Goggle-based chambers detect very low but dispersed tear evaporation rates of 0.14 ± 0.07 μL/min in the healthy eye, but 0.26 ± 0.16 μL/min for patients with meibomian gland dysfunction (MGD) [252]. Tear film thinning kinetics measured by interferometry suggests a higher tear evaporation rate of 1.137 μL/min in the healthy eye, which is a value similar to that of water [25,252]. The thickness of the lipid layer may not affect the evaporation rate unless it is very thin (less than 24 nm) or completely absent [24–27,67]. Nonetheless, increased expression of meibum in normal eyes correlates with reduced evaporation in both healthy individuals and patients with DED [233,253]. An abnormal tear film lipid layer is associated with increased evaporation [24–27,67]. Evaporation rates differ substantially between different studies, likely due to challenges in controlling environmental factors such as air flow and humidity [254]. Whether only evaporation from the ocular surface is being measured is also not always clear.
Such in vivo variability makes in vitro studies attractive [255]. Meibum films with physiological thickness do not retard evaporation in in vitro studies [255]. Evaporation found in studies using meibum films is in the range of 6–8% [225,256,257], or less in studies using artificial tear film lipid layers of up to 100 nm thick [253,254,257,258]. Only non-physiologically thick, 4 μm or ≥ 17 μm, meibum films can reduce the evaporation of water in laboratory experiments, but then by only 7 or 23% respectively [225,254]. Straight chain alcohols and acids and pure wax esters used alone can retard evaporation [259–261]. Monolayers of tear film-like wax esters can retard evaporation when used at temperatures 3 °C lower than their bulk melting temperature [258,260,261]. However, meibum has a much more complex composition, structure and thermotropic behavior compared to the model films, and the melting temperature of meibum (∼30 °C) [262–264] is lower than the ocular surface temperature (35 °C). Recently in vitro studies with the Interfacial Dewetting and Drainage Optical Platform (i-DDrOP) on silicone hydrogel (SiHy) contact lenses have shown that the mucoaqueous subphase beneath thinner parts of meibomian films thins and dewets faster compared to that seen below thicker “islands” in the meibomian layer [265]. Therefore, it might be possible that meibum may locally be an effective barrier to evaporation. Suppression of tear evaporation by the lipid layer is thus poorly understood.
Three evaporation suppression hypotheses have been proposed: (1) the effect is based on a certain organization of the lipid layer, as proposed previously [266]; (2) the lipid layer is not solely the structure that is responsible for the retardation of evaporation, but specific protein or mucin (or a combination) interactions are also needed [267]. However, whole tears collected by capillary tube, apparently with all tear lipid and protein species and at least some mucins [17,19,21,268–272], evaporate in vitro with water-like rates; (3) water evaporation from the ocular surface is not at all controlled by lipids and the lipid layer has other functions [257]. A thick duplex film enriched with unsaturated and branched chain lipids, as found in meibum, is a much less effective barrier to evaporation than tightly packed lipid monolayers [273].
If tears do evaporate at the same rate as water, eventually the concentration of secretory mucins and proteins in the lipid layer would increase, and approximate mucin near the surface of the cornea [274]. A dense polymer network of mucin has a high hydration “water holding” property and might suppress further tear evaporation [275]. It is also possible that mucins, together with proteins and polar lipids from tears, enhance spreading and structure of the tear film lipid layer. Thus all tear film key constituents may contribute to increased evaporative resistance.
The tear film lipid layer is derived predominantly from meibum from the meibomian glands and spreads upward over the mucoaqueous layer [8] with blinking. Thinning of the lipid layer superiorly may create a rise in surface tension as a driving force for spreading [8]. Spreading may initially involve interaction between polar lipids and the mucoaqueous layer [276]. Polar lipids then carry nonpolar lipids [277]. In the normal eye, spreading of the lipid layer can be observed clinically by interference video microscopy as an upwardly moving front of horizontally disposed, colored fringes (Fig. 2). Fringes reflect topographic variations in thickness of the lipid layer and are thereby reflective of its intermolecular organization. The lipid layer is compressed during the down stroke of the blink and restored in the upstroke, with only a moderate disturbance of its organization between consecutive blinks [4,26]. Spreading is rapid at first (about 10 mm s−1), although lagging behind the upper lid [278]. Spreading slows and stabilizes after one second or more, with the interference pattern showing remarkable stability over the remainder of the blink [34,278]. Spreading is slower and less laterally arranged in dry eye patients [34,278], with reduced stability of the lipid layer fringes [279], suggesting that intermolecular stability is lost.
