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Geraint Parfitt, Yilu Xie, Donald Brown, James Jester; Age-related changes in the meibomian gland revealed by Immunofluorescent Computed Tomography. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2671.
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Recent studies suggest that age-related meibomian gland (MG) dysfunction is defined by acinar atrophy and a decrease in cell proliferation and gland volume. We have developed a novel method, immunofluorescent computed tomography (ICT), which enables 3-D quantification of multiple antigens in large tissue volumes to validate and expand on these findings in the mouse MG.
Lower eyelids from a 5month and 2year old mouse were excised and fixed in 2% PFA for 24hrs. Lids were embedded in butyl-methyl methacrylate and polymerised under UV light at 4oC for 20hrs before serially sectioning at 2µm intervals for 1mm. Both data sets were then sequentially immunostained for the cytokeratins 1, 5 and 6 and the cell proliferation marker Ki67, prior to mounting with DAPI. Image acquisition was carried out using a 20x/0.75NA objective on a Leica DMI6000B microscope and alignment and 3-D reconstruction was performed using Amira software. Finally, quantification of gland volume, cell density and Ki67+ nuclei was carried out using ImageJ.
ICT of 3 young and 3 old mouse MGs confirmed acinar atrophy and revealed a mean gland volume loss of 77% with age. Furthermore, ICT visualised an elaborate ductal system in the young mouse MG that undergoes truncation of epithelial tissue with age and a subsequent decrease in the proximity of the gland to the Obicularis muscle. 3-D reconstructions also show that, in some cases, the orifice may narrow due to hyper-keratinisation. Interestingly, 3-D rendering of K5 and K6 reveals acini in younger MGs that largely consist of epithelial cells, suggesting dynamic acini turnover. ICT quantification of cell density and proliferation in both young and aged MGs showed no statistically significant differences in cell density (p = 0.28) and proliferation (p = 0.9) even though the total cell and proliferation counts throughout the MG are reduced with aging (75% and 73% loss, respectively).
ICT is an emerging and powerful technique to visualise multiple cell populations in large volumes at high resolution that enables us to better understand MG structure and function. The correlation of MG atrophy with decreases in cell and proliferation counts suggests a potential deficiency in the adult stem cell population and their differentiation. Further analysis with ICT may allow us to localise and quantify putative stem cells to understand the maturation process of the MG.
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