M. Zhao

Use of Poloxamers for Deswelling of Organ-Cultured Corneas

PURPOSE 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Endothelial morphometry by image analysis of corneas organ cultured at 31 degrees C.

PURPOSE 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Assessment of the human corneal endothelium: in vivo Topcon SP2000P specular microscope versus ex vivo sambacornea eye bank analyser

Comparison between assessment of donor tissue in eye banks and specular microscopy in the recipient is important to quantify the post-keratoplasty cell loss dynamics. Our aim was to determine the agreement between the in vivo non-contact specular microscope Topcon SP2000P and the computer-assisted eye bank endothelial analyser Sambacornea. We enrolled 51 future recipients of penetrating keratoplasty, and determined the endothelial cell density (ECD) and morphometry firstly in vivo with Topcon and then ex vivo with Sambacornea on the excised cornea stained with Alizarin Red. Specular microscopy was found to underestimate the ECD by 11%, (95% CI 6 to 15), whereas morphometric parameters did not differ. Endothelial cell loss after penetrating keratoplasty is commonly evaluated by non-contact specular microscopy. A comparison of tissue assessment in eye banks and that by specular microscopy in the recipient is important to quantify the postoperative cell loss dynamics. In Europe, where organ culture is common, preoperative endothelial cell density (ECD) is assessed either by manual counting by observation through a microscope reticule or by using computer-assisted analysers.1– …

Agreement between two non-contact specular microscopes: Topcon SP2000P versus Rhine-Tec

Endothelial cell density (ECD) assessment with the noncontact Topcon SP2000P specular microscope is known to be reliable when the automated mode is followed by manual corrections (touched-up mode). We compared its agreement with Rhine-Tec, a new noncontact specular microscope, in 270 eyes of 160 patients, by comparing the ECD measured in the automated and touched-up modes with that of Topcon touched-up. Good agreement existed between either touched-up modes with a mean difference of only 2 cells/mm2 95% CI (−27; 23) whereas agreement with the Rhine-Tec automated mode was poor with an overestimation by a mean of 226 cells/mm2 95% CI (172; 281). The Topcon SP2000P non-contact specular microscope (Topcon, Tokyo, Japan) is widely used to measure corneal endothelial cell density (ECD). It uses a cell contour recognition algorithm based on contrast differences, and ECD derived from an automated delineation of cell boundaries with manual correction of inaccurately drawn cells (“touched-up” mode) has been validated.1,2 A new commercially available non-contact specular microscope Rhine-Tec (Rhine-Tec, Krefeld, Germany) determines ECD by a cell-centre method …

020 Évaluation expérimentale de la viabilité endothéliale cornéenne par triple marquage Hoechst 33342, éthidium homodimère et calcéine-AM (HEC) : améliorations techniques par microscopie 3D

Introduction Une quantification fiable de la viabilite endotheliale est primordiale dans l’evaluation experimentale de toute nouvelle procedure (nouveaux milieux ou procedes de conservation) susceptible de modifier l’endothelium corneen. La mesure de la densite cellulaire endotheliale (DCE) seule ne renseigne pas sur la viabilite et la coloration vitale au bleu trypan sous-estime largement la mortalite. Le double marquage par calceine-AM (C) et ethidium homodimere (E) permet classiquement de reperer (1) les cellules viables dont les esterases non specifiques transforment la calceine en un substrat vert fluorescent et (2) les cellules mortes dont la membrane permeable laisse entrer l’ethidium qui colore les noyaux en rouge. But : presenter des ameliorations de cette coloration combinee au marquage nucleaire par Hoechst 33342 (H) par l’utilisation d’un microscope fluorescent 3D applique a l’evaluation experimentale de l’endothelium de cornees humaines entieres. Materiels et Methodes La face endotheliale etait incubee 45 min a 31°C avec 100 μL de C (2 μm), E (5 μm), H (10 μm). Apres montage a plat, pour chaque marqueur, les images de la totalite de la surface endotheliale (81 mm 2 ) etaient acquises par microscope IX81, a l’objectif ×4, par platine motorisee XY. Pour chaque position, une pile d’image en Z permettait de recuperer les differences de niveaux dues aux plis endotheliaux. La DCE etait mesuree par comptage automatique des noyaux H +, la mortalite par le rapport E +/H +. Les cellules H +/E-/C-, considerees comme non encore mortes mais metaboliquement inactives etaient denombrees a part. Resultats Le triple marquage HEC sur la totalite de la surface endotheliale et en 3D permettait une mesure fiable et rapide de la viabilite de la totalite de l’endothelium corneen, donnant acces a la DCE, au taux de mortalite et a une evaluation de l’activite metabolique. Discussion Le triple marquage HEC permet de mesurer objectivement les lesions endotheliales a un instant donne et de prendre en compte l’heterogeneite de repartition des lesions endotheliales. Conclusion Son utilisation ameliore et fiabilise l’evaluation experimentale de tout nouveau procede susceptible de modifier l’endothelium corneen ex vivo .

