Thesis (Ph. D.)--University of Hong Kong, 1991.

Thesis (Ph. D.)--University of Hong Kong, 1998. / Includes bibliographical references (leaves 98-126).

Thesis (Ph.D.) - University of Queensland, 2006. / Includes bibliography.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 91-101).

The general aim of the thesis was to deduce whether selected naturally occurring flavonoids (genistein, epicatechin gallate (EC), epigallocatechin gallate (EGCG), baicalin) attenuate various secondary insults that may cause death of ganglion cells in primary open angle glaucoma (POAG). An ischemic insult to the rat retina significantly causes the inner retina to degenerate indexed by changes of various antigens, proteins and mRNAs located to amacrine and ganglion cells. These changes are blunted in animals treated with genistein as has been shown for ECGC. Studies conducted on cells (RGC-5 cells) in culture showed that hydrogen peroxide, L-buthionine sulfoximine (BSO)/glutamate and serum deprivation (mimicking oxidative stress), rotenone, sodium azide (affecting mitochondria function in specific ways) and light (where the mitochondria are generally affected) all generated reactive oxygen species and caused death of RGC-5 cells. EGCG was able to attenuate cell death caused by hydrogen peroxide, sodium azide and rotenone. Only EC was able to attenuate BSO/glutamate-induced cell death, in addition to cell death caused by hydrogen peroxide and rotenone. Genistein had no positive effect on cell death in experiments carried out on RGC-5 cells. Exposure of RGC-5 cells to flavonoids showed that EC and EGCG increased the mRNA expression of endogenous antioxidants such as HO-1 (heme oxygenase 1) and Nrf-2 (nuclear erythroid factor-2-related factor 2). Light insult, rotenone and sodium azide activate the p38 (protein kinase 38) pathway, while only light and rotenone activate the JNK (c-Jun amino-terminal kinase) pathway. Serum deprivation affects mitochondrial apoptotic proteins causing an increase in the ratio of Bax/Bcl2 (Bax: Bcl-2-associated X protein; Bcl-2: B-cell lymphoma 2). An insult of light to RGC-5 cells, unlike that induced by sodium azide, is inhibited by necrostatin-1 and causes an activation of AIF (apoptosis-inducing factor) with alpha-fodrin being unaffected. These studies suggest that ganglion cell death caused by insults as may occur in POAG involves various cellular signaling pathways. The selected flavonoids have diverse actions in increasing cellular defense mechanisms, and in negating the effects of ischemia and specific types of oxidative stress. The results argue for the possible use of flavonoids in the treatment of POAG to slow down ganglion cell death.

Indiana University-Purdue University Indianapolis (IUPUI) / Retinal ganglion cells (RGCs) serve as a vital connection between the eye and the brain with damage to their axons resulting in loss of vision and/or blindness. Reti- nal organoids are three-dimensional structures derived from human pluripotent stem cells (hPSCs) which recapitulate the spatial and temporal differentiation of the retina, providing a valuable model of RGC development in vitro. The working hypothesis of these studies is that hPSC-derived RGCs are capable of extensive outgrowth and display target specificity and pathfinding abilities. Initial efforts focused on charac- terizing RGC differentiation throughout early stages of organoid development, with a clearly defined RGC layer developing in a temporally-appropriate manner express- ing a compliment of RGC-associated markers. Beyond studies of RGC development, retinal organoids may also prove useful to investigate and model the extensive axonal outgrowth necessary to reach post-synaptic targets. As such, additional efforts aimed to elucidate factors promoting axonal outgrowth. Results demonstrated significant enhancement of axonal outgrowth through modulation of both substrate composi- tion and growth factor signaling. Furthermore, RGCs possessed guidance receptors that are essential in influencing outgrowth and pathfinding. Subsequently, to de- termine target specificity, aggregates of hPSC-derived RGCs were co-cultured with explants of mouse lateral geniculate nucleus (LGN), the primary post-synaptic target of RGCs. Axonal outgrowth was enhanced in the presence of LGN, and RGCs dis- played recognition of appropriate targets, with the longest neurites projecting towards LGN explants compared to control explants or RGCs grown alone. Generated from xvii the fusion of regionally-patterned organoids, assembloids model projections between distinct regions of the nervous system. Therefore, final efforts of these studies focused upon the generation of retinocortical assembloids in order to model the long-distance outgrowth characteristic of RGCs. RGCs displayed extensive axonal outgrowth into cortical organoids, with the ability to respond to environmental cues. Collectively, these results establish retinal organoids as a valuable tool for studies of RGC develop- ment, and demonstrate the utility of organoid-derived RGCs as an effective platform to study factors influencing outgrowth as well as modeling long-distance projections and pathfinding abilities.

