O presente trabalho tem por objetivo analisar o efeito do metilmercúrio na retina de peixe tropical Hoplias malabaricus (Traíra) através de baixas doses agudas. As intoxicações foram realizadas, por meio de injeção intraperitoneal, nas doses de 0,01, 0,05, 0,1 e 1,0 g/g, com um período de quinze dias de depuração do MeHg. Após o término do período de depuração, os olhos foram enucleados e as retinas isoladas foram fixadas em PFA 4% por 3 horas. As retinas foram conservadas, até o momento do uso (ou por no mínimo 9 horas), em tampão PB 0,1M a 4ºC. Após os procedimentos imunohistoquímicos para marcação de células bipolares do tipo ON com estratificação na sublâmina b da CPI, as retinas foram aplanadas para confecção de montagens planas para a análise quantitativa de células bipolares ON imunorreativas a proteína cinase C _. A análise quantitativa das células da camada de células ganglionares (CCG) também foi realizada. Células da CCG foram coradas pela técnica de Nissl, as retinas foram aplanadas em lâminas gelatinizadas e submetidas a uma bateria de desidratação (com diferentes concentrações alcoólicas) e coloração, utilizando cresil violeta como corante. Estas análises foram realizadas em 3 ou 4 retinas para cada dose testada. Análises idênticas foram realizadas nas retinas controle. Todas as retinas foram dividas nos quadrantes dorsal, ventral, nasal, temporal e em centro e periferia. Campos foram fotografados por toda a retina com intervalos de 1 mm, com auxilio do programa Axio Vision por meio de uma câmera digital e um microscópio acoplados a um computador. Os campos amostrados foram contados com o auxilio do programa NIH Scion Imagem 2.0. A densidade média de células foi estimada para cada retina e os grupos intoxicados foram comparados com o grupo controle (Teste T-student). A partir dos dados de densidade celular, mapas de isodensidade foram confeccionados, além de permitir estimar o poder de resolução teórico da acuidade visual de cada um dos animais experimentais utilizados para análise de células da CCG a partir da densidade máxima de células. Evidenciamos que as baixas doses agudas testadas não causam diminuição na densidade célular de células bipolares ON e células da CCG, comparado ao grupo controle. Não houve reduções significativas na densidade de células para ambos os tipos celulares analizados em nenhuma das regiões retinianas nas doses de MeHg testadas. Assim, a intoxicação de MeHg por baixas doses agudas não alterou o poder de resolução teorio da acuiade visual dos animais testados / This study aims to examine the effects of low acute doses of methylmercury (MeHg) on the retina of the tropical fish Hoplias malabaricus (Thraira). Four levels of MeHg intoxication were induced by intraperitoneal injection of doses of either 0.01, 0.05, 0.1 or 1.0 g MeHg/g of body weight, followed by a fifteen day period of depuration of MeHg. After the depuration period, the eyes were harvested, and the retinas were isolated and fixed in 4% paraformaldehyde for 3 hours. The retinas were then stored (for at least for 9 hours) in 0.1 M sodium phosphate PB buffer at 4°C until the time of analysis. ON bipolar cells in sublamina b of the inner plexiform layer immunoreactive to protein Kinase C_ were immunohistochemically labeled, and the retinas were flattened to make whole mounts for quantitative analysis of ON bipolar cell densities. Quantitative analysis of cells in the retinal ganglion cell layer (GCL) was also performed. GCL cells were Nissl stained, and the retinas were flattened on gelatinized slides and subjected to another battery of dehydration (with different alcohol concentrations) and staining using cresyl violet. These analyses were carried out in 3 or 4 retinas for each dose tested. Identical analyses were performed on the control retinas. All retinas were divided into regions: dorsal, ventral, nasal, temporal, center and periphery. Sample retinal fields were photographed throughout the retina at intervals of 1 mm, with a digital camera attached to a microscope using Axio Vision software coupled to a computer. ON bipolar and GCL cells within the fields were counted with the help of the NIH Scion Image 2.0 software. The average density (mm2) of both types of cells was estimated for each retina and the data from each of the four MeHgintoxicated groups were compared with the control group values (Student t-test). From the density data we derived isodensity maps, permitting us to estimate the theoretical resolving power (maximum visual acuity) of each of the experimental animals used from the maximum density of cells in the ganglion cell layer. We showed that low acute doses of MeHg/g do not decrease cell densities of either ON bipolar cells or cells in the GCL, compared to controls. There were no significant decreases in cell density (counts) for either cell type in any of the retinal regions, for any of the MeHg doses tested. Thus, acute low-dose MeHg intoxication did not degrade the estimates of the animals theoretical resolving power

