Les agressions graves de la surface oculaire peuvent se compliquer d'ulcérations cornéennes épithéliales, de néovascularisation, d'opacification et d'inflammation chroniques, échappant à tout traitement médical. Les progrès dans la compréhension de la physiologie du renouvèlement de l'épithélium et du stroma cornéen ont permis d'introduire une approche thérapeutique, la greffe de cellules souches. L'objectif de notre étude est d'apporter une source de cellules souches cornéennes par thérapie cellulaire afin de restituer une fonction épithéliale et stromale permettant l'obtention d'une cornée claire chez l'animal. Les cellules souches épithéliales limbiques (LSC) et les cellules stromales limbiques (SSC) ont été isolées dans un milieu exempt de produits animaux tout en gardant leur caractère indifférencié. L'effet régénérateur des SSC a été étudié in vivo dans un modèle de fibrose cornéenne chez la souris. Les SSC ont été injectées dans le stroma cornéen et ont permis la restitution d'un stroma organisé et d'une cornée transparente. Le projet a aussi consisté à élaborer et mettre en forme une matrice à base de collagène I afin de favoriser la culture concomitante de tous les types cellulaires présents dans la cornée tout en préservant organisation, transparence et propriétés mécaniques. La connaissance des conditions physico-chimique du collagène a permis de produire des matrices transparentes qui ont pu être colonisées par les SSC et les LSC. Notre étude ouvre de nouvelles perspectives dans le traitement des pathologies provocant des opacités cornéennes afin de faire face au manque de dons de cornée. / Cornea, the outer part of the eye, features high transparency crucial for good vision. The maintenance of a healthy cornea is linked to the presence of corneal stem cells in the limbus (peripheral region of the cornea). Several diseases can lead to its opacification requiring transplantation of donor tissue to restore vision. Although corneal transplantation has achieved clinical success, there is a shortage of donor corneas worldwide. To solve this problem, cell therapy and tissue-engineered artificial cornea are promising approaches that could eventually outperform current treatment. Corneal epithelial stem cells (LSC) and stromal stem cells (SSC) were isolated in feeder free and xeno free medium. The therapeutic effect of SSC was investigated in vivo by their injection in mouse cornea treated with liquid nitrogen. SSC have the ability to restore the extracellular matrix organization and corneal transparency. Transparent collagen I fibrillated matrices have been synthesized and were evaluated for fibril organization, transparency, mechanical properties and their ability to be repopulated by corneal stromal stem cells and to support limbal epithelial stem cell growth. We show that the matrices were organized and transparent. SSC and LSC were able to repopulate and to grow into the collagen matrix. These cells present a potential for stem cell-based treatment of corneal blindness.

Made available in DSpace on 2015-07-22T20:49:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-05-30. Added 1 bitstream(s) on 2015-08-11T03:26:18Z : No. of bitstreams: 1 Publico-13221.pdf: 17673997 bytes, checksum: 5eccd3ef771fd6553a1cc32756fcef7a (MD5) / Objetivos: 1) Avaliar se o cross-linking do colágeno corneano antes da inserção de segmentos de anéis corneanos intraestromais altera o tratamento do ceratocone. 