Thomas J. Millar

The international workshop on meibomian gland dysfunction: report of the subcommittee on anatomy, physiology, and pathophysiology of the meibomian gland.

The tarsal glands of Meibom (glandulae tarsales) are large sebaceous glands located in the eyelids and, unlike those of the skin, are unassociated with hairs. According to Duke-Elder and Wyler,1 they were first mentioned by Galenus in 200 AD and later, in 1666, they were described in more detail by the German physician and anatomist Heinrich Meibom, after whom they are named. Lipids produced by the meibomian glands are the main component of the superficial lipid layer of the tear film that protects it against evaporation of the aqueous phase and is believed also to stabilize the tear film by lowering surface tension.2 Hence, meibomian lipids are essential for the maintenance of ocular surface health and integrity. Although they share certain principal characteristics with ordinary sebaceous glands, they have several distinct differences in anatomy, location, secretory regulation, composition of their secretory product, and function. Functional disorders of the meibomian glands, referred to today as meibomian gland dysfunction (MGD),3 are increasingly recognized as a discrete disease entity.4–8 In patients with dry eye disease, alterations in the lipid phase that point to MGD are reportedly more frequent than isolated alterations in the aqueous phase. In a study by Heiligenhaus et al.,9 a lipid deficiency occurred in 76.7% of dry eye patients compared with only 11.1% of those with isolated alterations of the aqueous phase. This result is in line with the observations by Shimazaki et al.10 of a prevalence of MGD in the absolute majority of eyes with ocular discomfort defined as dry eye symptoms. These observations noted that 64.6% of all such eyes and 74.5% of those excluding a deficiency of aqueous tear secretion were found to have obstructive MGD, or a loss of glandular tissue, or both.10 Horwath-Winter et al.11 reported MGD in 78% of dry eye patients or, if only non-Sjogren patients are considered, in 87% compared with 13% with isolated aqueous tear deficiency. It may thus be accepted that MGD is important, conceivably underestimated, and possibly the most frequent cause of dry eye disease due to increased evaporation of the aqueous tears.5,9–12 After some excellent reviews of MGD4,7,8,13,14 in the past, many new findings have been reported in recent years, and other questions remain to be identified and resolved. A sound understanding of meibomian gland structure and function and its role in the functional anatomy of the ocular surface15 is needed, to understand the contribution of the meibomian glands to dysfunction and disease. Herein, we seek to provide a comprehensive review of physiological and pathophysiological aspects of the meibomian glands.

Understanding and Analyzing Meibomian Lipids—A Review

Purpose: This review is intended to bring to the informed reader the current state of knowledge about meibomian lipids and the art for analyzing them. Methods: At the forefront of any endeavor, there are controversies, and these, along with future directions in the field, are brought to the readers attention. Results: Function and anatomy of meibomian glands are briefly covered, giving insight into possible mechanisms for secretory controls. Anatomically, some anomalies in meibomian gland distribution of different species, such as whales versus dolphins, are presented, and, for the first time, the structure of the meibomian glands in a selection of marsupials is presented. In attempting to make the literature more accessible, lipid structure and nomenclature are described, and these structures are related to their possible effects on the physicochemical properties of meibomian lipids. The advantages and disadvantages of various collection and storage techniques are described, as well as how gas chromatography and combined HPLC and mass spectrometry coupled with fragmentation are currently enabling us to determine the nature of the lipids in very small samples. Conclusions: This review extends to discussing the lipids in tears (as opposed to meibomian gland lipids) and briefly highlights new thoughts about the interactions between proteins of the tear film and meibomian lipids. A model that includes proteins in the outer layer of the tear film is also presented. This model is currently being critically analyzed by the ocular community. It concludes briefly by highlighting possible further areas of research in this area.

Tear film stability: A review

Tear film stability can be assessed via a number of tools designed for clinical as well as research purposes. These techniques can give us insights into the tear film, and allow assessment of conditions that can lead to dry eye symptoms, and in severe cases, to significant ocular surface damage and deterioration of vision. Understanding what drives tear film instability and its assessment is also crucial for evaluating existing and new therapies. This review examines various techniques that are used to assess tear film instability: evaluation of tear break-up time and non-invasive break-time; topographic and interferometric techniques; confocal microscopic methods; aberrometry; and visual function tests. It also describes possible contributions of different tear film components; namely meibomian lipids, ocular mucins and proteins, and factors such as age, contact lens wear, ocular surgery and environmental stimuli, that may influence tear film instability.

