Biophysical behaviors of meibum from mice with meibomian gland dysfunction

Abstract

Purpose : Meibomian gland dysfunction (MGD) is the most common cause of evaporative dry eye disease (EDED) due to the meibomian gland obstruction and/or alterations in tear lipid composition, leading to tear film instability. We recently identified a nonpolar lipid (rNPL593), derived from the rabbit Harderian gland (an accessory orbital gland), that was not present in mouse, dog or human tears. Due to the hyperstability of the rabbit tear film and the uniqueness of rNPL593, we hypothesize that it promotes tear film stability. In this study, we compared the interfacial properties and biophysical behaviors of abnormal meibum derived from a murine model of EDED (acyl-CoA: wax alcohol acyltransferase 2 (Awat2) knockout (KO) mice) with meibum from wildtype (WT) mice with and without the addition of rNPL593.

Methods : Six-month-old Awat2 KO and WT mice were euthanized and meibum was expressed from the eyelids using gentle pressure and collected with a #15 blade. The meibum lipids were dissolved in chloroform and rNP593 (MCAL Therapeutics) was added to determine its effect on the biophysical properties of abnormal and healthy meibum in vitro. Meibum spreading characteristics were assessed using confocal fluorescence microscopy. To enable lipid visualization, a trace concentration of a fluorescently labeled cholesterol ester or triglyceride was added to the meibum samples. The meibum phase characteristics were determined by measuring pressure-area isotherms using a Langmuir-Blodgett (LB) trough. LB trough results were also utilized to determine the interfacial stability of the meibum at the air-water interface.

Results : Confocal microscopy analysis determined that Awat2 KO meibum had decreased spreadability as large concretions of labeled cholesterol ester or triglyceride accumulated across the slide, whereas the WT meibum exhibited even spreading of small lipid droplets. The addition of rNPL593 decreased the droplet size of the Awat2 KO meibum, thus increasing its spreadability. In the LB pressure-area isotherms, the Awat2 KO meibum displayed increased compressibility compared to WT meibum.

Conclusions : Meibum from Awat2 KO mice demonstrated altered biophysical behaviors compared to WT meibum. Lipid concretions observed in Awat2 KO meibum were markedly decreased by the addition of rNPL593. The improvement in tear lipid dynamics of Awat2 KO meibum by rNPL593 suggests that it may have a beneficial effect in patients with EDED and MGD.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.