published_or_final_version / Anatomy / Master / Master of Philosophy

Mutations in BEST1, the gene encoding for Bestrophin-1 (Best1), cause five, clinically distinct inherited retinopathies: Best vitelliform macular dystrophy (BVMD), adult-onset vitelliform macular dystrophy (AVMD), autosomal recessive bestrophinopathy (ARB), autosomal dominant vitreoretinochoroidopathy (ADVIRC), and retinitis pigmentosa (RP). Little is known regarding how BEST1 mutations cause disease and why mutations cause multiple disease phenotypes. Within the eye, Best1 is a homo-oligomeric, integral membrane protein that is exclusively localized to the basolateral plasma membrane of the retinal pigment epithelium (RPE). Here, it regulates intracellular Ca2+ signaling and putatively mediates anion transport. Since defects in localization and oligomerization are known to underlie other channelopathies, we investigated how mutations causal for BVMD, AVMD, ARB, ADVIRC, and RP impact the localization and oligomerization of Best1. We generated replication-defective adenoviral vectors encoding for WT and 31 mutant forms of Best1 associated with these five diseases and expressed them in confluent, polarized Madin-Darby canine kidney and/or RPE cells. Localization was assessed via immunofluorescence and confocal microscopy. Oligomerization was examined using live-cell fluorescence resonance energy transfer (FRET) as well as reciprocal co-immunoprecipitation experiments. We report that all 31 BVMD, AVMD, ARB, ADVIRC, and RP mutants tested can reciprocally co-immunoprecipitate with and exhibit comparable FRET efficiencies to WT Best1, indicative of unimpaired oligomerization. While all RP and ADVIRC mutants were properly localized to the basolateral plasma membrane, many but not all AVMD, ARB, and BVMD mutants were mislocalized to intracellular compartments. When co-expressed with WT Best1, mislocalized mutants predominantly co-localized with WT Best1 in intracellular compartments. Studies involving four ARB truncation mutants reveal that the first 174 amino acids are sufficient to mediate oligomerization with WT Best1 and that amino acids 472-585 are not necessary for proper trafficking. We conclude that, although mislocalization is a common result of BEST1 mutation, it is not an absolute feature of any individual bestrophinopathy. Moreover, we show that some recessive mutants mislocalize WT Best1 when co-expressed, indicating that mislocalization cannot, on its own, generate a disease phenotype, and that the absence of Best1 at the plasma membrane is well tolerated.

Aquaporin proteins significantly increase water permeability across tissues and cell membranes. Ocular tissues, including the trabecular meshwork (TM) and retinal pigment epithelium (RPE), are especially reliant on aquaporin mediated water movement for ocular homeostasis. Even though bulk fluid movement is paracellular through the TM and transcellular through the RPE, both express aquaporin-1 (AQP1). The role and regulation of AQP1 as it relates to homeostasis in different ocular tissues is not well understood. I hypothesized that ocular tissues respond to external mechanical and molecular cues by altering AQP1 expression and function in order to regulate ocular fluid movement and maintain homeostasis.To test how AQP1 function is altered in response to external cues in order to maintain tissue-specific homeostasis, I addressed the following two aims. The first aim was directed at determining how mechanical strain, an external stimulus that routinely affects TM function, influences AQP1 expression and TM homeostasis. Primary cultures of human TM were subjected to static and cyclic stretch and then analyzed for changes in AQP1 expression by western blot and cell damage by activity of lactate dehydrogense (LDH) in conditioned media. The results show AQP1 expression and LDH release significantly increased with static stretch. Analysis of LDH release with respect to AQP1 expression revealed an inverse linear relationship (r² = 0.7780).The second aim was directed at characterizing signaling mechanisms responsible for regulating fluid transport in RPE, previously shown to be dependent upon AQP1. I treated primary cultures of human RPE with either atrial natriuretic peptide (ANP) or 8-bromo-cyclic guanosine monophosphate (8-Br-cGMP) in the presence or absence of Anantin (ANP-receptor inhibitor) or H-8 (Protein Kinase G inhibitor). The results show that ANP and 8-Br-cGMP significantly increased apical to basal net fluid movement (p < 0.05, n = 3). Inhibition of these effects was successful with Anantin treatment but not with application of H-8.The data presented demonstrate a novel role of protection for AQP1 in TM, and also expand upon cGMP dependent regulation of RPE fluid transport. The combined studies indicate tissue specific AQP1 regulation may offer new avenues to target water movement in treatment of ocular pathologies.