Surface pressure measurements of human tears and individual tear film components indicate that proteins are major contributors to the surface pressure

Purpose: 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. Methods: A Langmuir trough was used to measure and compare the surface activities of albumin, lipocalin, β-lactoglobulin, lactoferrin, lysozyme, secretory IgA, mucin, meibomian lipid, and tears. Results: All proteins exhibited surface activity. The surface pressure-area (Π-A) profiles of most protein films at equilibrium surface pressure (Πeq) were sigmoidal and showed hysteresis between the expansion and compression phases of the cycle. Πeq of most proteins took 4-9 hours to occur. By contrast, the Π-A profiles for meibomian lipid films were hyperbolic rather than sigmoidal and had little hysteresis, and Πeq was attained within 1 hour. The Π-A profiles of mucin films showed mostly hyperbolic characteristics with small hysteresis. The Π-A profiles of films of tears were sigmoidal, showed strong hysteresis, and reached Πeq at about 5 hours. Partitioning of the proteins and whole tears into the subphase also occurred. Conclusion: Comparison between the dynamic Π-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.

Barriers to clinical uptake of tear osmolarity measurements

Aim The aim of the study was to examine the possibilities of measuring tear osmolarity in a general clinical setting, and to identify the barriers preventing the uptake of new methodologies for its measurement. Methods Five non-contact-lens wearers were recruited to evaluate the diagnostic capability of the TearLab. Three osmolarity measurements were taken at 1 min intervals in the morning at 09:00, midday between 12:00 and 13:00 and afternoon at 16:00 for two consecutive days. Forty more osmolarity measurements were carried out at different times on one subject with low and one subject with high tear osmolarity over 4 months. The osmolarity of a standard solution, 290 mOsm/l, was measured 19 times alternatively with the TearLab by two examiners. Results Consecutive tear osmolarity readings in an individual varied up to 35 mOsm/l, but an average over three readings was found to be a reliable indicator of tear osmolarity at 95% confidence level. For population studies, a power analysis based on the variability of the data showed that three repeat measurements would be required to obtain reliable data for a study with <50 subjects, whereas one measurement would suffice for 490 or more subjects. There were no interobserver or interinstrumental differences, but readings obtained for the standard solution varied up to 89 mOsm/l. Conclusion Three consecutive readings are required with the TearLab to obtain a reliable measure of tear osmolarity. The variation in recorded tear osmolarity makes it difficult to use the technique for the diagnosis of mild dry eye.

The surface activity of purified ocular mucin at the air-liquid interface and interactions with meibomian lipids

Purpose: Ocular mucins are thought to contribute to the stability of the tear film by reducing surface tension. The purpose of this study was to compare the effect of different mucins and hyaluronic acid (HA) alone and mixed with meibomian lipids on the surface pressure at an air-liquid interface. Methods: A Langmuir trough and Wilhelmy balance were used to measure and compare the surface activity of bovine submaxillary gland mucin (BSM), purified BSM, purified bovine ocular mucin and HA, and mixtures of these with meibomian lipids, phosphatidylcholine, and phosphatidylglycerol. Their appearance at the surface of an air-buffer interface was examined using epifluorescence microscopy. Results: Purified ocular mucin had no surface activity even at concentrations that were 100 times more than normally occur in tears. By contrast, commercial BSM caused changes to surface pressure that were concentration dependent. The surface pressure-area profiles showed surface activity with maximum surface pressures of 12.3-22.5 mN/m depending on the concentration. Purified BSM showed no surface activity at low concentrations, whereas higher concentrations reached a maximum surface pressure of 25 mN/m. HA showed no surface activity, at low or high concentrations. Epifluorescence showed that the mucins were located at the air-buffer interface and changed the appearance of lipid films. Conclusion: Purified bovine ocular mucin and HA have no surface activity. However, despite having no surface activity in their own right, ocular mucins are likely to be present at the surface of the tear film, where they cause an increase in surface pressure by causing a compression of the lipids (a reorganization of the lipids) and alter the viscoelastic properties at the surface.

Adsorption of human tear lipocalin to human meibomian lipid films

PURPOSE Tear lipocalin (Tlc) is a major lipid binding protein in tears and is thought to have an important role in stabilizing the Meibomian lipid layer by transferring lipids to it from the aqueous layer or ocular surface, or by adsorbing to it directly. These possible roles have been investigated in vitro using human Tlc. METHODS Tlc was purified from human tears by size exclusion chromatography followed by ion exchange chromatography. Three additional samples of the Tlc were prepared by lipidation, delipidation, and relipidation. The lipids extracted from the purified Tlc were analyzed by HPLC-MS followed by fragmentation. Adsorption of these different forms of Tlc to a human Meibomian lipid film spread on the surface of an artificial tear buffer in a Langmuir trough were observed by recording changes in the pressure with time (Pi-T profile) and monitoring the appearance of the film microscopically. These results were compared with similar experiments using a bovine Meibomian lipid film. RESULTS The results indicated that Tlc binds slowly to a human Meibomian lipid film compared with lysozyme or lactoferrin, even at 37 degrees C. The adsorption of Tlc to a human Meibomian lipid film was very different from its adsorption to a bovine Meibomian lipid film, indicating the nature of the lipids in the film is critical to the adsorption process. Similarly, the different forms of Tlc had quite distinct adsorption patterns, as indicated both by changes in Pi-T profiles and the microscopic appearance of the films. CONCLUSIONS It was concluded that human Tlc was capable of adsorbing to and penetrating into a Meibomian lipid layer, but this process is very complex and depends on both the types of lipids bound to Tlc and the lipid complement comprising the Meibomian lipid film.