2) Avaliar se o cross-linking do colágeno corneano modifica as características citológicas da superfície ocular em portadores de ceratocone. Métodos: Trinta e nove olhos de 31 pacientes foram alocados em 2 grupos: 19 olhos foram submetidos ao tratamento do cross-linking com riboflavina e luz ultravioleta A, e 20 olhos receberam colírio de riboflavina 0,1% (w/v) em solução de 20% dextran 4 vezes ao dia por 30 dias. Após 3 meses, todos os pacientes foram submetidos à inserção de segmentos de anéis corneanos intraestromais pela técnica do laser de femtosegundo. Avaliações foram realizadas nos momentos pré-operatório, com 1 e 3 meses após o cross-linking ou colírio de riboflavina, e também com 1, 3, 6, 12 e 24 meses após a inserção de segmentos de anéis corneanos intraestromais. Os pacientes foram submetidos aos seguintes exames: acuidade visual sem correção e acuidade visual com correção; refração manifesta; biomicroscopia; sensibilidade ao contraste; topografia corneana; Orbscan IIz; Pentacam; tonometria de aplanação; tonometria dinâmica de contorno; paquimetria ultrassônica; biomecânica da córnea; tomografia de coerência óptica; microscopia especular da córnea; citologia de impressão e mapeamento de retina. Análise de covariância e o teste de Mann-Whitney foram realizados para comparação das variáveis do estudo entre os 2 grupos. Teste de Friedman foi utilizado para avaliação da citologia de impressão antes do tratamento, e com 1 e 3 meses após o cross-linking ou colírio de riboflavina, e novamente com 6, 12 e 24 meses após a inserção de segmentos de anéis corneanos intraestromais. Resultados: Todos os pacientes foram acompanhados por 24 meses. A média e variação de idade dos pacientes foram de 28,30 ± 9,3 anos (17-55 anos) no grupo cross-linking e 30,40 ± 9,1 anos (22-55 anos) no grupo colírio de riboflavina. Não houve diferença estatisticamente significante entre os grupos em relação à acuidade visual sem correção (p=0,70) ou acuidade visual corrigida (p=0,78). Com 24 meses de seguimento, não houve diferença estatística entre os grupos em relação ao equivalente esférico (p=0,94). Também não houve diferença estatística para os 3 parâmetros topográficos (mais plano-K1 [p=0,81], mais curvo-K2 [p=0,68] e curvatura média [p=0,52]). Os exames de sensibilidade ao contraste, microscopia especular, paquimetria, tonometria e propriedades biomecânicas da córnea não apresentaram diferença estatística entre os grupos com 24 meses de seguimento. Na citologia de impressão, alguns parâmetros conjuntivais (ex.: adesividade das células epiteliais, proporção núcleo:citoplasma, nível de organização da cromatina nuclear, células caliciformes e queratinização [p≥0,001]) apresentaram diferenças estatisticamente significantes. Conclusões: O cross-linking do colágeno corneano não altera o efeito de segmentos de anéis corneanos intraestromais para ceratocone antes de sua inserção em relação à refração, topografia, sensibilidade ao contraste, microscopia especular, paquimetria, tonometria e biomecânica da córnea com 24 meses de seguimento. A comparação do escore total de citologia de impressão entre os grupos não mostrou diferença estatisticamente significante, apesar de diferenças em alguns parâmetros conjuntivais. / Purpose: To report the impression cytologic results after corneal cross-linking and insertion of intrastromal corneal ring segments for keratoconus. Methods: Thirty-nine eyes were distributed into two groups: 1) cross-linking group (patients underwent corneal cross-linking procedure), and 2) riboflavin eyedrops group (patients received riboflavin 0.1% (w/v) eyedrops in 20% dextran solution for 1 month). After 3 months, all patients underwent insertion of intrastromal corneal ring segments. Impression cytologic specimens were obtained from all eyes at baseline, at 1 month and 3 months after cross-linking or riboflavin eyedrops, and again at 6 months, 1 year, and 2 years after intrastromal corneal ring segment insertion. Results: Patients in the cross-linking group demonstrated improvement in the cell-to-cell contact of epithelial cells and the nucleusto- cytoplasm ratio on the temporal conjunctiva after treatment (P = 0.008 and P = 0.047), respectively. On the superior conjunctiva, increases in goblet cell density (P = 0.037) and level of organization of nuclear chromatin (P = 0.010) after treatment were noted. Patients in the riboflavin eyedrops group demonstrated improvement in the cellto- cell contact of epithelial cells on the