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ABSTRACT: Maintenance of corneal transparency is crucial for vision and depends mainly on the endothelium, a non-proliferative monolayer of cells covering the inner part of the cornea. When endothelial cell density falls below a critical threshold, the barrier and "pump" functions of the endothelium are compromised which results in corneal oedema and loss of visual acuity. The conventional treatment for such severe disorder is corneal graft. Unfortunately, there is a worldwide shortage of donor corneas, necessitating amelioration of tissue survival and storage after harvesting. Recently it was reported that the ROCK inhibitor Y-27632 promotes adhesion, inhibits apoptosis, increases the number of proliferating monkey corneal endothelial cells in vitro and enhance corneal endothelial wound healing both in vitro and in vivo in animal models. Using organ culture human cornea (N = 34), the effect of ROCK inhibitor was evaluated in vitro and ex vivo. Toxicity, corneal endothelial cell density, cell proliferation, apoptosis, cell morphometry, adhesion and wound healing process were evaluated by live/dead assay standard cell counting method, EdU labelling, Ki67, Caspase3, Zo-1 and Actin immunostaining. We demonstrated for the first time in human corneal endothelial cells ex vivo and in vitro, that ROCK inhibitor did not induce any toxicity effect and did not alter cell viability. ROCK inhibitor treatment did not induce human corneal endothelial cells proliferation. However, ROCK inhibitor significantly enhanced adhesion and wound healing. The present study shows that the selective ROCK inhibitor Y-27632 has no effect on human corneal endothelial cells proliferative capacities, but alters cellular behaviours. It induces changes in cell shape, increases cell adhesion and enhances wound healing ex vivo and in vitro. Its absence of toxicity, as demonstrated herein, is relevant for its use in human therapy.
PLoS ONE 01/2013; 8(4):e62095. · 4.09 Impact Factor
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ABSTRACT: The control of corneal transparency depends on the integrity of its endothelial monolayer, which is considered nonregenerative in adult humans. In pathological situations, endothelial cell (EC) loss, not offset by mitosis, can lead to irreversible corneal edema and blindness. However, the hypothesis of a slow, clinically insufficient regeneration starting from the corneal periphery remains debatable. The authors have re-evaluated the microanatomy of the endothelium in order to identify structures likely to support this homeostasis model. Whole endothelia of 88 human corneas (not stored, and stored in organ culture) with mean donor age of 80 ± 12 years were analyzed using an original flat-mounting technique. In 61% of corneas, cells located at the extreme periphery (last 200 μm of the endothelium) were organized in small clusters with two to three cell layers around Hassall-Henle bodies. In 68% of corneas, peripheral ECs formed centripetal rows 830 ± 295 μm long, with Descemet membrane furrows visible by scanning electron microscopy. EC density was significantly higher in zones with cell rows. When immunostained, ECs in the extreme periphery exhibited lesser differentiation (ZO-1, Actin, Na/K ATPase, CoxIV) than ECs in the center of the cornea but preferentially expressed stem cell markers (Nestin, Telomerase, and occasionally breast cancer resistance protein) and, in rare cases, the proliferation marker Ki67. Stored corneas had fewer cell clusters but more Ki67-positive ECs. We identified a novel anatomic organization in the periphery of the human corneal endothelium, suggesting a continuous slow centripetal migration, throughout life, of ECs from specific niches. STEM CELLS2012;30:2523-2534.
Stem Cells 09/2012; 30(11):2523-34. · 7.78 Impact Factor
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Pierre-Loïc Cornut,
Gilles Thuret,
Catherine Creuzot-Garcher,
Max Maurin,
Andre Pechinot,
Alain Bron, Philippe Gain,
Anne Carricajo,
Philippe Denis,
Jean-Paul Romanet,
Francois Vandenesch,
Christophe Chiquet
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ABSTRACT: To correlate the initial ocular presentation with bacterial identification in 100 patients with acute postcataract endophthalmitis.
This was a prospective multicenter study. Demographic data, medical history, and the initial eye examination data were recorded on a standardized form. The relationship between bacterial identification and clinical factors at baseline was studied using univariate and multivariate analyses.
