Os processos que regem o desenvolvimento dos olhos compostos em insetos têm sido amplamente estudados em Drosophila melanogaster onde estes se originam a partir de discos imaginais. Pouco se sabe, porém, sobre o desenvolvimento do lóbulo óptico e da retina em outros insetos que, na sua grande maioria, não possuem discos imaginais de olhos separados do sistema nervoso central. Neste sentido, a análise comparada do desenvolvimento dos olhos de Apis mellifera pode contribuir não somente para aspectos evo-devo entre as grandes famílias dos insetos holometábolos, quanto pode elucidar questões de plasticidade de desenvolvimento pois os olhos compostos apresentam fortes características sexo e casta-específicas. Com o objetivo primário de elucidar os padrões de divisão e diferenciação celular durante o desenvolvimento do olho em A. mellifera realizamos análises histológicas e de imunomarcação durante o desenvolvimento pós-embrionário, juntamente com análise de expressão do gene roughest em tempo real. Para imunomarcação utilizamos o anticorpo anti-fosfo-histona H3 fosforilada que marca células em fase M do ciclo celular. Foram analisadas larvas operárias entre o terceiro instar larval (L3) até pupas de olho branco, rosa e marrom, com foco sobre o quinto instar larval que fica subdividida em fase de alimentação e crescimento (L5F), fases de tecelagem de casulo (L5S) e prepupa (PP). O desenvolvimento do lóbulo óptico em Apis mellifera ocorre por dobramento neuroepitelial, a partir de um centro de diferenciação, seqüencialmente gerando as camadas neurais do lóbulo óptico (lóbula, medula e lâmina). A lâmina (última a surgir) 6 apresentou-se com desenvolvimento mais lento e em duas fases antes da metamorfose: a primeira fase é o seu surgimento no começo do quinto instar larval acompanhando o primeiro pico de expressão de roughest e a segunda fase ocorre durante a tecelagem de casulo com o desenvolvimento do córtex acompanhando o segundo pico de expressão de roughest. Ainda durante o segundo pico de expressão de roughest os rabdômeros da retina começam a ficar visíveis, assim como os feixes axonais. Estes porém estarão completamente formados somente após a metamorfose.. O desenvolvimento completo da lâmina, lóbula e medula e da retina ocorre somente após a metamorfose. Durante a fase pupal as estruturas do lóbulo óptico estão prontas, porém na retina observa-se ainda gradual pigmentação, encurtamento dos feixes axonais e alongamento dos rabdômeros até atingirem o seu comprimento final logo antes da emergência. / The processes that drive compound eye development in insects have been broadly studied in Drosophila melanogaster in which they arise from imaginal discs. Little is known about optic lobe and retina development in other insects, most of which do not have imaginal eye discs attached to the nervous system. For this reason, a comparative analysis of eye development in the honey bee, Apis mellifera, not only contributes to evo-devo aspects comparing the major families of holometabolous insects, but also may elucidate questions about developmental plasticity because the compound eyes of the honeybee show strong sex and caste-specific differences. Since our primary objective was to elucidate the pattern of cellular differentiation and division during eye development we performed histological and immunolabelling analyses during the postembrionic stages of development, concomitant with a realtime analysis of roughest gene expression. For the immunolabelling experiments we used an anti-phospho-histone H3 antibody that labels cells in M phase. We analyzed eye development in worker larvae starting with the third instar until white, pink and browneyed pupae, paying special attention to the fifth instar which was subdivided into feeding phase (L5F), cocoon spinning phase (L5S) and prepupae (PP). Optic Lobe development in Apis mellifera occurs by neuroepithelial folding initiating from a differentiation center, in the larval brain. This center sequentially produces the neural layers of the optic lobe (medulla, lobula and lamina). Development of the lamina, which is the last layer to be formed, takes more time and happens in two steps before metamorphosis. The first step is emergence at the beginning of the fifth larval instar coinciding with the first peak of roughest gene expression. The second step 8 occurs during the cocoon spinning phase and is marked by its inner differentiation, again accompanied by a second peak of roughest expression. During this second peak of roughest expression the rabdomers in the retina become visible. These, however, cplete thir development only during the pupal stage. The development of the lamina, lobula and medulla is not complete until after metamorphosis, even though these optic lobe structures are structurally defined already at the beginning of the pupal phase. Retinal development in this phase is marked by gradual pigmentation, axonal bundle shortening and rabdomer elongation, which reach their final size just prior to emergence of the bees from their brood cells.

Nas últimas décadas uma série de experimentos realizados em vertebrados tem demonstrado a capacidade que alguns destes possuem de discriminar a luz ultravioleta, ao contrário do acreditado anteriormente. O peixe dourado (Carassius auratus), um bom modelo experimental para pesquisas sobre visão, apresentou em experimentos comportamentais altos níveis de discriminação de cor nas regiões espectrais do azul e do violeta, o que só seria possível com a existência de um receptor adicional para o ultravioleta (Neumeyer, 1985; Hawryshyn & Beauchamp, 1985; Neumeyer & Arnold, 1989; Neumeyer, 1992; Fratzer, Dörr & Neumeyer, 1994). Estes resultados foram confirmados pela determinação microespectrofotométrica (Bowmaker, Thorpe & Douglas, 1991) e eletrofisiológica (Palacios et al., 1998) da existência de cones com pigmentos específicos para luz ultravioleta, sem haver entretanto um estudo da codificação dessa entrada em neurônios de segunda ordem até o momento. Além disso, permanece ainda em discussão na literatura o papel das células horizontais retinianas no processamento cromático. O presente projeto teve por objetivo investigar qual a contribuição da entrada do receptor UV para os perfis de resposta eletrofisiológica das células horizontais e quais tipos celulares da retina externa do Carassius auratus são subjacentes à discriminação observada comportamentalmente. Para tanto, registros intracelulares de células horizontais e bipolares foram obtidos sob estimulaçãomonocromática de diferentes intensidades, ) diâmetros e comprimentos de onda, com o intuito de determinar suas respostas espectrais, bem como o campo receptivo e as possíveis interações entre estas e os cones. As células horizontais mono-, bi- e trifásica apenas hiperpolarizam na região do UV, não havendo oponência cromática entre as regiões do UV e do azul em nenhum destes tipos celulares; tampouco encontramos uma célula horizontal tetrafásica. Através de adaptações cromáticas, observamos serem as respostas eletrofisiológicas à luz das células horizontais mono- e bifásicas resultado de interações entre os sistemas de cones vermelho,verde e azul, não tendo sido identificada nenhuma entrada UV significativa nessas células. Por fim, encontramos uma célula bipolar oponente entre as regiões espectrais do UV e do azul, o que constitui uma base neural compatível com a discriminação comportamentalmente observada. Uma vez que canais oponentes são necessários para a discriminação de cor, e essa oponência não foi encontrada nas células horizontais, mais sim nas bipolares, acreditamos ser esse um indício de que as células horizontais não participam diretamente