The study purpose was to determine whether canine corneal cultures demonstrate superior growth when cultured in a fully defined epithelial selective medium, Epilife®, compared to Dulbecco's modification of Eagle's medium (DMEM) with fetal bovine serum (FBS), and to characterize cultured canine corneal cells. Superficial keratectomies were performed on three dogs. Samples were trypsinized to separate cell layers. Post-trypsinization, immunohistochemistry confirmed that epithelial cells had been released from the stroma. Both cell populations (presumed epithelial cells and stromal tissues) were cultured in DMEM with FBS or Epilife®. First passage cells were fixed for immunocytochemistry and prepared for PCR. Immunocytochemical staining for pancytokeratin, vimentin, and E-cadherin was evaluated, and immunofluorescence for zonula occludens-1 was attempted. Amplification of cytokeratin 5 (CK5) mRNA was assessed by PCR. Primary presumed epithelial cells grew faster when cultured in DMEM with FBS compared to Epilife®. Stromal tissue segments in Epilife® medium failed to adhere to culture plates, indicating that this medium may inhibit attachment and growth of non-epithelial tissues. Staining of corneal tissue segments confirmed that epithelial layers were pancytokeratin and E-cadherin positive, while stromal cells were vimentin positive. Immunocytochemistry of cultured cells revealed that epithelial cells stained positively for pancytokeratin, vimentin, and E-cadherin, while stromal cells remained only vimentin positive. Greater amplification of CK5 mRNA occurred from epithelial cells grown in Epilife® compared to epithelial cells in DMEM with FBS or the stromal cells. Based on PCR results, Epilife® medium may support retention of the epithelial characteristic of CK5 mRNA expression better than DMEM with FBS. / Master of Science

Yes / In the study of microbial keratitis, in vivo animal models often require a large number of animals, and in vitro monolayer cell culture does not maintain the three-dimensional structure of the tissues or cell-to-cell communication of in vivo models. Here, we propose reproducible ex vivo models of single- and dual-infection keratitis as an alternative to in vivo and in vitro models. / Wellcome Trust

This study aimed to create computer models to aid the diagnosis of keratoconus using an edge segmentation algorithm, wavelet transforms and machine-learning techniques based on biomechanical parameters of the cornea derived from Corvis ST. 102 normal eyes, and 73 eyes with keratoconus grade I and II for training and validation of the models were included. Initially the 31 original equipment parameters were studied to assess its power in separating the control groups and research group. Then, it was used the Canny algorithm for edges segmentation of the selected images, so that 400 central points of these images could be processed with the wavelet transforms and later with machine learning techniques. The best result was achieved using the Haar wavelet transform and a Multilayer Perceptron Neural Network, reaching the sensitivity of 84.93%, a specificity of 89.22% and an Area Under the ROC Curve (AUC) of 0932. It concludes that the developed models can help make the diagnosis of early keratoconus. / O presente trabalho objetivou criar modelos computacionais de auxílio ao diagnóstico de ceratocone, utilizando um algoritmo de segmentação de borda, transformadas de wavelet e técnicas de aprendizagem de máquina, baseados em parâmetros biomecânicos da córnea derivados do CorVis ST. Foram incluídos 102 olhos normais, e 73 olhos com ceratocone grau I e II para o treinamento e validação dos modelos criados. Inicialmente foram estudados os 31 parâmetros originais do equipamento, para avaliar seu poder em separar os grupos de controle e pesquisa. Foi então utilizado o algoritmo Canny para segmentação das bordas das imagens selecionadas, para que 400 pontos centrais extraídos dessas imagens pudessem ser processados com as transformadas de wavelet e posteriormente com técnicas de aprendizado de máquina. O melhor resultado foi alcançado utilizando a transformada de Wavelet do tipo Haar e uma Rede Neural Multilayer Perceptron, alcançando a sensibilidade de 84.93%, uma especificidade de 89.22% e uma Area Under de ROC Curve (AUC) de 0.932. Conclui-se que os modelos desenvolvidos podem contribuir para tornar o diagnóstico de ceratocone mais precoce.

