Hypothesis 1. MfERG is useful in the assessment of retinal dysfunction in patients following photodynamic therapy with verteporfin. / Hypothesis 2. MfERG is useful in the evaluation of retinal dysfunction in patients following safety-enhanced photodynamic therapy using half-dose verteporfin. / Hypothesis 3. MfERG is useful in the assessment of retinal dysfunction in patients receiving hydroxychloroquine therapy. / Hypothesis 4. MfERG findings correlate to certain extent with the visual field findings in patients receiving hydroxychloroquine therapy and mfERG might be more sensitive compared with 10-2 visual field testing in assessing retinal dysfunction associated with hydroxychloroquine therapy. / Hypothesis 5. MfERG is useful In the assessment of retinal dysfunction associated with intraoperative application of indocyaniue green for internal limiting membrane staining in epiretinal membrane surgery. / Multifocal electroretinography (mfERG) is an investigation which can provide objective assessment of retinal function, In contrast with full-field electroretinography which measures the mass electrical activity of the entire retina, mfERG allows simultaneous measurements of multiple retinal responses from the macula. / Several studies have demonstrated that mfERG might be useful in assessing retinal dysfunction caused by pharmacological agents and following laser therapy. This thesis aims to demonstrate the application of mfERG in the evaluation of retinal dysfunction associated with various ocular or systemic pharmacological agents. Three treatment modalities including photodynamic therapy with verteporfin, systemic use of hydroxychloroquine, and intraoperative application of indocyanine green dye were chosen for evaluation. These pharmacological agents were selected as they are associated with potential retinal dysfunction and are commonly encountered in the ophthalmic clinical practice. The thesis examines the following hypotheses: / Summary of studies arising from the thesis. Based on the findings from the above studies, it was demonstrated that mfERG can objectively evaluate the retinal dysfunction caused by a variety of ocular or systemic pharmacological agents. These included PDT with verteporfin, systemic therapy with hydroxychloroquine, as well as intraoperative application of ICG dye for ILM staining. The use of mfERG has enhanced the understanding of the underlying pathophysiology of drug-associated retinal toxicity. As mfERG becomes more widely available, its application will provide a valuable option for clinicians to assess toxic retinopathy objectively and enable safer administration of treatment to minimize potential retinal toxicity. (Abstract shortened by UMI.) / by Lai Yuk Yau, Timothy. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0951. / Thesis (M.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 161-183). / 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. / School code: 1307.

published_or_final_version / Anatomy / Master / Master of Philosophy

Ischemic stroke is a devastating cerebrovascular disease resulting in high mortality rate and distressing sequelae such as hemiplegia, ataxia and even visual impairment. Retinal ischemia refers to a common pathological feature shared by many blinding diseases including retinal vascular occlusions, diabetic retinopathy, glaucoma, and retinopathy of prematurity. Ischemia/reperfusion injury is implicated in both of these pathological conditions, which greatly impact on one’s daily life. The eventual consequence of the insult is irreversible neuronal cell death and functional deterioration. Apart from current symptomatic treatment for these diseases, researchers and clinicians are dedicated to look for ideal neuroprotectant to meet the clinical needs. Traditional Chinese medicine has been received accumulating attention in recent years, and Lycium barbarum is one of them. The polysaccharides (LBP) utilized in the present study are the rich extracts of the fruit of Lycium barbarum that has been shown to exert many biological effects. This study aims to evaluate its protective effects in cerebral and retinal ischemia, which has not yet been fully investigated. A well-established rodent model, middle cerebral artery occlusion, was utilized in the present study to mimic cerebral and retinal ischemia/reperfusion injury. In the study of cerebral ischemia, both pre-treatment and post-treatment of LBP were explored. Seven-day LBP pre-treatment revealed significant protection against neurological deficits and cerebral infarction. Besides, it attenuated cerebral edema and glial activation, as well as preserved blood-brain barrier integrity. Further study showed that these beneficial effects of LBP pre-treatment might act via anti-apoptosis, antioxidation and anti-inflammation. However, similar findings were not noted in LBP post-treatment experiments, possibly due to the timing of intervention. In the investigation of retinal ischemia, the observation time was prolonged to 7 days after the insult. Electroretinogram was used to evaluate the functional alternation of retinal neurons. Sustained retinal dysfunction was induced by two-hour ischemia. LBP pre-treatment with continuous daily supplementation effectively alleviated visual dysfunction and protected the retina from morphological impairment including neuronal death, glial activation and blood-retinal barrier disruption. Similarly, these protective effects might be associated with the involvement of attenuation of apoptosis and oxidative stress. In conclusion, LBP pre-treatment with continuous daily supplementation protected the brain and retina, both functionally and morphologically, from ischemia/reperfusion injury. This dosing regimen hold great promise in serving as a prophylactic neuroprotectant in patients at high risk for ischemic stroke, as well as preserving normal visual function and reducing irreversible neuronal death in ischemic retinopathies. Further studies on the active ingredients and underlying mechanisms would be informative for better application of LBP in clinical situation. / published_or_final_version / Ophthalmology / Doctoral / Doctor of Philosophy

