Tear film lipid layer and corneal oxygenation: a new function?

Abstract

The classic model of tear film is composed of mucin layer, aqueous layer and the outermost tear film lipid layer (TFLL). The complex mixture of different classes of lipids, mainly secreted by meibomian glands, gives the TFLL unique physicochemical properties. Based on these properties, several functions of TFLL have been found and/or proposed such as the resistance to evaporation and facilitating the formation of a thin film. However, the role of TFLL in the oxygenation of the cornea, a transparent avascular tissue, has never been discussed in the literature. The continuous metabolic activity of the corneal surface and the replenishment of atmospheric gas creates an O₂ gradient in the tear film. The molecules of O₂ must therefore be transferred from the gas phase to the liquid phase through the TFLL. This process is a function of the diffusion and solubility of the lipid layer as well as interface transfer, which is influenced by alterations in the physical state and lipid composition. In the absence of research on TFLL, the present paper aims to bring the topic into the spotlight for the first time based on existing knowledge on O₂ permeability of the lipid membranes and evaporation resistance of the lipid layers. The oxidative stress generated in perturbed lipid layers and the consequent adverse effects are also covered. The function of the TFLL proposed here intends to encourage future research in both basic and clinical sciences, e.g., opening new avenues for the diagnosis and treatment of ocular surface conditions.

Introduction

The tear film is a nourishing, lubricating and protecting layer over the ocular surface, which ensures ocular health and normal vision [1]. Classically, it is stratified into an inner mucus layer, an intermediate aqueous layer and an outer lipid layer (TFLL) at the eye-air interface [2]. The meibomian glands (MGs) [3], as the main source of TFLL [4], secrets a complex mixture of lipids of different classes [5]. According to the generally accepted model, these compounds form a two-layered structure comprising a bulky non-polar hydrophobic lipid layer on a thin polar amphiphilic lipid sub-layer [6]. The lipid contents also impart unique properties such as viscosity and surface tension to this layer. Several functions have been found and/or proposed for TFLL in the lid margin reservoir and layer covering the outer surface of the tear film (Fig. 1). For the latter, two crucial functions are facilitating the formation of a thin film and preventing its collapse onto the ocular surface [7].

Fig. 1. The known and/or proposed functions for tear film lipid layer [7, 19, 27, 45].

The aqueous layer and lipid layer are shown in blue and yellow, respectively.

Cornea (Fig. 2), a part of the ocular surface, is the major refractive surface of the visual system [8]. Its health depends on metabolic processes [9], and thus any deficiency in O₂ supply can lead to detrimental effects and eventually eye diseases [10]. This transparent avascular tissue receives the required supply of O₂ from different routes, but the atmosphere is the primary source when the eyes are open [11]. In order to be available for the corneal surface, O₂ molecules must be transferred from the gas phase (atmospheric environment) to the liquid phase (tear film) through the TFLL. The continuous metabolic activity of cells and the replenishment of atmospheric gas may create an O₂ gradient in the tear film. A reduced O₂ level in the vicinity of the corneal epithelium owing to the O₂ gradient in the tear film is consistent with the fact that mammalian cells are generally adapted to O₂ levels much lower than in the atmospheric (~21%) [12].

Fig. 2. Cross-section of the ocular anterior segment (left), the cornea (middle) and the three-layered model of the tear film (right).

The box shows the lipid composition of human tears, which has been listed for tear film lipid layer in the model proposed by Lam et al. [91].

The transport of O₂ across a lipid layer is a function of its diffusion and solubility. The alterations in the physical state and lipid composition of the layer influence this process [13]. Due to the lack of research on TFLL, studies on O₂ permeation in lipid membranes can potentially improve our understanding [14, 15]. Cholesterol and unsaturated fatty acids are examples of TFLL constituents that make the membrane a moderate barrier to O₂ diffusion [16] (Fig. 3). Another beneficial area is the assessment of tear film dynamics, mainly from studies on dry eye disease (DED) [17], an ocular condition commonly caused by excessive evaporation of tear fluid [18]. The presence of saturated long-chain hydrocarbon is considered essential for efficient anti-evaporative effects of lipids [19]. The diversity in lengths and saturations of TFLL lipids [5] may be subjected to qualitative or quantitative changes leading to adverse effects [2]. A consequence of increased levels of O₂ is overproduction of free radicals, leading to the occurrence of oxidative stress in cells [12], and indeed, several ocular surface inflammatory diseases are linked to oxidative stress [20].

