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Contact lens-related complications are common, affecting around one third of wearers, although most are mild and easily managed. Contact lenses have well-defined anatomical and physiological effects on the ocular surface and can result in other consequences due to the presence of a biologically active material. A contact lens interacts with the tear film, ocular surface, skin, endogenous and environmental microorganisms, components of care solutions and other antigens which may result in disease specific to contact lens wear, such as metabolic or hypersensitivity disorders. Contact lens wear may also modify the epidemiology or pathophysiology of recognised conditions, such as papillary conjunctivitis or microbial keratitis. Wearers may also present with intercurrent disease, meaning concomitant or pre-existing conditions unrelated to contact lens wear, such as allergic eye disease or blepharitis, which may complicate the diagnosis and management of contact lens-related disease. Complications can be grouped into corneal infection (microbial keratitis), corneal inflammation (sterile keratitis), metabolic conditions (epithelial: microcysts, vacuoles, bullae, tight lens syndrome, epithelial oedema; stromal: superficial and deep neovascularisation, stromal oedema [striae/folds], endothelial: blebs, polymegethism/ pleomorphism), mechanical (corneal abrasion, corneal erosion, lens binding, warpage/refractive error changes; superior epithelial arcuate lesion, mucin balls, conjunctival epithelial flaps, ptosis, discomfort), toxic and allergic disorders (papillary conjunctivitis, solution-induced corneal staining, incomplete neutralisation of peroxide, Limbal Stem Cell Deficiency), tear resurfacing disorders/dry eye (contact lens-induced dry eye, Meibomian gland dysfunction, lid wiper epitheliopathy, lid parallel conjunctival folds, inferior closure stain, 3 and 9 o'clock stain, dellen, dimple veil) or contact lens discomfort. This report summarises the best available evidence for the classification, epidemiology, pathophysiology, management and prevention of contact lens-related complications in addition to presenting strategies for optimising contact lens wear.

Purpose: To reveal the effect of cigarette smoking on corneal endothelial cells. Patients andmethods: this is a case control cross sectional study including 99 persons, 61 chronic cigarettesmokers and another age matched 38 control nonsmokers. Parameters used in our study werecell density (CD), polymegethism, pleomorphism, the average of cell size, and central cornealthickness (CCT). Results: The mean values of the CD were 2829.9±334.9 compared to3243.6±237.9, CV were 44.13±9.73 compared to 32.84±2.97, HEX were 40.44±6.95% compared to60.11±5.83% for smokers and non-smoker groups respectively, with a highly statistically significantvalue (p

Background To compare the endothelial cell density and morphology in the peripheral cornea before and after corneal cross-linking (CXL). Methods This study evaluated thirty-one eyes of 31 patients who were treated with standard CXL for progressive keratoconus. Preoperatively and 6 months postoperatively, we compared the corneal endothelial cell density (ECD), the coefficient of variation in cell size (CV), and the percentage of hexagonal cells (HEX), in the peripheral regions of the cornea, using a non-contact specular microscope (EM-3000, Tomey). Results All keratoconic eyes in this series were measurable in the peripheral regions. No significant differences were found in the peripheral ECD preoperatively and 6 months postoperatively at each point (Wilcoxon signed-rank test, superior, p = 0.16, nasal superior, p = 0.12, temporal superior, p = 0.17, inferior, p = 0.37, nasal inferior, p = 0.28, temporal inferior, p = 0.17). The mean percentage of the ECD loss was 1.3, 1.3, 1.0, 1.4, 0.7, and 1.4%, respectively. No significant differences in the peripheral CV or HEX were found preoperatively and 6 months postoperatively at each point. Conclusions Standard CXL does not cause significant changes in endothelial cell density, polymegethism, or polymorphism, in the peripheral regions of the cornea. It is suggested that CXL is a minimally invasive surgical approach for progressive keratoconus, even in terms of peripheral endothelial cells. Trial registration University Hospital Medical Information Network Clinical Trial Registry (000031162).

Purpose To assess variability in the coefficient of variation (COV) in cell area estimates when using different numbers of cells for endothelial morphometry. Methods Using non-contact specular microscopy images of the corneal endothelium, 4 sets of 20 cases were selected that included 200 cells and had overall (global) COV values of less than 30 (group 1), 31-40 (group 2), 41-50 (group 3) and over 50% (group 4). Subjects could be normal, or had ophthalmic disease (such as diabetes), a history of rigid or soft contact lens wear or were assessed after cataract surgery. A step-wise analysis was undertaken, 20 cells at a time, of the variability in cell area estimates when using different numbers of cells for the calculations. Results Variability in the average cell area values was higher if only 20-60 cells were used in the calculations and then tended to decrease. The standard deviation values on these average cell area values and the calculated COV showed the same overall trends and were more than twice as large for endothelia with marked polymegethism. Using more than 100 cells/image in markedly polymegethous endothelia only increased the variability in the calculations. Conclusions These analyses indicate that substantial region variability in cell area values can be expected in polymegethous endothelia. The analysis further confirm that using only small numbers of cells (e.g. less than 50/image) in such cases is likely to yield far less reliable estimates of COV.

Purpose To assess variability in endothelial cell density (ECD) estimates when polymegethism (variance in cell areas) is present. Methods Using noncontact specular microscope images of the corneal endothelium, 4 sets of 20 cases were selected, which included 200 cells and had coefficient of variation values of less than 30% (group 1), 31%-40% (group 2), 41%-50% (group 3), and over 50% (group 4). A stepwise analysis was undertaken, 20 cells at a time, of the ECD estimates when using different numbers of cells for the calculations. Results The net differences in ECD estimates when comparing sets of 20 cells with 200 cells were 5.0% ± 3.9%, 8.1% ± 7.3%, 11.3% ± 9.4%, and 14.5% ± 12.4% for groups 1 to 4, respectively. For measures on 100 cells per image, the predicted variances in ECD values were 5.6%, 8.8%, 11.1%, and 13.7% for the 4 groups. Conclusions Higher values of corneal endothelial polymegethism result in predictable increases in the variability (uncertainty) in ECD estimates, thus reducing the "accuracy" of ECD values. There is no obvious utility in assessing more than 100 cells in such endothelia.

