Comparative Biophysical Study of Meibomian Lipids of Wild Type and Soat1-Null Mice: Implications to Meibomian Gland Dysfunction and Dry Eye Disease

Abstract

Purpose: The biophysical roles of Meibomian lipids (MLs) played in health and meibomian gland dysfunction (MGD) are still unclear. The purpose of this research is to establish the composition-structure-functional correlations of the ML film (MLF) using Soat1-null mice and comprehensive in vitro biophysical simulations.

Methods: MLs were extracted from tarsal plates of wild type (WT) and Soat1 knockout (KO) mice. The chemical composition of ML samples was characterized using liquid chromatography - mass spectrometry. Comprehensive biophysical studies of the MLFs, including their dynamic surface activity, interfacial rheology, evaporation resistance, and ultrastructure and topography, were performed with a novel experimental methodology called the constrained drop surfactometry.

Results: Soat1 inactivation caused multiple alternations in the ML profile. Compared to their WT siblings, the MLs of KO mice were completely devoid of cholesteryl esters (CEs) longer than C18 to C20, but contained 7 times more free cholesterol (Chl). Biophysical assays consistently suggested that the KO-MLF became stiffer than that of WT mice, revealed by reduced film compressibility, increased elastic modulus, and decreased loss tangent, thus causing more energy loss per blinking cycle of the MLF. Moreover, the KO mice showed thinning of their MLF, and reduced evaporation resistance.

Conclusions: These findings delineated the composition-structure-functional correlations of the MLF and suggested a potential biophysical function of long-chain CEs in optimizing the surface activity, interfacial rheology, and evaporation resistance of the MLF. This study may provide novel implications to pathophysiological and translational understanding of MGD and dry eye disease.

Figure 1.

Schematics of the constrained drop surfactometry (CDS) as a versatile biophysical model for studying mouse MLFs. The CDS uses the air-water surface of a 4-mm sessile drop, constrained on a carefully machined pedestal with knife-sharp edges, to accommodate the spread MLF. The droplet is enclosed in an environmental control chamber that maintains a constant temperature, relative humidity, and ambient airflow rate. The spread MLF can be compressed and expanded at a highly dynamic rate of 20% relative area per second to simulate a blink, by regulating fluid flow into and out of the droplet using a motorized syringe. Dynamic surface tension of the film is determined with closed-loop axisymmetric drop shape analysis (CL-ADSA), which measures surface tension of the film remotely by analyzing the shape of the MLF-covered droplet in real-time. The CDS is capable of multiple biophysical assays of the MLFs, including dynamic surface tension, interfacial dilational rheology, evaporation resistance, and ultrastructure and topography of the MLFs when being used in conjunction with the Langmuir-Blodgett (LB) technique and atomic force microscopy (AFM).

Figure 2.

Ultrahigh-pressure liquid chromatography –mass spectrometry (UHPLC-MS) lipidomic characterization of the meibomian samples extracted from wild-type (WT) and Soat1-knockout (KO) mice. (a) Compositions of the MLs extracted from WT and KO mice. (b) Effect of Soat1 inactivation on the ML profiles of mice. Note the major shifts in the Chl/CE ratio in the KO MLs, and a much subtler effect of the mutation on TAGs and WEs.

Figure 3.

Dynamic surface activity of the MLFs of WT and KO mice at 34°C. (a) Typical compression-expansion isotherms of the WT and KO MLFs. (b) Statistical analysis of the compressibility (κ) of the WT and KO MLFs. (c) Statistical analysis of the hysteresis area, that is, the energy loss per cycle, for the WT and KO MLFs. *P < 0.05 represents statistically significant differences.

Figure 4.

Interfacial dilational rheological properties of the MLFs of WT and KO mice. (a) Elastic modulus (E_r) of the WT and KO MLFs. (b) Viscous modulus (E_i) of the WT and KO MLFs. (c) Loss tangent (tanφ) of the WT and KO MLFs. All interfacial rheological properties were determined at the characteristic surface pressure of 30 mN/m at 34°C.

Figure 5.

Effects of the MLFs of WT (a) and KO (b) mice, under various surface pressures, on water evaporation. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 6.

Ultrastructure and topography of the MLFs of WT (a-c) and KO (d-f) mice at three characteristic surface pressures, that is, 20, 30, and 40 mN/m. All AFM images shown in the 2 middle rows have the same scanning area of 20 × 20 µm, and the same z range of 50 nm. Images in the top and bottom rows show the 3D renderings of the WT and KO MLFs, respectively. Single-headed arrows indicate the heights of the structures, while double-headed arrows indicate the lateral dimensions of the structures. The bar represents 5 µm.