Fig. 3. The role of tear film lipid layer in corneal oxygenation and the adverse effects following its molecular and/or structural changes.

Normal tear film lipid layer (A) and its equivalent disrupted form (B) due to changes in the length of fatty acids (symbol: ) and the number of cholesterol units (symbol: ) as well as unsaturated fatty acids (symbol: ), leading to excessive oxygen diffusion and subsequent occurrence of oxidative stress. The order of molecules is based on the model proposed by Miyamoto et al. [92] and the details of the constituents is according to Butovich [5]. The background colour spectrum represents the lipid polarity gradient in the tear film lipid layer. Abbreviations: cholesteryl esters (Chl-E), wax esters (WE), wax diesters (WdiE), cholesteryl esters of (O-acyl)-ω-hydroxy fatty acids (Chl-OAHFA), triacylglycerols (TAG), phospholipids (Pl), cholesterol (Chl), free fatty acids (FFA), ceramides (Cer).

Despite the potential importance of the role of TFLL in the oxygenation of the cornea for both basic and clinical sciences, this topic has never been discussed in the literature. The current paper presents a novel hypothesis to address the abovementioned knowledge gap based on existing knowledge on O₂ permeability of lipid membranes and the evaporation resistance of lipid layers. This could encourage future basic and clinical research work, with the potential of opening new avenues for the diagnosis and treatment of ocular surface conditions.

Cornea

The anterior segment of the eye constitutes one-third of this organ, which comprises all the structures located between the ocular surface and the posterior capsule of the lens [21] (Fig. 2). The ocular surface is an interface between the eye and the environment, which includes the cornea, bulbar and palpebral conjunctiva and the lacrimal and MGs [2]. The cornea, a transparent avascular tissue, is horizontally oval [8], measuring ~11–12 mm horizontally and ~9–11 mm vertically. It covers one-sixth of the total surface area of the eyeball [22] and acts as a structural barrier for protecting against infections. Cornea and tear film together form the anterior refractive surface and ensures sharp vision acuity. It is alone responsible for two‑thirds of the refractive power of the eye. Cornea has five layers, including the corneal epithelium, Bowman’s layer, the corneal stroma, Descemet’s membrane and the endothelium [8].

Tear film

Tears provide the ocular surface with lubrication, nourishment and protection required for its health and helps in normal vision acuity. The large water content (~98%) gives this fluid a transparent appearance [1]. Tear film was described in 1946 by Wolff using a slit lamp to have a three-layered structure [23]. In this classic model (Fig. 2), the inner mucus layer on the epithelial surface with ~2.5–5 µm thickness contains chiefly sugar-rich glycosylated proteins. Goblet cells produce these high–molecular weight glycoproteins for coating the ocular surface and lowering epithelial cell hydrophobicity. The intermediate aqueous layer with ~4 µm thickness is mainly produced by lacrimal glands. It is composed of water and many water-soluble and insoluble components including electrolytes, peptides, proteins and small molecule metabolites. The outer lipid layer (TFLL) at the eye-air interface with ~0.015–0.160 µm thickness predominantly holds a complex mixture of lipids of different classes. Although the tear film in reality is not a layered and static unit due to physicochemical properties, flow, evaporation and blinking, yet considering each subunit separately could answer some questions and/or disagreements, e.g., in resolving the continuing debate about the composition, structure and function of TFLL despite advances in analytical technologies and instrumentation [2, 6].

Tear film lipid layer

Origin

The main source of TFLL secretion is MG [4]. First described in 1666 by Heinrich Meibum [24], MGs are sebaceous glands with ~30–40 (upper lid) and ~20–25 (lower lid) individual grape-like clusters located in the tarsal plate. The secretory acini of the MG follow a holocrine secretion mode, when the most central meibocytes and their lipid-rich contents (meibum) are released into a ductule. Individual ductules drain the meibum into the central duct, which is eventually released at the orifice located at the edge of the eyelids [25]. The process is regulated by the modulation of lipid synthesis or cell maturation, possibly through androgens, like other non-ocular sebaceous glands. Additionally, autonomic innervation and several neuropeptides have been shown in MG but the mechanisms are not well understood [26]. The quantity of meibum stored in each eyelid is estimated to be several hundred micrograms [27]. Mature meibum, particularly from clusters residing near orifices, is spontaneously released with slow and constant pace, generating secretory pressure. This process can be facilitated by normal complete unforced blinking that compresses the glands. As a result, the meibum is driven out of the orifice into the marginal lipid reservoir for eventually forming the TFLL [25].

