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标题：Absence of ductal hyper-keratinization in Mouse age-related meibomian gland dysfunction (ARMGD)
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作者：Geraint J. Parfitt ; Yilu Xie ; Mikhail Geyfman 等
期刊名称：Aging
出版年度：2013
卷号：5
期号：11
页码：825-834
出版社：U.S.Department of Health & Human Service
摘要：Meibomian.gland.dysfunction.(MGD).is.frequent.with.aging.and.is.the.primary.cause.of.dry.eye.disease,.the.mostprevalent. ocular. complaint.. We. used. a. novel. 3©\D. reconstruction. technique,. immunofluorescent. computed. tomography(ICT),. to. characterize. meibomian. gland. keratinization. and. cell. proliferation. in. a. mouse. model. of. age©\related. meibomiangland.dysfunction.(ARMGD)..To.visualize.the.changes.associated.with.ARMGD,.5©\month.and.2©\year.old.mouse.eyelids.were3©\D.reconstructed.by.ICT.using. antibodies.to.cytokeratin.(CK). 1,.5.and. 6. and.the. proliferation. marker.Ki67..We.quantifiedtotal. gland,. ductal. and. lipid. volume. from. the. reconstructions,. observing. a. dramatic. decrease. in. old. glands.. In. youngglands,. proliferative. ductules. suggest. a. potential. site. of. acinar. progenitors. that. were.found. to.be. largely. absent.in.aged,atrophic. glands.. In. the. aged. mouse,. we. observed. an. anterior. migration. of. the. mucocutaneous. junction. (MCJ). and. anabsence.of. hyper©\keratinization. with. meibomian.gland.atrophy..Thus,.we. propose. that.changes. in. the. MCJ. and. glandularatrophy. through. a. loss. of. meibocyte. progenitors. are. most. likely. responsible. for. ARMGD. and. not. ductal. hyper©\keratinization.and.gland.obstruction....www.impactaging.com. . . . ............825...............................AGING, .November.2013,.Vol..5.No.11 var currentpos,timer; function initialize() { timer=setInterval("scrollwindow()",10);} function sc(){clearInterval(timer); }function scrollwindow() { currentpos=document.body.scrollTop; window.scroll(0,++currentpos); if (currentpos != document.body.scrollTop) sc();} document.onmousedown=scdocument.ondblclick=initializeEvidence for obstructive MGD in human patients has been supported by the identification of 'keratotic' clusters of squamous cells detected in MGD excreta [22], and histopathological evidence showing isolated regions of abnormal keratinization, ductal dilation and enlarged acini [23]. While a recent study of gene expression patterns of MGD glands has detected increased expression of genes associated with keratinization [24], analysis of proteins from excreta of MGD subjects failed to detect cytokeratin (CK) 1/10, the biomarkers for epidermal keratinization, while there was a general increase in other CKs associated with non-keratinized epithelium [25]. Recently, meibomian gland dropout has been documented in wild-type mice over 1 year of age [26, 27]. Since meibomian gland dropout is highly correlated with changes in lipid quality and frequently observed in human subjects over the age of 50[1, 4, 28-30], study of this mouse model may help in identifying underlying pathogenic mechanisms of ARMGD and suggest novel and more effective therapeutic strategies for this widespread clinical problem. Immunofluorescent Computed tomography (ICT) is a novel technique based on butyl-methyl methacrylate (BMMA) embedding that allows for repeated antibody-based staining on serial tissue sections cut in the range of ultra-thin (0.1¦Ìm) to semi-thin (5¦Ìm) thickness while maintaining excellent morphological preservation of tissue[31]. This enables 3-D reconstruction of multiple antigens with more reliable immunostaining and higher axial resolution across a large volume (>1mm3) than possible with conventional immunohistochemistry methods. We used ICT to characterize the distribution of epithelial CK proteins (1, 5 and 6) in a young, healthy mouse eyelid and an old mouse eyelid with ARMGD to assess the connection between keratinization, gland plugging and acinar atrophy with aging. CKs were chosen based on a previous study which examined their distribution at the mucocutaneous junction of the eyelid [32]. Results obtained here further demonstrate how ICT can be used for probing tissue structure and function from the macro (whole meibomian gland) to the micro (meibocyte) level where conventional histopathologic and immunofluorescent approaches are limited due to sampling error during repetitive antigen probing and 3-D reconstruction. RESULTS Effect of Aging on Eyelid and Meibomian Gland Architecture Prior to BMMA embedding and immunofluorescence staining to compare the effects of aging, excised eyelids from a 5-month and 2-year old mouse were imaged under a dissecting microscope at 43x magnification (Fig. 1A and B, respectively) and the meibomian glands were identified as a whitish tissue underlying the palpebral conjunctiva (black arrows). Loss of lipid containing acinar tissue consistent with meibomian gland dropout was observed in the 2-year old (Fig 1B, asterisk) and not the younger 5-month old mouse eyelid tissue. Next, BMMA serial sections cut at 2¦Ìm were sequentially immunostained with CK1, 5, 6 and Ki67 and 0.64mm x 1.14mm x 1.25mm of the 5-month old mouse eyelid (Fig 1A, red box) and 0.58mm x 0.80mm x 1.1mm of the 2-year old mouse eyelid (Fig 1B, red box) were 3-D reconstructed by ICT protocol. The typical CK1, 5, 6 and Ki67 immuno-staining patterns of a young mouse eyelid are shown in Figure 1C-H. In the 5-month old mouse eyelid CK5 reconstruction (Fig. 1I), meibomian glands (green) are in close proximity to each other and sit between regularly organized eyelash cilia (red arrowhead). At the lid margin (Fig. 1J), CK1+cells of the epidermis (orange) define the mucocutaneous junction as the transition to CK6+ conjunctiva (green) posterior to meibomian gland orifices (white arrow). In contrast, ICT of the 2-year old mouse eyelid reveals migration of the mucocutaneous junction to the level of meibomian gland orifices (Fig. 1K, white arrow). We also observed prominent truncation or atrophy of meibomian glands in the old lid (Fig. 1L). These glands have become largely obscured from view by eyelash cilia and are positioned much further apart from each other compared to the younger lid. Three meibomian glands were segmented from each eyelid reconstruction for visualization (Fig. 2) and quantification (Fig. 3) of total gland, ductal and lipid volumes as well as quiescent and proliferative cells. The 5-month old mouse meibomian glands (Fig. 2A-D) are comprised of multiple, large acini branching from a long tubular central duct characterized by a CK6+epithelium (Fig. 2B). There are instances where CK6+ cells extend into an acinus (Fig. 2B, white arrowhead), implying that, in some cases the ductule, or terminal ends of the ductal epithelium, can form a significant component of an acinus. Acini are composed of terminally differentiated meibocytes filled with lipid (Fig. 2C) and Ki67+cycling cells in the peripheral basal layer (Fig. 2D). In comparison, an aged meibomian gland (Fig. 2E-H) had distinct shortening from the distal end and a marked loss in glandular and ductal volume (Fig. 2F) which reveals both ductal and acinar atrophy. Moreover, there is a visible reduction in the CK6+ductal component of the gland (Fig. 2F). There is a clear loss in acinar tissue and acini appear noticeably smaller when compared to the younger glands (Fig. 2G). Ki67+nuclei count is also largely reduced in the older meibomian gland and ..www.impactaging.com. . . . ............826................................AGING,.November.2013,.Vol..5.No.11