June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Histochemical and immunohistochemical characterization of lipid synthesis in meibomian glands
Author Affiliations & Notes
  • Anne McMahon
    Ophthalmology, UT Southwestern Medical Center, Dallas, Texas, United States
  • Jadwiga C Wojtowicz
    Ophthalmology, UT Southwestern Medical Center, Dallas, Texas, United States
  • Igor A Butovich
    Ophthalmology, UT Southwestern Medical Center, Dallas, Texas, United States
  • Footnotes
    Commercial Relationships   Anne McMahon, None; Jadwiga Wojtowicz, None; Igor Butovich, None
  • Footnotes
    Support  NIH Grants R01EY019480, P30EY020799 and R01EY024324; An Unrestricted Grant from Research to Prevent Blindness.
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3574. doi:
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    • Get Citation

      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)

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Abstract

Purpose : 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.

Methods : 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.

Results : 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.

Conclusions : 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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