Geraint J. Parfitt

The Architecture of the Cornea and Structural Basis of Its Transparency

The cornea is the transparent connective tissue window at the front of the eye. In the extracellular matrix of the corneal stroma, hybrid type I/V collagen fibrils are remarkably uniform in diameter at approximately 30 nm and are regularly arranged into a pseudolattice. Fibrils are believed to be kept at defined distances by the influence of proteoglycans. Light entering the cornea is scattered by the collagen fibrils, but their spatial distribution is such that the scattered light interferes destructively in all directions except from the forward direction. In this way, light travels forward through the cornea to reach the retina. In this chapter, we will review the macromolecular components of the corneal stroma, the way they are organized into a stacked lamellar array, and how this organization guarantees corneal transparency.

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.

Three-dimensional reconstruction of collagen-proteoglycan interactions in the mouse corneal stroma by electron tomography

Corneal transparency is fundamental to the visual system, and is directly related to the ordered collagen fibril architecture that the cornea maintains. Proteoglycans, through their protein core and highly anionic glycosaminoglycan side chains, are thought to regulate the collagen organisation in the corneal stroma. To understand the inter-relationships between proteoglycans and collagen fibrils in the cornea, adult mouse corneas were treated with cuprolinic blue and three-dimensional reconstructions of the anterior, mid and posterior corneal stroma were obtained. The reconstructions show regular diameters of collagen fibrils throughout the cornea and uniform interfibrillar spacing within each region. Both longitudinal and transverse reconstructions were obtained to establish a clear picture of proteoglycan organisation, yet no distinct regular pattern or symmetry of proteoglycan orientation was observed. Large, electron-dense proteoglycans (possibly chondroitin sulphate/dermatan sulphate proteoglycans) interconnecting two or often three adjacent collagen fibrils are seen, whilst another sub-population of smaller proteoglycans (of the keratan sulphate variety) interconnect only neighbouring fibrils. The reconstructions suggest a complex interaction between proteoglycans and collagen, which allows for the dynamic control of collagen fibril architecture in the cornea.

Meibomian gland dysfunction: hyperkeratinization or atrophy?

Meibomian gland dysfunction (MGD) is the major cause of evaporative dry eye disease (EDED) and dysfunction is widely thought to mechanistically involve ductal hyperkeratinization, plugging and obstruction. This review re-evaluates the role of hyperkeratinization in MGD based on more recent findings from mouse models. In these studies, eyelids from normal young and old mice or mice exposed to desiccating stress were evaluated by immunofluorescent tomography and 3-dimensional reconstruction to evaluate gland volume, expression of hyperkeratinization markers and cell proliferation or stimulated Raman scattering (SRS) microscopy to assess lipid quality. Results indicate that aging mice show dropout of meibomian glands with loss of gland volume and a forward migration of the mucocutaneous junction anterior to the gland orifice; similar age-related changes that are detected in human subjects. Atrophic glands also showed evidence of epithelial plugging of the orifice without the presence of hyperkeratinization. Mice exposed to desiccating stress showed hyperproliferation of the meibomian gland and ductal dilation suggesting a marked increase in lipid synthesis. Lipid quality was also affected in EDED mice with an increase in the protein content of lipid within the duct of the gland. Overall, age-related changes in the mouse show similar structural and functional correlates with that observed in clinical MGD without evidence of hyperkeratinization suggesting that gland atrophy may be a major cause of EDED. The response of the meibomian gland to desiccating stress also suggest that environmental conditions may accelerate or potentiate age-related changes.

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.

Electron tomography reveals multiple self-association of chondroitin sulphate/dermatan sulphate proteoglycans in Chst5-null mouse corneas.

The spatial distribution of collagen fibrils in the corneal stroma is essential for corneal transparency and is primarily regulated by extrafibrillar proteoglycans, which are multi-functional polymers that interact with hybrid type I/V collagen fibrils. In order to understand more about proteoglycan organisation and collagen associations in the cornea, three-dimensional electron microscopy reconstructions of collagen-proteoglycan interactions in the anterior, mid and posterior stroma from a Chst5 knockout mouse, which lacks a keratan sulphate sulphotransferase, were obtained. Both longitudinal and transverse section show sinuous, oversized proteoglycans with near-periodic, orthogonal off-shoots. In many cases, these proteoglycans traverse over 400nm of interfibrillar space interconnecting over 10 collagen fibrils. The reconstructions suggest that multiple chondroitin sulphate/dermatan sulphate proteoglycans have aggregated laterally and, possibly, end-to-end, with orthogonal extensions protruding from the main electron-dense stained filament. We suggest possible mechanisms as to how sulphation differences may lead to this increase in aggregation of proteoglycans in the Chst5-null mouse corneal stroma and how this relates to proteoglycan packing in healthy corneas.

Renewal of the holocrine meibomian glands by label-retaining, unipotent epithelial progenitors

Summary The meibomian and sebaceous glands secrete lipids to prevent desiccation of the ocular surface and skin, respectively. Precisely how these holocrine tissues regenerate is not well understood. To address this, we characterized keratin 5+ (K5) label-retaining cells (LRCs) and the lineage tracing of keratin 14 (K14) progenitors in mouse meibomian glands. Using the tet-off H2B-GFP/K5tTA mouse, H2B-GFP fluorescence dilutes 2-fold with every division in K5+ cell nuclei after doxycycline administration. In 3D reconstructions generated over a >28-day doxycycline chase, we observed LRCs at the acinus entrance where K6+ ductal epithelium terminates. For lineage tracing, K14CreERT2-Confetti mice were injected intraperitoneally with tamoxifen and euthanized at 23 and 59 weeks later. Meibomian gland acini in these mice were either monochromatic or dual-colored, whereas the duct exhibited multiple colors. In conclusion, LRCs are likely to direct meibomian gland turnover and may exist as two distinct unipotent progenitors that renew ductal and acinar tissue separately.

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

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Meibocyte differentiation and renewal: Insights into novel mechanisms of meibomian gland dysfunction (MGD) ☆

This paper reviews our current understanding of age-related meibomian gland dysfunction (MGD) and the role of the nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARγ), in the regulation of meibomian gland function, meibocyte differentiation and lipid synthesis. The studies suggest that PPARγ is a master regulator of meibocyte differentiation and function, whose expression and nuclear signaling coupled with meibocyte renewal is altered during aging, potentially leading to atrophy of the meibomian gland as seen in clinical MGD. Study of meibomian gland stem cells also suggest that there is a limited number of precursor meibocytes that provide progeny to the acini, that may be susceptible to exhaustion as occurs during aging and other environmental factors. Further study of pathways regulating PPARγ expression and function as well as meibocyte stem cell maintenance may provide clues to establishing cellular and molecular mechanisms underlying MGD and the development of novel therapeutic strategies to treating this disease.