This project explores the use of a novel sustained release mucoadhesive micelle-based drug delivery system in combination with 0.005% latanoprost (LTP) on our partially open angle mouse model of glaucoma (AP-2β TMR-KO). We previously tested for LTP treatment in our model and found a reduction in intraocular pressure (IOP) 20 minutes following treatment. This information led us to investigate the long-term effect of LTP treatment and micelle loaded with LTP (MLTP) treatment in our model. We hypothesized that the MLTP treatment would be more effective in reducing IOP and preventing glaucomatous effects than LTP treatment alone in the AP-2β TMR-KO mice. The MLTP groups of animals (wildtype and mutant) were treated every 3 days, and this was compared with animals treated with LTP daily as well as animals treated every 3 days with LTP alone for comparison’s sake for 60 days. IOP measurements were taken every 3 days. Following long term LTP treatment alone, mutant mice showed a consistent decrease in their baseline IOPs with a significant reduction in baseline IOP at 35 days of treatment across all cohorts (P<0.0001). In comparison, mutants treated with MLTP exhibited an even greater reduction in baseline IOP following long term treatment. After the treatment period, mice were euthanized, and their eyes were enucleated, fixed, sectioned, and stained for retinal ganglion cells (RGCs) using Brn3a. Mutant mice exhibited a significant decrease in RGC cell number when compared to wildtype, and this loss was not rescued by treatment with LTP. However, mutants treated with MLTP demonstrated significant RGC cell protection compared to eyes of untreated mutants, as well as everyday LTP treated mutants. / Thesis / Master of Science (MSc) / An effective treatment strategy is required to prevent irreversible blindness caused from glaucoma. Unfortunately, compliance with current medications is extremely poor, as they require frequent administration due to their low ocular bioavailability and short-term effect. As such, this thesis aims to explore an alternative drug delivery approach in a partially open angle mouse model of glaucoma to prevent the worsening of glaucoma and ultimately improve patient compliance.

No description available.