The dynamic nature of the lipid layer allows it to respond to the shear stress induced by the changing area of the air-tear interface during blinking. The lipid layer must be compressible during the down-sweep [280,281], but must spread rapidly after the up-sweep of the lid [26]. Polar lipids decrease the surface tension of the air-tear interface and increase the spreading rate of the lipid layer. Non-polar lipids increase the compressibility and stability of the lipid layer [281]. Imbalances in composition show as impaired viscoelastic properties.
Human meibum comprises ∼90% of the tear film lipid layer, and is often investigated as the archetype for the tear film lipid layer. Commercially available clinical devices (such as Yoshitomi's meibomian gland pressure forceps, or Arita's meibum collector) [120,282] ensure collection of ≤0.5 mg meibum per donor, thus allowing for multiple repetitions of experiments. The different meibum collection procedures (by glass microcapillary tube, Dacron swab, cytology micro brush, or spatula) and inherent intra- and inter-individual variability may contribute to variations in the amount and composition of the meibum samples [283–285].
The most representative tear film lipid layer mimic might be the lipid extract of whole human tears. However, to-date only one study has used it for in vitro analysis of surface tension [286], which is not surprising considering the difficulties in collecting a sufficient sample for reproducible experiments. Lipids extracted from deposits adsorbed on worn contact lenses have been used [287], although they contain higher amounts of phospholipids than meibum. Schirmer strips can be used to collect lipids [288]. The tear film contains ∼8% of amphiphilic (polar) lipids [289–291] that may be released from the lipid-binding protein lipocalin [292] from the mucoaqueous layer upon organic solvent extraction. These phospholipids are at very low levels in meibum [21,272], but as surfactants, if they are not bound to lipocalin, may have a significant role at the tear interface [255,285,293].
The tear film lipid layer lowers the tear surface tension, thus sustaining the tear film's high area-to-volume aspect ratio. Tear surface tension of healthy individuals is 43.6 ± 2.7 mN/m [294], rising to 53.0–55.5 mN/m after delipidation, which is remarkably close to the upper boundary of values observed in samples collected from patients with dry eye, of 49.6 ± 2.2 mN/m [286].
Supplementing delipidated tears with meibum lipids does not restore the surface tension [286,295]. Normal surface tension may thus rely on lipids and perhaps some proteins and glycoproteins from the mucoaqueous layer, such as lipocalin, lysozyme, and mucin, that are thought to intercalate with the lipid layer [272,276,296]. Increased surface tension in dry eye implicates abnormally enhanced protein content at the air/tear interface as a consequence of a compromised lipid layer. Proteins appear to take hours to penetrate a normal meibum lipid layer [297–300].
Data on the thermotropic phase transitions (via spectroscopy, differential scanning calorimetry and X-ray diffraction studies) [262,264,301–303] and interfacial and bulk rheology [29,301,302] of tear lipids in vitro and in vivo, suggest a thick viscoelastic duplex structure composed of a monomolecular layer of amphiphilic polar lipids at the mucoaqueous surface and a generally unstructured shear thinning lipophilic suspension at the lipid-air interface. The lipophilic suspension consists of lipid lamellar-crystallite particulates immersed in a continuous liquid phase with no long-range order. An alternative hypothesis rebrands the old folded trilayer model of McCulley and Shine and views the tear film lipid layer as a lamellar sandwich [304]. Lamellar models demand an ordered, almost rigid solid [302], which conflicts with the need for rapid reorganization during blinking.
