Human Tear Film Research Articles

Human tear film and meibum. Very long chain wax esters and (O-acyl)-omega-hydroxy fatty acids of meibum

Human meibum was targetly analyzed for the presence of intact wax esters (WEs) and related compounds by means of reverse-phase HPLC in combination with ion trap mass spectrometry. The major detected WEs were based on C(18:n) (n = 1-4) unsaturated FAs ranking in the following order of abundance: C(18:1)>C(18:2)>C(18:3)>C(18:4). The major fatty alcohols (FAls) found in WE were of saturated nature and varied from C(18:0) to C(28:0). The three most abundant species were C(18:1)-FA esters of C(24:0), C(25:0), and C(26:0)-FAl. Typically, a major compound based on C(18:1)-FA and a saturated FAl was accompanied by a few related compounds based on a C(18:2), C(18:3), and C(18:4)-FA. Contrary to previous reports, no epoxy-WEs or epoxy-FAs were detected in fresh and 1-year-old meibum samples. More than 20 (O-acyl)-omega-hydroxy-FAs (OAHFAs) were observed. The main detected OAHFAs were based on very long-chain omega-hydroxy-FA (C(30:1), C(32:1), and C(34:1)) acylated through their omega-hydroxyls by a C(18:1)-FA. Due to their amphiphilic anionogenic nature, OAHFAs may be responsible for stabilization of the tear film lipid layer by creating an interface between the vast pool of strictly nonpolar lipids of meibum (WEs, cholesteryl esters, etc.) and the aqueous subphase beneath it, a role previously attributed to phospholipids.

A Novel Method for Pachymetry Mapping of Human Precorneal Tear Film Using Pentacam with Fluorescein

To report a novel method for pachymetry mapping of human precorneal tear film using Pentacam. Precorneal tear film is routinely undetected by Pentacam but could be well visualized with the aid of fluorescein. Twenty patients with dry eye and 20 age-matched control subjects with normal eyes were enrolled in this prospective study. The right eye of each subject was scanned once with Pentacam and rescanned after instillation of 1 microL 0.1% fluorescein, and the differential map of corneal thickness between the two measurements was identified as the pachymetry map of tear film. Then the central tear film thickness was evaluated, and the pattern of each pachymetry map was determined. Mean central tear film thickness in dry eyes (22.2 +/- 4.5 microm) was less than in normal eyes (24.7 +/- 3.9 microm) (Student's t-test, P = 0.0614). Additionally, the tear film pachymetry map could be classified into three patterns: pattern 1, thickening upward; pattern 2, uniform distribution; pattern 3, thickening downward. Tear film pachymetry maps of normal eyes consisted of pattern 1 (40%), pattern 2 (40%), and pattern 3 (20%), whereas those of dry eyes consisted of pattern 1 (70%), pattern 2 (20%), and pattern 3 (10%). Dry eyes tended to have a higher proportion of pattern 1 pachymetry maps than normal eyes, though still no significant difference was found between two groups (Cochran-Mantel-Haenszel chi(2) test, P = 0.0852). This novel method feasibly could be used to map tear film thickness, and it provides a valuable means to investigate the spatial distribution of tear film.

The Meibomian puzzle: combining pieces together.

