Mammalian corneal endothelial cells are considered to be non-proliferative due to the arrest of cells at the G1 phase of the cell cycle. The purpose of this study was to determine whether the down regulation of cyclin dependant kinase inhibitors (p21cip1 and p27kip1) levels by Endothelin-1 (ET-1), would overcome the G1 phase arrest and promote cell cycle progression and proliferation in cultured BCECs (Bovine corneal endothelial cells). BCECs were isolated from bovine corneas and cultured in DMEM supplemented with 10% serum. 5-Bromo 2-deoxy Uridine (BrdU) incorporation was determined in serum starved cultures in 24-well plates as a measure of cell proliferation. Confluent serum starved cells grown in T-25 flasks were treated with 100nM Endothelin-1 in DMEM. The control cells were left untreated in serum free medium. Total cellular protein was isolated using RIPA buffer and quantified according to the Peterson modification of the Lowry method. The level of expression of p21cip1 and p27kip1 proteins relative to β-actin was determined by western blotting technique. Immuno fluorescent localization of p27kip1 was performed using polyclonal anti-p27kip1 and anti-p21cip1 antibodies in confluent and growing cells. An increase in cell proliferation was observed in sub-confluent cultures with Endothelin-1 treatment. This evidence was supported by an increase (~18%) in BrdU incorporation in response to Endothelin-1. Densitometry analysis of immunoblots revealed an increase in the expression of p27kip1 in confluent cell cultures when compared to sub-confluent, dividing cells. p21cip1 was almost undetectable in sub-confluent, actively dividing cultures. Immuno fluorescent analysis revealed that the nuclear staining of p27kip1 was apparently decreased with ET-1 treatment. In conclusion, Endothelin-1 treatment resulted in decrease in p27kip1 and p21cip1 expression in confluent cultures that was greatest at 30 hr of post incubation with Endothelin-1. Endothelin-1 appears to promote cell proliferation. Expression of p27kip1 and p21cip1 was greatly reduced in actively dividing BCECs. Endothelin-1 treatment down-regulated these cyclin dependent kinase inhibitors and may promote cell cycle progression via this mechanism.

This thesis presents a novel system that estimates the point-of-gaze (where a person is looking at) remotely while allowing for free head movements and minimizing personal calibration requirements. The point-of-gaze is estimated from the pupil and corneal reflections (virtual images of infrared light sources that are formed by reflection on the front corneal surface, which acts as a convex mirror) extracted from eye images captured by video cameras. Based on the laws of geometrical optics, a detailed general mathematical model for point-of-gaze estimation using the pupil and corneal reflections is developed. Using this model, the full range of possible system configurations (from one camera and one light source to multiple cameras and light sources) is analyzed. This analysis shows that two cameras and two light sources is the simplest system configuration that can be used to reconstruct the optic axis of the eye in 3-D space, and therefore measure eye movements, without the need for personal calibration. To estimate the point-of-gaze, a simple single-point personal calibration procedure is needed. The performance of the point-of-gaze estimation depends on the geometrical arrangement of the cameras and light sources and the method used to reconstruct the optic axis of the eye. Using a comprehensive simulation framework developed from the mathematical model, the performance of several gaze estimation methods of varied complexity is investigated for different geometrical system setups in the presence of noise in the extracted eye features, deviation of the corneal shape from the ideal spherical shape and errors in system parameters. The results of this investigation indicate the method(s) and geometrical setup(s) that are optimal for different sets of conditions, thereby providing guidelines for system implementation. Experimental results with adults, obtained with a system that follows those guidelines, exhibit RMS point-of-gaze estimation errors of 0.4-0.6º of visual angle (comparable to the best commercially available systems, which require multiple-point personal calibration procedures). Preliminary results with infants demonstrate the ability of the proposed system to record infants' visual scanning patterns, enabling applications that are very difficult or impossible to carry out with previously existing technologies (e.g., study of infants' visual and oculomotor systems).