512 Comparaison entre 3 microscopes spéculaires non-contact

Introduction Les microscopes speculaires (MS) non-contact Rhinetec®, SeaEagle® pour la station complete d’acquisition et de traitement de l’image et SeaEagle SL® pour la version simplifiee adaptable sur lampe a fente, mesurent la densite cellulaire endotheliale (DCE) par detection des centres cellulaires soit automatiquement soit apres retouche manuelle. But : determiner leur concordance avec le MS non-contact Topcon SP2000®, le plus utilise, mesurant la DCE par detection des contours apres retouche obligatoire. Materiels et Methodes Analyse de la DCE centrale de 270 (SeaEagle®) et de 108 cornees (SeaEagle SL®). Utilisation de la methode de Bland Altman pour determiner la concordance entre le comptage retouche de Topcon® (gold standard) et les comptages automatiques puis retouches de SeaEagle® et SeaEagle SL®. Resultats Pour SeaEagle®. Nombre de cellules prises en compte peu different (moyenne, ecart-type) : 137 (44) vs 129 (46) (Topcon®) (p Discussion Les comptages automatiques de SeaEagle® et SeaEagle SL® ne sont pas concordants avec le comptage retouche de Topcon®. Les comptages retouches sont concordants, meme s’il peut ponctuellement exister une variation l’ordre de 400 a 500 cell/mm2. Conclusion Le comptage automatique doit etre banni des bonnes pratiques de comptage endothelial. Le comptage retouche avec SeaEagle® a une concordance excellente avec Topcon® retouche, moindre dans sa version adaptable sur lampe a fente SeaEagle SL®. Cette derniere nous parait neanmoins acceptable pour une utilisation de routine, hors recherche clinique.

018 Morphométrie endothéliale par analyse d’image des cornées conservées en organoculture : étude prospective de 505 cornées consécutives

But Determiner les facteurs influencant la morphometrie endotheliale des cornees en organoculture (mesure par analyse d’image) : coefficient de variation de surface cellulaire (CV) et pourcentage de cellules hexagonales. Materiels et Methodes L’endothelium de 505 cornees consecutives conditionnees a la banque de cornee de l’EFS Loire/Auvergne a ete analyse en routine par l’analyseur Sambacornea sur 3 images grand champ, apres dilatation osmotique des espaces intercellulaires et comptage d’au moins 300 cellules. La qualite du set d’images etait classee en mauvaise, acceptable, ou bonne par un observateur independant. Parametres etudies : âge et sexe du donneur, statut cristallinien, delai postmortem, type de milieu de conservation, duree de conservation avant analyse, qualite intrinseque des images saisies. Resultats L’analyse d’image a ete possible sur 504 des 505 cornees evaluees. L’âge du donneur etait significativement correle a la densite cellulaire endotheliale (r =-0343) au CV (r = 0,221) et a l’hexagonalite (r =-0,314) (p Discussion Malgre les conditions difficiles de l’organoculture et la qualite des images de microscopie optique parfois mediocre, l’analyse par Sambacornea de l’endothelium des greffons fournit des parametres endotheliaux qui sont cliniquement fiables puisque superposables a celles connues de longue date en microscopie speculaire. La morphometrie (CV et hexagonalite) est susceptible d’apporter des informations supplementaires sur la fonction endotheliale, peut etre en particulier pour les greffons proches du seuil conventionnel de cession de 2000 cell/mm 2 . Conclusion Des essais cliniques prospectifs utilisant l’analyse d’image devront evaluer l’influence de la morphometrie endotheliale sur la survie du greffon a long terme chez le receveur.