da codificação da informação UV na retina do Carassius auratus. Essa idéia está de acordo com dados recentes da literatura, que atribuem às células horizontais papel relacionado aos mecanismos de constância e contraste simultâneo de cor, deixando para as células bipolares a função de codificar cores para os neurôniossubseqüentes (Kamermans, Kraaij & Spekreijse, 1998) / In the last decades a number of experiments in vertebrates has demonstrated the ability of some of these animals to discriminate ultraviolet light. Among them, the goldfish (Carassius auratus), considered a good model in vision research, presented in behavioural experiments high discrimination rates in the violet and blue spectral regions, which could only be accomplished through an additional UV receptor (Neumeyer, 1985; Hawryshyn & Beauchamp, 1985; Neumeyer & Arnold, 1989; Neumeyer, 1992; Fratzer, Dörr & Neumeyer, 1994). These results were confirmed by microspectrophotometrical (Bowmaker, Thorpe & Douglas, 1991) and electrophysiological (Palacios et al., 1998) determinations of the existence of cones with specific photopigments with a maximum in the UV region of the light spectrum. However, to date there are no data concerning the spectral coding of this input in second order neurons. Furthermore, the role of the retinal horizontal cells in chromatic processing remains under debate. The present study aimed at investigating the contribution of the UV input to the electrophysiological response profiles of the cone-driven horizontal cells, as well as determining which neurons in the outer retina of the goldfish could subserve vision in the UV range, as observed behaviourally. For that purpose, intracellular recordings of horizontal and bipolar cells under monochromatic stimuli of different intensities, diameters and wavelengths were obtained in order to determinetheir action spectra, receptive field sizes and characteristics as well as the possible interactions between them and the different cone systems. Mono-, bi- and triphasic horizontal cells always hyperpolarised in the UV spectral range: there was no chromatic opponency between the UV and the blue zones in any of these cell types. We have also failed to find a tetraphasic horizontal cell. We observed through chromatic adaptation experiments that the electrophysiological responses recorded from mono- and biphasic horizontal cells are the result of interactions between the red, green and blue cone systems. No relevant UV input to these cells could be found. Finally, we found a bipolar cell type with spectral opponency between the UV and blue regions, which could be the neural basis of the discrimination observed behaviourally. Colour-opponent channels are thought to be necessary for colour discrimination, and the absence of a horizontal cell type with opponency between the UV and blue spectral regions provides further evidence that this cell type might not play a major role in the chromatic processing in the goldfish retina. Since this opponency pattern was found in a bipolar cell, we think that this cell type, and not the horizontal cells, might underlie the coding of the UV information in this animal. This idea is in good agreement with some recent literature data that ascribe the horizontal cells a role in colour constancy and simultaneous colour contrast phenomena,leaving the function of colour codification to the bipolar cells (Kamermans, Kraaij & Spekreijse, 1998)

Em exposições especificamente relacionadas ao metilmercúrio (MeHg), o principal órgão alvo é o Sistema Nervoso Central (SNC). Os efeitos morfológicos do metilmercúrio nos fotorreceptores da retina de Hoplias malabaricus, traíra, foram investigados através de técnicas histopatológicas. Os exemplares de traíra foram distribuídos em três grupos: um grupo controle e dois grupos expostos ao MeHg. A contaminação trófica e subcrônica foi realizada através de exemplares vivos de Astyanax sp os quais receberam intraperitonealmente um volume de solução aquosa de MeHg correspondente a (0,075µg/g de traíra) ou (0,75µg/g de traíra). Quinze peixes por condição experimental foram sacrificados após 70 dias de exposição e os olhos foram coletados para as análises de microscopia de luz, microscopia eletrônica de transmissão e microscopia eletrônica de varredura. Através de análises químicas quantificamos o mercúrio em músculo e observamos em ambas as doses um acúmulo do metal. Para a maior dose do metal (0,75µg/g), as análises histopatológicas revelaram alterações nas membranas que unem os dois elementos de um cone pareado, degeneração da camada dos fotorreceptores e alterações morfológicas no segmento interno e segmento externo dos bastonetes. As análises também demonstraram alterações estruturais na membrana plasmática do segmento externo e alterações no processo de eliminação dos discos membranosos dos fotorreceptores. No momento da eliminação dos discos antigos foi observada a formação de vacúolos e também uma anormal eliminação dos discos membranosos pelas partes laterais do segmento externo. Para a menor dose do metal (0,075µg/g) além da alteração observada nas membranas que unem os cones pareados e da degeneração celular na camada dos fotorreceptores, observamos uma descontinuidade estrutural dos discos membranosos do segmento externo dos fotorreceptores. Todas estas alterações histopatológicas nos levam a concluir que o metilmercúrio induz alterações histopatológicas nas células da camada dos fotorreceptores podendo consequentemente trazer danos fisiológicos para toda a retina. / In exposures specifically related to methylmercury (MeHg), the main target organ is the Central Nervous System (CNS). In this study, morphological effects of methylmercury in retinal photoreceptors of Hoplias malabaricus, trahira, through histopathological techniques were investigated. Some mature fish were divided in three groups - one control group and the other two which were exposed to MeHg. The trophic and subchronic contamination was performed by live specimen of Astyanax sp which received a volume of aqueous solution of MeHg corresponding to (0,075µg /g trahira) or (0,75µg /g trahira). In an experimental condition, fifteen fish were sacrificed after 70 days of exposure and their eyes were collected for analysis of light microscopy, electron transmission microscopy and scanning electron microscopy. Through chemical analysis the amount of mercury found in muscle was quantified. In both doses an accumulation of the metal in the muscle of these animals was observed. For the highest dose of metal (0,75µg /g), the histopathological analysis revealed changes in the membrane that connects the two elements of a paired cone, cellular degeneration in the layer of photoreceptors and morphological changes in the internal and outer rod segments. The analysis also showed structural changes in the plasma membrane of the outer segment and changes in the process of removal of membranous discs in the apical region of a photoreceptor. At the time of disposal of old discs, the formation of vacuoles and also an abnormal membranous discs removal through the sides of the outer segment were observed. For the smallest dose of the metal (0,075µg/g) besides the morphological changes observed in the membrane which unites the paired cones and cellular degeneration throughout the layer of photoreceptors, a structural discontinuity of the membranous discs of the outer segment were observed in radial sections of rods. All these histopathological changes lead us to conclude that methylmercury induces morphological changes in cells of the layer of photoreceptors, and therefore causing physiological damage to the smooth functioning of the retina.