The retina is one of the most metabolically active tissue in the body, rendering it sensitive to vascular dysfunction. Consequently, diseases that disrupt normal retinal blood supply, including retinal vein occlusions (RVO) and diabetic retinopathy, are the leading causes of blindness in working-age adults. Despite available therapies, an estimated 50% of patients do not respond to treatment. We employed a mouse model of retinal vein occlusion (RVO), achieved by tail-vein injection of rose bengal, followed by laser photocoagulation of retinal veins. In vivo analyses – optical coherence tomography (OCT), fluorescein angiography, and electroretinograms (ERGs) - were conducted with the Micron IV system (Phoenix Research Labs). RVO induces acute retinal edema, which peaks during the first 24 hours following injury. Over a 7 day time course the edema resolves, revealing a permanent retinal thinning due to death of retinal neurons. We identified caspase-9, a protease traditionally associated with apoptosis, as an essential mediator of edema. Increased levels of activated caspase-9 were detected in vascular endothelial cells 1 hour following RVO. We tested RVO in mice with inducible endothelial-cell-specific deletion of caspase-9 (iC9 ECKO). Compared to littermate controls, iC9 ECKO mice develop less edema, and sustain less retinal degeneration after RVO injury. ERG analysis showed preservation of retinal function in iC9 ECKO mice. To study whether inhibiting caspase-9 would provide protection against RVO we utilized a highly specific caspase-9 inhibitor, which we can deliver to the retina using simple eyedrops. Treatment of wildtype mice with the caspase-9 inhibitor immediately after induction of RVO provided morphologic, biochemical and functional retinal protection. Inhibition of caspase-9 reduces edema, protects retinal morphology, and helps prevent vision loss following RVO injury. Our studies indicate that endothelial caspase-9 plays an essential role in regulating edema pathogenesis. Moreover, our novel cell permeant caspase-9 inhibitor abrogates the edema and may be a potential therapy for individuals suffering from vascular eye disease.

No description available.

Tese de mestrado. Engenharia Biomédica. Faculdade de Engenharia. Universidade do Porto. 2008

Heritable retinal degenerations dramatically affect individuals across the lifespan. Heritable degenerations with onset in childhood or young adulthood, such as the ABCA4- associated maculopathies, generally obey Mendelian segregation and are attributable to mutations within a single gene. Retinal degenerations with onset in late adulthood, such as age-related macular degeneration, are usually influenced by a complex constellation of genetic and environmental factors. This thesis applies several complementary, high-throughput genotyping platforms to identify relationships between specific heritable retinal phenotypes and genetic variation. This findings of this thesis will aid in the development of guidelines for inclusion in retinal gene therapy trials and help physicians refine their prognoses based on genetic information.

Electrical stimulation of retinal neurons is known to elicit visual sensations. When applied to the retina in a spatial pattern, electrical stimulation may be capable of providing rudimentary patterned vision that may be of benefit to sufferers of degenerative retinal disorders. No such device has yet been devised to provide for chronic study of the psychophysical perceptions elicited from a prosthesis for retinal stimulation. In this study, steps towards achieving this goal have been successfully carried out. Foregoing research was reviewed such that appropriate stimulation parameters were incorporated in the design of a 100 stimulation channel, complimentary metal oxide semiconductor (CMOS) integrated circuit, small enough in size so as to be capable of being implanted within the ocular anatomy or surrounding orbit. The device, and its associated external hardware and software were designed, modeled, fabricated, and interfaced with stimulating electrodes in acute testing in a highorder mammal (Ovis aries) so as to assess the capabilities of the device to elicit cortical potentials as a direct result of stimulation of the neural retina. Testing was performed under conditions similar to those anticipated in chronic in-situ configurations wherein radio-frequency telemetry was used to deliver power and configuration parameters to the device thus avoiding the passage of wires through tissue in order to communicate to the implant circuit. The results of the testing indicate that the circuit is indeed capable of eliciting physiological responses in the animal and evidence is present that these responses could be elicited in patterned form. Further work undertaken includes the development of surgical methods for implantation, and application of the prosthesis circuit in functional electronic stimulation.

The zebrafish visual system relies on positional information in the retina and optic tectum, so that the spatial fidelity of light signals that enter the eye are preserved for visual processing. This positional information is essential for ordered topographic mapping of retinal ganglion cell axons. Spatial information in the retina and tectum relies on discrete signaling pathways that regulate polarized expression of axon guidance molecules in distinct domains in both the retina and tectum, thereby ensuring that accurate topographic maps are created. In this thesis, I have investigated the function of two families of transcription factors, Pbx and Meis, as well as a growth factor of the Bmp family, Gdf6a, in specifying positional identity in the zebrafish visual system. I demonstrate that two partially redundant pbx genes, pbx2 and pbx4, along with members of the meis family, are required for patterning of the dorsal retina and tectum in zebrafish. Embryos lacking these critical transcription factors exhibit retinal ganglion cell axon outgrowth errors, which are likely the result of tectal mis-patterning. Bone morphogenetic protein (Bmp) growth factors regulate dorsal retinal identity in vertebrate models, but the developmental timing of this signaling remains unclear. In this thesis, I investigate the functions of two zebrafish Bmps, Gdf6a and Bmp4, during initiation of dorsal retinal identity. Knockdown of zebrafish Gdf6a blocks initiation of dorsal marker expression, while knockdown of Bmp4 produces no discernable retinal phenotype. These data, combined with analyses of embryos ectopically expressing Bmps, demonstrate that Gdf6a is necessary and sufficient for initiation of dorsal retinal identity, and loss of such identity leads to errors in retinal ganglion cell topographic mapping. Finally, I demonstrate that gdf6a is required for numerous embryonic processes in addition to dorsal retina specification. Gdf6a in required for eye growth, as loss of Gdf6a function leads to microphthalmia. I have obtained preliminary evidence that this growth factor is also required for development of the lens and axial skeleton. Furthermore, many of these phenotypes are similar to those seen in human patients with mutations in GDF6, highlighting the importance of understanding the function of this growth factor in model organisms. / Molecular Biology and Genetics