Some of data from lipidomic studies indicating difference in lipid composition of whole tear film fluid vs. meibum have challenged the long-held view that MG is the sole contributor to TFLL [6]. Among the proposed sources, minor secretion of lipid tear elements has been linked to Harderian (in rabbits especially) [4], Moll and Zeiss glands [11]. The last two glands are embryologically developed from the same origin as MG—surface ectoderm [28].

Composition

Our knowledge about the lipid components of tear film has been remarkably improved by analysis of meibum with different methods, especially lipidomics. Common difficulties in such investigations are small quantities of collected sample, potential contamination during sampling or preparation, biological variation, difference in separation and analysis techniques per lipid class, absence of some standards and presence of excessive amounts of particular lipid species in a sample [7].

A review of multiple studies [5] has listed the main lipid classes in whole human meibum: 1) Nonpolar lipids including wax esters (WE, 41%), cholesteryl esters (Chl-E, 31%), cholesteryl esters of (O-acyl)-ω-hydroxy fatty acids (Ch-OAHFA, 3%) and triacylglycerols (TAG, 1%); 2) Amphiphilic lipids consisting of (O-acyl)-ω-hydroxy fatty acids (OAHFA, 4%), cholesterol (Chl, 0.5%), free fatty acids (FFA, 0.1%), phospholipids (PL, 0.1%) and ceramides in various proportions (CER, 0.1%). An unknown fraction (19.2%) with nonpolar (e.g., diacylated α,ω-diols, diacylated a,b-diols and other more complex lipids), amphiphilic and non-lipid properties (e.g., denatured proteins, salts, etc.) has also been suggested [5]. Regarding TFLL, another review [7] has suggested the presence of 30–45 mol% of Chl-E with long acyl chains (mostly C22:1–C34:1), ~30–50 mol% of WE with a dominant C18:1 fatty acid chains combined with C18–C30 alcohol chains and ~4 mol% of OAHFAs from MG origin. For non-MG origin, the author has proposed ~13 mol% of PL with the primary presence of glycerophospholipids, lysophospholipids and sphingomyelins.

Structure

The generally accepted model of TFLL is a two-layered structure comprising a bulky non-polar hydrophobic lipid layer (33–40 nm) at the eye-air interface and a thin sub-layer of polar amphiphilic lipids (2–9 nm) that separates TFLL from the underlying aqueous phase (Fig. 3A). The key component of the polar sub-layer was thought to be PLs until the identification of other polar lipids, OAHFAs particularly, in meibum [6]. OAHFAs were shown to be very potent surfactants in vitro, even in small amounts [29].

Some other models have also been proposed based on e.g., MG lipid chemistry and biophysical properties of TFLL [27]. Eyelids also pay a role presumably through lubrication and/or oil layer collapse following compression [30, 31]. Examples describing the interactions between eyelids and TFLL are the pleated drape and rolled-up scum effects [32–34]. Considering experimental characteristics of TFLL, a multilayer or even more complicated structure could meet the requirements for an effective layer [6, 35].

Properties

Viscosity and surface tension are two key properties of the tear film, a non-Newtonian fluid. The former is defined as a measure of a fluid resistance to deformation at a given rate [19]. The viscosity is a result of complex molecular interactions between secretory and membrane-associated mucins, lipids and various proteins in tears (e.g., lipocalin). It is also influenced by other parameters such as shear rate and temperature [36]. For meibomian lipids, the role of temperature difference in viscosity between the lid margin reservoirs and the ocular surface has previously been reviewed [27]. The range of viscosity and melting range of samples from meibomian lipids are 9.7–19.5 Pa s [37] and 19.5–32·9 °C, respectively [38]. The latter is less than the temperature of the substance of lids (~37 °C) or close to that of the cornea (32–36 °C [39]) [27]. In general, viscosity is a function of the inverse of temperature in liquids [36, 40].

