Yilu Xie

Effect of Desiccating Stress on Mouse Meibomian Gland Function

PURPOSE Mice exposed to standardized desiccating environmental stress to induce dry eye-like symptoms have been used as a model to study the underlying mechanisms of evaporative dry eye. While studies have shown marked inflammatory and immune changes, the effect of such stress on meibomian gland function remains largely unknown. We sought to evaluate the effects of desiccating stress on meibocyte proliferation and meibum quality. METHODS Ten mice were treated with scopolamine and subjected to a drafty low humidity environment (30-35%). Five and ten days after treatment, eyelids were harvested and cryosections stained with Ki67 antibody to identify cycling cells. Sections were also imaged using stimulated Raman scattering (SRS) microscopy to characterize the gland compositional changes by detecting the vibrational signatures of methylene (lipid) and amide-I (protein). RESULTS Desiccating stress caused a 3-fold increase in basal acinar cell proliferation from 18.3 ± 11.1% in untreated mice to 64.4 ± 19.9% and 66.6 ± 13.4% after 5 and 10 days exposure, respectively (P < .001). In addition, SRS analysis showed a wider variation in the protein-to-lipid ratio throughout the gland, suggesting alterations in meibocyte differentiation and lipid synthesis. CONCLUSIONS These data are consistent with a model that a desiccating environment may have a direct effect on meibomian gland function, leading to a significant increase in basal acinar cell proliferation, abnormal meibocyte differentiation, and altered lipid production.

A Novel Immunofluorescent Computed Tomography (ICT) Method to Localise and Quantify Multiple Antigens in Large Tissue Volumes at High Resolution

Current immunofluorescence protocols are limited as they do not provide reliable antibody staining within large tissue volumes (mm3) and cannot localise and quantify multiple antigens or cell populations in the same tissue at high resolution. To address this limitation, we have developed an approach to three-dimensionally visualise large tissue volumes (mm3) at high resolution (<1 µm) and with multiple antigen labelling, for volumetric and quantitative analysis. This is made possible through computer reconstruction of serial sectioned and sequentially immunostained butyl-methyl methacrylate (BMMA) embedded tissue. Using this novel immunofluorescent computed tomography (ICT) approach, we have three-dimensionally reconstructed part of the murine lower eyelid that contains the meibomian gland and localised cell nuclei (DAPI), Ki67 and cytokeratin 1 (CK1), as well as performing non-linear optical (NLO) microscopy imaging of collagen, to assess cell density, cell proliferation, gland keratinisation and gland volume respectively. Antigenicity was maintained after four iterative stains on the same tissue, suggesting that there is no defined limit to the number of antigens that can be immunostained for reconstruction, as long as the sections remain intact and the previous antibody has been successfully eluted. BMMA resin embedding also preserved fluorescence of transgenic proteins. We propose that ICT may provide valuable high resolution, three-dimensional biological maps of multiple biomolecules within a single tissue or organ to better characterise and quantify tissue structure and function.

Immunofluorescence Tomography of Mouse Ocular Surface Epithelial Stem Cells and Their Niche Microenvironment

PURPOSE Currently, there are no definitive immunomarkers for epithelial stem cells (corneal and conjunctival) or their poorly understood niche microenvironment. The H2B-GFP/K5tTA mouse enables visualization of label-retaining cells (LRCs), which exhibit the functional marker of stem cell quiescence. We used immunofluorescence tomography to evaluate putative stem cell markers and LRCs of the mouse ocular surface. METHODS H2B-GFP/K5tTA mice were pulsed for 56 days and then chased with doxycycline to label LRCs. Limbus and eyelid tissue was 3-dimensionally (3-D) reconstructed using immunofluorescence tomography to identify and characterize LRCs using the putative stem cell markers sox9, keratin 19, lrig1, blimp1, and abcb5. RESULTS After 28 days of chase, LRCs were localized to the entire limbus epithelium and, infrequently, the anterior limbal stroma. Label-retaining cells comprised 3% of limbal epithelial cells after 56 days of chase. Conjunctival LRCs were localized to the fornix and comprised 4% of the total fornix epithelial cells. No stem cell immunomarker was specific for ocular surface LRCs; however, blimp1 enriched for limbal basal epithelial cells and 100% of green fluorescent protein-positive (GFP+) cells at the limbus and fornix were found to be lrig1-positive. CONCLUSIONS Label-retaining cells represent a larger population of the mouse limbus than previously thought. They decrease in number with increased doxycycline chase, suggesting that LRC populations with different cell cycle lengths exist at the limbus. We conclude that current immunomarkers are unable to colocalize with the functional marker of epithelial stem cell quiescence; however, blimp1 may enrich for limbal epithelial basal cells.