The purpose of this review was to summarize the available information on lipidomic analysis of human meibum and tear film, and critically evaluate the pertinent past and present analytical procedures and results obtained in various laboratories. Human meibum was shown to be a very complex mixture of lipids of various classes. For decades, their exact structures have remained elusive. Because of the limitations of the then-current techniques, most of the complex lipids that constitute meibum could not be analyzed as whole molecules and required prior hydrolysis and/or transesterification of the entire lipid pool. These procedures effectively made it very difficult, and often impossible, to reconstruct the complete structures of the original intact compounds, which prompted us to call this The Meibomian Puzzle. Modern techniques such as high-performance liquid chromatography in combination with mass spectrometry help in solving this puzzle by allowing a researcher to detect and analyze intact molecules of complex lipid compounds, even if present in extremely low concentrations. This current de-facto standard procedure in lipidomic analysis of natural lipids and their mixtures is compared with other experimental techniques such as nuclear magnetic resonance spectroscopy, infrared spectroscopy, gas chromatography, and thin layer chromatography, among the others. The results obtained by older techniques, and their limitations and deficiencies are discussed. It appears that some of the earlier findings did not withstand a scrupulous re-evaluation and need to be modified and/or corrected. The most intriguing development is the virtual absence in meibum of typical phospholipids - an important group of amphiphilic compounds whose role in the human tear film was thought to be to stabilize the entire tear film structure. Instead, another group of previously unidentified compounds, very long chain (O-acyl)-omega-hydroxy fatty acids, appears to be a stabilizing factor which might be related to tear film stability and deterioration. Thus, these compounds may become an important target in biochemistry and (patho)physiology of ocular surface and dry eye research.

Factors Affecting Evaporation Rates of Tear Film Components Measured In Vitro

With increasing age and in patients affected with dry-eye symptoms, the human tear film becomes more unstable and exhibits shorter tear break-up times. We examined whether the inclusion of proteins and lipids to water affected the evaporation rates measured in vitro and could account for the lower rates reported previously from in vivo measurements. The impact of temperature, air flow, and humidity on the evaporation rate of tears was measured in vitro. Lipid-protein interactions were measured using fluorescence spectroscopy and in vitro rates of evaporation were performed gravimetrically. Human reflex tears evaporated at a rate similar to that of water. A temperature increase from 25 degrees C to 34 degrees C caused a threefold increase in the evaporation rates of tears in still air. Further increases were observed under a current of dry air. Wax, mucin, lysozyme, or beta-lactoglobulin did not influence significantly the rates of evaporation measured in vitro. In contrast, lipid layered on the surface resulted in a 23% decrease in the rates. Environmental factors affect evaporation rates significantly and should be carefully controlled when performing in vivo measurements. The presence of a lipid layer lowers evaporation rates. The significantly lower rates of evaporation of tears measured in vivo suggest that with the lipid layer intact, the high reserve capacity of the lacrimal gland to provide both unstimulated and stimulated tear flow is more than enough to compensate for evaporative loss. However, with dry eye, increased rates of evaporation and decreased lacrimal tear flow could result in decreased break-up times.

An overset grid method for the study of reflex tearing

We present an overset grid method to simulate the evolution of human tear film thickness subject to reflex tearing. The free-surface evolution is governed by a single fourth-order non-linear equation derived from lubrication theory with specified film thickness and volume flux at each end. The model arises from considering the limiting case where the surfactant is strongly affecting the surface tension. In numerical simulations, the overset grid is composed of fine boundary grids near the upper and lower eyelids to capture localized capillary thinning referred to as 'black lines' and a Cartesian grid covers the remaining domain. Numerical studies are performed on a non-linear test problem to confirm the accuracy and convergence of the scheme. The computations on the tear film model show qualitative agreement with in vivo tear film thickness measurements. Furthermore, the role of the black lines in the presence of tear supply from the lid margins, reflex tearing, was found to be more subtle than a barrier to tear fluid flow between the anterior of the eye and the meniscus at the lid margin. During reflex tearing, tears may flow through the region normally containing the black line and drift down over the cornea under the influence of gravity.