The Royal College of Surgeons (RCS) rat was used as a model of human retinal degenerative disease, and for studying the efficacy of cell transplanation treatments. In order to characterize the spatial vision of the RCS strain, the visual acutiy and contrast sensitivity of adult non-dystrophic RCS rats was measured. The acuity and contrast sensitivity of these rats was normal. The acuity of dystrophic RCS rats was alos characterized to determine how photoreceptor degeneration affects vision. These rats progressively lost visual acuity from one month of age until elevn months of age when they were judged to be blind. The degeneration of vision in these animals was more protacted than would be predicted from previous anatomical and electrophysiological measures. Subretinal transplantation of human-derived Retinal Pigment Epithelial (RPE) cells and human Schwann cells into the dystrophic RCS rat significantly delayed the loss of visual acuity. These studies show that cell transplantation may be a viable method of limiting loss of vision in humans with retinal degenerative blinding diseases. / vii, 77 leaves ; 29 cm.

The established model used for evaluating potential therapies for retinal disease has significant limitations. A new model is proposed to account for these limitations: the visual adaptation model. The visual adaptation model was developed to provide a novel approach for testing potential treatments for retinal disease, and the work in this thesis provides empirical support for this model. Specifically, we evaluated two potential therapies for retinal degenerative disease and examined their effects on vision and retinal anatomy. In addition, the profile of retinal reorganization and its functional correlates were examined in RCS rats and transgenic rats which express a rhodopsin mutation; however, immunohistological work targeted one specific line (S334ter-4). Collectively, these studies provide evidence that supports the retinal adaptation model. These studies also provide a novel view of retinal and visual function in retinal disease which should be considered when evaluating treatments involving retinal degeneration. / xvii, 205 leaves : ill. ; 29 cm. --

The retina is one of the most metabolically active tissues in the body and the high energetic demand is met by a well-organized vascular network. Aberrant vasculature is a prominent feature of many vision-threatening diseases, and although angiogenic pathways have been extensively studied the limited efficacy of therapies currently available for the treatment of these diseases suggests that there is more to be elucidated. The caspase family of proteases is best known for their roles in programmed cell death and inflammation, however members of this family have been found to have essential functions independent of cell death. Caspase-8, in particular, has been previously shown to be essential for embryonic vascular development, however, a requirement for caspase-8 in postnatal vascular development has not been established and it is unclear how caspase-8 exerts its function. In this study, we investigate the cell specific roles of caspase-8 in the development of the retinal vasculature using the postnatal mouse retina as our model and identified endothelial caspase-8 as a mediator of canonical Wnt signaling. Inducible endothelial cell-specific caspase-8 knockout (Casp8 iECKO) resulted in a delay in early angiogenesis and barrier establishment, and an increase in inflammation and premature vascular remodeling compared to littermate controls. Assessment of Lef1, a downstream effector of the Wnt pathway, confirmed that this phenotype was a result of inhibited Wnt signaling. We additionally show that caspase-8 mediates this pathway through degradation of its substrate HDAC7. HDAC7 has been shown previously to bind to β-catenin blocking its nuclear translocation. Caspase-8 mediated HDAC7 degradation restores β-catenin translocation and downstream Wnt signaling. We also explore the function of caspase-8 in myeloid cells – microglia and macrophages – during angiogenesis. We used an inducible myeloid-specific caspase-8 knockout (Casp8 imGKO) mouse and found that loss of caspase-8 in these cells did not affect angiogenesis. However, Casp8 imGKO resulted in a reduction in microglia number and a change in their morphology specifying a role for caspase-8 in mediating cell survival and activation in microglia. Altogether we show that caspase-8 exerts cell specific functions during retinal angiogenesis that are independent of cell death. We elucidate a novel role of caspase-8 in mediating Wnt/β-catenin signaling, and implicate caspase-8 as a potential therapeutic target in pathological angiogenesis.