Aim To compare the corneal endothelial cells after posterior chamber phakic intraocular lens with and without a central hole (hole implantable collamer lens (ICL) and conventional ICL) implantation for the correction of moderate to high myopia. Methods This retrospective study evaluated 34 eyes of 34 patients who underwent hole ICL implantation and 25 eyes of 25 patients who underwent conventional ICL implantation. Preoperatively and 3 months, and 1 and 2 years postoperatively, we compared the central corneal endothelial cell density (ECD), coefficient of variation in cell size (CV) and the percentage of hexagonal cells (HEX) between the two groups using a non-contact specular microscope (EM-3000, Tomey). Preoperatively and 2 years postoperatively, we also compared them in the peripheral regions. Results The mean central ECD loss was 0.3% and 1.1%, 2 years after hole ICL and conventional ICL implantation, respectively (Mann-Whitney U test, p=0.72). There were no significant changes in central ECD, CV or HEX at any time points either after hole ICL or conventional ICL implantation, and a significant decrease only in terms of ECD in the superior regions after conventional ICL implantation. Conclusions Both ICLs do not induce a significant change in the density, polymegethism or polymorphism of corneal endothelial cells even 2 years postoperatively, except for the density in the superior regions after conventional ICL implantation. However, hole ICL implantation may have advantages over conventional ICL implantation only in terms of the density in the superior regions, possibly because preoperative laser iridotomies are unnecessary.

Fuchs endothelial corneal dystrophy (FECD), estimated to affect 4% of the US population [1], is the leading cause of endogenous corneal endothelial degeneration and the primary indication for keratoplasty worldwide [2]. FECD is a slowly progressive bilateral corneal disease that is characterized by abnormal corneal endothelial morphology, such as pleomorphism and polymegethism, decreased cell density, thickening of the Descemet membrane (DM), and formation of extracellular matrix (ECM) excrescences called guttae [3], which lead to derangements in endothelial cell function. Quality of vision is slowly compromised by corneal edema secondary to gradual failure of corneal endothelial pump action and guttae-induced visual disturbances such as decreased contrast sensitivity [4] and higher order aberrations [5]. Its pathogenesis is proposed to be an interaction between endogenous and exogenous factors [6]. The disease manifests in the early third to late fifth decade of life and has a female preponderance [7–9]. Since the first description of the disease by Ernest Fuchs more than 100 years ago [10], major advances in understanding the nature and pathogenesis of FECD have been made and are described in this chapter.

To evaluate the ocular surface, meibomian gland alterations, and in vivo confocal microscopy (IVCM) parameters through correlation with nicotine dependency level (NDL) and duration of smoking (DS) in chronic smokers. This cross-sectional study included 50 eyes of 50 chronic smokers, and 50 eyes of 50 age- and gender-matched healthy nonsmokers. To examine the ocular surface and tear film, corneal sensitivity (CS) measurement with Cochet-Bonnet esthesiometer, tear film break-up time (TBUT), lissamine green (LG) staining, Schirmer I test with anesthesia, and Ocular Surface Disease Index (OSDI) questionnaire were performed consecutively. Basal epithelial cell, keratocyte, and endothelial cell density, the percentage of endothelial polymegethism/pleomorphism, and subbasal nerve plexus were evaluated using IVCM. Meibomian gland density was evaluated with a meibography unit. The Fagerstrom test was used to measure NDL, and DS was recorded. In the chronic smokers group, the basal epithelial cell density, anterior and posterior keratocytes, endothelial cell density, and long and total subbasal nerve numbers were lower, and LG staining, meiboscore, and OSDI scores were higher, compared with the control group, whereas other parameters were similar. The percentage of polymegethism was higher and the percentage of pleomorphism was lower in the chronic smokers group. In the correlation analysis, no significant relationship was found between the DS, NDL and ocular surface, and IVCM findings. Decreased corneal basal epithelium, anterior and posterior keratocytes, endothelial cell density, meibomian gland density, and subbasal nerve numbers were found in chronic smokers. The results of the study show that smoking has an adverse effect on ocular surface parameters.

PURPOSE: To identify donor and recipient factors, including eye bank tissue observations, predictive of operative complications in the Cornea Preservation Time Study. METHODS: One thousand three hundred thirty study eyes undergoing Descemet stripping automated endothelial keratoplasty for Fuchs dystrophy or pseudophakic/aphakic corneal edema were randomized to receive a donor cornea with preservation time (PT) of 0 to 7 days (N = 675) or 8 to 14 days (N = 655). Donor factors included demographics, prelamellar corneal and postlamellar lenticule dissection thickness, central endothelial cell density, and tissue processing time. Recipient factors included demographics, intraocular pressure, and glaucoma medications or surgery (trabeculectomy, laser trabeculoplasty). Eye bank observations included donor tissue folds, pleomorphism/polymegethism, and endothelial cell abnormalities. Possible tissue-related operative complications were recorded including difficult donor lenticule unfolding and positioning. Multivariable logistic regression with backward selection was used to identify statistically significant (P < 0.01) associations between factors and operative complications. RESULTS: The only factor predictive of operative complications [58 (4.4%) of 1330 surgeries] was prelamellar dissection donor corneal thickness (P = 0.002). For every 50 μm of donor corneal thickness prior to lamellar dissection, operative complication odds increased by 40% (odds ratio [99% confidence interval (CI)]: 1.40 [1.06–1.83]) adjusting for PT and whether the epithelium was on or off. The estimated mean prelamellar dissection donor corneal thickness for PT 0 to 7 days was 537 μm (99% CI: 516 μm–558 μm) compared with 567 μm (99% CI: 546 μm–588 μm) for PT 8 to 14 days (P < 0.001). CONCLUSIONS: Thicker donor tissue (prelamellar dissection) is associated with operative complications and should be considered in tissue selection for Descemet stripping automated endothelial keratoplasty lenticule preparation.

We report on a pathway for Gabor domain optical coherence microscopy (GD-OCM)-based metrology to assess the donor’s corneal endothelial layers ex vivo. Six corneas from the Lions Eye Bank at Albany and Rochester were imaged with GD-OCM. The raw 3-D images of the curved corneas were flattened using custom software to enhance the 2-D visualization of endothelial cells (ECs); then the ECs within a circle of 500-μm-diameter were analyzed using a custom corner method and a cell counting plugin in ImageJ. The EC number, EC area, endothelial cell density (ECD), and polymegethism (CV) were quantified in five different locations for each cornea. The robustness of the method (defined as the repeatability of measurement together with interoperator variability) was evaluated by independently repeating the entire ECD measurement procedure six times by three different examiners. The results from the six corneas show that the current modality reproduces the ECDs with a standard deviation of 2.3% of the mean ECD in every location, whereas the mean ECD across five locations varies by 5.1%. The resolution and imaging area provided through the use of GD-OCM may help to ultimately better assess the quality of donor corneas in transplantation.