superior conjunctiva after treatment (P = 0.021). On the temporal conjunctiva, an improvement in the cell-to-cell contact of epithelial cells (P < 0.001) and increases in the nucleus-to-cytoplasm ratio (P < 0.001), goblet cell density (P = 0.001), and less keratinization (P = 0.011) were noted. No changes were identified on the cornea for either group. Fisher’s exact test comparison of the impression cytologic total scores after treatment revealed no difference between groups. Conclusion: Despite changes in some conjunctival parameters (e.g., cell-tocell contact of epithelial cells, nucleus-to-cytoplasm ratio, level of organization of nuclear chromatin , goblet cell density, and keratinization), comparison of the total impression cytologic scores revealed no difference between groups. / TEDE / BV UNIFESP: Teses e dissertações

Portadores de diabetes parecem ter mais queixas de olho seco do que o resto da população. Acredita-se que isto possa estar associado a uma forma de neuropatia diabética expressa por uma redução na sensibilidade corneana desses pacientes. Nossos principais objetivos neste estudo foram avaliar a influência da diabetes melito tipo 2 na sensibilidade corneana central e verificar se há uma associação entre a sensibilidade corneana central e a síndrome do olho seco em indivíduos com a doença. Assim, 62 portadores de diabetes tipo 2 foram submetidos a um exame oftalmológico de rotina, a uma ceratoestesiometria e a testes específicos para avaliar olho seco e polineuropatia distal simétrica. Num outro grupo, 20 voluntários saudáveis tiveram seus olhos avaliados da mesma forma, exceto pela não realização dos testes específicos para disfunção lacrimal. Entre os indivíduos diabéticos avaliados, foram observados 53.2% com hipoestesia corneana, 54.2% com retinopatia diabética, 45.9% com polineuropatia distal simétrica e 51.6% com a síndrome do olho seco. Entre os principais achados, observamos associações significativas envolvendo: diabetes tipo 2 e hipoestesia corneana central, síndrome do olho seco e hipoestesia corneana central, produção lacrimal reflexa (avaliada pelo teste de Schirmer II) e sensibilidade corneana central e retinopatia diabética proliferativa e sensibilidade corneana central. Uma possível associação foi encontrada envolvendo síndrome do olho seco retinopatia diabética proliferativa. Os autores discutem os resultados obtidos e os mecanismos envolvidos. / Diabetes bearers seem to have more complaints of dry eye than the rest of the population. It`s believed that this fact might be associated to a kind of diabetes neuropathy wich is represented by a reduction in corneal sensibility of these patients. Our main target in this study was to evaluate the influence of type 2 diabetes mellitus in central corneal sensibility and to determine if there is an association among central corneal sensibility and the dry eye syndrome in individuals suffering of this disease. Therefore, 62 type 2 diabetic patients were submitted to an ophthalmological routine examination, to corneal esthesiometry and to specific tests to evaluate dry eye and peripheral polineurophaty. In other group, 20 healthy volunteers had their eyes evaluated in the same way, except for the non accomplishment of the specific tests for dry eye. Among the examined diabetic individuals, 53.2% had corneal hypoesthesia, 54.2% presented diabetic retinopathy, 45.9% presented periferal polineuropathy and 51.6% presented the dry eye syndrome. Among the main findings, we observed associations between: type 2 diabetes and central corneal hypoesthesia, dry eye syndrome and central corneal hypoesthesia, reflex tear production (evaluated by Schirmer 2 test) and central corneal esthesiometry and also between proliferative diabetic retinopathy and central corneal sensibility. A possible association was found involving dry eye syndrome and proliferative diabetic retinophaty. The authors discuss the results obtained and the involved mechanisms.

