Experimental Eye Research xxx (2015) 1e14

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The real reason for having a meibomian lipid layer covering the outer surface of the tear film e A review Thomas J. Millar*, Burkhardt S. Schuett University of Western Sydney, School of Science and Health, Locked Bag 1797, Penrith, NSW 2751, Australia

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 January 2015 Received in revised form 5 May 2015 Accepted in revised form 8 May 2015 Available online xxx

This review critically evaluates a broad range of literature in order to show the relationship between meibum, tear lipids and the tear film lipid layer (TFLL). The relationship of meibum composition to dry eye syndrome is briefly discussed. The review also explores the interactions between aqueous and the TFLL by examining the correlations between meibomian lipids and lipids extracted from whole tears, and by considering protein adsorption to the TFLL from the aqueous. Although it is clear to the authors that a normal tear film resists evaporation, an emerging idea from the literature is that the main purpose of the TFLL is to allow the spread of the tear film and to prevent its collapse onto the ocular surface, rather than to be an evaporative blanket. Current models on the possible structure of the TFLL are also examined. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Dry eye Thin films Tear film Ocular surface Evaporation Mucin Blepharitis

1. Introduction The tear film lipid layer (TFLL) covering the surface of the tear film has been detected using interference methods (Goto and Tseng, 2003a, 2003b; King-Smith et al., 2013a). Seven different functions were ascribed to the TFLL in a review by Bron et al., 2004 (their Table 4). Of these, retarding evaporation has been the most commonly mentioned in publications, particularly those concerning dry-eye syndrome (see Nichols et al., 2011 - Special report on meibomian gland dysfunction; Lemp et al., 2007 - DEWS report). Although Bron et al. (2004) stated that one of the roles of the lipid layer is to lower free energy and impart stability to the tear film, the literature quoted in his review bases this increased stability on the premise that the aqueous layer was exposed to the atmosphere following a blink and the spreading lipid layer would decrease the surface tension of the exposed aqueous layer meaning that the aqueous would not form droplets in the presence of a lipid layer i.e.

Abbreviations: Ch-Es, cholesteryl esters; IR, infrared; MGD, meibomian gland dysfunction; MS, mass spectrometry; NMR, nuclear magnetic resonance; OAHFAs, (O-acyl)-u-hydroxy fatty acids; TFLL, tear film lipid layer; WEs, wax esters. * Corresponding author. E-mail addresses: [email protected] (T.J. Millar), [email protected] (B.S. Schuett).

a more stable film is formed. Current evidence indicates the aqueous is never exposed to the atmosphere following a blink and so when the term stability is used in publications, it is essential that it be defined so that the context can be understood. The review by Bron et al. (2004), also states that a purpose of the TFLL is to thicken the aqueous sub-phase on the principal of the Marangoni effect. This again seems to be on the premise of there being an aqueous air interface at the end of a blink and a full dissertation about this supposed effect is beyond the scope of this review and so has not been discussed further. Other functions mentioned in the review by Bron et al. (2004) were: to be able to spread across the aqueous subphase, provide a smooth optical surface, and to provide some anti-microbial activity. Possible functions of the TFLL not explicitly stated in the review by Bron et al. (2004) were that the TFLL forms a structure that resists collapse of the tear film onto the ocular surface, and that the TFLL enables the tear film to form a thin layer. The latter was indicated in early papers by Holly (Holly and Lemp, 1971; Holly, 1973), and the concept of the structure of TFLL actually enabling it to resist collapse is based on recent experimental evidence showing that a meibomian lipid layer spread on water film resists collapse of the thin film. These will be discussed later in this review. In reading the literature for this review, it was necessary for us to maintain an awareness of the terminology e.g. were the authors

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referring to a meibomian lipid layer or a TFLL; or to whole tears or just the aqueous component. Here: TFLL will be used when referring to the in vivo lipid film covering the tear film; meibum will refer to the secretion from meibomian glands and refer to the bulk; a meibomian lipid film will refer to films made from meibum spread on an aqueous subphase; and lipids of the tear film refers to lipids that are collected from whole tears, and not just the aqueous. 2. Composition of meibum One of the major advances in understanding the TFLL has been a thorough and exacting analysis of lipids found in meibum.1 Such effort was predicated on the concept that dry-eye disease associated with an ineffective TFLL was likely to be caused by a change to the composition of meibum (Brown et al., 2013; Butovich, 2009a, 2013; Green-Church et al., 2011; Lam et al., 2011; Nicholaides and Santos, 1985; Pucker and Nichols, 2012; Saville et al., 2011; Shrestha et al., 2011). It is worthwhile noting that to analyse the lipids in meibum and in tears (see below) is extraordinarily difficult because of small sample sizes, ease of contamination, biological variation, major differences in lipid classes (each class needs different separation and analysis techniques), lack of standards, and the plethora of individual lipid species within a single sample (something only truly appreciated by those attempting the analysis). Overlayed on this complexity has been a confusion generated because on some occasions contaminants from plastics or solvents have been attributed to meibum (Butovich, 2009a; Nichols et al., 2007), and because the composition of rabbit meibum (used as an animal model) appears to be markedly different from that of other animals including humans (Butovich et al., 2012). It is therefore unsurprising that the analysis of the lipids has been a topsy-turvy ride with considerable variation in the data. The analysis of lipid components of meibum has been extensively reviewed (Butovich, 2009a, 2013; Pucker and Nichols, 2012) and the current evidence is that human meibum is ~30e45 mol% cholesterol esters (Ch-Es); the acyl chains of these are generally long (mainly C22:1 e C34:1); and that Ch-Es are present in all normal and dry-eye subjects (Brown et al., 2013; Butovich, 2009b; Lam et al., 2011). This contradicts an earlier study (Shine and McCulley, 1991), where it was reported that some normal subjects did not have Ch-Es. Wax esters (WEs) form ~30e50 mol% of human meibomian lipids (Butovich, 2009a, 2013; Pucker and Nichols, 2012). Prominent in these are C18:1 fatty acid based esters with C18 e C30 alcohols. Using different methodology, Borchman et al. (2013a) showed a WE:Ch-E ratio of 1:0.57 which did not alter with age. While these data are consistent with those reported above, they also highlight the wide variance obtained between research groups. The main amphiphilic molecules secreted from the meibomian glands are currently reported to be (O-acyl)-u-hydroxy fatty acids (OAHFAs). In vitro studies have shown that these molecules are strong surfactants in that small amounts readily spread to cover relatively large surface areas of an aqueous subphase, and such films resist collapse under high compression (Schuett and Millar, 2013). Esters of u-hydroxy fatty acids in meibum were first described by Nicholaides and Santos (1985). It is important note that these were identified by Nicholaides and Santos (1985) as components of non-polar molecules: di- or tri-esters formed with fatty alcohols and fatty acids. By contrast, it was the pioneering work of Butovich et al. (2009) using more modern techniques that revealed the presence of monoesters

