Glaucoma is characterized by retinal ganglion cell apoptosis leading to a corresponding loss of the visual field. Elevated intraocular pressure is the principal clinical association of this disease and its reduction remains the mainstay of current therapy. This research established an in-vitro glaucoma model and investigated the direct effects of increased hydrostatic pressure on retinal ganglion cell survival as well as the cellular response to changes in pressure. In the first part of this thesis (chapter 3) the direct effects of pressure on retinal ganglion cell survival was established. The differentiated RGC5 cell line was subjected to elevated pressure 100 mmHg for a period of two hours in a pressure chamber. Cell apoptosis was then detected by TdT-mediated dITP Nick-End Labelling (TUNEL). Quantitative analysis of the percentage of apoptotic cells between the control and pressure groups by Laser Scanning Cytometry (LSC) revealed that pressure alone induced significant apoptosis. Furthermore, caspase-3 cleavage was detected in the pressure treated cells by Western blot analysis. The next three chapters investigated how the applied pressure may be mediated through cellular mechno-sensitive structures. TWIK Related Arachiodonic Acid stimulated K+ channel (TRAAK) is a mechano-gated neuronal potassium channel, which can be opened by pressure and arachidonic acid. In chapter 4, TRAAK was identified as expressed on the rat RGC5 cell line. This was determined by both immunostaining and RT-PCR. Opening this channel by arachidonic acid induced significant apoptosis in RGC5 neurons; elevated extracellular K+ concentration and blockage of TRAAK by gadolinium inhibited both arachidonic acid and pressure-induced apoptosis. These results indicated that elevated pressure resulted in opening of the outward potassium channel-TRAAK and consequently potassium ion efflux and apoptotic volume decrease (AVD). Data from chapter 5 revealed that pressure also caused actin reorganization with both F- and G-actin shifts. At the early stage (following 2 hours pressure treatment), actin polymerization led to G-actin pool decrease and disinhibition of DNase1 in the cytoplasm. This has been suggested to lead to DNase1 nuclear translocation and contribution to DNA fragmentation associated with apoptosis. The preliminary microarray results of chapter 6 revealed pressure effects on gene expression Included in the many up- and down-regulated genes was; down-regulation of antiapoptotic gene- BcL-x and up- regulation of Damage-Induced Neuronal Endopeptidase (DINE) after pressure treatment. This study showed that elevated pressure induced RGC5 apoptosis and affected multi cellular mechanosnesitive structures. These results may indicate new mechanisms of RGC neuron apoptosis and further therapeutic strategies.

Thesis (Ph.D.)--Texas Christian University, 2007. / Title from dissertation title page (viewed Apr. 25, 2007). Includes abstract. Includes bibliographical references.

In this dissertation, I investigated some fundamental issues related to medical image segmentation, registration, and watermarking. I used color retinal fundus images to perform my study because of the rich representation of different objects (blood vessels, microaneurysms, hemorrhages, exudates, etc.) that are pathologically important and have close resemblance in shapes and colors. To attack this complex subject, I developed a divide-and-conquer strategy to address related issues step-by-step and to optimize the parameters of different algorithm steps. Most, if not all, objects in our discussion are related. The algorithms for detection, registration, and protection of different objects need to consider how to differentiate the foreground from the background and be able to correctly characterize the features of the image objects and their geometric properties. To address these problems, I characterized the shapes of blood vessels in retinal images and proposed the algorithms to extract the features of blood vessels. A tracing algorithm was developed for the detection of blood vessels along the vascular network. Due to the noise interference and various image qualities, the robust segmentation techniques were used for the accurate characterization of the objects’ shapes and verification. Based on the segmentation results, a registration algorithm was developed, which uses the bifurcation and cross-over points of blood vessels to establish the correspondence between the images and derive the transformation that aligns the images. A Region-of-Interest (ROI) based watermarking scheme was proposed for image authenticity. It uses linear segments extracted from the image as reference locations for embedding and detecting watermark. Global and locally-randomized synchronization schemes were proposed for bit-sequence synchronization of a watermark. The scheme is robust against common image processing and geometric distortions (rotation and scaling), and it can detect alternations such as moving or removing of the image content.