INTRODUÇÃO: Comparar duas soluções viscoelásticas, ácido hialurônico 1,6%/sulfato de condroitina 4% (DisCoVisc®, Alcon Laboratórios, EUA) e hidroxipropilmetilcelulose 2% (HPMC, Ophthalmos, Brasil), com relação ao desempenho durante a facoemulsificação (FACO) e o implante de lente intraocular. MÉTODOS: Foi realizado um ensaio clínico randomizado, envolvendo 78 olhos (39 pacientes) submetidos à FACO bilateral, por um único cirurgião. Os pacientes foram randomizados para receber DisCoVisc ou HPMC no primeiro olho. O olho contralateral foi operado mais tarde recebendo a outra solução viscoelástica em todas as etapas da cirurgia. Examinadores mascarados realizaram as avaliações pré e pósoperatórias (5, 24 e 48 horas; 7 e 14 dias; 3 e 6 meses), incluindo a medida da pressão intraocular (PIO), espessura cornena central (ECC) e acuidade visual corrigida. A densidade endotelial foi realizada no pré-operatório e ao final do seguimento (6 meses). Variáveis intraoperatórias incluíram medidas da quantidade total de solução viscoelástica, tempo de ultrassom durante a FACO e tempo para a retirada completa da solução do olho. RESULTADOS: A densidade endotelial foi estatisticamente superior com DisCoVisc (2.214 ± 372 cel/mm2) do que com HPMC (2.032 ± 460 cel/mm2) ao final do seguimento (p = 0,001). Uma redução média de 7% e de 15%, respectivamente. Não houve diferença estatística entre as soluções viscoelásticas com relação a densidade da catarata (p = 0,363) e o tempo de ultrassom (p = 0,456). O tempo de aspiração da HPMC (0,22 ± 0,09 min) foi significativamente maior que o DisCoVisc (0,17 ± 0,06 min) (p = 0,001) e a quantidade de viscoelástico utilizada foi maior com a HPMC (1,35 ± 0,20 ml) do que com DisCoVisc (0,89 ± 0,11 ml) (p < 0,001). Não houve diferença estatisticamente significativa entre as soluções quanto a ECC e a PIO durante o seguimento. CONCLUSÕES: A densidade endotelial ao final do estudo foi significativamente maior com o uso de DisCoVisc, o que pode representar uma melhor proteção endotelial. Foi necessário uma menor quantidade de DisCoVisc e um tempo de aspiração menor durante a FACO com essa solução viscoelástica / INTRODUCTION: To compare two ophthalmic viscosurgical devices (OVDs), 1.6% hyaluronic acid/4% chondroitin sulfate (DisCoVisc®, Alcon Lab., USA) and 2% hydroxypropylmethylcellulose (HPMC, Ophthalmos, Brazil), in terms of their overall performance during phacoemulsification (PHACO) and intraocular lens implantation. METHODS: This prospective, randomized clinical trial comprised 78 eyes (39 patients) that underwent PHACO by the same surgeon. Patients were randomly assigned to receive DisCoVisc or HPMC on the first eye. The other eye was treated later and received the other OVD. Masked examiners measured intraocular pressure (IOP), central corneal thickness (CCT) and best-corrected visual acuity. The corneal endothelial cell density was measured at baseline and at 6 months postoperatively. Intraoperative variables include the total amount of the OVD, ultrasound and washout times. RESULTS: Corneal endothelial cell density was significantly higher with DisCoVisc (2.214 ± 372 cell/mm2) than HPMC (2.032 ± 460 cell/mm2) at the end of follow-up (p = 0.001). A mean reduction of 7% and 15%, respectively. There were no statistically significant differences between OVDs in terms of cataract density (p = 0.363) or ultrasound time (p = 0.456). Regarding washout time, it took longer to remove HPMC (0.22 ± 0.09 min) than DisCoVisc (0.17 ± 0.06 min) (p = 0.001), and the amount of viscoelastic material used was greater with HPMC (1.35 ± 0.20 ml) than DisCoVisc (0.89 ± 0.11 ml) (p < 0.001). There were no statistically significant differences between the two OVDs regarding CCT or IOP measurements at any point in time. CONCLUSION: The corneal endothelial cell loss was significantly less with DisCoVisc than HPMC, which can improve corneal endothelium protection. DisCoVisc was easier to remove after IOL implantation and fewer amounts were necessary during PHACO

O presente trabalho trata-se do desenvolvimento de um programa para de análise de imagens de topografia corneana de sistemas comerciais, para ser implementado no topógrafo corneano para Lâmpada de Fenda, em desenvolvimento no Laboratório de Instrumentação Oftálmica - EESC/USP e no Laboratório de Física Oftálmica - FMRP/USP. O programa foi desenvolvido em C++, utilizando a plataforma Windows, e fornece mapas axiais de topografia corneana. O programa foi testado em esferas de calibração e em olhos humanos, apresentando um fator de correlação de 0,9998 para as medidas em esferas e um erro inerente estimado em 3%. Os mapas de topografias axiais em olhos humanos foram comparados com os mapas gerados por sistemas comerciais e o padrão visual de forma e relevo estão em concordância. / This work is about a software for the analisys of corneal topography images provided by commercial available systems to be implemented in a corneal topographer for slit lamps under evelopment at Laboratório de Instrumentação Oftálmica . EESC/USP e no Laboratório de Física Oftálmica . FRMP/USP. The software was developed in Borland C++ Builder for Windows and provides the corneal topography axial maps. The software has been tested in calibration spheres and in human eyes, presenting a correlation factor of 0,9998 for the measurements performed in the spheres and an inherent error of 3%. The axial topographic maps form the exams performe in human eyes have been compared to the axial maps provided by the commercial available system and the visual pattern as well as the relief are in accordance.