1 In this review we have avoided developing an extensive reference list that includes older references where these have been adequately covered in recent review articles unless there is a specific point that needs to be referred to. This is in no way meant to imply that the work is not of great value.

of u-hydroxy fatty acids with fatty acids to give the polar OAHFAs. It is now recognised that these comprise ~4 mol% of meibomian lipids (Brown et al., 2013; Butovich et al., 2009; Lam et al., 2011). Triacylglycerides are ~2% of meibomian lipids, free cholesterol is likely to be less than 0.5%, and phospholipids either within the baseline noise or less than 0.01% (Brown et al., 2013). The presence of small amounts of cholesteryl sulfate in meibum (and tears) from dry-eye syndrome patients has recently been reported (Lam et al., 2014a, 2014b), but the role of this amphiphile in meibum, tears or TFLL is still to be determined. However, due to its common association with epithelia, where it may have a secondary messenger role (Strott and Higashi, 2003), the cholesteryl sulfate detected in Lam et al.'s samples may have originated from meibomian ductal cells. 2.1. Correlation of meibum composition and tear lipid composition with dry eye syndrome Consideration of the science presented in the literature indicates that there is no correlation between dry eye syndrome and an increase or decrease of a particular meibomian lipid or whole tear lipid. One reason for this might be that it simply does not exist, but other reasons are associated with the reliability of the techniques being used: inability to gain a consensus on values for normal subjects; clear and consistent binning of patients into different severities of dry eye; differences in collection techniques; values for many lipids, particularly phospholipids, that are at detection limits; a relativity problem of the techniques e when there is certainty about the individual lipid species there is uncertainty about the amount and vice versa; and rabbits being used as models. Lam et al. (2011) examined the composition of meibum obtained from an Asian population and found statistically significant differences between some saturated triglycerides, sphingolipids and ChEs in dry eye patients compared with normal subjects. They used Student's T test, which relies on a normal distribution, for the statistical analysis but there is no evidence of the data being normally distributed e this includes the severity of dry eye and the distribution of lipid classes. Given this, that the lipid classes where the differences were claimed represent tiny mole fractions (this is difficult to tell for triglycerides because the ordinate on their Fig. 3C duplicates numbers) and there is no consistent trend when going from mild, moderate to severe dry eye, it appears that a much larger cohort would need to be tested before any conclusions could be made. They also recognise this because their cautious suggestion that a decrease in OAHFAs may be an indicator of dry eye progression was qualified by a caveat that a larger sample size would be required in order to determine this. It is interesting to note that Lam et al. (2014b) more recently showed that there was a trend of OAHFAs to increase with age, which is counter intuitive to the concept of increasing dry eye with age. Borchman and his colleagues used a number of techniques to also examine age related changes in the composition of meibomian lipids and compared these with subjects having meibomian gland dysfunction (MGD; Borchman et al., 2013a, 2012a; 2012b, 2011; 2010a, 2010b; 2007a; Shrestha et al., 2011). The basic approach by Borchman and his co-workers was to use infrared (IR) analysis, which is excellent for identification of key functional groups within meibum, complemented with nuclear magnetic resonance (NMR), which is excellent for quantitative measurements, or relative quantitative measurements of different lipid species. However, overall it is problematic to assign specific lipids to any of these. The authors combined these data with transition temperature profiles of bulk meibum. Their thesis was that there may be a relationship between changes to molecules in the main lipid classes and a particular aged population or MGD; and that these changes would correlate to a change in the “stiffness” of meibum as reflected in different

Please cite this article in press as: Millar, T.J., Schuett, B.S., The real reason for having a meibomian lipid layer covering the outer surface of the tear film e A review, Experimental Eye Research (2015), http://dx.doi.org/10.1016/j.exer.2015.05.002

