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Quantitative Assessment of Central and Limbal Epithelium After Long Eye (2016) 30, 979–986 © 2016 Macmillan Publishers Limited All rights reserved 0950-222X/16 www.nature.com/eye 1,5 1,5 1 Quantitative RK Prakasam , BS Kowtharapu , K Falke , CLINICAL STUDY K Winter2,3, D Diedrich4, A Glass4, A Jünemann1, assessment of central RF Guthoff1 and O Stachs1 and limbal epithelium after long-term wear of soft contact lenses and in patients with dry eyes: a pilot study Abstract Purpose Analysis of microstructural Eye (2016) 30, 979–986; doi:10.1038/eye.2016.58; alterations of corneal and limbal epithelial published online 22 April 2016 cells in healthy human corneas and in other ocular conditions. Introduction Patients and methods Unilateral eyes of three groups of subjects include healthy The X, Y, Z hypothesis1 explains cell mechanism volunteers (G1, n = 5), contact lens wearers that is essential for the renewal and maintenance 1Department of (G2, n = 5), and patients with dry eyes of the corneal epithelium. This hypothesis Ophthalmology, University = proposes that the loss of corneal epithelial of Rostock, Rostock, (G3, n 5) were studied. Imaging of basal Germany (BC) and intermediate (IC) epithelial cells surface cells (Z) can be maintained by the from central cornea (CC), corneal limbus proliferation of basal epithelial cells (X), and the 2Faculty of Medicine, centripetal movements of the peripheral (CL) and scleral limbus (SL) was obtained by Institute of Anatomy, epithelial cells (Y). By utilizing this mechanism, University of Leipzig, in vivo confocal microscopy (IVCM). An it is also possible to categorize both disease and Leipzig, Germany appropriate image analysis algorithm was therapies according to the specific component 3 used to quantify morphometric parameters involved.1 Therefore it is vital to understand the Institute for Medical including mean cell area, compactness, Informatics, Statistics and cellular structures of both central and limbal Epidemiology (IMISE), solidity, major and minor diameter, and epithelial cells in normal and in various corneal University of Leipzig, maximum boundary distance. disease conditions. Leipzig, Germany Results The morphometric parameters of BC Non-invasive in vivo confocal imaging2 of the 4 and IC demonstrated no significant living cornea is a novel clinical technique for Institute for Biostatistics, differences (P40.05) between groups. the study of corneal cellular morphology. University of Rostock, Rostock, Germany Comparison between three corneal locations Earlier, the systematic approach in acquiring (CC, CL, and SL) within the groups showed high quality and reproducible confocal images 5These authors contributed significant differences (Po0.05) with mean and subsequent precise cell quantification were equally to this work. values of cell area, compactness, solidity, and the major challenges in the field of confocal major and minor diameter of BC that increase microscopy. Today, due to the significant Correspondence: from CC to limbus. The BC were round and advancements in the optical properties of the RK Prakasam, Department regular in the central cornea (Po0.05) confocal microscopy, the image quality has been of Ophthalmology, University of Rostock, compared with CL and SL. dramatically improved and the digital 18055 Rostock, Germany Conclusions IVCM enables high-quality computing systems enable compression and Tel: +49 381 494 8501; confocal images from central corneal and storage of huge data sets for the further Fax: +49 381 494 8502. limbal epithelium. This quantitative study processing of the images for the quantification of E-mail: [email protected] – demonstrated morphological differences in cellular structural alterations.3 5 the basal and intermediate epithelium Recently, in vivo confocal microscopy (IVCM) Received: 13 October 2015 Accepted in revised form: between limbus and central cornea, and was used in studying the anatomical structures 11 February 2016 4 found no differences between contact lens of the corneal and limbal epithelium Published online: wearers, dry eyes, and normal subjects. particularly the palisades of Vogt (POV) 22 April 2016 Quantitative assessment of central and limbal epithelium RK Prakasam et al 980 including inter-palisadal epithelial rete pegs. POV6 are Germany) with a planar surface with a refractive clinically visible radially oriented fibrovascular ridges index of 1.49. observed along the upper and the lower corneoscleral limbus. Inter palisadal region serve as a repository for Image identification corneal epithelial cells. These interesting morphological structures of the limbus also thought to provide the We have used the internal volume scan modality with microenvironment or niche for progenitor or limbal stem a back and forth oscillation option that allows automatic