Eye / Vision
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21711
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Browsing Eye / Vision by Author "Bermudez, Jaclyn"
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Item Perfusion-cultured bovine anterior segments as a model for studying TGFB2-induced ocular hypertension and glaucoma(2016-03-23) Bermudez, Jaclyn; Clark, Abbot; Mao, Weiming; Wileman, JustinPurpose: Primary open angle glaucoma (POA) is a leading cause of blindness worldwide. The most important risk factor is elevated intraocular pressure (IOP), which is due to glaucomatous damage to the trabecular meshwork (TM). Damaged TM does not function properly and creates an obstruction to aqueous humor outflow, and therefore elevates IOP. Transforming growth factor beta 2 (TGFβ2) is elevated in about 50% POAG patients, and is known to increase IOP in several experimental models. The purpose of this study was to determine if TGFβ2 induces IOP elevation in perfusion cultured bovine eyes, which are an important glaucoma research model. Methods: Fresh bovine eyes were obtained, transferred to the lab and carefully dissected. Vitreous humor, uvea, retina, retinal pigment epithelium, and lens were removed. The remaining anterior segment tissue, which contained the sclera, cornea, and TM, was mounted and sealed on a custom-made plexiglass dish with an O-ring using four screws. Perfusion medium was infused by a syringe pump at a constant infusion rate of 5 ul/min. After baseline IOP was established, bovine eyes were perfused with or without 10ng/ml TGFB2 for up to 7 days. IOP was recorded by a pressure transducer and a computerized system. Changes in IOP were calculated by subtracting baseline IOP from IOP post treatment. Conditioned medium was collected for Western immunoblotting (WB). Results: TGFβ2 increased IOP in treated bovine eyes by about 4.5 mmHg while the fellow control eye did not show significant changes in IOP. WB showed that fibronectin, a TGFβ2-inducible protein, was increased in perfusate collected from the TGFβ2 treated eyes. Conclusion: Our study showed that TGFβ2 is able to induce ocular hypertension in perfusion cultured bovine eyes. This will provide researchers a useful model to study POAG.Item The role of canonical Wnt signaling and K cadherin in the regulation of intraocular pressure(2016-03-23) Bermudez, Jaclyn; Millar, J. Cameron; Clark, Abbot F.; Mao, Weiming; Webber, HannahPurpose: Primary open angle glaucoma (POAG) is the most prevalent form of glaucoma and has been associated with pathological changes in the trabecular meshwork (TM), the primary site of aqueous humor outflow in the eye. We have found that inhibition of canonical Wnt signaling in the TM raises intraocular pressure (IOP), and restoration of Wnt signaling normalizes IOP, though the mechanisms by which Wnt signaling maintains TM homeostasis are unknown. We hypothesize that the canonical Wnt signaling pathway in the TM regulates IOP via cadherins junctions. Materials and methods: We studied five cadherin isoforms abundant in the TM as shown by exome sequencing of normal and glaucomatous human TM (NTM and GTM, respectively) tissues. For in vitro studies, NTM cells (gift from Novartis) were treated with or without recombinant 100ng/ml Wnt3a or 1ug/ml sFRP-1 or both for 4-48 hours. Membrane protein fractions were isolated for western immunoblotting (WB) and probed for the cadherin isoforms. TM cells were also immunostained for cadherin isoforms or β-catenin. RNA was extracted from TM cells for cDNA synthesis and qPCR analysis of cadherins. Ad5.CMV recombinant adenoviruses encoding E cadherin, K cadherin, and/or sFRP-1 were injected unilaterally into the eyes of 4-6 month old female BALB/cJ mice (n=6). Conscious IOP of both eyes was then non-invasively measured for up to 35 days. Results: WB showed that Wnt3a TM cell membrane associated K-cadherin, which was inhibited with the addition of the Wnt antagonist sFRP-1. Immunostaining showed that -catenin accumulated on TM cell membrane upon Wnt3a treatment, and filopodia-like connections formed between TM cells. qPCR showed that Wnt3a also significantly increased K cadherin expression (n=3, p Conclusion: Our results suggested that cadherins play a role in the regulation of TM homeostasis and IOP via the Wnt signaling pathway.