D-Serine is regarded as an obligatory co-agonist required for the activation of NMDA-type glutamate receptors (NMDARs). In the retina D-serine and a second NMDAR coagonist, glycine, are present at similar concentration and the cells that produce and release them are in close apposition. This arrangement allows for an abundant supply of coagonists and under certain conditions the NMDAR coagonist binding site could be saturated. There is also evidence suggesting that D-serine can act in an inhibitory manner at AMPA/kainate-type glutamate receptors (GluRs). Glutamate receptor activation can lead to direct and indirect elevation of intracellular calcium (Ca2+) concentration ([Ca2+]i). Therefore, in this thesis, I predominantly used Ca2+ imaging techniques to study the effect of D-serine on GluR activation in the mammalian retina. I first describe a novel method I developed to load retinal cells with Ca2+ indicator dye using electroporation and show that retinas remain viable and responsive following electroporation. This technique was used to explore the excitatory role of D-serine at NMDARs and its potential inhibition of AMPA/kainate receptors using cultured retinal ganglion cells (RGCs) and isolated retina preparations. Using cultured RGCs I demonstrated that D-serine and glycine enhance NMDAR-mediated Ca2+ responses in a concentration-dependent manner and are equally effective as coagonists. In isolated retinas I showed that D-serine application enhanced NMDA-induced responses consistent with sub-saturating endogenous coagonist concentration. Degradation of endogenous D-serine reduced NMDAR-mediated Ca2+ responses supporting the contribution of this coagonist to NMDAR activation in the retina. Using imaging and two different electrophysiological approaches, I found that D-serine reduced AMPA/kainate receptor-mediated responses in cultured RGCs and isolated retinas at concentrations that are saturating at NMDARs. Antagonist experiments suggest that the majority of inhibition is due to D-serine acting on AMPA receptor activity. Degradation of endogenous D-serine enhanced AMPA/kainate-induced responses of some cells in isolated retina suggesting that, under these conditions, D-serine concentration may be sufficient to inhibit AMPA receptor activity. Overall, the work in this thesis illustrates the utility of electroporation as a method to load Ca2+-sensitive fluorescent dyes into retinal cells and highlights the potential role for D-serine as a modulator of ionotropic GluRs in the CNS.

Purpose: Retinal ganglion cell (RGC) loss is a measure of the progression of many visual disorders. It is important to identify RGCs with good specificity, so RGC numbers can be reliably analyzed. The purpose of this study was to analyze the effectiveness of two current RGC markers: Brn3a immunohistochemistry and the expression of Thy1-CFP in the Thy1-CFP transgenic mouse. Methods: Rhodamine-?-isothiocyanate (RITC) retrograde labeling, immunohistochemistry, wholemount retinal imaging, western blot, cross sectional analysis and cell densities in uninjured control animals and 3, 5, 7 and 14 days post-optic nerve crush (ONC) or transection (ONT) were tabulated. Results: Brn3a positive (Brn3a+) cell density was significantly less than RITC positive (RITC+) cell density in control mice. After ON injury, Brn3a+ cell density did not decrease at the same rate as RITC+ cell density. The density of RGCs that express Brn3a was significantly less than the individual Brn3a+ and RITC+ cell density at all experimental time points. Thy1-CFP positive (Thy1-CFP+) cell density was significantly less than RITC+ in control mice and significantly more than RITC+ cell density 14 days after ON injury. Thy1-CFP co-localized with ChAT positive (ChAT+) cells 7 days after ONT. Conclusion: Brn3a and Thy1-CFP are not reliable markers of RGCs. Retrograde labeling remains one of the most reliable methods of labeling RGCs in mice.

