La cécité d’origine cornéenne est Ia deuxième cause de cécité dans le monde. Son traitement de choix est la kératoplastie. ll est aujourd’hui nécessaire de réfléchir à de nouvelles solutions pour remplacer la kératoplastie dans sa forme actuelle qui bien que satisfaisante, n’est pas totalement parfaite. En effet, les techniques actuelles de kératoplastie utilisent une allogreffe. Le greffon va subir chez le donneur une diminution de la densité des cellules endothéliales (CE) du greffon au fil du temps. Ce travail présente successivement les solutions abordées par le BiiGC pour produire des CE en masse ainsi que les différents supports en vue d’une kératoplastie lamellaire postérieure. Les solutions envisagées par le laboratoire BiiGC doivent permettre un transfert à l’être humain dans des conditions de sécurité et d’acceptabilité maximales. Les différents contrôles de l’identité des cellules obtenues et les contrôles de sécurité nécessitent une connaissance robuste de la biologie, du morphotype et du phénotype des CE cornéennes. Cette thèse présente les différentes exigences réglementaires et de qualité nécessaires à l’obtention de greffons cornéens bioengineerés. Le choix du BiiGC devant se conformer d’emblée à ces exigences en vue d’un transfert à l’être humain. Dans une première partie, nous présentons une revue de la littérature sur le bioengineering de greffons cornéens. La production de CE et de stroma seront ensuite exposés avec les différentes approches abordées par le BiiGC. Enfin, les différentes techniques de validation morphologiques et fonctionnelles de greffons obtenus seront présentées. / Corneal diseases are the first leading cause of blindness worldwide. Its treatment relies on keratoplasty which in its actual form is a satisfying but not a perfect solution. We have to think about new solutions for corneal graft. Today, corneal graft is often an allograft. With this technique, there is a loss of endothelial cell (EC) density through time. This work presents the solutions which BiiGC laboratory is working on for mass production of EC and different carriers for these cells for posterior keratoplasty. These solutions must be possible on human being with safety conditions. Controls of identity, of obtained cells and security controls need a strong knowledge of biology, morphology and phenotype of EC. This work presents the legal conditions of quality needed to obtain bioengineered corneal grafts. The choice of BiiGC laboratory must involve these conditions for a transfer to human being. In a first part, we present a review of the literature about corneal bioengineering. Production of EC and of corneal stroma are discussed with different approaches by BiiGC laboratory. In the end, the different techniques of morphological controls of corneal grafts are discussed.