Purpose To assess the impact of using different numbers of cells in calculations of the coefficient of variation (COV) value for normal and polymegethous endothelia Methods Four sets of 20 non-contact specular microscope images obtained from Caucasian individuals were assessed, and categorized according to the extent of polymegethism, i.e. grade 0 (none), grade 1 (mild), grade 2 (moderate) and grade 3 (substantial). Cell areas were measured manually and then values for between 2 and 100 cells were progressively added and averaged to generate COV estimates. These were then assessed in terms of their relative values (as percentages +/- SD) in relation to the value obtained on over 100 cells. Results For the 4 sets of endothelia with group-mean COV values of 25.8, 33.1, 45.1 and 56.8%, the reliability of the COV estimates realized asymptotic values of close to ±1.0, ±2.7, ±3.6 and ±5.0% with 90 cells, but with greater uncertainty with few numbers of cells, e.g. only to within ±3.4, ±5.3, ±6.1 and ±7.9% with 75 cells. Conclusions COV estimates for the corneal endothelium are dependent on the number of cells used in the calculations. It is recommended that every effort should be made to not only assess 75–100 cells per endothelial image, but that this number should be the same or very similar for all endothelial images in a particular data set so that the uncertainly (or estimated proportional error) in the estimates is balanced.

To evaluate corneal endothelial cell density (ECD) and morphology after cataract surgery using coaxial ultrasound (US) phacoemulsification or a recently introduced coaxial nanosecond laser technique.Department of Ophthalmology, Medical University of Sofia, Sofia, Bulgaria.Prospective cohort study.Coaxial US phacoemulsification was performed in 1 eye (US group) and coaxial nanosecond laser-assisted cataract surgery in the contralateral eye (laser group) of the same patient. Nuclear sclerosis was graded from nuclear opalescence (NO) 3, nuclear color (NC) 3 to NO4, NC4 using the Lens Opacities Classification System III. The central ECD, coefficient of variation (CoV) in cell size (objective measure of polymegethism), and percentage of hexagonal cells (an index of pleomorphism) were evaluated.Eighty-two eyes (41 patients) had uneventful surgery. The mean ECD was 2517 cells/mm(2) ± 137 (SD) preoperatively and 2287 ± 155 cells/mm(2) at 2 years in the US group and 2521 ± 233 cells/mm(2) and 2420 ± 292 cells/mm(2), respectively, in the laser group (both P .001). The mean CoV was 0.27 ± 2.4 preoperatively and 0.30 ± 2.4 at 2 years in the US group and 0.27 ± 2.8 and 0.27 ± 2.0, respectively, in the laser group (both P .001). The mean percentage of hexagonal cells was 42.3% ± 3.6% preoperatively and 37.74% ± 3.54% at 2 years in the US group and 42.8% ± 3.2% and 43.00% ± 2.68%, respectively, in the laser group (both P .001).Nanosecond laser phacoemulsification had advantages over US phacoemulsification in terms of endothelial cell structure preservation.Dr. Kanellopoulos is consultant to Alcon Surgical, Inc., Allergan, Inc., Avedro, Inc., Keramed, Inc., Optovue, Inc., i-Optics Corp., and Carl Zeiss Meditec AG. Neither of the other authors has a financial or proprietary interest in any material or method mentioned.

Purpose. Ocular aging is a natural process of functional decline in vision. When the process reaches a point that compromised vision affects normal daily activity, it manifests as age-related ocular diseases, such as age-related macular degeneration, cataracts, glaucoma, and pseudoexfoliation syndrome. We previously reported that repressed Wnt signaling accelerated the maturation of corneal epithelium during tissue development. Here, we explore the hypothesis that repressed Wnt signaling is associated with accelerated aging in mouse eyes.Methods. Wnt ligand antagonist secreted frizzled-related protein 1 (sFRP1) was expressed in the corneal stroma by a tissue-specific, inducible, bitransgenic system. Tissue structure was analyzed for signs of aging. Signal transduction analysis was performed to determine the cellular response to sFRP1.Results. Mouse eyes with sFRP1 expression showed signs of accelerated aging, resembling those found in pseudoexfoliation (PEX) syndrome, a known age-related disease. Specific findings include granular deposition on the surface of the anterior lens capsule, pigment loss from the anterior surface of the iris, the presence of fibrillary material in the anterior chamber, and changes in cell size (polymegethism) and shape (pleomorphism) of the corneal endothelial cells.In vitrostudies demonstrated that sFRP1 did not inhibit Wnt5a function and that cells responded to sFRP1 and Wnt5a in a very similar manner.Conclusion. The expression of sFRP1 accelerates the aging process in mouse eyes and future studies are warranted to elucidate the underlying mechanisms.

BackgroundCorneal endothelium (CE) images provide valuable clinical information regarding the health state of the cornea. Computation of the clinical morphometric parameters requires the segmentation of endothelial cell images. Current techniques to image the endothelium in vivo deliver low quality images, which makes automatic segmentation a complicated task. Here, we present two convolutional neural networks (CNN) to segment CE images: a global fully convolutional approach based on U-net, and a local sliding-window network (SW-net). We propose to use probabilistic labels instead of binary, we evaluate a preprocessing method to enhance the contrast of images, and we introduce a postprocessing method based on Fourier analysis and watershed to convert the CNN output images into the final cell segmentation. Both methods are applied to 50 images acquired with an SP-1P Topcon specular microscope. Estimates are compared against a manual delineation made by a trained observer.ResultsU-net (AUC=0.9938) yields slightly sharper, clearer images than SW-net (AUC=0.9921). After postprocessing, U-net obtains a DICE=0.981 and a MHD=0.22 (modified Hausdorff distance), whereas SW-net yields a DICE=0.978 and a MHD=0.30. U-net generates a wrong cell segmentation in only 0.48% of the cells, versus 0.92% for the SW-net. U-net achieves statistically significant better precision and accuracy than both, Topcon and SW-net, for the estimates of three clinical parameters: cell density (ECD), polymegethism (CV), and pleomorphism (HEX). The mean relative error in U-net for the parameters is 0.4% in ECD, 2.8% in CV, and 1.3% in HEX. The computation time to segment an image and estimate the parameters is barely a few seconds.ConclusionsBoth methods presented here provide a statistically significant improvement over the state of the art. U-net has reached the smallest error rate. We suggest a segmentation refinement based on our previous work to further improve the performance.