In another study, hyperthermic treatment was applied to study whether HSP27 expression would be induced in un-injured RGCs, and whether this treatment performed after axotomy would have effects on HSP27 expression, RGC survival and/or regeneration into PN graft. Brief duration of heat shock that elevate the body temperature to 42&deg;C did not up-regulate HSP27 in normal retina. About 8-10% increase in RGC survival in the hyperthermia group was observed compared to those received a 37&deg;C treatment at one week post-axotomy and it depended on the number of post-injury heat treatments applied. At the same time, the number of HSP27-RGCs was also doubled, although the same increase occurred was irrespective of the number of hyperthermic treatments. Multiple heat shock application also significantly enhanced RGC regeneration into PN graft through increased the number of HSP27 regenerating RGCs. These results suggest that post-injury hyperthermic treatment enhance HSP27 induction in RGCs and lead to their successful regeneration into the PNG, whereas further studies are necessary to determine whether the protective effect on survival by heat shock is due to the increase in a subset of HSP27-RGCs. (Abstract shortened by UMI.) / In the second study, different neurotrophic factors were injected into the vitreous to enhance RGC survival and/or regeneration. Brain-derived neurotrophic factor (BDNF) significantly reduced RGC death transiently at 14 days after ON cut, but the expression of HSP27 was reduced compared to bovine serum albumin-injected controls. In peripheral nerve (PN)-grafted retina, BDNF suppressed RGC regeneration via reducing the number of HSP27-RGCs regenerating into the PN graft. In ciliary neurotrophic factor (CNTF)-injected group, although there was only a 10% increase in RGC survival, a 5-fold drastic increase in the number of RGCs which expressed HSP27 was observed, and some of these were found to undergo intra-retinal sprouting similar to VPN-transplanted retina. Combined treatment of intra-vitreal CNTF injection with PNG resulted in a 5 fold-increase in the number of regenerating RGCs as well as increasing the proportion of cells which expressed HSP27 from about 60% to about 80%. The data indicates that HSP27 participates in axonal regrowth especially under synergistic interaction of CNTF and PNG. Intra-vitreal injection of hepatocyte growth factor (HGF) significantly sustained RGC survival compared to BDNF at 28 days after axotomy, but the HSP27 expression in RGCs did not change correspondingly. In the PN-ON grafted retina, HGF promoted more RGCs regenerate without altering the number of HSP27-RGCs regrowing into the PNG. Such results indicate that some trophic factors can specifically enhance or suppress RGC regeneration by modulating HSP27 expression, while other trophic factors promote regeneration which is independent to HSP27. Therefore, it suggests that RGCs may regenerate through at least two different mechanisms. / In this study, the detailed in vivo expression of HSP27 in retinal ganglion cells (RGCs) of golden hamster following axotomy and regeneration stimulated by peripheral nerve grafting and neurotrophic factors have been examined. / In whole-mount normal retinas, HSP27 was constitutively expressed in astrocytes and blood vessels, but not in RGCs. Three days after optic nerve (ON) transection, a small subset of surviving RGC began to express HSP27, the number of which peaked at 7 days and dropped to a minimal level at two weeks post-axotomy. When axotomy was done more proximally to their cell bodies, RGCs survival was significantly decreased but HSP27 expression did not change. This suggests the HSP27 expression does not correlate with cell survival after axonal injury. When a viable peripheral nerve (VPN) was transplanted intravitreally into the eye after ON cut, it induced intra-retinal sprouting of RGCs. Although it did not promote RGC survival, VPN prolonged HSP27 expression up to 56 days after surgery and significantly increased the number of HSP27-RGCs. This protein was localized in the cell body, and especially, in dendritic sprouts and growth cones, indicating that it was transported to active growing sites where it may have a functional role associated with regenerative sprouting. / Wong, Wai Kai. / Adviser: Eric Cho. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3270. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 159-198). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Despite that the RGC axons closely associated with astrocytes, the role of astrocytes in RGC regeneration was uncertain. In view of this, the effect of cultured adult astrocytes on RGC regeneration through an extensive ON lesion segment was studied. Adult ON astrocytes were prepared by sub-culturing of cells migrating out of ON explants. A small hole in the ON was punctured by 27G needle and about 0.5 to 1.0mul (1000 cells) cultured astrocytes was injected into the extensive ON lesion segment. We found that cultured adult astrocytes promoted significant RGC axon regeneration in the extensive ON lesion. / Finally, co-transplantation of intravitreal PN followed by transplantation of astrocytes into the extensive lesion has a synergistic effect on the regrowth of RGC axons, as indicated by the maximum distance achieved by regenerating axons and integrated intensity of staining of the CTB-labeled axons. Transplanatation of VPN+AST, VPN+NAST and NPN+AST as 3.9, 2.5 and l.9 times more potent in inducing regeneration than that of NPN+NAST as shown by integrated intensity measurement. However, co-transplantation of PN and astrocytes could not enhance RGC survival. (Abstract shortened by UMI.) / In this study, we have established an extensive lesion paradigm to study the behavior of injured retinal ganglion cell (RGC) axons after ON crush in adult golden hamster. We found that RGC axons regenerated in the extensive lesion for 406.8mum at 1 week post-crush to 1174.0mum at 4 weeks post-crush. RGC axons were able to regenerate the entire lesion segment but they terminated precisely at the interface between the lesion and the distal segment of the ON. Regrowing axons were intimately associated with astrocytes which repopulated the lesion segment. Repopulated oligodendrocytes were scattered in the lesion segment and myelin debris was significantly decreased in the lesion segment with time. / It is commonly believed that central nervous system (CNS) neurons are unable to regenerate after injury. Recently, there have been several lines of evidence showing that damaged CNS neurons can undergo axonal regeneration under appropriate conditions. Since the retina and optic nerve (ON) are regarded as part of the CNS, therefore, they are used as a model to study CNS regeneration. / Kong Wai Chi. / "July 2005." / Adviser: Y.P. Cho. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3616. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 96-115). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.