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. --

No description available.

<p><span lang="EN-GB" style="font-size:12.0pt / font-family:&quot / Times New Roman&quot / ,&quot / serif&quot / mso-fareast-font-family:&quot / Times New Roman&quot / mso-ansi-language:EN-GB / mso-fareast-language: EN-ZA">Literature shows that cataracts are the leading cause of blindness globally and nationally. Retinal detachment has also been a substantial problem both globally and nationally. Both of these conditions are prevalent in patients of 50 years and older. The treatment for both conditions is for surgery to be performed. In the Western Cape the three leading hospitals do not have ophthalmic pre-operative and post-operative protocols<span style="mso-bidi-font-weight:bold">.</span>Review question:What are the best practices to manage pre-operative and post-operative nursing care in patients waiting for cataract and retinal detachment surgery? bjectives:1. To determine the best practice in pre-operative and post-operative care in patients who have undergone cataract and/or retinal detachment surgery regarding: health education offered by nurses, counselling to prevent psychological effects, and positioning to prevent physical complications. 2. To develop a framework based on systematic reviews for pre-operative and post-operative ophthalmic nursing care in South Africa. Methodology: </span><span lang="EN-GB" style="font-size: 12.0pt / font-family:&quot / Times New Roman&quot / ,&quot / serif&quot / mso-ansi-language:EN-GB">A systematic review using the guide by the Centre for Reviews and Dissemination was done, and <span style="mso-bidi-font-weight:bold">studies were </span>identified by searching various electronic databases and visually scanning reference lists from the relevant studies. Studies that were included were evidence-based. All study types were considered and the studies were selected based on the title and, where available, the abstract. These were then assessed against the inclusion criteria. A narrative synthesis was used. Finally the evidence was summarised and a framework was drawn up, focusing on pre-operative and post-operative nursing care for cataract and retinal detachment surgery</span></p> <p>&nbsp / </p>

A high speed, high resolution spectral domain optical coherence tomography (SD-OCT) system was used to study in-vivo early morphological changes and optical nerve head (ONH) blood flow in the Long Evans rat retina, induced by administration of sodium iodate (NaIO3). Linear and circular scanned OCT images were acquired at the same location in the retina from healthy control rats and from rats injected with 40mg/kg of NaIO3 solution at 1, 3, 6 12, 24, 72 and 168 hours post drug administration. Morphological OCT images showed changes in the optical reflectance and layer thickness of the photoreceptor IS and OS. The formation of a new low reflective layer between the photoreceptor OS and the RPE was observed in all tested rats. This new layer appeared as early as 1 hour, increased in thickness after 6 hours, and disappeared by 12 hours post NaIO3 injection. The low optical reflectance and the dynamics of this new layer suggest that it was most likely fluid accumulation. Comparison with H&E stained histological sections and IgG immunohistochemistry revealed minimal photoreceptor OS cell swelling at hour 1, detachment of the OS from the RPE by hour 3, and breaking of the blood-retina barrier with significant fluid accumulation by hour 6 post NaIO3 injection. The Doppler Optical Micro-Angiography (DOMAG) algorithm was used to carry out quantitative analysis of the ONH blood flow. Estimation of flow rate on each ONH vessel was done by measurements of the Doppler angle, vessel size and the axial velocity. This study has demonstrated that the capability of UHR-OCT to study optical reflectance and layer thickness changes, rearrangement and detachment of the photoreceptor OS and RPE layers, together with flow rate estimation of retinal blood vessels. Therefore, it can serve as markers in future non-invasive, in-vivo studies of disease or drug induced retinal degeneration in ophthalmic research.