Les vaisseaux rétiniens sont accessibles par la visualisation du fond d'œil grâce notamment aux rétinophotographies. Cette visualisation donne donc un accès direct à l'étude de la microcirculation humaine. Depuis 1999, le logiciel IVAN® permet une analyse «semi-automatique» du calibre vasculaire rétinien. L'analyse de la littérature retrouve que les variations du calibre vasculaire rétinien sont le reflet cumulatif du poids de naissance, de facteurs génétiques, de l'âge, des facteurs de risques cardiovasculaires, de la fonction rénale et de l'inflammation. Les méta-analyses d'études épidémiologiques suggèrent un rôle prédictif des calibres vasculaires rétiniens sur les évènements cardiovasculaires : un calibre artériel rétinien étroit serait prédictif de maladie coronaire chez les femmes et augmenterait la mortalité cardiovasculaire globale ; un large calibre veineux serait prédictif d'accident vasculaire cérébral. Cette thèse a pour objectif d'approfondir les connaissances sur les déterminants des calibres vasculaires rétiniens chez les sujets indemnes d'athérosclérose clinique (antécédent de maladie coronaire, d'accident vasculaire cérébral, d'artériopathie des membres inférieurs). Depuis 2007, en partenariat avec l'université de Melbourne, nous avons acquis le logiciel IVAN® et l'agrément permettant son utilisation en recherche clinique et épidémiologique a été obtenu en 2008. Dans un premier article, nous avons mis en évidence une relation linéaire entre calibre artériel et veineux et fonction rénale mesurée par des méthodes gold standard (filtration glomérulaire mesurée par la clairance de 99mTc-DTPA et ratio albuminurie/créatinurie) sur 80 sujets de la cohorte de médecine interne-hypertension artérielle de l'hôpital Lapeyronie, suggérant des déterminants communs de ces lésions précoces d'organes cibles. Dans un deuxième article, nous avons tout d'abord confirmé le lien entre dilatation veineuse rétinienne et inflammation systémique, puis observé une relation entre calibre artériel étroit et stress oxydatif sur les participants de la cohorte POLA (pathologies oculaires liées à l'âge). Cela est d'une importance particulière car cela suggère que la microcirculation rétinienne, dont des associations avec la macrocirculation carotidienne et cardiaque ont été retrouvées, serait particulièrement sensible au stress oxydatif et à l'inflammation, de façon indépendante des facteurs de risques cardiovasculaires. Dans un troisième article, nous avons retrouvé une relation entre malnutrition biologique et dilatation veineuse rétinienne sur les participants de la cohorte POLA. Cela suggère que des remaniements microcirculatoires précoces pourraient être un mécanisme associé à l'augmentation du risque cardiovasculaire observé chez les sujets âgés souffrants de malnutrition. Les caractéristiques techniques du logiciel IVAN® limitent sa généralisation en médecine, mais son utilisation permet d'approfondir les connaissances sur les déterminants des variations de calibre des vaisseaux de la rétine. Le futur challenge des études sur la vascularisation rétinienne utilisera probablement l'étude de la géométrie vasculaire rétinienne afin d'améliorer la compréhension de microcirculation humaine. / Retinal photography, by allowing a direct observation of retinal vessels, may thus constitute a practical and noninvasive method for the examination of early changes in human microcirculation. Since 1999-2000, IVAN ® software allows for a "semi-automatic" retinal vascular caliber analysis. In the literature, changes in the caliber of retinal vessels have been shown to reflect the cumulative effects of birth weight, genetic factors, aging process, cardiovascular risk factors, renal function, and inflammation. In meta analyses from epidemiological studies, wider retinal venules and narrower arterioles were associated with an increased risk of coronary heart disease in women and an increased risk of global cardiovascular mortality, while wider retinal venular caliber predicted stroke. The aim of the current research is thus to improve the knowledge of