A retina é formada por camadas de células que a estrutura, os contatos sinápticos e as características fisiológicas são específicas para cada tipo de célula. O processamento da informação visual, após a ativação dos fotorreceptores pela luz, ocorre em mecanismos pós-receptorais que formam diferentes circuitos de processamento, entre eles as vias ON, OFF, de luminância e de oponência cromática. As doenças que afetam a retina podem estar relacionadas com alterações específicas de um determinado circuito, como acontece com a distrofia muscular de Duchenne (DMD) que na alteração do gene dmd da retina afeta a comunicação entre os fotorreceptores e as células bipolares, e na diabetes Mellitus (DM) o dano celular ocorre principalmente na camada mais interna da retina. O objetivo deste estudo foi avaliar os efeitos da DMD e da diabetes Mellitus Tipo 1 (DM1) em diferentes mecanismos visuais. A amostra deste trabalho contou com três grupos: controle com 18 participantes (13,5, ±7,6), DMD com 9 participantes (16, ±5,9) e DM1 com 12 partcipantes (13,8, ±2,5). Os testes eletrorretinográficos foram aplicados nas condições mesópica, fotópica com estímulos que enfatizam respostas ON ou OFF, e flicker heterocromático (verde-vermelho) em 12Hz (para enfatizar a atividade da via de oponência cromática) e 36Hz (para enfatizar a atividade da via de luminância), e testes psicofísicos com estímulos que enfatizam os mecanismos ON e OFF com contraste de luminância e a via de oponência cromática por um estímulo com contraste cromático (isoluminante) verde-vermelho. O grupo DMD apresentou alteração no tempo de resposta e redução na amplitude da onda-a e onda-b nas condições mesópicas ON e OFF, redução da amplitude da onda-b na condição fotópica ON e alteração da via cromática. Enquanto o grupo DM1 apresentou aumento na amplitude e tempo da ond-a e onda-b das via ON e OFF na condição mesópica, mudança na fase da resposta na via cromática e na via de luminância. Os resultados estão de acordo com diversos estudos anteriores que mostram que as duas doenças causam alterações funcionais na retina, mas cada doença com características específicas. O presente estudo demonstrou que as duas doenças afetam as vias visuais ON e OFF independente do prejuízo neural ocorrer principalmente nas camadas mais externas ou nas camadas mais internas da retina. A possibilidade de identificar prejuízos funcionais em pacientes sem alteração clínica é uma valiosa ferramenta para o acompanhamento de doenças e para avaliar a eficácia de tratamentos. O presente estudo poderá contribuir para o estabelecimento de novos protocolos mais sensíveis e capazes de identificar alterações assimétricas no caso da DMD ou alterações que precedem o diagnóstico clínico no caso da DM1 / The retina is formed by layers of cells that the structure, synaptic contact and physiology characters are specific for each type of this cells. The processing of visual information, after the photoreceptor activation by light is separate by post-receptor mechanisms that constitute the ON, OFF, luminance and chromatic pathways. The retina diseases have specific alteration in this tissue, thus it happens with the Duchenne muscular dystrophy (DMD), that have communication problems among photoreceptors and bipolar cells because of damage in dmd gene, and the Diabetes Mellitus (DM) that has cells damage mainly in the inner layer of retina. The purpose of this study was to evaluate the effects of DMD and Type 1 diabetes Mellitus (DM1) on different visual mechanisms. The sample had three grups: control with 18 subjects (13.5, ±7.6), DMD with 9 subjects (16, ±5.9) and DM1 with 12 subjects (13,8, ±2,5). The ERG recordings were applied in of the mesopic and photopic conditions with stimuli that laid emphasis on ON and OFF responses and heterochromatic (red-green) flicker modulation in 12Hz (laid emphasis on chromatic pathway) and 36Hz (laid emphasis on luminance pathway), and psychophysical tests laid emphasis on ON and OFF mechanisms with luminance contrast and the chromatic opponent pathway by stimulus with red-green chromatic contrast (isoluminance). As a result the DMD group presented alteration in the response time and reduction in the amplitude of the a- and b-wave in ON and OFF mesopic conditions, reduction of the amplitude of the a- and b-wave in ON photopic condition and alteration of the chromatic pathway. While the DM1 group showed an increase in the amplitude and time of the a- and b-wave in ON and OFF pathways of the mesopic condition, change in phase of the chromatic pathway and luminance pathway. These results were in agreement with previous studies that showed the two diseases present functional changes in the retina, but each disease with specific characteristics. The present study showed that two diseases affect the ON and OFF pathways regardless of the neural damage occur mainly in outer layer or inner layer of the retina. The possibility identify functional damage in patients without clinic injuries is the valuable tool for the following the dieses and evaluated treatment efficiency. The present study may contribute to establishment of the new sensitive and able protocols to identify asymmetric changes in DMD or changes that precede the clinical diagnosis in DM1

青光眼作為一種慢性進展性視神經病變，已經成為世界眼科病變中導致不可逆盲的首要原因。青光眼的早期診斷和治療對於降低疾病進展的風險至關重要。光學相干斷層掃描（OCT）可以提供在體視網膜橫斷面的視圖，從而實現了對視網膜神經纖維層（RNFL）改變的客觀測量，這些改變已經被證明了與青光眼引起的視神經損害相關，並已成為診斷青光眼的重要參考依據。 / 頻域OCT是最新一代的光學相干斷層掃描，它具有比時域OCT更快的掃描速度和更高的圖像解析度，因此，頻域OCT可以提供更可靠的RNFL厚度測量和RNFL缺損評估。本文的研究目的在於評估頻域OCT對RNFL厚度的重測再現性，以及探討影響RNFL厚度測量的因素，這些因素包括（1）影像平均法的應用，（2）RNFL分層錯誤，和（3）視網膜血管的影響。此外，由於RNFL攝影是一個評估青光眼RNFL缺損的臨床參考標準，我們還將其對RNFL缺損的測量與頻域OCT的RNFL厚度偏差圖所作出的測量進行了比較。 / 首先，為了評估頻域OCT對RNFL厚度測量的重測再現性，15名正常人和15名青光眼患者連續四周每週均接受一次OCT掃描。正常組和青光眼組的RNFL厚度再現性係數分別為4.77-12.65微米和4.53-16.66微米，由於組內相關性係數均大於0.773，說明頻域OCT所作出的RNFL厚度測量是具備可重複性的。 / 其次，通過分析54隻眼（25名正常志願者和29名青光眼患者）的RNFL厚度測量值，本文對圖像平均法的應用是否會影響RNFL厚度的測量這一問題進行了探討。分析中，每一隻眼均接受了3次OCT掃描，3次掃描的圖像分別使用2、8、和16張連續的圖像進行影像平均。結果顯示，除了青光眼組的鼻下象限RNFL厚度測量值之外（P=0.036），不同的圖像幀數並不會對兩組的總體和其它各象限的RNFL厚度測量值產生顯著的影響（P≥0.055）。雖然圖像平均法的應用對RNFL厚度測量的影響並不顯著，但是視網膜血管和RNFL分層錯誤對青光眼,尤其是對RNFL非常薄的晚期青光眼患者的RNFL厚度測量有影響。結論來自對60個正常人，66個輕至中度青光眼（MD≥-6 dB）患者和54個嚴重青光眼（MD<-6 dB）患者的共180張OCT圖像的分析。視網膜血管相對於平均RNFL厚度的比例均值在正常組，輕至中度青光眼組，和嚴重青光眼組分別為11.2±2.3，12.6±2.5，和16.6±3.9。在人為調整了RNFL界限以糾正RNFL分層錯誤的前後，總體RNFL厚度的差異範圍在正常組為-3.0-2.5微米，輕至中度青光眼組為-2.5-5.0微米，嚴重青光眼組為-11.0-9.5微米組。 / 最後，通過對41名青光眼患者的51隻眼的RNFL缺損面積，位置，和覆蓋角度進行測量，本文將頻域OCT作出的測量結果和共焦鐳射掃描檢眼鏡（CSLO）RNFL反射影像圖的測量結果進行了比較，結果顯示：OCT不但可以檢測到所有出現在CSLO的RNFL反射影像圖上的RNFL缺損，更重要的是，OCT還可以檢測出額外的並未在RNFL反射影像圖上出現的RNFL缺損。 / 總之，頻域OCT是一種可提供高再現性RNFL厚度測量的影像方法。對青光眼,尤其是晚期青光眼的RNFL厚度測量值的詮釋，應當考慮到視網膜血管和RNFL分層錯誤的影響。OCT具備對RNFL缺損進行多維度量化（包括厚度，面積，位置，和覆蓋角度）的能力，在青光眼RNFL改變的檢測和監測方面，相對于傳統的RNFL攝影，OCT無疑是更有效的選擇。 / Glaucoma, a chronic progressive optic neuropathy, is the leading cause of irreversible blindness in the world. An early diagnosis and treatment of glaucoma is vital to reduce the risk of disease progression. Providing a cross-sectional view of the retina in vivo, optical coherence tomography (OCT) can objectively measure the changes of retinal nerve fiber layer (RNFL), which has been shown to be of relevance and importance in detecting glaucomatous damage of the optic nerve. / The latest generation of OCT, the spectral-domain OCT, has a faster scan speed and a higher image resolution compared to the time-domain OCT. It is expected that the spectral-domain OCT would allow a more reliable measurement of the RNFL thickness and assessment of RNFL defects. The objectives of this research project were to examine the test-retest reproducibility of spectral-domain OCT RNFL measurement and investigate factors including (1) image averaging, (2) segmentation failure, and (3) contribution of retinal blood vessels that might affect the measurement of RNFL thickness. As RNFL photography is a reference standard to evaluate RNFL defects in glaucoma, we also evaluated whether RNFL defects measured in the spectral-domain OCT RNFL thickness map would be comparable to those detected in RNFL photographs. / To evaluate the test-retest reproducibility of RNFL measurements obtained by the spectral-domain OCT, 15 normal individuals and 15 glaucoma patients were followed and imaged weekly for 4 consecutively weeks. The reproducibility coefficients of RNFL thicknesses ranged between 4.53 and 16.66 μm for the normal group, and 4.77 and 12.65 μm for the glaucoma group. The intraclass correlation coefficients were all above 0.773, indicating RNFL measurement with spectral-domain OCT was reproducible. / We then investigated if multiple-image averaging would influence the measurement of RNFL thickness. A total of 54 eyes from 25 normal volunteers and 29 glaucoma patients with RNFL images captured and averaged with 2, 8, and 16 consecutive image frames were analyzed. For both groups, there were no significant differences in global or sectoral RNFL thicknesses among the image series averaged with different number of image frames (all with P≥0.055) except for the inferonasal sector in the glaucoma group (P=0.036). Although the impact of image averaging on RNFL measurement was insignificant, the presence of retinal blood vessels and segmentation errors were influential on the measurement, particularly in advanced glaucoma patients when the RNFL was thin. Analyzing a total of 180 eyes from 60 normal individuals, 66 mild to moderate (MD≥-6 dB) and 54 advanced (MD<-6 dB) glaucoma patients, the mean proportion of retinal blood vessels relative to the average RNFL thickness was 11.2±2.3%, 12.6±2.5% and 16.6±3.9%, respectively. After correcting the segmentation errors by manually refining the RNFL boundaries, the differences in average RNFL thickness ranged from -3.0 to 2.5 m in the normal, -2.5 to 5.0 m in the mild to moderate glaucoma and -11.0 to 9.5 m in the advanced glaucoma groups. / Finally, we compared the area, the angular location, and the angular width of RNFL defects from 51 eyes of 41 glaucoma patients measured with the spectral-domain OCT and RNFL reflectance images obtained by a confocal scanning laser ophthalmoscope (CSLO). OCT was able to detect areas of RNFL abnormalities in all eyes with RNFL defects which were evident in the CSLO RNFL reflectance images. More important, OCT could identify additional RNFL thinning not apparent in RNFL reflectance images. / In summary, spectral-domain OCT could offer an effective approach in measuring RNFL with high reproducibility. Interpretation of RNFL measurement should take the contribution of the retinal blood vessels and segmentation errors into consideration, particularly in advanced glaucoma when the RNFL is thin. With the ability to quantify multiple dimensions of RNFL defects (thickness, area, angular location, and angular width), OCT could provide a useful alternative to detect and monitor RNFL changes in glaucoma. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Ye, Cong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 117-130). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / ABSTRACT --- p.i / 摘要 (ABSTRACT IN CHINESE) --- p.v / DEDICATION --- p.viii / ACKNOWLEDGEMENT --- p.ix / TABLE OF CONTENTS --- p.x / PUBLICATIONS --- p.xiv / ABBREVIATIONS --- p.xvi / Chapter CHAPTER 1: --- INTRODUCTION --- p.1 / Chapter 1.1 --- Glaucoma --- p.2 / Definition of Glaucoma --- p.2 / Epidemiology of Glaucoma --- p.3 / Pathogenesis of Glaucoma --- p.4 / Diagnosis of Glaucoma --- p.7 / Chapter 1.2 --- Retinal Nerve Fiber Layer --- p.13 / Anatomy of Retinal Nerve Fiber Layer --- p.13 / Visualization of Retinal Nerve Fiber Layer --- p.14 / Retinal Nerve Fiber Layer Defect in Glaucoma --- p.16 / Significance of Detecting Retinal Nerve Fiber Layer Defect in Glaucoma --- p.18 / Chapter 1.3 --- Optical Coherence Tomography --- p.20 / Principle of Optical Coherence Tomography --- p.20 / Retinal Nerve Fiber Layer Imaging with OCT --- p.21 / Optic Nerve Head Imaging with OCT --- p.27 / Advantages and Disadvantages of Optical Coherence Tomography --- p.29 / Chapter 1.4 --- Research Objectives --- p.30 / Chapter CHAPTER 2: --- GENERAL MATERIALS AND METHODS --- p.32 / Chapter 2.1 --- Subject Enrollments --- p.33 / Chapter 2.2 --- Clinical Ophthalmic Examination --- p.34 / Chapter 2.3 --- Visual Field Examination --- p.35 / Definition of Normal and Glaucoma Groups --- p.35 / Chapter 2.4 --- Optical Coherence Tomography Imaging --- p.37 / Cirrus HD-OCT Imaging --- p.37 / Spectralis OCT Imaging --- p.37 / Chapter 2.5 --- Statistical Analysis --- p.39 / Chapter CHAPTER 3: --- RETINAL NERVE FIBER LAYER IMAGING WITH SPECTRAL-DOMAIN OPTICAL COHERENCE TOMOGRAPHY --- p.40 / Chapter 3.1 --- Reproducibility and Agreement of Retinal Nerve Fiber Layer Measurement --- p.41 / Introduction and Study Objectives --- p.41 / Methods --- p.42 / Results --- p.45 / Discussion --- p.47 / Tables and Figures --- p.51 / Chapter 3.2 --- Effect of Multiple B-scans Averaging on Retinal Nerve Fiber Layer Measurement --- p.58 / Introduction and Study Objectives --- p.58 / Methods --- p.59 / Results --- p.61 / Discussion --- p.62 / Tables and Figures --- p.67 / Chapter 3.3 --- Impact of Blood Vessels and Segmentation Failure on Retinal Nerve Fiber Layer Measurement --- p.73 / Introduction and Study Objectives --- p.73 / Methods --- p.75 / Results --- p.78 / Discussion --- p.80 / Tables and Figures --- p.84 / Chapter 3.4 --- Agreement of Localized Retinal Nerve Fiber Layer Defect Assessment with Confocal Scanning Laser Ophthalmoscopy --- p.95 / Introduction and Study Objectives --- p.95 / Methods --- p.97 / Results --- p.101 / Discussion --- p.103 / Tables and Figures --- p.108 / Chapter CHAPTER 4: --- GENERAL CONCLUSIONS --- p.115 / REFERENCES --- p.117

