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An in vitro phagocytosis assay system was developed and characterized for studying the specific receptor-mediated phagocytosis of bovine ROS by bovine RPE cells. The phagocytosis of ROS was detected qualitatively by electron microscopy and quantitatively by treating RPE cells with radioiodinated ROS or by probing ROS-treated RPE cells with a radiolabeled antirhodopsin monoclonal antibody. The binding sites for various antirhodopsin monoclonal antibodies were localized as an essential step in their application as immunochemical probes for analysis of the structure and function of rhodopsin. Five monoclonal antibodies raised against rhodopsin have been shown to be directed against the N-terminal regions on the basis of their reactivity to an immunoaffinity purified 2-39 glycopeptide, a 2-16 tryptic glycopeptide and a 1-16 synthetic peptide as measured by radioimmune competition assays. Limited proteolysis, immunogold-dextran labeling and competitive inhibition studies identified two antirhodopsin monoclonal antibodies which bound to internal cytoplasmic loop regions of rhodopsin. Finally, the binding sites for these and other C-terminal specific antirhodopsin monoclonal antibodies were used to elucidate the proposed transmembrane helical model of rhodopsin. An antirhodopsin monoclonal antibody (rho 4D2), which bound to rhodopsin in glutaraldehyde-fixed ROS plasma membranes, was employed as an immunocytochemical probe in studying the possible role of rhodopsin in the binding and phagocytosis of rod outer segments. An immunoaffinity purified 2-39 N-terminal rhodopsin glycopeptide, a synthetic 1-16 peptide analogue of rhodopsin and phospholipid vesicles reconstituted with rhodopsin were all found to be ineffective in inhibiting the phagocytosis of ¹²⁵I-labeled ROS by RPE cells. In essence, these results provided compelling evidence that rhodopsin in the ROS plasma membrane does not function as the ligand for recognition by RPE cells. The molecular properties of the ROS cell surface ligand(s), which are involved in recognition by bovine RPE cells, were studied by limited-proteolytic digestion in conjunction with quantitative phagocytosis assays. Mildly trypsin-treated ROS were found to be less effectively phagocytized than untreated ROS by bovine RPE cells. Moreover, the glycopolypeptides (34kD and 24kD) released from the ROS cell surface by trypsin were capable of inhibiting ROS phagocytosis. The ROS plasma membrane specific, ricin-binding, 230kD glycoprotein was observed by SDS-gel electrophoresis and western blotting to be highly trypsin sensitive under these conditions. Hence, ricin affinity chromatography and immunoaffinity chromatography were employed in an attempt to purify this 230kD glycoprotein from ROS membranes. Enriched preparations of the 230kD glycoprotein were reconstituted into phospholipid vesicles and effectively used to inhibit the phagocytosis of ROS by RPE cells. In summary, a ROS plasma membrane specific, 230kD glycoprotein has been identified and isolated; this protein may act as a ligand in specific ligand-receptor interactions between ROS and RPE cells. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The interplexiform cells(IP cells) are the most recently discovered neurons in the retina and their function is to provide centrifugal feedback in retina. The anatomical structure of the IP cells has been well studied, but the function of these neurons is largely unknown. I systematically studied the excitatory and inhibitory inputs from IP cells in salamander retina. I found that L-EPSCs in IP cells are mediated by AMPA and NMDA receptors; in addition, L-IPSCs are mediated by glycine receptors and GABAC receptors. In response to light, IP cells reaction potentials transiently at the onset and onset of light stimulation. The major neural transmitter of IP cells in salamander retina is glycine. We also studied the distribution and function of glycine transporters. Our result indicates that GlyT1- and GlyT2-like transporters were present in Muller cells and neurons. The glycine feedback at outer plexiform layer (OPL) has effects on both the bipolar cell dendrites and rod photoreceptor terminals. At bipolar cell dendrites, glycine selectively depolarizes rod-dominant On-bipolar cells, and hyperpolarizes Off- bipolar cells. At rod photoreceptor terminals, 10 M glycine activates voltage-gated Ca2+ channels. These effects facilitated glutamate vesicle release in photoreceptors. It increases the sEPSC in OFF bipolar cells. The combined effect of glycine at rod terminals and bipolar cell dendrites leads to enhanced dim light signal transduction in the rod photoreceptor to ganglion cell pathway. This study provides a model that displays the function of centrifugal feedback through IP cells in the retina. / by Zheng Jiang. / Thesis (Ph.D.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