retinal vascular calibers determinants in subjects free of clinical evidence of atherosclerosis (either stroke or coronary and peripheral artery disease). In 2007, Melbourne University provided us IVAN ® software and the agreement for its use in clinical and epidemiological research was obtained in 2008. In a first article, an inverse linear relationship between retinal arterial and venous caliber and renal function measured with gold standard methods (glomerular filtration rate from urinary clearance of 99mTc-DTPA and urinary albumin ⁄ creatinine ratio.) was observed in 80 subjects from the cohort of internal medicine-arterial hypertension-Lapeyronie hospital, suggesting common determinants of these preclinical target organ damages. In a second article, we first confirmed the relationship between a wider retinal venular caliber and inflammation, as well as provided evidence for a novel association between wider retinal arteriolar caliber and oxidative stress quantified by GPx-3 activity in the participants of the POLA (Pathologies Oculaires Liées à l'Age) cohort. This finding is of particular importance as it suggests that retinal microvasculature, which has been related to carotid arterial stiffness and cardiac remodeling, may be particularly sensitive to systemic oxidative stress and systemic inflammation, independently of known cardiovascular risk factors. In a third article based on POLA cohort participants, a retinal venular dilatation appears to be strongly associated with malnutrition biomarkers (albumin and transthyretin). This suggests that early microvascular changes may be one of the mechanisms associated with the observed increased risk of cardiovascular disease among elderly subjects with malnutrition. Technical characteristics of IVAN® software limit its generalization in medicine, but its improved our knowledge about the caliber of retinal vessels changes. The future challenge for retinal microcirculation research will probably use parameters of retinal vascular geometry for a better understanding of human microcirculation.

PURPOSE: This study aims to quantify the retinal vascular blood flow in eyes affected by unilateral central retinal vein occlusions (CRVO) or branch retinal vein occlusions (BRVO). We created and explored a new, unitless metric for the severity of these diseases: relative blood flow (RBF). We then contextualized RBF in terms of patient demographics, ocular presentation and other systemic conditions, as well as explored its efficacy as a predictor of future outcomes. METHODS: Data was collected from 20 control subjects and 32 patients with clinically diagnosed retinal vein occlusions (15 CRVO and 17 BRVO). Laser speckle flowgraphy was then used to quantify retinal vascular blood flow in terms of mean blur rate, a metric shown to be highly heterogeneous between patients but fairly consistent in intra-patient repeated measurements over time. After confirming this and establishing a strong correlation between a healthy patient’s two eyes, we used an RVO patient’s fellow eye as a nondiseased expectation and presented relative blood flow as the ratio between their diseased and healthy eye. We then correlated this data with demographic variables and disease characteristics from patients’ medical history. RESULTS: We found an average blood flow decrease of 26% in CRVO eyes relative to healthy eyes in the same patients and an average decrease of 7% in BRVO eyes. In CRVO, duration of occlusion, central macular thickness, intraocular pressure, diabetes, previous laser and injection treatments, and an injection within three months after blood flow measurement were significantly associated with relative blood flow. In BRVO, no demographic variables or disease characteristics were significantly associated with relative blood flow. CONCLUSIONS: Relative blood flow represents a promising new, consistent and informative metric for quantifying the severity of unilateral retinal vein occlusions. With both descriptive and predictive properties in eyes with CRVO, future work should explore its great potential.