目的1)發現與中風相闋的視網膜特徵2) 利用視網膜特徵建立統計模型對老年人中風風險進行分類。 / 方法:配對病例對照研究。病例為中風患者，一部分中風患者來自於糖尿病眼病的篩查項目，另外一部分是腦內科的中風患者。對照是沒有中風的老年人。對照來自糖尿病眼病篩查項目內沒有患中風的患者及在眼科門診沒有中風及特殊眼病的患者。對照與病例在年齡及是否患有糖尿病進行匹配。所有研究對象均來自香港威爾斯親王醫院。我們收集所有研究對象的中風危險因素，包括年齡，性別，吸煙，及是否患有糖尿病，高血壓，缺血性心髒病，心房顫動，高血脂。所有研究對象的彩色視網膜照片都被採集。我們應用軟件“ImageJ"分析並記錄視網膜動靜脈直徑，血管分叉係數，分叉角度，分叉對稱性，視乳頭周長。我們也記錄其他視網膜特徵，如動靜脈壓跡，出血，硬性滲出，動脈阻塞及血管彎曲性。獨立t檢驗用於對連續變量的單因素分析，卡方檢驗用於對分類變量的單因素分析。Logistic 回歸用於建立統計模型對中風風險進行分類。所有統計方法均應用SPSS16.0 軟件。 / 結果:本研究納入122 中風患者及122 例患者做對照。每組分別有81 例糖尿病患者， 41 例非糖尿病患者。視網膜特徵包括動靜脈直徑，血管彎曲度，出血，硬性滲出，動靜脈壓跡在兩組中有顯著性差異。我們建立風險模型對兩組患者進行風險分類。分類準確度最高達的模型裡面包括的因子有：1)中風相關危險因素包括:高血壓，糖尿病，心房顫動2) 視網膜特徵包括:動脈直徑，血管彎曲性，出血，動靜脈壓跡跟靜脈對稱性；3) 視網膜特徵間的交立作用包括:動脈直徑與靜脈對稱性，動脈直徑與出血，靜脈對稱性與血管彎曲度。分類的準確度為80 .4%。只包括視網膜特徵的分類模型的準確度為74.5% 。 / 結論:彩色視網膜照相可成為中風風險的分類工具。與中風相關的視網膜特徵包括血管直徑，血管彎曲度，血管對稱性，出血，動靜脈壓跡。視網膜特徵與中風之間的聯繫存在交互作用。 / Objective: 1) To detect retina characteristics that associated with stroke; 2) To develop a statistics model with variables of retina characteristics for classifying patients with stroke from those without stroke in aged population. / Method: Matched case control study. Patients with stroke from the diabetic retinopathy screening program and stroke patients from Acute Stroke Unit were selected as stroke cases. Controls (patients without history of stroke) with matched diabetes status and age were selected from the diabetic retinopathy screening program and eye outpatient clinics. All subjects in this study were from Prince of Wales Hospital, Hong Kong. Risk factors of stroke from all subjects were collected, including age, gender, diabetes, hypertension, hyperlipidemia, history of ischemic heart disease, atrial fibrillation and smoking. Color retina images of each subject were collected and analyzed. The retina characteristics, including diameters of arterioles and venules, bifurcation coefficients, bifurcation angles, branch symmetry, optic disc perimeter were extracted from the color retina images by software "ImageJ". Other retina characteristics including arteriole-venule nicking, hemorrhages, exudates, arteriole occlusion, and vessel tortuosity were also recorded. Independent t test and Chi-squire test were used to compare the continuous and categorical retina characteristics respectively between patients with stroke and those without stroke. Logistic model combining the risk factors of stroke and retina characteristics was established to classify patients with stroke from those without stroke. All data analysis was by SPSS 16.0. / Results: there were 122 stroke cases and 122 controls recruited in this study. There were 41 patients without diabetes and 81 patients with diabetes in each group. Retina characteristics including diameters of arterioles and venules, vessel tortuosity, hemorrhages, exudates, arteriole-venule nicking were significantly different between the two groups. We established risk models to classify patients with stroke from those without stroke. The risk model with highest accuracy of classification included 1) stroke risk factors including hypertension, diabetes and atrial fibrillation; 2) retina characteristics, including arteriole diameters, vessel tortuosity, hemorrhages, arteriolevenule nicking and venule symmetry; 3)interaction between retina characteristics, including arteriole diameters by venule symmetry, arteriole diameters by hemorrhage,and venule symmetry by vessel tortuosity. The accuracy of classification was 80.4%. Using retinal characteristics alone achieved an accuracy of 74.5%. / Conclusion: color retina images are a potential tool for stroke risk stratification. Useful characteristics found in the retinal images included vessel diameters, vessel tortuosity, vessel symmetry, hemorrhage, arteriole-venule nicking. The association between the retinal characteristic and stroke was modified by other retinal characteristics. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Li, Qing. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 139-148). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iii / Acknoledgements --- p.v / Chapter Chapter 1 --- Introduction and review of the Literature --- p.1 / Chapter Section 1: --- Stroke prevention and risk assessment tools --- p.1 / Chapter Section 2: --- Rationale of relationship of vascular circulation between retina and brain --- p.9 / Chapter Section 3: --- Manifestation of hypertensive retinopathy and diabetic retinopathy --- p.12 / Chapter Section 4: --- Retina characteristics related to stroke --- p.15 / Chapter Section 5: --- How to make retina as a tool of risk stratification for stroke --- p.28 / Chapter Section 6: --- Rationale to do study to further explore the useful information in color retina images to make it as tool for stroke risk stratification --- p.31 / Chapter Chapter 2 --- Research hypothesis and general design --- p.33 / Chapter Chapter 3 --- Methods of retia characteristics extraction --- p.34 / Chapter Chapter 4 --- A Study of the Reliability of manual measurement of Retinal characteristics using ImageJ --- p.46 / Chapter Chapter 5 --- A study of comparison of retina characteristics between patients with stroke and patients without stroke --- p.55 / Chapter Section 1: --- Method --- p.56 / Chapter Section 2: --- Result-univariate analysis --- p.62 / Chapter Section 3: --- Results-stratification analysis --- p.68 / Chapter Section 4: --- Result-risk model building for stroke risk stratification --- p.79 / Chapter Chapter 6: --- Discussion --- p.118 / Chapter Chapter 7: --- Limitation of this study --- p.133 / Chapter Chapter 8: --- Future development and application of the study results --- p.134 / Appendix --- p.136 / Reference --- p.139