Made available in DSpace on 2014-06-11T19:27:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-03Bitstream added on 2014-06-13T18:31:58Z : No. of bitstreams: 1 conceicao_lf_me_jabo.pdf: 2281445 bytes, checksum: c6ad72b6b6c930106b1b1fa9f6e7ea2a (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Estudou-se os efeitos do cetorolaco de trometamina 0,5%, sem conservante, sobre a inflamação ocular, avaliando-se a reparação epitelial e a sensibilidade corneal, em coelhos submetidos à ulceração corneal química com hidróxido de sódio (NaOH) 1 mol/L. Constituíram-se dois grupos (n=12). O primeiro grupo recebeu 30 μl de cetorolaco de tometamina 0,5%, sem conservante (OT), a cada seis horas, em uma das córneas ulceradas e 30 μl de solução salina fisiológica 0.9% (OC) na córnea adelfa, totalizando 4 aplicações diárias, por um período de 24 horas. O limiar de sensibilidade corneal foi avaliado por estesiometria antes e posteriormente a lesão ser realizada, a intervalos regulares de quatro horas. Avaliou-se ainda o edema corneal, a hiperemia conjuntival, o blefarospasmo e o diâmetro das úlceras, empregando-se, biomicroscópio com lâmpada em fenda, teste do tingimento pela fluoresceína, imagem digitalizada para posterior avaliação em programa Image J, até a reepitelização completa. Decorridas 24 horas da abrasão nos animais do primeiro grupo (n=6) e 55 horas nos animais do segundo grupo (n=6), estes foram submetidos à eutanásia. Ambas as córneas (OT e OC) foram avaliadas histologicamente e à microscopia eletrônica de varredura. Utilizou-se Análise de Variância para Medidas Repetidas, considerando significativos os valores de p ≤ 0,05. Conclui-se que mesmo sem a adição de conservantes e a despeito do bom efeito analgésico, o cetorolaco de trometamina a 0,5%, sem conservante, retarda a reparação cicatricial do epitélio corneal em coelhos submetidos a abrasões com hidróxido de sódio 1 mol/L. / Studied the effects of ketorolac tromethamine 0.5% without preservatives on ocular inflammation, evaluating the sensitivity and corneal epithelial repair in rabbits subjected to chemical corneal ulceration with sodium hydroxide (NaOH) 1 mol / L. Constituted two groups (n = 12). The first group received 30 μl of tometamine ketorolac 0.5%, without preservative (OT) every six hours in one of ulcerated corneas and 30 μl of saline solution 0.9% (OC) in the other cornea a total of four applications daily for a period of 24 hours. The threshold of corneal sensitivity was assessed by esthesiometry before and after the injury to be held at regular intervals of four hours. It was also evaluated corneal edema, conjunctival hyperemia, blepharospasm and the diameter of the ulcers, using, biomicroscopy with slit lamp, the fluorescein dye test, the scanned image for further evaluation in program Image J, until complete reepithelialization. After 24 hours of abrasion in the animals of first group (n = 6) and 55 hours in animals of the second group (n = 6), they were euthanized. Both corneas (OT and OC) were evaluated histologically and scanning electron microscopy. We used ANOVA for repeated measures, considering significant p values ≤ 0.05. We conclude that even without the added preservatives and despite the good analgesic effect of ketorolac tromethamine 0.5%, without preservative, slowing the repair of corneal epithelium wound healing in rabbits subjected to abrasion with sodium hydroxide 1 mol/L.

Se analizaron los errores teóricos cometidos en el cálculo de la potencia corneal central en ojos con queratocono cuando se utilizaba la estimación queratométrica. Además, estos resultados fueron analizados clínicamente para confirmar este error de estimación. Posteriormente se calculó el índice queratométrico exacto que hacía cero el error cometido y se validó clínicamente el uso de un índice queratométrico variable que minimizaba el error cometido en el cálculo de la potencia corneal queratométrica. De esta manera se indicaron los posibles errores que podían cometerse en la clasificación del queratocono cuando se utilizaba una potencia corneal queratométrica en dichas clasificaciones. Además, se evaluó la influencia del error queratométrico en la estimación de la potencia de las lentes intraoculares en pacientes con queratocono, y a partir de ello, se desarrollaron y evaluaron de forma preliminar clínica los algoritmos necesarios para minimizar dicho error. Finalmente, se realizó un análisis teórico y clínico de los errores asociados al cálculo de la potencia corneal usando la estimación queratométrica en una población de queratoconos después de una cirugía de crosslinking y se obtuvo un modelo para la estimación del índice queratométrico ajustado para minimizar estos errores.

Yes / Background and Objective Corneal endothelial cell abnormalities may be associated with a number of corneal and systemic diseases. Damage to the endothelial cells can significantly affect corneal transparency by altering hydration of the corneal stroma, which can lead to irreversible endothelial cell pathology requiring corneal transplantation. To date, quantitative analysis of endothelial cell abnormalities has been manually performed by ophthalmologists using time consuming and highly subjective semi-automatic tools, which require an operator interaction. We developed and applied a fully-automated and real-time system, termed the Corneal Endothelium Analysis System (CEAS) for the segmentation and computation of endothelial cells in images of the human cornea obtained by in vivo corneal confocal microscopy. Methods First, a Fast Fourier Transform (FFT) Band-pass filter is applied to reduce noise and enhance the image quality to make the cells more visible. Secondly, endothelial cell boundaries are detected using watershed transformations and Voronoi tessellations to accurately quantify the morphological parameters of the human corneal endothelial cells. The performance of the automated segmentation system was tested against manually traced ground-truth images based on a database consisting of 40 corneal confocal endothelial cell images in terms of segmentation accuracy and obtained clinical features. In addition, the robustness and efficiency of the proposed CEAS system were compared with manually obtained cell densities using a separate database of 40 images from controls (n = 11), obese subjects (n = 16) and patients with diabetes (n = 13). Results The Pearson correlation coefficient between automated and manual endothelial cell densities is 0.9 (p < 0.0001) and a Bland–Altman plot shows that 95% of the data are between the 2SD agreement lines. Conclusions We demonstrate the effectiveness and robustness of the CEAS system, and the possibility of utilizing it in a real world clinical setting to enable rapid diagnosis and for patient follow-up, with an execution time of only 6 seconds per image.