Portadores de diabetes parecem ter mais queixas de olho seco do que o resto da população. Acredita-se que isto possa estar associado a uma forma de neuropatia diabética expressa por uma redução na sensibilidade corneana desses pacientes. Nossos principais objetivos neste estudo foram avaliar a influência da diabetes melito tipo 2 na sensibilidade corneana central e verificar se há uma associação entre a sensibilidade corneana central e a síndrome do olho seco em indivíduos com a doença. Assim, 62 portadores de diabetes tipo 2 foram submetidos a um exame oftalmológico de rotina, a uma ceratoestesiometria e a testes específicos para avaliar olho seco e polineuropatia distal simétrica. Num outro grupo, 20 voluntários saudáveis tiveram seus olhos avaliados da mesma forma, exceto pela não realização dos testes específicos para disfunção lacrimal. Entre os indivíduos diabéticos avaliados, foram observados 53.2% com hipoestesia corneana, 54.2% com retinopatia diabética, 45.9% com polineuropatia distal simétrica e 51.6% com a síndrome do olho seco. Entre os principais achados, observamos associações significativas envolvendo: diabetes tipo 2 e hipoestesia corneana central, síndrome do olho seco e hipoestesia corneana central, produção lacrimal reflexa (avaliada pelo teste de Schirmer II) e sensibilidade corneana central e retinopatia diabética proliferativa e sensibilidade corneana central. Uma possível associação foi encontrada envolvendo síndrome do olho seco retinopatia diabética proliferativa. Os autores discutem os resultados obtidos e os mecanismos envolvidos. / Diabetes bearers seem to have more complaints of dry eye than the rest of the population. It`s believed that this fact might be associated to a kind of diabetes neuropathy wich is represented by a reduction in corneal sensibility of these patients. Our main target in this study was to evaluate the influence of type 2 diabetes mellitus in central corneal sensibility and to determine if there is an association among central corneal sensibility and the dry eye syndrome in individuals suffering of this disease. Therefore, 62 type 2 diabetic patients were submitted to an ophthalmological routine examination, to corneal esthesiometry and to specific tests to evaluate dry eye and peripheral polineurophaty. In other group, 20 healthy volunteers had their eyes evaluated in the same way, except for the non accomplishment of the specific tests for dry eye. Among the examined diabetic individuals, 53.2% had corneal hypoesthesia, 54.2% presented diabetic retinopathy, 45.9% presented periferal polineuropathy and 51.6% presented the dry eye syndrome. Among the main findings, we observed associations between: type 2 diabetes and central corneal hypoesthesia, dry eye syndrome and central corneal hypoesthesia, reflex tear production (evaluated by Schirmer 2 test) and central corneal esthesiometry and also between proliferative diabetic retinopathy and central corneal sensibility. A possible association was found involving dry eye syndrome and proliferative diabetic retinophaty. The authors discuss the results obtained and the involved mechanisms.