Surface enhanced Raman scattering of herpes simplex virus in tear film

Conjunctivitis is currently diagnosed using white light microscopy of corneal or conjunctival cells in culture. This takes time and is subjective. An ideal test would be sensitive, specific, near-patient and quick. Raman spectroscopy is the measurement of inelastic light scatter that produces unique molecular spectra. Infected human tear film can be analysed for viral and bacterial particles. However, the concentration of these and the constituents of the tear are very low. Surface enhanced Raman scattering can be used to amplify the Raman signal. For this experiment two different SERS substrates were used: silver mirror reaction glass and gold thin film. Heat denatured herpes simplex virus in transport medium culture and transport medium only were added to different ratios of a synthetic tear. The synthetic tear was modelled on the aqueous layer of the human tear film. Linear discriminant analysis was performed on each spectral dataset for each ratio of mixture on both substrates. From the classification tables, the sensitivity and specificity for silver mirror reaction glass were 75.5+/-13.8%, 77.3+/-8.3%, and for gold thin film 75.5+/-5.9% and 78.3+/-6.2% respectively. The results show proof of principle that SERS could potentially be used to detect the presence of herpes simplex viral particles in an aqueous solution such as the tear film.

Changes in aberrations and retinal image quality due to tear film dynamics

A reliable and objective method to measure aberration changes due to the tear film is essential in improving clinical assessment of the tear film and in vivo retinal imaging. The tear film of 11 subjects are studied by acquiring continuous wavefront measurements in real-time with a customized Shack-Hartmann wavefront sensor. The device has a high resolution lenslet array (190 mum) and a topographer unit with an infrared pupil illuminator (940 nm). A Fourier transform reconstructor algorithm [1] was used to estimate the eyes' wavefront aberrations from slope measurements. Increasing irregularities in the tear film produced observable wavefront variations. The temporal behavior of tear induced aberrations and retinal image quality was evaluated by the root mean squared (RMS) error of the residual wavefront and the volume modulation transfer function (MTF). Similar trends were observed from both metrics. Our analysis demonstrates the applicability of the SH wavefront sensor to assessing the dynamics of the human tear film.

The adhesion of Pseudomonas aeruginosa to high molecular weight human tear film species corresponds to glycoproteins reactive with Sambucus nigra lectin

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 α2–6) but not WGA lectin ( N-acetylglucosamine, sialic acid α2–3). 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.

Refractive power of a multilayer rotationally symmetric model of the human cornea and tear film

Optical models of the human cornea and tear film typically employ a single homogeneous cornea with an average refractive index. I propose to use a more realistic multilayer model based on morphological data from the literature. The mathematical methodology to derive the refractive power equation of this model is presented. Special attention is given to the axial gradient index of the refraction structure of the stroma layer because of its optical implications. The importance of considering this multilayer model is quantified in a specific example (orthokeratology) with the help of the derived power equation.

Glycoprotein 340 in Normal Human Ocular Surface Tissues and Tear Film

The tear film is a complex mixture of secreted fluid, ions, proteins, glycoproteins, and lipids that lubricates and protects the ocular surface. Recently, several antimicrobial peptides have been described in the tear fluid. In this study, we describe the presence of the large secreted glycoprotein gp340 in the tear film. Western blot analysis showed that gp340 is abundant in secreted tears and in the lacrimal glands. Lesser amounts of gp340 were detected in the cornea and conjunctiva. Consistent with Western blot data, reverse transcription-PCR and real-time quantitative PCR showed that gp340 transcripts were abundant in lacrimal gland tissue and were also present in the cornea and conjunctiva. Immunohistochemistry localized gp340 to the acinar cells of the lacrimal gland and the deeper layers of the conjunctival epithelium. gp340 was not detected in conjunctival goblet cells. In the cornea, gp340 was present only in a peripheral band of basal epithelial cells, suggesting that gp340 may play a role in the cycle of corneal epithelial renewal. To determine if tear film gp340 may function as a bacterial agglutinin as it does in saliva, tears were incubated with streptococcal cells and the formation of bacterial aggregates was monitored. Addition of tears to late-exponential-phase Streptococcus mutans cells resulted in time- and dose-dependent aggregation of the bacteria. Furthermore, Western blot analysis confirmed the presence of cell-associated gp340 in isolated bacterial aggregates. The ocular pathogen Staphylococcus aureus, but not Pseudomonas aeruginosa, also aggregated when incubated with tears. These results suggest that gp340 is a normal component of the tear film and that the glycoprotein may function as a bacterial agglutinin.