Aim To measure the parameters of endothelium in the central and peripheral parts of the cornea and evaluate the influence of wearing the hard and soft contact lenses on the mentioned parameters. Methods A specular microscope was used to measure the corneal endothelium parameters in both eyes of 139 Caucasians (a total of 278). All participants were divided into three groups: soft lens wearers, hard lens wearers and a control group. Factors, such as age, smoking, types of lens material, duration of lens wear and lens air permeability were assessed to determine their impact on the morphometric parameters of the endothelium. Results A lower percentage of hexagon-like cells and higher cell variation than in other groups were determined in hard contact lens wearers. The difference in density of endotheliocytes between the groups was not observed. The measurements of the morphometric parameters in soft contact lens wearers did not depend neither on the duration of lens wear, nor on air permeability. The relation between the patients' age and the variation of endothelium parameters was determined in the group of hard contact lens wearers. Conclusion Wearing hard contact lenses provokes pleomorphism and polymegethism of the corneal endothelium, while soft contact lenses do not impact any parameters of the endothelium, most likely due to higher air permeability.

We aimed to compare the effect of accelerated and conventional corneal collagen cross-linking (CXL) on visual, refractive, and topographic parameters in patients with progressive keratoconus. Between December 2014 and February 2016, at Imam Khomeini Hospital, Ahvaz Jundishapur University of Medical Sciences, Iran, we compared 37 eyes of 21 patients treated by conventional CXL (CCXL; 3 mW/cm2 in 30 minutes) with 34 eyes of 18 patients treated by accelerated CXL (ACXL; 18 mW/cm2 in 5 minutes) based on generalizing estimation equation analysis in terms of corrected distance visual acuity (CDVA), uncorrected distance visual acuity (UDVA), corneal endothelial cell indices, and topographic parameters before and at 3, 6 and 12 months after the operation. The mean UDVA and spherical equivalent changes were similar in the two groups, but an improvement in CDVA was only observed in the CCXL group (P = 0.003). Keratometry (minimum and maximum) was significantly decreased in the CCXL group (P = 0.043 and P = 0.008, respectively). Indices of keratoconus progression—surface asymmetry index (SAI), keratoconus prediction index (KPI), and keratoconus index (KCI)—were significantly lower in the CCXL group than in the ACXL group (P = 0.002, P < 0.001, and P < 0.001, respectively). The thinnest corneal thickness (TCT) was not significantly different between the two groups (P = 0.15). The reduction of corneal endothelial cell density was also similar between the two groups; however, polymorphism and polymegethism were significantly lower in the ACXL group than in the CCXL group. In conclusion, we showed that although ACXL at 18 mW/cm2 slowed keratoconus progression safely during a 1-year follow-up, CCXL at 3 mW/cm2 may be superior in the prevention of keratoconus progression.

Objective: To compare corneal morphological parameters between diabetics and age matched non-diabetic control subjects and to evaluate the correlation of these parameters in relation to duration of diabetes mellitus (DM), glycemic status and severity of diabetic retinopathy.(DR). Methods: This cross sectional comparative study was conducted at the Department of Ophthalmology, PNS Shifa Karachi from February 2016 to January 2017. Patients with ages between 10 to 80 years of either gender who were diagnosed to have DM were recruited in the study. Control group comprised of age matched healthy volunteers who did not have DM. Corneal morphological parameters (CED, Average cell size, CV of cell size and hexagonality) was evaluated in each subject with non-contact specular microscope and findings were endorsed on a pre devised proforma. Results: Data of 298 eyes (149 diabetic patients and 149 healthy controls) was evaluated. Mean corneal endothelial cell density (CED) of diabetic population was 2494.47 ± 394.10 cells/mm2, while mean CED of control group was 2574.46 ± 279.97 cells/mm2 [p = 0.04]. Between group differences in mean average cell size, CV of cell size and hexagonality was statistically not significant. Analysis of corneal endothelial parameters among subgroups of patients with no DR, with NPDR and PDR did not show statistically significant difference. Moreover, patients with diabetes of more than 10 years duration had significantly lower CED (p

This review examines problems associated with in vivo assessment of corneal endothelium with special reference to keratoconus (KC) corneas. The main difficulty appears to be that a single assessment of less than 1/400 of the total number of endothelial cells in any eye may not detect any abnormal levels of heterogeneity. A diagnosis of polymegethism, pleomorphism, corneal guttae, or incipient Fuchs dystrophy may be missed if the sample observed is not representative. Evaluation of the endothelium may be more important in KC when the endothelium may be more susceptible to ultraviolet radiation due to stromal thinning and/or be more susceptible to damage from wearing thicker contact lenses that place hypoxic demands on endothelial deturgescing function. Also, assessment appears to be a greater problem for KC corneas for which central observations are likely to miss any changes in the region of an eccentric cone or in noncentral areas that may be affected by ruptures of the posterior limiting lamina. Apart from genetic influences and ethnic and/or racial considerations, factors such as exposure to ultraviolet and other geographic or vocational variables, atopy, or specific ocular allergies, which may be related to chronic habits of abnormal rubbing and any associated damage due to elevated intraocular pressure, may all contribute to variable presentations of the endothelium. Because of the phenotypic diversity of KC, the status of the endothelium may vary according to many other criteria including age of onset, rate of progression, degree of asymmetry, the type and number of biomicroscopic signs, degree of thinning, and the location, type, and area of the cone, for example. For in vivo assessments, observation of multiple sites is required, but for KC, it should include the cone area to increase the chance of detecting any significant differences from normal.