cells.7,8 Any degree of disease or destruction to image acquisition of up to 160 continuous optical sections corneoscleral limbus leads to the formation of opaque with three cycles of oscillation (160 × 3 = 480 scans). Image conjunctival sheet of cells on the cornea causing acquisition from central cornea and inferior limbus disruption of smooth and clear optical surface leading to (includes corneal limbus and scleral limbus) produced 960 visual impairment or blindness. (480 scans × 2) serial images of full-thickness corneal and fi In vivo corneal confocal studies have also quanti ed the inferior limbal epithelium from single eye including all characteristic features of the central and limbal epithelial the cell layers. The distance between two optical sections cells using inbuilt or commercial software tools that was about 0.4 μm, as determined earlier.5 From 960 fi require manual intervention and identi cation of cells, images, a single cycle (160 images) of best sequence has which may cause random and/or systemic error. been selected from each examined eye. For the automatic To minimize these errors, we have used a fully automated cell analysis, two consecutive optical sections of software base5 with advanced image acquisition protocols intermediate cell layer and two consecutive sections of and segmentation routines for the quantification of basal cell layer were selected from each sequence. features of the epithelial cells. Therefore total of 60 images (4 images × 15 subjects = 60) The present work is the further step on the clinical was processed for automated cell analysis. The scanned application of the developed quantitative methods area of an image was 400 × 400 μm, 8-bit grey scale with of the cellular alterations.5 Here we have studied two 384 × 384 pixels. different corneal conditions that include contact lens wearing subjects9,10 whoarepronetodevelop mechanical or hypoxia-related corneal changes Automatic cell segmentation and subjects with dry eyes where corneal epithelium Automatic cell segmentation process involved several is stressed due to tear film insufficiency and frequent crucial steps.5 Initially, the images were magnified by a application of artificial tear drops.11 The purpose of factor of 4 and the central 60% of overall dimension was this study was to quantify and compare the morphological features of the central and limbal used for cell segmentation to exclude image borders with epithelium between normal and other corneal low contrast. Inverted images were smoothed and fi fi conditions. enhanced using different image lters, nally image artefacts and incorrectly segmented cells were deleted for quantification. The segmented cells were labelled and Materials and methods number of their pixels was counted to measure different In vivo confocal microscopy cell parameters. The quantified cell parameters (Table 1) include cell area, compactness, solidity, major and minor IVCM was performed that works on the principle of cell radius, and maximal boundary distance of both Heidelberg Retina Tomograph II in combination with intermediate and basal cell layers. The compactness and Rostock Cornea Module12 (HRT II/RCM; Heidelberg solidity are the shape factors that explain the shape and Engineering, Heidelberg, Germany). As already described by our group,13 this system enables both automated internal and manual external z-scan device to move the Table 1 Definition of quantified cell parameters focal plane on to the corneal surface and thus allow Parameters Definition imaging of cell layers at any depth with an axial μ fi Cell Area (μm2) Number of pixels × area of a pixel (1 μm2) resolution of about 1 m. High magni cation was Cell compactness (Boundary length2/area)/(4*π) achieved by using an immersion lens with a short focal Cell solidity Cell convex hull area/number of cell pixels length and a high numerical aperture (Achroplan × 63W/ Cell major radius (μm) Length of the semi-major axis if ellipse fitted into a cell NA 0.95/AA 2.00 mm, 670 nm/Fa C Zeiss). The Cell minor radius (μm) Length of the semi-minor axis if ellipse distance between corneal surface and the objective fitted into a cell lens was kept stable using a single-use contact element Cell maximal boundary Maximum distance from centre to boundary distance (μm) (Tomocap; Heidelberg Engineering, Heidelberg, Eye Quantitative assessment of central and limbal epithelium RK Prakasam et al 981 regularity of the cells, whereas other parameters measure (CC), corneal (CL) and scleral (SL) sides of the the size of the epithelial cells. inferior limbus. Subjects Statistical analysis Three different groups of subjects (15 with each All data were stored and analysed using the SPSS group having 5) were studied including normal or statistical package 15.0 (SPSS Inc. Chicago, IL, USA). The control
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