La transparence et les propriétés optiques du greffon cornéen sont des qualités essentielles pour le résultat post opératoire après la kératoplastie perforante et la kératoplastie lamellaire antérieure qui sont les 2 greffes les plus utilisées au monde. Pourtant, la transparence est difficile à mesurer avec précision avec les techniques d’eye banking actuelles et les propriétés optiques ne le sont pas du tout car lors de la conservation cornéenne à 4°C et plus encore en organoculture, la cornée séparée du globe est moins transparente que sur le sujet vivant et comporte des plis. Nous avons développé un outil de conservation, un bioréacteur, permettant de simuler les conditions physiologiques de la cornée pendant la conservation, permettant ainsi la caractérisation optique du greffon. L’objectif de cette thèse a été la conception et la réalisation des instruments de caractérisation des propriétés optiques et tissulaires des greffons conservés en BR. Ces propriétés sont la densité cellulaire endothéliale, la transparence, la puissance, l’épaisseur et la détection d’interfaces cicatricielles / The optical properties of a corneal graft, namely its transparency, are pivotal to the post-operative result of penetrating keratoplasty and deep anterior lamellar keratoplasty, which are today the world’s two most performed grafts. However, transparency is difficult to accurately measure with modern eye banking techniques, and other optical properties are not measurable at all, as with both hypothermic storage and organ culture the enucleated cornea is less transparent than in a living eye, and presents endothelial folds. We developed a corneal storage device, a bioreactor, to simulate the eye’s physiological conditions during storage, thus allowing the measurement of the graft’s optical properties. The goal of this thesis was to create a set of instruments to be used in eye banking for the optical characterization of corneal grafts. The parameters to be measured are endothelial cell density, transparency, dioptric power, thickness and the presence of scar tissue

La bioingénierie de greffons endothéliaux cornéens est une des solutions réalistes en cours de développement dans quelques laboratoires pour palier au grand déséquilibre entre pénurie mondiale de dons de cornée et besoins immenses et croissant des populations. Cette véritable médecine régénérative consistera à injecter des cellules endothéliales (CEs) dans la chambre antérieure aux stades précoces des dystrophies endothéliales et, pour les stades avancées des pathologies endothéliales, à reconstruire in vitro des greffons endothéliaux composés d'un support transparent et biocompatible colonisé par une monocouche des CEs. Dans les 2 cas, la première étape obligatoire est la production de masse de CEs ou de "CE-like". Dans ce travail, nous avons exploré les différentes possibilités pour obtenir suffisamment de CEs fonctionnelles pour envisager des applications cliniques : 1- L'expansion de CEs natives prélevées sur des cornées de donneurs, ou culture primaire, se heurtent aux capacités prolifératives limitées des CEs in vitro. Un des prérequis étant la compréhension des mécanismes d'arrêt de la prolifération des CEs humaines, nous avons fait la synthèse bibliographique des connaissances sur leur cycle cellulaire et leur sénescence, et présentons 2 articles originaux: le premier utilise un microarray spécifique de 112 gènes de contrôle du cycle cellulaire pour comparer les profils transcriptionnels des CEs de 6 modèles biologiques comportant théoriquement un stimulus prolifératif croissant: in vivo, post mortem, organoculture, culture primaire confluente, culture primaire non confluente et lignée immortalisée. Nous identifions de nombreux acteurs impliqués dans l'arrêt du cycle, en particulier ceux impliquant une réponse à des dommages oxydatifs de l'ADN. Le second article explore les capacités prolifératives résiduelles des CEs de donneurs âgés de plus de 50 ans, sont les plus nombreux en Europe et donc les pourvoyeurs habituels de CEs pour la bioingénierie. Nous montrons qu'une optimisation des techniques de culture permet d'obtenir in vitro une mosaïque endothéliale de 2000 cellules/mm2. Enfin, un troisième article montre qu'un stimulus physique inattendu constitué d'un train d'impulsion électrique permet d'obtenir un très grand nombre de figures mitotiques mais uniquement dans des zones de faible DCE, démontrant encore une fois l'importance majeur de l'inhibition de contact dans l'arrêt prolifératif. 2- La sélection et l'expansion, par culture en sphère, des progéniteurs endothéliaux de la périphérie endothéliale pourrait être un moyen de contourner la sénescence de la majorité des CEs. La synthèse bibliographique montre que la plupart des travaux publiés émanent d'une seule équipe japonaise. Dans notre second article, nous avons montré l'existence de rares "label-retaining cells" dans les cornées de donneurs âgés, qui pourraient correspondre à ces progénieteurs. Nous avons aussi montré qu'il était possible d'obtenir des sphères avec ces donneurs. 