　　Proliferative vitreoretinopathy (PVR) remains the major cause of failure of retinal detachment surgery. Retinal pigment epithelial (RPE) cells have been suggested to play a major role in the pathogenesis of PVR. Numerous studies have employed pharmacological means to modulate cellular activities in attempts to inhibit the process. Recent attempts using adjunctive therapy during PVR surgery that consisted of 5-fluorouracil (5-FU) and low molecular weight heparin showed some promise in preventing PVR but the concern is that prolonged 5-FU treatment may have a toxic effect. On the other hand, lectin from the edible mushroom Agaricus bisporus (ABL) was found to inhibit growth of RPE cells in a potent manner without apparent cytotoxicity. This lectin could be a candidate to modulate anomalous proliferation of RPE cells while the mechanism for the observed inhibition is unknown. 　　In our study, we investigated whether RPE cells treated with 5-FU or ABL would attenuate cellular proliferation, cell migration, cell adhesion and cell-mediated contraction rates. Further, we investigated if complementary inhibition for the above cellular activities could be obtained when RPE cells were treated with ABL after the short treatment using 5-FU. We also explored the possible mechanisms through which ABL inhibited RPE cell proliferation. 　　ARPE-19 and primary human RPE cells were treated with 5-FU or vehicle for 10 minutes. Cells were then maintained in culture medium supplemented with or without ABL. The rate of cellular proliferation was measured by a tetrazolium salt assay. Effects on cell adhesion were investigated through loading RPE cells onto the strips coated with collagen I or fibronectin. Cell migration was investigated using a scratch wound model. The effect on cell-mediated contraction was assessed using a free floating collagen I matrix. Cytotoxicity of 5-FU and ABL was determined by the live/dead assay. 　　To elucidate the mechanism through which ABL inhibited RPE cell proliferation, we investigated cell cycle distribution patterns using flow cytometry. Phosphorylation statuses of Erk, Jnk, p38, Akt as well as p53 and Cyclin D expression level were investigated by Western blotting. 　　Both 5-FU and ABL inhibited RPE cell proliferation. Only ABL promoted cell adhesion towards collagen I in hRPE3 cells. ABL was found to attenuate the rate of cell migration. Cell-mediated collagen gel contraction was attenuated by 5-FU only. Complementary inhibition in cellular proliferation and cell-mediated collagen gel contraction was observed when both 5-FU and ABL were applied. No significant cell death was observed after treatment with 5-FU, ABL or both. 　　ABL was found to reduce the amount of cells present at S phase. Akt and Erk were found to be hypo-phosphorylated and hyper-phosphorylated respectively after ABL treatment. The expression levels of phosphorylated-Jnk, phosphorylated-p38, p53, and Cyclin D1 were not altered when compared with the control. 　　These results showed that 5-FU and ABL complement with each other on inhibiting the wound healing activities of RPE cells in vitro without apparent cytotoxicity. They suggested a possible new treatment modality for PVR. ABL hypo-phosphorylated Akt and this observation is in line with the fact that ABL could attenuate cell proliferation. / published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Neurons are able to maintain membrane potential and synaptic integrity by an intricate equilibrium of membrane transporter proteins and ion channels. Two membrane proteins of particular importance in the vertebrate retina are the excitatory amino acid transporters (EAATs) which are responsible for the reuptake of glutamate into both glial and neuronal cells and the sodium potassium chloride cotransporters (NKCCs) that are responsible for the uptake of chloride ions into the cell. NKCCs are electro-neutral with the uptake of 2 Cl- coupled to an exchange of a potassium and Na+ ion into the cells. Therefore, there is little change of cell membrane potential in the action of NKCCs. In this study the localization and function of EAATs in the distal retina is investigated. Whole cell patch clamp recordings in lower vertebrate retina have demonstrated that EAAT2 is the main synaptic EAATs in rod photoreceptors and it is localized to the axon terminals. Furthermore, the action of the transporter seems to be modified by intracellular calcium concentration. There is also evidence that EAAT2 might be regulated by feedback from the neuron network by glycinergic and GABAergic mechanisms. The second half of this study investigates expression of NKCCs in the retina by western blot analysis and quantitative polymerase chain reaction. There are two forms of NKCCs, NKCC1 and NKCC2. NKCC1 is mostly expressed in the central nervous system and NKCC2 was thought to only be expressed in the kidneys. NKCC1 is responsible for the majority of chloride uptake into neuronal and epithelial cells and NKCC1 is expressed in the distal retina where photoreceptors synapse on second order horizontal and bipolar cells. This study found the expression of NKCC1 in the distal retina to be regulated by temporal light and dark adaptation. Light adaptation increased phosphorylated NKCC1 expression (the active form of the cotransporter). The increase in NKCC1 expression during light adaptation was modulated by dopamine. Specifically, a D1 receptor agonist increased phosphorylated NKCC1 expression. Dopamine is an essential chemical and receptor known for initiating light adaptation in retina. Finally, an NKCC1 knockout mouse model was examined and it revealed that both forms of NKCC are expressed in the vertebrate retina. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection

published_or_final_version / Anatomy / Master / Master of Philosophy

[Truncated abstract] During development of the visual system, topographic connections between the retina and the superior colliculus are established using guidance molecules. The EphA family of tyrosine kinase receptors and their ephrin-A ligands are important for establishing topography between the temporo-nasal axis of the retina and the rostro-caudal axis of the superior colliculus. After injury to the visual system via unilateral optic nerve transection, adult mammalian retinal ganglion cells fail to regenerate axons spontaneously to their main visual centre, which in rodents, is the superior colliculus. The EphA5 gradient is down-regulated from a temporalhigh to nasallow gradient to a uniform low level in the few surviving retinal ganglion cells, but ephrin-A2 is upregulated back to a significant rostrallow to caudalhigh gradient in the superior colliculus, similar to that seen during development. In this thesis, a number of experiments have been undertaken to investigate further how EphA5 and ephrin-A2 are regulated after injury and how they may play a role once regeneration has been encouraged through surgical intervention. In the first study, targeted unilateral retinal laser lesions were used to ablate either dorso-nasal or ventro-temporal quadrants of the retina. ... Surviving and regenerating retinal ganglion cells in the retina, and axons in the optic nerve, were analysed. The data suggest that metallothionein-I/II increases axonal regeneration through the optic nerve injury site but, at the dose administered, had no neuroprotective effects on retinal ganglion cells. This thesis provides further insight into the response of guidance molecules to injury, and the potential of metallothionein-I/II as a neuroregenerative factor in the adult mammalian visual system. The regulation of both EphA5 and ephrin-A2 through transsynaptic connections may be a response common to other guidance molecules. Such connectivity now needs to be studied further to understand how it may impact on various treatments designed to increase re-connectivity after other brain injuries, including stroke. The ectopic expression of ephrin-A2 at the insertion site of a peripheral nerve graft in the superior colliculus, implicate this guidance molecule in the glial scar for the first time. Therefore, to overcome inhibition by the glial scar, axons must also overcome ephrin-A2 mediated inhibition, potentially by the addition of EphA5 fusion proteins. Metallothionein-I/II?s effect of increasing axonal regeneration through the optic nerve injury site suggests that it could be used to increase the number of regenerating axons reaching their target. Such strategies to increase the absolute number of regenerated axons should enable these axons to better use the EphA5 and ephrin-A2 topographic gradients to optimize regenerative success.

Diabetic retinopathy is an ischemic retinal neovascular disease causing vision loss among adults. The studies presented involve the design and testing of a gene therapy vector to inhibit retinal revascularization, similar to that found in diabetic retinopathy. Gene therapy has proven to be an effective method to introduce therapeutic proteins to treat retinal diseases. Targeting a specific cell type and expression of therapeutic proteins according to the tissue microenvironment should have an advantage over traditional gene therapy by avoiding unwanted transgene expression. Hypoxia plays a significant role in the pathophysiology of many retinal ischemic diseases. Retinal Mèuller cells provide structural and functional support to retinal neurons, as well as playing a significant role in retinal neovascularization. Targeting Mèuller cells may be an effective strategy to prevent retinal neovascularization under pathological conditions. ... The hypoxia regulated, glial specific vector successfully reduced the abnormal neovascularization in the periphery by 93% and reduced the central vasobliterated area by 90%. A substantial amount of exogenous endostatin was produced in the retinas of P17 OIR mice. A significant increase in human endostatin protein and reduced vascular endothelial growth factor (VEGF) were identified by Western blot and ELISA, respectively. These findings suggest hypoxia-regulated, glial cell-specific scAAV mediated gene expression may be useful to prevent blindness found in devastating retinal diseases involving neovascularization. / by Manas Ranjan Biswal. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.