Interfacial viscoelasticity of human meibomian lipid films

Abstract

Purpose: The meibomian lipid layer is a thin, mutilayered structure which forms the outermost layer of the tear film. Remarkably, this layer is able to adapt and maintain its structure, even during blink cycles when the lipid film is under stress. Stress arises from either the dragging of the eyelids across the surface, or the opening and closing motion of the eyelids which rapidly compresses and expands the film. As such, this layer must have properties of viscosity and elasticity, which would provide the film with both the necessary strength and flexibility to maintain its integrity under these enormous forces. Therefore, the purpose of this study was to determine the viscoelasticity of human meibomian lipid films at the air-liquid interface. Meibomian lipids from various animal species which have different lipid compositions were also measured and compared to human meibomian lipid films. The effect of whole tears, commercial proteins and a diblock copolymer, EOBO was also measured and compared to human meibomian lipid films. Methods: Viscoelasticity of meibomian lipid films were measured under both oscillatory shear and dilatational methods. The shear properties (G) were measured with the ISR, which utilises a commercial Langmuir trough. Here, both human and animal meibomian lipids were spread on the surface of the ATB subphase. Films were compressed to a desired surface pressure and viscoelasticity was measured as a function of frequency. The dilatational properties (E) were measured using the pendant drop rheometer. Here, an aqueous drop was suspended from the tip of stainless steel needle in air giving a characteristic pendant shape. Human meibomian lipids were spread on the surface of the drop where viscoelasticity was measured as a function of amplitude, frequency, time and temperature. The subphase of the drop was also varied to using whole tears, as well as commercial proteins and EOBO dissolved in ATB. Complementary information on meibomian lipid films were obtained from -A profiles carried out on the Langmuir trough. The appearances of these films were monitored using BAM and fluorescence microscopy. Experiments were conducted at both 20°C and 37°C. Results: Shear viscoelasticity of human meibomain lipid films increased overall with surface pressure, where the film transitioned from a Newtonian fluid-like film at low pressures (only G present, with frequency dependant modulus) to a viscoelastic gel with increasing pressure (G' and G' moduli present with G' greater than G' and moduli independent of frequency). Animal meibomian lipid films showed similar trends to human lipid films with subtle differences. The exception was the kultarr lipid film which demonstrated Newtonian behaviour at all surface pressures. -A profiles and BAM of these lipid films demonstrated similar findings. The absence or presence of particular lipid species between meibomian lipid samples between humans and animals could not be correlated to a particular difference in viscoelasticity, surface pressure or film morphology. Dilatational viscoelasticity also demonstrated that human meibomian lipid films were capable of forming a viscoelastic gel at the air-liquid interface however, moduli (E' and E') demonstated slight frequency dependence, indicating rearrangement of the lipid molecues on the surface. Aging of the film demonstrated differences in the first 30 minutes, but remained stable afterwards. Lowering the temperature to 20°C made the film more rigid, however the overall characteristics observed at 37°C were retained. The adsoprtion of commercial proteins to the lipid interface enhanced the viscoelasticity of the meibomian lipid film, indicated by the overall increase of the dilatational moduli. Surprisingly, the use of whole tears as the subphase did not mimick the values obtained from the use of commercial proteins however, similar trends were observed. This indicates that proteins from whole tears contribute towards the viscoelasticity of the meibomian-protein interface in vivo. The presence of EOBO lowered the dilatational viscoelasticity of the meibomian lipid film, indicating that this molecule was interrupting important lipid-lipid and lipid-protein interactions that occur at the meibomian lipid interface. However, -A profiles and fluorescence microscopy demonstrated EOBO integrating into the meibomian lipid layer and enhancing surface pressure, suggesting that EOBO needs further analysis and characterisation. Conclusions: The information reported in this thesis has provided a sound basis for understanding the mechanical properties of the meibomian lipid interface in vivo. Shear and dilatational measurements provided complementary information where the formation of a viscoelastic gel at the surface layer of the tear film is most likely a mechanism where the lipid film can maintain its integrity. This is supported by clinical studies that show limited rearrangements of the surface of the tear film between blink cycles. For a better understanding of how different components, such as proteins and lipids effect the viscoelasticity of the meibomian lipid layer, a more systematic approach of analysing these molecules need to be considered for future experiments. Analysing these lipid and lipid-protein/polymer films at higher resolutions, such as neutron scattering experiments would also allow a better understanding of the overall biophysical properties of the meibomian lipid layer and would particularly provide complementary information about the viscoelastic properties of the meibomian lipid interface.

Date of Award	2011
Original language	English