In the developing embryo, the timely fusion of opposing epithelial sheets into one uniform layer denotes the completion of several developmental events. Failure of this epithelial sheet fusion event (ESF) within the choroid fissure (CF) is associated with the congenital disorder Ocular Coloboma, and is one of the leading causes of pediatric blindness. A requirement for a highly coordinated dismantling of the basement membrane (BM) to allow for fusion to occur is undoubted, however the underlying mechanisms of this process are poorly understood. Due to its BM crosslinking capabilities, I have hypothesized that the regulation of nidogen plays a crucial role in the disassembly of the BM prior to ESF. Whole mount in situ hybridization for all four BM components has revealed that expression of nidogen decreases prior to that of other BM components. Additionally, preliminary IHC data has revealed nidogen and collagenIV deposition within the CF. Further, knock-down of nidogen1a and 1b, or the expression of dominant negative nidogen1b resulted in gross morphological, as well as BM organization defects in developing eyes. Together, these data suggest that nidogen plays a role in regulating the integrity of the BM of the eye and may play a role in its disassembly prior to ESF.

Delivering drugs to effectively treat diseases of the back of the eye can be a challenging task. Although pharmacological therapies exist, drug delivery devices and techniques are not very effective at targeting delivery of drugs to the diseased tissues. This work introduces a novel approach to effectively deliver drugs to target tissues such as the choroid and retina. The approach involves a device, a hollow microneedle, to administer the drug formulation into a unique location in the eye, the suprachoroidal space. This new route of administration and a device to accomplish the delivery may provide an effective way to treat diseases of the choroid and retina. The first part of the work determines the ex-vivo feasibility of delivering materials within the suprachoroidal space. The results show that fluids and particles can be delivered into the suprachoroidal space of rabbit, pig and human eyes using a hollow microneedle. It further examines the important parameters for injection of the particles within the suprachoroidal space. The data shows that injection pressure and microneedle length are important parameters for effective delivery of particles. The results lead to a theory on the mechanism by which the particles are delivered into the suprachoroidal space. The second part of the research aims to develop a reliable in vivo delivery device and study the surface area coverage of materials injected into the suprachoroidal space. A hollow glass microneedle device is developed and for the first time shown to be effective in delivering a fluid into the suprachoroidal space in vivo. Up to 100 µL of India ink could be delivered into rabbit eyes in vivo and the spread within the suprachoroidal space is characterized. The results show that a single microneedle injection can cover a significant percentage of the available suprachoroidal space. This is the first study to examine the spread of a material injected into the suprachoroidal space of a live animal. A hollow metal microneedle device is also developed and shown to be effective. The device was able to inject up to 150 µL of latex into suprachoroidal space of fresh human cadaver eyes. The spread of latex is characterized and the results also show that a significant portion of the suprachoroidal space can be covered. The final part of the study examines the clearance of materials injected into the suprachoroidal space of rabbit eyes in vivo. First a comparison of a suprachoroidal injection to a conventional intravitreal injection shows that a suprachoroidal injection is more targeted to the chorioretinal tissues. In addition hollow microneedles are shown to effectively target macromolecules and a therapeutic antibody to the chorioretinal tissues. A study of the clearance kinetics show half lives within the suprachoroidal space on the order of several hours. Nano- and microparticles were also injected into the suprachoroidal space and showed very effective targeting. These non-degradable particles are shown to be present in the suprachoroidal space for months. Basic visual safety assessments identified no adverse effects from the injection of these materials. This represents the first study to compare intraocular clearance kinetics between a suprachoroidal injection and an intravitreal injection. It is also the first study to examine the clearance of a variety of materials from within the suprachoroidal space. Overall this work shows that microneedles have the capability to deliver a variety of materials into the suprachoroidal space of rabbit, pig, and human eyes. The injection can be done in a minimally invasive way with the proper design of an injection device and can target the chorioretinal tissues more effectively than the currently used method. In addition particles have long residence times in the suprachoroidal space, so a particle based drug formulation could provide sustained delivery to the eye. This work represents the first comprehensive study on using the suprachoroidal space as a drug delivery route and also the first study to use hollow microneedles to deliver formulations into the eye in vivo.

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 140-156). Also available online.