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transition (melting) temperatures. The structural basis for this is that longer and less saturated acyl chains in the lipids are more ordered in their packing and hence have higher transition temperatures. Essentially, their studies indicated that there was an increase ordering of the molecules in MGD relative to age matched controls, and a decrease in lipid order with age (Borchman et al., 2011). The likely cause for this in MGD was an increase in saturated lipids and a decrease in unsaturated lipids rendering the lipid mix of meibum to be more ordered (the opposite occurring with ageing; Borchman et al., 2010a). They claimed from their NMR analysis that there was a statistically significant difference between, methyl moieties, lipid oxidation products and methine moieties of normal versus those with MGD (Borchman et al., 2012b). However, some aspects need more explanation (also see commentary by Butovich, 2013). The 1.26 ppm signal that they refer to in their discussion as being due to methyl groups (eCH3) may have been a mistake because the signal is too far downfield for protons of a methyl group. Instead the signal is most likely due to protons of methylene groups (eCH2e). They also use the strong signal at 1.39 ppm to normalise their data. This resonance will not only be a signal from the methylene groups of meibomium lipids, but also include a signal from un-deuterated solvent, which is an unavoidable contaminant of deuterated cyclohexane. To compensate for this, the data in Borchman et al., 2012b were normalised against another prominent NMR-peak at 4.1 ppm, which was later assigned to the methylene group neighbouring the ester of a WE (Borchman et al., 2013a; Borchman personal communication). In addition, principal component analysis of resonances from 0 to 1.34 ppm (i.e. not including the potential solvent peak was used (Borchman et al., 2012b) which allowed discrimination between meibum from normal subjects from those with MGD. However, in general, the use of solvents such as chloroform or tetrachloroethylene would eliminate the need for such compensation and have the additional advantage of not relying on the presence of a second resonance that was constant across samples from different populations. Overall the pioneering work Borchman's group provides a basis for an alternative method to mass spectrometry (MS) for studying meibum and tear lipid composition. Compared with MS (see below) quantitative data of functional groups using protonNMR are easy to gain and they are absolute and so changes to their signal levels represent changes to the lipidome. A limitation of this approach is that definitive assignment of resonances of these complex NMR spectra to functional groups within complex mixtures is problematic, and to assign it to a particular lipid species is not generally possible. For example it is not possible to assign a double bond of in an acyl chain to a particular position, and nor is it possible to determine whether the acyl chain with the double bond is part of a WE, Ch-E, OAHFA, etc. It is also not possible to assign the double bond resonance to a particular component of a WE molecule i.e. the fatty acid, the fatty alcohol or both. Assignment of squalene to ~5.0 ppm (Borchman et al., 2013b) is an exception indicating that squalene is present on the eyelid, and a tiny amount is present in meibum. However, similar molecules such as the carotenoids have been shown to have quite different peak assignments (Borchman et al., 2013b), which exemplifies the difficulties of being definite about peak assignment to compound groups. In another follow-up study using high resolution equipment, Borchman et al. (2013a) were able to identify and distinguish the neighbouring protonsignals of the ester-groups found in WEs and Ch-Es in native meibum. This accomplishment allowed them to calculate the WEs to Ch-Es ratio, although with the caveat that signals of the same protons in OAHFAs could be part of the WE signal. With regards to the impact of changes to phospholipids correlating with dry eye, it is evident that almost all of the phospholipids found in whole tears are not sourced from meibum (Butovich, 2013,


€ki et al., 2011a; Saville et al., 2008; Lam et al., 2014a, 2014b; Rantama 2011). This implies that any correlation between changes to phospholipid levels and dry eye syndrome would not be due to changes in meibum composition but due to a change in another ocular tissue. The ocular tissue that is the source of the phospholipids found in whole tears is currently unknown. Given that detection for many individual phospholipid species in meibum and whole tears is already at the limitations of the technology, then a disease state would need to show a significant and consistent increase in a particular phospholipid to be certain of a correlation and to date this has not occurred. Likely partitioning of phospholipids into the TFLL and hence possible effects of phospholipids in dry eye syndrome associated with the TFLL is discussed below. 3. The effect of lipids on structure or performance of meibomian lipid films Another tack taken to understand how changes to lipid profile may affect the TFLL has been to examine the effect of deliberately seeding meibomian lipid films with specific lipids (Georgiev et al., 2012, 2011; 2010; Schuett and Millar, 2013, 2012; Raju et al., 2013). These experiments were based on the fact that there are vast numbers (in the thousands) of individual lipid species in meibum and therefore it is difficult to determine by lipid analysis if a specific lipid has a strong influence over the structure or performance of the TFLL or a meibomian lipid film. Instead, this information might be gleaned if lipids were deliberately added to meibomian lipid films and changes to their structure or performance measured with an expectation that very influential lipids would cause large changes even in small concentrations. A general outcome from these studies has been that meibomian lipid films are extremely tolerant to seeding with various lipids. A minor effect on pressure area profiles was seen using a very unsaturated WE, linolenyl linolenate, at ~17% by mass whereas the less unsaturated lipids such as arachidyl oleate and oleyl stearate had little effect at ~17% by mass (Schuett and Millar, 2012). Similarly, viscosity and elasticity of meibomian films were not strongly altered by adding cholesterol or b-carotene at 1 mol% (Raju et al., 2013). A small effect on meibomian lipid film pressure area profiles was observed if surfactants such as sphingomyelin or dipalmitoylphosphotidylcholine (a phospholipid) at 20 mol% were used (Georgiev et al., 2010). The implication from these studies, and those from Borchman showing age related changes to meibum in normal subjects (Borchman et al., 2013a), is that small changes to any lipid component of meibum are likely to be tolerated, or difficult to delineate as a real change from normal changes in an ageing spectrum. Therefore exhaustive examinations of meibomian lipid profiles in MGD are unlikely to be fruitful in identifying a specific lipid as cause for dry eye syndrome. A possible exception to this gloomy prediction comes from Georgiev et al. (2011, 2012) who showed that benzalkonium chloride (a common preservative in tear formulations), but not other preservatives (SofZia or Polyquad) can interfere with meibomian lipid films. They proposed that benzalkonium chloride was capable of displacing meibomian lipids off the surface. Therefore, extensive analysis of meibomian lipids, or lipids in the tear film might reveal some components of the tear film having a similar property, i.e. displace lipids from the TFLL rather than become a component of the TFLL. However, a relatively large compositional change relative to the total lipids in the TFLL would most probably be needed. 4. Measurement and levels of lipids in whole tears and the relationship of this to the TFLL In recent times, there has been a focus on collecting and

Please cite this article in press as: Millar, T.J., Schuett, B.S., The real reason for having a meibomian lipid layer covering the outer surface of the tear film e A review, Experimental Eye Research (2015), http://dx.doi.org/10.1016/j.exer.2015.05.002