Due to the abundance (≥90%) of non-polar lipids, both meibum and contact lens extracts do not spread as monolayers at the air/water surface, but instead form thick (10 nm to >100 nm) duplex films. They are thick enough to display bulk properties with two separate interfaces, but thin enough to neglect the effects of gravity) [257,285,288,301,302,305]. Uniform spreading of meibum is enhanced by polymers that contain polyanionic polysaccharide moieties, such as hyaluronic acid and secretory mucin glycoproteins [296,305], that form polymer interfacial gel-like networks due to hydrogen bonding of the polymer moieties with each other and with the phospholipid head groups. As a consequence, film viscosity is enhanced, and a more uniform 2-dimensional (2D) distribution of lipids and water is achieved [257,296,305].
Films of meibum and contact lens lipid extracts are non-collapsible, which helps them to withstand the dramatic area changes during the blink. In some studies, meibum layers show almost full surface pressure (π)/area reversibility (i.e. very low π/area hysteresis), which point to meibum's capacity to rapidly reorganize during area cycling (as a model for the interblink period) [29,296,305–307]. Interestingly, meibum films expand and thin at lower temperatures (23-25 °C), but shrink and thicken at physiological (≈35 °C) temperatures [29,263,301,305,308]. Such behavior agrees with the liquid suspension model of the non-polar lipids of the lipid layer. The non-polar lipid oily cap might act as a lipophilic solvent [29,301,309–311] to accommodate polar lipids, which may form inverse micelles within it, during temperature-induced interface/bulk redistribution of the polar layer and during film compression. In other words, it may prevent expulsion of polar lipids into the aqueous phase such that polar lipids can promptly return to the mucoaqueous interface at film expansion.
Meibum interfacial properties and structure are largely insensitive to changes in osmolarity [312,313]. However, both meibum and contact lens lipid extracts are sensitive to the presence of (glyco)proteins (lactoferrin, lysozyme, mucin, lipocalin, serum albumins, lactoglobulin, lactoferrin, secretory IgA, keratin, lung surfactant proteins) [297–300,307,314] and pharmaceutical agents (hyaluronic acid, benzalkonium chloride, SofZia, Polyquad, whole eye drops and lens care solutions) [305,315–317] in the film subphase. However, even in sub-physiologically “diluted” (5–10 mN/m vs. ∼30 mN/m) meibum films, the kinetics of the penetration of such substances is much slower (>1 h) than the physiological time scale [297–300].
An alternative mechanism has been proposed, whereby the proteins or pharmaceuticals dissolved in the mucoaqueous subphase do not have to insert into the meibum or contact lens lipid extracts films. Instead, they need only to interact with polar lipid head groups aligned at the mucoaqueous interface. This is a rapid interaction that immediately alters the structure and dynamic interfacial properties of the layers [287,305,311]. These data, along with studies evaluating the impact of lipid (or lipophilic) inclusions directly into meibum layers, demonstrate that non-surface active or lipophilic substances are well tolerated by tear lipid films [318–322], while polar surface active ingredients can disrupt the film's structural integrity and surface properties [314–317]. Interactions between the tear film lipid layer and pharmaceutical agents are of particular pharmacokinetic importance as they may impart long term effects at the ocular surface, in light of the much slower in vivo turnover rate of the tear film lipid layer compared to the mucoaqueous layer (0.93 ± 0.36%/min vs. 10.3 ± 3.7%/min respectively) [323].
Shear rheology experiments have demonstrated the viscoelastic nature of meibum films, which were able to stabilize tear film-mimicking films and to reduce the critical film thickness for dewetting [28]. The protective effect of meibum layers was stronger than that of Newtonian arachidonic acid and primarily viscous dipalmitoylphosphatidylcholine films, in line with the classic paradigm that viscoelastic surfactants are particularly effective at stabilizing thin films [324].
Meibum and contact lens lipid extract films also display viscoelastic behavior in dilatational rheology studies [29–31], with predominantly elastic properties, thereby enabling resistance to deformation during tear film breakup. Whereas films of meibum collected from healthy donors are continuous, thick and predominantly elastic, films from patients with meibomian gland dysfunction are discontinuous, patchy and show viscoelasticity compromised at time scales (ie at low frequencies) encompassing the durations of blink and interblink intervals found in vivo [29]. The in vitro data correlate well with the impaired spreading and heterogeneous structure of the tear film lipid layer of patients with MGD seen in vivo [29]. Similar characteristic differences in the viscoelastic properties are reported between films of contact lens lipid extracts from Caucasians versus dry eye-susceptible Asians [30,325]. More attention should be paid to the capacity of the lipid layer to elastically stabilize the air/tear interface.