Submitted by (ana.araujo@ufrpe.br) on 2016-05-20T15:32:16Z No. of bitstreams: 1 Antonio Jose de Oliveira.pdf: 823994 bytes, checksum: 52b6217fffe3ac44f9d20524a5afd1bd (MD5) / Made available in DSpace on 2016-05-20T15:32:16Z (GMT). No. of bitstreams: 1 Antonio Jose de Oliveira.pdf: 823994 bytes, checksum: 52b6217fffe3ac44f9d20524a5afd1bd (MD5) Previous issue date: 2006-02-23 / The analysis of the fundus of the eye photographic images has permitted the obtaining of an accurate diagnoses for several diseases related to the human retina vascularization. This work proposes the morphormetric analysis and the fractal dimension as methods capable to parameterize the retinal vascular process. A hand made segmentation of the retina vessels done in this work allowed the obtaining of high defined image of the retina vessels. These images made possible the parametrization of the retina vessels using the shape indexes and fractal dimension. The shape indexes like perimeter, area,circularity and rectangularity were used to describe the shape of the tributary venules of the human retina central vein. The statistical analysis showed that both the perimeter and the area of the tributary venules of the central vein of human retina are significantly different between normal and with non proliferative diabetes individuals. But the parameter circularity and rectangularity of these tributary venules were not significantly different. Then,the parameters perimeter and area can be used to discriminate the normal from the ill retina, while the parameters circularity and rectangularity are inadequate to distinguish them. The fractal dimension of the vascularization human retina,in the normal and disease conditions, differed significantly between themselves, independently of the method applied to calculate it.Therefore, the fractal dimension is a parameter which can be used to distinguish the normal from the disease state of the human retina. / A análise de imagens fotográficas do fundo do olho tem tornado possível à obtenção de diagnósticos precisos para diversas enfermidades que envolvem a vascularização da retina humana. A análise morfométrica e a dimensão fractal são propostas no presente trabalho como métodos capazes de parametrizar esse processo vascular. A segmentação manual dos vasos da retina, realizada nesse trabalho, permitiu a obtenção de imagens bem definidas desses vasos, que possibilitaram a sua parametrização através de cálculos de índices de formas e dimensão fractal. Os índices de formas, perímetro e área usados para descrever a geometria das vênulas tributárias da veia central da retina humana,diferiram significativamente nas retinas normal e com retinopatia diabética não proliferativa, podendo dessa maneira, serem usados para diferenciar uma retina normal de outra doente. No entanto, os índices circularidade e retangularidade das vênulas tributárias da veia central da retina não mostraram diferenças significativas entre os estados normal e doente da retina, sendo,portanto, parâmetros inadequados para descriminarem alterações vasculares da retina. A dimensão fractal do processo de vascularização da retina humana nos estados normal e com retinopatia diabética não-proliferativa diferiram significativamente entre si,independente do método usado no cálculo dessa dimensão. Portanto, a dimensão fractal é um parâmetro que pode ser usado para diferenciar estados normal e patológico da retina humana.

La dystrophine Dp71, issue du gène DMD impliqué dans la dystrophie musculaire de Duchenne intervient dans le maintien de l'homéostasie rétinienne et de la barrière hémato-rétinienne. Nous avons mis en évidence une localisation rétinienne jusque-là méconnue de la Dp71 au sein des astrocytes rétiniens. Nous avons également étudié l'effet in vivo de l'absence de Dp71, sur le développement vasculaire et observé une plus faible densité et une morphologie astrocytaire différente comparativement aux souris contrôles, à l'origine d'un développement vasculaire retardé. Compte tenu de la spécificité du réseau vasculaire rétinien dont l'étanchéité des parois est maintenue en partie par les pieds des CGM et des astrocytes, nous avons émis l'hypothèse, étant donné l'implication de la Dp71 dans la stabilisation de ces cellules, d'une altération de cette protéine dans les phénomènes de rupture de la BHR. Ainsi dans un modèle transitoire de rupture de la BHR in vivo relativement " pur ", sans ischémie ni injection de VEGF, nous avons observé une diminution de l'expression de la dystrophine Dp71 ainsi que du canal aqueux AQP4 et une délocalisation du canal potassique Kir4.1 dans les CGM. L'injection intra-vitréenne de Dexaméthasone dans ce modèle a permis de prévenir les modifications d'expression et de localisation de ces protéines. / Dp71, a dystrophin produced from DMD gene, is involved in retinal homeostasis and maintenance of blood retinal barrier. We have