Glaucoma is the second leading cause of blindness worldwide. The clinical standard for monitoring the functional deficits in the retina that are caused by glaucoma is the visual field test. In addition to monitoring the functional loss, evaluating the disease-related structural changes in the human retina also helps with diagnosis and management of this progressive disease. The characteristic changes of retinal structures such as the optic nerve head (ONH) are monitored utilizing imaging modalities such as color (stereo) fundus photography and, more recently, spectral-domain optical coherence tomography (SD-OCT). With the inherent subjectivity and time required for manually segmenting retinal structures, there has been a great interest in automated approaches. Since both fundus and SD-OCT images are often acquired for the assessment of glaucoma, the automated segmentation approaches can benefit from combining the multimodal complementary information from both sources. The goal of the current work is to automatically segment the retinal structures and extract the proper parameters of the optic nerve head related to the diagnosis and management of glaucoma. The structural parameters include the cup-to-disc ratio (CDR) which is a 2D parameter and is obtainable from both fundus and SD-OCT modalities. Bruch's membrane opening-minimum rim width (BMO-MRW) is a recent 3D structural parameter that is obtainable from the SD-OCT modality only. We propose to use the complementary information from both fundus and SD-OCT modalities in order to enhance the segmentation of structures of interest. In order to enable combining information from different modalities, a feature-based registration method is proposed for aligning the fundus and OCT images. In addition, our goal is to incorporate the machine-learning techniques into the graph-theoretic approach that is used for segmenting the structures of interest. Thus, the major contributions of this work include: 1) use of complementary information from SD-OCT and fundus images for segmenting the optic disc and cup boundaries in both modalities, 2) identifying the extent that accounting for the presence of externally oblique border tissue and retinal vessels in rim-width-based parameters affects structure-structure correlations, 3) designing a feature-based registration approach for registering multimodal images of the retina, and 4) developing a multimodal graph-based approach to segment the optic nerve head (ONH) structures such as Internal Limiting Membrane (ILM) surface and Bruch's membrane surface's opening.

ABSTRACT A multitude of complex cellular changes are required throughout development in order for a single cell to transform into a fully functioning organism. Cellular events including proliferation, migration, and differentiation have to be carefully controlled in order for development to proceed correctly. In order to study such dynamic processes, in vivo models are often utilized. Using the zebrafish (Danio rerio) as a model system, we have investigated the role of an axon guidance signaling pair, Semaphorin6A (Sema6A) and PlexinA2 (PlxnA2), in neurodevelopment. A previous investigation into the developmental expression patterns of sema6A and plxnA2 in zebrafish, revealed overlapping expression in the developing eye. At this early stage, the cells in the optic vesicles are undifferentiated retinal precursor cells (RPCs) and therefore do not require Sema/Plxn signaling for their canonical axon guidance role. To understand what the function of this early expression was, we knocked down both sema6a and plxna2 and observed 1) a loss of cohesion of RPCs within optic vesicles, and 2) a decrease in RPC proliferation (Ebert et al., 2014). Because these phenotypes were seen at an early stage and given that many developmental processes are dependent on genetic regulation, we hypothesized that Sema6A/PlxnA2 signaling could be regulating transcription of downstream target genes. To investigate this, we performed a microarray experiment and uncovered 58 differentially regulated genes (Emerson et al., 2017a). Prior to our study, it was not known that Sema/Plxn signaling led to changes in gene transcription. In an effort to understand the contribution of identified candidate genes to early sema6A/plxnA2 knockdown phenotypes, candidate genes with predicted functions in proliferation and migration were investigated. First, we show that rasl11b is important for regulation of RPC proliferation in the developing optic vesicles. Second, we show that shootin-1 is important in optic vesicle migration, retinal pigmented epithelium formation and optic tract patterning. Furthermore, PlxnA2 regulation of shootin-1 levels is important in sensory and motor axon patterning and branching in the peripheral nervous system. Belonging to a large family of proteins with the ability to cross talk, Semas and Plxns rely on spatially and temporally differential expression patterns to perform their tissue-specific roles. Here, we used in situ hybridization to comprehensively uncover the neuronal expression patterns of the PlxnA family in the early developing zebrafish (Emerson et al., 2017b). In addition, we present for the first time that zebrafish have two genes for PlxnA1, A1a and A1b, which show divergent expression patterns. Semas and Plxns are critical for many aspects of development and together, this body of work provides further insight into the downstream signaling mechanisms and roles of these essential developmental signaling proteins.