Cytotoxicity of Multi-Purpose Solutions (MPS) is commonly tested on cells using diluted MPS or extracts from MPS soaked contact lenses. There is evidence that lens type will affect uptake and release of compounds contained in MPS. To assess the cytotoxicity of agents contained in MPS that would be released by contact lens, an in vitro “onlay” model was used, whereby MPS soaked silicone hydrogel lenses were directly set onto a confluent monolayer of corneal cells. Chapter 4 describes the impact of MPS released from contact lenses on immortalized human corneal epithelial cells. MPS-soaked lens interactions with cells were characterized by studying cell viability, cell adhesion and caspase assays. In Chapter 5, mechanisms of cell death induced by exposure to MPS from contact lenses were determined through evaluation of apoptotic markers, such as activation of caspase 3 and 9. In Chapter 6, the impact of the physical properties of silicone hydrogel lenses, specifically surface treatments, on cytotoxicity of MPS were investigated. The development of methods for characterizing the release of MPS from lenses, using absorbance spectra, is also described. The results indicate that exposure to contact lenses soaked in Opti-Free Express (OFX) and ReNu not only induces cell death in vitro, but also has an adverse effect on adhesion phenotype, suggesting that the remaining cells may have a compromised epithelial structure. Borate- buffered MPS were found to be more cytotoxic than phosphate-buffered base solutions. Investigation of the mechanisms of cell death revealed that ReNu and OFX induced corneal epithelial cell death in vitro using different pathways, whereby ReNu induced a necrotic pathway while OFX-induced cell death was mediated by the intrinsic pathway of apoptosis. The in vitro model was also able to identify differences between silicone hydrogels with different surface treatments: the different surface treatments and chemistries of silicone hydrogels lens will affect the release profile of MPS and hence their potential cytotoxicity. By investigating the induction of cell death processes by solution-lens combinations in vitro, we aim to prevent potential adverse effects in the cornea, which may ultimately compromise various visual and barrier functions. The findings indicate the wealth of information in vitro cytotoxicity testing can provide when evaluating the toxicological profile of MPS.

Cytotoxicity of Multi-Purpose Solutions (MPS) is commonly tested on cells using diluted MPS or extracts from MPS soaked contact lenses. There is evidence that lens type will affect uptake and release of compounds contained in MPS. To assess the cytotoxicity of agents contained in MPS that would be released by contact lens, an in vitro “onlay” model was used, whereby MPS soaked silicone hydrogel lenses were directly set onto a confluent monolayer of corneal cells. Chapter 4 describes the impact of MPS released from contact lenses on immortalized human corneal epithelial cells. MPS-soaked lens interactions with cells were characterized by studying cell viability, cell adhesion and caspase assays. In Chapter 5, mechanisms of cell death induced by exposure to MPS from contact lenses were determined through evaluation of apoptotic markers, such as activation of caspase 3 and 9. In Chapter 6, the impact of the physical properties of silicone hydrogel lenses, specifically surface treatments, on cytotoxicity of MPS were investigated. The development of methods for characterizing the release of MPS from lenses, using absorbance spectra, is also described. The results indicate that exposure to contact lenses soaked in Opti-Free Express (OFX) and ReNu not only induces cell death in vitro, but also has an adverse effect on adhesion phenotype, suggesting that the remaining cells may have a compromised epithelial structure. Borate- buffered MPS were found to be more cytotoxic than phosphate-buffered base solutions. Investigation of the mechanisms of cell death revealed that ReNu and OFX induced corneal epithelial cell death in vitro using different pathways, whereby ReNu induced a necrotic pathway while OFX-induced cell death was mediated by the intrinsic pathway of apoptosis. The in vitro model was also able to identify differences between silicone hydrogels with different surface treatments: the different surface treatments and chemistries of silicone hydrogels lens will affect the release profile of MPS and hence their potential cytotoxicity. By investigating the induction of cell death processes by solution-lens combinations in vitro, we aim to prevent potential adverse effects in the cornea, which may ultimately compromise various visual and barrier functions. The findings indicate the wealth of information in vitro cytotoxicity testing can provide when evaluating the toxicological profile of MPS.