T.J. Millar, B.S. Schuett / Experimental Eye Research xxx (2015) 1e14

analysing lipids in whole tears (Brown et al., 2013; Borchman et al., €ki 2007a; Butovich, 2013, 2008; Lam et al., 2014a, 2014b; Rantama et al., 2011a; Saville et al., 2011). The overall purpose behind these studies has been to determine the nature of lipids in whole tears from normal subjects, and if changes in the lipid profile of whole tears correlate with changes to the tear film due to a particular variable e.g. ethnicity, sex, age, dry-eye, contact lens wear, meibum lipid profile. 4.1. Analysis of lipids in whole tears An outcome of examining normal whole tears has been that there is a broader array of lipids in whole tears than in meibum, but all of the lipids found in meibum including OAHFAs have been detected in whole tears. In particular, the most modern technologies and recent publications have been consistent in demonstrating a relative abundance of phospholipids of ~10 mol% in whole tears, whereas in meibum, phospholipids have a relative abundance of ~0.006e0.1 mol% (Brown et al., 2013; Butovich, 2013, 2008; Lam €ki et al., 2011a; Saville et al., 2011; et al., 2014a, 2014b; Rantama see also Table 1). The importance of finding a relative abundance of phospholipids in whole tears is that these could form part of the TFLL, particularly the surfactant layer (as described in the McCulley and Shine, 1997 model); and if this were true, then changes in phospholipids could affect the performance of the TFLL and therefore may be a cause of dry eye syndrome. In order to establish the association of changes to the lipid profile of whole tears and specific ocular conditions such as dry eye syndrome it is important to consider: the reliability of measurements of lipids in whole tears and hence what are the normal values; the origin of the phospholipids; and the likelihood that

phospholipids arising from sources other than meibum are likely to become the surfactant layer of the TFLL and if they were to, whether they would be in sufficient quantities to affect the performance of the TFLL. A confounding element in determining the answers to these questions is that the literature in this area has many inconsistencies, which for the most part have not been dealt with at length by the authors, nor have they been identified in the manuscript peer review process. Foremost in understanding this literature is that nobody has been able to demonstrate from which compartment or compartments of the tear film that the lipids originate (meibum, TFLL, aqueous, skin of the eyelid margin, epithelial cells of the ocular surface), although this has been partially addressed. A sophisticated comparison between meibum and lipids of the whole tears of individuals (Brown et al., 2013) showed that, except for phospholipids, the classes and ratios of lipids in whole tear samples reflected the classes of lipids and ratios found in meibum. This is a very important observation because it indicates that the meibomian lipids detected in whole tear film samples are most likely derived from meibum, and that the phospholipids must be from another source. This was also supported by Lam et al. (2014a) who showed that except for an enhanced phospholipid content, whole tears had a similar lipid profile to meibum and this was independent of whether or not capillary tubes or Schirmer strips were used to collect the tears. Irrespective of methodologies (collection and lipid profile evaluation), there are vast differences in this area of the literature. Comparing values obtained between groups and even within groups is not an easy task because of the different ways that have been used to report the results and so this has been summarised (Table 1). If the CE/WE ratios are taken as an example and it is

Table 1 Molar ratios and concentration of selected lipid classes in tears. Lipid

Ch-E (mol %) WE (mol %) Cholesterol (mol %) OAHFA (mol %) PLs (mol %): PL total Glycero-PL Lyso PL SM PL (pmol/mL): PL total Glycero-PL Lyso PL SM Total lipid (pmol/mL) Collection method

Brown et al., Saville et al., 2013 2010,a 39 43 n.a

n.a. n.a. n.a.


n.a. n.a.

Saville et al., 2011,a

Basal Reflex Flush

Lam et al. 2014b,g

Lam et al., 2014c,h

Dean and Glasgow 2012

€ki et al., Rantama 2011a

54.8 35.7 29.1 18.6 8.2 17.2

33.0 44.8 (26)d 30.0 35.2 (59)d ~20.0 5.9

43 30 8

33 46 8

n.a. n.a. n.a.

n.a.i n.a.i n.a.j

~2.5 60, and there was an expected slightly higher level of casual meibum in males than females up to the age of ~70; presumably due to androgen effects (Sullivan et al., 2002). Given the challenges of measuring evaporation rates from the ocular surface and likely variances in the population, the hypothesis of a TFLL reducing evaporation has been explored under controlled conditions. An often cited paper to support the blanket preventing evaporation theory is that of Iwata et al. (1969). Basically, a rabbit's eye was held open and the evaporation rates were measured from: the bare corneal surface with epithelium intact; the intact tear film covering the corneal surface; the surface (presumably comprising an artificial tear solution) after removing the lipid layer by copiously rinsing the corneal surface with a physiological artificial tear solution; and a control water solution. They showed in their Fig. 2 that the evaporation rate after removing the lipid layer by copious washing was higher than with the intact tear film. Indeed, the evaporation rate was almost identical to that of water. This would actually be the expected result if the copious washing removed the whole tear film and not just the lipid layer. However, they do not comment on this possibility, and nor do authors who subsequently quote them. By contrast, Brown and Dervichian (1969) were unable to show that a spread film of human meibomian lipids at 37  C prevented evaporation. Herok et al. (2009) also found that in vitro a meibomian lipid layer did not prevent evaporation. This contrasted with an earlier publication from the same group where it was shown that bovine meibomian lipids reduced evaporation from a pendant drop (Miano et al., 2004). Millar has since repeated these experiments by coating a pendant drop of collected human tears or a pendant drop of saline with human meibomian lipids and did not find resistance to evaporation (unpublished data). Subsequently, Cerretani et al. (2013) have carried out a series of much more elegant in vitro studies to investigate this problem more thoroughly. Although they were able to demonstrate that a meibomian lipid film at ~35  C and subjected to a strong air flow of ~2.5 ms1could prevent evaporation from a saline solution, it was minimal (~8%) until it was greater than 100 nm thick (beyond normal physiological thicknesses). A slightly different approach was taken by Holopalainen's group €ki et al., 2013) (Kulovesi et al., 2014; Paananen et al., 2014; Rantama who investigated the effects of emulated tear film lipid layers using individual WEs and mixtures of lipids at different temperatures on evaporation. While they showed that only few WEs e.g. behenyl palmitoleate, could form films that reduced evaporation, they concluded, “In multicomponent lipid layers, the evaporation


