quaternary ammonium surfactants. These quaternary ammonium surfactants were found to be better promoters of two-dimensional order than PC lipid layers mixed with stearylamine. In addition, the length of the hydrocarbon chain was found to influence the achievement of 2-D order. Lipid layers composed of dilauryl-PC and didodecyldimethylammonium bromide, which are saturated C12 lipids, promoted 2-D crystallization better than mixtures of dipalmitoyl-PC, a saturated C16 lipid, and dioctadecyldimethylammonium bromide, a saturated C18 lipid. Thus, the hydrocarbon chain length, which influences lipid layer fluidity, had a significant effect on paracrystal formation. We suggest that quaternary ammonium surfactants may have advantages in some cases for forming ordered arrays on lipid layers. In addition to investigating the effect of lipid layer composition on paracrystal formation, we found that the injection of G-actin rather than F-actin under a fluid lipid layer into a polymerizing solution produced better ordered paracrystals.     

Kinetics of liposome disintegration from foam film studies: effect of the lipid bilayer phase state

The kinetics of interfacial liposome breakdown is investigated in the thin liquid film microinterferometric set up of Scheludko et al. Suspensions of small unilamellar vesicles of dimyristoylphosphatidylcholine are studied at temperatures above and below the temperature of the main gel-liquid crystal first order phase transition. The experimentally established time traces of the velocity of thinning of foam films are used to estimate the kinetic (rate) constants of interfacial liposome disintegration. New and previously established data for other lipids are summarized and compared with results from kinetic measurements of lipid monolayer formation. The thin film experiments confirm the existence of interfacial liposomal aggregates. A change in the kinetic behaviour is observed, due to the 'melting' of the hydrophobic tails in the lipid aggregates. This may have various consequences of biological and pharmacological importance.     

Parinaroyl and pyrenyl phospholipids as probes for the lipid surface layer of human low density lipoproteins

A simple protocol employing lipid transfer proteins was developed to label human low density lipoprotein (LDL) in a controlled manner with parinaroyl and pyrenyl phosphatidylcholines. In order to study the lipid fluidity in the surface lipid layer of LDL, the temperature-dependence of both polarization (parinaroyl probes) and excimer to monomer (E/M) intensity ratio (pyrenyl probes) were analyzed. A series of pyrenyl phosphatidylcholines containing a pyrenyl fatty acid varying from 6 to 14 carbons in length at the sn-2 position were inserted into LDL to investigate the lateral distribution of different phosphatidylcholines in the lipoprotein surface at 37 degrees C. Both polarization and E/M vs. temperature plots displayed discontinuities in the region of 22-32 degrees C, which coincides with the melting of the neutral lipid core, indicating that the latter induces an ordered to more disordered phase transition in the surface lipid layer. Determination of the E/M intensity ratio as a function of pyrene lipid concentration in LDL showed a linear relationship for the pyrenyl hexanoate and octanoate species, whereas a slope discontinuity was observed for the lipids containing a longer pyrenyl chain. These data suggest that two lipid domains with distinct properties exist in the surface layer and secondly, pyrenyl lipids partition between these domains in a chainlength-dependent manner. This is consistent with measurement of the tryptophan to pyrene energy transfer efficiency vs. pyrenyl lipid concentration, which showed a biphasic relationship for the long-chain pyrenyl lipids. These measurements further indicate that two surface lipid domains correspond to the protein-lipid boundary and the bulk lipid phase, respectively. The fact that relatively small changes in chainlength have a marked influence on the partitioning of pyrenyl lipids between the boundary and the bulk phase suggests also that native phospholipid species may not be randomly distributed in the surface lipid layer of LDL.     

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.     

Lipidomics of human Meibomian gland secretions: Chemistry, biophysics, and physiological role of Meibomian lipids

Human Meibomian gland secretions (MGS) are a complex mixture of diverse lipids that are produced by Meibomian glands that are located in the upper and the lower eyelids. During blinking, MGS are excreted onto the ocular surface, spread and mix with aqueous tears that are produced by lachrymal glands, and form an outermost part of an ocular structure called “the tear film” (TF). The main physiological role of TF is to protect delicate ocular structures (such as cornea and conjunctiva) from desiccating. Lipids that are produced by Meibomian glands are believed to “seal” the aqueous portion of TF by creating a hydrophobic barrier and, thus, retard evaporation of water from the ocular surface, which enhances the protective properties of TF. As lipids of MGS are interacting with underlying aqueous sublayer of TF, the chemical composition of MGS is critical for maintaining the overall stability of TF. There is a consensus that a small, but important part of Meibomian lipids, namely polar, or amphiphilic lipids, is of especial importance as it forms an intermediate layer between the aqueous layer of TF and its upper (and much thicker) lipid layer formed mostly of very nonpolar lipids, such as wax esters and cholesteryl esters. The purpose of this review is to summarize the current knowledge on the lipidomics of human MGS, including the discussions of the most effective modern analytical techniques, chemical composition of MGS, biophysical properties of Meibomian lipid films, and their relevance for the physiology of TF. Previously published results obtained in numerous laboratories, as well as novel data generated in the author’s laboratory, are discussed. It is concluded that despite a substantial progress in the area of Meibomian glands lipidomics, there are large areas of uncertainty that need to be addressed in future experiments.     

角膜上皮细胞代谢的研究进展

角膜上皮层位于角膜表面,外邻泪膜,内与角膜前弹力层相连。角膜上皮细胞代谢所需营养及氧分主要通过泪膜、房水和角膜缘毛细血管运送。正常的角膜上皮细胞代谢是维持角膜上皮细胞正常增殖与分化状态的关键。角膜上皮细胞代谢异常可导致上皮损伤或变性,是多种角膜疾病的病理基础。本文就近年来关于角膜上皮细胞代谢相关的组织结构、营养来源、细胞增殖分化以及相关疾病的研究进展进行综述。