One hundred patients were admitted to the hospital with a median delay of 6 days after cataract surgery. The main symptoms were loss of vision (94.9%) and pain (75.5%). Major clinical signs were hypopyon (72%), pupillary fibrin membrane (77.5%), and loss of fundus visibility (90%). Baseline factors significantly associated with microbiologic identification were as follows: diabetes mellitus, a shorter delay of onset, initial visual acuity limited to light perception, higher intraocular pressure, chemosis, pupillary fibrin membrane, loss of the red reflex, and reduced fundus visibility. As compared with other bacteria, the identification of Streptococcus species (n = 19) was more frequently associated with male gender, diabetes mellitus, initial visual acuity limited to light perception, and pain. The Staphylococcus aureus and Staphylococcus lugdunensis group (n = 14) differed from other coagulase-negative Staphylococcus groups (n = 33) in that those patients had greater hypopyon height.
The baseline features of acute endophthalmitis after cataract surgery in the era of phacoemulsification are similar to those reported in the Endophthalmitis Vitrectomy Study 15 years ago and differ according to the bacterial species. The association between the clinical signs and the microbiologic identification suggests that initial characteristics other than visual acuity may be useful in identifying patients presumed to be infected with a virulent species.
Retina (Philadelphia, Pa.) 03/2012; 32(3):549-57. · 2.93 Impact Factor
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Pierre-Loïc Cornut,
El Bichara Youssef,
Alain Bron,
Gilles Thuret, Philippe Gain,
Carole Burillon,
Jean-Paul Romanet,
François Vandenesch,
Max Maurin,
Catherine Creuzot-Garcher,
Christophe Chiquet
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ABSTRACT: Purpose: Study the clinical and microbiological characteristics and the prognostic factors of post-traumatic endophthalmitis. Methods: Seventeen eyes were included between 2004 and 2010, with clinical and microbiological data collected prospectively. Conventional cultures and panbacterial PCR were performed on aqueous and vitreous samples. Results: Clinical signs of endophthalmitis were observed soon after trauma (1.5 ± 2.5 days). Laceration with an intraocular foreign body (IOFB) was noted in 53% of the patients. At admission, all patients had aqueous humour (71%) and/or vitreous (53%) samples. Fifteen patients (88%) underwent a pars plana vitrectomy. Bacteria were identified in 77% of the cases: Staphylococcus epidermidis (n = 5), Streptococcus (n = 4), Bacillus (n = 2), Pseudomonas stuzeri (n = 1), and Streptococcus salivarius and Gemella haemolysans (multibacterial infection, n = 1). Progression toward phthisis was observed in 35% of the cases; 41% of the patients recuperated visual acuity (VA) ≥20/40. A good final visual prognosis (≥20/40) was significantly associated with initial VA better than light perception (0% versus 70%, p = 0.01) and absence of pupillary fibrin membrane (80% versus 20%, p = 0.05). There was no correlation between visual prognosis and age, the type of laceration (corneal or scleral) or presence of an IOFB. We found a statistical trend toward an association between bacterial virulence and poor final VA. Conclusion: This series showed that better final VA outcomes were associated with initial VA better than light perception, S. epidermidis or culture-negative cases and absence of retinal detachment during the clinical course.
Acta ophthalmologica 02/2012; · 2.44 Impact Factor
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ABSTRACT: The assessment of blood lipids is very frequent in clinical research as it is assumed to reflect the lipid composition of peripheral tissues. Even well accepted such relationships have never been clearly established. This is particularly true in ophthalmology where the use of blood lipids has become very common following recent data linking lipid intake to ocular health and disease. In the present study, we wanted to determine in humans whether a lipidomic approach based on red blood cells could reveal associations between circulating and tissue lipid profiles. To check if the analytical sensitivity may be of importance in such analyses, we have used a double approach for lipidomics.
