Eye / Vision
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/31256
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Browsing Eye / Vision by Subject "Glaucoma"
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Item COMPLEMENT AND GLIAL ACTIVITY IN THE RETINOCOLLICULAR PATHWAY OF MICE USING A NOVEL MODEL OF GLAUCOMA(2013-04-12) Silverman, SeanPurpose: Glaucoma is a leading cause of irreversible visual impairment and blindness throughout the world. C1q is responsible for axonal pruning in early ocular development and is upregulated in glaucomatous eyes of mice, non-human primates, and humans. We used an inducible mouse model of human primary open angle glaucoma with elevated intraocular pressure (IOP) to examine expression levels of C1q in the retina and superior colliculus (SC), as well as identify changes in cellular homeostasis. Methods: Anesthetized A/J mice were given a single intravitreal injection of Ad5.MYOC.Y437H (5x107 pfu), a mutant glaucoma gene, or Ad5.null control virus. Following injections, conscious IOPs were measured weekly, using a TonoLab tonometer (iCare). Mice were sacrificed at time points between 3 days and 8 weeks. Brains and retinas were harvested for immunofluorescence or immunoblotting studies. Microglia and astrocytes cells were identified using Iba1 and GFAP, respectively. All quantifications were performed using ImageJ Analysis software(NIH). Results: IOPs were significantly increased in the Ad5.MYOC.Y437H eyes (p<0.01) compared to the contralateral un-injected eye and eyes receiving Ad5.null. Clq expression was significantly upregulated in retinas receiving Ad5.MYOC.Y437H (2.69-fold±0.38, p<0.0001) compared to contralateral control retinas (0.7-fold±0.29). Clq upregulation was additionally observed in SC hemispheres receiving neural connections from injected eyes. Mice given Ad5.null vector displayed no elevation of Clq in the visual axis. Additionally, colocalization studies demonstrated significant increases of inner retinal microglia density beginning 2 weeks post injection (0.61%±0.07, p<0.001) and continuing at 4 weeks (0.87%±0.09, p<0.0001) compared to untreated retinas (0.4l%±0.03 and 0.44%±0.03, respectively). No signs of astrogliosis were detected. Conclusions: C1q is actively upregulated in the retina and SC, following mutant myocilin induced ocular hypertention, whereas adenovirus alone had no effect. An increased microglial population in the retina accompanied these changes. This suggests that microglia may sense the increased IOP and play a role in upregulating endogenous C1q. Early glaucoma pathogenesis may result from the reactivation of the ocular developmental roles of C1q and microglia, suggesting new therapeutic targets for future neuroprotective studies.Item DETERMINATION OF PROTEINS INVOLVED IN THE FORMATION OF CROSS-LINKED ACTIN NETWORKS IN THE TRABECULAR MESHWORK(2013-04-12) Bermudez, Jaclyn Y.Purpose: Glaucoma is a leading cause of blindness worldwide and the primary risk factor of glaucoma is increased intraocular pressure (IOP). IOP is determined by the equilibrium of aqueous humor (a fluid that fills the anterior segment of the eye) production and outflow. In glaucoma patients, the outflow resistance through the trabecular meshwork (TM), a special tissue located at the angle between the cornea and iris, is abnormally elevated. Inside TM cells, actin proteins form cross-linked actin networks (CLANs). Excessive formation of these unique structures can be found in the glaucomatous TM. They can also be induced by dexamethasone (DEX) and TGFβ2. It is suggested that CLANs increase cell rigidity and therefore elevate aqueous humor outflow resistance. However, the proteins that are involved in CLANs formation are not fully identified. We hypothesize that by comparing CLANs enriched and un-enriched TM cells, we will be able to identify the proteins that are involved in CLANs formation. Methods: We treated cultured mouse TM cells with DEX (100 nM), TGFβ2, (5 ng/mL), or ethanol (as vehicle control), evaluated CLANs formation, and separated the cell proteins by 2D gel electrophoresis. Differentially expressed proteins were analyzed by the Redfin software, and gel spots were picked and assessed by spectrometry analysis. Western blotting was