Objective To demonstrate the corneal morphologic aspects obtained with in vivo confocal microscopy (CM) and light and electron microscopy of specimens obtained from the same patients with macular corneal dystrophy (MCD). Design Case series. Participants Five consecutive patients affected by MCD undergoing penetrating keratoplasty (PK) in 1 eye. Methods The patients were examined with the slit-lamp, optical pachymetry, and CM before undergoing PK. The corneal buttons were processed for light, transmission, and scanning electron microscopy. Main Outcome Measures Corneal in vivo CM, corneal light, and electron microscopy. Results Confocal microscopy showed areas of altered reflectivity in basal epithelial cells, which appeared hyperreflective or completely white. In the anterior stroma, rectilinear hyperreflective areas were shown. The stroma was characterized by a granular appearance of both keratocytes and extracellular matrix. Dark striae of different length and orientation were present in the middle and posterior stroma. The corneal endothelium showed polymegethism and cells containing bright granules in their cytoplasm. The histopathologic study demonstrated areas of thickened Bowman's layer covered by an epithelium reduced in height. The Bowman's layer thickenings were due to the accumulation of free or vesiculated material of different electron density. The keratocytes showed intracytoplasmatic vesicles, whereas the extracellular matrix presented a large quantity of intercellular electron-lucent material and parallel lamellae with an undulated course. Occasional macrophages, filled with vesicles of granular-filamentous material and evident podosomes, were observed. Descemet's membrane was formed by a normal anterior banded zone and a posterior nonbanded zone of honeycombed aspect. The endothelial cells showed a large number of intracytoplasmic vesicles. Conclusions The structural changes observed with the histopathologic methods give an account and provide an explanation for the pathologic changes demonstrated by CM in the course of MCD. This may contribute to the understanding of in vivo imaging, allowing a better, noninvasive study of the disease evolution.

The aim was to assess the morphology of the Asian corneal endothelium, particularly the index of polymegethism and its relationship to endothelial cell density assessments.The central regions of the corneal endothelium of 72 healthy, normal weight, non-contact lens wearers were assessed by non-contact specular microscopy. There were six groups of persons, aged between 18 and 27 years, whose ethnic origins were self-reported as Asian (Mainland China, Hong Kong, India, Malaysia, Pakistan or other Asian groups). Single images including at least 100 contiguous cells were obtained from each subject and were processed by planimetry after manual border marking to minimise errors. Calculations were made of the coefficient of variation of cell area (COV; polymegethism) as well as the endothelial cell density and percentage of six-sided cells.The COV values ranged from 18 to 30.4 per cent (group mean 24.5 ± 2.8 per cent). The corneas had essentially normal thickness (0.530 ± 0.026 mm), with the endothelia showing a high percentage of six-sided cells (66.3 ± 7.1 per cent) and none showing even mild signs of polymegethism. Endothelial cell density estimates based on average cell areas ranged from 2,020 to 3,623 cells per mm(2) and from 2,157 to 3,835 with calculations based on individual cell areas. The difference between the two estimates of endothelial cell density was strongly related to the coefficient of variation (r = 0.765).This result is consistent with several older reports indicating that it is uncommon to find polymegethism in healthy young adult Asian individuals. Nonetheless, even low coefficient of variation values can substantially affect the estimations of the cell density.

PURPOSE To assess the corneal endothelium, particularly the polymegethism feature, using the Topcon SP-3000P specular microscope with newer center-dot software. METHODS Forty-eight healthy, normal weight, noncontact lens wearers of Asian ethnicity were assessed. Single endothelial images from each subject were processed with center-dot software, reevaluated after correction of obvious errors, and then by manual border marking and planimetry. Endothelial cell density based on average cell area and the coefficient of variation (COV) of cell area (polymegethism) were calculated. RESULTS Error sources are associated with erroneous location of cell borders (usually creating larger or smaller "cells") or failure to assign cell borders to a marked cell. On the initial application of the center-dot software, the endothelial cell density values ranged from 1822 to 3244 cells per square millimeter (mean, 2644 cells/mm); this range was reduced (eg, 1955-3054 cells/mm; mean 2690 cells/mm on editing or in manual planimetry). The COV values ranged from 17% to 39% (mean, 27.5% ± 5.5%), with one third of the endothelia yielding COV values of greater than or equal to 30%. On editing or in manual planimetry, the COV values were reduced to between 17% and 29% (mean, 24.5% ± 3.2%; P < 0.001). CONCLUSIONS In the use of a center-dot endothelial analysis program with cell border identification, it is likely that at least 1 set of editing steps is required to produce reasonable results.

A detailed microscopic assessment of corneal quality is a crucial step in determining the suitability of a cornea for grafting. This chapter describes in detail the assessment of quantitative and qualitative corneal/endothelial parameters and their irregularities as observed by light and specular microscopy, together with links to images presented in the atlas (see Chap. 6). Endothelial cell density, the most important quantitative parameter of corneas intended for grafting, should generally be at least 2000 cells/mm2. In this regard, the detection of dead endothelial cells is important for preventing the overestimation of endothelial cell density. Polymegethism (variation in cell size) and pleomorphism (variation in cell shape), two basic qualitative morphologic parameters of the endothelium, together with the presence of Descemet membrane folds, cornea guttata, intracellular changes, and foreign bodies or precipitates are also assessed. The tissue should also be checked for the presence of severe epithelial defects or stromal opacities as signs of various stromal pathologies. In addition to initial cornea quality, changes affecting stored corneas or alterations arising as a consequence of the preparation of various types of endothelial lamellae may influence the quality and survival of a graft. Corneal alterations characteristic of certain corneal pathologies (endothelial dystrophies) that may be seen during the microscopic assessment of a donor cornea are described and linked to images of corneal explants (light microscopy) or patients’ corneas (specular microscopy) in the atlas.

Purpose To assess if polymegethism and pleomorphism were evident in corneal endothelium after medium-term rigid gas permeable (RGP) contact lens wear. Methods In a cross-sectional observational study over 12 years, single images of the central region of the corneal endothelium of one eye of 46 subjects were taken with a non-contact specular microscope, along with a measure of central corneal thickness (CCT). The images were printed onto A3-sized paper and 100 cells/image measured by planimetry. Results Subjects aged between 20 and 32 years, with an average cumulative RGP wear of 6.0 +/− 1.6 years (range 3–9 years) were assessed; 26 of the subjects were Caucasian and 20 were Asian. The mean CCT was 0.515 +/− 0.027 mm. The group cell area value was 401 +/− 42 sq micron to give an estimated endothelial cell density (ECD) of 2520 +/− 273 cells/sq mm. As compared to a historical database, most endothelia (37/46) showed some changes with the mean coefficient of variation on cell area (COV) being 36.7 +/− 8.0% and the percentage of 6-sided (HEX) being 51.8 +/− 8.8%. There were modest correlations between years of RGP wear and both COV (p = 0.009, r spearman = 0.424) and HEX (p = 0.025, r spearman = −0.291), but not for ECD or CCT. Conclusions Corneal endothelial polymegethism appears to be a commonplace consequence of RGP lens wear with the magnitude of the change being related to the cumulative duration of the lens wear.