Red blood cells, retinas and optic nerves were collected from 9 human donors. The lipidomic analyses on tissues consisted in gas chromatography and liquid chromatography coupled to an electrospray ionization source-mass spectrometer (LC-ESI-MS). Gas chromatography did not reveal any relevant association between circulating and ocular fatty acids except for arachidonic acid whose circulating amounts were positively associated with its levels in the retina and in the optic nerve. In contrast, several significant associations emerged from LC-ESI-MS analyses. Particularly, lipid entities in red blood cells were positively or negatively associated with representative pools of retinal docosahexaenoic acid (DHA), retinal very-long chain polyunsaturated fatty acids (VLC-PUFA) or optic nerve plasmalogens.
LC-ESI-MS is more appropriate than gas chromatography for lipidomics on red blood cells, and further extrapolation to ocular lipids. The several individual lipid species we have identified are good candidates to represent circulating biomarkers of ocular lipids. However, further investigation is needed before considering them as indexes of disease risk and before using them in clinical studies on optic nerve neuropathies or retinal diseases displaying photoreceptors degeneration.
PLoS ONE 01/2012; 7(4):e35102. · 4.09 Impact Factor
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ABSTRACT: The determination of endothelial cell density (ECD) is a crucial activity in eye banks for the assessment of corneal tissue quality. These cells are responsible for corneal transparency, and ECD correlates with graft survival. ECD is mainly assessed with a manual "naked-eye" procedure under a transmitted light microscope in Europe and using a specular microscope in the United States. Interbank and intrabank variability has been previously demonstrated. In order to facilitate training and continuing education of technicians and reliability assessment of eye banks' ECD determination, we use micro-optics technologies to fabricate test mosaics that exactly reproduce the image of human corneal endothelium. The description of the fabrication process is detailed, and comparisons are made between amplitude and phase mosaics.
Optics Letters 01/2012; 37(1):22-4. · 3.40 Impact Factor
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ABSTRACT: To present an experimental method for determining the viable cell pool of corneal endothelia and its application to assessing predissected endothelial grafts.
The endothelial cell density (ECD) of five pairs of human organ cultured corneas was determined using a standard counting method with a calibrated image analysis system. A thin posterior graft (30-50 μm) was manually predissected from a cornea chosen at random. Predissected and control corneas were shipped to the remote center, where standard ECD determination was repeated and was immediately followed by a triple Hoechst/ethidium/calcein labeling coupled with image analysis of the whole graft surface. Numeration of nuclei (H+), dead cells (E+), and total area covered by viable cells (C+) allowed the calculation of viable ECD corresponding to the cell density that the cornea may have after redistribution of viable cells over the whole Descemet surface.
The median (range) viable ECD was lower than the standard ECD determined immediately earlier in predissected and control corneas: 1628 (1138-2379) and 2065 (1492-2876) cells/mm(2) (P = 0.043), corresponding to -20% (-1%-38%) and -12% (-3%-26%), respectively (P = 0.08).
Standard counting by eye banks overestimates the actual pool of viable endothelial cells. This may be the main explanation for the initially rapid decrease in ECD universally described in patients after all types of keratoplasty. Early low postoperative ECD may indicate that surgeons graft fewer living cells than the eye banks' ECD let suppose, rather than a massive pre- and postoperative cell death. The novel concept of viable ECD can be useful for assessing all types of corneal processing.
Investigative ophthalmology & visual science 06/2011; 52(8):6018-25. · 3.43 Impact Factor
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ABSTRACT: To compare the efficacy of the diabetic retinopathy (DR) screening with digital camera by endocrinologists with that by specialist and resident ophthalmologists in terms of sensitivity, specificity, and level of "loss of chance."
In a cross-sectional study, 500 adult diabetic patients (1,000 eyes) underwent three-field retinal photography with a digital fundus camera following pupillary dilatation. Five endocrinologists and two ophthalmology residents underwent 40 h of training on screening and grading of DR and detection of associated retinal findings. A κ test compared the accuracy of endocrinologist and ophthalmology resident screening with that performed by experienced ophthalmologists. Screening efficiency of endocrinologists was evaluated in terms of "loss of chance," i.e., missed diagnoses that required ophthalmologist referrals.