No description available.

An analytical method was developed for the determination of the â-agonists clenbuterol, cimaterol and salbutamol in bovine retina. The method involved extraction into a pH 8.5 tris-HCl buffer, followed by protease enzyme digestion and immunoaffinity column cleanup before analysis by liquid chromatography with tandem mass spectrometry detection LC/MS/MS. The LOD for clenbuterol, salbutamol and cimaterol were 0.64, 1.20 and 1.92 ng/g respectively. The identities of the analytes were able to be confirmed to an acceptable standard. An analytical method was also developed for the analysis of the â-agonists clenbuterol, salbutamol, cimaterol, ractopamine, and mabuterol in bovine urine and emu muscle. The urine and muscle samples were digested with â-glcuronidase enzyme and cleaned up using a Bond Elute Certify SPE. The extracts were analysed by LC/MS/MS. Deuterated internal standards were used for quantitation. The LOD for urine [less than] 1ng g and for emu muscle it was [less than] 0.3 ng/g. The last part of the work describes the simultaneous gas chromatography-mass spectrometric analysis of diethylstilbestrol DES, hexestrol HEX, dienestrol DIEN, zeranol ZER, taleranol TAL and zeralenone ZON in fresh full cream and fresh skim milk. The analytes were analysed as their trimethyl silane (TMS) derivatives. A three phase solvent system was used for extraction and the extract was cleaned up using a combination of the anion exchange and hydrophobic properties of an anion exchange SPE. The detection limits for DES, DIEN, HEX, ZER, TAL and ZON were 9.6, 9.6, 16.8 , 7.2 , 13.5 and 34.8 ng/L respectively. / Doctor of Philosophy(PhD)

Doctor of Philosophy (Medicine) / Background: Diabetic vascular complications affect both the macro- and microvasculature. Microvascular pathology in diabetes may be mediated by biochemical factors that precipitate cellular changes at both the gene and protein levels. In the diabetic retina, vascular pathology is found mainly in microvessels, including the retinal precapillary arterioles, capillaries and venules. Macular oedema secondary to breakdown of the inner blood-retinal barrier is the most common cause of vision impairment in diabetic retinopathy. Müller cells play a critical role in the trophic support of retinal neurons and blood vessels. In chronic diabetes, Müller cells are increasingly unable to maintain their supportive functions and may themselves undergo changes that exacerbate the retinal pathology. The consequences of early diabetic changes in retinal cells are primarily considered in this thesis. Aims: This thesis aims to investigate the effect of perivascular cells (Müller cells, RPE, pericytes) on retinal endothelial cell permeability using an established in vitro model. Methods: Immunohistochemistry, cell morphology and cell growth patterns were used to characterise primary bovine retinal cells (Müller cells, RPE, pericytes and endothelial cells). An in vitro model of the blood-retinal barrier was refined by coculturing retinal endothelial cells with perivascular cells (Müller cells or pericytes) on opposite sides of a permeable Transwell filter. The integrity of the barrier formed by endothelial cells was assessed by transendothelial electrical resistance (TEER) measurements. Functional characteristics of endothelial cells were compared with ultrastructural morphology to determine if different cell types have barrier-enhancing effects on endothelial cell cultures. Once the co-culture model was established, retinal endothelial cells and Müller cells were exposed to different environmental conditions (20% oxygen, normoxia; 1% oxygen, hypoxia) to examine the effect of perivascular cells on endothelial cell permeability under reduced oxygen conditions. Barrier integrity was assessed by TEER measurements and permeability was measured by passive diffusion of radiolabelled tracers from the luminal to the abluminal side of the endothelial cell barrier. A further study investigated the mechanism of laser therapy on re-establishment of retinal endothelial cell barrier integrity. Müller cells and RPE, that comprise the scar formed after laser photocoagulation, and control cells (Müller cells and pericytes, RPE cells and ECV304, an epithelial cell line) were grown in long-term culture and treated with blue-green argon laser. Lasered cells were placed underneath confluent retinal endothelial cells growing on a permeable filter, providing conditioned medium to the basal surface of endothelial cells. The effect of conditioned medium on endothelial cell permeability was determined, as above. Results: Co-cultures of retinal endothelial cells and Müller cells on opposite sides of a permeable filter showed that Müller cells can enhance the integrity of the endothelial cell barrier, most likely through soluble factors. Low basal resistances generated by endothelial cells from different retinal isolations may be the result of erratic growth characteristics (determined by ultrastructural studies) or the selection of vessel fragments without true ‘barrier characteristics’ in the isolation step. When Müller cells were co-cultured in close apposition to endothelial cells under normoxic conditions, the barrier integrity was enhanced and permeability was reduced. Under hypoxic conditions, Müller cells had a detrimental effect on the integrity of the endothelial cell barrier and permeability was increased in closely apposed cells. Conditioned medium from long-term cultured Müller cells and RPE that typically comprise the scar formed after lasering, enhanced TEER and reduced permeability of cultured endothelial cells. Conclusions: These studies confirm that bovine tissues can be used as a suitable model to investigate the role of perivascular cells on the permeability of retinal endothelial cells. The dual effect of Müller cells on the retinal endothelial cell barrier under different environmental conditions, underscores the critical role of Müller cells in regulating the blood-retinal barrier in health and disease. These studies also raise the possibility that soluble factor(s) secreted by Müller cells and RPE subsequent to laser treatment reduce the permeability of retinal vascular endothelium. Future studies to identify these factor(s) may have implications for the clinical treatment of macular oedema secondary to diseases including diabetic retinopathy.