Ocular neovascularization (NV) associated with corneal NV, ischemic retinopathies and age-related macular degeneration is a leading cause of severe vision loss. While numerous contributing factors have been identified, the potential role of the CD36 scavenger receptor has been largely overlooked notwithstanding its crucial involvement in normal retinal function. Accordingly, the central aim of this work was to elucidate the contribution and regulation of CD36 during ocular NV using the cornea as a model. / Initial work investigating the role of CD36 10 maintaining corneal avascularity, an important feature of the normal cornea, revealed that genetic ablation of CD36 elicits age-related corneal NV. Subsequent studies using a pathophysiologically relevant model of inflammatory corneal NV showed constitutive expression of CD36 in the normal cornea with marked induction in the neovascularized cornea. Importantly, activation of CD36 suppressed and induced regression of corneal NV, effects that proceeded via concerted inhibition of VEGFA, JNK-1, and cJun. / Because hypoxia is a fundamental stimulus for angiogenesis, it was pertinent to explore the role and regulation of CD36 during hypoxia. We demonstrate that CD36 expression was significantly elevated in hypoxia-exposed corneal and retinal tissue and in hypoxic retinal pigment epithelial cells. Essential contributions of hypoxia-inducible factor (HIF)-1 and reactive oxygen species were also established. Functional consequences were depicted by augmentations in CD36 phagocytic and anti-angiogenic activities. / Collectively, data disclose CD36 as an important modulator of corneal avascularity and inflammatory corneal NV; this imparts several interesting avenues for future research on the involvement of CD36 in neovascular diseases of the eye. Novel data further identify CD36 as a hypoxia and HIF-1 regulated gene thus creating a framework for future elucidation of the regulatory aspects of this receptor.

Access to primary human corneal endothelial cells (HCEC) is limited and donor-derived differences between cultures exacerbate the issue of data reproducibility, whereas cell lines can provide sufficient numbers of homogenous cells for multiple experiments. An immortalized HCEC population was adapted to serum-free culture medium and repeated cloning was performed. Clonally grown cells were propagated under serum-free conditions and growth curves were recorded. Cells were characterized immunocytochemically for junctional proteins, collagens, Na,K-ATPase and HCEC-specific 9.3.E-antigen. Ultrastructure was monitored by scanning and transmission electron microscopy. Two clonal cell lines, HCEC-B4G12 and HCEC-H9C1, could be isolated and expanded, which differed morphologically: B4G12 cells were polygonal, strongly adherent and formed a strict monolayer, H9C1 cells were less adherent and formed floating spheres. The generation time of B4G12 cells was 62.26 ± 14.5 h and that of H9C1 cells 44.05 ± 5.05 h. Scanning electron microscopy revealed that B4G12 cells had a smooth cell surface, while H9C1 cells had numerous thin filopodia. Both cell lines expressed ZO-1 and occludin adequately, and little but well detectable amounts of connexin-43. Expression of HCEC-specific 9.3.E-antigen was found commensurately in both cell lines, while expression of Na,K-ATPase α1 was higher in H9C1 cells than in B4G12 cells. B4G12 cells expressed collagen IV abundantly and almost no collagen III, while H9C1 cells expressed both collagens at reasonable amounts. It is concluded that the clonal cell line B4G12 represents an ideal model of differentiated HCEC, while H9C1 may reflect features of developing or transitional HCEC. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Nous proposons de construire un atlas numérique 3D contenant les caractéristiques moyennes et les variabilités de la morphologie d’un organe. Nos travaux seront appliqués particulièrement à la construction d'un atlas numérique 3D de la totalité de la cornée humaine incluant la surface antérieure et postérieure à partir des cartes topographiques fournies par le topographe Orbscan II. Nous procédons tout d'abord par normalisation de toute une population de cornées. Dans cette étape, nous nous sommes basés sur l'algorithme de recalage ICP (iterative closest point) pour aligner simultanément les surfaces antérieures et postérieures d'une population de cornée vers les surfaces antérieure et postérieure d'une cornée de référence. En effet, nous avons élaboré une variante de l'algorithme ICP adapté aux images (cartes) de cornées qui tient compte de changement d'échelle pendant le recalage et qui se base sur la recherche par voisinage via la distance euclidienne pour établir la correspondance entre les points. Après, nous avons procédé pour la construction de l'atlas cornéen par le calcul des moyennes des élévations de surfaces antérieures et postérieures recalées et leurs écarts-types associés. Une population de 100 cornées saines a été utilisée pour construire l'atlas cornéen normal. Pour visualiser l’atlas, on a eu recours à des cartes topographiques couleurs similairement à ce qu’offrent déjà les systèmes topographiques actuels. Enfin, des observations ont été réalisées sur l'atlas cornéen reflétant sa précision et permettant de développer une meilleure connaissance de l’anatomie cornéenne. / We propose to build a 3D digital atlas which contains the average characteristics and variability of the morphology of an organ. In particular our work consists in the construction of a 3D digital atlas of the entire human cornea including anterior and posterior surfaces. The atlas was built using topographies provided by the Orbscan II system. First, we normalized the given population of corneas using a variant of the ICP (iterative closest point) algorithm for shape registration to fit simultaneously the anterior and posterior surfaces with the anterior and posterior surfaces of a reference cornea. Indeed, we developed a specific algorithm for corneas topographies that considers scaling during registration and which is based on neighborhood search via the Euclidean distance to find the correspondence between points. After that, we built the corneal atlas by averaging elevations of anterior and posterior surfaces and by calculating their associated standard deviations. A population of 100 healthy corneas was used to construct the normal corneal atlas. To illustrate the atlas, we used topographic color maps like those already offered by existing topographic systems. Finally, observations were made on the corneal atlas that reflects its precision and allows to develop a better understanding of corneal anatomy.

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Added 1 bitstream(s) on 2015-08-11T03:25:36Z : No. of bitstreams: 4 Publico-026a.pdf: 72550 bytes, checksum: afe0787528f227bc8c799cf1cd67fcc7 (MD5) Publico-026b.pdf: 2073659 bytes, checksum: b85cc4a971ac414e9e8aa01daa4ad8ed (MD5) Publico-026c.pdf: 2083167 bytes, checksum: 806646b87026c136bf8b329233fe315b (MD5) Publico-026d.pdf: 895113 bytes, checksum: 37de279e78fead2b9aa3c17fd44ab0f0 (MD5) / Objetivos: Comparar os resultados de acuidade visual, refração estática, aberrometria e sensibilidade ao contraste em olhos com miopia moderada submetidos à ceratectomia fotorrefrativa (PRK) com mitomicina C (MMC) ou à ceratomileuse assistida por excimer laser in situ (LASIK) em cirurgias guiadas por frente de onda durante acompanhamento de um ano. Avaliar o aspecto biomicroscópico nos dois grupos durante seguimento. Avaliar subjetivamente percepção de dor, queixas visuais e satisfação com resultado cirúrgico nos dois grupos durante acompanhamento. Analisar índices de microscopia especular nos dois grupos antes e seis meses após cirurgia. Comparar propriedades biomecânicas da córnea nos dois grupos um ano após o procedimento cirúrgico. Métodos: Quarenta e quatro pacientes (88 olhos) com miopia moderada e cálculo de consumo corneano maior que 50 μm na plataforma LADARWave 4000 (Alcon) em ambos os olhos foram selecionados para receber aleatoriamente LASIK em um olho e PRK com aplicação de MMC 0,002% durante um minuto no olho contralateral em cirurgias guiadas por frente de onda. Topografia corneana (EyeSys 2000, EyeSys e Orbscan