The mean weighted κ of DR screening performed by endocronologists was similar to that of ophthalmology residents (0.65 vs. 0.73). Out of 456 DR eyes, both endocrinologists and ophthalmology residents misdiagnosed only stage 1 DR (36 and 14, respectively), which did not require ophthalmologist referral. There were no significant differences between endocrinologists and ophthalmology residents in terms of diabetic maculopathy and incidental findings except for papillary cupping and choroidal lesions, which were not the main purpose of the study or of the training.
The endocrinologist with specific training for DR detection using a three-field digital fundus camera with pupillary dilatation can perform a reliable DR screening without any loss of chance for the patients when compared with identical evaluation performed by experienced ophthalmologists.
Diabetes care 01/2011; 34(3):580-5. · 8.09 Impact Factor
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ABSTRACT: En face observation of corneal endothelial cells (ECs) using flat-mounted whole corneas is theoretically much more informative than observation of cross-sections that show only a few cells. Nevertheless, it is not widespread for immunolocalization (IL) of proteins, probably because the endothelium, a superficial monolayer, behaves neither like a tissue in immunohistochemistry (IHC) nor like a cell culture in immunocytochemistry (ICC). In our study we optimized IL for ECs of flat-mounted human corneas to study the expression of cell cycle-related proteins.
We systematically screened 15 fixation and five antigen retrieval (AR) methods on 118 human fresh or stored corneas (organ culture at 31 °C), followed by conventional immunofluorescence labeling. First, in an attempt to define a universal protocol, we selected combinations able to correctly localize four proteins that are perfectly defined in ECs (zonula occludens-1 [ZO-1] and actin) or ubiquitous (heterogeneous nuclear ribonucleoprotein L [hnRNP L] and histone H3). Second, we screened protocols adapted to the revelation of 9 cell cycle proteins: Ki67, proliferating cell nuclear antigen (PCNA), minichromosome maintenance protein 2 (MCM2), cyclin D1, cyclin E, cyclin A, p16(Ink4a), p21(Cip1) and p27(Kip1). Primary antibody controls (positive controls) were performed on both epithelial cells of the same, simultaneously-stained whole corneas, and by ICC on human ECs in in vitro non-confluent cultures. Both controls are known to contain proliferating cells. IL efficiency was evaluated by two observers in a masked fashion. Correct localization at optical microscopy level in ECs was define as clear labeling with no background, homogeneous staining, agreement with previous works on ECs and/or protein functions, as well as a meaningful IL in proliferating cells of both controls.
The common fixation with 4% formaldehyde (gold standard for IHC) failed to reveal 12 of the 13 proteins. In contrast, they were all revealed using either 0.5% formaldehyde at room temperature (RT) during 30 min alone or followed by AR with sodium dodecyl sulfate or trypsin, or pure methanol for 30 min at RT. Individual optimization was nevertheless often required to optimize the labeling. Ki67 was absent in both fresh and stored corneas, whereas PCNA was found in the nucleus, and MCM2 in the cytoplasm, of all ECs. Cyclin D1 was found in the cytoplasm in a paranuclear pattern much more visible after corneal storage. Cyclin E and cyclin A were respectively nuclear and cytoplasmic, unmodified by storage. P21 was not found in ECs with three different antibodies. P16 and p27 were exclusively nuclear, unmodified by storage.
IL in ECs of flat-mounted whole human corneas requires a specific sample preparation, especially to avoid overfixation with aldehydes that probably easily masks epitopes. En face observation allows easy analysis of labeling pattern within the endothelial layer and clear subcellular localization, neither of which had previously been described for PCNA, MCM2, or cyclin D1.