retardant interactions between carbon chains decrease and therefore these lipid layers do not retard evaporation”(Rantam€ aki et al., 2013). Therefore, their data support the concept that meibomian lipids do not retard evaporation, because meibum is a multicomponent lipid mixture (Butovich, 2009a). A similar finding was reported by Borchman et al. (2013a) that an artificial lipid layer made of a mixture of a WE and a Ch-E can reduce evaporation, but it needed to be 500 times the thickness of the TFLL to have a notable effect. Even if evaporation were the cause of collapse of the tear film onto the ocular surface, Holly noted some 40 years ago that under average conditions it would take about 10 min to cause drying (much slower than blink intervals), water influx from the cornea due to increased osmolality would increase the drying time, and the patch-like drying and the contact angles at the boundaries of such dry spots were not commensurate with an evaporation theory (Holly, 1973). 6.2. Enabling the formation of a thin film Experimental data indicate that the meibomian lipid layer in conjunction with the mucin layer at the ocular surface, allow the aqueous to spread to form a thin film (Holly and Lemp, 1971; Holly, 1973) and more recently that the lipid layer prevents collapse of the film once it has formed Rosenfeld and Fuller (2012). In order to understand that formation of a thin film is not trivial, it is important to have a concept of the thickness of the precorneal tear film. Current estimates place it at ~4 mm thick (see King-Smith et al., 2004 for a review). If one imagines a human eye ~2 m in diameter (~80 times linear size), then the tear film would have a mere thickness of ~0.3e0.4 mm spread over a ~2 m2 surface, and such a film would have to remain stable without collapsing down onto the ocular surface. Holly and Lemp (1971) explained that to enable the film to spread requires the contact angle of water to the ocular surface to be reduced to zero or close to zero. This is accomplished by two things: increasing the adhesion tension of the ocular surface (the ocular surface has to be sticky for water); and decreasing the surface tension of the water so that it spreads out rather than forming a drop. Membrane bound mucins on the apical surface of the corneal epithelium (Gipson et al., 2004) increase the adhesion tension, and this helps to explain why alterations in mucin is associated with dry eye syndrome. This will not be discussed further in this review. Decreasing the surface tension of the aqueous of the tear film is accomplished by the lipid layer where the surfactant molecules, primarily the OAHFAs, play a key role (Butovich, 2009a). As stated above, the proteins including mucins of the aqueous part of the tear film and possibly proteins of meibum are also likely to form integral parts of the lipid layer of the tear film and contribute to lowering the surface tension (Holly and Lemp,  and Tiffany, 1999; Millar et al., 1977; Miano et al., 2005; Nagyova 2006). An interesting finding in this context was the demonstration that mucins can cause a disordering of WEs (Faheem et al., 2012). However, this was a very artificial situation with buffer covering a bulk mixture of two WEs spread over a crystal, and therefore the types of molecular interactions between mucins and the TFLL still remain to be demonstrated. Experiments by Rosenfeld and Fuller (2012) have given insight into the prominence of meibomian lipids to preventing collapse of the tear film. They developed a technique that enabled sampling of a thin film of water covered by a lipid layer, and then drew the water out from underneath the lipid layer until the film collapsed. Their studies show that a thin aqueous film can be formed using different lipid surfactants: fatty acids, phospholipids or meibomian lipids. However, unlike the other surfactant lipids, they were able to show that the meibomian lipids actually resisted collapse of the thin film and so enabled the formation of a much thinner film than

Please cite this article in press as: Millar, T.J., Schuett, B.S., The real reason for having a meibomian lipid layer covering the outer surface of the tear film e A review, Experimental Eye Research (2015), http://dx.doi.org/10.1016/j.exer.2015.05.002


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either films with fatty acids or phospholipids as the surfactants. These exciting findings have to be treated with caution because they were carried out at ~20  C, which is lower than the transition temperature of meibomian lipids (Borchman et al., 2010a, 2007b), and the compression rate of the film was much slower than that which would occur in vivo during a blink. Nevertheless, the results of Rosenfeld and Fuller (2012), if proven to be true at higher temperatures are very compelling. It is consistent with known but poorly understood concepts about meibum: why meibum has its particular lipid composition (Butovich, 2009a); and why it has the very unusual feature of having a phase transition temperature range (Borchman et al., 2010a, 2007b) close to that of the ocular surface temperature (Geiser et al., 2004). The lipid composition of meibum and its transition temperature is such that it is liquid enough to enable it to spread across the ocular surface, but solid enough to resist collapse i.e. it has viscous (liquid) and elastic (solid) properties at the same time. During a blink, the stiffness of the TFLL would also make it difficult for the aqueous to breach the lipid layer and overflow onto the lids and thereby ensure that the aqueous flowed towards the puncta. A remarkable feature of the composition of meibum is the abundance of Ch-Es. A well-known characteristic of cholesterol and Ch-Es is that they are able to form liquid crystals (Barrall et al., 1966; McMillan, 1972). With this in mind, Millar (2013) proposed a mechanism involving the formation of liquid crystals to explain the workings of meibomian lipid films. Essentially, while surfactants in meibomian lipids enable the spread of meibomian lipid films, the formation of liquid crystals by Ch-Es or mixed with WEs orthogonal to the film would give the film elastic properties that would resist collapse of the film onto the ocular surface. Although, this mechanism was speculative, it has been supported by Rosenfeld et al. (2013) and Butovich (2013). Rosenfeld et al. (2013) used small and wide angle diffraction to analyse bulk meibomian lipids and this led to model of a meibomian lipid film which included lamellar crystals. Butovich et al. (2014) used cross-polarized microscopy to demonstrate the presence of liquid crystals in bulk meibum and these were present at 32  C and disappeared as the temperature increased to 40  C. Therefore, it seems that these crystals are likely to exist, but the speculation by Millar (2013) that they might be ChEs, although attractive, was not supported by the X-ray diffraction results of Leiske et al. (2012). However, their measurements were on meibomian lipid films that had not been conditioned by heating and multiple compression cycles, which cause the lipids to reorganise from their initial arrangement when spread with a solvent. Speculatively, an absence or deficiency of these crystalline structures may mean, that the TFLL is unable to prevent the collapse of the tear film onto the ocular surface. Given that a main role of the TFLL is to prevent collapse of the tear film, then the stiffness (elastic modulus) of this layer is critical and will be governed in part by the transition temperature of the meibum forming the TFLL. In this regard, the extensive studies on transition temperature of meibomian lipids by Borchman and his colleagues may have some significance to formation of a stable tear film. Borchman and his co-workers found differences in different populations: the transition temperature in bulk meibum was higher in children and lower in elderly people (Borchman et al., 2010a), and higher in patients with MGD than for age matched normal (Borchman et al., 2011). Whether these differences in transition temperatures translate to differences in the TFLL, and in particular affecting the ability of the TFLL to prevent collapse of the tear film still has to be established. 7. Structural characteristics of the TFLL The TFLL has a flexible structure that allows it to be compressed