Made available in DSpace on 2014-06-11T19:31:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-05-07Bitstream added on 2014-06-13T19:41:02Z : No. of bitstreams: 1 duqueortiz_jp_dr_jabo.pdf: 3328277 bytes, checksum: 59130c5ee41b6047be8d1bef9884147b (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Schering do Brasil / Estudaram-se os efeitos do BandVet® na reparação corneal em cães após ceratectomia superficial. Utilizaram-se 16 cães adultos, sem raça definida, machos ou fêmeas, sendo oito tratados com o BandVet® (BV) e os demais, com cloreto de sódio a 0,9%, (CO) subdivididos em grupos para avaliação clínica diária durante os 3, 7, 14 e 28 dias de pós-operatório. Foram atribuídos escores para a avaliação clínica quanto ao blefarospasmo, congestão conjuntival, vascularização, pigmentação e edema corneais. Encontraram-se blefarospasmo e fotofobia nos períodos iniciais de pósoperatório. Houve diferença significativa quanto à congestão conjuntival e secreção ocular aos sete dias de pós-operatório. A vascularização manteve-se constante desde os períodos iniciais, desaparecendo no período final, apenas no grupo tratado. O edema corneal esteve sempre presente em ambos os grupos, diferindo estatisticamente aos 14 dias de pós-operatório. Ao término dos períodos, os olhos ceratectomizados foram enucleados e as córneas submetidas a estudo morfométrico (contagem de células inflamatórias, contagem de pontos de colágeno, contagem de fibroblastos e mensuração da espessura do epitélio e estroma corneais), histoquímico (Alcian Blue pH 1.0 e Alcian Blue pH 2.5), de análise digital (avaliação cromática tridimensional de superfície) e imunoistoquímico (expressão da fibronectina e da tenascina). Relativamente à contagem das células inflamatórias, observou-se maior quantitativo nos animais do grupo BV, durante os quatro períodos avaliados. Relativamente aos pontos de colágeno, observou-se nas córneas dos animais do grupo BV, um maior número de pontos nos quatro períodos avaliados, havendo diferença estatística, comparativamente aos valores das áreas... / The effects of Bandvet® on corneal repair was studied after superficial keratectomy. Sixteen adult cross-breed dogs were used, being eight treated with Bandvet® (BV) and eight with saline 0.9% (CO). Daily clinical evaluations were accomplished 3, 7, 14 and 28 days in the post-operative period. Blepharospasm, conjuntival congestion, ocular discharge, photofobia, vascularization, pigmentation, and corneal edema were assessed by means of clinical scores. Blepharospasm and photofobia were noted at early stages of the post-operative period. Conjunctival congestion and ocular discharge changed significantly at day 7 of the post-operative period. Vascularization advanced axially in a constant manner in the early post-operative periods, disappearing in the late stages, only in the treated group. Corneal edema was observed in all time points in both of groups, changing significantly at day 14 of the postoperative period. At the completion of the study, keratectomized eyes were enucleated and corneas submitted to morphometrical evaluation (inflammatory cell count, collagen patches count, fibroblast count, and corneal epithelium and stromal thickness measurement) histochemistry (Alcian Blue pH 1.0 and Alcian Blue pH 2.5), digital analysis (tridimensional chromatic evaluation of the surface), and immunohistochemistry (fibronectin and tenascin labeling). During the 4 time points evaluated, inflammatory cell count was increased in the BV group. An enhanced number of collagen patches were observed in the corneas of the animals of the BV group, in the four time points evaluated, being significant in comparison to values observed in the transitional areas (P = 0.040). A higher fibroblast count was noted in the BV group at days 3, 14, 28, and 28. At day 7, however, the CO group showed higher... (Complete abstract click electronic access below)

Orientador: José Luiz Laus / Banca: Paola Castro Moraes / Banca: Cristiane dos Santos Honsho / Resumo: 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. / Abstract: 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. / Mestre

Purpose: Clinically evaluate a modified applanating surface Goldmann tonometer prism designed to substantially negate errors due to patient variability in biomechanics. Methods: A modified Goldmann prism with a correcting applanation tonometry surface (CATS) was mathematically optimized to minimize the intraocular pressure (IOP) measurement error due to patient variability in corneal thickness, stiffness, curvature, and tear film adhesion force. A comparative clinical study of 109 eyes measured IOP with CATS and Goldmann prisms. The IOP measurement differences between the CATS and Goldmann prisms were correlated to corneal thickness, hysteresis, and curvature. Results: The CATS tonometer prism in correcting for Goldmann central corneal thickness (CCT) error demonstrated a reduction to <+/- 2 mmHg in 97% of a standard CCT population. This compares to only 54% with CCT error <+/- 2 mmHg using the Goldmann prism. Equal reductions of similar to 50% in errors due to corneal rigidity and curvature were also demonstrated. Conclusion: The results validate the CATS prism's improved accuracy and expected reduced sensitivity to Goldmann errors without IOP bias as predicted by mathematical modeling. The CATS replacement for the Goldmann prism does not change Goldmann measurement technique or interpretation.

No description available.