Molecular vision 01/2011; 17:3494-511. · 2.20 Impact Factor
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ABSTRACT: Purpose: The outer limiting membrane (OLM) is considered to play a role in maintaining the structure of the retina through mechanical strength. However, the observation of junction proteins located at the OLM and its barrier permeability properties may suggest that the OLM may be part of the retinal barrier. Material and methods: Normal and diabetic rat, monkey, and human retinas were used to analyze junction proteins at the OLM. Proteome analyses were performed using immunohistochemistry on sections and flat-mounted retinas and western blotting on protein extracts obtained from laser microdissection of the photoreceptor layers. Semi-thin and ultrastructure analyses were also reported. Results: In the rat retina, in the subapical region zonula occludens-1 (ZO-1), junction adhesion molecule (JAM), an atypical protein kinase C, is present and the OLM shows dense labeling of occludin, JAM, and ZO-1. The presence of occludin has been confirmed using western blot analysis of the microdissected OLM region. In diabetic rats, occludin expression is decreased and glial cells junctions are dissociated. In the monkey retina, occludin, JAM, and ZO-1 are also found in the OLM. Junction proteins have a specific distribution around cone photoreceptors and Müller glia. Ultrastructural analyses suggest that structures like tight junctions may exist between retinal glial Müller cells and photoreceptors. Conclusions: In the OLM, heterotypic junctions contain proteins from both adherent and tight junctions. Their structure suggests that tight junctions may exist in the OLM. Occludin is present in the OLM of the rat and monkey retina and it is decreased in diabetes. The OLM should be considered as part of the retinal barrier that can be disrupted in pathological conditions contributing to fluid accumulation in the macula.
Clinical Ophthalmology 01/2010;
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ABSTRACT: To describe an innovative device that allows gene electrotransfer to human corneal endothelial cells (EC) during storage in organ culture.
Customized electrodes without endothelial contact were developed. Two plasmids containing the cytomegalovirus promoter and reporter genes [enhanced green fluorescent protein (eGFP) or beta-galactosidase (beta-gal)] were electroporated in 2 series of human corneas with eight 1-Hz 100-ms pulses of 125 mA square current. Controls were exposed to naked DNA without electric pulses. eGFP-transduced corneas were used to determine the transgene expression kinetics, whereas beta-gal measured transfection efficiency using image analysis tools. Overall, endothelial toxicity was determined by: (1) cytotoxicity tests using triple staining with Hoechst 33342, ethidium homodimer III, and calcein AM, 3 h and 3 and 14 days after electroporation on the series of 15 eGFP-transfected paired corneas; (2) anti-ZO-1 staining to assess tight junctions' integrity.
All electroporated corneas carried transfected ECs, whereas the controls carried none. eGFP expression was observed 3 h after electrotransfer, and was then present from days 1 to 28. Transfection efficiency determined on 63 corneas transfected with beta-gal ranged from 0.1 to 54% of the transfected ECs (mean +/- SD: 7 +/- 11%, median: 2.9%) with significant reproducibility for paired corneas from the same donor. Electroporation produced low early EC death. Anti ZO-1 staining revealed no dramatic change in EC mosaic continuity, neither 1 and 3 nor 28 days after electroporation.
Gene electrotransfer to the endothelium of organ-cultured human corneas with custom-designed electrodes allows rapid and easy EC transfection. However, further optimization is required to ensure reproducible results.
Ophthalmic Research 10/2009; 43(1):43-55. · 1.56 Impact Factor
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ABSTRACT: To determine the factors influencing endothelial morphometry by using image analysis of corneas stored in organ culture to determine the coefficient of variation (CV) in cell area and percentage of hexagonal cells.
The endothelia of 505 of the 559 corneas consecutively stored at the eye bank were routinely analyzed with Sambacornea image-analysis software (ver. 1.2.10; Tribvn, Châtillon, France) on three large-field images of 750 x 1000 microm, obtained after osmotic dilation of the intercellular spaces with 0.9% sodium chloride. Analysis was performed on at least 300 cells. The quality of the three-image set was graded poor, average, or good by an independent observer. The studied parameters were donor age and sex, lens status, storage time, and intrinsic quality of captured images. Statistics were analyzed by nonparametric tests.
Image analysis was possible for 504 of the 505 assessed corneas. Donor age correlated significantly with endothelial cell density (ECD; r = -0.343), CV (r = 0.221), and hexagonality (r = -0.314; P < 0.001 for the three). Image quality significantly influenced these three parameters. ECD and hexagonality decreased parallel to image quality, whereas the CV increased. In the 258 corneas assessed twice (on average, at day [D] +4, then D +14) ECD, CV, and hexagonality decreased during storage.