Objetivos: Avaliar as alterações retinianas em modelo experimental de hipóxia em coelhos aclimatizados em ambiente hipóxico-normobárico e investigar os efeitos do tratamento com bevacizumabe intravítreo (IV). Métodos: Vinte e dois coelhos New Zealand, com pesos entre 2,4 a 3,8 kg, foram divididos em quatro grupos. Os grupos S12% (n=5) e B12% (n=5) foram aclimatizados durante três dias consecutivos em concentrações de oxigênio (O2) a 12%. Os grupos S8% (n=5) e B8% (n=7) foram aclimatizados durante três dias consecutivos, com reduções graduais da concentração de O2, até atingir o nadir de 8%. Os olhos direitos (OD) foram mantidos como controle e os olhos esquerdos (OE) dos animais dos grupos S12% e S8% receberam injeção IV de 0,05 ml de solução salina balanceada (SSB), enquanto os OE dos grupos B12% e B8% receberam 0,05 ml (1,25 mg) de bevacizumabe IV. Foram realizados exames de tomografia de coerência óptica (OCT) para avaliação da espessura dos segmentos retinianos (SR) e coroidianos (SC), angiografia por fluoresceína sódica (AF) para observação da presença ou ausência de vasodilatação e tortuosidade da vasculatura retiniana e quantificação do Vascular Endothelial Growth Factor (VEGF) do humor aquoso e soro no primeiro dia (D0), antes do tratamento, no terceiro dia de hipóxia (D7) e no décimo primeiro dia (D11), utilizando-se a técnica do Luminex®. Após, os animais foram sacrificados e amostras do tecido retiniano foram avaliadas por histologia e imuno-histoquímica (IHQ). Resultados: Comparando-se os cortes horizontais dos OD (controle) nos períodos D0 e D7, notou-se redução de 8% (p<0,0001) e 10% (p<0,0001) da espessura do SR nas concentrações de O2 a 12% e 8%, respectivamente. Comparando-se os cortes verticais nos mesmos períodos, verificou-se redução da espessura do SR de 7%, tanto nas concentrações a 12% (p<0,0001) como a 8% (p<0,0001). Nos olhos tratados com bevacizumabe, a redução das médias das espessuras do SR para os cortes horizontais entre os períodos D0 e D7 foi de 6% (<0,0001) e 9% (<0,0001), para as concentrações de O2 a 12 e 8%, respectivamente. Enquanto que nos olhos tratados com SSB no mesmo período, observou-se redução de 8% (<0,0001) e 6% (<0,0001) para as concentrações de O2 a 12 e 8%, respectivamente, nos cortes horizontais. Nos cortes verticais, para os olhos tratados com bevacizumabe, houve redução de 5% (p=0,0005) e 8% (<0,0001) para concentrações de O2 a 12% e 8%, respectivamente; e para os olhos tratados com SSB foi encontrada redução de 7% (<0,0001) e 8% (<0,0001) nas concentrações de O2 a 12% e 8%, respectivamente. As espessuras dos SC não apresentaram alterações. O grupo B8 apresentou diferença estatisticamente significativa na análise da proporção dos olhos que não evidenciaram vasodilatação e tortuosidade dos vasos retinianos durante o período hipoxêmico, e não foram observados neovasos retinianos. A histologia e IHQ dos olhos tratados com SSB e bevacizumabe não demonstraram alterações quando comparados com os controles. Conclusões: A aclimatização de coelhos em ambiente hipóxico-normobárico resultou na redução da espessura do SR no terceiro dia de hipóxia. Notou-se, ainda, aumento da tortuosidade e vasodilatação. O bevacizumabe IV não inibiu a redução da espessura retiniana, mas sim a vasodilatação e tortuosidade vascular. / Objectives: Evaluate retinal changes in experimental model of hypoxia in rabbits acclimatized in normobaric-hypoxic environment and to investigate the effects of the treatment by intravitreal (IV) bevacizumab drug. Methods: Twenty two New Zealand rabbits weighing between 2,4 to 3,8 kg were divided into 4 groups. The groups S12 (n=5) and B12 (n=5) were acclimatized for 3 consecutive days in oxygen concentration (O2) to 12%. The groups S8 (n=5) and B8 (n=7) were acclimatized for 3 consecutive days with gradual reductions in O2 concentration until the nadir of 8%. The right eye (RE) were kept as controls and the left eye (LE) of the animals belonging to S12 and S8 groups received IV injection of 0,05 ml of balanced salt solution (BSS), while the LE belonging to groups B12 and B8 received 0,05 ml (1,25 mg) of bevacizumab IV. Optical coherence tomography (OCT) to evaluate the thickness of the retinal segments (RS) and choroidalsegments (CS), sodium fluorescein angiography (FA) for evaluation of the presence or absence of vasodilation and tortuosity of the retinal vasculature and quantification of VEGF in the aqueous fluid and peripheral blood sample were conducted at the first day (D0) before treatment, on the third day of hypoxia (D7) and day 11 (D11) using the Luminex® technique. After the animals were sacrificed, the retinal tissue samples were evaluated by histology and immunohistochemistry (IHC). Results: Comparing the horizontal sections of the RE (control) in D0 and D7 periods, a reduction of 8% (p<0,0001) and 10% (p<0,0001) the thickness of the RS in O2 concentration at 12% and 8%, respectively. Comparing the vertical cuts in the same period, there was reduced RS thickness of 7% in both concentrations to 12% (p<0,0001) and 8% (p<0,0001). In the eyes treated with bevacizumab, to reduce the average thickness of the retinal segment for horizontal cuts between D0 and D7 periods were 6% (<0,0001) and 9% (<0,0001) for O2 concentrations to 12 and 8%, respectively. While in the eyes treated with BSS in the same period, there was an 8% reduction (<0,0001) and 6% (<0,0001) for the O2 concentration at the 12% and 8%, respectively, in the horizontal cuts. In the vertical sections is observed for the eyes treated with bevacizumab, 5% reduction (p=0.0005) and 8% (<0,0001) O2 concentration at 12% and 8%, respectively; and BSS treated eyes was reduced by 7% (<0,0001) and 8% (<0,0001) in the O2 concentrations of 12% and 8%, respectively. The thickness of the CS did not show changes. The B8 group showed statistical difference in the analysis of the eyes that did not have vasodilation and tortuosity of the retinal vessels during the hypoxic period. Retinal neovascularization were not observed. Histology and IHC of the eyes treated with BSS and bevacizumab showed no changes compared to the control eyes. Conclusions: The acclimatization of the rabbits in normobaric-hypoxic environment has the effect of reducing the thickness RS on the third day of hypoxia. It is observed also increased tortuosity and vasodilation. The intravitreal bevacizumab does not inhibit retinal thickness decrease, but inhibits vasodilation and vascular tortuosity.