A hallmark of retinal diseases is degeneration of neural cells, leading to subsequent vision loss. For such diseases, replenishment of functional neural cells may be an optimal therapy. Unlike humans, the adult red-spotted newt, Notophthalmus viridescens, possesses the remarkable ability to regenerate a complete retina following its removal or injury. The purpose of this study was to develop a reproducible model of retinal damage and regeneration in the newt to understand the process of retinal regeneration. Intense light, shown in other organisms to be a relevant model of visual cell loss, was tested in the newt and resulted in variable loss of retinal function, correlating with the appearance of apoptotic cells. Due to the variability of damage observed, surgical removal of the retina was used to complement the light-damage model. A novel and non-invasive protocol using full-field electroretinography was developed to assess retinal function in vivo following damage. Measures of retinal function with the electroretinogram protocol successfully showed that photoreceptor function is initially lost and subsequently restored during regeneration. These results enhance our understanding of retinal regeneration in the adult newt and serve as a starting point for further studies aimed at determining the molecular mechanisms involved in the regeneration process.

The term “stem cell” is often broadly applied to a range of cell types that are relatively undifferentiated and have some capacity for proliferation. In this thesis, I employ a strict definition of stem cells as cells that are capable of both self-renewal and multilineage differentiation. These properties are tested in single precursor cells from the forebrain and its derivative, the retina, using clonal assays. Poor survival is a common problem in single cell cultures, and I show that low oxygen dramatically improves viability in neural stem cells clonally derived from mouse embryonic stem cells, as well as in cultured forebrain neural stem cells. Caspase-dependent and apoptosis-inducing factor-dependent cell death pathways were found to be differentially influenced in low oxygen culture of early, primitive and later, definitive neural stem cells. I isolate precursors from 2 separate regions of the adult mouse forebrain, the lateral ventricle and the hippocampus and argue that only cells resident in the lateral ventricle can be classified as stem cells while the hippocampus contains restricted progenitor cells. Unlike neural stem cells, the very existence of retinal precursors in the adult mammal is controversial. I investigate methods to prospectively identify a rare stem cell population in the pigmented ciliary epithelium of the adult mouse eye and show that, although this population intrinsically gives rise to all retinal cell types, cells can be directed specifically towards a photoreceptor fate by the addition of exogenous factors to the culture media. Pigmentation of retinal stem cells is used as a convenient marker to isolate a retinal stem cell from human embryonic stem cells differentiating under conditions known to promote neural differentiation. Retinal stem cells derived from human embryonic stem cells have highly similar properties to those directly isolated from the eye, and their progeny can similarly be driven to differentiate into photoreceptors. The findings presented in this thesis help to define intrinsic properties of adult neural and retinal precursors and provide a basis for manipulating these cells, potentially for future use in clinical applications.

The term “stem cell” is often broadly applied to a range of cell types that are relatively undifferentiated and have some capacity for proliferation. In this thesis, I employ a strict definition of stem cells as cells that are capable of both self-renewal and multilineage differentiation. These properties are tested in single precursor cells from the forebrain and its derivative, the retina, using clonal assays. Poor survival is a common problem in single cell cultures, and I show that low oxygen dramatically improves viability in neural stem cells clonally derived from mouse embryonic stem cells, as well as in cultured forebrain neural stem cells. Caspase-dependent and apoptosis-inducing factor-dependent cell death pathways were found to be differentially influenced in low oxygen culture of early, primitive and later, definitive neural stem cells. I isolate precursors from 2 separate regions of the adult mouse forebrain, the lateral ventricle and the hippocampus and argue that only cells resident in the lateral ventricle can be classified as stem cells while the hippocampus contains restricted progenitor cells. Unlike neural stem cells, the very existence of retinal precursors in the adult mammal is controversial. I investigate methods to prospectively identify a rare stem cell population in the pigmented ciliary epithelium of the adult mouse eye and show that, although this population intrinsically gives rise to all retinal cell types, cells can be directed specifically towards a photoreceptor fate by the addition of exogenous factors to the culture media. Pigmentation of retinal stem cells is used as a convenient marker to isolate a retinal stem cell from human embryonic stem cells differentiating under conditions known to promote neural differentiation. Retinal stem cells derived from human embryonic stem cells have highly similar properties to those directly isolated from the eye, and their progeny can similarly be driven to differentiate into photoreceptors. The findings presented in this thesis help to define intrinsic properties of adult neural and retinal precursors and provide a basis for manipulating these cells, potentially for future use in clinical applications.