Despite the sometimes mediocre quality of the transmitted light microscopy images, endothelial parameters supplied by the analyzer were clinically reliable, since variations similar to those long known in specular microscopy were found. Endothelial morphometry (CV and hexagonality) is likely to provide further information on the endothelial function of the graft tissue, perhaps particularly for grafts of borderline ECD, close to the discard threshold.
Investigative ophthalmology & visual science 10/2009; 51(3):1356-64. · 3.43 Impact Factor
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ABSTRACT: Accumulation of lipids within Bruch's membrane (BrM) and between BrM and retinal pigment epithelium (RPE) accounts for one of the biological changes associated with normal aging and may contribute to the development of age-related maculopathies. The origin of these lipids is still being actively investigated. The relative contribution of plasma lipids and lipids coming from the neural retina remains a matter of controversy. Low-density lipoproteins (LDLs) have been reported to significantly participate in the retina's lipid supply, after active remodeling within RPE. Meanwhile, RPE expresses the enzymatic machinery for synthesizing lipoprotein-like particles. The objective of this study was to establish associations between the fatty acid profile of the ocular structures and adipose tissue as a surrogate for the subjects' past dietary intake. Lipids and fatty acids were analyzed from the neural retina, retinal pigment epithelium (RPE)/choroid, the lacrimal gland, and adipose tissue, collected from 27 human donors (19 women, eight men) aged 59-95 years. DHA concentrations in the neural retina were positively associated with the concentrations in cholesteryl esters (CEs) from RPE/choroid and negatively associated with DHA concentrations in phospholipids (PLs) from RPE/choroid. DHA in orbital fat was positively associated with DHA in the lacrimal gland. No significant association was observed in the other ocular structures. Linoleic acid in orbital fat was positively associated with linoleic acid in the lacrimal gland, followed by the neural retina and CEs from RPE/choroid; it was slightly correlated with PLs from RPE/choroid. Other fatty acids that originate exclusively from the diet such as trans fatty acids were detected in orbital fat, the lacrimal gland, PLs, and CEs from RPE/choroid. DHA in the neural retina was poorly associated with its dietary intake, contrary to other fatty acids such as linoleic acid. Within this context, CEs may be important carriers of fatty acids entering the retina. Although epidemiological studies have reported the benefit of DHA in the prevention of age-related macular degeneration, the leading cause of blindness in Western countries, the relevance of supplementing patients with DHA is questioned.
Experimental Eye Research 09/2008; 87(6):521-8. · 3.26 Impact Factor
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ABSTRACT: Dextran T500, routinely used as a deswelling supplement in organ culture (OC), has been suspected of being toxic to corneal endothelial cells (ECs). This study was conducted to evaluate the innovative use of poloxamers compared with dextran for deswelling OC corneas.
Five poloxamers (P124, P188, P237, P338, and P407) were dissolved respectively in a standard OC medium to reach 350 mOsmol/kg. In vitro cytotoxicity of these media was tested by MTT assay on human corneal epithelial and endothelial cell lines and on primary human corneal fibroblasts. Paired human corneas stored in OC for at least 21 days were assigned for 48 hours to a poloxamer medium or to a standard deswelling medium containing 5% dextran T500. Corneal EC density, morphometry, visualization, mortality, stromal thickness, transparency, and folding were evaluated before and after deswelling. Corneas were finally cut into three parts for histologic and ultrastructural observation.
Besides similar corneal transparency improvement and thickness deswelling, poloxamers (except P124) reduced EC loss and facilitated endothelial visualization, but improved stromal folding less than dextran. The similar ultrastructures observed in the two groups were epithelial shedding, normal collagen fiber diameter and organization, uptake of deswelling agents by ECs, vacuolization but normal organelles in ECs and keratocytes, and endothelial surface modifications.
P188, P237, P338, and P407 performed similarly in preserving ECs, improving EC visualization, deswelling corneal stroma and inducing moderate injuries to corneal ultrastructure. They appear superior to dextran for corneal deswelling in OC.