3- La différenciation de cellules souches embryonnaires, mésenchymateuses ou des cellules pluripotentes induites est enfin la troisième voie qui pourrait permettre de produire des CEs en grand nombre. La méthode d'induction de la différenciation pourrait reproduire le schéma physiologique et désormais bien caractérisé de formation de l'endothélium à partir du mésenchyme périoculaire dérivé de la crête neurale et que nous rappelons au début de notre mémoire bibliographique. Nous rappelons également les méthodes utilisables pour caractériser les cellules obtenues: vérification de leur identité par immunomarquage d'un panel de protéines caractéristique (en absence de marqueur unique spécifique) et vérification de leur fonctionnalité par mesures de leurs capacités de pompage ionique, au minimum de façon indirecte sur chambre d'électrophysiologie de type Ussing, au mieux de façon directe en quantifiant la déturgescence d'une cornée humaine conservée dans le bioréacteur breveté du BiiGC / Corneal endothelial engineering is becoming a more and more realistic solution to restore vision from corneal edema. This method focus to regenerate corneal endothelium by direct injection of corneal endothelial cells (ECs) into patient anterior chamber at the early stage of endothelial dystrophies, or by grafting a transparent biocompatible material covered by a monolayer of ECs. These two techniques require both in vitro isolation and amplification of ECs or endothelial-like cells. In this thesis, different strategies to obtain a high quantity of functional ECs for clinical application are explored: 1- Due to the limit proliferative capacity of EC, the first strategy consists to analyze mechanisms implicated EC cell cycle arrest and then to optimize protocol for native EC isolation or for cell proliferation activation ex vivo. This is summarized in three publications. The first publication describes the cell cycle regulation by comparing transcriptional expression of 112 genes in 6 biological models of EC with different proliferative profile: in vivo, postmortem, organ-culture, confluent primary culture, non confluent primary culture and immortalized cell line. , The key molecular actors identified using the combining microarray analysis and gene ontology methods are consistent with previous findings about oxidative DNA damage mechanism. The second publication characterizes EC differentiation process and its impact on EC proliferative capacity in old donor corneas. Analyses of differentiation/progenitor markers and of proliferative capacity underline the differentiation process of EC from the centre to the peripheral corneal endothelium. Thereby, an optimized culture protocol was developed, allowing the formation of high-density monolayer (> 2000 cells/mm2) with stable endothelial morphology. We proved the possibility to make profit from a majority of old-donor cornea grafts invalidated for penetrating graft In the third publication, the activation of endothelial cell cycle by electric pulses directly in corneal graft was characterized. We confirm the activation of endothelial cell cycle at different phases but also the damage of tissue during electroporation. 2- Second strategy consists of the amplification of ECs from potential EC progenitors. Using sphere forming culture and a new method to detect slow-cycling cells, we demonstrate the existence of "young" ECs population with higher proliferative capacity in corneal periphery. The isolation of ECs by sphere formation is one possible step for ECs selection in vitro. 3- The differentiation of embryonic stem cells, mesenchymal stem cells or induced pluripotent stem cells into corneal endothelial cells is the third approach considered by our laboratory. The manipulation of stem cells differentiation would be based on the molecular mechanisms implicated in the formation of corneal endothelium from periocular mesenchymal cells described in the first part of the bibliography. Finally, in order to validate the quality of endothelial cell mass obtained, we revisited recent methods for the evaluation of corneal endothelial identity (immunolocalisation of specific markers), for the measurement of pump activity of cell monolayer (Ussing chamber, perfusion chamber) or directly in deswelled cornea using the bioreactor patented by the BiiGC laboratory