Master of Science / In vivo many forms of glia utilise both intercellular and extracellular pathways in the form of IP3 permeable gap junctions and cytoplasmic ATP diffusion to produce calcium waves. We introduce a model of ATP and Ca2+ waves in clusters of glial cells in which both pathways are included. Through demonstrations of its capacity to replicate the results of existing theoretical models of individual pathways and to simulate experimental observations of retinal glia the validity of the model is confirmed. Characteristics of the waves resulting from the inclusion of both pathways are identified and described.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

<p>The focus of this thesis is the life and death of adult retinal ganglion cells (RGCs). RGCs are neurons that convey visual information from the retina to higher centers in the brain. If the optic nerve is transected (ONT), adult RGCs die by a form of cell death called apoptosis, and a general hypothesis is that neurotrophic factors can support the survival of injured neurons.</p><p>With the intention to gain knowledge about systems that can be used to decrease RGC death after ONT, we have studied growth factor receptors belonging to the tyrosine kinase family of receptors (RTK), known to mediate important cell survival signals. We found that the RTK Ret and its coreceptor GFRα1 were expressed by RGCs, and to test the above-mentioned hypothesis, we intraocularly administered glial cell-line derived factor, which activates a Ret-GFRα1 complex, and found transiently mediated RGC survival after ONT. </p><p>To identify new, potential neurotrophic factor receptors expressed by RGCs, with the aim to improve RGC survival after ONT, we developed a method for the molecular analysis of acutely isolated RGCs. The method involves retrograde neuronal tracing, mechanical retinal layer-separation, and isolation of individual RGCs under UV-light for RT-PCR analysis. Using this method, in combination with degenerate PCR directed towards the tyrosine kinase domain, several RTKs were identified. Axl, Sky, VEGFR-2, VEGFR-3, CSF-1R, and PDGF-βR are expressed by adult RGCs, and considered to be receptors with potential neurotrophic activity. Other results have shown that RGCs may require depolarization or increase in intracellular cAMP levels in order to fully respond to exogenously added trophic factors. We found that melanocortin receptors (MCRs) were expressed by RGCs, and MCRs can mediate elevation of intracellular AMP. We observed that α-MSH induced neurite outgrowth from embryonic retinal cells, indicating that MCR ligands have direct effects on retinal cells. RTKs and their ligands may be involved in endogenous systems for neuronal repair within the visual system. BDNF, NT-3, FGF2, and HGFR all increased in the retina after ONT and may be a part of an activated system for neuronal repair locally within the retina. </p><p>Adult axotomized RGCs die by apoptosis, therefore we examined the regulation of apoptotic genes after ONT. Bim and Bax increased in the retina after ONT, and may promote death of axotomized RGCs, whereas the increase in Bcl-2 may contribute to limit RGC apoptosis after ONT. </p><p>All in all, this thesis provides insights into the expression and regulation of molecules involved in the death and survival of RGCs. The results have revealed a number of potential neurotrophic receptors expressed by RGCs, and both identified RTKs and MCRs will serve as new targets in therapeutic approaches aiming at counteraction of RGC death after injury.</p>