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 140-156). Also available in print.

published_or_final_version / Anatomy / Master / Master of Philosophy

The choroid plexus is an organ within each brain ventricle comprised of elaborate folds of epithelium (CPe) and vasculature. It performs numerous functions essential for brain development and health, including secretion of cerebrospinal fluid (CSF) and acting as the blood-CSF barrier. Functionality requires: (1) that CPe and vasculature develop in register and in close proximity, so that the CPe ensheaths the vasculature at a high surface area to volume ratio, which permits efficient CSF secretion; and (2) that CPe barrier integrity is sustained throughout choroid plexus expansion. Genetic experiments in mouse embryos have identified a central role for Sonic hedgehog (Shh) in coordinating these developmental challenges. Specifically, Shh is secreted by differentiated CPe and drives choroid plexus expansion. In the absence of Shh, a hypoplastic choroid plexus forms, which is deficient in CPe, vasculature, and villous folds. Two choroid plexus cell populations respond to Shh: (1) rhombic lip-resident CPe progenitor cells and (2) vascular pericytes. Here, I present evidence that canonical Shh signaling to CPe progenitors alone is insufficient to fully drive their proliferation at normal rates. Rather, Shh-triggered pericytes appear to secondarily boost CPe progenitor cell proliferation, in addition to acting in vascular development. Shh-triggered pericytes also appear necessary for formation of the characteristic folds of the choroid plexus. Thus, pericytes coordinate the expansion of choroid plexus epithelium and vasculature. Notch signaling was also explored and was found to inhibit the differentiation of CPe progenitors, maintaining them in a proliferative state. Notch activation in CPe progenitors leads to invaginated tubules from the overproliferating CPe progenitor domain, without associated vascular growth or villous folds. Folding morphogenesis may thus be regulated by vascular components such as pericytes, and require that vascular growth match CPe growth. To identify Shh-induced pericyte signaling programs that might underlie these developmental processes, expression profiling was performed on dsRed-labeled pericytes isolated from Shh-deficient versus wild-type choroid plexuses. Candidate genes, including several involved in lipid metabolism, were identified. Collectively, this work points to pericytes as central in orchestrating the coordinated elaboration of multiple choroid plexus cell types, producing the complex tissue architecture required for efficient CSF production.

During optic cup morphogenesis, the neuroectodermal layers of the optic vesicle (OV) invaginate ventrally, and fuse at the choroid fissure (CF) along the proximo-distal axis such that the retina and retinal pigment epithelium (RPE) are confined within the cup. Failure of CF closure results in colobomas, which are characterized by the persistence of a cleft or hole at the back of the eye. While CF closure is a critical aspect of ocular development, the molecular and cellular mechanisms underlying this process are poorly understood. My research examined CF closure and colobomas using zebrafish as a model system. In the first study, I determined that early cell fate changes within the eye field could cause colobomas using the zebrafish mutant blowout. Colobomas in blowout resulted from defects in optic stalk morphogenesis whereby the optic stalk extended into the retina and impeded the edges of the CF from meeting and fusing. Positional cloning of blowout identified a nonsense mutation in patched1, a negative regulator of the Hedgehog pathway. Up-regulation of Hedgehog pathway activity causes disruption in the patterning of the OV into proximal and distal territories, revealing that cell fate determination, mediated by Hedgehog signaling, is intimately involved in regulating CF closure. In the second study, I examined Bcl6 function and regulation during zebrafish eye development. bcl6 encodes a transcriptional repressor expressed in the ventral retina during zebrafish eye development. Loss of Bcl6 function leads to colobomas along with up-regulation of p53, a previously known Bcl6 target, and an increase in the number of apoptotic cells in the retina, demonstrating that Bcl6 plays a critical role in preventing apoptosis in the retina during early eye development. I also showed that Vax1 and Vax2 act upstream of bcl6 in the ventral retina. Furthermore, I identified functional interactions between Bcl6, Bcor and Hdac1 during eye development, demonstrating that Bcl6 functions along with Bcor and Hdac1 to mediate cell survival by regulating p53 expression. Together my studies expand the gene regulatory network involved in cell fate determination and cell survival during CF closure and ventral retina formation, and provide mechanistic insight into coloboma formation. / text