Characterization of Quiescent Epithelial Cells in Mouse Meibomian Glands and Hair Follicle/Sebaceous Glands by Immunofluorescence Tomography

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Light transmission/absorption characteristics of the meibomian gland

PURPOSE While meibography has proven useful in identifying structural changes in the meibomian gland (MG), little is known regarding the MG spectral transmission and absorption characteristics. The purpose of this study was to measure the transmission/absorption spectra of the MG compared to other eyelid tissues. METHODS Human and rabbit eyelids were fixed in paraformaldehyde, serial sectioned (50 μm) using a cryotome and imaged by brightfield and reflectance microscopy. Eyelid regions (MG, muscle, tarsus and dermis) were then illuminated by a 100 μm spot using a infrared enhanced white light source. Transmission spectra over a 550-950 nm range were then measured using a spectrometer and differences compared using two-way analysis of variance. RESULTS Brightfield microscopy of both human and rabbit eyelid tissue showed a marked decrease in light transmission for MG acini compared to other eyelid tissues. In rabbit, the dermis showed 5× and the muscle showed 2× more light transmission compared to MG (P < .001 and P < .001, respectively). For human, the muscle showed 14× and the tarsus showed 84× more light transmission compared to MG (P < .01 and P < .001, respectively). No specific spectral region of light absorption could be detected in either rabbit or human MG. Loss of light transmission in MG was localized to acini containing small lipid droplets, averaging 2.7 ± 0.8 μm in diameter. CONCLUSIONS The data suggest that light transmission is dramatically reduced in the acini due to light scattering by small lipid droplets, suggesting that Meibography detects active lipid synthesis in differentiating meibocytes.

PPARγ regulates meibocyte differentiation and lipid synthesis of cultured human meibomian gland epithelial cells (hMGEC)

PURPOSE To evaluate the role of PPARγ in regulating meibocyte differentiation and lipid synthesis in a human meibomian gland epithelial cell line (hMGEC). METHODS HMGEC were exposed to the PPARγ agonist, Rosiglitazone, from 10-50 μM. Cultures were also exposed to specific PPARγ antagonist, T0070907, to block PPARγ receptor signaling. Cells were then stained with Ki-67 and LipidTox to determine the effects on cell cycling and lipid synthesis, respectively. Expression of meibocyte differentiation related proteins, ADFP, PPARγ, ELOVL4, and FABP4, were evaluated by quantitative PCR and western blotting. A human corneal epithelial cell line (hTCEpi) was used as a control. RESULT Rosiglitazone significantly decreased Ki-67 staining within 2 days in a dose-dependent manner (P = 0.003) and increased lipid accumulation in hMGEC in a dose dependent manner. T0070907 suppressed both lipid droplet synthesis and cell cycle exit. Rosiglitazone significantly upregulated expression of ADFP, PPARγ, ELOVL4, and FABP4 by 9.6, 2.7, 2.6, and 3.3 fold on average (all P < 0.05 except for FABP4, P = 0.057) in hMGEC. T0070907 significantly abrogated rosiglitazone-induced upregulation of these genes when treated prior to rosiglitazone treatment (all P < 0.05). The observed lipogenic differentiation response was not duplicated in hTCEpi after exposure to rosiglitazone. CONCLUSION Rosiglitazone induced cell cycle exit and upregulation of lipogenic gene expression leading to lipid accumulation in hMGEC. These effects were suppressed by PPARγ antagonist indicating that PPARγ signaling specifically directs lipogenesis in hMGEC. These findings suggest that PPARγ plays a critical role in meibocyte differentiation.