Investigative Ophthalmology & Visual Science 03/2008; 49(2):550-9. · 3.60 Impact Factor
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ABSTRACT: To compare two semiautomated methods of evaluating endothelial cells of eye bank corneas.
Using a commercially available semiautomatic endothelial analyzer, seven observers determined the endothelial cell density (ECD), coefficient of variation (CV) of cell area, and the percentage of hexagonal cells (hexagonality) of the light microscopic images of the endothelium of 30 organ-cultured corneas. The image quality was graded as good, average, and poor. Border (contour detection and manual retouch) and center (indicating cell centers) methods for identifying endothelial cells were compared. The interobserver variability in ECD determination (indicating reproducibility) and morphometry was statistically analyzed by using the two methods. The importance of accurate pointing of cell centers was assessed by counting on 10 standard photolithographic mosaics and noting the time taken.
There was no significant difference in the interobserver variability or between ECDs obtained by the border and center methods. Decrease in image quality had a similar influence on both methods. Although measurement of hexagonality was acceptable by both methods, the CV was reliable only with the border method, with a significant underestimation by the center
However, an accurate indication of cell center slightly improved the CV estimation.
Although both the border and center methods of semiautomatic evaluation of eye bank corneas measure similar ECD with a similar reproducibility, only the border method gives a reliable morphometry.
Investigative Ophthalmology & Visual Science 08/2007; 48(7):3077-82. · 3.60 Impact Factor
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ABSTRACT: To investigate the reproducibility of endothelial assessment of organ-cultured corneas with the computer-assisted Sambacornea analyzer in comparison with manual
methods. Seven observers of two eye banks determined the endothelial cell density (ECD) of 30 corneas through a grid overlay placed on endothelial photographs using two manual modes, unaided (naked-eye) and pointing (point-out). ECD was measured with the analyzer, first in automated mode, where analysis was completely machine determined, and then in touched-up mode, where the observer selected the analysis zone and corrected poorly drawn cell borders. Interobserver variability of ECD for the different methods was compared. Reproducibility of morphometry parameters was determined for the touched-up mode.
Interobserver variability was +/-19.2% (95% confidence interval [CI], 13.0-25.4) and +/-17.6% (95% CI, 11.9-23.3) for the naked-eye and point-out mode, respectively, whereas the touched-up mode gave the least variability of +/-9.6% (95% CI, 6.5-12.7), confirmed by the highest intraclass correlation coefficient of 0.95 (95% CI, 0.91-0.97). Interobserver variability increased with worsening image quality. Manual modes underestimated ECD (naked-eye by a mean 10.7% [SD, 2.9%]; point-out by a mean 6.9% [SD, 2.3%]), whereas the automated mode overestimated ECD by a mean 14.7% (SD, 24.3%). Reproducibility of morphometric parameters by the touched-up mode was acceptable but was influenced by endothelial pleomorphism.
Manual counting shows systematic underestimation of ECD with high interobserver variability. The analyzer in automated mode overestimates ECD and is absolutely unreliable. Detection of cell contours by the specific algorithm, combined with manual correction by a skilled technician, appears to be the most reliable method of ECD and morphometry determination.
Investigative Ophthalmology & Visual Science 06/2007; 48(5):2062-7. · 3.60 Impact Factor
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VISAPP 2007: Proceedings of the Second International Conference on Computer Vision Theory and Applications, Barcelona, Spain, March 8-11, 2007 - Volume 2; 01/2007
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ABSTRACT: To develop standard microscopic hexagonal mosaics mimicking the human corneal endothelium for quality control of endothelial cell density (ECD) measurement and verification of cell counting strategy by light microscopy in eye banks using organ culture.
A standard slide, the Keratotest, was developed with 10 laser-engraved mosaics and different predetermined "cell" densities representing the range of ECDs observed routinely. Horizontal and vertical micrometric scales were etched adjacently to each mosaic, and a standard microscopy resolution test pattern was included. The Keratotest was applied to assess the reliability of a computer-assisted analyzer developed for corneal endothelial evaluation based on light microscopy images.