PURPOSE To compare corneal endothelial cells following strabismus surgery in children. METHODS Thirty-four children, 18 boys and 16 girls (mean age, 8.21 years) who had undergone strabismus surgery were included in a prospective study from September 2007 to November 2008. Following non-contact specular microscopy (type Topcon SP 2000P), endothelial cell density (ECD), endothelial pleomorphism (hexagonality (H)) and area cells (polymegethism) were measured in a 3 x 6-cm area. Four groups were established according to the type of surgery: group 1 (n=8), isolated posterior fixation on the medial rectus; group 2 (n=18), posterior fixation with recession of the medial rectus; group 3 (n=19), recession of the lateral rectus muscle; group 4 (n=5), resection of the superior oblique muscle. RESULTS The mean preoperative values for ECD, hexagonality (H) and cell areas were 3413.8 cells/mm(2), 68.4 % and 11.62 % for 100 microm(2), 39.56 % for 200 microm(2), 36.66 % for 300 microm(2), 8.78 % for 400 microm(2), 1.59 % for 500 microm(2) and 1.79 % for 600 microm(2), with homogenous distribution. The mean postoperative values were 3391.9 cells/mm(2), 63.95 % (-6.16 %, statistically significant). Postoperatively, we found a nonsignificant increasing percentage for 200 and 300microm(2) and decreasing for 400 and 500 microm(2). Group 1 showed mean pre- and postoperative ECD at 3501.7 + or - 399.7 and 3499.7 + or - 243.28 cells/mm(2), respectively; H was + 0.43 %; group 2 showed 3498.3 + or - 182.8 and 3422.8 + or - 274.2 cells/mm(2), respectively; -13.66 % for H (p=0.039); group 3 had 3238 + or - 385.5 and 3366 + or - 334.4 cells/mm(2), respectively; -3.29 % for H; and group 4 had 3264 + or - 325.36 and 3328 + or - 124.42 cells/mm(2), respectively; -6.2 % for H. A decrease in H was found in group 2 (p

International audience; Background/aims. A computer program for the automatic estimation of morphometric parameters (cell density, pleomorphism, polymegethism) in alizarine red stained images is presented and evaluated. Methods. Images of corneal endothelium from 30 porcine eyes stained with alizarine red were acquired with an optical microscope and saved as grey-level digital images. Each image was at first pre-processed for luminosity correction and contrast enhancement. An artificial neural network was used to classify all pixels as cell contour or cell body pixels. The segmented cell contours were then used to obtain estimates of morphometric parameters. The central area was assessed and the mean area per cornea was 0.54±0.07 mm2. The whole system was implemented as a computer program using the Matlab® language. Estimated parameters were compared with the corresponding values derived from manual contour detection on the same images used for the automatic estimation. Results. On the 30 images of our data set, mean differences of automatic parameters vs. manual ones were -12±52 cells/mm2 (range -103 to +145) for density; 0.5±2.6 per cent (range -5.6 to +5.6) for pleomorphism; -0.7±1.9 per cent (range -4.1 to +2.8) for polymegethism. Conclusion. The evaluation of the automatic system on 30 images from porcine eyes confirmed its capability of reliably estimating morphometric parameters with respect to parameter values derived by manual analysis.

PURPOSE: To validate tissue quality parameters for donor corneas designated for emergency grafting to preserve the globe. METHODS: In a longitudinal cohort follow-up study, 151 emergency grafts in the Netherlands were studied. Grafts were performed with a pool of organ-cultured donor corneas designated for emergency grafting and prepared for immediate use with all safety tests performed. Assignation criteria were corneas with a small superficial stromal opacity but meeting all selection criteria for penetrating keratoplasty tissue and corneas without stromal opacity but an endothelial cell density from 1800 to 2300 cells per square millimeter or mild polymegethism or pleomorphism. Cox multivariate regression analysis, Kaplan-Meier survival, and log rank test were applied. RESULTS: All requests for corneal tissue were honored within 24 hours. Ninety-one patients showed a complete and 60 an imminent perforation. One hundred thirty-one penetrating grafts and 20 lamellar grafts have been used. The globes were saved in 140 of the 151 patients (92.7%). Globe preservation was not significantly related to the absence of penetrating keratoplasty quality of the donor endothelium, the type of grafting, the degree of vascularization in the host cornea, or diabetic disease in the recipient. The main risk factor for globe preservation was the presence of a systemic autoimmune disease in the recipient (P = 0.0021). CONCLUSIONS: A selected pool of donor corneas designated for emergency grafting, that does not interfere with the scheduled procedures, allows more efficient and safe use of donor tissue in case of an imminent perforation. Globe preservation rates justify the quality criteria for designation of this tissue.

Purpose: To compare the outcome measures from two different methods for estimating the coefficient of variation (COV) in cell area for human corneal endothelia. Methods: A single non-contact specular micrograph was obtained from the central region of the corneal endothelium of 100 healthy non-contact lens-wearing white European subjects, aged from 32 to 62 years. The captured image file was either assessed using a machine-based algorithm in which 21 cells were marked and their areas reported (designated as the TOPCON ‘centre-dot’ method), or by an overlay and semi-automated computer-based image analysis system of the entire image of around 200 cells (designated as the AUTO method). The average cell area values were used to calculate the endothelial cell density (ECD), while the COV was calculated from the standard deviation of the cell area measures. Results: The TOPCON and AUTO methods yielded comparable data for average cell area (395 vs 391 μm2) and estimated ECD (2566 vs 2575 cells mm−2) that were not statistically different (p ≥ 0.351), although there was a slight bias between the two methods. However, the mean COV values were very different at 43.5 and 31.4% (p

Specular microscopy can provide a non-invasive morphological analysis of the corneal endothelial cell layer from subjects enrolled in clinical trials. The analysis provides a measure of the endothelial cell physiological reserve from aging, ocular surgical procedures, pharmaceutical exposure, and general health of the corneal endothelium. The purpose of this review is to discuss normal and stressed endothelial cell morphology, the techniques for determining the morphology parameters, and clinical trial applications.