used to confirm 2D gel results. Results: We found a subset of proteins that are differentially expressed in CLANs-enriched TM cells, compared to non-enriched samples. Among these proteins, ferritin heavy chain, glial fibrillary acidic protein (GFAP), and integrin alpha V, were confirmed by mass spectrometry and Western immunoblot. Conclusions: We found a subset of proteins that are differentially expressed in CLANs-enriched mouse TM cells. Their contributions and involvements in CLANs formation and regulation of TM cell morphology and functions are being investigated. They may provide new insight of the pathogenesis of glaucoma.Item EFFECT OF CELLULAR AND PLASMA FIBRONECTIN ISOFORMS ON NORMAL HUMAN TRABECULAR MESHWORK CELLS(2013-04-12) Medina-Ortiz, Wanda E.Purpose: The expression of cellular (cFN) and plasma (pFN) fibronectin isoforms are induced by TGF-β2 in human trabecular meshwork (HTM) cultured cells. Expression of specific FN isoforms can alter ECM homeostasis, ECM-cell interactions, and gene expression. Our purpose is to determine cFN levels in HTM tissues and to explore the impact of FN isoforms on HTM cells by studying changes in adhesion, cytoskeletal organization and gene expression. Methods: Differences between cFN levels in normal (NTM) and glaucomatous (GTM) tissues were obtained by immunohistochemistry. NTM cell strains were cultured for 24-48 hrs on surfaces coated with cFN or pFN, and the responses were compared to PBS controls. Changes in formation and redistribution of F-actin fibers and adhesion proteins were analyzed by phalloidin staining, Western immunoblots, and immunocytochemistry. Gene expression changes were analyzed using PCR arrays. Results: GTM tissues exhibited significantly greater cFN levels (1.7-fold, p<0.05). NTM strains exposed to both FN isoforms showed increased F-actin formation and redistribution; however, the F-actin pattern and distribution was different between cFN and pFN. Similarly, adhesion molecules such as talin, vinculin, paxillin and integrin beta 1 were increased and redistributed. Both FN isoforms changed gene expression, including alpha-smooth muscle actin-2, metalloproteases and their inhibitors, inflammatory cytokines, and TGF-β related genes. Conclusions: Our results show that GTM tissues expressed more cFN and that NTM cells respond differently depending on the FN isoform. The relationship between TGF-β2 modulation of FN isoform expression and the effect of FN isoforms on NTM cells suggests that this type of ECM remodeling may contribute to the TM changes associated with glaucoma.Item EFFECTS OF TGF-BETA2, FOLLISTATIN AND ACTIVIN A ON EXTRACELLULAR MATRIX IN NORMAL HUMAN TRABECULAR MESHWORK CELLS AND TISSUES.(2013-04-12) Fisher, AndrewPurpose: Primary open angle glaucoma (POAG) is characterized as a group of eye diseases resulting in optic nerve head damage and irreversible blindness. A major risk factor for developing POAG is increased intraocular pressure (IOP) leading to decreased outflow of aqueous humor (AH) through the trabecular meshwork (TM). Transforming growth factor-beta2 (TGF-β2) is increased in the AH of glaucoma patients, and causes increased extracellular matrix (ECM) protein synthesis in the TM. Bone morphogenetic protein-4 (BMP-4) has been shown to inhibit TGF-β2 actions. Follistatin (FST) is an antagonist of BMP-4 and is elevated in the glaucomatous TM. Elevated levels of FST in the TM may block BMP-4 ability to attenuate TGF-β2 induction of ECM proteins. FST may also have a direct role in regulating ECM protein expression in human TM (HTM) cells. HTM cells also express Activin A (Act A). The purpose of this study was to assess the role of FST and Act A in HTM cells as related to TGF-β2/BMP-4 signaling. Understanding these interactions may provide possible new therapeutic targets for the treatment of glaucoma. Methods: Normal HTM cell lines were cultured and treated with TGF-β2 (5ng/ml), FST-315, FST-288, or Act A (each at 50ng/ml) alone and/or simultaneously for 24 and 48 hrs. Western blot analysis was used to evaluate the effects of FST-315/288, Act A, and TGF-β2 on ECM protein synthesis including fibronectin (FN), PAI-1, and collagen1A. Results: TGF-β2 induced expression of PAI-1 and FN.. ACT-A mildly induced PAI-1 and