Surface tension is defined as a measure of interfacial forces in the surface of a liquid. Compared to water (72.8 mN/m at 20 °C [41]), this parameter has low values for tears (35–40 mN/m [42]) due to interactions between proteins (e.g., mucins and lipocalin [7]) and lipids [19]. In this respect, meibomian lipids play an important role by reducing free energy at the surface after being spread over the tear film and by interacting with the aqueous layer along with other components [27]. Surfactants in MG excretion, principally OAHFAs, play a central role in this process [43]. It is thought that surface tension gradients driven by lipid layer produces a Marangoni flow [44].

Functions

Depending on the location of the lipids, either in the lid margin reservoir or in the layer covering the outer surface of the tear film, multiple roles have been found and/or proposed (Fig. 1) [27]. The former is responsible for maintaining the hydrophobic state of the lid skin to prevent maceration of the lid skin as well as overflow of tears and contamination with sebum. The layer covering the outer surface of the tear film contributes to diverse functions such as producing a smooth optical surface to improve the refraction of light, providing lubrication for blinks and eye movements, thickening the aqueous sub-phase due to Marangoni effect, sealing the lid margins during prolonged eye closure and defending against the external environment (e.g., foreign particles and microbes) [19, 27, 45]. In addition, providing resistance to evaporation, enabling the formation of a thin film and preventing its collapse are the crucial functions of TFLL. However, an article reviewing ideas from the literature pointed out the last two roles as the main reasons for the presence of an outer lipid layer rather than an evaporative blanket [7].

Oxygenation of cornea

Metabolism

Metabolic processes contribute to the health of the cornea. For instance, the metabolism-dependent active fluid transport system maintains the state of corneal hydration, especially the structural integrity of the epithelium and stroma [9]. Therefore, corneal O₂ requirement in humans has become the centre of attention [10]. Over the past decades, attempts have been made to estimate O₂ demand by evaluating the epithelium, stroma and endothelium using a wide variety of methods, such as biochemical, structural and functional changes [46]. Alternatively, mathematical modelling of data collected, e.g., by placing a microelectrode onto a human cornea, has been performed to estimate O₂ uptake [10]. In spite of these initiatives, an ideal method for revealing the true value of O₂ consumption under normal corneal metabolism has not yet been established.

Oxygen delivery

Oxygen deficiency in the cornea may lead to detrimental effects such as oedema, acidosis, hypoesthesia, epithelial microcysts, stromal and epithelial thinning, and increased endothelial polymegathism [10]. Transparency of the cornea for perfect transmission of visible light is ensured by its avascular structure [11]. Several mechanisms have evolved in anterior segment of the eye to supply the O₂ required for aerobic metabolism in cornea.

When the eyes are open, the atmosphere is the primary O₂ source. At sea level, the tear film presumably reaches a saturation of 155 mm Hg before being available to the anterior corneal surface. This method of oxygenation is responsible for ~70 % of ATP generation in the corneal epithelium, whereas the rest of the oxygen requirement is fulfilled by palpebral conjunctival blood vessels with 55 mm Hg saturation during eye closure [11, 47]. Aqueous humour also provides O₂ to the cornea and other nonvascularized tissues at the posterior region of the eye, including the lens and the chamber-angle structures [48]. The O₂ delivered from aqueous humour to the cornea is predominantly consumed by endothelial and stromal cells [9]. The rate of O₂ consumption differs among layers, with the lowest for endothelium (21%) and the highest for epithelium (40%) and stroma (39%) in terms of the total O₂ used in the cornea [49].

Tear film and oxygen diffusion

Oxygen gradient in tear film

The O₂ gradient is formed across the tear film due to normal metabolic processes of the corneal surface and continuous O₂ replenishment from the atmosphere. Although the saturation levels of O₂ have been estimated (see “Oxygen Delivery”) and frequently used in mathematical models [50], the presence of O₂ gradient in tears has remained unclear. Research to date has focused on measuring O₂ diffusion through contact lenses made of different permeable materials by, e.g., phosphorescent probes installed at the posterior surface [51–54]. Additionally, the quantification of corneal O₂ uptake in situ using membrane-covered micropolarographic Clark electrode has been of interest [10]. A patent approved in 2015 has also described a wireless electrochemical oxygen sensor embedded in a polymeric material configured for mounting on the surface of the eye [55]. However, these methods do not determine the O₂ gradient established in tears of uncovered eyes and are, therefore, unable to resemble in vivo conditions. Future development in instrumentation engineering may shed some light on this topic. For instance, fibreoptic probes presently used to measure O₂ in the eye [56] can be merged with novel sensing technologies on micro- or nanoscale to measure the thinness of tear film [57].