II, Orbtek/Bausch & Lomb), acuidade visual sem correção (AVSC, tabela Early Treatment Diabetic Retinopaty Study), refração estática, acuidade visual com correção (AVCC), aberrometria (LADARWave 4000), paquimetria ultrassônica corneana central (Sonogage) e exame oftalmológico completo foram realizados no pré-operatório e no seguimento de um, três, seis e doze meses pós-operatório. Sensibilidade ao contraste fotópica e mesópica (Optec 6500, F.A.C.T.; Stereo Optical) com correção foram realizadas nos dois olhos antes da cirurgia e três, seis e doze meses após. Questionário subjetivo de dor foi aplicado no pós-operatório recente, e questionário de sintomas visuais e satisfação com o procedimento em cada olho foi aplicado nas visitas de acompanhamento com um, três, seis e doze meses de pós-operatório. Biomicroscopia de segmento anterior foi realizada no período pós-operatório recente e nas visitas de acompanhamento sempre como último exame do dia (examinador mascarado para procedimento cirúrgico). Microscopia especular (Topcon SP 2000p) foi realizada antes e seis meses após cirurgia. Avaliação biomecânica da córnea (ORA, Reichert) foi realizada um ano após o procedimento cirúrgico. Os testes ANOVA para medidas repetidas e t de student foram utilizados para análise estatística. Resultados: A média de idade dos pacientes do estudo foi de 31,7 anos (variou entre 21 e 54 anos). Não houve diferença significativa entre os grupos antes da cirurgia quanto a AVSC, AVCC, aberrometria, sensibilidade ao contraste ou microscopia especular. O equivalente esférico (EE) médio programado nos olhos que receberam LASIK foi de - 3,99±1,20 dioptrias (D) e de - 3,85±1,12 D nos olhos que receberam PRK com MMC (p>0,05). A profundidade de ablação média foi de 73,09±14,55 μm e 70,70±14,07 μm, no grupo LASIK e no grupo PRK com MMC, respectivamente (p>0,05). Quarenta e dois pacientes (95,5%) completaram um ano de acompanhamento. Os olhos que receberam PRK com MMC apresentaram média de AVSC significativamente superior aos olhos que receberam LASIK com três, seis e doze meses de pós-operatório. A média de AVCC também foi estatisticamente superior no grupo PRK com MMC na visita de um ano de pós-operatório (p<0,05). Não houve diferença estatística entre os grupos quanto ao EE ao longo do acompanhamento. Todos os olhos que receberam PRK com MMC completaram a reepitelização corneana em até cinco dias após o procedimento, e nenhum olho apresentou haze maior que grau 1 (escala de Fantes). Os olhos que receberam LASIK apresentaram valores de aberrações de baixa e alta ordem estatisticamente superiores aos olhos que receberam PRK com MMC durante todo o acompanhamento (p<0.05). Os olhos que receberam PRK com MMC obtiveram desempenho superior no teste de sensibilidade ao contraste em condições fotópicas e mesópicas quando comparados ao grupo LASIK durante seguimento (p<0,05). Até o quinto dia de pós-operatório, o grupo PRK com MMC apresentou índices de dor superiores ao grupo LASIK. O grupo PRK com MMC foi melhor avaliado no questionário subjetivo de queixas visuais e satisfação cirúrgica. Não houve diferença estatística entre os grupos quanto à microscopia especular (p>0,05). Na avaliação biomecânica da córnea, o grupo LASIK apresentou valores de fator de resistência corneana (CRF) e histerese (CH) significativamente superiores ao grupo PRK com MMC (p<0,05). Conclusões: Os olhos que receberam PRK com MMC apresentaram melhores valores de AVSC e AVCC, melhor correção de aberrações de baixa ordem e menores valores de aberrações de alta ordem em relação aos olhos que receberam LASIK. O grupo PRK com MMC também apresentou valores superiores de sensibilidade ao contraste e foi melhor avaliado em questionário subjetivo de satisfação cirúrgica. Não houve presença de haze clinicamente significativo no grupo PRK com MMC. O grupo PRK com MMC apresentou maiores índices de dor no período pósoperatório recente. Não houve diferença entre os índices de microscopia especular nos dois grupos. O grupo LASIK apresentou índices superiores de CRF e CH. / Purpose: To compare visual acuity results, cycloplegic refraction, aberrometry and contrast