Yes / The quantification of corneal endothelial cell (CEC) morphology using manual and semi-automatic software enables an objective assessment of corneal endothelial pathology. However, the procedure is tedious, subjective, and not widely applied in clinical practice. We have developed the CellsDeepNet system to automatically segment and analyse the CEC morphology. The CellsDeepNet system uses Contrast-Limited Adaptive Histogram Equalization (CLAHE) to improve the contrast of the CEC images and reduce the effects of non-uniform image illumination, 2D Double-Density Dual-Tree Complex Wavelet Transform (2DDD-TCWT) to reduce noise, Butterworth Bandpass filter to enhance the CEC edges, and moving average filter to adjust for brightness level. An improved version of U-Net was used to detect the boundaries of the CECs, regardless of the CEC size. CEC morphology was measured as mean cell density (MCD, cell/mm2), mean cell area (MCA, µm2), mean cell perimeter (MCP, µm), polymegathism (coefficient of CEC size variation), and pleomorphism (percentage of hexagonality coefficient). The CellsDeepNet system correlated highly significantly with the manual estimations for MCD (r = 0.94), MCA (r = 0.99), MCP (r = 0.99), polymegathism (r = 0.92), and pleomorphism (r = 0.86), with p

Yes / Corneal ulcers, a leading cause of blindness in the developing world are treated inappropriately without prior microbiology assessment because of issues related to availability or cost of accessing these services. In this work we aimed to develop a device for identifying the presence of Gram-positive or Gram-negative bacteria or fungi that can be used by someone without the need for a microbiology laboratory. Working with branched poly (N-isopropyl acrylamide) (PNIPAM) tagged with Vancomycin, Polymyxin B, or Amphotericin B to bind Gram-positive bacteria, Gram-negative bacteria and fungi respectively, grafted onto a single hydrogel we demonstrated specific binding of the organisms. The limit of detection of the microbes by these polymers was between 10 and 4 organisms per high power field (100X) for bacteria and fungi binding polymers respectively. Using ex vivo and animal cornea infection models infected with bacteria, fungi or both we than demonstrated that the triple functionalised hydrogel could pick up all 3 organisms after being in place for 30 min. To confirm the presence of bacteria and fungi we used conventional microbiology techniques and fluorescently labelled ligands or dyes. While we need to develop an easy-to-use either a colorimetric or an imaging system to detect the fluorescent signals, this study presents for the first time a simple to use hydrogel system, which can be applied to infected eyes and specifically binds different classes of infecting agents within a short space of time. Ultimately this diagnostic system will not require trained microbiologists for its use and will be used at the point-of-care. / We gratefully acknowledge support for this research by the Well- come Trust which provided funding for Shivshetty, Swift and Pinnock (Grant 0998800/B/12/Z).