Yes / Endothelial dysfunction and damage underlie cerebrovascular disease and ischemic stroke. We undertook corneal confocal microscopy (CCM) to quantify corneal endothelial cell and nerve morphology in 146 patients with an acute ischemic stroke and 18 age-matched healthy control participants. Corneal endothelial cell density was lower (P<0.001) and endothelial cell area (P<0.001) and perimeter (P<0.001) were higher, whilst corneal nerve fbre density (P<0.001), corneal nerve branch density (P<0.001) and corneal nerve fbre length (P=0.001) were lower in patients with acute ischemic stroke compared to controls. Corneal endothelial cell density, cell area and cell perimeter correlated with corneal nerve fber density (P=0.033, P=0.014, P=0.011) and length (P=0.017, P=0.013, P=0.008), respectively. Multiple linear regression analysis showed a signifcant independent association between corneal endothelial cell density, area and perimeter with acute ischemic stroke and triglycerides. CCM is a rapid non-invasive ophthalmic imaging technique, which could be used to identify patients at risk of acute ischemic stroke. / Qatar National Research Fund Grant BMRP20038654

Yes / Diabetic Peripheral Neuropathy (DPN) is one of the most common types of diabetes that can affect the cornea. An accurate analysis of the nerve structures can assist the early diagnosis of this disease. This paper proposes a robust, fast and fully automatic nerve segmentation and morphometric parameter quantification system for corneal confocal microscope images. The segmentation part consists of three main steps. First, a preprocessing step is applied to enhance the visibility of the nerves and remove noise using anisotropic diffusion filtering, specifically a Coherence filter followed by Gaussian filtering. Second, morphological operations are applied to remove unwanted objects in the input image such as epithelial cells and small nerve segments. Finally, an edge detection step is applied to detect all the nerves in the input image. In this step, an efficient algorithm for connecting discontinuous nerves is proposed. In the morphometric parameters quantification part, a number of features are extracted, including thickness, tortuosity and length of nerve, which may be used for the early diagnosis of diabetic polyneuropathy and when planning Laser-Assisted in situ Keratomileusis (LASIK) or Photorefractive keratectomy (PRK). The performance of the proposed segmentation system is evaluated against manually traced ground-truth images based on a database consisting of 498 corneal sub-basal nerve images (238 are normal and 260 are abnormal). In addition, the robustness and efficiency of the proposed system in extracting morphometric features with clinical utility was evaluated in 919 images taken from healthy subjects and diabetic patients with and without neuropathy. We demonstrate rapid (13 seconds/image), robust and effective automated corneal nerve quantification. The proposed system will be deployed as a useful clinical tool to support the expertise of ophthalmologists and save the clinician time in a busy clinical setting.