previously shown that Dp71, part of a complex called Dystrophin associated protein, is involved in the localization of aqueous and potassic channels in Muller glial cell (MGC), particularly around blood vessels, allowing maintenance of retinal homeostasis. Based on the knowledge that growing retinal vessels migrate on an astrocyte template during development, we highlighted the expression of Dp71 in retinal astrocytes. In absence of Dp71, in vivo, we observed a lower density and a different morphology of retinal astrocytes compared to control retina in mice, responsible for a delayed vascular development. Due to the role of barrier of the retinal vascular network, insured also by astrocytes, we studied a model of post surgical BRB breakdown and found a decreased of Dp71 protein expression associated with Kir4.1 delocalization and AQP4 decrease in MGC. Intravitreal Dexamethasone prevents these protein expression changes. We suggest here that the membrane associated cytoskeletal protein, Dp71, expressed in astrocytes is involved in the maintenance of astrocytes density and morphology necessary as a template for retinal vascular development. This protein insure also a key role in retinal homeostasis by localizing and maintaining aqueous and potassic channels in MGC. Moreover when this protein is altered, Dexamethasone seems to be capable to promote Dp71 expression which could have wide clinical implications in retinal diseases treatment and the target for retinal neuroprotection under pathological conditions seems to be the macroglial cells.

Dans la rétine, la protéine dystrophine Dp71 est principalement exprimée dans les cellules gliales de Müller (CGM), qui contribuent à la stabilisation de la barrière hémato-rétinienne (BHR). Les CGM sont aussi les principales sources de facteurs inflammatoires. Ainsi, nous avons étudié les effets de l’absence de Dp71 sur l’homéostasie potassique et aqueuse, ainsi que sur l’expression de médiateurs de l’inflammation et la perméabilité vasculaire rétinienne.L'absence de Dp71 diminue l'expression de la protéine AQP4 et induit la redistribution de Kir4.1 tout le long des CGM. Par ailleurs, nous avons également constaté que le décollement expérimental de la rétine chez les souris WT induit une diminution de Dp71 associée à une délocalisation de Kir4.1, une régulation à la baisse de la protéine AQP4 dans les CGM.Nos données montrent clairement que l'absence de la Dp71 entraîne une augmentation de l'expression du VEGF, d’ICAM-1, une augmentation du nombre de leucocytes adhérents rétiniens, une dégénérescence accrue des capillaires associée à une forte perméabilité vasculaire chez les souris Dp71-null.L’ensemble de nos résultats a mis en évidence le rôle de la Dp71 dans les mécanismes visant à réguler l'homéostasie rétinienne et à assurer la stabilisation de la BHR. Nous apportons la preuve que la perte de Dp71 favorise l'inflammation vasculaire rétinienne et la dégénérescence des capillaires associée à une perméabilité vasculaire. Ensemble, ces observations suggèrent que la souris Dp71-null serait un modèle approprié pour étudier les pathologies vasculaires rétiniennes telles que la rétinopathie diabétique, l’uvéite rétinienne et l’occlusion veineuse rétinienne. / In the retina, the Dp71 dystrophin protein is mainly expressed in Müller glial cells (MGC), which contribute to the stabilization of the blood-retinal barrier (BRB). MGC are also the main sources of inflammatory factors. Thus, in our thesis project we studied the effects of the absence of the Dp71 protein on potassium and water homeostasis, as well as the expression of inflammatory mediators and retinal vascular permeability.The absence of the Dp71 protein decreased the expression of AQP4 protein and induces the redistribution of Kir4.1, initially restricted to the end-feet of MGC and around vessels, all along the cell membrane. Moreover, we have also shown that the experimental retinal detachment in WT mice induces a reduction of Dp71 which is associated with Kir4.1 mislocation, a down regulation of AQP4 protein in MGC.Our data clearly demonstrate that the absence of the Dp71 leads to increased retinal VEGF and ICAM-1 expression in Dp71-null mouse compared to WT mouse strain. There is also an increase of the number of retinal adherent leukocytes, capillary degeneration associated with high BRB permeability observed in Dp71-null mice.Our findings highlight Dp71 as an important component in the mechanisms leading to the regulation of retinal homeostasis; and to the maintaining of the BRB stabilization. We provide evidence that deficiency of Dp71 promotes retinal vascular inflammation and significantly exacerbated degeneration of capillaries and BRB breakdown. Together these results suggest that the Dp71-null mouse could be a good model to study retinal vascular diseases such as diabetic retinopathy, retinal uveitis and retinal vein occlusion.