A retinopatia da prematuridade é uma doença ocular do desenvolvimento associada a um crescimento vascular retiniano anormal e ocorre somente em recém-nascidos com menos de 32 semanas de gestação e submetidos a longos períodos em incubadoras, tipicamente ricas em oxigênio. A doença possui duas fases: (1) a inibição do desenvolvimento normal de vasos na retina, decorrente da hiperóxia induzida pelo ambiente da incubadora; (2) neovascularização retiniana devido ao aumento de fatores de crescimento desencadeados pela pouca disponibilidade de oxigênio fora da incubadora. O objetivo deste trabalho é avaliar a função visual em um modelo animal de retinopatia da prematuridade, comparando-o a animais sadios. Eletrorretinogramas (ERGs) de campo total foram realizados em camundongos (Mus musculus) controle e camundongos submetidos hiperoxigenação em câmara hiperbárica (75% de oxigênio) durante o desenvolvimento retiniano pós-natal para entender as perdas visuais que ocorrem na retinopatia da prematuridade. Foram utilizados 122 animais, divididos em dois grupos: no grupo controle¸ ERGs foram realizados em P17 (n = 32), P30 (n = 26) e P60 (n = 44) com estimulação em comprimentos de onda distintos (LEDs com pico de emissão em 365 nm, 459 nm, 525 nm e 635 nm) para avaliação da atividade funcional da retina ao longo do desenvolvimento, não passando por nenhuma manipulação experimental adicional; no grupo experimental, os animais foram submetidos à hiperoxigenação de P7 a P12 para indução da angiogênese e avaliados com ERGs com estimulação em 459 nm em P17 (n = 7), P30 (n = 6) e P60 (n = 7), para acompanhar o desenvolvimento retiniano. Todos os animais foram adaptados ao escuro por pelo menos duas horas antes da realização dos experimentos. Em sessão de 40 a 60 minutos de duração, os animais foram submetidos a flashes de luz em intensidades crescentes. Amplitudes e latências das ondas a, b e potenciais oscilatórios foram medidas, e relações intensidade-resposta ajustadas com modelos matemáticos diferentes para comparação entre indivíduos e entre grupos. Os parâmetros obtidos com os ajustes foram comparados através de ANOVA e testes T de Student com as devidas correções de Bonferroni. Nos níveis luminosos testados, a onda b do ERG murino é majoritariamente dominada pela atividade dos bastonetes e a onda a majoritariamente dominada pelos cones. Os registros em animais em P17 e P30 do grupo controle foram similares aos realizados em P60 quanto à latência e amplitude de resposta, bem como quanto à sensibilidade e grau de cooperação entre os distintos elementos do ERG. Entretanto, as amplitudes máximas de resposta foram ligeiramente maiores em P30 para todos os comprimentos de onda e houve ligeira redução da sensibilidade absoluta ao longo do desenvolvimento. Para o grupo experimental, os animais em P17 sofreram as maiores perdas, com diminuições nas amplitudes de todos os componentes do ERG, sem prejuízo das latências ou sensibilidade. Também houve uma sucessiva recuperação das respostas ao longo do desenvolvimento animal. Esses achados indicam que o modelo de ROP em camundongos reproduz aspectos essenciais ao quadro patológico severo humano / The retinopathy of prematurity is an ocular developmental disorder associated with abnormal retinal vascular growth and occurs only in neonates younger than 32 weeks of gestation and undergoing long periods in incubators, typically rich in oxygen. The disease has two phases: (1) the inhibition of the normal development of vessels in the retina, due to the hyperoxia induced by the incubator environment; (2) retinal neovascularization due to the increase in growth factors triggered by the low availability of oxygen outside the incubator. The objective of this study is to evaluate the visual function in an animal model of retinopathy of prematurity, comparing it to healthy animals. Full-field electroretinogram (ERGs) were performed on control mice (Mus musculus) and that were exposed to a hyperbaric chamber (75% oxygen) during post-natal retinal development to understand the visual losses that occur in retinopathy of prematurity. We used 122 animals, divided into two groups: in the control group, ERGs were performed at P17 (n = 32), P30 (n = 26) and P60 (n = 44) with stimulation at different wavelengths (LEDs with peak of emission at 365 nm, 459 nm, 525 nm and 635 nm) for evaluation of the functional activity of the retina throughout the development, without any additional experimental manipulation; in the experimental group, animals were submitted to hyperoxogenation of P7 to P12 for induction of angiogenesis and evaluated with ERGs with stimulation at 459 nm in P17 (n = 7), P30 (n = 6) and P60 (n = 7) to follow up retinal development. All animals were dark adapted for at least two hours prior to the experiments. In sessions with 40 to 60 minutes, the animals were subjected to flashes of light at increasing intensities. Amplitudes and latencies of a-waves, b-waves and oscillatory potentials were measured, and intensity-response relationships adjusted with different mathematical models for comparison between individuals and between groups. The parameters obtained with the adjustments were compared through ANOVA and Student\'s T tests with the appropriate Bonferroni correction. In the light levels tested, the murine ERG b-wave is dominated by rod activity and the a-wave is dominated by the cones. The records in animals at P17 and P30 of the control group were similar to those performed at P60 regarding the latency and amplitude of response, as well as the sensitivity and degree of cooperation between the different ERG elements. However, the maximum response amplitudes were slightly higher at P30 at all wavelengths and there was slight reduction of absolute sensitivity throughout development. For the experimental group, the animals at P17 suffered the greatest losses, with decreases in the amplitudes of all the components of the ERG, without prejudice to the latencies or sensitivity. There was also a successive recovery of responses throughout animal development. These findings indicate that the ROP model in mice reproduces aspects essential to the severe human pathology

Este trabalho apresenta as metodologias que foram empregadas no desenvolvimento de um equipamento de retinografia digital. O sistema proposto foi inteiramente desenvolvido, e manufaturado no Brasil, tornando-se o primeiro retinógrafo digital com tecnologia nacional. O equipamento descrito nesta dissertação é capaz de realizar tanto retinografias, que são imagens capturadas para observação do fundo de olho, quanto angiografias, que são exames nos quais, por meio da administração de contrastes, é possível destacar estruturas específicas da retina. Estes procedimentos permitem o diagnóstico de diversas patologias relacionadas não só à retina, mas também a diversas outras estruturas do olho humano, como por exemplo, a coróide. Esta dissertação demonstra alguns dos desafios e métodos empregados no desenvolvimento do equipamento proposto, além de uma visão geral de cada um dos sistemas que o compõem. São descritos os sistemas mecânico, óptico, eletrônico e de software, sendo cada um deles detalhados quanto às principais funções que desempenham no equipamento. Também são descritas as ferramentas de desenvolvimento utilizadas durante as etapas de análise e projeto. Ainda, são apresentados os testes de desempenho do equipamento, bem como um comparativo entre o sistema proposto e alguns dos equipamentos que atualmente são considerados referência entre médicos e operadores. / This work presents the methodology applied to the development of a digital fundus camera. The proposed system was entirely developed and manufactured in Brazil, becoming the first digital fundus camera with national technology. The described equipment can perform fundus images, which are images captured from eye fundus observation, and also angiograms, which are exams performed through the admission of dyes, where particular retinal structures are enhanced. Those procedures allow the diagnosis of several pathologies not only related to the eye fundus, but also many different structures of the human eye, like the choroid. This dissertation shows some of the challenges and methods that were applied to the development of the proposed system, in addition to an overview of each of the systems that composes the equipment. The mechanical, optical, electronic and software systems are described, being each one of them detailed regarding its main features performed in the equipment. Development tools used during the design are also described. Still, the functional and performance tests and results are described, and also a comparative evaluation between the proposed system and some of the equipments currently defined as reference among physicians and users.