La dystrophie cornéenne endothéliale de Fuchs (FECD, pour l’abréviation du terme anglais « Fuchs endothelial corneal dystrophy ») est une maladie de l'endothélium cornéen. Sa pathogenèse est mal connue. Aucun traitement médical n’est efficace. Le seul traitement existant est chirurgical et consiste dans le remplacement de l’endothélium pathologique par un endothélium sain provenant de cornées de la Banque des yeux. Le traitement chirurgical, en revanche, comporte 10% de rejet immunologique. Des modèles expérimentaux sont donc nécessaires afin de mieux comprendre cette maladie ainsi que pour le développement de traitements alternatifs. Le but général de cette thèse est de développer un modèle expérimental de la FECD en utilisant le génie tissulaire. Ceci a été réalisé en trois étapes. 1) Tout d'abord, l'endothélium cornéen a été reconstruit par génie tissulaire en utilisant des cellules endothéliales en culture, provenant de patients atteints de FECD. Ce modèle a ensuite été caractérisé in vitro. Brièvement, les cellules endothéliales cornéennes FECD ont été isolées à partir de membranes de Descemet prélevées lors de greffes de cornée. Les cellules au deuxième ou troisième passages ont ensuite été ensemencées sur une cornée humaine préalablement décellularisée. Suivant 2 semaines de culture, les endothélia cornéens reconstruits FECD (n = 6) ont été évalués à l'aide d'histologie, de microscopie électronique à transmission et d’immunomarquages de différentes protéines. Les endothélia cornéens reconstruits FECD ont formé une monocouche de cellules polygonales bien adhérées à la membrane de Descemet. Les immunomarquages ont démontré la présence des protéines importantes pour la fonctionnalité de l’endothélium cornéen telles que Na+-K+/ATPase α1 et Na+/HCO3-, ainsi qu’une expression faible et uniforme de la protéine clusterine. 2) Deux techniques chirurgicales (DSAEK ; pour « Descemet stripping automated endothelial keratoplasty » et la kératoplastie pénétrante) ont été comparées pour la transplantation cornéenne dans le modèle animal félin. Les paramètres comparés incluaient les défis chirurgicaux et les résultats cliniques. La technique « DSAEK » a été difficile à effectuer dans le modèle félin. Une formation rapide de fibrine a été observée dans tous les cas DSAEK (n = 5). 3) Finalement, la fonctionnalité in vivo des endothélia cornéens reconstruits FECD a été évaluée (n = 7). Les évaluations in vivo comprenaient la transparence, la pachymétrie et la tomographie par cohérence optique. Les évaluations post-mortem incluaient la morphométrie des cellules endothéliales, la microscopie électronique à transmission et des immunomarquage de protéines liées à la fonctionnalité. Après la transplantation, la pachymétrie a progressivement diminué et la transparence a progressivement augmenté. Sept jours après la transplantation, 6 des 7 greffes étaient claires. La microscopie électronique à transmission a montré la présence de matériel fibrillaire sous-endothélial dans toutes les greffes d’endothelia reconstruits FECD. Les endothélia reconstruits exprimaient aussi des protéines Na+-K+/ATPase et Na+/HCO3-. En résumé, cette thèse démontre que les cellules endothéliales de la cornée à un stade avancé FECD peuvent être utilisées pour reconstruire un endothélium cornéen par génie tissulaire. La kératoplastie pénétrante a été démontrée comme étant la procédure la plus appropriée pour transplanter ces tissus reconstruits dans l’œil du modèle animal félin. La restauration de l'épaisseur cornéenne et de la transparence démontrent que les greffons reconstruits FECD sont fonctionnels in vivo. Ces nouveaux modèles FECD démontrent une réhabilitation des cellules FECD, permettant d’utiliser le génie tissulaire pour reconstruire des endothelia fonctionnels à partir de cellules dystrophiques. Les applications potentielles sont nombreuses, y compris des études physiopathologiques et pharmacologiques. / Fuchs endothelial corneal dystrophy (FECD) is a primary disease of the corneal endothelium. Its pathogenesis is poorly understood. No medical treatment is effective. Surgical treatment (the only available treatment) carries 10% of immunogenic rejection. Experimental models are needed in order to better understand the disease and to investigate potential autologous treatments (to prevent immunogenic rejection). The overall goal of this thesis is to develop an experimental model for FECD using tissue engineering. This was achieved in three steps. 1) An in vitro tissue-engineered FECD model was created and characterized. Briefly, Descemet’s membranes from patients with late-stage FECD undergoing Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK) were used to isolate and culture FECD endothelial cells. Second or third-passaged FECD endothelial cells were seeded on a previously decellularized human cornea. After 2 weeks in culture, TE-FECD corneas (n=6) were assessed using histology, transmission electron microscopy (TEM) and immunofluorescence labeling of various proteins. TE-FECD endothelium yielded a monolayer of polygonal cells well adhered to Descemet’s membrane. The TE-FECD corneal endothelium expressed the function-related proteins Na+-K+/ATPase α1 and Na+/HCO3-. Clusterin expression was faint and uniform. 2) In order to determine the best surgical procedure to transplant the TE-FECD corneas in the feline model, a DSAEK procedure was evaluated and compared to penetrating keratoplasty technique. DSAEK assessments included surgical challenges and clinical outcomes. DSAEK technique was challenging to perform in the feline model. Rapid fibrin formation was observed in all DSAEK cases (n=5). 3) The in vivo functionality of the TE-FECD corneas was assessed. TE-FECD corneas were grafted in the feline model (n=7) using penetrating keratoplasty procedure and observed for seven days. In vivo assessments included transparency, pachymetry, optical coherence tomography, endothelial cell morphometry, TEM and immunostaining of function-related proteins. After transplantation, pachymetry gradually decreased and transparency gradually increased. Seven days after transplantation, 6 out of 7 grafts were clear. Post-mortem TEM showed subendothelial loose fibrillar material deposition in all TE-FECD grafts. The TE grafted endothelium expressed Na+-K+/ATPase and Na+/HCO3-. This thesis demonstrates that endothelial cells from late-stage FECD corneas can be used to engineer a corneal endothelium. Compared to DSEAK, penetrating keratoplasty is a more appropriate procedure for corneal transplantation in the feline model, since the DSAEK procedure in the feline model presently yields inconsistent clinical results. Restoration of corneal thickness and transparency demonstrates that the TE-FECD grafts are functional in vivo. This novel FECD living model suggests a potential role of tissue engineering for FECD cell rehabilitation. Potential applications are numerous, including pathophysiological and pharmacological studies.