The interfacial viscoelastic properties and structures of human and animal Meibomian lipids.

As the interface between the aqueous layer of the tear film and air, the lipid layer plays a large role in maintaining tear film stability. Meibomian lipids are the primary component of the lipid layer; therefore the physical properties of these materials may be particularly crucial to the functionality of the tear film. Surface pressure versus area isotherms, interfacial shear and extensional rheology, and Brewster angle microscopy (BAM) were used to characterize the Meibomian lipids from different species known to have different lipid compositions. The isotherms of humans, bovinae, wallabies, rabbits and kultarrs (a small desert marsupial) were qualitatively similar with little hysteresis between compression and expansion cycles. In contrast, several isocycles were necessary to achieve equilibrium behavior in the koala lipids. With the exception of kultarr lipids, the interfacial complex viscosity of all samples increased by one or two orders of magnitude between surface pressures of 5 mN/m and 20 mN/m and exhibited classic gel behavior at higher surface pressures. In contrast, the kultarr lipids were very fluid up to 22 mN/m; the behavior did not depend on surface pressure. Human lipids were very deformable in extensional flow and the BAM images revealed that the film became more homogeneous with compression as the elasticity of the film increased. The morphology of the kultarr lipids did not change with compression indicating a strong correlation between film structure and behavior. These results suggest that the lipid layer of the tear film forms a gel in vivo, which may aid in mechanically stabilization of the tear film.

Surfactant properties of human meibomian lipids.

PURPOSE Human meibomian lipids are the major part of the lipid layer of the tear film. Their surfactant properties enable their spread across the aqueous layer and help maintain a stable tear film. The purpose of this study was to investigate surfactant properties of human meibomian lipids in vitro and to determine effects of different physical conditions such as temperature and increased osmolarity, such as occur in dry eye, on these properties. METHODS Human meibomian lipids were spread on an artificial tear solution in a Langmuir trough. The lipid films were compressed and expanded to record the surface pressure-area (Π-A) isocycles. The isocycles were recorded under different physical conditions such as high pressure, increasing concentration and size of divalent cations, increasing osmolarity, and varying temperature. RESULTS Π-A isocycles of meibomian lipids showed that they form liquid films that are compressible and multilayered. The isocycles were unaffected by increasing concentration or size of divalent cations and increasing osmolarity in the subphase. Temperature had a marked effect on the lipids. Increase in temperature caused lipid films to become fluid, an expected feature, but decrease in temperature unexpectedly caused expansion of lipids and an increase in pressure suggesting enhanced surfactant properties. CONCLUSIONS Human meibomian lipids form highly compressible, non-collapsible, multilayered liquid films. These lipids have surfactants that allow them to spread across an aqueous subphase. Their surfactant properties are unaffected by increasing divalent cations or hyperosmolarity but are sensitive to temperature. Cooling of meibomian lipids enhances their surfactant properties.

Effect of Kainic Acid and NMDA on the Pattern Electroretinogram, the Scotopic Threshold Response, the Oscillatory Potentials and the Electroretinogram in the Urethane Anaesthetized Cat.

Kainic acid (KA, 12.5-100 nmol) or N-methyl-D-aspartate (NMDA 25-250 nmol) was injected into the vitreous of one eye of urethane anaesthetized cats. Pattern electroretinograms (PERGs) were recorded to transient contrast reversing bars. Scotopic luminance electroretinograms (ERGs) were recorded to blue flashes. All doses of KA reduced the oscillatory potentials (OPs), PERG and focal ERG (FERG). At 50 nmol KA, the b-wave and scoptic threshold response (STR) were normal. At 100 nmol KA, the STR was absent and the b-wave reduced by over 50%. OPs and STRs were reduced in all NMDA injected eyes. NMDA at 25 nmol enhanced the FERG, PERG, and b-wave and high doses (above 150 nmol) reduced them. Light microscopic examination of retinas showed 25 nmol KA only damaged dendrites of ganglion cells. NMDA damage was slight with < 200 nmol. These data show that the cat PERG has a proximal component which is very sensitive to low doses of KA; the PERG and FERG are very similar; the STR and PERG are generated by different structures and that the OPs and the FERG and PERG are all generated close to the ganglion cell layer, proximal to the STR.