FN proteins as compared to TGF-β2. TGF-β2 and Act A treatment appeared to have a synergistic effect on the expression of PAI-1 and FN protein as compared to individual treatment (Tx) of TGF-β2 or Act A. FST 288 does not seem to change the Act A + TGF-β2 synergism. FST 315 inhibits the synergism of Act A + TGF-β2 showing a decrease in PAI-1 and FN protein. Conclusions: FST-315 decreased the induction of ECM proteins by TGF-β2 and Act-A. FST-288 increased induction of ECM proteins in cells treated with TGF-β2 and Act A. FST-288 treatment for 24 hours induced increased ECM proteins PAI-1 and FN, but there was no induction at 48 hours. FST-315 treatment for 24 hours slightly induced ECM PAI-1 and FN, but there was no induction at 48 hours. Act A treatment for 24 and 48 hours increased induction of PAI-1 or FN. These results further our knowledge of the potential role of BMP antagonists in the human TM and their potential roles in the pathogenesis of glaucoma.Item ENDOTHELIN A RECEPTOR EXPRESSION IN RAT MODEL OF OCULAR HYPERTENSION(2013-04-12) McGrady, NolanPurpose: The endothelin system of peptides and their receptors have been implicated for their neurodegenerative role in glaucoma. The purpose of this study was to determine changes in ETA receptor expression in the retina in the Morrison's elevated IOP model of glaucoma in rats. Methods: IOP was elevated in the left eye of adult male retired breeder Brown Norway rats using the Morrison's model of glaucoma (by injection of hypertonic saline through episcleral veins) while the contralateral eye served as the corresponding control. The rats were maintained for two weeks following IOP elevation and sacrificed. Retinal sections were obtained from both the control and IOP-elevated eyes and analyzed for changes in ETA receptor expression using immunohistochemistry. ETA receptor immunostaining was co-localized with β-III-Tubulin, which is selectively expressed in retinal ganglion cells. Results: Rat eyes with IOP elevation showed an increase in immunostaining ETA receptor in several retinal layers including retinal ganglion cells, the inner plexiform layer, and the outer plexiform layer. An increased co-immunostaining of ETA receptors with β-III-Tubulin was observed both in retinal ganglion cells and inner plexiform layer. Conclusions: Elevated intraocular pressure results in an increase in ETA receptor expression. Increased endothelin receptor expression is associated with neurodegeneration in glaucoma.Item OVEREXPRESSION OF POU DOMAIN TRANSCRIPTION FACTOR, BRN3B CAUSES NEURITE OUTGROWTH IN CULTURED PC 12 CELLS(2013-04-12) Phatak, NitashaPurpose: Brn3b is a POU domain transcription factor shown to play key role in regulating retinal ganglion cell axon outgrowth during development of the retina. The purpose of this study was to determine if overexpression of Brn3b could promote neurite outgrowth in cultured PC 12 cells. Methods: Rat Pheochromocytoma cells ( PC 12) were seeded and grown on Poly-D-lysine coated 100 mm dish and transfected either with pCMV6-Brn3b (an expression vector encoding Brn3b cDNA) or pCMV6-MCS (empty vector). Medium changed to complete medium with NGF (100ng/ml) after 6 hours of transfection. Protein extracts were isolated from these cells and analyzed for Brn3b and GAP43, TUBA-1 protein expression in 24 hours by immunoblot analysis. In another set of experiments, PC 12 cells were seeded on Poly-D-Lysine coated 25mm cover slip and transfected with either pCMV6-Brn3b or pCMV6 -MCS. Medium changed to complete medium with NGF (100ng/ml) after 6 hours of transfection. Brn3b, GAP43 and TUBA-1 expression in 24 hours were analyzed by using immunocytochemistry in the transfected cells. Morphological changes in PC 12 cells transfected with Brn3b were studied by using confocal microscopy. Results: Immunoblot analysis showed overexpression of Brn3b in PC12 cells transfected with Brn3b cDNA. Overexpression of transcription factor Brn3b in PC12 cells produced morphological changes including increased neurite outgrowth. An increased immunostaining for Brn3b and neurite-specific GAP43, TUBA-1 were also observed in PC12 cells overexpressing Brn3b. Conclusions: The POU domain transcription factor, Brn3b, could promote neurite outgrowth in PC12 cells.