and then re-spread during a blink cycle. Understanding the structural properties of the TFLL that allow it to be compressed and then respread appears to be clinically important because a slow respreading after a blink may be a measure of aqueous dry-eye (Georgiev et al., 2014; Goto and Tseng, 2003a, 2003b; Ring et al., 2012; Yokoi et al., 2008). If only hydrophobic lipids e.g. WEs and Ch-Es, were present, then the lipids would simply form lenses and not spread (Cerretani et al., 2013; Rantam€ aki et al., 2011b; Rosenfeld and Fuller, 2012). Therefore, the nature of the surfactants is likely to be critical to the performance of the tear film lipid layer. On occasions, the TFLL has been compared with lung sur€ki et al., 2011b) in order to glean information that factant (Rantama might be relevant to developing a model of the TFLL. However, the differences between lung surfactant and meibum are vast and it seems that lung surfactant is a completely different mechanism for solving the problem of lowering surface tension and forming a thin film (Perez-Gill and Weaver, 2010). Lung surfactant appears to be formed by type II epithelial cells by intracellular construction of multi-lamellar bodies. These bodies are mainly formed by phospholipids (mainly dipalmitylphosphatidylcholine and some phosphatidylglycerol), some cholesterol and lung surfactant proteins SP-B and SP-C i.e. a suite of surfactant molecules. These are secreted into the airways where they merge with the surface surfactant. On compression, they leave the airway surface and the components are taken up by cells in multi-vesicular bodies to be recycled. That is, lung surfactant is designed to flow from an aqueous environment to the airway surface and then be retrieved back through an aqueous environment. This is completely different from meibum, which is mainly composed of non-surfactant molecules and designed to emerge from a lipophilic environment to spread over an aqueous environment. As such, the model prominent for the TFLL is that described by McCulley and Shine first in 1997 and then expounded on in 2001. Essentially it is a layer of surfactant molecules at the aqueous surface coated with multiple layers of WEs and Ch-Es. This model does not take into account the dynamic nature of the tear film and there were a number of critical considerations associated with their model that were presented by McCulley and Shine (2001). They indicated that: there was a critical temperature needed to form the TFLL and once reached, it would remain stable even below the critical temperature; the ratio of divalent (Ca2þ) and monovalent cations (including Hþ; pH) in the subphase would influence the organisation of the surfactant molecules (interaction with themselves and with water); through various mechanisms, rather than a superlattice as portrayed in their model, the lipids of the TFLL may form into domains making the TFLL less stable; and mucins and enzymes could interact with the TFLL. So the model represented the TFLL unencumbered by a number of physiological influences. It is also notable that their model used phospholipids and fatty acids as the main surfactant molecules, rather than OAHFAs which are now known to be the major surfactants in meibum. More recently, in silico modelling of the TFLL enabled a dynamic perspective (Kulovesi et al., 2012; Telenius et al., 2012; Wizert et al., 2014). As with McCulley and Shine (2001), phospholipids and free fatty acids were used as the surfactant molecules, and the models used Ch-Es in a much lower molar percentage than the other lipids, and neither WEs nor OAHFAs were used. This is quite contrary to the available evidence regarding the composition of meibum and so the relevance of this modelling for a TFLL needs to be treated cautiously. Nevertheless, a feature of their models was the formation of clusters of hydrophobic lipids into lenses under high surface pressures. These resemble the spots seen using high resolution lipid layer microscopy of the TFLL in vivo (King-Smith et al., 2011) and of meibomian lipid films in vitro (Millar and King-Smith, 2012). The formation of such lenses may be critical for allowing the spread

Please cite this article in press as: Millar, T.J., Schuett, B.S., The real reason for having a meibomian lipid layer covering the outer surface of the tear film e A review, Experimental Eye Research (2015), http://dx.doi.org/10.1016/j.exer.2015.05.002