Die Funktionsweise des invertierten Aufbaus der Netzhaut im Wirbeltierauge ist ein altes Rätsel der Wissenschaft. Das beim Sehvorgang auf die Netzhaut einfallende Licht muss erst alle Netzhautschichten durchdringen, bevor es die Photorezeptorzellen erreicht, welche sich auf der lichtabgewandten Seite des Gewebes befinden. Die vorgelagerten Gewebsschichten enthalten zahlreiche lichtstreuende Bestandteile und müssten den Sehvorgang der Wirbeltiere theoretisch negativ beeinflussen. Diese Annahme steht jedoch im Widerspruch zu dem beeindruckenden Sehvermögen der meisten Wirbeltiere. Die Müllerschen Radialgliazellen stellen eine Lösung für diesen scheinbaren Widerspruch dar. Aufgrund der auffälligen morphologischen Struktur dieser Gliazellen, welche die gesamte Dicke der Netzhaut säulenförmig durchspannen, wurde die Hypothese aufgestellt, dass Müllerzellen nach dem Prinzip der Lichtleitung arbeiten und so das Licht zu den Photorezeptoren transportieren. Diese Theorie konnte jedoch bisher noch nicht bewiesen werden, da die bisherigen experimentellen Messmethoden auf der Basis von isolierten Müllerzellen ungeeignet sind, um diese Funktion im lebenden Gewebe nachzuweisen. Die vorliegende Arbeit beweist erstmalig, dass die Müllerschen Gliazellen als lebende Lichtleiter im Netzhautgewebe funktionieren. Um diese Aufgabe den Müllerzellen eindeutig zuzuordnen, wurde eine neuartige Methode entwickelt, welche gleichzeitig mehrere für den Nachweis unverzichtbare Parameter erfassen kann. Aufgrund einer fluoreszenzbasierten Visualisierung der Müllerzellen in der intakten Netzhaut konnte mit Hilfe eines auf Glasfaseroptik basierenden Aufbaus die Beleuchtung einzelner Müllerzellen erfolgen. Zeitgleich war es möglich, sowohl den Weg des Lichtes von der lichtzugewandten Seite bis zu den Photorezeptoren als auch die Transmission hinter dem Gewebe zu detektieren. Die Komplexität dieses Messverfahrens erlaubte eine detaillierte Charakterisierung des Einflusses der Müllerzelle auf die Streueigenschaften der verschiedenen retinalen Schichten sowie des sich ergebenden Lichtsignals an den Rezeptorzellen. Mittels eines speziellen Analyseverfahrens konnte umfassendes Datenmaterial erhoben und so die Müllerzelle eindeutig als Lichtleiter identifiziert werden. Darauf aufbauend wird in dieser Arbeit außerdem gezeigt, dass alle Müllerzellen gemeinsam und damit in ihrer Gesamtheit mittels ihrer Lichtleitfunktion das an den Photorezeptoren ankommende Lichtmuster beeinflussen, was zu einer verbesserten Bildqualität führt. Dies wird zusätzlich durch morphologische Untersuchungen gestützt, die zeigen, dass die für das Kontrastsehen verantwortlichen Zapfen-Photorezeptorzellen lokal hinter den Müllerzellen angeordnet sind. Demnach ist jeder Zapfen mit einem ihm vorgelagerten Lichtleiter ausgestattet. Zusammenfassend liefert diese Arbeit eine Erklärung, wie trotz des invertierten Aufbaus der Netzhaut die visuelle Information als Grundlage für das Sehen der Wirbeltiere erhalten bleibt.

The helper-dependent adenoviral (HD-Ad) vector is the latest generation of Ad vectors. It ameliorates the vector-associated immunogenic problems with increased capacity for carrying DNA because all viral coding genes are removed. I hypothesize that HD-Ad vectors can be effective vehicles for retinal gene delivery. The objectives of this study are to determine if HD-Ad vectors can deliver reporter genes, GFP or lacZ, driven by a CMV or a MOPS promoter, into specific retinal layers. Subretinal injections were performed and eyes removed at time points from 1 week to 3 months, processed for fluorescent microscopy, X-gal staining, and H&E staining. Transgene expression was detected for at least 3 months. A dose dependent relationship was revealed between the level of transgene expression and viral vector dose. Distinctively, the MOPS promoter drove photoreceptor cell specific expression. Notably, no sign of inflammation or tissue toxicity was detected, demonstrating the benefits of the HD-Ad vector.