Retinal degenerative diseases are diseases that may severely affect vision of people at different ages. These include retinitis pigmentosa (RP) and age-related macular degeneration (AMD). The current treatments for these diseases are limited. Since dysfunction and atrophy of the RPE are the key factors in the development of retinal degenerative diseases, transplantation of healthy retinal pigment epithelial (RPE) cells might be a promising therapeutic strategy. However, homologous RPE cells may lead to host immune rejection and harvesting autologous RPE cells may cause severe complications. Autologous iris pigment epithelial (IPE) cells, which are relatively easy to obtain, possess the same embryonic origin and share similar characteristics as RPE cells. Therefore, they may be used as a substitute of RPE cells for transplantation. Increasing interests have been demonstrated with the use of substrate to support cell attachment, proliferation and differentiation, so that transplanted cells could maintain the differentiated phenotype and perform their normal functions. However, degradation of biodegradable substrates may cause the breakdown of functional cell monolayer and produce toxic byproducts. Therefore, the aim of current study is to investigate the in vitro characteristics of rat IPE cells cultured on surface modified non-degradable expanded-polytetrafluorethylene (ePTFE) substrates and host response to the substrates without cells. Primary pure IPE cells were successfully isolated from rat eyes, which provided abundant cells for subsequent experiments. IPE cells harvested from both Long Evans rats and Dark Agouti rats proliferated and reached confluence on fibronectin coated n-heptylamine modified (F-HA) ePTFE substrates. These cells exhibited cuboidal or polygonal morphology with heavy pigmentation. In addition to the typical epithelial cell morphology, rat IPE cells grown on F-HA ePTFE substrates were able to form a cell monolayer with functional formation of tight junctional complex between neighboring cells. The IPE cell monolayers also demonstrated increased phagocytosis of photoreceptor outer segments (POS) with time and expression of cellular retinylaldehyde-binding protein (CRALBP) that served an important role in the conversion of all-trans-retinal to 11-cis-retinal in visual cycle. In the in vivo study, F-HA ePTFE substrate was successfully transplanted into the subretinal space of Royal College of Surgeons (RCS) rat, which is a well-recognized animal model of retinal degeneration. The F-HA ePTFE substrate remained flat up to 4 weeks after transplantation and did not induce significant up-regulation of pro-inflammatory cytokines TNFα and IL1β as well as activation of Müller cells and astrocytes which occurred in response to retinal inflammation. In conclusion, rat IPE cells that were grown on F-HA ePTFE substrate were able to establish a monolayer with functional tight junctions and RPE-specific functions. The F-HA ePTFE substrate demonstrated good biocompatibility in the subretinal space of RCS rats. These findings provide a potential therapeutic strategy for retinal degeneration. / published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

No description available.

No description available.

Glaucoma is characterized by retinal ganglion cell apoptosis leading to a corresponding loss of the visual field. Elevated intraocular pressure is the principal clinical association of this disease and its reduction remains the mainstay of current therapy. This research established an in-vitro glaucoma model and investigated the direct effects of increased hydrostatic pressure on retinal ganglion cell survival as well as the cellular response to changes in pressure. In the first part of this thesis (chapter 3) the direct effects of pressure on retinal ganglion cell survival was established. The differentiated RGC5 cell line was subjected to elevated pressure 100 mmHg for a period of two hours in a pressure chamber. Cell apoptosis was then detected by TdT-mediated dITP Nick-End Labelling (TUNEL). Quantitative analysis of the percentage of apoptotic cells between the control and pressure groups by Laser Scanning Cytometry (LSC) revealed that pressure alone induced significant apoptosis. Furthermore, caspase-3 cleavage was detected in the pressure treated cells by Western blot analysis. The next three chapters investigated how the applied pressure may be mediated through cellular mechno-sensitive structures. TWIK Related Arachiodonic Acid stimulated K+ channel (TRAAK) is a mechano-gated neuronal potassium channel, which can be opened by pressure and arachidonic acid. In chapter 4, TRAAK was identified as expressed on the rat RGC5 cell line. This was determined by both immunostaining and RT-PCR. Opening this channel by arachidonic acid induced significant apoptosis in RGC5 neurons; elevated extracellular K+ concentration and blockage of TRAAK by gadolinium inhibited both arachidonic acid and pressure-induced apoptosis. These results indicated that elevated pressure resulted in opening of the outward potassium channel-TRAAK and consequently potassium ion efflux and apoptotic volume decrease (AVD). Data from chapter 5 revealed that pressure also caused actin reorganization with both F- and G-actin shifts. At the early stage (following 2 hours pressure treatment), actin polymerization led to G-actin pool decrease and disinhibition of DNase1 in the cytoplasm. This has been suggested to lead to DNase1 nuclear translocation and contribution to DNA fragmentation associated with apoptosis. The preliminary microarray results of chapter 6 revealed pressure effects on gene expression Included in the many up- and down-regulated genes was; down-regulation of antiapoptotic gene- BcL-x and up- regulation of Damage-Induced Neuronal Endopeptidase (DINE) after pressure treatment. This study showed that elevated pressure induced RGC5 apoptosis and affected multi cellular mechanosnesitive structures. These results may indicate new mechanisms of RGC neuron apoptosis and further therapeutic strategies.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available in print.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.