Permeability of lipid layer

Oxygen permeation in lipid membranes has been a matter of interest over the past decades [14, 15], but almost no discussion on the oxygen permeability of TFLL is present in the literature. The permeability of lipid bilayers to O₂ depends on its diffusion, solubility and interface transfer under the influence of alterations in their physical state as well as composition, especially the content of cholesterol and unsaturated fatty acids [13]. The presence of cholesterol in cell membranes has been suggested as an adaptation to restrict O₂ diffusion under the elevated O₂ pressure in the atmosphere during the evolution of aerobic life [58]. However, recent experimental data have indicated that permeability of O₂ in lipid bilayers would increase with the increased presence of cholesterol in membranes [59]. Therefore, the cholesterol content in phospholipid membranes, such as erythrocyte membranes, has well been adapted to supply O₂ enough for physiological needs and sufficient for complete blood oxygenation in the lungs [59].

The level of cholesterol in different cell systems has postulated a non-random distribution pattern [58]. One extensively studied example to understand O₂ diffusion is the membrane of lens fibre cells. During maturation, the loss of subcellular structures in lens fibre cells leaves these cells with only plasma membranes. This structural modification prevents excessive light scattering and ensures lens transparency. The altered bilayer structure of fibre cells has a particular lipid composition with very high cholesterol content, a high sphingomyelin level and some traces of polyunsaturated fatty acids. These provide unique biochemical characteristics and physicochemical properties (e.g., rigidity and resistance to peroxidation) to membranes for becoming a moderate barrier to O₂ diffusion [16]. Other examples extend from the pulmonary membrane to red blood cells and peripheral tissues, where O₂ flux is directly linked to transmembrane diffusion with the key role of cholesterol [58]. Interestingly, the swim bladder in deep-sea fishes, which helps in adjusting buoyancy, has high cholesterol levels in its lipid-rich membranes. Buoyancy is adjusted by combining the lipids in the membranes with O₂ and then releasing at extreme depths. Other commonly used lipids for buoyancy purposes by marine fish are wax esters, squalene, alkyldiacylglycerols and triacylglycerols [60].

TFLL is composed of several lipid components, all of which may affect O₂ permeation. Although research on this topic has not been conducted, the dynamics of tear film has been of particular interest due to the prevalence of dry eye [17] (see earlier reviews for the influence of polar [61] and non-polar [62] lipids on tear film dynamics). Dry eye, a multifactorial disease and one of the most common ocular conditions, is typically caused by excessive evaporation of tear fluid from the ocular surface [18]. For efficient evaporation resistance of the lipids, two main characteristics are generally considered [19]: (1) the presence of saturated hydrocarbon chains relative to unsaturated ones for packing tighter in order to effectively prevent the leakage of water molecules [35]; (2) the presence of hydrocarbon chains of longer length relative to those with short ones [63]. TFLL contains a mixture of lipids with different lengths and saturations [5]. As an example, the ratios of saturated to unsaturated WE and Chl-E, which are major lipid classes of normal human meibum, have been shown to be 1:4.6 and 4:1, respectively [5, 64]. Recently, several attempts have been made to understand the mechanisms underlying the evaporation resistance of TFLL. For example, one study showed that crystalline WEs regulate the process [65] and another study revealed the self-assembly of OAHFAs and WEs at the air-water interface to form an efficient anti-evaporative barrier [66].

As mentioned above, a well-ordered structure containing tightly packed molecules is necessary for an effect barrier [19]. Temperature is an important factor affecting the fluidity of lipids in the TFLL, which influences their distribution and functional performance. For instance, a uniform layer of WEs capable of resisting evaporation has been reported close to the melting temperature of this dominant class of lipid in the whole human meibum [67]. Any changes to the composition of the layer destabilize the order and disrupt the function. Meibomian oil with mixed lipid compositions has a melting range, which can be lowered by alcohols as well as branched and unsaturated fatty acids [27]. This range is close to that of the ocular surface temperature, making the liquid mixture viscous enough for spreading across and solidifying sufficiently for resisting any collapse [7]. The temperature of the cornea itself is affected by environmental conditions such as temperature and air movement [27]. Under conditions of low ambient temperature or surface cooling, the constancy and stability of lipid layer are firm due to higher rigidity. In contrast, these two parameters are less frequent at high ambient temperature when TFLL is more fluid [27].

