盖雨刮区的印象细胞学
Summary
We collected, stained and imaged cells from the conjunctiva of the inner eyelid margin of human subjects. By characterizing cell morphology and metabolic activity, this method may further our understanding of dry eye and the role that friction between the ocular surfaces may play in perceiving ocular discomfort.
Abstract
Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The optimization of a method of collecting, staining and imaging cells from the LW region using impression cytology (IC) is described in this study. Cells are collected from the inner surface of the upper eyelid of human subjects using hydrophilic polytetrafluoroethylene (PTFE) membranes, and stained with cytological dyes to reveal the presence of goblet cells, mucins, cell nuclei and various degrees of pre- and para-keratinization. Immunocytochemical dyes show cell esterase activity and compromised cell membranes by the use of a confocal scanning laser microscope. Up to 100 microscopic digital images are captured for each sample and stitched into a high-resolution, large scale image of the entire IC span. We demonstrate a higher sensitivity of IC than reported before, appropriate for identifying cellular morphologies and metabolic activity in the LW area. To our knowledge, this is the first time this selection of fluorescent dyes was used to image LW IC membranes. This protocol will be effective in future studies to reveal undocumented details of the LW area, such as assessing cellular particularities of contact lens wearers or patients with dry eye or lid wiper epitheliopathy.
Introduction
人的上眼睑周围执行每天1万闪烁，只用1 – 2 mm窄结膜区域反对眼球。由于眨眼在其擦拭动作，睑缘的这一部分被称之为“盖雨刮器”区域2。假定摩擦惯常闪烁期间在该区域增加，由于该盖抹遍布全球。这可能在眼睛舒适一个显著的作用。隐形眼镜配戴者特别是经验眼部不适，这是主要的原因停止配戴镜片3之一。当接触透镜被放置在眼睛上，透镜材料和眼表面之间的摩擦系数已经显示出改变4。有证据表明，干症状可能与此改变的摩擦2,5,6。
此关联，也可以在临床上观察到的现象的反射，尤其是盖的Wi每上皮病变（LWE），也称为上盖余量染色（ULMS）6。 LWE是对眼有刺激性的早期征兆和干眼病的潜在指标。据观察，并通过上，下眼睑边缘区域的活体染色测定。虽然这个分级系统2及其临床有效性（即与主观症状相关性）仍在争论，眼部不适仍然为临床医师和科学家都一个难题，以及最重要的是，对于患者的不便。
最新的，鲜为人知的是，临床相关的变化盖子雨刮区7的（子）细胞的解剖和生理，只有一个报告中运用的方法细胞学8。印象细胞学检查（IC）已雇用了超过40年由膜9,10的应用程序来收集细胞延髓或睑结膜上皮表面。取出后，贴壁细胞经历Çytological染色和显微成像。由于在盖刮水器区域的解剖和细胞的差异，这种技术需要优化。
Protocol
Representative Results
Discussion
印象细胞学检查是在球结膜通常进行的，使用混合纤维素酯膜。样品切成尺寸从散装材料片，消毒，使用镊子结膜表面施加和除去。使用相同的膜材料，活塞控制的IC器件最近已设计，以配合球结膜的表面几何形状，和维护应用程序13之间是一致的压力。这些方法是低效的窄，突出地弯曲的盖刮水器区域（ 见图1）。此外，混合的纤维素酯膜不提供对设置必要的透明度，其中亮…
Divulgations
The authors have nothing to disclose.
Acknowledgements
We would like to acknowledge our colleague Dr. Jalaiah Varikooty who provided insight and expertise on the topic of the lid wiper, that greatly assisted this research.
Materials
| Alcian Blue, 1% in 3% Acetic Acid | Sigma | B8438-250ML | |
| Hematoxylin, Gill 1 | Sigma | GHS116-500ML | |
| Papanicolaou stain, OG-6 | Sigma | HT40116-500 ml | |
| Papanicolaou stain, Modified EA | Sigma | HT40232-1L | |
| Live/Dead Viability/Cytotoxicity Kit | Life Technologies | L-3224 | contains ethidium homodimer-1 and Calcein AM dyes |
| Alcaine (0.5% proparacaine hydrochloride) | Alcon | ||
| Millicell Cell Culture Insert, 12 mm, hydrophilic PTFE, 0.4 µm | EMD Millipore | PICM01250 | |
| 35 mm Glass Bottom Dishes | MatTek Corporation | P35G-0-20-C |
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