Central nervous system (CNS) tissues are highly metabolically active which makes them particularly susceptible to vascular injury. Disruption to the supply of oxygen and nutrients by damaged vasculature can result in neurodegeneration in both the eye and brain. The retina is an accessible part of the CNS that can be taken advantage of to study neurovascular diseases through live, non-invasive visualization of vascular and neuronal conditions upon injury. Retinal vein occlusion (RVO) is a common neurovascular disease of the eye and is the second leading cause of blindness in working age adults. While pathophysiology is well described and can be determined by retinal edema, breakdown of the blood-retina-barrier (BRB), inflammation, and neurodegeneration, the underlying signaling pathways behind the pathology is not well understood. To understand the mechanism of disease in RVO, the Troy lab has employed a mouse model to investigate pathways. Previous studies in the lab determined that as early as 1 hour post RVO, there was a large induction of caspase-9, a known cell death protease, in endothelial cells. When further investigated, it was confirmed that these cells were not dying despite the high expression of caspase-9, implying a non-apoptotic role. Deletion of endothelial caspase-9 was sufficient to protect against the development of retinal edema, capillary ischemia, and neuronal death, indicating caspase-9 is a key player in the mechanism of disease. This thesis work aims to investigate which signaling events drive non-apoptotic endothelial caspase-9 signaling by investigating upstream and downstream mechanisms of endothelial caspase-9. To interrogate this question, the mouse model of RVO was optimized, limiting the variability previously observed to ensure accurate and reproducible results. Then, we used a tamoxifen inducible endothelial cell Apaf-1 (apoptosis protease activating factor-1) knock out (Apaf-1 iECKO) mouse line in order to investigate the contribution of upstream activation of non-apoptotic endothelial caspase-9 signaling. Apaf-1 iECKO mice and WT littermates were subjected to RVO. Then, expression of caspase-9 and -7, retinal edema, capillary ischemia, neuronal death, vision dysfunction, and BRB integrity were measured. The deletion of endothelial Apaf-1 resulted in reduced expression of cl-caspase-9 and caspase-7, indicating endothelial caspase-9 was activated by Apaf-1. Apaf-1 deletion also resulted in protection against some of the pathologies seen after RVO including retinal edema, capillary ischemia, and neurodegeneration. Lastly, in order to elucidate the signaling pathway further, experiments using endothelial cell-specific AAVs (adeno-associated virus) packaged with a downstream caspase-7 inhibitor were proposed and described. In sum, this thesis work reveals that endothelial caspase-9 is canonically activated by Apaf-1, but still leads to non-apoptotic signaling, indicating downstream caspase-9 substrates could be the source for non-apoptotic function within endothelial cells.

MRI can provide anatomical, functional, and physiological images at relatively high spatial resolution and is non-invasive and does not have depth limitation. However, the application of MRI to study the retina is difficult due to the very small size of the retina. This thesis details the development of MRI methods to image blood flow (BF), anatomy, and function of the retina and choroid, and their application to two diseases of the retina: diabetic retinopathy and retinal degeneration. A unique continuous arterial spin labeling technique was developed to image BF in mice and tested by imaging cerebral BF. This method was then applied to image layer-specific BF of the retina and choroid in mice, and to acquire BF functional MRI of the retina and choroid in response to hypoxic challenge. Additionally blood oxygen level dependent functional MRI of the mouse retina and choroid in response to hypoxic challenge was obtained using a balanced steady state free precession sequence which provides fast acquisition, has high signal to noise ratio, and does not have geometric distortion or signal dropout artifacts. In a mouse model of diabetic retinopathy, MRI detected reduced retinal BF in diabetic animals. Visual function in the diabetic mice, as determined by psychophysical tests, was also reduced. Finally, in a mouse model of retinal degeneration, BF and anatomical MRI detected reductions of retinal BF and the thickness of the retina. The studies detailed in this thesis demonstrate the feasibility of layer-specific MRI to study BF, anatomy, and function, in the mouse retina. Further, these methods were shown to provide a novel means of studying animal models of retinal disease in vivo.