Background: Little has been published on the reliability of estimates of the coefficient of variation (COV) in cell area for human corneal endothelia. The present study compares two methods. Methods: A non-contact specular micrograph (Topcon SP-2000P) was obtained from the central region of the corneal endothelium of 20 healthy myopic white European subjects, aged from 32 to 53 years, half of whom were successful long-term soft contact lens wearers. The captured image file was either assessed using a machine-based algorithm, in which 25 cells in the middle of the image were marked and their areas reported (designated as ‘centre-dot’ method) or by a manual method, by which all the cells in the image were outlined on very high magnification prints of the endothelia and the cell areas measured by a manual digitiser in stream mode. The average cell area was used to calculate the endothelial cell density (ECD), while the COV was calculated from the standard deviation (SD) of the cell area measures. Results: Identical mean cell area values were found (392 µm2) with the two methods, a marginally higher ECD estimate (2,594 versus 2,569) with the centre-dot method (p = NS) but a much higher COV with the centre-dot method (43.8 versus 29.0 per cent). This highly statistically significant difference in COV (p

Purpose Endothelial assessment is crucial in the release of corneas for grafting. We retrospectively analysed the role of endothelial morphology parameters in predicting endothelial cell loss during organ culture. Methods Human donor corneas were cultured in minimal essential medium with 2% fetal calf serum and antibiotics. Initial endothelial morphology was assessed microscopically using score parameters polymegethism (POL), pleomorphism (PLE), granulation (GRA), vacuolization (VAC), segmentation of cell membranes (SEG), Descemet's folds (DF), trypan blue-positive cells (TBPC) and endothelial cell-free areas (ECFA). Some corneas were primarily rejected based on endothelial assessment. Endothelial cell density (ECD) was assessed at the beginning (I-ECD) and end of culture. Corneas were then placed in dehydration medium (as above + 5% dextran 500). In a subgroup, ECD was reassessed after dehydration. Endothelial cell loss during culture (ECL@Culture) and culture+dehydration (ECL-Culture&Dehydration) were calculated. Data were given as mean ± SD and analysed using multiple linear and logistic regression. Odds ratios (OR) and 95% confidence intervals (CI) were calculated. Result I-ECD was 2812 ± 360/mm2 (n = 2356). The decision to reject a cornea due to endothelial assessment was associated negatively with I-ECD (OR = 0.77/100 cells, CI 0.7-0.82) and positively with ECFA (OR = 2.7, CI 1.69-4.35), SEG (OR =1.3, CI 1.01-1.68) and donor age (OR = 1.26/decade, CI 1.33-1.41). ECL@Culture was 153 ± 201/mm2 (n = 1277), ECL@Culture&Dehydration was 169 ± 183/mm2 (n = 918). ECL@Culture was associated positively with donor age, I-ECD, GRA and TBPC, and negatively with PLE, and DF. ECL@Culture&Dehydration was associated positively with age, sex, initial ECD, POL, PLE, VAC and TBPC. Conclusion Morphological parameters displayed associations with the exclusion of corneas from culture and with endothelial cell loss. Appropriate parameter selection for screening purposes may help improve graft quality.

Purpose Corneal images acquired by in-vivo microscopy provide clinical information on the cornea endothelium health state. The reliable estimation of the 3 clinical morphometric parameters (endothelial cell density, pleomorphism, polymegethism) requires the accurate detection of cell contours in a large number of cells. Thus for the practical application of this analysis in clinical settings an automated method is needed. Methods The contours of cells are detected using a genetic algorithm. It randomly modifies individuals from the current population to produce the children for the next generations, which evolve toward an optimal solution. We start with a small set of vertexes forming regular hexagons, which in successive generations evolve into polygons with possibly different number and positions of vertexes. Each vertex is positioned by considering both its correspondence with the actual image (pixels intensity) and the regularity of the resulting polygons. The goal is to obtain a final population of vertexes forming polygons that best fit the cells contours in the actual image. Results 15 images were acquired with a specular endothelial microscope (SP-3000P, Topcon Co, Japan) in healthy and pathological subjects. Ground truth values for the 3 parameter were obtained from manually drawn cell contours. Differences between this manual estimation of the parameters and the automated one were always less than 8%. Conclusions These preliminary results show the ability of the proposed algorithm to adapt to different shapes and sizes of cells and to allow the reliable estimation of the morphometric parameters used in clinical practice.

Corneal images acquired by in-vivo microscopy provide clinical in- formation on the cornea endothelium health state. The reliable estimation of the clinical morphometric parameters requires the accurate detection of cell con- tours in a large number of cells. Thus for the practical application of this analy- sis in clinical settings an automated method is needed. We propose the automat- ic segmentation of corneal endothelial cells contour through an innovative technique based on a genetic algorithm, which combines information about the typical regularity of endothelial cells shape with the pixels intensity of the actu- al image. Ground truth values for the clinical parameters were obtained from manually drawn cell contours. Results show that an accurate automatic estima- tion is achieved: for each parameter, the mean difference between its manual estimation and the automated one is always less than 4%, and the maximum difference is always less than 7%. The normal human corneal endothelium is a single layer of uniformly sized cells with a predominantly hexagonal shape, but this regular tessellation is affected by age and pathologies (1). Thus, the analysis of the main morphometric parameters of corneal endothelium provides clinical information capable to describe the cornea health state. Namely, endothelial cell density (ECD), polymegethism (differences in cell size ex- pressed as fractional standard deviation of cell areas), and pleomorphism or hexago- nality coefficient (fraction of hexagonal cells over the total number of cells) are commonly used as parameters to quantitatively characterize the endothelial cells' condition. In order to make this analysis practical in clinical settings, a computerized method that fully automates the segmentation procedure would be needed. The fundamental problem with automated endothelial analysis is to correctly identify the cells' contour, a necessary prerequisite for the estimation of the clinical parameters (2). The automat- ic recognition of the cell boundaries is a challenging task because of the noise that make the contour difficult to be distinguished even by an expert, and the substantial differences between an image and the other as regards size and appearance of the