Research on O₂ permeation in lipid layers is lacking in the literature. Therefore, exploring lipid and O₂ gradient dynamics using molecular dynamic simulations [6] and computational modelling [68] may be useful alternative approaches. The application of mathematical models and in silico simulations enables the assessment of system properties that are hard to measure (e.g., O₂ gradients). Detailed descriptions of mechanisms and system processes and virtual experiments can be useful tools for these assessments [68].

Oxidative stress caused by perturbed lipid layer

The health of the anterior segment of the eye is partly dependent on the existence of a normal tear film. TFLL, the outermost layer at the eye-air interface, plays crucial roles in this process. Any qualitative or quantitative alteration in its composition and/or structure may lead to adverse effects [2] (Fig. 3). The occurrence of oxidative stress in cells exposed to elevated levels of O₂ is a known adverse outcome [12]. Oxidative stress is caused by the overproduction of reactive oxygen (and/or nitrogen) species or the exhaustion of antioxidant mechanisms [69]. Oxidative stress is involved in the pathogenesis of ocular surface conditions such as keratoconus [70], infectious keratitis (e.g., fungal keratitis [71]), Fuch’s corneal endothelial dystrophy [70, 72], conjunctivitis and keratoconjunctivitis sicca (dry eye) [20]. Dry eye disease (DED) is one of the most common ocular conditions leading patients to seek eye care [2].

There is no study that has directly compared the level of O₂ in tears of patients with perturbed TFLL with that of controls. However, measuring the markers of oxidative stress and antioxidants can be informative. For example, a recent meta-analysis has systematically reviewed the levels of these biomarkers in DED and healthy subjects from nine independent studies [73]. The results indicated that oxidative stress markers, and probably antioxidants, were dysregulated in DED, thus creating a local oxidative environment in tears, conjunctival cells and tissues. Although other sources of free radicals in the eye could be external (e.g., particular matter, exhausts and solar ultraviolet radiations) [73] and internal (e.g., mitochondrial respiratory chain, xanthine/xanthine oxidase and NADPH oxidase) [21], a less restricted flux of O₂ through TFLL is an important contributor [58].

Eyes exposed for prolonged periods to O₂ at levels higher than normal (e.g., O₂ therapy) lead to discomfort, irritation and dryness [74], some of which are reported by asymptomatic and symptomatic patients with DED [75]. The chronic state of oxidative stress is linked to inflammatory changes at the ocular surface, which are responsible for diseases like DED [21]. This is of concern in the diagnosis and classification of the disease as the perturbed lipid layer-induced oxidative stress can add more complexity. In general, the diverse symptoms and the lack of a single reliable clinical test make the accurate clinical evaluations of the DED challenging [76]. Therefore, ophthalmologists and eye care practitioners should be mindful of this when assessing DED.

The proposed molecular and/or structural changes in lipid layers, which generate oxidative stress, may be challenged by the results of some studies. The inter-individual differences in meibomian lipids of normal subjects, especially the low levels of cholesterol and esters of unsaturated fatty acids, have been reported [77]. However, no data is available to compare the extent of oxidative stress produced by such differences, if any. Both normal groups have had lower levels of cholesterol and esters of unsaturated fatty acids as compared with most of the patient groups with chronic blepharitis [77], an inflammatory disease consistently linked to DED [7]. Chronic blepharitis is often associated with systemic diseases that provoke MG disfunction (MGD), particularly obstructive MGD, which are the most common reason of TFLL deficiency [78]. Other studies have doubted the water loss suppression by the mixture of meibomian lipids, challenging the permeability properties [6]. For example, an in vitro model system has indicated that only certain WEs retard evaporation at physiological temperatures [79]. Similarly, reduced evaporation was reported using a mixture of WE and Ch-E [80]. However, a substantially thicker layer than normal was required for remarkable effects.