sensitivity in eyes that underwent photorefractive keratectomy (PRK) with mitomycin C (MMC) or laser in situ keratomileusis (LASIK) for wavefront-guided myopic corrections during one year follow-up. To evaluate slit-lamp microscopy in both groups during follow-up. To evaluate subjective pain, visual complains and satisfaction with visual results in the two groups during follow-up. To analyse specular microscopy values before and six months after surgeries in both groups. To compare biomechanical properties of the cornea in the two groups one year after surgeries. Methods: Forty-four patients (88 eyes) with moderate myopia and an estimated ablation depth greater than 50 μm using the LADARWave 4000 (Alcon Laboratories) platform in both eyes were randomized to receive LASIK in one eye and PRK with application of MMC 0.002% for one minute in the fellow eye in wavefront-guided surgeries. Corneal topography (EyeSys 2000, EyeSys and Orbscan II, Orbtek/Bausch & Lomb), uncorrected visual acuity (UCVA, Early Treatment Diabetic Retinopaty Study table), cycloplegic refraction, best spectacle-corrected visual acuity (BSCVA), aberrometry (LADARWave 4000), central ultrasound corneal pachymetry (Sonogage Inc) and a comprehensive ophthalmologic examination were performed before surgeries and at one, three, six and twelve months postoperative visits. Photopic and mesopic contrast sensitivity (Optec 6500, F.A.C.T.; Stereo Optical Co) with BSCVA was performed in both eyes before surgeries and at three, six and 12 months follow-up. A subjective pain questionnaire was applied at early postoperative visits and another visual complain and satisfaction questionnaire with surgery in each eye was applied one, three, six and twelve months after surgical procedures. Slit-lamp anterior segment microscopy was performed at early postoperative visits and at follow-up visits always as the last examination (blind examiner for surgical procedure). Specular microscopy (Topcon SP 2000p, Topcon) was performed before and six months after surgeries. Biomechanical properties of the cornea (ORA, Reichert) were evaluated one year after surgeries. The tests ANOVA for repeated measures and the student’s t test were used for statistical analyses. Results: The mean age was 31.7 years (range, 21-54 years). There was no statistically significant between-group difference in UCVA, BSCVA, aberrometry, contrast sensitivity or specular microscopy before surgeries. The mean attempted spherical equivalent (SE) was - 3.99±1.20 diopters (D) in LASIK eyes and - 3.85±1.12 D in PRK with MMC eyes (p>0.05). The mean ablation depth was 73.09±14.55 μm and 70.70±14.07 μm in LASIK and PRK with MMC eyes, respectively (p>0.05). Forty-two patients (95.5%) completed one year follow-up. The PRK with MMC eyes presented statistically significant better mean UCVA values than LASIK eyes at three, six and 12 months visits. The mean BSCVA was also statistically significant better in PRK with MMC eyes than in LASIK eyes one year after surgeries (p<0.05). There was no between-groups statistical difference in SE during one year follow-up. All PRK with MMC eyes reepithelialized within five days after surgical procedure and no eye presented more than grade 1 haze (Fantes scale). The LASIK eyes presented statistically significant higher lower and higher order aberrations values than PRK with MMC eyes during follow-up (p<0.05). The PRK with MMC group showed better results in photopic and mesopic contrast sensitivity than LASIK eyes during one year follow-up (p<0.05). Until the fifth postoperative day, PRK with MMC eyes presented higher pain scores than LASIK eyes. PRK with MMC eyes were better rated in terms of subjective visual symptoms and visual satisfaction. There were no statistical differences between the groups in specular microscopy (p>0.05). LASIK eyes showed statistically significant higher corneal resistance factor (CRF) and corneal hysteresis (CH) values than PRK with MMC eyes (p<0.05). Conclusions: The PRK with MMC eyes presented better UCVA, BSCVA, better correction of lower order aberrations and lower higher order aberration values than LASIK eyes. It also showed better contrast sensitivity results and was better rated in terms of visual satisfaction. There was no clinically significant haze in PRK with MMC eyes. The PRK with MMC eyes presented higher pain scores at early postoperative visits. There was no between groups differences in specular microscopy. LASIK eyes presented higher CRF and CH values one year after surgeries. / TEDE / BV UNIFESP: Teses e dissertações