Submitted by Nathalya Silva (nathyjf033@gmail.com) on 2017-05-30T15:33:28Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AcaoHidroxicloroquinaNeuronios.pdf: 1481946 bytes, checksum: a9d07b8a4773b23720d1d74dd905cc64 (MD5) / Approved for entry into archive by Edisangela Bastos (edisangela@ufpa.br) on 2017-06-06T21:33:02Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AcaoHidroxicloroquinaNeuronios.pdf: 1481946 bytes, checksum: a9d07b8a4773b23720d1d74dd905cc64 (MD5) / Made available in DSpace on 2017-06-06T21:33:02Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AcaoHidroxicloroquinaNeuronios.pdf: 1481946 bytes, checksum: a9d07b8a4773b23720d1d74dd905cc64 (MD5) Previous issue date: 2017-03-22 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Hidroxicloroquina (HCQ), classicamente empregado no tratamento da malária e de doenças autoimunes, tem sido proposta como fármaco de escolha para outros fins terapêuticos. Entretanto, este fármaco é conhecido por causar efeitos colaterais, como distúrbios visuais, que podem ser irreversíveis, sendo que os mecanismos que levam a essas desordens não são completamente conhecidos. A toxicidade produzida na retina pelo uso da HCQ pode ser devida a sua alta taxa metabólica, sendo o tecido muito susceptível à ação de xenobióticos e danos oxidativos. Assim, este trabalho tem como objetivo avaliar os efeitos do fármaco HCQ sobre células da retina, bem como seus possíveis mecanismos de citotoxicidade. Como modelo de estudo, utilizamos culturas de células da retina de embrião de galinha. Para avaliar a viabilidade celular foi usado o ensaio de medida de atividade mitocondrial por MTT. A função lisossomal foi avaliada pela taxa de incorporação do corante vermelho neutro. Os níveis de espécies reativas de oxigênio geral e de ânion superóxido foram avaliados pela sonda CellROX e por Nitro Blue Tetrazolium (NBT), e os níveis de glutationa total foram quantificados usando o reagente de Ellman. A viabilidade foi testada em culturas mistas (glia e neurônios), ou culturas enriquecidas com neurônios ou glia, após tratamento com HCQ. Para comparação foi utilizado a cloroquina (CQ), fármaco da qual a HCQ é derivada. As células foram expostas às concentrações de 25μM, 50μM e 75μM por 24 horas. Os resultados demostram que culturas mistas tratadas com CQ apresentaram redução na viabilidade de 36 e 61% para as concentrações de 50μM e 75μM, respectivamente, enquanto HCQ não altera a viabilidade em nenhuma das concentrações testadas. No entanto, quando culturas enriquecidas com células gliais foram expostas a HCQ por 24 horas, a concentração de 75μM teve uma pequena redução na viabilidade das células, sendo as reduções nas células neuronais mais acentuadas, 20, 33 e 56% para as concentrações de 25μM, 50μM e 75μM, respectivamente. Mesmo um tempo menor de tratamento (6 horas) causou perda de viabilidade em neurônios retinianos. A capacidade de incorporação do corante supravital vermelho neutro, também foi alterada em culturas neuronais tratadas com HCQ por 24 horas, tendo redução de 19 e 32%, comparadas ao controle, para as concentrações de 50μM e 75μM, respectivamente. HCQ reduziu significativamente os níveis de espécies reativas de oxigênio produzidas pelas células neuronais, principalmente ânion superóxido, 43, 52 e 61% para as concentrações de 25μM, 50μM e 75μM de HCQ em 24 horas de tratamento, respectivamente. Os níveis de glutationa total em células neuronais apresentaram redução de 37 e 53% quando tratados com 50μM e 75μM de HCQ por 24 horas, respectivamente, comparado ao controle. Quando o meio condicionado de células gliais foi utilizado em células neuronais tratadas com HCQ por 6 horas, este reverteu completamente o processo de citotoxicidade causado pelo fármaco. E quando os níveis de glutationa total foram mensurados em culturas enriquecidas com glia, tratadas com HCQ por 24 horas, não foram observadas quaisquer alterações. Estes resultados sugerem ação citotóxica de CQ em cultura mista de células da retina de embrião de galinha, o que não é observado no tratamento com HCQ. Entretanto, HCQ mostrou ação citotóxica quando as células são cultivadas separadamente, principalmente sobre neurônios, que é revertida por algum fator liberado pelas células gliais no ambiente extracelular, sendo a glutationa uma possível candidata a exercer essa função neuroprotetora. / Hydroxychloroquine (HCQ) is currently used in the treatment of malaria and autoimmune diseases and others therapeutic purposes. However, this drug is known to cause side effects, including producing visual disturbances, which may be irreversible. The mechanisms that produce these visual disorders are not completely known. HCQ - related retinal toxicity may be due to high metabolic rate, being very susceptible to the action of xenobiotics and oxidative damages. Thus, this work aims to evaluate the effects of the HCQ on retinal cells, as well as their possible mechanisms of cytotoxicity. The model used in this work was of cultures of retina cells from chicken embryo. To evaluate cell viability, mitochondrial activity was measured by MTT. The lysosomal function was evaluated by the incorporation rate of the neutral red dye. The levels of reactive species of general oxygen and superoxide anion were evaluated by the CellROX probe and by Nitro Blue Tetrazolium (NBT) and total glutathione levels were quantified using the Ellman reagent. Viability was tested in mixed cultures (glia and neurons) or enriched cultures of neurons and glia after treatment with HCQ and compared with chloroquine (CQ). Cells were exposed to concentrations of 25μM, 50μM and 75μM for 24 hours. The results show that mixed cultures treated with CQ presented a reduction in viability of 36 and 61% at concentrations of 50μM and 75μM, respectively, whereas HCQ did not alter viability at any of the concentrations tested. However, when cultures enriched with glial cells were exposed to HCQ for 24 hours, the concentration of 75μM had a small reduction in cell viability, while that the reduction in neuronal cells was of 20, 33 and 56% at the concentrations of 25μM, 50μM and 75μM, respectively. Even a shorter treatment time (6 hours) there was loss of viability in retinal neurons. The incorporation of neutral red supravital dye was also altered in neuronal cultures treated with HCQ for 24 hours, with reduction of 19 and 32%, compared to the control for the concentrations of 50μM and 75μM, respectively. HCQ significantly reduced the levels of reactive oxygen species produced by the neuronal cells, mainly superoxide anion, 43, 52 and 61% for the concentrations of 25μM, 50μM and 75μM of HCQ in 24 hours of treatment, respectively. In concentrations of 50μM and 75μM of HCQ for 24 for hours, the levels of total glutathione in neuronal cells presented a reduction of 37 and 53%, respectively. When the glial cell conditioned medium was used in neuronal cells for 6 hours after treatment with HCQ, it completely reversed the drug-induced cytotoxicity. When total glutathione levels were measured in culture of glia treated with HCQ for 24 hours no changes were observed. These results suggest cytotoxic action of CQ in mixed culture of chicken embryo retina cells which is not observed in HCQ treatment. However, HCQ showed cytotoxic action when cells are cultured separately, mainly on neurons, which is reversed by some factor released by glial cells in the extracellular environment, and glutathione is a possible candidate to exert this neuroprotective function.