[Truncated abstract] Microenvironmental changes associated with apoptotic neural degeneration may instruct a proportion of newly transplanted donor cells to differentiate towards the fate of the deteriorating host cellular phenotype. In the work described in this thesis, this hypothesis was tested by inducing apoptotic retinal ganglion cell (RGC) death in neonatal and adult rats and mice, and then examining whether intravitreally grafted cells from a range of sources of donor neural tissue became incorporated into these selectively depleted retinae. Donor tissues were: a postnatal murine cerebellar-derived immortalised neural precursor cell line (C17.2); an adult rat hippocampal-derived clonal stem-like line (HCN/GFP); mouse embryonic day 14 (E14) primary dissociated retinal cells (Gt[ROSA]26); and adult mouse ciliary pigmented margin-derived primary neurospheres (Gt[ROSA]26). In neonates, rapid RGC death was induced by removal of the contralateral superior colliculus (SC), and in adults, delayed RGC death was induced by unilateral optic nerve (ON) transection. Some adult hosts received ON transection coupled with an autologous peripheral nerve (PN) graft. Donor cells were injected intravitreally 6-48 h after SC ablation (neonates) or 0, 5, 7 or 14 days after ON injury (adults). Cells were also injected into non-RGC depleted neonatal and adult retinae. At 4 or 8 weeks, transplanted cells were identified, quantified and their differentiation fate within host retinae was assessed. Transplanted male C17.2 cells were identified in host retinae using a Y-chromosome marker and in situ hybridisation, or by their expression of the lacZ reporter gene product Escherichia coli beta-galactosidase (beta-gal) using Xgal histochemistry or a beta-gal antibody. No C17.2 cells were identified in axotomised adult-injected eyes undergoing delayed RGC apoptosis (n = 16). Donor cells were, however, stably integrated within the retina in 29% (15/55) of mice that received C17.2 cell injections 24 h after neonatal SC ablation; 6-31% of surviving cells were found in the RGC layer (GCL). These NSC-like cells were also present in intact retinae, but on average there were fewer cells in GCL. In SC-ablated mice, most grafted cells did not express retinal-specific markers, although occasional donor cells in the GCL were immunopositive for beta-III tubulin (TUJ1), a protein highly iii expressed by, but not specific to, developing RGCs. Targeted rapid RGC depletion thus increased C17.2 cell incorporation into the GCL, but grafted C17.2 cells did not appear to differentiate into an RGC phenotype.

The eye contains closely related but widely different tissues, offering a unique opportunity to investigate the phenotype and function of monocyte-derived cell populations within functionally unique microenvironments in a single complex organ. The uveal tract and retina contain rich networks of immune cells that reside and traffic through the eye, these cells having been implicated in various ocular inflammatory processes and immune-mediated diseases. One such inflammatory condition is human posterior uveitis, an autoimmune disease mainly affecting the retina. As current treatments for posterior uveitis only serve to slow down disease progression, studies using animal models, namely, experimental autoimmune uveoretinitis (EAU), have focused on determining the key cellular and molecular mediators involved in disease initiation in order to expand the potential for novel therapeutic applications. The overall purpose of experiments in this thesis was to explore monocyte-derived cell populations of the uveal tract and retina, this being achieved by utilising a novel transgenic mouse model. Cx3cr1gfp/gfp transgenic mice on both BALB/c and C57Bl/6 backgrounds contain an enhanced green fluorescent protein (eGFP) encoding cassette knocked into the Cx3cr1 gene, disrupting its expression but facilitating GFP expression under the control of the Cx3cr1 promoter. Heterozygous (Cx3cr1+/gfp) mice were generated by crossing Cx3cr1gfp/gfp mice to wild-type (WT) mice. This transgenic model allowed for the exquisite visualisation of Cx3cr1-bearing monocyte-derived dendritic cells (DC) and macrophages in ocular tissues, whilst also enabling the investigation of a potential role for Cx3cr1 in recruiting monocyte-derived cells to the eye in steady-state and inflammatory conditions.