Clinical relevance

The use of TFLL constituents in producing artificial tears for treatment of patients with perturbed lipid layer may arrest the influx of O₂ to the anterior parts of the eye and subsequent generation of oxidative stress. To date, no study has yet defined any formula for such purpose. Artificial tears are currently a first line of treatment in all stages of DED for lubricating, spreading and wettability. The improvement of symptoms is one of the concerns; however, the therapy aims to prevent their build-up [81]. Artificial tears are developed in both water-based and lipid-based forms, but the latter is thought to be more effective due to better resemblance with aqueous and lipid contents of tears. Although the design of lipid-based products intends to mimic the tear film, the number of incorporated lipids is limited and it remains uncertain whether the lipids are fully replenished [82]. In addition to DED, other corneal pathologies with affected lipid layer (e.g., keratoconus [83] and persistent epithelial defects [84]) can be controlled by restoring TFLL to achieve normal corneal oxygenation.

Increasing knowledge on the lipid profile of TFLL using lipidomics takes us a step closer to personalized medicine e.g., by tailoring the formulations of artificial tears. The treatment of perturbed lipid layer-induced oxidative stress (and other abnormalities associated with lipid layer) can be remarkably improved by identifying affected lipid components and supplying these to the TFLL using topical delivery systems. For satisfactory results, the composition of tear substitutes may be combined with other ingredients such as antioxidants, viscosity-enhancing agents, electrolytes and osmo-protectants [85]. The topical and systemic application of e.g., antioxidants has been used for the treatment of oxidative stress-related ocular surface disease, especially DED [86, 87]. A relevant concept known as tear film-oriented therapy has already been introduced for tailoring the treatment of DED according to the analysis of tear film dynamics for detecting damaged components [88]. Although artificial tears are a first-line therapy, they are not necessarily adequate or effective management of the dry eye (and probably perturbed lipid layer-induced oxidative stress). Therefore, owing to variable properties of TFLL across individuals, personalized management for the best treatment choices has been recommended [89]. It involves clinical assessments of tear film and meibomian gland function along with the medical and treatment history of patient.

Single lipid molecules, including intact and modified forms (e.g., oxidized forms), the ratios of saturation to unsaturation and distinct lipidomic profiles determined in patients with perturbed lipid layer can potentially be used as biomarkers. It provides the clinicians with valuable information at an early stage or during monitoring of disease progression, thus enhancing prognosis, diagnosis and the choice of therapy. Moreover, it may improve our understanding of the underlying aetiology and pathology of both ocular (e.g., DED, MGD, keratoconus and persistent epithelial defects) and systemic diseases (e.g., multiple sclerosis), as both influence the lipid profile [2, 90]. The relations between lipid constituents of TFLL and O₂-associated contents of tear film, including O₂ levels, oxidative stress biomarkers and antioxidant molecules, are unknown to us. Further research on this topic would be of great help in treatment and/or preventive strategies.

Conclusions and future work

Several functions of TFLL have been discussed in the literature, except for its involvement in the oxygenation of the cornea. This transparent avascular tissue receives the required O₂ for aerobic metabolism from atmosphere (~70%), palpebral conjunctival blood vessels and aqueous humour. Corneal cells, like other mammalian cells, are generally adapted to O₂ levels much lower than atmospheric conditions (~21%). Based on the permeation of O₂ in lipid membranes as well as the evaporation resistance of the lipid layers, this paper hypothesizes the role of TFLL in the regulation of O₂ levels to meet the demands of corneal cells. In this process, the composition and structure of the lipid mixture at the eye-air interface are key factors. Therefore, any qualitative or quantitative alteration may lead to adverse effects such as the generation of oxidative stress. The chronic state of oxidative stress is associated with inflammatory changes at the ocular surface, which is responsible for diseases like DED. Treatment strategies can be remarkably improved in light of personalized medicine, e.g., by tailoring formulations of artificial tears, one of the primary and effective treatment options for DED. Future research should focus on the role of different classes of TFLL lipids, considering, e.g., their degree of oxidation and the ratios of saturation and unsaturation. In addition, the contributions of proteins to the permeability of O₂ in the layer need to be explored. The mechanism of O₂ transport through the TFLL is also an interesting field of research. The development of instrumentation engineering (e.g., micro- or nanoscale sensors) is necessary for advancing fundamental knowledge in these directions.

Competing interests

The author declares no competing interests.