En France, les dysfonctions endothéliales représentent environ la moitié des indications de greffes de cornée réalisées chaque année. Cependant, les problématiques liées à la pénurie de greffon, aux difficultés des techniques chirurgicales de greffes endothéliales ainsi qu’aux risques d’échec ou de rejet de greffe poussent les chercheurs à développer de nouvelles thérapies moins invasives et plus efficaces. La thérapie cellulaire cornéenne endothéliale est une des voies de recherche actuellement explorées dont le but est de s’affranchir des aléas de la greffe de cornée. La cornée humaine est un tissu idéal pour la thérapie cellulaire. Grâce à ses caractéristiques d’organe à la fois avasculaire et immunitairement privilégié, les cellules transplantées sont ainsi bien mieux tolérées par rapport aux autres tissus et organes vascularisés. Les avancées dans le domaine des cellules souches, de l'ingénierie, particulièrement avec l’arrivée des greffes de cellules souches épithéliales pour le traitement des pathologies sévères de la surface oculaire, ont suscité un intérêt massif afin d’adapter ces techniques aux cellules endothéliales / In France, around half of all corneal keratoplasties are performed to treat corneal endothelial dysfunction each year. However, the use of endothelial keratoplasty is limited by the technical difficulty of the procedure, a shortage of available grafts, and the potential for graft failure or rejection. These limitations are driving researchers to develop new, less invasive, and more effective therapies. Corneal endothelial cell therapy is being explored as a potential therapeutic measure, to avoid the uncertainty associated with grafting. The human cornea is an ideal tissue for cell therapy as owing to its avascular characteristics, transplanted cells are better tolerated compared with other vascularized tissues and organs. Advances in the field of stem-cell engineering, particularly the development of corneal epithelial stem cell therapy for the treatment of severe diseases of the ocular surface, have aroused a massive interest in adapting cell-therapy techniques to corneal endothelial cells

No description available.

Objetivo: Verificar resposta após debridamento epitelial de córneas humanas. Métodos: Córneas normais foram submetidas a debridamento antes da cirurgia de enucleação. Realizou-se histologia, TUNEL, Ki67, SMA e microscopia eletrônica. Resultados: Seis córneas foram debridadas e preservadas entre ½ e 65 horas, apresentando apoptose nos ceratócitos do estroma anterior. Células estromais em proliferação foram observadas apenas no tempo de 65 horas. Miofibroblastos não foram encontrados. Uma córnea serviu de controle. Conclusões: Os eventos observados em córneas humanas após debridamento epitelial, apoptose e proliferação dos ceratócitos, foram semelhantes aos descritos em animais de experimentação / Purpose: To examine the early wound healing response to epithelial scrape in human corneas. Methods: Normal corneas had epithelial scrape prior to enucleation. Histology, TUNEL assay, Ki67, SMA and transmission electron microscopy were performed. Results: Epithelial scrape was performed in six corneas from ½ to 65 hours prior to preservation. Keratocyte apoptosis was detected in the anterior stroma in all scraped corneas. Keratocyte proliferation was detected exclusively 65 hours after scrape. No myofibroblast was detected. One cornea was not scraped (control). Conclusion: Results obtained in human corneas (keratocyte apoptosis and proliferation) were similar to animal models

Objetivo: Verificar resposta após debridamento epitelial de córneas humanas. Métodos: Córneas normais foram submetidas a debridamento antes da cirurgia de enucleação. Realizou-se histologia, TUNEL, Ki67, SMA e microscopia eletrônica. Resultados: Seis córneas foram debridadas e preservadas entre ½ e 65 horas, apresentando apoptose nos ceratócitos do estroma anterior. Células estromais em proliferação foram observadas apenas no tempo de 65 horas. Miofibroblastos não foram encontrados. Uma córnea serviu de controle. Conclusões: Os eventos observados em córneas humanas após debridamento epitelial, apoptose e proliferação dos ceratócitos, foram semelhantes aos descritos em animais de experimentação / Purpose: To examine the early wound healing response to epithelial scrape in human corneas. Methods: Normal corneas had epithelial scrape prior to enucleation. Histology, TUNEL assay, Ki67, SMA and transmission electron microscopy were performed. Results: Epithelial scrape was performed in six corneas from ½ to 65 hours prior to preservation. Keratocyte apoptosis was detected in the anterior stroma in all scraped corneas. Keratocyte proliferation was detected exclusively 65 hours after scrape. No myofibroblast was detected. One cornea was not scraped (control). Conclusion: Results obtained in human corneas (keratocyte apoptosis and proliferation) were similar to animal models