Study of the dynamic aberrations of the human tear film

The dynamic aberrations introduced by the human tear film are studied by measuring the topography of the tear film surface on 14 subjects using a curvature sensing setup. The RMS wavefront error variation of the data obtained is presented showing the non-negligible contribution of the tear film to overall eye aberrations. The tear film wavefronts are decomposed in their constituent Zernike terms, showing stronger contributions from 4th order terms and terms with vertical symmetry, and the temporal behaviour of these aberrations is analysed.

Surface Pressure Measurements of Human Tears and Individual Tear Film Components Indicate That Proteins Are Major Contributors to the Surface Pressure

Tear film stability has been associated with a low surface tension (high surface pressure), which has been attributed to a variety of tear film components. In this study, we examined the contribution of various tear proteins, mucin, and meibomian lipids to the surface pressure of human tears. A Langmuir trough was used to measure and compare the surface activities of albumin, lipocalin, beta-lactoglobulin, lactoferrin, lysozyme, secretory IgA, mucin, meibomian lipid, and tears. All proteins exhibited surface activity. The surface pressure-area (Pi-A) profiles of most protein films at equilibrium surface pressure (Pieq) were sigmoidal and showed hysteresis between the expansion and compression phases of the cycle. Pieq of most proteins took 4-9 hours to occur. By contrast, the Pi-A profiles for meibomian lipid films were hyperbolic rather than sigmoidal and had little hysteresis, and Pieq was attained within 1 hour. The Pi-A profiles of mucin films showed mostly hyperbolic characteristics with small hysteresis. The Pi-A profiles of films of tears were sigmoidal, showed strong hysteresis, and reached Pieq at about 5 hours. Partitioning of the proteins and whole tears into the subphase also occurred. Comparison between the dynamic Pi-A profiles of tears and those of individual tear film components shows that tear film proteins not only are capable of surface activity but also are major contributors to the surface activity of the tear film.

An Interferometric Imaging Method for Studying the three Dimensional Structure of the Human Precorneal Tear Film

An Interferometric Imaging Method for Studying the three Dimensional Structure of the Human Precorneal Tear Film

Physical/Chemical Characteristics of the Human Tear Film That Impact Performace and Efficacy of Artificial Tear Products

Physical/Chemical Characteristics of the Human Tear Film That Impact Performace and Efficacy of Artificial Tear Products

Black-line formation and the "perched" human tear film

Introduction. McDonald and Brubaker observed that after instillation of fluorescein, dark lines appear near the upper and lower lid margins of the human eye immediately following a blink. Methods. This paper numerically models the dynamic black-line formation under the lubrication approximation at low Reynolds number, including gravity and surface tension forces. Results. The hydrodynamic model predicts that menisci at the lid margins draw liquid from the tear film, resulting in local thinning close to the lids (i.e., black lines) due to capillary suction. Once the film thins locally, resistance to further thinning increases. No vertical gravity-induced drainage occurs between blinks, and the tear film is trapped in its initially deposited state or "perched" between the two black lines. Conclusions. We demonstrate quantitatively that due to rapid formation of black lines, the tear film is trapped for extended periods. Lid action is therefore required to disturb the perched tear film.

Evaporation from the human tear film studied by interferometry.

Previous measurements of evaporation from the human tear film have been based on measuring humidity in an open chamber1,2 or a closed chamber.3–5 The presence of the chamber restricts natural air and convection currents, and therefore the measured evaporation rate may be less than in unobstructed conditions.

EVAPORATION FROM THE HUMAN TEAR FILM STUDIED BY INTERFEROMETRY.

Previous measurements of evaporation from the human tear film have been based on measuring humidity in an open chamber1,2 or a closed chamber.3–5 The presence of the chamber restricts natural air and convection currents, and therefore the measured evaporation rate may be less than in unobstructed conditions.