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of the lipid layer following a blink. Rather than a simple balling-up/ condensation of the hydrophobic molecules as suggested by the in silico modelling of Kulovesi et al. (2012), King-Smith et al. (2013b) provided a novel and insightful concept of a folded lipid layer to explain how the lipid layer is able to spread, and take up the possibility of how the WEs and Ch-Es might align into different domains. Although this folding model incorporates some of the features of the liquid crystal model proposed by Millar (2013) and would also resist collapse of the tear film, it has a distinct advantage in that it incorporates a mechanism for spreading the lipid layer following a blink. This would be particularly true if the folding were mainly associated with the surfactant molecules laminating. Such foldings in local regions could account for the lens like structures seen in high resolution microscopy of the TFLL and meibomian lipid films. It is also attractive to believe that the long acyl chains of the OAHFAs interacting with the hydrophobic lipids would cause these to fold so that they would be sandwiched between the OAHFA lamina. This is not a feature of the King-Smith et al. (2013b) model, which shows lamination of the polar and non-polar laminae as separate entities. Aqueous would also be trapped in the laminae between the hydrophilic head groups of the OAHFAs. The expectation would be that after a blink, the spots would actually unfold from spreading the lipids from one side of the spot giving the appearance of a spreading tongue out from one side of the initial spot. At the same time the spot would become depleted and eventually disappear. King-Smith (personal communication) has observed such structures. Based on his pictures (personal communication), which show different thicknesses of unfolding in the tongues, the folding includes super laminated structures (laminar folds that are folded). Having the OAHFA's very long acyl chains rather than phospholipids may be critical to such a model because their very long chains would enable them to integrate with the WEs and Ch-Es and hence prevent OAHFAs from forming micelles or vesicles that would leave the TFLL and enter the aqueous. A more simple, though conceptually similar model, has been proposed by Georgiev et al. (2014). Their model proposed that the WEs and Ch-Es would form a reservoir for the polar lipids so that the polar lipids would move into this reservoir under high pressure and move back onto the aqueous surface under low pressure. There is some indirect evidence of the importance of the OAHFAs for forming a stable tear film. Butovich et al. (2011) found that the composition of dog meibum was similar to that of humans except for a greater abundance of OAHFAs and suggested that the extra OAHFAs was a possible reason for longer interblink times in dogs compared with humans. In addition, Rosenfeld and Fuller (2012) were able to show that phospholipids or fatty acids spread on an aqueous subphase enabled the formation of thin films, but only meibomian lipid films (containing OAHFAs) resisted collapse. 8. Summary and conclusion Evidence from the literature is indicating that the role of the TFLL is to enable the formation of a thin film, to prevent collapse of this thin film and to form a strong barrier that forces the aqueous to flow to the puncta rather than being squeezed over the eyelids during a blink. The prevention of collapse is an important concept because a defective lipid layer, for whatever reason, would lead to premature collapse of the tear film onto the ocular surface. The appearance of such collapse would be exactly the same as that claimed for evaporative dry eye. The prevailing, strong and simplistic belief that the collapse of the tear film in dry eye syndrome is due to increased evaporation due to a defective lipid layer is not supported by scientific evidence. The evidence from studies of evaporation from the ocular surface in vivo irrespective of the method, show enormous variability between test re-test, variability


within blink cycles, variability between research groups, and has often been inconsistent with expected changes when physical parameters (temperature and humidity) are changed. This variability and inconsistency means that these are not scientifically valid tests for demonstrating a correlation between the collapse of the tear film and evaporation from the ocular surface. In addition, evaporative experiments in vitro overall indicate that the tear film lipid layer does not prevent evaporation and where researchers have found that it reduces evaporation, this reduction is so small or the lipid layer is so thick that it would not be a consideration in terms of evaporative dry eye. Although there is a clear and consistent link between blepharitis and dry eye syndrome, the assumption has been that a reduction in the quantity or quality of meibum, and hence the TFLL, leads to increased evaporation of the tear film and premature collapse. This correlation (evaporation rate and premature collapse of the tear film onto the ocular surface in blepharitis) has never been demonstrated. It is at least as plausible, but has not been demonstrated, that a defect in meibum in blepharitis leads to the TFLL being unable to resist collapse of the tear film. To understand this concept better, experiments using the methodology of Rosenfeld and Fuller (2012) could be carried out e.g. the effects of keratin, inflammatory cytokines and lipases on the ability of meibomian lipid films to resist collapse could be tested. Our knowledge of the composition of meibum and hence of the lipids of the TFLL (and the aqueous) has become extensive and detailed, but despite this, no correlation between dry eye syndrome and a specific lipid class or species had been convincingly demonstrated. Indeed, it appears that the TFLL has been designed to tolerate considerable variation in lipid species. Emerging from these studies has been the strong indication that OAHFAs are major lipid surfactants of the TFLL and being part of meibum, they are simply spread from the meibomian glands into the TFLL during blinking. The role of phospholipids as surfactants in the TFLL is scientifically unconvincing. Although they are present in whole tears, modern techniques indicate that they are virtually absent in meibum. This observation means that any models incorporating phospholipids as surfactants in the TFLL should also be able to explain the mechanism by which these molecules pass from the aqueous compartment into the TFLL and demonstrate that their final concentrations in the TFLL are such that they influence the TFLL. Logic based on the amounts of phospholipids detected in whole tears and applied to our knowledge of lipid binding proteins in tears would suggest that it is highly unlikely that significant amounts of phospholipids are being transported from the aqueous into the TFLL. However, this will remain irresolute until the methodology for whole tear film analysis (collection as well as lipid identification and quantification) become more reliable and experiments are carried out to determine likely compartmentalisation of the lipids in whole tears. Efforts in understanding the TFLL are still in their infancy with regards to the effects of proteins both within meibum e.g. keratins, and from the tear film, particularly mucins and proteins associated with inflammation. Mucins are specifically mentioned here because whenever the effects of proteins of meibomian lipids has been reported, mucins always emerge as having a dominant effect. While there has been strong progress in understanding the relationship between lipids in the aqueous and the lipids of the TFLL, this is still a developing field. Currently there is considerable inconsistency in data from these studies due to the difficult nature of this work. The past 10 years has enormously enhanced our knowledge of the lipid composition of meibum resulting in subtle, but important conceptual changes about the possible structure and function of the TFLL. A result has been an emergence of more detailed models that expand the McCulley and Shine (1997, 2001) model. These models

Please cite this article in press as: Millar, T.J., Schuett, B.S., The real reason for having a meibomian lipid layer covering the outer surface of the tear film e A review, Experimental Eye Research (2015), http://dx.doi.org/10.1016/j.exer.2015.05.002