Differential scanning calorimetry reveals that the phase transitions in meibum start at 10–15 °C and end at 35–36 °C, with a melting temperature (Tm) of ∼30 °C [264,302]. Thus, it is reasonable to assume that in the meibomian gland orifices meibum is both disorganized and liquid to facilitate excretion. When exposed to the cooler (33-35 °C) ocular surface, meibum solidifies to a partially melted liquid crystalline state, thereby enhancing lipid layer in-plane elasticity, and via denser 2D molecular packing, ensuring a barrier to water evaporation.
Combining differential scanning calorimetry with small- and wide-angle X-ray diffraction points to meibum as a liquid suspension consisting of lipid lamellar-crystallite particulates immersed in a continuous liquid phase, with no long-range order at physiological temperature [262,264,301–303]. Hot stage cross-polarized light microscopy confirms such a view and reveals that in meibum from patients with MGD there is an increased presence of non-lipid, non-melting, chloroform-insoluble inclusions of protein, including cytokeratin [262]. Spectroscopy studies also confirmed that MGD meibum contains more protein and relatively less methyl groups (CH3) and cis double bonds (cisCH) compared to normal meibum [303]. The increased content of non-melting proteinaceous particles that are poorly miscible with lipid can account for the impaired spreading and discontinuous structure of meibum films in vitro and for their worsened viscoelasticity and evaporation suppression capability in vitro and in vivo [29–31].
Bulk shear rheology evaluation suggests that meibum is a shear thinning liquid of extremely high viscosity [302]. At 35 °C, the shear viscosity of bovine and human meibum is approximately 105 greater than that of water and 3–4 orders of magnitude more viscous than mineral oil. However, such high bulk viscosity is inconsistent with the high mobility of fluorescent probes reported in meibum layers [296] and with the rapid reorganization of meibum films during area cycling [29,296,305–307]. More research needs to be performed to resolve this issue.
Research on the bulk rheology of non-stimulated (or mildly stimulated) tears is relatively old, but in light of the new findings on the tear lipidome, there is a need for reappraisal. Human reflex tears collected after cold air stimulation are non-Newtonian and shear thinning [326]. Their viscosity at rest is ∼9 cP and when shear is applied it rapidly drops to 1 cP (at shear rates ≥ 100 s−1) [327,328]. This indicates that the tear constituents, and particularly the compounds dissolved in the mucoaqueous layer, when at rest form a “transient” intermolecular network via weak non-covalent interactions (hydrogen-bonding, hydrophobic and/or electrostatic interactions etc.). Thus, they increase the mucoaqueous layer viscosity in the open eye, which raises its resistance to thinning and enhances tear film stability. At blink, the high shear applied by the eyelid breaks these weak interactions, the intermolecular network disintegrates and the aqueous can flow like water, thus preventing any damage that high viscosity may cause to the underlying corneal epithelium. The exact nature of the compounds involved in the transient intermolecular network remains unclear. Pure monocomponent solutions of mucin or tear proteins in physiological concentrations are low viscosity and/or Newtonian fluids. Also, if the lipocalin-bound lipids are extracted, then tears become a low viscosity Newtonian fluid. This suggests that lipid/protein interactions, lipocalin and possibly lysozyme and lactoferrin play important roles in the shear thinning property of tears.
An interesting study found that when whole human tears are subjected to shear (at rates of 2–160 sec−1), dry eye tears need >10 times longer relaxation times compared to normal tears (2.8 ± 0.14 s vs. 0.26 ± 0.12 s) in order to equilibrate after the shear is ceased [328]. This suggests that a longer period should be needed for dry eye tear film, compared to a healthy one, to stabilize at the ocular surface after a blink. This demonstrates the potential importance of elasticity for tear film stability.