022 Développement d’un dispositif de quantification de la transparence, du plissement et du gérontoxon pour les cornées conservées en organoculture

Aim To develop an image analysis device devoted to the measurement of donor corneal transparency (T), folding (F) and clear corneal diameter excluding gerontoxon (corneal arcus) (G) during organ culture. Materials and Methods High resolution digital images (2592×1944 pixels) of a retroilluminated test chart comprising parallel horizontal lines viewed through the cornea placed in a petri dish were captured and analysed using a dedicated software. T (%) was a ratio of local contrast of the test chart and F (%) was a ratio of mean of line profiles of the test chart, each measured with and without the cornea. Area of clear cornea outside gerontoxon was calculated from a circle delineated by the observer using a specific software tool. On each image, three independent experts classified T, F and G according to 3-level score (++/+/0) which served as reference. Human corneas (fresh, organ cultured before and after deswelling) (n=179) were consecutively analysed to include a broad spectrum of T and F. Results The device was able to discriminate between the three classes of T, F and G, established by the experts. T was 20±4, 36±8 and 51±10% respectively for transparency deemed poor, average and excellent (P Discussion This simple and original device provides reliable quantitative objective measures of corneal transparency, folding and corneal arcus. It will help standardizing quality assessment of corneas among eye banks. Conclusion A trial designed to assess the ability of the device to select corneas before delivery in the setting of the routine practice of our cornea bank is ongoing (PHRC, appel d’offre local du CHU 2007).

Stratégies de comptage par analyse d'image de l'endothélium des cornées conservées en organoculture : détection de contour versus pointage du centre . Counting strategies for endothelial assessment of organ cultured corneas using image analysis: comparison of border versus center method

Aim To compare two different cell count strategies, border and center methods for endothelial assessment during corneal storage. Material and Methods Seven observers determined the endothelial cell density (ECD), coefficient of variation (CV) of cell area, and percentage hexagonality of 30 organ cultured corneas by the border (contour detection and manual retouch) and center method (indicating cell centers) using Sambacornea analyser. Interobserver variability for ECD and agreement between the two methods was determined. The accuracy of the center method was verified by counting on ten standard photolithographic mosaics (called keratotest) by border (reference method) and center methods, first as fast and next as precise as possible (fast and slow modes respectively) and the time noted. Data between border and center methods was compared using nonparametric paired tests. Results For stored corneas, interobserver variability was ±9.6% (95%CI [6.5-12.7]) for border method and ±9.3% (95%CI[6.3-12.3]) for center method. ECD [mean±SD (range), median] was [2948±565 (1644-3878), 3081] and [2961±568 (1736-3914), 3037] respectively and showed excellent correlation (Pearson coefficient r=0.998, p<0.001). The center method underestimated the CV by a mean 9.5%95%CI [8.3-10.7] and overestimated the hexagonality by a mean 2.5% (95%CI [1.4-3.7])(p<0.001 for both). Precision of indication of cell center influenced only the CV (mean for slow and fast modes being 3.6±0.7% and 7.4±1.3% respectively, p<0.001) but was more time consuming (p=0.005). Discussion The center method gives accurate and reproducible measurement of ECD and is comparable to the border method (reference). However, morphometric evaluation is not fully reliable. Conclusion Given that center method is easier to use in poor quality images, we recommend its use only for these cases.

005 Analyses histologiques et ultra structurales des cornées humaines conservées en Corneamax et déturgées durant 48 heures dans les poloxamers

Aim Poloxamers (P) are various synthetic block copolymers widely used in pharmaceutic industry. We previously described the efficiency of P188, 237, 338 and 407 to deswell human corneas at the end of organ culture (OC) and their ability to reduce the endothelial cell loss compared to 5% T500 Dextran in CorneaMax (with 2% foetal calf serum). We aimed to further assess the cellular effects of 48 hours incubation in P using histology and electron microscopy (EM). Materials and Methods The P were dissolved in CMax (Eurobio, Les Ulis, France) at 350 mOsmol/L. Five paired human corneas with more than 2000 endothelial cells/mm 2 used for each poloxamer. After 3 weeks of OC in CMax, one cornea was incubated for 48 hours in CMax + poloxamer, and the mate cornea in CorneaJet (CMax+ 5% T500 Dextran). Controls: fresh corneal button from keratoconus and lattice dystrophy (for normal unstored endothelium) and organ cultured corneas without deswelling. Corneas were divided into 3 parts for cross sectional histology (epithelial thickness measurement and counting of keratocytes), transmission EM of the posterior stroma and endothelium (cell morphology and interfibrillar spaces measurement) and scanning EM of the endothelium (morphology). Results Cross sectional histology showed differences in epithelial thickness. For both types of macromolecules (poloxamers and dextran) TEM revealed cytoplasmic vacuoles in endothelial cells and keratocytes and SEM confirmed the integrity of endothelial monolayer. Discussion Histological and ultrastructural findings, along with capacity of thinning, transparency improvement and endothelial cell preservation will allow selecting poloxamers to be added to CorneaMax for a future clinical application. Conclusion This complementary validation using light and electron microscopy reasonably confirms the promising use of Poloxamer to replace T500 Dextran to deswell corneas during organ culture.