Purpose We provided the first report of an association between changes in corneal endothelial cells, retina, and choriocapillaris in a choroideremia family. Methods Four members of an Asian choroideremia family, comprising two affected patients and two carriers, were evaluated. All participants underwent complete eye examinations, including visual acuity (VA), slit-lamp examination, ophthalmoscopy, perimetry, and electrophysiology tests. In addition, images of corneal endothelium were captured with a noncontact specular microscope. Genomic DNA amplification and whole-genome cytogenic array analysis were used to confirm the diagnosis of choroideremia and determine the molecular basis of the phenotype. Results In the affected patients, funduscopy revealed characteristic features of RPE and chorioretinal atrophy. The slit-lamp biomicroscopy disclosed unexpected pigmented punctate lesions in the corneal endothelium in one of them. Surprisingly, specular microscopy detected decreased endothelial cell density (ECD) with features of pleomorphism and polymegethism. Genomic DNA analysis revealed large deletion (~4.5 mega base pairs) of the entire CHM gene and encompassed region. In carriers, funduscopy revealed stippling pigmentary change despite normal electrophysiological results. Specular microscopy also disclosed reduced ECD with features of pleomorphism and polymegethism. Conclusions To our knowledge, this is the first description of corneal changes in choroideremia patients. The loss of corneal ECD is conspicuous and is accompanied by pleomorphism and polymegethism in this family. The observed changes in corneal endothelium may be associated with larger encompassed regions of the CHM gene defect or dysfunction in the blood-aqueous barrier. It warrants further investigation and clarification of the pathophysiology and associations between retinal and corneal changes in choroideremia.

Endothelial cell function is essential to maintain corneal transparency, but unfortunately the regenerative capacity of the endothelium is limited. There are only a few reports describing the effect of age on morphologic appearance of corneal endothelial cells of dogs. Studies of normal corneal endothelial cells in humans and dogs have shown a decrease in endothelial cell density (ECD) and an increase in pleomorphism and polymegethism with advancing age. The purpose of this study was to investigate the effect of age on ECD and endothelial cell morphology in dogs. A total of 30 dogs were divided into three groups (10 dogs/group) based on age: group 1 (2-12 months old), group 2 (24-72 months old), and group 3 (84 months or older). Corneas were processed for light and scanning electron microscopy. Results showed only difference in cell density between group 1 and groups 2 and 3, showing an initial decrease in cell density as the animal matured. Whereas there was significantly greater variation in cell size within the dogs in group 3 than there was within the other two groups, suggesting that there was increased polymegethism and pleomorphism with advancing age.

Purpose: Low gas-permeable contact lens wear of polymethyl methacrylate or hydroxyethyl methacrylate material is known to cause morphologic abnormalities in the corneal endothelial cell layer. These tenses were widely prescribed and successfully worn until their use was actively discouraged in the late 1980s and early 1990s. This study was designed to investigate whether discontinuation of low gas-permeable contact lens wear leads to an improvement of corneal endothelial cell morphology. Methods: At the time of discontinuation and at least 5 years after discontinuation of low gas-permeable contact lens wear, noncontact specular photographs of the central corneal endothelium were made in 66 patients (14 male and 52 female, mean age 37.7 +/- 8.4, range 24.6-69.0). By computer analysis of endothelial photographs, parameters for polymegethism and pleomorphism were calculated, as well as cell density. Results: Mean follow-up time between photographs was 6.8 years (SD 1.1). Sixty-one patients were refitted with rigid high gas-permeable contact lenses or high-water-content soft lenses, and 5 patients switched to spectacle wear. A small but significant recovery of the corneal endothelial cell morphology was found for the mean coefficient of variation of cell area, from 37.5 to 35.7 (P = 0.022), and for the coefficient of variation of the number of sides, from 13.1 to 12.4 (P = 0.004). The mean percentage of hexagonal cells increased from 54.2 to 56.2 (P = 0.013). Although the corneal endothelial cell morphology improved significantly on cessation of LGP contact lens wear, the values did not return to levels observed in normal, noncontact lens wearing individuals. During follow-up, the mean endothelial cell density decreased significantly (P - 0.001) from 2994 to 2890 (a 3.5% cell loss in 6.8 years), which is similar to the known normal age-related cell loss of 0.6% per year in non-contact lens wearing healthy individuals. Conclusion: Endothelial polymegethism and pleomorphism caused by PMMA or HEMA contact lens wear is partly reversible

Purpose To evaluate the long-term endothelial cell changes in phakic eyes after implantation of a posterior chamber phakic intraocular lens to correct high ametropia. Setting Department of Ophthalmology, University Hospital of Vienna, Vienna, Austria. Methods Thirty-four eyes of 21 patients having implantation of an implantable contact lens (ICL, Staar Surgical) for high myopia or hyperopia were prospectively examined. The minimum follow-up was 2 to 4 years. Preoperative and serial postoperative specular microscopy (Noncon Robo SP 8000, Konan) was performed to evaluate the long-term endothelial cell changes. Results The mean preoperative endothelial cell density was 2854 cells/mm2. The mean endothelial cell loss from preoperatively was 1.8% at 3 months, 4.2% at 6 months, 5.5% at 12 months, 7.9% at 2 years (n = 34), 12.9% at 3 years (n = 13), and 12.3% at 4 years (n = 11). All other endothelial cell characteristics remained stable during the 4-year follow-up. Conclusions Continuous endothelial cell loss was observed after ICL implantation during a 4-year follow-up. There was rapid cell loss until 1 year postoperatively, after which the rate of loss was no longer statistically significant. The percentage of hexagonal cells (polymorphism) and the coefficient of variation (polymegethism) remained stable during the 4-year follow-up.

Purpose. To determine whether the eyes of high-Dk soft contact lens wearing subjects can be discriminated from non–contact lens wearing subjects. Methods. This study was a prospective masked assessment of 32 subjects, 16 of whom wore experimental high-Dk soft contact lenses and 16 of whom did not wear contact lenses. Subjects wore high-Dk lenses on a 30-night replacement schedule for an average of 9 months. Tear film characteristics, staining and vascularization of the cornea, conjunctival staining, and the presence of microcysts in the corneal epithelium were assessed using slitlamp microscopy. The endothelium was examined for polymegethism. Results. No differences were found between the two groups in any of the variables that were examined (p > 0.05) except that the high-Dk lens wearing group had about twofold more tear film debris and 2.5-fold more severe conjunctival staining (p Conclusions. Hypoxia-associated effects were not apparent in the eyes of subjects wearing experimental high-Dk soft contact lenses. Conjunctival staining can generally distinguish lens wearers from non–lens wearers and can be used to discriminate between high-Dk lens wearing subjects and non–lens wearing subjects.