Background: Diabetes mellitus and vascular diseases have a significant impact on the eye. / Aim: To determine the prevalence, risk factors, and racial/ethnic differences of major eye conditions, particularly retinal conditions, associated with diabetes and vascular diseases. / Scope: To date, the majority of studies have examined the association of retinal vascular calibre and diabetes in predominantly white Caucasian populations. Further elucidation of ethnic differences in effects of hyperglycaemia on early microvascular disease is relevant, particularly amongst Asians where diabetes is likely to see the largest increase in prevalence over the next decade. We therefore examined these findings from three Asian population-based studies, the Singapore Malay Eye Study (n=3280), Singapore Prospective Cohort Study and Singapore Cardiovascular Cohort Study 2 (n=3748). / Results: The prevalence of diabetic retinopathy in the Singapore Malay Eye Study was 35%, and associated with longer duration of diabetes, poorer glycemic and blood pressure control. More importantly, 9.0% had vision-threatening retinopathy, and retinopathy was found in 6.0% of people without diabetes. Retinal vascular calibre changes were incriminated in diseases such as diabetes and hypertension, independent of traditional cardiovascular risk factors. Wider venular calibre was independently associated with early age-related macular degeneration. We also found a novel association between peripheral artery disease and glaucoma, stronger in persons with diabetes, independent of vascular risk factors, supporting the vascular theory of glaucoma. / Implications: Subtle changes in retina, including retinal vascular calibre may be early markers of widespread microvascular changes in diabetes, resulting from chronic hyperglycaemia and other pathogenic processes. These results will have broad implications for understanding the impact of both microvascular and macrovascular complications of diabetes in the Asia Pacific region and targeting relevant therapeutic interventions.

Perivascular cells of microvascular niches are the prime candidates for being a reservoire of mesenchymal stem cell (MSC)-like cells in many tissues and organs that could serve as a potential source of cells and a target of novel cell-based therapeutic approaches. In the present study, by utilising typical markers of pericytes (neuronal-glial antigen 2, NG2, a chondroitin sulphate proteoglycan) and those of MSCs (CD146 and CD105) and primitive pluripotent cells (sex-determining region Y-box 2, Sox2), the phenotypic traits and the distribution of murine and rat retinal perivascular cells were investigated in situ. Our findings indicate that retinal microvessels of juvenile rodents are highly covered by NG2-positive branching processes of pericytic (perivascular) cells that are less prominent in mature capillary networks of the adult retina. In the adult rodent retinal vascular bed, NG2 labeling is mainly confined to membranes of the cell body resulting in a pearl-chain-like distribution along the vessels. Retinal pericytes, which were identified by their morphology and NG2 expression, simultaneously express CD146. Furthermore, CD146-positive cells located at small arteriole-tocapillary branching points appear more intensely stained than elsewhere. Evidence for a differential expression of the two markers around capillaries that would hint at a clonal heterogeneity among pericytic cells, however, is lacking. In contrast, the expression of CD105 is exclusively restricted to vascular endothelial cells and Sox2 is detected neither in perivascular nor in endothelial cells. In dissociated retinal cultures, however, simultaneous expression of NG2 and CD105 was observed. Collectively, our data indicate that vascular wall resident retinal pericytes share some phenotypic features (i.e. CD146 expression) with archetypal MSCs, which is even more striking in dissociated retinal cultures (i.e. CD105 expression). These findings might have implications for the treatment of retinal pathologies.