Made available in DSpace on 2014-06-11T19:27:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-03-18Bitstream added on 2014-06-13T20:57:20Z : No. of bitstreams: 1 candioto_cg_me_jabo.pdf: 1268753 bytes, checksum: 33fa5ad0387bc80fb30d39654299dced (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O bulbo do olho é uma parte do organismo pouco estudada e que carece de mais valores fisiológicos nas diversas espécies de animais. Neste sentido, o objetivo deste trabalho foi avaliar os cortes histológicos dos olhos de Falco tinnunculus (peneireiro-de-dorso-malhado), por meio da morfometria da córnea, retina, coróide e esclera. As aves eram adultas e de vida livre. Os resultados obtidos foram comparados entre machos e fêmeas e entre os olhos direito e esquerdo. Após enucleação dos olhos, os mesmos foram fixados em solução tamponada de formaldeído por 48 horas e posteriormente, descalcificados. Os olhos direitos foram cortados verticalmente (dorsoventral), e os esquerdos horizontalmente (temporonasal). Após inclusão em parafina, foram confeccionados cortes de 5m de espessura e corados pelas técnicas rotineiras de hematoxilina e eosina. Para a análise dos cortes, foram definidos e medidos, seis pontos (1 a 6) na retina, coróide e esclera, e três pontos referentes na córnea (A, B e C). Com um software de análise de imagens, avaliou-se a espessura em micrômetros da córnea total, e suas camadas (epitelial, “camada média”, endotelial), além da retina, coróide e esclera nos seus respectivos pontos pré-determinados. Na córnea as maiores espessuras foram na periferia (ponto A e C) e as menores na área central (ponto B). As comparações entre as médias foram feitas pelo teste de Tukey (p<0,05). Nas fêmeas a retina foi mais espessa que nos machos. O dados descritos no trabalho complementam os conhecimentos básicos da histomorfologia do olho de Falco tinnunculus necessários para avaliação de cortes histopatológicos / The bulb of the eye is not a well-studied part of the body and needs more physiological values for various animal species. In this manner, the objective of the study was to evaluate the histological eye Falco tinnunculus (commom kestrel), through measurements of the cornea, retina, choroid and sclera. The birds were adults from wild life. The results were compared between males and females and between the right and left eyes. After enucleation of the eyes, they were fixed in buffered formaldehyde for 48 hours and subsequently decalcified. The right eyes were cut vertically (dorsoventral), and left horizontally (temporonasal). After paraffin embedding, sections were prepared from 5m thick and stained using routine hematoxylin and eosin. For the analysis of the cuts, six points (1-6) in the retina, choroid and sclera, and three points on the cornea (A, B and C) have been defined and measured. With an image analyzing software, the corneal thickness and its layers (epithelium, middle layer, endothelial), were evaluated in total microns, as well as the retina, choroid and sclera in their respective pre-determined points. Corneal thickness was greater in the periphery (A and C) and lower in the central area (B). Comparisons between means were made using the Tukey test (p <0.05). In females retina was thicker than the males. The data described in the paper complement the basic knowledge of histomorphology eye Falco tinnunculus for the assessment of histopathology

Orientador: Karin Werther / Banca: Fabiano Montiani-Ferreira / Banca: Rosimeri de Oliveira Vasconcelos / Resumo: O bulbo do olho é uma parte do organismo pouco estudada e que carece de mais valores fisiológicos nas diversas espécies de animais. Neste sentido, o objetivo deste trabalho foi avaliar os cortes histológicos dos olhos de Falco tinnunculus (peneireiro-de-dorso-malhado), por meio da morfometria da córnea, retina, coróide e esclera. As aves eram adultas e de vida livre. Os resultados obtidos foram comparados entre machos e fêmeas e entre os olhos direito e esquerdo. Após enucleação dos olhos, os mesmos foram fixados em solução tamponada de formaldeído por 48 horas e posteriormente, descalcificados. Os olhos direitos foram cortados verticalmente (dorsoventral), e os esquerdos horizontalmente (temporonasal). Após inclusão em parafina, foram confeccionados cortes de 5m de espessura e corados pelas técnicas rotineiras de hematoxilina e eosina. Para a análise dos cortes, foram definidos e medidos, seis pontos (1 a 6) na retina, coróide e esclera, e três pontos referentes na córnea (A, B e C). Com um software de análise de imagens, avaliou-se a espessura em micrômetros da córnea total, e suas camadas (epitelial, "camada média", endotelial), além da retina, coróide e esclera nos seus respectivos pontos pré-determinados. Na córnea as maiores espessuras foram na periferia (ponto A e C) e as menores na área central (ponto B). As comparações entre as médias foram feitas pelo teste de Tukey (p<0,05). Nas fêmeas a retina foi mais espessa que nos machos. O dados descritos no trabalho complementam os conhecimentos básicos da histomorfologia do olho de Falco tinnunculus necessários para avaliação de cortes histopatológicos / Abstract: The bulb of the eye is not a well-studied part of the body and needs more physiological values for various animal species. In this manner, the objective of the study was to evaluate the histological eye Falco tinnunculus (commom kestrel), through measurements of the cornea, retina, choroid and sclera. The birds were adults from wild life. The results were compared between males and females and between the right and left eyes. After enucleation of the eyes, they were fixed in buffered formaldehyde for 48 hours and subsequently decalcified. The right eyes were cut vertically (dorsoventral), and left horizontally (temporonasal). After paraffin embedding, sections were prepared from 5m thick and stained using routine hematoxylin and eosin. For the analysis of the cuts, six points (1-6) in the retina, choroid and sclera, and three points on the cornea (A, B and C) have been defined and measured. With an image analyzing software, the corneal thickness and its layers (epithelium, "middle layer", endothelial), were evaluated in total microns, as well as the retina, choroid and sclera in their respective pre-determined points. Corneal thickness was greater in the periphery (A and C) and lower in the central area (B). Comparisons between means were made using the Tukey test (p <0.05). In females retina was thicker than the males. The data described in the paper complement the basic knowledge of histomorphology eye Falco tinnunculus for the assessment of histopathology / Mestre

