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Anne McMahon, Jadwiga C Wojtowicz, Igor A Butovich; Histochemical and immunohistochemical characterization of lipid synthesis in meibomian glands. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3574.
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© ARVO (1962-2015); The Authors (2016-present)
Meibum is composed of a complex mixture of lipid species with expression analysis suggesting in situ synthesis in meibomian glands (MG). This study seeks to identify the sites of lipid accumulation and cellular and subcellular location of key lipid synthesis enzymes identified by expression analysis.
Eyelid tissue, freshly harvested from mice, or human autopsy, were fixed in 4% paraformaldehyde and embedded in OCT or in 10% buffered formalin and embedded in paraffin blocks. Lipids in OCT embedded tissues were stained with either Nile red or HCS LipidTOX stains. Primary antibodies specific to mouse and/or human proteins, including markers for subcellular compartments, were incubated with tissue sections and binding detected using Alexa Fluor® conjugated secondary antibodies. Imaging used both a Zeiss fluorescent and a Leica SP2 confocal microscope.
Lipid staining revealed initial accumulation within meibocytes in ascini in MG with subsequent release into the ascinar ducts upon holocrine rupture. Limited numbers of lipid drops (LD) were seen in basal meibocytes with their numbers increasing in density and size as differentiation progressed. Both PLIN2 and 5 were observed in association with LD. Defining characteristics of meibum lipids are: 1) high abundance of fatty acyl components ≥ C18 requiring endoplasmic reticulum (ER) fatty acid elongation; Both ELOVL3 and ELOVL4 were highly expressed in meibocytes; ELOVL3 (substrates C16-C22) staining was strong in both basal and differentiating meibocytes. ELOVL4 (substrates ≥ C22) in contrast was most strongly expressed in differentiating meibocytes: 2) lipids containing cholesterol; expression of both SQLE and FDFT1 was observed in meibocytes suggesting endogenous synthesis: 3) lipids containing odd-numbered fatty acyl chain residues. Punctate staining throughout ascinar meibocytes for both BCKDHA and DBT, mitochondrial components of the BCKDH complex suggests a role for branched chain amino acids in their synthesis.
The observed cellular and subcellular expression patterns of proteins, whoses mRNAs are highly expressed in the tarsus, and whose functions/enzymatic activities strongly suggest a role in lipid synthesis, start to define the cellular means by which the MG elaborates the meibum lipidome. This provides a base on which to understand the qualitative/quantitative changes in meibum lipids with MG pathologies.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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