Made available in DSpace on 2016-05-02T13:55:15Z (GMT). No. of bitstreams: 1 Paulo Afonso da Silveira Ferreira.pdf: 81766 bytes, checksum: 29bb4f9fdb5b85f4aa4538727ae69dbe (MD5) Previous issue date: 2005-02-22 / Coordenacao de Aperfeicoamento de Pessoal de Nïvel Superior / Two surgical techniques were compared for yhe reparation of deep corneal lesions in dogs the flap from the upper bulbar conjunctiva and the superficial keratectomy (homologous transplant) Tem dog of both sexes with no defined breeds were used Considered healthy after a period of adaptation profylaxis and clinical evaluation the dogs were separated into two groups of five animal each They were clinically analized after the experiment through subjective observation with regard to blepharospasm opacities neovascularization adhesiveness and secretion (qualitavely and quantitatively) Both techniques were efficient for the reparation fo corneal lesions / Foi realizada a comparação de dois métodos cirúrgicos o flap a partir da conjuntiva bulbar superior e a ceratectomia superficial (transplante homólogo) para reparação de lesões corneanas profundas em cães Foram utilizados 10 cães de ambos os sexos sem raça definida considerados hígidos após um período de adaptação profilaxia e avaliação clínica Os animais foram divididos em dois grupos com 5 animais cada Foram analizados clinicamente após o experimento através de observação subjetiva em relação a blefaroespasmo opacidade neovascularização adesividade e secreção (qualitativamente e quantitativamente) Não houve diferença significativa em relação aos parâmetros mensurados Ambas as técnicas mostraram-se eficientes para a reparação de lesões corneanas

La kératite à Acanthamoeba (KA) est une kératite infectieuse rare et grave,potentiellement cécitante. L'infection est causée par Acanthamoeba spp., unprotozoaire ubiquitaire présent dans le sol, l'air et l'eau. Jusqu'à 85% des cas de KAsont associés au port de lentilles cornéennes, et plus rarement suite à untraumatisme.Actuellement, aucune molécule n’a d’autorisation de mise sur le marché danscette indication dans l'Union européenne et aux États-Unis. Ces dernières années,des combinaisons d'agents anti-amibiens tels que les biguanides et les diamidinesont été utilisées comme traitement de référence. Cependant, les schémasthérapeutiques et les concentrations d'agents actifs reposent sur des donnéesempiriques. Récemment, le voriconazole, antifongique triazolé, a été utilisé avecsuccès pour traiter des KA humaines. Malgré cela, la communauté ophtalmologiquese heurte le plus souvent dans les formes sévères à de grandes difficultés de prise encharge et se retrouve parfois en situation d’impasse thérapeutique. La pertefonctionnelle et anatomique de l’oeil est encore possible.A partir d’un modèle de KA chez le rat, plusieurs molécules et voiesd’administration ont été testées. Dans une première partie, en lien avec projeteuropéen ODAK (Orphan drug for Acanthamoeba Keratitis), nos travaux ont suggéréqu’une concentration de collyre PHMB supérieure ou égale à 0,04% devait êtrepréférée. Dans une deuxième partie, nous avons pu montrer la supériorité duvoriconazole en collyre par rapport à la voie orale. Enfin, l’étude de lapharmacocinétique du voriconazole et du posaconazole après injections directesintracornéennes, démontre leur faible utilité en clinique humaine du fait de lafréquence nécessaire de réinjection, bien que des analyses complémentairesconcernant le posaconazole en collyre pour confirmer son intérêt soient nécessaires.L'ensemble de ces travaux pourrait permettre d’adapter les protocolesthérapeutiques de la KA. / Acanthamoeba keratitis (AK) is a rare and severe form of infectious keratitis,which is potentially sight-threatening. The infection is caused by Acanthamoeba spp.a common protozoan present in soil, air and water. Up to 85% of AK cases areassociated with contact lens wearing, more rarely after corneal injury.Currently, there are no agents approved for the treatment of AK in theEuropean Union or in the United States of America. In recent years, combinations ofunlicensed anti-amoebic agents such as biguanides and diamidines have been usedas the reference treatment. Treatment regimens and concentrations of active agentsare based on empirical data. Recently, voriconazole, a mono-triazole, wassuccessfully used to treat cases of human AK. Despite this, the ophthalmologicalcommunity is most often faced with severe forms of the disease with severemanagement difficulties and sometimes with a situation of therapeutic impasse. Thefunctional and anatomical loss of an eye can occur.Several agents and routes of administration have been tested in a rat model ofAK. First, as part of the European ODAK project (Orphan drug for AcanthamoebaKeratitis), our work suggested that a concentration of PHMB eye drops greater thanor equal to 0.04% should be preferred. Second, we were able to show the superiorityof voriconazole in eye drops compared to the oral route. Finally, our study on thepharmacokinetics of voriconazole and posaconazole after intrastromal injections,demonstrates their low utility in human because of the need for frequent reinjection.Nevertheless, additional analyses are necessary to confirm the interest ofposaconazole eye drops. All of this work could make it possible to adapt thetherapeutic protocols of AK.