Le vieillissement et la dégénérescence du cerveau, associés à des déficits cognitifs, comportementaux et neurologiques, représentent aujourd'hui un problème majeur de santé publique. L'une des principales maladies liées à l'âge est la maladie d'Alzheimer (MA). L'une des caractéristiques de la MA est l'apparition de plaques amyloïdes, résultant de l'agrégation du peptide ßA4. Physiologiquement, le précurseur de la protéine amyloïde (APP) est clivé par une alpha-sécrétase qui génère un fragment soluble de l'APP (sAPP), important pour la formation de nouvelles cellules nerveuses (neurogenèse). Ce clivage en prévient deux autres, par les béta- et gamma-sécrétases, impliqués dans la MA, et conduisant à la formation du ßA4 toxique. Une analyse du plexus choroïde (PCh) a mis en évidence la forte expression de l’APP par cette structure cérébrale. Le PCh est une structure facilement accessible et produisant le liquide cérébro-spinal : son impact peut donc être répercuté à l’ensemble du cerveau. Il pourrait être une source cérébrale importante d’APP, et contribuer fortement à la pathologie. Mon projet de thèse s'inscrivait dans la possibilité de réguler génétiquement l'expression des formes sauvages et mutées de l'APP au niveau de cette source, et suivre les conséquences sur la neurogenèse adulte et la formation des plaques amyloïdes, marqueur histopathologique de la MA. Par l’utilisation de la thérapie génique pour moduler l’expression de l’APP dans les PCh, nous avons confirmé l’importance de l’APP soluble provenant des PCh dans la neurogenèse adulte. Les PCh semble être une source importante d’APP dans le cerveau, et pourraient avoir un rôle clé dans la maladie d’Alzheimer. / Aging and degeneration of the brain with cognitive decline and neurologic symptoms are major individual and societal problems. The major age-related brain degeneration disease is Alzheimer’s disease (AD) with about 40 million people affected in 2015.Physiologically, the Amyloid Precursor Protein (APP) is cleaved by an alpha-secretase, releasing soluble APP (sAPP) an important regulator of adult neurogenesis. This cleavage prevents two others in positions beta and gamma that generate the ßA4 toxic peptide, a hallmark of Alzheimer Disease.Next generation RNA-sequencing has revealed that APP is the 16th most expressed genes in the choroid plexus (CP), suggesting that it may be a major source of sAPP and ßA4 in the cerebrospinal fluid (CSF). If so, adult neurogenesis in the SVZ and hippocampus may be regulated by the choroid plexus and impeded in mutations favoring ßA4 production. My thesis project fell under the possibility to regulate App expression in the CP, and follow consequences on adult neurogenesis and plaques formation in AD. Using viral vectors to modulate App expression in the CP, we confirmed the importance of sAPP coming from CP in adult neurogenesis. With so, CP seems to be an important source of APPin the brain, and could have a key role in AD.