T.J. Millar, B.S. Schuett / Experimental Eye Research xxx (2015) 1e14

often include proteins as part of their structure, possible mosaic distributions of structural elements and many attempts have been made to take into account the effects of squeezing and respreading of the TFLL during blink cycles. The advantage and availability of these models means that they form a strong base for experimental testing. In this regard, it is exciting that the enormous computing power now available means that much of this testing could be done in silico, which will enable us to come closer to understanding the enigma of the TFLL. Acknowledgements Thomas Millar and Burkhardt Schuett were supported by the Australian Government CRC grant “Vision CRC”. We would also like to thank George Herok and Shiwani Raju for critically reviewing this manuscript and making helpful suggestions. References Barrall II, E.M., Porter, R.S., Johnson, J.F., 1966. Temperatures of liquid crystal transitions in cholesteryl esters by differential thermal analysis. J. Phys. Chem. 70, 385e390. rtek, J., B s, Z., Ba artkov a, J., Taylor-Papadimitriou, J., Rejthar, A., Kovarík, J., Luka sek, B., 1986. Differential expression of keratin 19 in normal human Vojte epithelial tissues revealed by monospecific monoclonal antibodies. Histochem. J. 18, 565e575. Borchman, D., Foulks, G.N., Yappert, M.C., Tang, D., Ho, D.V., 2007a. Spectroscopic evaluation of human tear lipids. Chem. Phys. Lipids 147, 87e102. Borchman, D., Foulks, G.N., Yappert, M.C., Ho, D.V., 2007b. Temperature-induced conformational changes in human tear lipids hydrocarbon chains. Biopolymers 87, 124e133. Borchman, D., Foulks, G.N., Yappert, M.C., Kakar, S., Podoll, N., Rychwalski, P., Schwietz, E., 2010a. Physical changes in human meibum with age as measured by infrared spectroscopy. Ophthal. Res. 44, 34e42. Borchman, D., Yappert, M.C., Foulks, G.N., 2010b. Changes in human meibum lipid with meibomian gland dysfunction using principal component analysis. Exp. Eye Res. 91, 246e256. Borchman, D., Foulks, G.N., Yappert, M.C., Bell, J., Wells, E., Neravetla, S., Greenstone, V., 2011. Human meibum lipid conformation and thermodynamic changes with meibomian-gland dysfunction. Invest. Ophthalmol. Vis. Sci. 52, 3805e3817. Borchman, D., Foulks, G.N., Yappert, M.C., Milliner, S.E., 2012a. Changes in human meibum lipid composition with age using nuclear magnetic resonance spectroscopy. Invest. Ophthalmol. Vis. Sci. 53, 475e482. Borchman, D., Foulks, G.N., Yappert, M.C., Milliner, S.E., 2012b. Differences in human meibum lipid composition with meibomian gland dysfunction using NMR and principal component analysis. Invest. Ophthalmol. Vis. Sci. 53, 337e347. Borchman, D., Yappert, M.C., Milliner, S.E., Duran, D., Cox, G.W., Smith, R.J., Bhola, R., 2013a. 13C and 1H NMR ester region resonance assignments and the composition of human infant and child meibum. Exp. Eye Res. 112, 151e159. Borchman, D., Yappert, M.C., Milliner, S.E., Smith, R.J., Bhola, R., 2013b. Confirmation of the presence of squalene in human eyelid lipid by heteronuclear single quantum correlation spectroscopy. Lipids 48, 1269e1277. €rgermann, J., Pleyer, U., Tsokos, M., Paulsen, F.P., 2007. Detection and Br€ auer, L., Bo localization of the hydrophobic surfactant proteins B and C in human tear fluid and the human lacrimal system. Curr. Eye Res. 32, 931e938. Br€ auer, L., Paulsen, F.P., 2008. Tear film and ocular surface surfactants. J. Epith. Biol. Pharmacol. 1, 62e67. Bron, A.J., Tiffany, J.M., 2004. The contribution of meibomian disease to dry eye. Ocul. Surf. 2, 149e164. Bron, A.J., Tiffany, J.M., Gouveia, S.M., Yokoi, N., Voonin, L.W., 2004. Functional aspects of the tear film lipid layer. Exp. Eye Res. 78, 347e360. Brown, S.H.J., Kunnen, C.M.E., Duchoslav, E., Dolla, N.K., Kelso, M.J., Papas, E.B., Lazon de la Jara, P., Willcox, D.P., Blanksby, S.J., Mitchell, T.W., 2013. A comparison of patient matched meibum and tear lipidomes. Invest. Ophthalmol. Vis. Sci. 54, 7417e7423. Brown, S.I., Dervichian, D.G., 1969. The oils of the meibomian glands. Physical and surface characteristics. Arch. Ophthalmol. 82, 537e540. Butovich, I.A., 2008. On the lipid composition of human meibum and tears: comparative analysis of nonpolar lipids. Invest. Ophthalmol. Vis. Sci. 49, 3779e3789. Butovich, I.A., 2009a. The meibomian puzzle: combining pieces together. Prog. Ret. Eye Res. 28, 483e498. Butovich, I.A., 2009b. Cholesteryl esters as a depot for very long chain fatty acids in human meibum. J. Lipid Res. 50, 501e513. Butovich, I.A., 2013. Tear film lipids. Exp. Eye Res. 117, 4e17. Butovich, I.A., Wojtowicz, J.C., Molai, M., 2009. Human tear film and meibum. Very long chain wax esters and (O-acyl)-omega-hydroxy fatty acids of meibum. J. Lipid Res. 50, 2471e2485.

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Please cite this article in press as: Millar, T.J., Schuett, B.S., The real reason for having a meibomian lipid layer covering the outer surface of the tear film e A review, Experimental Eye Research (2015), http://dx.doi.org/10.1016/j.exer.2015.05.002

The real reason for having a meibomian lipid layer covering the outer surface of the tear film - A review.

This review critically evaluates a broad range of literature in order to show the relationship between meibum, tear lipids and the tear film lipid lay...
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