The effects of human tear electrolytes on the viability and hydrogel colonization of Pseudomonas aeruginosa and Staphylococcus aureus

This study was designed to evaluate the effects of the inorganic electrolytes present in human tear film on the viability and colonization of bacteria to hydrogels. Pseudomonas aeruginosa and Staphylococcus aureus were used in these experiments. A D-value test was performed to investigate any bacteriostatic effect by measuring the reduction of viable test microorganisms over time when exposed to the inorganic electrolyte solution. No D-value was calculable for S. aureus in electrolyte solution whereas a D-value of 8.1 h was obtained for P. aeruginosa in electrolyte solution. The D-value data indicate that staphylococci have a greater survivability potential in a hypertonic environment than do pseudomonads. Bacterial adhesion to high water, ionic hydrogels was studied using the Modified Robbins Device (MRD). The data for P. aeruginosa recovered from the lenses showed an 82% decrease in bacterial counts in electrolyte solution as compared to bacteria incubated in control solution. In contrast there were slight increases in S. aureus counts recovered from the lenses. Journal of Industrial Microbiology & Biotechnology (2000) 25, 17–19.

Immunohistochemical evidence for estrogen receptors in meibomian glands

PurposeTo look for sex hormone receptor distribution in three structures contributing to the normal human tear film: the conjunctiva, the accessory lacrimal glands, and the meibomian glands. DesignAn immunohistochemical study. Tissues and controlsForty-one upper eyelid specimens were collected from 15 male and 26 female patients (age range, 1.5–85 years) during blepharoptosis surgery via posterior tarsoconjunctival mullerectomy (Fasanella-Servat or Gavaris). In addition, control sections of histologically normal breast, prostate, and skin tissue were obtained. MethodsImmunohistochemical staining using mouse monoclonal antibodies against estrogen, progesterone, and androgen receptors was performed on all tissues and controls. Quantitation of the receptors was performed and expressed as percentage nuclear positivity. Specimens were divided into three groups based on the age of the patient: <12 years (n = 9); 18–55 years (n = 1); >55 years (n = 12). ResultsForty-one specimens contained conjunctiva. All were negative for estrogen, progesterone, and androgen receptors. Twenty-four specimens contained accessory lacrimal glands of Wolfring. All were negative for the three receptors. Twenty-two specimens contained meibomian glands. All were positive for estrogen receptors; one was positive for progesterone receptors and one for androgen receptors. Using Minitab statistical software (Minitab Inc. State College, PA), analysis of variation revealed no statistical difference between sexes or between age groups studied. The sebaceous glands of skin were uniformly positive for androgen receptors. Sebaceous glands of the face and scalp (3 of the 15 skin samples) were also positive for estrogen receptors. ConclusionsEstrogen receptors are present in the meibomian glands of the upper eyelid. Unlike sebaceous glands elsewhere on the skin, the meibomian glands lack androgen receptors. Estrogen receptors may play a role in modulation of the lipid layer of the tear film, and their activity may be linked to meibomian gland dysfunction and dry eye syndrome.

Application of Twyman–Green Interferometer for Evaluation of In Vivo Breakup Characteristic of the Human Tear Film

The paper presents an interferometric method of assessing the in vivo stability of the precorneal tear film. To observe dynamic effects on a human cornea the Twyman-Green interferometer with television frame speed digital registration synchronized with a laser flash was used. The instrument was applied to the human cornea in vivo. The results of the experiment, both tear film distribution and its dynamics, are presented. The proposed interferometric setup can be used to evaluate the breakup characteristics of the tear film, its distribution, and to examine its dynamic changes. The breakup profiles and their cross sections calculated from the interferogram analysis are presented. The depth of recorded breakup, calculated on the basis of interferogram analysis, amounts to about 1.5 μm. The proposed method has the advantage of being noncontact and applies only a low-energy laser beam to the eye. This provides noninvasive viewing of human cornea in vivo and makes it possible to observe the kinetics of its tear film deterioration. © 1999 Society of Photo-Optical Instrumentation Engineers.