The Keratotest consisted of 10 microlithographic homogeneous mosaics of 1-mm2 printed area and 1.2-microm cell boundary thickness. The micrometric scale associated with each mosaic aided in simultaneous verification of microscope calibration, and the test pattern aided in checking the microscope resolution. The design was unalterable and reproducible, and the glass slide incorporated in a carbon fiber support ensured easy handling and safe transport. Evaluation of the Keratotest mosaics by the computer-assisted analyzer found a high level of agreement (error margin between +0.12 and -0.46%) with the laser-engraved cell density.
This prototype device enabled assessment of reliability of ECD measurement in eye banks. It also allowed verification of the calibration and resolution of light microscopes. Periodic validation of counting procedure in eye banks with mosaics of known "cell" densities should be useful for standardization of donor corneal tissue quality control.
Investigative Ophthalmology & Visual Science 11/2006; 47(10):4373-7. · 3.60 Impact Factor
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ABSTRACT: Transmission of donor malignancy to the recipient could be one of the most disastrous complications of corneal grafting. Because of the scarcity of donor tissue and the lack of sufficient scientific evidence, the harvest of donor tissues from deaths due to systemic malignancy is permitted. This study was conducted to investigate the possible transmission of donor metastatic disease via corneal tissue preserved in organ culture (OC) conditions.
The viability of four frequent human cancer cell lines (lung, breast, skin, and colon) was studied in OC. Various inoculums of cancer cells labeled with the membrane marker PKH67 were seeded on donor corneas and preserved in OC, followed by cell-tracking studies, histopathology, and immunohistochemistry. HLA matching of the dissected Descemet's membrane (DM) of preserved corneas was conducted, to demonstrate cell adherence. Primary cell culture was performed to confirm the viability of adherent tumor cells.
Viability tests showed a poor but persistent survival of cancer cells after 2 weeks in OC. Cell tracking, histopathology, and immunohistochemistry demonstrated cancer cell adherence to donor endothelium. HLA typing of the DM of preserved corneas revealed the presence of cancer cell alleles. Primary culture of the DM showed cell proliferation that was identical with the original cancer cell line, according to HLA studies.
The findings demonstrate that under laboratory conditions, metastatic cancer cells adhere to donor corneal tissue, survive, and retain proliferative capacity during storage in OC. Cell lines differ in their viability potential, as well as the pattern of adherence to donor endothelium. Further in vivo experimentation in laboratory animals is need to determine the safety of such harvests.
Investigative Ophthalmology & Visual Science 05/2006; 47(4):1339-47. · 3.60 Impact Factor
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ABSTRACT: Retinal detachment surgery is frequently associated with significant postoperative pain and emesis in adults. In this randomized, double-blind, controlled study we sought to demonstrate that 1% ropivacaine peribulbar (PB) block in conjunction with general anesthesia (GA) improves operative conditions and postoperative analgesia compared with GA combined with subcutaneous normal saline injection into the inferior eyelid. Thirty-one patients were included in each group. Anesthesia was performed with target-controlled infusion propofol and continuous remifentanil infusion adjusted to maintain bispectral index values between 40 and 50. Postoperative analgesia included fixed-dose IV infusion of propacetamol and IV injection of nefopam via a patient-controlled analgesia device. Tramadol was infused IV as rescue medication. Demographic data were comparable between the groups and bispectral index values were maintained at the objective target. In the PB group, fewer patients presented an oculocardiac reflex (6 versus 17; P < 0.01); bleeding interfering with the surgical field was reduced (1 versus 11 patients; P < 0.01); mean time to first nefopam request was longer (148 +/- 99 versus 46 +/- 58 min; P < 0.01); mean nefopam consumption was diminished during the first 6 h after tracheal extubation (18.9 +/- 13.9 versus 28.5 +/- 14.7 mg; P < 0.05); immediate postoperative pain scores were lower; and fewer patients required rescue medication (5 versus 23; P < 0.01). The two groups were similar with respect to the incidence of postoperative nausea and vomiting. Overall, PB block combined with GA improved operating conditions and postoperative analgesia in retinal detachment surgery.
Anesthesia and analgesia 04/2006; 102(4):1082-7. · 3.08 Impact Factor