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

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The bone marrow (BM)adult stem cell, mainly the mononuclear cell (MC) fraction, calls great interest in tissue regeneration researches in veterinary medicine, among those, works with experimental corneal ulcers. It is believed that the limbus represents the main stem cell source to replace damaged corneal cells, however, the number of animals showing limbal deficiency with subsequent conjunctivalization increases every day. This condition can be developed for several reasons, where the alkali burn is the most common. Aiming to conduct a study about transplanted BM MC presence confirmation, the homing process and to histopathologically compare the treated and sham groups, an experimental alkali corneal ulcer model, associated with the autologous MC transplant was used. Sixteen male or female, stray dogs, weithing around 10kg were submitted to experimental corneal ulcer, where the sodium hydroxide soaked filter discs alkali burn model was used. After the lesions, the animal were then submitted to subonjunctival autologous BM MC transplantation. The mononuclear fraction was isolated by density gradient centrifugation at 678G for 30 minutes and marked with nanocrystals to engrafment and homing follow-up. The pos-operatory evaluation was made with imunofluorescence exams in the sixth day after the transplant and for histopathology after 15 days of the same procedure where it could be noticed that the MC fixed in the damaged area, there was no homing phenomenom and despite they reduce the local inflamation, the MC did not help the corneal epithelium cicatrization process in this short term evaluation. Thus BM MC transplant studies in corneal wounds are indicated. / As células-tronco adulta da medula óssea (MO), em particular a fração de células mononucleares (CM), despertam grande interesse nas pesquisas que visam regeneração tecidual na medicina veterinária, dentre elas, estudos com úlceras de córnea experimentais. Atualmente, acredita-se que o limbo representa a fonte de células-tronco para a reposição de células corneanas lesadas, no entanto, o número de animais que apresentam deficiência límbica com conjuntivalização subseqüente aumenta a cada dia. Essa condição pode ser desenvolvida por diversas afecções, em que a queimadura por base é a mais comum. Visando realizar um estudo sobre a confirmação da presença das CM da MO transplantadas, da ocorrência de quimiotaxia (homing) das mesmas e comparar histopatologicamente os grupos tratados e controles, utilizou-se um modelo experimental de úlcera de córnea associado ao autotransplante de CM. Dezesseis cães machos ou fêmeas, sem raça definida, com peso ao redor de 10kg foram submetidos à ulceração experimental de córnea, em que o modelo de queimadura ocular por álcali com papel filtro embebido em hidróxido de sódio foi utilizado. Após a realização das lesões, os animais foram submetidos ao transplante subconjuntival de CM da MO, previamente isoladas. A fração mononuclear foi separada por centrifugação da MO com gradiente de densidade a 678G por 30 minutos e marcada com nanocristais para posterior acompanhamento dos processos de pega e quimiotaxia. A avaliação pós-operatória foi realizada por exames de imunofluorescência no sexto dia após o transplante e por histopatologia passados 15 dias do mesmo procedimento, quando pode se notar que as CM fixaram-se na região lesionada, não sofreram quimiotaxia e, apesar de diminuírem a inflamação do local, não auxiliaram no processo de cicatrização do epitélio corneano em curto prazo. Assim, sugere-se estudos adicionais no transplante de CM da MO na cicatrização da córnea .