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Masataka Ito, Rika Shirakawa, Reiko Arita, Yoko Karasawa, Junko Imaki, Shiro Amano, Ysushi Kobayashi, Masaru Takeuchi; Observation of Whole-mount Meibomian Glands from Cadaveric Eyelids using a Fructose-based Optical Clearing Agent.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4442.
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© ARVO (1962-2015); The Authors (2016-present)
The chronic dysfunction of meibomian glands known as meibomian glands dysfunction (MGD) is one of the causes of dry eye syndrome and is known to be related to aging. Meibomian glands are clinically observed using meibography, however, those in formalin-fixed cadavers are difficult to be observed in whole-mount because of the stiffness and decreased transparency of the eyelids. A recently-developed method using fructose-based optical clearing agent (SeeDB method) enabled observation of the deep structures of the brain in formalin-fixed experimental animals. We applied this method to human meibomian glands. The purpose of this study was to establish the methods to observe the morphology of whole-mount meibomian glands of the formalin-fixed cadaveric eyelids.
All cadavers were perfused by 3.3% formalin in 67% ethanol within 4 days after death and stored in the same fixative more than 6 months. 13 upper eyelids were dissected from 9 cadavers (69- to 98-year-old, male and female). After skin and subcutaneous tissues were manually removed, the samples were serially incubated in 30%, 60% and 80% (wt/vol) fructose each for 12-24h at room temperature. Samples were then incubated in SeeDB (80.2% wt/wt fructose) for 24h at 37°C. Between each step, samples were observed by a dissecting microscope under transmitted light. Photographs of the glands were taken by a digital camera and the contrast between the glands and surrounding connective tissues were compared. In some cases, observation with meibography was performed.
In cadaveric eyelids, tarsal plates were best cleared by overnight incubation with 80% fructose solution, but 24h incubation in SeeDB decreased the contrast of the glands. Observation with meibography could be done after clearing process with fructose, and the degeneration of the gland could be scored (meiboscore) in all cases tested.
The fructose-based optical clearing agent successfully cleared the palpebral tissues and enhanced the contrast of meibomian glands in cadavers. This method could be applied for the evaluation of the morphology of meibomian glands in formalin-fixed human eyelids.
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